JPH08242859A - Gene coding for protein phosphoenzyme derived from human mast cell - Google Patents

Abstract

PURPOSE: To obtain the subject gene useful for mass-producing a protein phosphoenzyme useful in screening of an antiallergic agent and diagnosis of various allergies. CONSTITUTION: This gene codes for a protein phosphoenzyme derived from a human mast cell, e.g. the one coding for the protein phosphoenzyme, derived from the human mast cell and having the substantially the same amino acid sequence as that of the formula. The gene having the amino acid sequence of the formula is obtained by preparing a polyA-RNA from a human mast cell strain KU812, then preparing a double-stranded cDNA based on the prepared polyA-RNA, inserting the resultant double-stranded cDNA into a λ phage vector, preparing a cDNA library, synthesizing a pair of primers based on the structure of a syk gene derived from a porcine B cell, providing a partial sequence of the cDNA according to a polymerase chain reaction (PCR) and isolating the gene from the cDNA library using the partial sequence as a probe.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a human mast cell-derived protein phosphatase gene.

[0002]

2. Description of the Related Art It is considered that an immediate (anaphylactic) allergy (or also called type I allergy) reaction in humans is caused by the following mechanism. When an allergic antigen invades the body, a specific IgE antibody against the antigen is produced in the body, and the produced IgE is bound through a specific receptor (FcεRI) existing on the surface of basophils and mast cells. To do.

[0003] When IgE bound to FcεRI specifically reacts with an antigen, receptor agglutination occurs, and active amines such as histamine accumulated in cells are released, followed by leukotriene, prostaglandin and various other amines. , The cytokines are released, and various allergic reactions are induced by these substances. It is no exaggeration to say that FcεRI is one of the key substances in this series of reactions, but what mechanism does the series of reactions proceed after the antigen binds to IgE bound to FcεRI? Not clear.

FcεRI is one molecule of α chain and one molecule of β
Chain exists on the cell membrane as a tetramer composed of two molecules of γ chain. It is known that the FcεRI aggregation occurs when the antigen binds to the FcεRI-bound IgE on the cell membrane. Intracellular phosphorylation by FcεRI stimulation has also been investigated, and Benhamou
Et al. Showed that the FcεRI and the 72 kD tyrosine phosphorylated protein crosslink (Proc. Nat.
ul.Acad.Sci.USA 87, p5327-5330 [1990]).

Furthermore, in addition to the 72 kD, phospholipase C-γ1 isoform (PLC
(γ1), FcεRIβ, and γ chain have also been revealed to be phosphorylated (Mol.Cell.Biol.12, p3176-3182 [199
2]). Since phosphorylation of these proteins is observed very early (within 10 seconds), protein tyrosine phosphatase (PTK) is of great importance in the signal transduction system upon antigen stimulation. However, the protein phosphorylase that plays a role in the signal transduction system by antigen stimulation has not been identified.

Identification of this protein phosphatase is required for elucidating the signal transduction system and developing antiallergic agents and allergy diagnostic agents corresponding thereto. 1 molecule of F
Among the α chain, 1 molecule of β chain, and 2 molecules of γ chain constituting cεRI, FcεRI γ chain is B-type other than mast cells.
It has been shown to be expressed on cells and is also associated with a low affine IgG Fc receptor (FcγRIIa). Furthermore, the γ chain is a cytotoxic T cell line (cytoto
xic T cell line (CTL) cell line, which forms a hetero-dimer with the ζ and η chains of the T cell receptor complex (TCR complex) (trans / splicing products of the ζ chain gene). , Ζ-γ
Hetero-dimers have been shown to associate with TCR (Nature 347, p189-191 [1990]), suggesting a common role in ζ, η, γ chain signaling. It was

That is, it is suggested that there is a molecule having a common function with the signal transduction pathway of the TCR complex. T cell receptor complex (TCR complex) (αβγδε ₂ ζ ₂ )
Although it has not yet been clarified as to the pathway by which the signal transduces, the ζ chain of TCR and phospholipase C (PLC) are known to be phosphorylated upon stimulation. ZAP-70 is a protein newly found as a phosphorylated protein that associates with the ζ chain of stimulated TCR (Proc. Natul. Acad. Sci. USA88, p9166).
-9170 [1991]). Since this association is observed only when a stimulus is applied and is a very early reaction that occurs within 15 seconds after the stimulus, it is considered to be one of the substances that play an important role in the TCR signal transduction system.

Recently, the ZAP-70 gene has been changed to Chan.
Etc. and its structure was clarified (Cell 7
1, 469-662 [1992]). However, expression of this product is not
Since it is cell-specific and does not exist in mast cells that cause allergic reaction, it is not considered that ZAP-70 is a protein kinase that plays a role in a signal transduction system by antigen stimulation.

The present inventors have confirmed that mast cells can be treated with ZAP-7.
To isolate a protein phosphatase with a structure similar to that of 0 and to isolate a gene encoding a protein phosphatase that is present in the poly-A RNA of mast cells in a very small amount. It succeeded and came to complete this invention.

