JPH1072495A - Immunity-related factor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new immunity-related factor comprising a polypepetide which participates in immune response having a specific amino acid sequence, specifically expressing in a lymph node tissue cell and useful for researches and treatment, etc., of immunological diseases related to immune response and infectious diseases. SOLUTION: This immunity-related factor comprises a new polypeptide which participates to immune response and contains an amino acid sequence represented by the formula and is useful for researches for and treatment of immunological diseases related to immune response and infections diseases, because the factor is specifically expressed in a lymph node tissue cell. The polypeptide is obtained by screening λ phage cDNA library of human lymph node tissue by plaque hybridization using cDNA specifically expressing in the lymph node as a probe, integrating a gene obtained from a positive clone into a vector to prepare a recombinant DNA, transducing the DNA into a host cell to transform the cell, culturing the transformant and expressing the gene.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to immune-related factors, and more particularly, to a novel polypeptide expressed by human-derived cells and a method for producing the same. The present invention also relates to the gene of the polypeptide cloned using genetic engineering techniques. The present invention also relates to a DNA fragment and an RNA fragment synthesized from the amino acid sequence of the polypeptide. The present invention also relates to a recombinant DNA comprising a DNA encoding the polypeptide produced by applying genetic engineering techniques and a vector DNA. The present invention also relates to a transformed cell that expresses the polypeptide produced by applying a genetic engineering technique. Furthermore, the present invention
The present invention relates to a method for producing the polypeptide by applying a genetic engineering technique. Furthermore, the present invention relates to an antibody that specifically binds to the polypeptide.

[0002]

2. Description of the Related Art The lymphatic system is composed of lymphocytes, epithelial cells and supporting cells, and forms an organ or tissue to carry out an immune reaction. The lymphoid organs create a primary lymphoid organ, where lymphocytes mature from lymphoid stem cells to differentiate and proliferate into functional cells, and an environment in which lymphocytes can react with each other or with antigens. They are classified as secondary lymphoid organs that serve to spread the immune response that has occurred.

[0003] Lymph nodes are one of the main organs that function as secondary lymphoid organs, and are located in the cortex and paracortex (T cell area).
And a central cord. Germinal centers (secondary follicles) and follicular dendritic cells (Follicular de
It is thought that Ndritic cells (FDC) are deeply involved in the induction of T cell-dependent antigen stimulation-specific proliferation and differentiation of B cells.

It is considered that cell-cell interaction is important for lymphocyte activation. In cell-cell interaction between lymphocytes, CD40 / CD4 together with cytokines is considered.
0L, CD80, CD86 and their ligand CD2
It is becoming clear that signals through adhesion molecules such as 8 play an important role. However, the mechanism of activation of lymph nodes, which are involved in lymphocyte activation as supporting tissues of lymphocytes, including the properties of tissues and cells, is not well understood.

[0005]

In order to control the immune response, it is important to analyze the cell-cell interaction between antigen-presenting cells and responsive cells. Although lymph nodes play an important role in the secondary immune response, the factors involved in cell-cell interactions between lymph node tissue cells and lymphocyte cells have not yet been elucidated. Accordingly, an object of the present invention is to provide a novel polypeptide which is specifically expressed in lymph node tissue cells and is involved in an immune response.

[0006]

Means for Solving the Problems Cloning of a gene specifically expressed in a specific tissue cell can be performed, for example, by a subtraction cloning method or a differential display method (P. Liangand AB Pardee,
Science, 257, 967, 1992), a differential screening method, and a cross-hybridization method using an appropriate probe.

[0007] In the subtraction cloning method, cDNA of a gene specifically expressed in a specific cell is obtained,
This is a method of cloning a gene by screening a cDNA library using the cDNA as a probe. That is, messenger RNA (mRNA) is purified from specific cells of interest, that is, lymph nodes,
Then, RNA is also extracted from control cells, that is, cells of the same species but different lineages, for example, peripheral blood, thymus, spleen, and the like.

Next, cDNA is synthesized with reverse transcriptase using mRNA purified from lymph nodes as a template. CDNA can be labeled by adding α- ³² PdNTP during synthesis. Labeled cDNA and mRN as template
A forms a stable double-stranded DNA-RNA hybrid.
Only RNA is degraded to purify single-stranded cDNA. This single-stranded cDNA is mixed with RNA extracted from control cells,
When allowed to stand under appropriate conditions, a stable double-stranded DNA-RNA hybrid is formed depending on the complementarity of the base sequence.

That is, mRNA which is also expressed in control cells
CDNA forms a hybrid while using as a template, but cDNA using mRNA specifically expressed in lymph nodes as a template remains single-stranded. The double-stranded DNA-RNA hybrid is separated from the single-stranded cDNA using a hydroxyapatite column, and only the single-stranded cDNA is purified. By repeating this step, cDNA specific to the target cell can be concentrated. Since the concentrated specific cDNA is labeled with a radioisotope, it can be used as a probe for screening a cDNA library.

In the differential display method, R
NAmapTM Kit (GenHunter Corp.)
Oration Co., Ltd.) according to the attached protocol. That is, an anchor primer [T1
2MG, T12MA, T12MT, T12MC (M:
A, G, C mixture)], and then use this cDNA as a template to randomize
PCR is performed in the presence of ³⁵ S using er primers (AP1 to AP20) and anchor primers.

The PCR product obtained here is separated by acrylamide gel electrophoresis. Identify bands strongly amplified in lymph nodes by comparing electrophoresis images obtained by performing the same operation on control cells, such as RNA extracted from peripheral blood, thymus, spleen, etc., with the above-mentioned electrophoresis images. Then cut out the gel at the band position. DNA is extracted from the excised gel fragment, the band is amplified by PCR using the DNA as a template, and a suitable vector such as pT7
The base sequence can be determined by subcloning into a Blue vector (Novagen).

The differential screening method comprises:
CDNA prepared from mRNA purified from target cells
The library was synthesized from the cells of interest mRNA ³²
In this method, screening is performed using a P-labeled cDNA probe and a probe prepared from mRNA of a control cell, and a clone that hybridizes only with the probe of the target cell is selected. That is, a cDNA library is prepared from mRNA purified from lymph nodes according to a conventional method. Two sets of replica filters are made from the library.

Next, cDNA is synthesized with reverse transcriptase using mRNA purified from lymph nodes as a template. Α during synthesis
The cDNA can be labeled by adding ³² PdNTP. Labeled cDNA and mRNA as template
Has formed a stable double-stranded DNA-RNA hybrid, but the mR
Only NA is degraded and single-stranded cDNA is purified. Similarly, single-stranded c labeled with ³² P using mRNA purified from control cells such as peripheral blood, spleen, and thymus as a template.
Make DNA.

Hybridization is performed with a filter prepared from a lymph node library using both labeled cDNAs as probes. By comparing the autoradiographic images of the X-ray film and selecting a clone that hybridizes only to the lymph node cDNA probe, a gene specifically expressed in the lymph node can be cloned.

In the cross-hybridization method, a cDNA library of a target cell is hybridized with an appropriate DNA as a probe under conditions of low stringency to obtain a positive clone. Using the obtained clone as a probe, Northern hybridization is performed to select a clone that is expressed only in the target cell. The present inventors attempted the above-described subtraction screening method and differential display method to obtain a novel polypeptide gene specifically expressed in the lymph node of the present invention, but did not result in cloning of the target gene. .

Next, for the following reasons: 1) CD40
L, 2) VCAM-1, 3) Htk ligand (hereinafter Ht ligand)
KL) (International Publication Number WO96 / 1121)
Screening by cross-hybridization of a lymph node cDNA library was attempted using the gene fragments of 2) as probes.

1) CD40L is a type II membrane glycoprotein belonging to the TNF superfamily (D. Holle).
nbaugh, et al. , EMBO J .; , 11,
4313, 1993). Expressed on activated T cells, mature B
It is thought that the interaction with CD40 expressed on cells transmits a costimulatory signal important in T cell-dependent B cell activation, and as a result, B cell proliferation and antibody production are induced. Therefore, it is conceivable that there is a factor associated with CD40L that is specifically expressed in the lymph node and is involved in the immune response.

2) VCAM-1 is an adhesion molecule which has a lymphocyte adhesion function and is expressed on vascular endothelial cells upon stimulation of cytokines, and is involved in leukocyte recirculation and infiltration of leukocytes into inflamed areas, and is constantly involved in FDC. Express and bind activated B cells to form lymphoid follicles and B in germinal centers
It is thought to be involved in the selection of cells (A.S.
Freeman, et al. , Science, 2
49, 1030, 1990). But on the other hand,
It was thought to play an important role in the inflammatory response of the living body, but because of low expression of vascular endothelial cells at the site of inflammation, it is also thought that it is not related to infiltration into the site of inflammation. Therefore, it is conceivable that there is a factor associated with VCAM-1 that is specifically expressed in lymph nodes and is involved in an immune response.

3) Htk-L is one of the tyrosine kinase ligands belonging to the Eph family,
Based on the finding that it is involved in the differentiation and proliferation of undifferentiated cells of the hematopoietic system, it is considered that the ligand family may be involved in the maturation and differentiation of immunocompetent cells.

As a result of screening using the above-mentioned gene fragment of CD40L and VCAM-1 as a probe, a plurality of positive clones were obtained, but a new clone which was specifically expressed in lymph nodes could not be obtained. But,
Screening of a lymph node cDNA library using a part of the Htk-L gene fragment as a probe was performed, 48 positive clones were isolated, and Northern hybridization was performed on each clone to analyze the expression tissues. The novel polypeptide gene of the present invention, which is specifically expressed in the appendix, was successfully cloned.

The present inventors immediately obtained a cDNA containing the full length of the polypeptide gene, determined the nucleotide sequence, and determined the full length amino acid sequence of the polypeptide. next,
An expression system for the polypeptide was prepared using the full-length cDNA of the polypeptide to obtain a recombinant polypeptide. Furthermore, an antibody was prepared using the recombinant polypeptide as an immunogen, a purification method was established, and the present invention was completed.

That is, according to the present invention, SEQ ID NO: 1 in the sequence listing
The novel polypeptide containing the amino acid sequence represented by these is provided. According to another aspect of the present invention, there is provided a novel polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1 and comprising the amino acid sequence represented by SEQ ID NO: 2. According to another aspect of the present invention, there is provided a DNA encoding a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing. According to another aspect of the present invention, a DN encoding a polypeptide containing the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing
A is provided.

According to still another aspect of the present invention, there is provided a nucleotide sequence containing a DNA encoding a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing, and a base sequence which can be expressed in host cells. A recombinant DNA body obtained by ligating with a vector DNA is provided. According to still another embodiment of the present invention, there is provided a nucleotide sequence containing a DNA encoding a polypeptide containing the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing, and a vector DNA that can be expressed in a host cell. Are provided. According to still another aspect of the present invention, a DN encoding a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing.
A cell transformed with a recombinant DNA obtained by ligating a base sequence containing A with a vector DNA that can be expressed in a host cell is provided.

