JP2000262290A - Novel protein that controls defense response to bacteria and DNA encoding the same - Google Patents

Abstract

(57) [Summary] (Modified) [Problem] MD-2 molecule that interacts with TLR and regulates the expression of inflammatory protein and encodes it
Provide DNA. Kind Code: A1 A gene having homology to the amino acid sequence of mouse MD-1 or human MD-1 is searched in an EST database, and mouse kidney cDN is identified based on the identified EST nucleotide sequence.
From the A library or the human uterus cDNA library, the full length encoding human MD-2 which is the following human protein (A) or (B) and mouse MD-2 which is the following mouse protein (C) or (D) Obtain cDNA. (A) A protein having a specific amino acid sequence. (B) NF by changing the amino acid sequence in the sequence of (A)
-A protein having properties involved in increasing KB activation. (C) a protein having an amino acid specific sequence different from those described above. (D) By changing the amino acid sequence in the sequence of (C),
A protein having properties involved in increasing F-κB activation.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a novel protein that controls a protective response to bacteria against bacteria, a DNA encoding the same, and use thereof.

[0002] The protein of the present invention and its encoding
DNA can be used as a therapeutic or diagnostic agent for diseases such as autoimmune diseases, allergic diseases, asthma, and atopic dermatitis.

[0003]

2. Description of the Related Art Leucine-rich repeat (LRR) is a protein-
It is a protein motif related to protein interaction. The motif is present in various proteins, both in function and distribution within the cell. Some have a role in protecting against pathogens. LfR is a Cf-2 and Cf-9 receptor that transmits a signal to induce defense responses to mold in tomato
Carrying a motif in the extracellular domain, LRR recognizes specific pathogen molecules and activates plant defense responses. Similarly, Toll receptor, an LRR molecule of Drosophila
Elicits a protective response to mold. Recently, a human homolog of Toll (Toll like receptors, TLRs) has been cloned (R. Medzhitov, P. Preston-Hurlburt, C.
A. Janeway Jr. Nature, 388, 349 (1997)), and five more TLRs (TLR1 to TLR5) were identified as Toll-like rece
The ptor family has been reported (F.
L. Rock, G. Hardiman, JC Timans, RAKastelei
n, JFBazan, Proc. Natl. Acad. Sci. USA 95, 58
8 (1998)). Its extracellular LRR recognizes the ligand, and its extracellular domain triggers the activation of NF-κB, one of the transcriptional regulators of RNA polymerase II, and induces gene expression of multiple inflammatory proteins ( R. Med
zhitov, P. Preston-Hurlburt, CA Janeway Jr. Natu
re, 388, 349 (1997)). Thus, the LRR molecule plays a fundamental role in various types of immune systems, but the details have not been analyzed at present. RP105 is an LRR molecule expressed on mouse B lymphocytes. This was identified by the inventor by establishing an RP antibody that protects splenic B cells from radiation-induced apoptosis. This extracellular LRR is similar to TRL. RP105, when cross-linked with an antibody, transmits an activation signal that is resistant to stimuli that trigger apoptosis. Interestingly, these activated and proliferating B cells suppress proliferation and undergo apoptosis when stimulated by antigen receptors. Thus, RP105 is involved in B cell proliferation and regulation of antigen-induced apoptosis. The present inventors have determined that RP10 on cells
A molecule that associates with five molecules and is predicted to interact with the LRR was isolated. Further, the gene was cloned to obtain a cDNA of about 1 kb in length, and the entire nucleotide sequence was determined. When the database was searched again using all amino acid sequences, MD-1 which is one of the v-myb regulated genes showed high homology, and this molecule was identified as a mouse homolog of MD-1. (J. Immunology, 161: 1348-135
3, 1998) The present inventors have cloned a gene encoding a human MD-1 molecule by performing a homology search based on the nucleotide sequence of the mouse MD-1 molecule (Japanese Patent Application No. 10-286470).
issue). Furthermore, it has been shown that the human MD-1 molecule is required for the expression of the RP105 molecule on the cell surface (Blood, 92 (8): 2
815-2822, 1988).

[0004]

As described above, the MD-1
The molecule interacts with the RP105 molecule and is involved in normal B cell proliferation and apoptosis.
Regulates the physiological function of the molecule. The present invention relates to RP105
An object of the present invention is to provide an MD-2 molecule that interacts with a TLR having an LRR structure similar to that of a molecule and regulates the expression of an inflammatory protein, and a DNA encoding the same.