[0010]

Accordingly, the present invention provides a gene system encoding a protein phosphorylating enzyme derived from human mast cells, and a method for producing the enzyme using the gene.

[0011]

The present invention provides a gene encoding a protein mastase derived from human mast cells, for example, an enzyme having an amino acid sequence substantially the same as the amino acid sequence shown in Sequence Listing: 3. The present invention further provides a method for producing protein phosphatase, which comprises culturing a host transformed with an expression vector containing the above gene and collecting protein phosphatase from the culture. The invention further provides an expression vector comprising said gene. The present invention also provides a host transformed with the expression vector.

[0012]

[Specific Description] In order to isolate the gene of the present invention,
PolyA RNA is prepared from any human mast cell producing the present enzyme, for example, human mast cell line KU812, by a conventional method. Based on this poly A RNA, double-stranded c
Prepare DNA. A cDNA library, for example, a KU812 cell cDNA library is prepared by inserting the prepared cDNA into a lambda phage vector.

CDNA of interest from this library
Appropriate probes are needed to obtain In the present invention, the following method is established and executed in order to obtain this suitable probe. That is, the gene to be obtained as described above is a gene encoding a protein kinase having a structure similar to that of ZAP-70. When a gene database was searched based on ZAP-70, ZAP-70 was found to be a syk gene derived from porcine B cells (J. Biol. Chem. 266, p15790-15796.
[1991]) was found to be very similar.
Therefore, a pair of primers was synthesized based on the structure of the syk gene, and the cD prepared by polymerase chain reaction (PCR reaction) using the primers.
The partial sequence is obtained from NA.

The target gene is obtained from the previously prepared cDNA library by using the obtained partial sequence as a probe. The determined sequence and the encoded amino acid sequence are shown in SEQ ID NO: 1. The obtained cDNA
The gene has a total length of 2541 base pairs and has one reading frame (o
a pen reading frame (ORF). The protein encoded by this ORF has a molecular weight of 69 kDa consisting of 612 amino acids.

Comparing the amino acid sequence from the nucleotide sequence of the obtained cDNA with the amino acid sequences from the ZAP-70 and porcine syk genes, the homology is 57% and 93%, respectively, and these proteins are very good. It became clear that it had a similar structure. When the amino acid sequence is analyzed in more detail, two src homology 2 (SH
2) It was found to have a domain and a kinase domain on the C-terminal side, and it became clear that this gene encodes a protein belonging to the so-called tyrosine kinase family.

[0016] Northern hybridization was carried out using the obtained gene as a probe to obtain KU812 cells or R
Since it reacts with poly A RNA prepared from BL-2H3B cells, it is clear that the obtained gene is expressed in mast cells. It can be confirmed as follows that the cDNA obtained as described above certainly encodes the target protein phosphorylating enzyme.

The isolated cDNA fragment is inserted into an appropriate expression vector. The host used for expression may be bacteria, yeast, mammalian cells, night-light moth cells, etc., but mammalian cells are preferable for assaying the function. Further, when mammalian cells are used, viral vectors such as vaccinia virus vector can be considered as the expression vector, but plasmid vectors having promoters such as SV40 promoter, modified SV40 promoter and Ad2 promoter, or Mul-cos vector. And the like are preferable in that the cells do not die.

The phosphorylated protein can be confirmed by detecting the expressed protein by Western method using an anti-phosphotyrosine antibody. That is, the DNA inserted into the expression vector is transferred to mammalian cells (COS cells, C
HO cells), calcium phosphate method, DEAE-Dextr
It is introduced by a method such as an method or electroporation. After introduction 4
After culturing for 8 hours, the introduced cells are disrupted. The extract obtained by crushing is fractionated by SDS polyacrylamide gel electrophoresis, and after fractionation is completed, the fractionated protein is transferred to a nitrocellulose membrane or a nylon membrane.

The transferred membrane is reacted with an anti-phosphotyrosine antibody, and the membrane-bound anti-phosphotyrosine antibody
Detect with secondary antibody. Due to the presence of the 69 kd band that is specifically detected in the introduced cells, the introduced cDNA
It is confirmed that the A fragment encodes a phosphorylated protein. Furthermore, it can be confirmed that the introduced cDNA fragment has protein phosphatase activity by the plurality of bands which are detected specifically in the introduced cells.

Furthermore, the fact that this gene is involved in signal transduction can be shown by the following method. The expression vector having the gene inserted therein is introduced into a cell into which a high affinity receptor gene has been introduced (Blank et al., Natu.
re 337, p187-189). The cells into which this receptor gene has been introduced have the ability to bind to IgE, but do not cause a series of phosphorylation reactions and changes in calcium concentration caused by IgE. An expression vector into which the gene fragment of the present invention has been inserted is introduced into this cell,
After reacting the obtained transformant with IgE, it is shown that the phosphorylated protein is involved in signal transduction by examining the phosphorylated protein by the above-mentioned Western method or intracellular calcium concentration. Is possible.