Further, according to another aspect of the present invention, a nucleotide sequence containing a DNA encoding a polypeptide containing the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing, and a base sequence that can be expressed in host cells A cell transformed with a recombinant DNA obtained by ligating the vector DNA is provided.
Furthermore, according to another aspect of the present invention, cells capable of producing a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 in the sequence listing are cultured in a medium, and There is provided a method for producing a polypeptide, comprising producing a peptide, collecting a culture supernatant from a culture solution, and separating and purifying the polypeptide from the collected culture supernatant.

According to still another aspect of the present invention, cells capable of producing a polypeptide containing the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing are cultured in a medium, and the cells are collected. After the disruption, a method for producing the polypeptide comprising separating and purifying the polypeptide by immunoprecipitating the cell extract with an antibody characterized by specifically binding to the polypeptide. Provided. According to still another aspect of the present invention, there is provided an antibody that specifically binds to at least a part of a polypeptide containing the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing.

According to still another aspect of the present invention, there is provided a sense DNA comprising at least 18 consecutive nucleotide sequences of the nucleotide sequence of SEQ ID NO: 4 in the sequence listing,
Isolating a DNA fragment selected from the group consisting of an antisense DNA complementary to said sense DNA and said antisense DNA to methylate, methylphosphate, thiophosphate or deaminate, respectively. And an isolated DNA fragment selected from the group consisting of the sense DNA and the derivative of the antisense DNA.

According to still another aspect of the present invention, at least 18 nucleotides complementary to the nucleotide sequence of SEQ ID NO: 4 in the sequence listing are provided.
Antisense RNA containing a contiguous base sequence,
An isolated RNA fragment selected from the group consisting of a sense RNA complementary to the antisense RNA, and the antisense RNA and the sense RNA are respectively methylated, methylphosphated, thiophosphated or deaminated. The present invention provides an isolated RNA fragment selected from the group consisting of the antisense RNA and the derivative of the sense RNA.

The method for cloning the novel polypeptide gene of the present invention will be described in detail below. The gene for the novel polypeptide of the present invention can be cloned from a gene library of a tissue or cell producing the polypeptide, for example, a lymph node. A gene library is obtained by purifying mRNA from a target cell,
It can be prepared by synthesizing a single-stranded complementary DNA (cDNA) from NA and then a double-stranded DNA, and transforming the complementary DNA into a host using a phage or a plasmid.

RNA from human lymph nodes was obtained by the guanidine thiocyanate method (T. Maniatis et al.
l. , Molecular cloning 2nd
edition, Cold Spring Harbo
r Lab. , 1989). Next, mRNA is obtained according to a conventional method. On this occasion,
The mRNA Purification Kit (Pharmacia) can be used. Using the mRNA thus obtained as a template, the method of Okayama Berg (M
olecular and Cellular Bio
logic, 2,161, 1982 and the same magazine, 3, 28
0, 1983), and the resulting cD
Incorporate NA into plasmid.

As a plasmid incorporating the cDNA,
For example, pBR322, pUC18, pUC derived from Escherichia coli
19, pUC118 and pUC119 (all sold by Takara Shuzo Co., Ltd.), and any other types can be used as long as they can be replicated and propagated in the host. Examples of the phage vector into which the cDNA is incorporated include λgt10 and λgt11. Other phage vectors can be used as long as they can be propagated in the host. The plasmid thus obtained can be used in a suitable host, for example Escherichia (Esch
erichia), Bacillus
It is introduced into the genus using a calcium chloride method.

Examples of the genus Escherichia include Escherichia coli K12HB101, MC1061, LE
392 and JM105. Examples of the bacterium of the genus Bacillus include Bacillus subtilis MI114. The phage vector can be prepared, for example, by using an in vitro packaging method [Proc. N
atl. Acad. Sci. (1978) 71, 244
2].

According to the above method, c of the polypeptide of the present invention
A cDNA library of the polypeptide-producing cells containing DNA can be prepared. Methods for cloning DNA encoding the polypeptide from the cDNA library include, for example, a phage vector λg
The polypeptide cDNA library prepared using t10 and the Htk ligand gene (International Publication No. WO9
6/11212: deposit number FERM BP-5008)
EcoRI digested DNA fragment (SEQ ID NO: 13 in Sequence Listing)
Plaque hybridization method using DNA as a probe.

The expression tissues of the clones that have become positive in the above hybridization are analyzed by, for example, Northern blot hybridization, and clones that specifically express in the lymph nodes can be obtained. The nucleotide sequence of the DNA thus obtained is determined, for example, by the method of Sanger et al. [Sanger, F. et al. , Et al. Proc. Nat
l. Acad. Sci. (1977) 74, 5463].
Can be determined by As described above, a DNA encoding the polypeptide is obtained. The nucleotide sequence of the DNA containing the DNA encoding the polypeptide is shown in SEQ ID NO: 4 in the sequence listing.

This DNA sequence had an open reading frame capable of encoding a 532 amino acid sequence from the start codon (ATG) starting at position 98 to the stop codon (TGA) ending at position 1696. The amino acid sequence translated from the open reading frame is shown in SEQ ID NO: 2 in the sequence listing. The amino acid sequence is
-Doolittle's method (J. Mol. Biol. 1)
57: 105, 1982), the hydrophobic portion and the hydrophilic portion were analyzed from the amino acid sequence. As a result, the polypeptide of the present invention was expected to be expressed on cells as a cell membrane protein. The novel polypeptide cD
A transformant obtained by introducing the plasmid p # 53-4 containing the entire nucleotide sequence of NA into E. coli JM109 (manufactured by Toyobo) was named JM109-pUCLIC. This transformant strain was sent to E.I.
coli: JM109-pUCLIC, 1996
Deposited on 30th October, Accession No. FERM P-15
921.

That is, according to the analysis results, the amino acid sequence of the polypeptide is a signal peptide composed of 16 amino acids corresponding to the -16th to -1st amino acids of the amino acid sequence of SEQ ID NO: 2 in the sequence listing, and the sequence of the sequence listing. Extracellular portion composed of 436 amino acids corresponding to Nos. 1 to 436 of the amino acid sequence of No. 2; transmembrane portion composed of 23 amino acids corresponding to Nos. 437 to 459 of the amino acid sequence of SEQ ID No. 2 in the sequence listing; Of the amino acid sequence of SEQ ID NO: 2 consisting of 57 amino acids corresponding to positions 460 to 516. However,
Each part is a domain configuration predicted from the amino acid sequence to the last, and it is sufficiently considered that the actual form on a cell and in a solution may be slightly different from the above-described configuration. It is also conceivable for the amino acids to be around 5 to 10 amino acid sequences.

The amino acid sequence set forth in SEQ ID NO: 3 in the Sequence Listing is an amino acid sequence corresponding to the 50th to 159th amino acids in the amino acid sequence of SEQ ID NO: 2 in the Sequence Listing.
The amino acid sequence of the domain having the highest homology to the g receptor is shown. In addition, the amino acid sequence described in SEQ ID NO: 1 in the sequence listing corresponds to the amino acid sequence of SEQ ID NO: 2 in the sequence listing.
The amino acid sequence of the portion corresponding to No. 428 to No. 428, that is, the amino acid sequence of the above-mentioned extracellular portion excluding the signal peptide is shown.

As expected from the amino acid sequence,
The portion to which the sugar chain is added is a portion to which N-acetyl-D-glucosamine can be N-glycoside-bonded, and examples thereof include asparagine residues 151 and 260 in the amino acid sequence of SEQ ID NO: 2 in the sequence listing. Also, N-acetyl-D-
The portion where the O-glycosidic bond of galactosamine is estimated may be a frequently occurring portion of a serine or threonine residue. Particularly, as a highly probable portion, a serine residue at position 160 in the amino acid sequence of SEQ ID NO: 2 in the sequence listing is considered. Groups. It is considered that the protein to which these sugar chains have been added is generally more stable to degradation in vivo than the polypeptide itself, and has stronger physiological activity.

The gene sequence and amino acid sequence of the polypeptide disclosed in the present invention were
bank, October 1995, release 91), the most similar substance was poly I
g receptor (polymeric immunogl)
obulin receptor) (Mostov,
K. E. FIG. et al. Nature, 308, 37, 1
984: Krajci, P .; et al. , Bioc
hem. Biophys. Res. Commun. Fifteen
8, 783, 1989, Jean-Pierre, Patent Publication No. 5-501118), but no significant homology was found as the full length, and the most similar part was No. 50 in SEQ ID NO: 2 in the sequence listing. And the region corresponding to No. 159, but the homology in this region was extremely low at about 45%.

Therefore, the polypeptide of the present invention is a completely novel substance. Also, Htk-L used as a probe
The homology of the molecule to the cDNA is shown in SEQ ID NO: 4
Nos. 511 to 700 found only about 47%, and had only 19% homology as an amino acid.

The poly Ig receptor in which this similarity was found is a transmembrane protein having five immunoglobulin-like domains, and a dimeric Ig in the extracellular first domain.
Binds to A and takes it up into epithelial cells. Next, secretory IgA is secreted into the intestinal lumen by moving to the intestinal lumen side of the epithelial cells as intracellular transport vesicles and merging with the intestinal side cell membrane. Upon secretion, the poly-Ig receptor is cleaved near the cell membrane by proteolytic enzymes and secretory components (secr
ethory component (SC) as IgA
And secretory IgA together with the J chain. Secreted IgA
Exerts a protective function in the intestinal mucosa by the mechanism described below. That is, the secretory IgA easily causes cross-linking with the specific antigen, and as a result, the movement and growth of the microorganism to which the secretory IgA is bound are remarkably suppressed.

Also, since secretory IgA has SC and is highly hydrophilic, it is considered that it binds to an antigen to form a complex having high hydrophilicity and enhances the affinity of the antigen into the mucus layer. This also leads to a decrease in the affinity of the antigen for the cell membrane, which is rich in hydrophobicity, and inhibits bacteria and the like from adhering to epithelial cells. Secretory IgA is often an antibody against bacterial surface molecules, and exerts a protective function by blocking adhesion molecules to epithelial cells present on bacterial surfaces.
In addition, secretory IgA and secretory IgA antigen complex bind to lymphocytes, macrophages, monocytes, and the like via Fc receptors, further enhance antigen-specific IgA immune responses, and maintain secondary immune responses in mucous membranes. It is believed to be working (Maliszewski, CR et al.,
J. Exp. Med. , 172, 1665, 199
0).

In the intestinal mucosa, dimeric IgA is secreted form I
In order to exert the above-mentioned immunity as gA, a poly Ig receptor is indispensable, the mucous membrane is a septum dividing the inside and outside of a living body like the skin, and the mucosal defense mechanism against invasion from the outside is extremely important for the living body. Because of that,
Poly Ig receptor is one of important molecules from the viewpoint of infection protection.

A detailed comparison of the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing with the amino acid sequence of the above poly Ig receptor showed that the first domain, which is the antibody binding domain of the poly Ig receptor, and the sequence listing of the novel polypeptide were SEQ ID NO: 3 showed the highest homology and was about 45%, suggesting that the polypeptide had antibody binding activity. In addition, the four amino acids 469 to 472 including threonine of SEQ ID NO: 2 in the intracellular sequence listing are poly Igs.
Nos. 662 to 665 containing serine 664, which is a phosphorylation site involved in receptor transcytosis
No. 4 amino acids (Hirt, RP et al., C
ell, 74, 245, 1993).

The molecular interaction between proteins can be examined, for example, by immunoprecipitation, enzyme immunoassay, or BIAcore (Pharmacia Biotech) which is a surface plasmon resonance sensor. The immunoprecipitation method is an experimental method in which a target protein is purified and analyzed on a minute scale using an antibody. In other words, an antibody and protein A beads are added to a solution containing the target protein,
Forming an antigen-antibody-protein A bead complex,
Centrifuge and separate. SDS-PAG for this complex
E, Western blotting and the like can be performed to search for proteins that bind to the target protein.

The detection of an intermolecular interaction in BIAcore can be performed as follows. First, one of the interacting molecules is immobilized on a sensor chip as a ligand, and the other molecule is injected on the sensor chip as an analyte. The injected analyte is supplied onto the sensor chip at a constant flow rate, and diffuses into the immobilized ligand. If affinity is present in both, the analyte binds to the ligand, and the analyte is concentrated on the sensor chip. The mass change of the sensor chip at that time is optically detected, a sensorgram is drawn in real time, and it can be observed as a binding curve (Experimental Medicine Separate Volume Bio Manual UP Series Protein Interaction Experiment Method for Proteins Yodosha) .

The present inventors detected the molecular interaction between the novel polypeptide and a human antibody using the enzyme immunoassay described in Example 10. That is, the extracellular partial protein of the novel polypeptide of the present invention obtained according to the method described in Example 8 was used as a 96-well immunoplate (manufactured by Nunc).
And then reacting with a solution containing human immunoglobulin to form a complex of the novel polypeptide and human immunoglobulin. Next, a peroxidase-labeled anti-human immunoglobulin goat antibody that specifically recognizes human immunoglobulin is allowed to react, and a substrate for peroxidase is allowed to act, and the absorbance is measured. The absorbance of extracellular proteins of the novel polypeptide was significantly higher than that of the control, indicating that the novel polypeptide of the present invention has an activity of binding to a human antibody. Therefore, the novel polypeptide of the present invention can be used in the secondary lymph organs, lymph nodes and appendix,
It was suggested that the possibility of acting as one of the factors inducing an antigen-specific secondary immune response by binding to the antigen-antibody complex was extremely high.

The polypeptide of the present invention comprises a polypeptide having an amino acid sequence represented by SEQ ID NO: 3 in the sequence listing as a constituent, preferably SEQ ID NO: 1 in the sequence listing, and more preferably SEQ ID NO: 2 in the sequence listing. A polypeptide having the amino acid sequence of the present invention is a component. Further, the polypeptide of the present invention may lack a part of the amino acid sequence of SEQ ID NO: 1, 2 or 3 in the sequence listing, and may be partially mutated by a point mutation technique. In any case, those which do not lose the properties of SEQ ID NO: 1, 2 or 3 in the sequence listing of the polypeptide of the present invention are included in the present invention.

In addition, the N-terminal or C
Some amino acid residues and peptide residues may be added to the terminal. Furthermore, in the amino acid sequence, N-
When a sugar chain such as acetyl-D-glucosamine or N-acetyl-D-galactosamine is converted to N-glycoside or O-
What consists of a glycosidic bond is also included in the present invention. The homology of the amino acid sequence between the above mutant of the polypeptide and the polypeptide is usually preferably 60% or more, particularly preferably 70% or more, further preferably 80% or more, particularly preferably 90% or more. Certain cases are preferred.

The DNA encoding the polypeptide of the present invention cloned as described above can be used as it is, or digested with a restriction enzyme if desired. A region to be expressed is excised from the cloned DNA and ligated downstream of a promoter in a vector suitable for expression to obtain an expression type vector.

As described in Examples 3 and 5, as a form for expressing the polypeptide of the present invention, a method using cDNA encoding the polypeptide having the amino acid sequence of SEQ ID NO: 2 in the sequence listing may be used. . However, in this state, it is a membrane-bound type, and as described in Examples 4 and 6, the amino acid sequence described in SEQ ID NO: 1, Only the extracellular portion of the polypeptide can be expressed, and is preferred. Therefore, it is possible to express and produce a form containing an extracellular portion in secretion form using DNA encoding the amino acid sequence of SEQ ID NO: 4 in the sequence listing.

Further, the form may be a single compound, but a polypeptide having a complex form is also possible. The “complex” used in the present invention is not a mixture obtained by simply mixing two or more kinds of substances, but a compound, a conjugate, and a compound in which one or more kinds of compounds have any binding mode including a covalent bond. Or a generic term for a complex. Examples of such a case include a complex through covalent bonding of a chimeric protein to an immunoglobulin by a disulfide bond, or an antigen-antibody reaction between a polypeptide having a FLAG sequence prepared in Example 6 and an anti-FLAG antibody. Examples of the form include a complex.

Further, a method of expressing the antibody as a chimeric protein with the Fc portion of human IgG and expressing it as a multimer having a disulfide bond at the hinge portion of the antibody, and a method of expressing the antibody recognition site at the C-terminus or N-terminus And expressing the polypeptide containing the extracellular portion of the expressed polypeptide at the C-terminus or N-terminal.
A method of forming a multimer by reacting with an antibody that specifically recognizes the terminal antibody recognition site can be used.

Further, as another method, a method of expressing a fusion protein with only the hinge region portion of an antibody to form a dimer by a disulfide bond, or any other effect on the activity of the polypeptide Multimeric polypeptide having a high specific activity of at least a dimer composed of a fusion protein prepared so as to express a peptide capable of forming a disulfide bond in a C-terminal, N-terminal or other site in a non-existent manner You can also get

Further, there is a method in which two or more DNAs encoding the amino acid sequence of SEQ ID NO: 1 in the sequence listing are arranged in series in a frame-fitting manner to express the multimeric structure when they are translated into amino acids. Other 2 currently known
Any method that has a multimeric structure that is equal to or larger than a multimer is applicable. Therefore, the polypeptide in a dimeric or higher form by genetic engineering techniques,
Alternatively, the present invention also includes a compound containing a part or all of the amino acid sequence described in SEQ ID NO: 3 or 1 in the sequence listing. By using the complex thus obtained, the biological activity of a novel immune-related factor can be searched for as a research reagent. The expression of the immune-related factor is specifically high in lymph nodes and appendix. By isolating tissues at these sites or by using the various types of cell lines to cause the complex to act, it is possible to search for in vitro physiological activities. Further, by applying this assay system, it is possible to screen for a compound that inhibits the action of the complex.

D encoding the novel polypeptide of the present invention
NA has a translation initiation codon at its 5 'end and may have a translation termination codon at its 3' end. These translation initiation codon and translation termination codon can also be added using a suitable synthetic DNA adapter. Further, the DN
To express A, a promoter is connected upstream thereof. As the vector, the plasmid derived from Escherichia coli described above,
Bacillus subtilis-derived plasmids, yeast-derived plasmids, bacteriophages such as λ phage, and animal viruses such as retroviruses and vaccinia viruses.

The promoter used in the present invention may be any promoter as long as it is appropriate for the host used for gene expression. When the host used for the transformation is Escherichia, a tac promoter, a trp promoter, a lac promoter, or the like is preferable. When the host is a Bacillus, an SPO1 promoter, an SPO2 promoter, or the like is preferable. When the host is a yeast, the host is a yeast. Are preferably PGK promoter, GAP promoter, ADH promoter and the like.

When the host is an animal cell, SV40
Derived promoter, retroviral promoter,
A metallothionein promoter, a heat shock promoter and the like can be used, respectively. Using the thus constructed expression plasmid containing the DNA encoding the polypeptide, a transformant is produced. Examples of the host include Escherichia, Bacillus, yeast, animal cells and the like. Examples of animal cells include monkey cells such as COS-1, Vero, Chinese hamster cells CHO, and silkworm cells SF9.

Thus, a transformant transformed with the expression plasmid containing the DNA encoding the novel polypeptide of the present invention is obtained. The polypeptide can be produced by culturing each transformant in a suitable medium under suitable culture conditions by a known method. The polypeptide can be separated and purified from the culture by, for example, the following method.

When the polypeptide is extracted from the cultured cells or cells, after the culture, the cells or cells are collected by a known method, suspended in an appropriate buffer, and subjected to ultrasound, lysozyme and / or freezing. After disrupting the cells or cells by thawing or the like, a method of obtaining a crude extract of the polypeptide by centrifugation or filtration may be used as appropriate. The buffer may contain a protein denaturant such as urea or guanidine hydrochloride, or a surfactant such as Triton X-100. When the polypeptide is secreted into the culture solution, after the culture is completed, the supernatant is separated from the cells or cells by a method known per se, and the supernatant is collected. The culture supernatant thus obtained,
Alternatively, the polypeptide contained in the extract was obtained in Example 9
Purification using an antibody against the polypeptide obtained by the method described in (1).

The novel polypeptide thus obtained is
A carrier, diluent or excipient, which can be administered as a research reagent or as the polypeptide alone or as at least one drug, is added to an appropriate dosage form to control lymphocyte activation, It is also used as an agent for treating related diseases and infectious diseases. Target immune-related diseases include:
Examples include immunodeficiency such as severe combined immunodeficiency, and autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus. When the polypeptide of the present invention is used as a pharmaceutical, it is preferable to use a freeze-dried product of the polypeptide of the present invention after dissolving or suspending the same in distilled water for injection. For example, 0.1 to 1000 μg /
It is convenient to provide injections and drops prepared at a concentration of ml. In an experiment in which 1 mg / kg of the polypeptide of the present invention was intraperitoneally administered to mice, no death cases of the mice were confirmed, so that it was confirmed that the polypeptides can be used as pharmaceuticals.

The dose of the novel polypeptide of the present invention per adult dose varies depending on age, sex, body weight, symptoms and the like, but is generally about 0.1 μg to 100 mg / kg, and is preferably once or more than once a day. Can be administered several times depending on the dose. When the novel polypeptide of the present invention is used as an injection, a thickener such as sucrose, glycerin, methylcellulose, carboxymethylcellulose and the like, a pH adjuster for various inorganic salts, and the like can be added as additives.

Further, using the novel polypeptide gene of the present invention, a promoter sequence capable of controlling the expression of the polypeptide gene can be obtained from the genome, and a reporter such as a luciferase gene can be obtained downstream of the promoter. The gene is ligated, transfected into appropriate cells, and the cells are stimulated with various specimens, for example, peptides having any sequence, or various compounds, or fermentation products of various microorganisms, and the like, and the reporter gene By comparing the expression of the above, it is possible to screen for a factor that induces or inhibits the expression of the polypeptide gene.

The novel polypeptide of the present invention can be immobilized as an adaptation to a cell therapy method.
As a method for immobilization, the amino group or carboxyl group of the polypeptide can be used, an appropriate spacer can be used, or the above-mentioned crosslinking agent can be used to covalently bind the polypeptide to a culture vessel. . Therefore, it has a form existing on a solid surface, further has at least a novel polypeptide activity, and further has a part or all of the amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2 and 3 in the sequence listing. The present invention also includes compounds containing the contained polypeptide.