The gene of the present invention has, for example, the sequence listing: 3
However, the present invention is not limited to this. That is, the gene of the present invention also includes a gene consisting of an arbitrary codon encoding the amino acid sequence shown in SEQ ID NO: 3. Furthermore, the gene of the present invention is not limited to the one encoding the amino acid sequence shown in SEQ ID NO: 3, and the gene encoding the amino acid sequence substantially the same as the amino acid sequence shown in SEQ ID NO: 3 is also the gene of the present invention. . Here, the amino acid sequence substantially the same as the amino acid sequence shown in SEQ ID NO: 3 means the same amino acid sequence as the amino acid sequence shown in SEQ ID NO: 3, or the modified amino acid sequence shown in SEQ ID NO: 3. Sequences that still maintain the desired biological activity, ie, protein phosphorylation activity.

Here, amino acid modification includes removal of one or more amino acids, substitution of one or more amino acids with other amino acids, and addition of one or more amino acids.
As well as combinations thereof. Here, the number of amino acids involved in the modification is, for example, the number of site-directed mutagenesis (site-directed mutagenesis).
s) and the like can be modified according to a conventional method, for example, 1
The number is about 15 and more preferably about 1 to 10.

The gene encoding the modified amino acid sequence can be obtained by subjecting the DNA encoding the amino acid sequence set forth in SEQ ID NO: 3 to conventional mutagenesis means such as site-directed mutagenesis, restriction enzyme cleavage and ligase. Linking,
It can be prepared by applying a conventional gene manipulation means such as shortening by exonuclease, addition of oligonucleotide, and the like. The present invention also includes a gene that can hybridize with the base sequence shown in SEQ ID NO: 3 and encodes a protein having a desired protein phosphorylating enzyme activity.

In the present specification, a method for cloning a cDNA encoding a target enzyme will be described in Example 1.
However, the method for producing the gene of the present invention is not limited to this. That is, once the amino acid sequence of a gene is determined or designed, the base sequence can be designed based on it and chemically synthesized according to a conventional method.

The gene of the present invention can also be obtained by screening genomic DNA from human mast cells using, for example, a DNA having the nucleotide sequence set forth in SEQ ID NO: 3 or a fragment thereof as a probe. If a probe is available, genomic DNA encoding the enzyme of interest
Can be obtained by a conventional method. For example, extracting genomic DNA from human mast cells, fragmenting it with, for example, one or more kinds of restriction enzymes, screening the generated fragments with a probe, and comparing the nucleotide sequence of the positive clone with the nucleotide sequence of Sequence Listing: 3. The target gene can be confirmed by.

The present invention also provides an expression vector containing the above gene, a host transformed with the expression vector, and a method for producing a target protein phosphorylating enzyme using the host. For this purpose, prokaryotic hosts and eukaryotic hosts can be used as hosts. Examples of prokaryotic hosts include bacteria such as Escherichia coli ( Escheme).
richia coli ), Bacillus
s ) microorganisms such as Bacillus subtilis ( Baci)
illus zubtilis ) or the like can be used. Eukaryotic hosts include lower eukaryotes such as yeast,
For example, Saccharomyces (Saccharomyces)
Yeasts such as Saccharomyces cerevisiae ( Sacc
haromyces cerevisiae ) and the like.

As a higher eukaryotic host, animal cells,
For example, CHO cells, Hela cells, COS cells and the like can be used. Further, insect cells such as silkworm cells such as night lantern moth cells can be used. In addition, adult insects can also be used.

As the expression vector, a plasmid, phagemid, phage, virus or the like can be used depending on the host. For example, plasmids or phagemids are used for bacterial hosts, plasmids are used for yeasts, and viruses such as vaccinia virus, baculovirus, etc. are used for animal or insect cells.

The expression vector contains, in addition to the structural gene encoding the protein phosphorylating enzyme, an expression control region operably linked to the structural gene, such as a promoter, an enhancer and a terminator. . Examples of bacterial promoters include:
Tryptophan operon, Tac promoter, Trc
Used promoter is, for yeast promoters, for example, TDH ₃ promoter, ADHI promoter, GALI-7 promoter, PGK promoter, etc. are used, as the animal cell promoter such as, SV40 promoter, Ad2 promoter, vaccinia 75K A promoter or the like is used, and as the insect promoter, for example, a polyhedrin promoter or the like is used.

The culture of the transformed host and the invention of the target gene can be carried out according to a conventional method depending on the host, promoter and the like used. In addition, isolation and purification of the target enzyme from the culture can be performed by a conventional method for isolating and purifying the enzyme, such as filtration, centrifugation, salting out, column chromatography, electrophoresis, affinity chromatography, etc. It can be done by combining.