As shown in Example 2, when a DNA encoding a part or all of the gene sequence of SEQ ID NO: 4 in the sequence listing is used, a Northern blot can be performed. Therefore, as a method for examining the expression of the present gene, a complementary nucleic acid of 12 mer to 16 mer or more, preferably 18 mer or more having a partial gene sequence of SEQ ID NO: 4 in the sequence listing, that is, antisense DNA, RNA, and This can be done by methylated, methylphosphated, deaminated, or thiophosphated derivatives. The expression of the present gene can also be examined by PCR using these sense and antisense oligonucleotides as primers.

Furthermore, a homolog of the present gene of another organism such as a mouse or a rat can be detected or cloned by a method similar to the method shown in Example 2, or a PCR method. Further, cloning of genes on the genome including humans is also possible. Therefore, using these cloned genes, it is possible to reveal more detailed functions of the polypeptide. For example, using recent gene manipulation techniques, transgenic mice, gene targeting mice,
Any method such as double knockout in which the gene related to the present gene is inactivated can be used. If there is an abnormality in the genome of this gene, genetic diagnosis,
Application to gene therapy is also possible.

Further, by using the antibody specifically recognizing the polypeptide of the present invention shown in Example 9, the polypeptide of the present invention can be detected and measured. It can be used as a diagnostic for diseases such as In addition, a specific immunocompetent cell group can be separated using the present antibody. In addition, the present invention includes the polypeptide in any form described in the present invention, that is, a polypeptide containing a part or all of the amino acid sequence of the amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2 and 3 in the sequence listing. Compounds obtained by combining a compound and a complex composed thereof are included in the present invention.

The general procedures necessary for handling DNA and Escherichia coli as a recombinant host described in the present specification are routinely performed by those skilled in the art.
experimental manual (T. Maniatis, etc.)
et al. , Molecular Cloning
A Laboratory Manual, Cold
Spring Harbor Laboratory
1982, 1989). Commercially available enzymes and reagents can be used for all the enzymes and reagents to be used. Unless otherwise specified, these objects can be completely achieved according to the use conditions specified for the products.

[0068]

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described in detail with reference to embodiments, but the present invention is not limited to these embodiments. Example 1 cDNA Cloning and Identification (1) Human HtkL Gene (International Publication No. WO96 /
11212) was digested with EcoRI, followed by agarose gel electrophoresis to cut out a gel fragment containing a DNA fragment of about 0.25 kb (SEQ ID NO: 13 in the sequence listing). DNA was extracted from the gel fragment using Geneclean II (manufactured by BIO101) according to the attached protocol, and the obtained DNA fragment was subjected to DNA labeling kit (Megapr).
im DNA labeling system: A
It was labeled with radioisotope ³² P using mersham Co.) as a probe for cDNA screening.

That is, 5 μl of a primer solution and deionized water were added to 25 ng of DNA to make the total volume 33 μl, and a boiling water bath was carried out for 5 minutes. Thereafter, 10 μl of a reaction buffer containing dNTP, 5 μl of α- ³² P-dCTP, and T4DN
Add 2 μl of A polynucleotide kinase solution and add
C. for 10 minutes in a water bath, and thereafter, a Sephadex column (Quick Spin Column Sepha).
dex G-50: Boehringer Mannheim), and after boiling in a boiling water bath for 5 minutes, ice-cooling for 2 minutes before use.

(2) λ phage cD of human lymph node tissue
A part of the phage solution of the NA library (manufactured by CLONTECH) was taken and SM buffer (0.1 M NaCl,
_{8mMMgSO 4 · 7H2O, 50mM Tris-} H
C. (pH 7.5, 0.01% gelatin) at several dilutions. coli NM51
4 and infected with the phage plating cell prepared in No. 4, L medium (1% tryptone, 0.5% yeast extract,
As a result of titration by forming plaques on an agar plate (0.5% NaCl), the titer was 2 × 10 ⁵ pfu / ml.

Approximately 400,000 clones of the λgt10 recombinant of the above library were screened by plaque hybridization using the DNA probe prepared in (1) to obtain 48 positive clones. That is,
2 ml of the phage solution of the above library was added to E. coli. coli
4 × 10 ⁵ plaques formed on an L medium agar plate by infection with NM514 were transferred to a nylon filter (Hybond N +: manufactured by Amersham), and the transferred nylon filter was treated with alkali (1.
The mixture was allowed to stand on a filter paper impregnated with 5 M NaCl and 0.5 M NaOH for 7 minutes), and then neutralized (1.5 M
NaCl, 0.5 M Tris-HCl (pH 7.
2) twice for 3 minutes on a filter paper impregnated with 1 mM EDTA, and then SSPE solution (0.36 M N
aCl, 0.02 M sodium phosphate (pH 7.
7) After shaking for 5 minutes in a 2 × solution of 2 mM EDTA), the plate was washed and air-dried. Then, it was left for 20 minutes on filter paper impregnated with 0.4 M NaOH,
After shaking for 5 minutes with the E solution, the plate was washed and air-dried again.

Next, hybridization was performed with the DNA probe prepared in (1) under the following conditions.
In the hybridization, an SSPE solution having a final concentration of each component of 5 times (hereinafter referred to as 5 ×), a Denhardt solution (manufactured by Wako Pure Chemical Industries, Ltd.), 0.5% SDS (sodium dodecyl sulfate), and It is immersed in a prehybridization solution that is denatured salmon sperm DNA in a 20 μg / ml boiling water bath, shaken at 50 ° C. for 2 hours, and then mixed with a prehybridization solution containing a probe labeled with ³² P by the above-described method. It was immersed in a hybridization solution of the same composition and shaken at 50 ° C. for 16 hours.

Next, the filter was immersed in a 2 × SSPE solution containing 0.1% SDS and washed by shaking at room temperature for 15 minutes, and the washing was performed four times. After the washing, the filter was subjected to autoradiography for 24 hours using an intensifying screen. As a result, phages were extracted from the agar region corresponding to the strongly exposed portion, and plaque hybridization was performed again according to the above-described steps, thereby obtaining 48 types of purified λgt10 recombinants. From these 48 kinds of recombinants, the method of Grossberger et al.
er et al. , Nucleic Acids R
λgt10 recombinant DNA was extracted according to E. search (1987) 15, 6737).

(3) One of the λgt10 recombinant DNAs obtained in the above (2) was cleaved with EcoRI and subjected to agarose gel electrophoresis.
DNA was observed, and a gel fragment containing this cDNA fragment was cut out. Next, from the gel fragment, Geneclean I
DNA was extracted using I (manufactured by BIO101) according to the attached protocol. The obtained DNA fragment of about 2 kb was subcloned into the EcoRI site of puc18 (Takara Shuzo) to obtain plasmid p # 53-4. Deletion mutant (dele
Tion Mutant) was prepared.

A mutant kit for kilosequence (Takara Shuzo) was used for preparing the mutant. The nucleotide sequence of each mutant was determined by the method of Sanger et al.
r, F. , Et al. Proc. Natl. Aca
d. Sci. (1977) 74, 5463), and the entire base sequence of the 2 kb cDNA fragment was determined. The determined base sequence is shown in SEQ ID NO: 4 in the sequence listing. The nucleotide sequence is determined by a DNA fluorescence sequencer (Applied Biosystem) (Applied Biosystem).
And according to the attached manual.

The present DNA sequence had an open reading frame capable of encoding a 532 amino acid sequence from the start codon (ATG) starting at position 98 to the stop codon (TGA) ending at position 1696. The amino acid sequence translated from the open reading frame is shown in SEQ ID NO: 2 in the sequence listing. The direction of the gene of the plasmid p # 53-4 is opposite to the direction of the lacZ gene of the puc18 vector, that is, the 5 'side shown in SEQ ID NO: 4 in the sequence listing is Hi of the puc18 vector multicloning site.
ndIII side.

Example 2 Expression of mRNA by Northern Blotting To examine the expression of mRNA of the immune-related factor of the present invention,
Clontech Human Multiple, a filter to which mRNA has been transcribed in advance
Tissue Northern Blot, Huma
n Multiple Tissue Norther
n Blot II, Human Multiple T
issue Northern Blot III, Hum
an Immun System Multiple
Using Tissue Northern Blot,
Plasmid p # 53-4 was digested with restriction enzyme PstI, and electrophoresed on a 1% agarose gel.
p (120 to 614 of SEQ ID NO: 4 in the sequence listing)
First, cut out Geneclean II (BIO
101) (DN 101).
A Labeling Kit (MegaPrime DNA I
abeling system: examined the ³² P-labeled expressed in the above-mentioned method by Amersham).

As a result, remarkable expression was observed in lymph nodes and appendix among human tissues. Expression was also observed in the small intestine, and extremely weak expression was observed in the stomach, kidney, thymus and spleen. However, heart, brain, placenta, lung, liver, skeletal muscle, pancreas, prostate, testis, ovary, large intestine, peripheral blood leukocytes, thyroid, spinal cord, trachea, adrenal gland, bone marrow,
No expression was observed in fetal liver. Based on the above, the present immunity-related factor is specifically expressed in the tissue in charge of immunity, and in particular, markedly expressed specifically in lymph nodes, and not expressed in peripheral blood leukocytes. It was thought to be related to the secondary immune response and to be a factor specific to the tissue cells of the lymph nodes but not the lymphocytes themselves. Furthermore, expression in the small intestine is presumed to be derived from Peyer's patch, an immune system in the intestinal tract.

Example 3 Preparation of Plasmid for Expression of Full-length Gene of Immune-Related Factor In order to introduce the full-length gene of the polypeptide obtained by the method described in Example 1 into animal cells, a plasmid represented by SEQ ID NO: 4 in the sequence listing was prepared. Using a peptide-encoding DNA and Kodak IBI FLAG, 8 amino acids at the C-terminal of the amino acid sequence of SEQ ID NO: 2 in the sequence listing, that is, Asp TyrLys Asp as the amino acid sequence
A DNA encoding a polypeptide to which a polypeptide having Asp Asp Asp Lys (hereinafter referred to as FLAG, SEQ ID NO: 12 in the sequence listing) was added was converted into a plasmid pSV2-
dhfr (ATCC 37146) was separately ligated to the dhfr gene site to prepare full-length expression plasmids pSV2Fu and pSV2FuFLAG for immune-related factors, respectively.

In preparing an expression vector for a polypeptide-expressing cell containing the amino acid sequence of SEQ ID NO: 2 in the Sequence Listing, a restriction enzyme HindIII site was added to the 5'-terminal sequence except for the 5 'non-transcribed region of SEQ ID NO: 4 in the Sequence Listing. -ACAA
GCTTACGTTCACCAGCAGGAGGGCA-
Oligo DNA having 3 ′ sequence (primer 1, SEQ ID NO: 5 in Sequence Listing), 5′-GGTGCCTGTTGACA
PCR was performed using oligo DNA having the sequence of GATGAG-3 ′ (primer 2, SEQ ID NO: 6 in the sequence listing) as a primer and p # 53-4 as a template. After confirming that a DNA of about 310 bp was amplified by agarose gel electrophoresis, the PCR product was purified by the method described in Example 1 (3), and the restriction enzyme Hi was purified.
p treated with ndIII and HincII and treated similarly
uc18 vector (Takara Shuzo).

Thereafter, plasmid DNA was purified from the colonies of the four clones and sequenced to confirm the gene sequence.
That is, it was confirmed that the fragment had a sequence from the 98th to the 367th of the DNA sequence of SEQ ID NO: 4 in the sequence listing. Hereinafter, the plasmid having this fragment is called pL
IC-1.

Next, the restriction enzyme BamHI was removed except for the 3 'non-transcribed region of the novel polypeptide gene of SEQ ID NO: 4 in the sequence listing.
5'-GAAAGGGTCAC to add site
Oligo D which is a sequence of CTTAATTCAGAT-3 '
NA (Primer 3, SEQ ID NO: 7 in Sequence Listing), 5′-T
CGGATCCTCAGGGTCCTGGGATTTCT
Using oligo DNA having the sequence of CTC-3 '(primer 4, SEQ ID NO: 8 in the sequence listing) as a primer,
Using plasmid p # 53-4 as a template,
R was performed. After confirming that about 150 bp of DNA was amplified by agarose gel electrophoresis, this PCR
The product was purified by the method described in Example 1 (3), and pT7B
lue vector (Novagen).

Thereafter, the plasmid DNA was purified from the colonies of the three clones, sequenced and the gene sequence was confirmed.
That is, it was confirmed that the fragment was a fragment having the sequence from No. 1552 to No. 1696 of the DNA sequence of SEQ ID NO: 4 in the sequence listing. Hereinafter, the plasmid having this fragment is referred to as pLIC-2.

Furthermore, 8 amino acids at the C-terminus of the full-length amino acid sequence of the novel polypeptide of SEQ ID NO: 2 in the sequence listing, ie, Asp Tyr Lys Asp
In preparing an expression vector having a gene sequence encoding a polypeptide to which a polypeptide having Asp Asp Asp Lys (FLAG, SEQ ID NO: 12 in the sequence listing) is added, the above primers 3 and 5 ′ are prepared in the same manner.
-AAGGATCCTCATTTATCATCATCA
TCTTTAATAATCGGGTCCTGGATTTC
Oligo DNA having the sequence of TCCTCTGG-3 '(primer 5, SEQ ID NO: 9 in the sequence listing) was used as a primer
CR was performed, subcloned into the pT7Blue vector, 4 clones were sequenced, and the gene sequence was confirmed. The target gene sequence, that is, 3 ′ of the sequence from nucleotides 1552 to 1693 of the DNA sequence of SEQ ID NO: 4 in the sequence listing, was confirmed. 5'-GATTATAAAGATGATGAT at the end
It was confirmed that GATAAAATGA-3 ′ (FLAG: SEQ ID NO: 12 in the sequence listing) was a fragment having a connected sequence. Hereinafter, the vector having this fragment is referred to as pLIC-3.

Next, p # 53-4 was replaced with the restriction enzyme HindII.
PLIC-1 was digested with I and HincII and purified by the same method as in Example 1 (3), and pLIC-1 was digested with restriction enzymes HindIII and HindIII in the same manner as described above.
The 310 bp fragment obtained by digestion with HincII was ligated to DNA
Ligaion Kit Ver. 2 (manufactured by Takara Shuzo Co., Ltd.) to obtain a plasmid pLIC-4. Furthermore, pLIC-4 was replaced with restriction enzymes HindIII and Eco.
An about 1.8 kbp DNA fragment obtained by digestion with RI and purification by the same method as in Example 1 (3) was obtained using pBlues.
scriptII KS + vector (STRATAGENE
Plasmid pLIC-5 was obtained by subcloning into HindIII and EcoRI sites of the same company.

The thus prepared pLIC-5 was digested with the restriction enzymes BstPI and BamHI and subjected to agarose gel electrophoresis to obtain a fragment of about 4.4 kbp, that is, the 98th fragment of the DNA sequence of SEQ ID NO: 4 in the sequence listing. PBluesc at the 5 'end of the sequence from position 1557 to
from restriction site HindIII to Bam
Cut out the fragment connected to the part up to HI,
Purified as described above. This gene fragment is designated as F1.
Similarly, pLIC-2 and pLIC-3 were digested with the restriction enzymes BstPI and BamHI, and each was digested for about 1 hour.
A 50 bp, approximately 180 bp DNA fragment was purified.

These gene fragments are called F2 and F3, respectively. Then, F1 and F2, and F1 and F3 are each connected by the above-mentioned method, and the vector pBSFu1 and the restriction enzymes HindIII and BamHI from which the gene fragment of the portion encoding the polypeptide of SEQ ID NO: 2 in the sequence listing is cut out with the restriction enzymes HindIII and BamHI. A vector pBSFu2 from which a gene fragment corresponding to a portion encoding a polypeptide having a FLAG amino acid sequence at the C-terminus of the polypeptide of SEQ ID NO: 2 in the sequence listing was prepared.

The vector pBS thus prepared
Fu1 and pBSFu2 were replaced with restriction enzymes HindIII and Bam.
Digested with HI and electrophoresed to approximately 1.6 kbp of D
An NA fragment, that is, a DNA fragment containing a DNA fragment encoding the amino acid sequence of the novel polypeptide was separated and purified. These two types of DNA fragments were combined with the pSV2-dh described above.
fr is treated with restriction enzymes HindIII and BglII,
The product excluding the dhfr gene was ligated with T4 DNA ligase (Takara Shuzo Co., Ltd.) to construct a novel polypeptide expression vector. The vector prepared without the FLAG sequence as described above is called pSV2Fu, and the vector containing the FLAG sequence is called pSV2FuFLAG.

Example 4 Preparation of Plasmid for Expression of Extracellular Part Gene of Immune-Related Factor The extracellular part of the polypeptide encoded by the gene obtained by the method described in Example 1, that is, the polypeptide represented by SEQ ID NO: 1 in the sequence listing Expression plasmid pSV2ECFL for producing animal-containing polypeptides containing a peptide
AG was constructed. That is, 8 amino acids at the C-terminal of the polypeptide having the extracellular partial amino acid sequence of the polypeptide of SEQ ID NO: 1 in the sequence listing, that is, Asp as the amino acid sequence
TyrLys Asp Asp Asp Asp Ly
s-containing polypeptide (FLAG: SEQ ID NO: 1 in the sequence listing)
In preparing an expression vector having a gene sequence encoding a polypeptide to which 2) was added, 5′-AAAGGTCCTAGGAACCA was prepared in the same manner as in the case of full length.
Oligo DNA having the sequence of TTGGG-3 ′ (primer 6, SEQ ID NO: 10), 5′-AAGGATCCTCCA
TTTATCATCATCATCTTTTATAATCT
PCR was performed using oligo DNA (primer 7, SEQ ID NO: 11 in the sequence listing) having the sequence of TCTGGAAAAGTACGCTTCACG-3 ′ as a primer and p # 53-4 containing the gene of SEQ ID NO: 4 in the sequence listing as a template.

After confirming that a DNA of about 360 bp was amplified by agarose gel electrophoresis, this PCR was performed.
The product was purified by the method described in Example 1 (3) to give pT7
It was subcloned into the Blue vector. Then 5
Purification of plasmid DNA from clone colonies,
The sequence of the gene was confirmed by sequencing, and there was no error in the gene sequence, ie, 5′-GATTATAAAGATGATGAT at the 3 ′ end of the target gene sequence, that is, the DNA sequence of SEQ ID NO.
GATAAATGA-3 ′ (SEQ ID NO: 12 in the sequence listing) was confirmed to be a fragment having a continuous sequence, and was designated as plasmid pLIC-6.

The above-mentioned plasmid pLIC-5 was digested with restriction enzymes BlnI and BamHI (manufactured by Takara Shuzo Co., Ltd.), and a DNA fragment of about 4 kbp was obtained by removing a DNA fragment having a sequence of No. 1083 and after of the DNA sequence of SEQ ID NO: 4 in the sequence listing. Similarly, pLIC-6 was added to the fragments with the restriction enzymes BlnI and BamHI.
A DNA fragment of about 360 bp obtained by digestion with T4 DN
Ligation with A ligase yielded plasmid pBSEC. The vector pBSEC thus prepared is digested with restriction enzymes HindIII and BamHI and contains a DNA fragment of about 1.4 kbp by electrophoresis, that is, a DNA sequence encoding the amino acid sequence of the extracellular region of the novel polypeptide. The DNA fragment was separated and purified.

This DNA fragment was ligated with the plasmid pS
V2-dhfr was treated with restriction enzymes HindIII and BglII to remove the dhfr gene, ligated with T4 DNA ligase (manufactured by Takara Shuzo), and C-terminal of the amino acid sequence of the novel polypeptide (SEQ ID NO: 1 in the sequence listing). FL
A plasmid capable of producing a polypeptide having an AG sequence was prepared. The expression plasmid prepared as described above is referred to as pSV2ECFLAG.

Example 5 Transformation of Balb / 3T3 cells with a plasmid Mouse fibroblast Balb / 3T3 clone A3
No. 1 (available from RIKEN, Cell Development Bank, No.
RCB0005) and expression plasmids pSV2Fu and pSV2FuFLAG obtained by the method described in Example 3.
Was used for transformation. That is, D-MEM (Dulbecco's modified MEM medium, GIBCO-BRL) 10% F
On the day before the transformation, the medium was exchanged with the cells cultured in the CS, the number of cells was changed to 5 × 10 ⁶ cells per 10 cm cell culture dish, and 10 ml of the above medium was added and cultured overnight. The next day, the cells were precipitated by centrifugation and
After centrifugal washing twice with S (-), 1 mM MgCl ₂ , P
Cells were prepared in BS (−) at a concentration of 1 × 10 ⁷ cells / ml.

Gene transfer was performed by an electroporation method using a gene transfer device manufactured by Bio-Rad.
500 μl of the above cell suspension was placed in a cell (0.4 cm) dedicated to electroporation, and the expression vector pSV2
Fu or pSV2FuFLAG and pSV2-ne
o (ATCC 37150) was added in an amount of 20 μg, and the mixture was allowed to stand on ice for 5 minutes. Thereafter, the interval was left at room temperature for 1 minute, and a voltage of 3 μF and 450 V was applied twice. afterwards,
After standing on ice for 5 minutes, the cells were cultured in a 10-cm diameter cell culture dish in 10 ml of the above medium for 3 days.

Three days later, G418 (Sigma) was added to the above medium.
a) was exchanged with a medium supplemented with 400 μg / ml, and thereafter, every 2 days, 1/3 of the medium was exchanged for 1 day.
Culture was continued for about 0 days. After 10 days, the colonies formed on the dish were removed using a trypsin solution (GIBCO-BR).
(Manufactured by L Company) to obtain a cell line stably expressing the cloned gene. Hereinafter, the cell line stably expressing the polypeptide containing the amino acid sequence represented by SEQ ID NO: 2 in the Balb / 3T3 sequence listing prepared in this manner is referred to as B.
alb / FULL, FLA was added to the C-terminal of the polypeptide containing the amino acid sequence of SEQ ID NO: 2 in the sequence listing.
A cell line stably expressing a polypeptide having a G amino acid sequence is designated as Balb / FULLFLAG. At the same time, a control transformed cell line into which only pSV2-neo was introduced by the same method was prepared, and this was used for Balb / CO.
Shown as N.