[0031]

EFFECTS OF THE INVENTION By using the gene of the present invention, protein phosphatase can be easily produced in a large amount as described above, and the enzyme can be used to screen various antiallergic agents. become. It is also useful for diagnosing various allergies. Also, the obtained gene is artificially Fc
By introducing into cells expressing εRI, allergic reaction model cells can be prepared, and by using this, it becomes possible to screen antiallergic agents and diagnose various allergies.

Further, by using the protein phosphatase encoded by the obtained gene, it becomes possible to identify the protein which is the substrate of this enzyme. In addition, it becomes possible to purify the substrate by using a column in which protein phosphatase is combined, and by using this found substrate, development of anti-allergic agents and diagnostic agents for various allergies can be achieved. It becomes possible to carry out development.

[0033]

The present invention will be described in more detail with reference to the following examples. Example 1 Preparation of KU812 cell cDNA library Human mast cell line KU812 was supplemented with 10% fetal bovine serum R
The cells were cultured in PMI-1640 medium (Gibco Oriental Lifetech) to obtain 4 × 10 ⁷ cells. From the obtained cells, mRNA Separator Kit (Clonte
ch company) according to the method recommended by the manufacturer.
NA was prepared to obtain 40 μg of poly A RNA.

Of these, 3.6 μg was used and TimeSa
Using the cDNA ver. cDNA synthesis kit (Pharmacia), using oligo (dT) _12-18 as a primer, and cD according to the method recommended by the manufacturer.
NA was synthesized, and EcoRI was added to both ends of the synthesized cDNA.
150 μl of a dscDNA solution to which an adapter was bound was obtained.

25 μl and 10 μl of the obtained cDNA solution
4 μl (0.5 μg / μl) of dephosphorylated lambda phage vector (ZAPIEco-
CIP: Stratagene) was added, and 1/20 amount of 3M sodium acetate (pH 5.2) and 2.5 times amount of ethanol were added to precipitate DNA. Collect DNA by centrifugation and
l DNA ligase reaction solution (66 mM Tris-HCl
[PH 7.5], 6.6 mM MgCl ₂ , 10 mM DT
T, 1 mM ATP, 1 mM spermidine), and add 1 μl
T4 DNA ligase (Pharmacia) was added,
The reaction was carried out at 0 ° C for 3 hours. Using 4 μl of reaction solution, G
IGA-Pack Gold Kit (Stratagene)
In vitro packaging reaction was carried out by the method recommended by the manufacturer.

After the reaction is completed, a part of the reaction solution is added with maltose.
Added NZY medium (10 g NZ amine, 5 g Ye
ast extract, 5g NaCl, 2g Mg
SO _Four ・ 7H₂ O / ll), 10 mM MgSO 4
_Four To OD₆₀₀ E. coli X resuspended to be 1.0
By infecting L1-Blue (Stratagene)
Package prepared from 25 μl and 10 μl cDNA
4.6 × 10 in each aging reaction solution^Five , 3.
1 × 10^Five The presence of infectious phages
It became clear.

[0037] The plaque was formed by sprinkling so that the plaque was 2-3 x 10 ⁴ plaques per 150 mm dish. The two plates were combined into one, and phages were diluted from each with a phage dilution solution (50 mM Tris-HCl [pH 7.5],
10 mM MgCl ₂ , 100 mM NaCl, 0.02%
Gelatin), and a library containing about 5 to 6 × 10 ⁴ different recombinant phages was prepared.

Example 2 Preparation of Probe A primer having the following sequence was synthesized. SYKL1: 5′-TTCGGGACTG TGAAGAAGGG GTACTACCAG ATG-3 ′ (SEQ ID NO: 1) SYKL2: 5′-CGGAGCGTAC CATTTCACGG GCCACTTCCC GTG-3 ′ (SEQ ID NO: 2) Of the cDNA solution (150 μl) obtained in Example 1 A portion of 10 μl of the above SYKL1 primer,
Using SYKL2 primer, 100 μl of reaction solution (1
μM SYKL1 primer, 1 μM SYKL2 primer, 10 mM

Tris-HCl (pH 8.3), 50 mM
KCl, 1.5 mM MgCl ₂ , 0.001% gel
atin, 200 μM dATP, 200 μM dCT
P, 200 μM dGTP, 200 μM dTTP,
2.5U AmpliTaq DNA polymer
se [Takara Shuzo]) and prepare DNA Thermal C
Using a cycler, the reaction was performed at 94 ° C. for 30 seconds, 50 ° C. for 1 minute, and 72 ° C. for 2 minutes for 30 cycles.

A 40 μl portion of the reaction solution was applied to 1% agarose and electrophoresed. The separated DNA fragment of about 0.5 kb was separated from agarose using a GeneClean kit and 10 μl of TE solution [10 mM Tris-HC
1 (pH 7.5), 1 mM EDTA]. 0.5 μl (50 ng / μl) in 5 μl of recovered DNA solution
PGEM-T vector (Promega) and 1 μl of 10
× Ligation buffer (Promega), 1 μl T4
Add DNA ligase (Takara Shuzo), and the total amount is 10μ
The TE solution was added so that the amount became 1 and the reaction was carried out at 16 ° C. for 1 hour.