Example 6 Transformation of COS-7 Cells with Plasmid COS-7 (available from RIKEN, Cell Development Bank, RCB0539) was performed using plasmid pSV2ECFLAG obtained by the method described in Example 4. Transformation was carried out in the same manner as described in Example 5. That is, D-
MEM (Dulbecco's modified MEM medium, GIBCO-BR
The culture medium was changed on the day before the transformation of the cells cultured in 10% FCS (manufactured by L Company), the number of cells was changed to 5 × 10 ⁶ cells per 10 cm diameter cell culture dish, and 10 ml of the medium was added overnight. Cultured. The next day, the cells were precipitated by centrifugation, washed twice with PBS (-),
1 × 10 ⁷ cells / m in MgCl ₂ , PBS (-)
The cells were prepared so as to be 1. Gene transfer is Bi
This was performed by an electroporation method using a gene transfer device manufactured by o-Rad.

[0097] 500 µl of the above cell suspension is placed in a cell (0.4 cm) dedicated to electroporation, 20 µg of the expression vector pSV2ECFLAG is added, and the mixture is left on ice for 5 minutes. Thereafter, the interval was left at room temperature for 1 minute, and a voltage of 3 μF and 450 V was applied twice. Then, after leaving to stand on ice for 5 minutes, the cells were cultured in a 10-cm diameter cell culture dish in 10 ml of the above medium. On the day after the gene transfer, the medium was replaced with serum-free D-MEM (manufactured by GIBCO-BRL). The medium was replaced every four days thereafter, and the culture supernatant was collected over two weeks. Separated culture supernatants were respectively buffered from the culture medium with Centricon 30 (manufactured by Amicon) in PBS.
Exchanged to (-) and concentrated 10-fold.

Example 7 Detection of Recombinant New Polypeptide Using Western Blot Polypeptide containing polypeptide shown in SEQ ID NO: 1 in the supernatant of a transformed cell prepared by the method described in Example 6 In order to confirm that the peptide was produced, it was confirmed by Western blot as follows. That is, the concentrated culture supernatant was subjected to SDS-PAG manufactured by ACI Japan.
Using an electrophoresis tank for E and a polyacrylamide gel for SDS-PAGE (gradient gel 5 to 20%), SDS-PAGE was performed according to the attached instruction manual.
Samples were obtained for a sample under reducing conditions in which 2-mercaptoethanol (2-ME) was added and heat-treated, and a sample under non-reducing conditions in which the treatment was not performed. As a marker, a rainbow marker manufactured by Amersham (for high molecular weight) ), And a sample buffer and an electrophoresis buffer were prepared according to the attached instruction manual. After completion of SDS-PAGE, acrylamide gel
B to VDF membrane filter (BioRad)
Transcription was performed using a mini-trans blot cell manufactured by ioRad.

[0099] The filter thus produced was made 5% B
SA (manufactured by Sigma), TBS-T (20 mM Tr
is, 137 mM NaCl (pH 7.6), 0.1%
Blocking was performed by shaking overnight at 4 ° C in Tween 20). Mouse monoclonal antibody An as primary antibody
ti-FLAG M2 (manufactured by Kodak), a peroxidase-labeled anti-mouse Ig sheep antibody (Amer
sham). The reaction time of each antibody was 1 hour at room temperature, and between each reaction, the operation of shaking and washing with TBS-T for 10 minutes at room temperature was repeated three times.

After the last wash, the filter was
It was immersed in a reaction solution of an ECL Western blotting detection system manufactured by Am Inc. for 5 minutes, wrapped in Saran wrap, and exposed to an X-ray film. As a result, a protein of about 50 kDalton was detected in both the reducing and non-reducing condition samples. From the above results, a cell producing a polypeptide having a FLAG sequence at the C-terminus of the amino acid sequence of SEQ ID NO: 1 in the target sequence listing could be obtained.

Example 8 Purification of Novel Polypeptide Extracellular Partial Protein 5 L of the culture supernatant of the COS-7 transformed cells obtained by the method described in Example 6 was prepared. These culture supernatants were added to Kodak Anti-FLAG M2 Affini.
The recombinant polypeptide was adsorbed to the column through a ty Gel column. The size of the column is 1 cm × 3 cm, the volume is about 2 ml, and the flow rates are all 1 ml / mi.
n. I went in. After the adsorption, the column is washed with 20 ml of PBS (-).
And then 0.5M Tris-glycine (pH
3.0). The eluted fraction was collected in 2 ml portions,
200 μl each of 0.5 M Tris-HCl (pH 9.5) was added, and each fraction was subjected to SDS-PAGE by the method described above.
Was done.

After the electrophoresis, staining was carried out with Wako Pure Chemical Industries silver staining kit Wako II according to the attached instructions. as a result,
It was confirmed that the purified protein was obtained in a band having the same size as the result of the Western blot described in Example 7. From these results, a polypeptide having a FLAG sequence at the C-terminus of the amino acid sequence described in SEQ ID NO: 1 in the sequence listing could be obtained as a pure product. In order to obtain a polypeptide with higher purity, the recombinant polypeptide purified by the above method was purified by gel filtration.

The eluate from the affinity column was concentrated and centrifuged with Amicon Centricon 30 and the buffer was exchanged with PBS (-). Gel filtration was performed using a Pharmacia FPLC system.
Performed on a column. The main peak eluted at the same molecular weight position as the result of the Western blot of Example 7 was collected. Hereinafter, SEQ ID NO: 1 of the sequence listing thus purified
The polypeptide having a FLAG sequence at the C-terminus of the amino acid sequence described in 1) is referred to as EXFLAG-PTN.

Example 9 Establishment of a Monoclonal Antibody Recognizing a Novel Polypeptide Using purified EXFLAG-PTN as an immunogen, a mouse monoclonal antibody was prepared according to the method described in the written literature. That is, the purified recombinant polypeptide EXF prepared in Example 8
LAG-PTN was immunized subcutaneously or intradermally with Balb / c mice (manufactured by SLC Japan) at 10 μg per animal. After the second immunization, blood was collected from the fundus and an increase in the antibody titer in the serum was observed. After the third immunization, the spleen cells of the mouse were removed, and the mouse myeloma cell line P3X63A was obtained.
Cell fusion was performed with g8 (ATCC TIB9) by the polyethylene glycol method.

Hybridomas were selected in a HAT medium (manufactured by Japan Institute of Immunology), and a hybridoma strain producing an antibody recognizing the extracellular portion of the polypeptide in the medium was isolated by an enzyme-linked immunosorbent assay. A hybridoma strain producing a mouse monoclonal antibody that specifically recognizes the polypeptide has been established. The culture supernatant of the hybridoma thus established was purified using Pharmacia's Mab Tr.
Using apGII, the anti-polypeptide monoclonal antibody was purified and prepared according to the attached protocol.

The polypeptide purified in Example 8 using this monoclonal antibody was subjected to Western blot under the conditions described in Example 7. As a result, a single band having the same molecular weight as the result of the Western blot shown in Example 7 could be detected, and it was revealed that the present monoclonal antibody could specifically recognize the polypeptide.

Example 10 Using EXFLAG-PTN, an extracellular partial protein of the novel immune-related factor obtained by the method described in Example 8, the binding to human antibodies was confirmed. That is, EXFLAG-PTN (100 n) was placed on a 96-well immunoplate (manufactured by Nunc).
g / ml) was dispensed in an amount of 100 μl. After leaving still at room temperature for 1 hour or more, the solution was rinsed and washed twice with PBS (-) containing 0.05% Tween 20 (hereinafter abbreviated as PBS / Tween). Next, 1-time concentration of Block Ace
(Manufactured by Dainippon Pharmaceutical Co., Ltd.) in 100 μl aliquots at room temperature.
Let stand for hours. After washing the solution and washing twice with PBS / Tween, H diluted 1000 times with PBS / Tween
UMANIMMUNOGLOBULIN STANDA
100 μl of RD (manufactured by Cappel) was dispensed and left at room temperature for 1 hour.

Next, the solution was washed twice with PBS / Tween, and 100 μl of a peroxidase-labeled anti-human immunoglobulin goat antibody (manufactured by EY Laboratories) diluted 500 times with PBS / Tween was dispensed. It was left at room temperature for 1 hour. After washing four times with PBS / Tween, 1 mg of tetramethylbenzidine (manufactured by Sigma) in 5 ml of a buffer consisting of 0.1 M Na ₂ HPO ₄ and 0.05 M citric acid, and 50 μl of 30% hydrogen peroxide 50 μl of the coloring solution prepared by adding
The mixture was dispensed at room temperature and allowed to stand at room temperature for 5 to 10 minutes. The reaction was stopped by dispensing 50 μl each of 2N sulfuric acid. As a target experiment 1, 100 μl of bovine serum albumin (100 ng / mg) was used in place of EXFLAG-PTN, and the others were operated according to the above method. As a control experiment 2, HU
MAN IMMUNOGLOBULINSTANDAR
Bovine serum albumin (100 ng / ml) 1 in place of D
The operation was performed in the same manner as described above except for using 00 μl.

The samples containing the two kinds of control experiments were subjected to a plate reader (manufactured by BIO-RAD) at 450
The absorbance at nm was measured. The absorbance is EXFLAG-P
TN, control 1 and control 2 for 1.2, 0.
EXFLAG-PTN was 9, 1.0, showing significantly higher absorbance than the control. That is, it was shown that the novel immune-related factor of the present invention binds to human immunoglobulin.

[0110]

Industrial Applicability The immune-related factor of the present invention is specifically expressed in lymph nodes, so that it can be usefully used for research and treatment related to immune diseases and infectious diseases related to immune response.