Using 3 μl of the reaction solution, Escherichia coli SURE strain (Stratagene) was subjected to the method of Hanahan [DNA clonin].
g: A practical approach (ed.DMGlover), vol.1, p109
-, IRCpress, (1885)] and X-G
By plating on L-amp plates containing al and selecting white colonies, colonies resistant to ampicillin and lacking β-galactosidase were selected.

Selected colonies were transferred to 2 × YT-amp medium (1.6% bacto-tryptone, 1% yeast extract, 0.
5% NaCl, 100 μg / ml) was used to culture
cPrep DNA miniprep kit (Prom
DNA according to the manufacturer's recommended method using ega)
Was prepared. Prepared DNA is DyeTeminato
r cycle sequence kit (Applied Biosystems) using M13M4 primer (Takara Shuzo) according to the conditions recommended by the manufacturer, and the reaction product is the DNA sequencer Model 3
It was analyzed using 73A to determine the partial base sequence of the inserted fragment, and it was confirmed that the target fragment was inserted.

1 ng of plasmid DNA whose sequence was confirmed
100 μl of reaction solution (1 μM SYKL1 primer, 1 μM SYKL2 primer, 10 mM Tris)
-HCl (pH 8.3), 50 mM KCl, 1.5 mM M
gCl ₂ , 0.001% gelatin, 200 μM
dATP, 200 μM dCTP, 200 μM dGT
P, 200 μM dTTP, 2.5U AmpliTa
q DNA polymerase [Takara Shuzo])
Using a Thermal Cycler, the reaction was carried out for 30 cycles of 94 ° C. for 30 seconds, 50 ° C. for 1 minute, and 72 ° C. for 2 minutes. The reaction solution was applied to 1% agarose and electrophoresed.

The separated DNA fragment of about 0.5 kb was separated from agarose using a GeneClean kit and 50 μl of TE solution [10 mM Tris-HCl (pH 7.5), 1
mM EDTA]. Recovered DNA from Photo
oGene DNA labeling Kit (New
It was labeled with biotin according to the method recommended by the manufacturer using England Biolab). After the labeling reaction, Nick Colu
The labeled DNA was purified using mn (Pharmacia) (800 μl). This was used as a probe.

Example 3 Isolation of cDNA and determination of nucleotide sequence Phage DNA was prepared from 0.6 ml of the prepared library.
Method using EAE-cellulose (TJ Silhav
y et al., “Experiments with gene fusions”, Cold
Spring Harbor laboratory, 1984), 50μ
1 of TE solution.

Using 5 μl of this DNA solution, 100 μl
Reaction solution (1 μM SYKL1 primer, 1 μM S
YKL2 primer, 10 mM Tris-HCl (pH
8.3), 50 mM KCl, 1.5 mM MgCl ₂ ,
0.001% gelatin, 200 μM dATP, 200
μM dCTP, 200 μM dGTP, 200 μM
dTTP, 2.5U AmpliTaq DNA polymerase (Takara Shuzo)) was prepared and DNA Thermal
Using Cycler, 94 ℃ for 30 seconds, 50 ℃
30 cycles of 1 minute at 72 ° C. and 2 minutes at 72 ° C.

The reaction solution was applied to 1% agarose and electrophoresed. As a result, it was confirmed that a DNA fragment of about 0.5 kb was amplified when the DNA recovered from the library 25 + 26 was used. Library 2
5 + 26 was infected with XL1-Blue and seeded at 5 × 10 ⁴ plaques per 150 mm dish (4
Plaques) were allowed to form. Hyd the formed plaque
Transfer to a bond-N membrane (Amersham), 0.5M
2 minutes with NaOH, 0.5M NaCl, 0.5M T
ris-HCl (pH 7.5), 0.5M NaCl 5
After processing for 0.1 minutes, 0.1 × SSC, 0.1M

After being thoroughly washed with NH ₄ OAc, it was air dried. After immobilizing DNA on the membrane by UV treatment, prehybridization solution (6 x SSC, 5 x Denh
alt's solution, 20 mM Tris-HCl [pH 7.
5], 50% formamide, 0.5% SDS, 100
μg / ml denatured salmon sperm DNA) and kept at 42 ° C. for 2 hours. The biotin-labeled probe was treated at 100 ° C. for 5 minutes, and then rapidly cooled to change to a prehybridization solution to which the denatured one was added, and the temperature was further kept at 42 ° C. overnight.