Sequence Listing SEQ ID NO: 1 Sequence Length: 428 Sequence Type: Amino Acid Topology: Linear Sequence Type: Peptide Sequence Leu Pro Gln Lys Arg Pro His Pro Arg Trp Leu Trp Glu Gly Ser Leu 1510 15 Pro Ser Arg Thr His Leu Arg Ala Met Gly Thr Leu Arg Pro Ser Ser 20 25 30 Pro Leu Cys Trp Arg Glu Glu Ser Ser Phe Ala Ala Pro Asn Ser Leu 35 40 45 Lys Gly Ser Arg Leu Val Ser Gly Glu Pro Gly Gly Ala Val Thr Ile 50 55 60 Gln Cys His Tyr Ala Pro Ser Ser Val Asn Arg His Gln Arg Lys Tyr 65 70 75 80 Trp Cys Arg Leu Gly Pro Pro Arg Trp Ile Cys Gln Thr Ile Val Ser 85 90 95 Thr Asn Gln Tyr Thr His His Arg Tyr Arg Asp Arg Val Ala Leu Thr 100 105 110 Asp Phe Pro Gln Arg Gly Leu Phe Val Val Arg Leu Ser Gln Leu Ser 115 120 125 Pro Asp Asp Ile Gly Cys Tyr Leu Cys Gly Ile Gly Ser Glu Asn Asn 130 135 140 Met Leu Phe Leu Ser Met Asn Leu Thr Ile Ser Ala Gly Pro Ala Ser 145 150 155 160 Thr Leu Pro Thr Ala Thr Pro Ala Ala Gly Glu Leu Thr Met Arg Ser 165 170 175 Tyr G ly Thr Ala Ser Pro Val Ala Asn Arg Trp Thr Pro Gly Thr Thr 180 185 190 Gln Thr Leu Gly Gln Gly Thr Ala Trp Asp Thr Val Ala Ser Thr Pro 195 200 205 Gly Thr Ser Lys Thr Thr Ala Ser Ala Glu Gly Arg Arg Thr Pro Gly 210 215 220 Ala Thr Arg Pro Ala Ala Pro Gly Thr Gly Ser Trp Ala Glu Gly Ser 225 230 235 240 Val Lys Ala Pro Ala Pro Ile Pro Glu Ser Pro Pro Ser Lys Ser Arg 245 250 255 Ser Met Ser Asn Thr Thr Glu Gly Val Trp Glu Gly Thr Arg Ser Ser 260 265 270 Val Thr Asn Arg Ala Arg Ala Ser Lys Asp Arg Arg Glu Met Thr Thr 275 280 285 Thr Lys Ala Asp Arg Pro Arg Glu Asp Ile Glu Gly Val Arg Ile Ala 290 295 300 Leu Asp Ala Ala Lys Lys Val Leu Gly Thr Ile Gly Pro Pro Ala Leu 305 310 315 320 Val Ser Glu Thr Leu Ala Trp Glu Ile Leu Pro Gln Ala Thr Pro Val 325 330 335 Ser Lys Gln Gln Ser Gln Gly Ser Ile Gly Glu Thr Thr Pro Ala Ala 340 345 350 Gly Met Trp Thr Leu Gly Thr Pro Ala Ala Asp Val Trp Ile Leu Gly 355 360 365 Thr Pro Ala Ala Asp Val Trp Thr Ser Met Glu Ala Ala Ser Gly Glu 370 375 380 Gly SerAla Ala Gly Asp Leu Asp Ala Ala Thr Gly Asp Arg Gly Pro 385 390 395 400 400 Gln Ala Thr Leu Ser Gln Thr Pro Ala Val Gly Pro Trp Gly Pro Pro 405 410 415 Gly Lys Glu Ser Ser Val Lys Arg Thr Phe Pro Glu 420 425

SEQ ID NO: 2 Sequence length: 532 Sequence type: amino acid Topology: Linear Sequence type: Peptide sequence Met Pro Leu Phe Leu Ile Leu Cys Leu Leu Gln Gly Ser Ser Phe Ala -15 -10- 5 Leu Pro Gln Lys Arg Pro His Pro Arg Trp Leu Trp Glu Gly Ser Leu 1 5 10 15 Pro Ser Arg Thr His Leu Arg Ala Met Gly Thr Leu Arg Pro Ser Ser 20 25 30 Pro Leu Cys Trp Arg Glu Glu Ser Ser Phe Ala Ala Pro Asn Ser Leu 35 40 45 Lys Gly Ser Arg Leu Val Ser Gly Glu Pro Gly Gly Ala Val Thr Ile 50 55 60 Gln Cys His Tyr Ala Pro Ser Ser Val Asn Arg His Gln Arg Lys Tyr 65 70 75 80 Trp Cys Arg Leu Gly Pro Pro Arg Trp Ile Cys Gln Thr Ile Val Ser 85 90 95 Thr Asn Gln Tyr Thr His His Arg Tyr Arg Asp Arg Val Ala Leu Thr 100 105 110 Asp Phe Pro Gln Arg Gly Leu Phe Val Val Arg Leu Ser Gln Leu Ser 115 120 125 Pro Asp Asp Ile Gly Cys Tyr Leu Cys Gly Ile Gly Ser Glu Asn Asn 130 135 140 Met Leu Phe Leu Ser Met Asn Leu Thr Ile Ser Ala Gly Pro Ala Ser 145 150 155 160 Thr Leu Pro Thr Ala Thr Pro Ala Ala Gly Glu Leu Thr Met Arg Ser 165 170 175 Tyr Gly Thr Ala Ser Pro Val Ala Asn Arg Trp Thr Pro Gly Thr Thr 180 185 190 Gln Thr Leu Gly Gln Gly Thr Ala Trp Asp Thr Val Ala Ser Thr Pro 195 200 205 Gly Thr Ser Lys Thr Thr Ala Ser Ala Glu Gly Arg Arg Thr Pro Gly 210 215 220 Ala Thr Arg Pro Ala Ala Pro Gly Thr Gly Ser Trp Ala Glu Gly Ser 225 230 235 240 Val Lys Ala Pro Ala Pro Ile Pro Glu Ser Pro Pro Ser Lys Ser Arg 245 250 255 Ser Met Ser Asn Thr Thr Glu Gly Val Trp Glu Gly Thr Arg Ser Ser 260 265 270 Val Thr Asn Arg Ala Arg Ala Ser Lys Asp Arg Arg Glu Met Thr Thr 275 280 285 Thr Lys Ala Asp Arg Pro Arg Glu Asp Ile Glu Gly Val Arg Ile Ala 290 295 300 Leu Asp Ala Ala Lys Lys Val Leu Gly Thr Ile Gly Pro Pro Ala Leu 305 310 315 320 Val Ser Glu Thr Leu Ala Trp Glu Ile Leu Pro Gln Ala Thr Pro Val 325 330 335 Ser Lys Gln Gln Ser Gln Gly Ser Ile Gly Glu Thr Thr Pro Ala Ala 340 345 350 Gly Met Trp Thr Leu Gly Thr Pro Ala Ala Asp Val Trp Ile Leu Gly 355 360 365 Thr Pro Ala Ala Asp Val Trp Thr Ser Met Glu Ala Ala Ser Gly Glu 370 375 380 Gly Ser Ala Ala Gly Asp Leu Asp Ala Ala Thr Gly Asp Arg Gly Pro 385 390 395 400 Gln Ala Thr Leu Ser Gln Thr Pro Ala Val Gly Pro Trp Gly Pro Pro 405 410 415 Gly Lys Glu Ser Ser Val Lys Arg Thr Phe Pro Glu Asp Glu Ser Ser 420 425 430 Ser Arg Thr Leu Ala Pro Val Ser Thr Met Leu Ala Leu Phe Met Leu 435 440 445 Met Ala Leu Val Leu Leu Gln Arg Lys Leu Trp Arg Arg Arg Thr Ser 450 455 460 Gln Glu Ala Glu Arg Val Thr Leu Ile Gln Met Thr His Phe Leu Glu 465 470 475 480 480 Val Asn Pro Gln Ala Asp Gln Leu Pro His Val Glu Arg Lys Met Leu 485 490 495 495 Gln Asp Asp Ser Leu Pro Ala Gly Ala Ser Leu Thr Ala Pro Glu Arg 500 505 510 Asn Pro Gly Pro 515

SEQ ID NO: 3 Sequence length: 110 Sequence type: amino acid Topology: linear Sequence type: peptide sequence Gly Ser Arg Leu Val Ser Gly Glu Pro Gly Gly Ala Val Thr Ile Gln 1 5 10 15 Cys His Tyr Ala Pro Ser Ser Val Asn Arg His Gln Arg Lys Tyr Trp 20 25 30 Cys Arg Leu Gly Pro Pro Arg Trp Ile Cys Gln Thr Ile Val Ser Thr 35 40 45 Asn Gln Tyr Thr His His Arg Tyr Arg Asp Arg Val Ala Leu Thr Asp 50 55 60 Phe Pro Gln Arg Gly Leu Phe Val Val Arg Leu Ser Gln Leu Ser Pro 65 70 75 80 Asp Asp Ile Gly Cys Tyr Leu Cys Gly Ile Gly Ser Glu Asn Asn Met 85 90 95 Leu Phe Leu Ser Met Asn Leu Thr Ile Ser Ala Gly Pro Ala 100 105 110