The membrane was treated with 2 × SSC, 0.5% SDS solution at room temperature for 15 minutes, 68 ° C. for 15 minutes, and then 0.1 × SSC,
After washing with 0.1% SDS solution at 68 ° C for 15 minutes and 68 ° C for 15 minutes, PhotoGene Detection
Biotin-labeled DNA hybridized to the DNA was detected using Kit (New England Biolab) according to the method recommended by the manufacturer. For exposure, HyperFil
mmP (Amersham) was used.

A plaque corresponding to the detected region was picked up, plaque was formed again, and the plaque was isolated by the hybridization method. Thus, the lambda phage in which the DNA fragment of interest is inserted, λ
I got syk11.

The isolated lambda phage was treated with XL1-blue.
A high titer phage solution was obtained by infecting e and incubating in NZY medium overnight at 37 ° C. Using this, R408 phage was used as a helper phage, and phagemid (Ph
Agemid) was collected. This is XL-1 blue
Escherichia coli strain syk11 having phagemid DNA was obtained by infecting Escherichia coli and culturing on an L-amp plate. From this strain, phagemid DNA, pKUsyk1
1 was collected.

5 μg of the recovered phagemid DNA was added to S
Cleavage with acI and XbaI, or KpnI and EcoRV, Kilo-Sequencing deletion
A deletion mutant was prepared using nkit (Takara Shuzo) according to the method recommended by the manufacturer. Deletion mutant phagemid D
NA for DyePrimer cycle sequence
Using ce kit (Applied Biosystems),
-21M13 primer or M13RP1 primer was used for reaction according to the conditions recommended by the manufacturer, and the reaction product was analyzed using a DNA sequencer Model 373A to determine the nucleotide sequence.

From the determined nucleotide sequence, it was revealed that a 2541 bp cDNA fragment was inserted into pKUsyk11. The cDNA region of the phagemid is shown schematically in FIG.

[0054]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 33 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA Sequence TTCGGGACTG TGAAGAAGGG GTACTACCAG ATG 33

SEQ ID NO: 2 Sequence Length: 33 Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Synthetic DNA Sequence CGGAGCGTAC CATTTCACGG GCCACTTCCC GTG 33

SEQ ID NO: 3 Sequence length: 2541 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Hypothetical: NO antisense: NO sequence GAGGAAGAGC CGCGGGCCCG GCGGCTGAGG CCACCCCGGC GGCGGCTGGA 50 GAGCGAGGAG GAGCGGGTGG CCCCGCGCTG CGCCCGCCCT CGCCTCACCT 100 GGCGCAGGTG GACACCTGCG CAGGTGTGTG CCCTCCGGCC CCTGAAGC 148 ATG GCC AGC AGC GGC ATG Gla Ala CAC Ser Gle Ala CAC Ser Glyta TTT TTC GGC AAC ATC ACC CGG GAG GAG GCA GAA GAT TAC CTG GTC 238 Phe Phe Gly Asn Ile Thr Arg Glu Glu Ala Glu Asp Tyr Leu Val 20 25 30 CAG GGG GGC ATG AGT GAT GGG CTT TAT TTG CTG CGC CAG AGC CGC 283 Gln Gly Gly Met Ser Asp Gly Leu Tyr Leu Leu Arg Gln Ser Arg 35 40 45 AAC TAC CTG GGT GGC TTC GCC CTG TCC GTG GCC CAC GGG AGG AAG 328 Asn Tyr Leu Gly Gly Phe Ala Leu Ser Val Ala His Gly Arg Lys 50 55 60 GCA CAC CAC TAC ACC ATC GAG CGG G AG CTG AAT GGC ACC TAC GCC 373 Ala His His Tyr Thr Ile Glu Arg Glu Leu Asn Gly Thr Tyr Ala 65 70 75 ATC GCC GGT GGC AGG ACC CAT GCC AGC CCC GCC GAC CTC TGC CAC 418 Ile Ala Gly Gly Arg Thr His Ala Ser Pro Ala Asp Leu Cys His 80 85 90 TAC CAC TCC CAG GAG TCT GAT GGC CTG GTC TGC CTC CTC AAG AAG 463 Tyr His Ser Gln Glu Ser Asp Gly Leu Val Cys Leu Leu Lys Lys Lys 95 100 105 CCC TTC AAC CGG CCC CAA GGG GTG CAG CCC AAG ACT GGG CCC TTT 508 Pro Phe Asn Arg Pro Gln Gly Val Gln Pro Lys Thr Gly Pro Phe 110 115 120 GAG GAT TTG AAG GAA AAC CTC ATC AGG GAA TAT GTG AAG CAG ACA 553 Glu Asp Leu Lys Glu Asn Leu Ile Arg Glu Tyr Val Lys Gln Thr 125 130 135 TGG AAC CTG CAG GGT CAG GCT CTG GAG CAG GCC ATC ATC AGT CAG 598 Trp Asn Leu Gln Gly Gln Ala Leu Glu Gln Ala Ile Ile Ser Gln 140 145 150 AAG CCT CAG CTG GAG AAG CTG ATC GCT ACC ACA GCC CAT GAA AAA 643 Lys Pro Gln Leu Glu Lys Leu Ile Ala Thr Thr Ala His Glu Lys 155 160 165 ATG CCT TGG TTC CAT GGA AAA ATC TCT CGG GAA GAA TCT GAG CAA 688 Met Pro Trp Phe His Gly Lys Ile Ser Arg Glu Glu Ser Glu Gln 170 175 180 ATT GTC CTG ATA GGA TCA AAG ACA AAT GGA AAG TTC CTG ATC CGA 733 Ile Val Leu Ile Gly Ser Lys Thr Asn Gly Lys Phe Leu Ile Arg 185 190 195 GCC AGA GAC AAC AAC GGC TCC TAC GCC CTG TGC CTG CTG CAC GAA 778 Ala Arg Asp Asn Asn Gly Ser Tyr Ala Leu Cys Leu Leu His Glu 200 205 210 GGG AAG GTG CTG CAC TAT CGC ATC GAC AAA GAC AAG ACA GGG AAG 823 Gly Lys Val Leu His Tyr Arg Ile Asp Lys Asp Lys Thr Gly Lys 215 220 225 CTC TCC ATC CCC GAG GGA AAG AAG TTC GAC ACG CTC TGG CAG CTA 868 Leu Ser Ile Pro Glu Gly Lys Lys Phe Asp Thr Leu Trp Gln Leu 230 235 240 GTC GAG CAT TAT TCT TAT AAA GCA GAT GGT TTG TTA AGA GTT CTT 913 Val Glu His Tyr Ser Tyr Lys Ala Asp Gly Leu Leu Arg Val Leu 245 250 255 ACT GTC CCA TGT CAA AAA ATC GGC ACA CAG GGA AAT GTT AAT TTT 958 Thr Val Pro Cys Gln Lys Ile Gly Thr Gln Gly Asn Val Asn Phe 260 265 270 GGA GGC CGT CCA CAA CTT CCA GGT TCC CAT CCT GCG TCC TCC CCT 1003 Gly Gly Arg Pro Gln Leu Pro Gly Ser His Pro Ala Ser Ser Pro 275 280 28 5 GCC CAA GGG AAC CGG CAA GAG AGT ACT GTG TCA TTC AAT CCG TAT 1048 Ala Gln Gly Asn Arg Gln Glu Ser Thr Val Ser Phe Asn Pro Tyr 290 295 300 GAG CCA GAA CTT GCA CCC TGG GCT GCA GAC AAA GGC CCC CAG AGA 1093 Glu Pro Glu Leu Ala Pro Trp Ala Ala Asp Lys Gly Pro Gln Arg 305 310 315 GAA GCC CTA CCC ATG GAC ACA GAG GTG TAC GAG AGC CCC TAC GCG 1138 Glu Ala Leu Pro Met Asp Thr Glu Val Tyr Glu Ser Pro Tyr Ala 320 325 330 GAC CCC GAG GAG ATC AGG CCC AAG GAG GTT TAC CTG GAC CGA AAG 1183 Asp Pro Glu Glu Ile Arg Pro Lys Glu Val Tyr Leu Asp Arg Lys 335 340 345 CTG CTG ACG CTG GAA GAC AAA GAA CTG GGC TCT GGT AAT TTT GGA 1228 Leu Leu Thr Leu Glu Asp Lys Glu Leu Gly Ser Gly Asn Phe Gly 350 355 360 ACT GTG AAA AAG GGC TAC TAC CAA ATG AAA AAA GTT GTG AAA ACC 1273 Thr Val Lys Lys Gly Tyr Tyr Gln Met Lys Lys Val Val Lys Thr 365 370 375 GTG GCT GTG AAA ATA CTG AAA AAC