SEQ ID NO: 4 Sequence length: 1911 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to mRNA Origin Organism name: Human Tissue type: Lymph Node Sequence Characteristic Characteristic symbol: CDS Location: 98. . 1693 Method for determining feature: S Symbol representing feature: sig peptide Location of occurrence: 98. . 145 Method for determining feature: S Symbol representing feature: mat peptide Location: 146. . 1693 Method for determining characteristics: S sequence GGAAGTG GTGAGGAGAG GAAGAGAAGT GGACTACTCC 37 AGGCTCATTG CTGGCACTTT ACCACAATCT CACGTCACCA GCAGGAGGGC AGGATGGAAA 97 ATG CCC CTC TTC CTC ATA CTG TGC CTG CTA CAA GGT TCT Lec Tet GTC Lec G Lec G Ser Ser Phe Ala -15 -10 -5 CTT CCA CAA AAA AGA CCC CAT CCG AGA TGG CTG TGG GAG GGC TCT CTC 193 Leu Pro Gln Lys Arg Pro His Pro Arg Trp Leu Trp Glu Gly Ser Leu 1 5 10 15 CCC TCC AGG ACC CAT CTC CGG GCC ATG GGA ACA CTC AGG CCT TCC TCG 241 Pro Ser Arg Thr His Leu Arg Ala Met Gly Thr Leu Arg Pro Ser Ser 20 25 30 CCC CTC TGC TGG CGG GAG GAG AGC TCC TTT GCA GCT CCA AAT TCA TTG 289 Pro Leu Cys Trp Arg Glu Glu Ser Ser Phe Ala Ala Pro Asn Ser Leu 35 40 45 AAG GGC TCA AGG CTG GTG TCA GGG GAG CCT GGA GGA GCT GTC ACC ATC 337 Lys Gly Ser Arg Leu Val Ser Gly Glu Pro Gly Gly Ala Val Thr Ile 50 55 60 CAG TGC CAT TAT GCC CCC TCA TCT GTC AAC AGG CAC CAG AGG AAG TAC 385 Gln Cys His Tyr Ala Pro Ser Ser Val Asn Arg His Gln Arg L ys Tyr 65 70 75 80 TGG TGC CGT CTG GGG CCC CCA AGA TGG ATC TGC CAG ACC ATT GTG TCC 433 Trp Cys Arg Leu Gly Pro Pro Arg Trp Ile Cys Gln Thr Ile Val Ser 85 90 95 ACC AAC CAG TAT ACT CAC CAT CGC TAT CGT GAC CGT GTG GCC CTC ACA 481 Thr Asn Gln Tyr Thr His His Arg Tyr Arg Asp Arg Val Ala Leu Thr 100 105 110 GAC TTT CCA CAG AGA GGC TTG TTT GTG GTG AGG CTG TCC CAA CTG TCC 529 Asp Phe Pro Gln Arg Gly Leu Phe Val Val Arg Leu Ser Gln Leu Ser 115 120 125 CCG GAT GAC ATC GGA TGC TAC CTC TGC GGC ATT GGA AGT GAA AAC AAC 577 Pro Asp Asp Ile Gly Cys Tyr Leu Cys Gly Ile Gly Ser Glu Asn Asn 130 135 140 ATG CTG TTC TTA AGC ATG AAT CTG ACC ATC TCT GCA GGT CCC GCC AGC 625 Met Leu Phe Leu Ser Met Asn Leu Thr Ile Ser Ala Gly Pro Ala Ser 145 150 155 160 ACC CTC CCC ACA GCC ACT CCA GCT GCT GGG GAG CTC ACC ATG AGA TCC 673 Thr Leu Pro Thr Ala Thr Pro Ala Ala Gly Glu Leu Thr Met Arg Ser 165 170 175 TAT GGA ACA GCG TCT CCA GTG GCC AAC AGA TGG ACC CCA GGA ACC ACC 721 Tyr Gly Thr Ala Ser Pro Val Ala Asn Arg Trp Thr Pro Gly Thr Thr 180 185 190 CAG ACC TTA GGA CAG GGG ACA GCA TGG GAC ACA GTT GCT TCC ACT CCA 769 Gln Thr Leu Gly Gln Gly Thr Ala Trp Asp Thr Val Ala Ser Thr Pro 195 200 205 GGA ACC AGC AAG ACT ACA GCT TCA GCT GAG GGA AGA CGA ACC CCA GGA 817 Gly Thr Ser Lys Thr Thr Ala Ser Ala Glu Gly Arg Arg Thr Pro Gly 210 215 220 GCA ACC AGG CCA GCA GCT CCA GGG ACA GGC AGC TGG GCA GAG GGT TCT 865 Ala Thr Arg Pro Ala Ala Pro Gly Thr Gly Ser Trp Ala Glu Gly Ser 225 230 235 240 GTC AAA GCA CCT GCT CCG ATT CCA GAG AGT CCA CCT TCA AAG AGC AGA 913 Val Lys Ala Pro Ala Pro Ile Pro Glu Ser Pro Pro Ser Lys Ser Arg 245 250 255 AGC ATG TCC AAT ACA ACA GAA GGT GTT TGG GAG GGC ACC AGA AGC TCG 961 Ser Met Ser Asn Thr Thr Glu Gly Val Trp Glu Gly Thr Arg Ser Ser 260 265 270 GTG ACA AAC AGG GCT AGA GCC AGC AAG GAC AGG AGG GAG ATG ACA ACT 1009 Val Thr Asn Arg Ala Arg Ala Ser Lys Asp Arg Arg Glu Met Thr Thr 275 280 285 ACC AAG GCT GAT AGG CCA AGG GAG GAC ATA GAG GGG GTC AGG ATA GCT 1057 Thr Lys Ala Asp Arg Pro Arg Glu As p Ile Glu Gly Val Arg Ile Ala 290 295 300 CTT GAT GCA GCC AAA AAG GTC CTA GGA ACC ATT GGG CCA CCA GCT CTG 1105 Leu Asp Ala Ala Lys Lys Val Leu Gly Thr Ile Gly Pro Pro Ala Leu 305 310 315 320 GTC TCA GAA ACT TTG GCC TGG GAA ATC CTC CCA CAA GCA ACG CCA GTT 1153 Val Ser Glu Thr Leu Ala Trp Glu Ile Leu Pro Gln Ala Thr Pro Val 325 330 335 TCT AAG CAA CAA TCT CAG GGT TCC ATT GGA GAA ACA ACT CCA GCT GCA 1201 Ser Lys Gln Gln Ser Gln Gly Ser Ile Gly Glu Thr Thr Pro Ala Ala 340 345 350 GGC ATG TGG ACC TTG GGA ACT CCA GCT GCA GAT GTG TGG ATC TTG GGA 1249 Gly Met Trp Thr Leu Gly Thr Pro Ala Ala Asp Val Trp Ile Leu Gly 355 360 365 ACT CCA GCT GCA GAT GTG TGG ACC AGC ATG GAG GCA GCA TCT GGG GAA 1297 Thr Pro Ala Ala Asp Val Trp Thr Ser Met Glu Ala Ala Ser Gly Glu 370 375 380 380 GGA AGC GCT GCA GGG GAC CTA GAT GCT GCC ACT GGA GAC AGA GGT CCC 1345 Gly Ser Ala Ala Gly Asp Leu Asp Ala Ala Thr Gly Asp Arg Gly Pro 385 390 395 400 400 CAA GCA ACA CTG AGC CAG ACC CCG GCA GTA GGA CCC TGG GGA CCC CCT 1393 Gln Ala Thr Leu Ser Gln Thr Pro Ala Val Gly Pro Trp Gly Pro Pro 405 410 415 GGC AAG GAG TCC TCC GTG AAG CGT ACT TTT CCA GAA GAT GAA AGC AGC 1441 Gly Lys Glu Ser Ser Val Lys Arg Thr Phe Pro Glu Asp Glu Ser Ser 420 425 430 TCT CGG ACC CTG GCT CCT GTC TCT ACC ATG CTG GCC CTG TTT ATG CTT 1489 Ser Arg Thr Leu Ala Pro Val Ser Thr Met Leu Ala Leu Phe Met Leu 435 440 445 445 ATG GCT CTG GTT CTA TTG CAA AGG AAG CTC TGG AGA AGG AGG ACC TCT 1537 Met Ala Leu Val Leu Leu Gln Arg Lys Leu Trp Arg Arg Arg Thr Ser 450 455 460 CAG GAG GCA GAA AGG GTC ACC TTA ATT CAG ATG ACA CAT TTT CTG GAA 1585 Gln Glu Ala Glu Arg Val Thr Leu Ile Gln Met Thr His Phe Leu Glu 465 470 475 480 GTG AAC CCC CAA GCA GAC CAG CTG CCC CAT GTG GAA AGA AAG ATG CTC 1633 Val Asn Pro Gln Ala Asp Gln Leu Pro His Val Glu Arg Lys Met Leu 485 490 495 495 CAG GAT GAC TCT CTT CCT GCT GGG GCC AGC CTG ACT GCC CCA GAG AGA 1681 Gln Asp Asp Ser Leu Pro Ala Gly Ala Ser Leu Thr Ala Pro Glu Arg 500 505 510 AAT CCA GGA CCC 1693 Asn Pro Gly Pro 515 TGAGGGACAG AGAGATGAA C TGCTCAGTTA CCATGGGAGA AGGACCAAGA TCAAAGGCCT 1753 TCAGGACCCC AGCCTCTTTC CATCATCCTT CCTCCACCTG TGGGAAGAGA AGCTGATGCA 1813 GCCGGTGCTC CACCCATGGA AGAAAGGCTG GCTGTCCTTG GGCCCAAGAA AGTCAAGCAT 1873 TATCCACGGAGAGG

SEQ ID NO: 5 Sequence length: 28 Sequence type: nucleic acid Number of strands: single stranded Topology: linear Sequence type: other nucleic acid; oligonucleotide sequence 5'-ACAAGCTTACGTCACCAGCAGGAGGGCA-3 '

SEQ ID NO: 6 Sequence length: 20 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acids; oligonucleotide sequence 5'-GGTGCCTGTTGACAGATGAG-3 '

SEQ ID NO: 7 Sequence length: 23 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid; oligonucleotide sequence 5'-GAAAGGGTCACCTTAATTCAGAT-3 '

SEQ ID NO: 8 Sequence length: 29 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acids; oligonucleotide sequence 5'-TCGGATCCTCAGGGTCCTGGATTTCTCTC-3 '

SEQ ID NO: 9 Sequence length: 56 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid; oligonucleotide sequence 5'-AAGGATCCTCATTTATCATCATCATCTTTATAATCGGGTCCTGGATTTCTCTCTGG-3 '

SEQ ID NO: 10 Sequence length: 22 Sequence type: Nucleic acid Number of strands: Single stranded Topology: Linear Sequence type: Other nucleic acids; Oligonucleotide sequence 5'-AAAGGTCCTAGGAACCATTGGG-3 '

SEQ ID NO: 11 Sequence length: 57 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acid; oligonucleotide sequence 5'-AAGGATCCTCATTTATCATCATCATCTTTATAATCTTCTGGAAAAGTACGCTTCACG-3 '

SEQ ID NO: 12 Sequence length: 27 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: other nucleic acids; oligonucleotide

SEQ ID NO: 13 Sequence length: 245 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA sequence AATTCCTCGA ACTCCAAATT TCTACCTGGA CAAGGACTGG TACTATACCC ACAGATAGTAGGA 60 GACAAATTGG ATATTATTTG CCCCAAAGTGCTGCAAAA GTATGAATAT 120 TATAAAGTTT ATATGGTTGA TAAAGACCAA GCAGACAGAT GCACTATTAA GAAGGAAAAT 180 ACCCCTCTCC TCAACTGTGC CAAACCAGAC CAAGATATCA AATTCACCAT CAAGTTTCAA 240 GAATT 245

──────────────────────────────────────────────────の Continuation of the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI Technical display location C12P 21/02 C12N 5/00 B // A61K 38/00 ABA 9282-4B 15/00 ZNAA C12P 21 / 08 A61K 37/02 ABA (C12N 5/10 C12R 1:91) (C12P 21/02 C12R 1:91) (C12P 21/08 C12R 1:91)

Claims (16)

[Claims] 1. A polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1. 2. The polypeptide according to claim 1, wherein the polypeptide comprises the amino acid sequence represented by SEQ ID NO: 1. 3. The polypeptide according to claim 1, which has the amino acid sequence represented by SEQ ID NO: 2, including the amino acid sequence represented by SEQ ID NO: 1. 4. A polypeptide comprising the amino acid sequence represented by SEQ ID NO: 3. 5. The polypeptide according to claim 1, comprising an amino acid sequence represented by SEQ ID NO: 1 to which a sugar chain is bound. 6. A polypeptide having a complex form of a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 1 and another polypeptide. 7. A DNA encoding a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1. 8. A DNA encoding a polypeptide comprising the amino acid sequence represented by SEQ ID NO: 2. 9. A recombinant D obtained by ligating a DNA encoding a polypeptide containing the amino acid sequence represented by SEQ ID NO: 1 with a vector DNA that can be expressed in a host cell.
NA body. 10. A recombinant DNA obtained by ligating a DNA encoding a polypeptide containing the amino acid sequence represented by SEQ ID NO: 2 to a vector DNA that can be expressed in a host cell. 11. A cell transformed with the recombinant DNA according to claim 9 or 10. 12. A method for producing a polypeptide, comprising culturing cells capable of producing the polypeptide according to claim 1 in a medium, and collecting the produced polypeptide. 13. The method for producing a polypeptide according to claim 12, wherein the cell is the cell according to claim 11. (14) an antibody which specifically binds to the polypeptide according to (3); 15. At least one nucleotide sequence of SEQ ID NO: 4
An isolated DNA fragment selected from the group consisting of a sense DNA containing eight consecutive base sequences, an antisense DNA complementary to the sense DNA, and the sense DNA
And an isolated DNA selected from the group consisting of the sense DNA and the derivative of the antisense DNA, which is obtained by methylating, methylphosphorizing, thiophosphorating, or deaminating the antisense DNA, respectively. fragment. 16. An antisense RNA containing at least 18 consecutive nucleotide sequences complementary to the nucleotide sequence of SEQ ID NO: 4, a sense RN complementary to the antisense RNA.
A, wherein the isolated RNA fragment selected from the group consisting of A An isolated RNA fragment selected from the group consisting of a sense RNA and a derivative of the sense RNA.