GAG GCC AAT GAC CCC GCT CTT 1318 Val Ala Val Lys Ile Leu Lys Asn Glu Ala Asn Asp Pro Ala Leu 380 385 390 AAA GAT GAG TTA TTA GCA GAA GCA AAT GTC ATG CAG CAG CTG GAC 1363 Lys Asp Glu Leu Leu Ala Glu Ala Asn Val Met Gln Gln Leu Asp 395 400 405 AAC CCG TAC ATC GTG CGG ATG ATC GGG ATA TGC GAG GCC GAG TCC 1408 Asn Pro Tyr Ile Val Arg Met Ile Gly Ile Cys Glu Ala Glu Ser 410 415 420 TGG ATG CTG GTT ATG GAG ATG GCA GAA CTT GGT CCC CTC AAT AAG 1453 Trp Met Leu Val Met Glu Met Ala Glu Leu Gly Pro Leu Asn Lys 425 430 435 TAT TTG CAG CAG AAC AGA CAT GTC AAG GAT AAG AAC ATC ATA GAA 1498 Tyr Leu Gln Gln Asn Arg His Val Lys Asp Lys Asn Ile Ile Glu 440 445 450 CTG GTT CAT CAG GTT TCC ATG GGC ATG AAG TAC TTG GAG GAG AGC 1543 Leu Val His Gln Val Ser Met Gly Met Lys Tyr Leu Glu Glu Ser 455 460 465 AAT TTT GTG CAC AGA GAT CTG GCT GCA AGA AAT GTG TTG CTA GTT 1588 Asn Phe Val His Arg Asp Leu Ala Ala Arg Asn Val Leu Leu Val 470 475 480 ACC CAA CAT TAC GCC AAG ATC AGT GAT TTC GGA CTT TCC AAA GCA 1633 Thr Gln His Tyr Ala Lys Ile Ser Asp Phe Gly Leu Ser Lys Ala 485 490 495 CTG CGT GCT GAT GAA AAC TAC TAC AAG GCC CAG ACC CAT GGA AAG 1678 Leu Arg Ala Asp Glu Asn Tyr Tyr Lys Ala Gln Thr His Gly Lys 500 505 510 TGG CCT GTC AAG TGG TAC GCT CCG GAA TGC ATC AAC TAC TAC AAG 1723 Trp Pro Val Lys Trp Tyr Ala Pro Glu Cys Ile Asn Tyr Tyr Lys 515 520 525 TTC TCC AGC AAA AGC GAT GTC TGG AGC TTT GGA GTG TTG ATG TGG 1768 Phe Ser Ser Lys Ser Asp Val Trp Ser Phe Gly Val Leu Met Trp 530 535 540 GAA GCA TTC TCC TAT GGG CAG AAG CCA TAT CGA GGG ATG AAA GGA 1813 Glu Ala Phe Ser Tyr Gly Gln Lys Pro Tyr Arg Gly Met Lys Gly 545 550 555 AGT GAA GTC ACC GCT ATG TTA GAG AAA GGA GAG CGG ATG GGG TGC 1858 Ser Glu Val Thr Ala Met Leu Glu Lys Gly Glu Arg Met Gly Cys 560 565 570 CCT GCA GGG TGT CCA AGA GAG ATG TAC GAT CTC ATG AAT CTG TGC 1903 Pro Ala Gly Cys Pro Arg Glu Met Tyr Asp Leu Met Asn Leu Cys 575 580 585 TGG ACA TAC GAT GTG GAA AAC AGG CCC GGA TTC GCA GCA GTG GAA Trp Thr Tyr Asp Val Glu Asn Arg Pro Gly Phe Ala Ala Val Glu 590 595 600 CTG CGG CTG CGC AAT TAC TAC TAT GAC GTG GTG AAC TAA CCGCTCCCGC 1997 Leu Arg Leu Arg Asn Tyr Tyr Tyr Asp Val Val Asn 605 6 10 ACCTGTCGGT GGCTGCCTTT GATCACAGGA GCAATCACAG GAAAATGTAT 2047 CCAGAGGAAT TGATTGTCAG CCACCTCCCT CTGCCAGTCG GGAGAGCCAG 2097 GCTTGGATGG AACATGCCCA CAACTTGTCA CCCAAAGCCT GTCCCAGGAC 2147 TCACCCTCCA CAAAGCAAAG GCAGTCCCGG GAGAAAAGAC GGATGGCAGG 2197 ATCCAAGGGG CTAGCTGGAT TTGTTTGTTT TCTTGTCTGT GTGATTTTCA 2247 TACAGGTTAT TTTTACGATC TGTTTCCAAA TCCCTTTCAT GTCTTTCCAC 2297 TTCTCTGGGT CCCGGGGTGC ATTTGTTACT CATCGGGCCC AGGGACATTG 2347 CAGAGTGGCC TAGAGCACTC TCACCCCAAG CGGCCTTTTC CAAATGCCCA 2397 AGGATGCCTT AGCATGTGAC TCCTGAAGGG AAGGCAAAGG CAGAGGAATT 2447 TGGCTGCTTC TACGGCCATG AGACTGATCC CTGGCCACTG AAAAGCTTTC 2497 CTGACAATAA AAATGTTTTG AGGCTTTAAA AAGAAAAAAA AAAA 2541

[Brief description of drawings]

FIG. 1 shows c in the phagemid pKUsyk11.
The DNA insert is shown.

 ─────────────────────────────────────────────────── ─── Continuation of front page (72) Inventor Chiyasu Rao 2-14-13 Hanazono, Hanamigawa-ku, Chiba-shi, Chiba

Claims (5)

[Claims] 1. A gene encoding a human mast cell-derived protein phosphorylating enzyme. 2. A gene encoding a human mast cell-derived protein phosphatase having an amino acid sequence substantially the same as the amino acid sequence shown in SEQ ID NO: 3. 3. An expression vector comprising the gene according to claim 1 or 2. 4. A host transformed with the expression vector according to claim 4. 5. A method for producing protein phosphatase, which comprises culturing a host transformed with the expression vector according to claim 4 and collecting protein phosphatase from the culture.