[0005]

Means for Solving the Problems As described above, the present inventors have found that a cD encoding a mouse MD-1 protein has already been achieved.
NA has been isolated and its entire nucleotide sequence has been determined. (J. Immu
nology 161: 1348-1353, 1998) Based on the gene encoding this mouse MD-1 molecule, repeated studies aimed at isolating human MD-1 molecules suitable for application to pharmaceuticals, etc.
The gene encoding the human MD-1 molecule was successfully cloned. The present inventors furthermore, based on the amino acid sequence of the MD-1 molecule and the nucleotide sequence of the gene encoding the MD-1 molecule, to isolate an MD-2 molecule suitable for application to pharmaceuticals, etc. Research on human and mouse MD
We succeeded in cloning the gene encoding -2 molecules. That is, in order to isolate the mouse MD-2 molecule, a homology search was performed based on the nucleotide sequence of the mouse MD-1 molecule to obtain a gene encoding the mouse MD-2 molecule portion. All genes were successfully cloned. Furthermore, a gene encoding human MD-2 was successfully cloned based on the nucleotide sequence of the human MD-1 molecule.

That is, the present invention provides the following (A) or (B)
Or a protein having a part thereof. (A) a protein having the amino acid sequence of SEQ ID NO: 2; (B) the amino acid sequence represented by SEQ ID NO: 2 having an amino acid sequence having one or more amino acid substitutions, deletions, insertions or additions, and NF-mediated through a TLR4 molecule;
A protein having properties involved in increasing κB activation.

[0007] The present invention further relates to a DNA encoding the above human protein or a DNA having at least a part thereof.
I will provide a. Specifically, the following DNA (a)
Or the DNA shown in (b). (A) DNA containing at least the base sequence consisting of base numbers 174 to 605 in the base sequence described in SEQ ID NO: 1 in the sequence listing; (b) At least base number in the base sequence described in SEQ ID NO: 1 in the sequence listing Hybridizes with a base sequence consisting of 174 to 605 under stringent conditions, and
DNA encoding a human protein having properties involved in increasing NF-κB activation via the R4 molecule.

[0008] The present invention also provides a mouse protein of the following (C) or (D) or a protein having a part thereof. (C) a protein having the amino acid sequence of SEQ ID NO: 4; (D) the amino acid sequence represented by SEQ ID NO: 4 having an amino acid sequence having one or more amino acid substitutions, deletions, insertions or additions, and NF-mediated through a TLR4 molecule;
A protein having properties involved in increasing κB activation.

[0009] The present invention further provides a DNA having the DNA encoding the above mouse protein or at least a part thereof.
Provide A. Specific examples of the DNA include the DNAs shown in (c) and (d) below. (A) DNA containing at least the base sequence consisting of base numbers 98 to 532 in the base sequence described in SEQ ID NO: 3 in the sequence listing; (b) DNA containing at least base number in the base sequence described in SEQ ID NO: 3 in the sequence listing Hybridizes with a base sequence consisting of 98 to 532 under stringent conditions, and
DNA encoding a mouse protein having properties involved in increasing NF-κB activation through four molecules.

[0010] The present invention also provides a recombinant DNA comprising any one of the above DNAs. Further, the present invention provides a transformant transformed with any of the above DNAs or the above recombinant DNA.

[0011]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail. <1> DNA of the present invention

The first DNA of the present invention is a DNA encoding the protein (A) or (B) (hereinafter referred to as "human MD-2") or a DNA having at least a part thereof. Specific examples of the DNA of the present invention include a DNA having the base sequence shown in SEQ ID NO: 1, particularly a DNA having the base sequence represented by base numbers 174 to 605 in SEQ ID NO: 1. Since it is known that there are 1 to 6 types of DNA triplets for each type of amino acid, the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 2 is not limited to the nucleotide sequence shown in SEQ ID NO: 1. .

The second DNA of the present invention is a DNA encoding the protein (C) or (D) (hereinafter referred to as “mouse MD-2”) or a DNA having at least a part thereof. Specific examples of the DNA of the present invention include a DNA having the nucleotide sequence shown in SEQ ID NO: 3, particularly a DNA having the nucleotide sequence represented by nucleotide numbers 98 to 532 in SEQ ID NO: 3, and generally, the DNA encoding the amino acid It is known that there are 1 to 6 types of DNA triplets for each type of amino acid, and thus the nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO: 4 is not limited to the nucleotide sequence shown in SEQ ID NO: 3. .

The "part" means (A) or (B)
Means any part of the DNA encoding the protein of (C) or (D), and "having at least a part" means the protein of (A) or (B), or (C) ) Or a DNA encoding the protein of (D), wherein the DNA comprises a sequence in which one or more bases are added to one or both of the 5 ′ end and the 3 ′ end of all or a part of the DNA. Also means good. Here, the added base may be any base as long as it does not cause a shift in the coding frame, for example, a linker sequence, a base sequence encoding a signal peptide, a base sequence encoding another protein, or a DNA. An example is a sequence added for the purpose of increasing the detection sensitivity when a probe or the like is produced.

When the DNA of the present invention is prepared from a genomic library, it is highly possible that the DNA contains an intron. 2 is included in the DNA of the present invention.

The DNA of the present invention may be cDNA or chromosomal DNA as long as it has at least a part of the nucleotide sequence encoding the amino acid sequence of the novel protein MD-2.
However, due to the ease of handling in genetic engineering techniques such as ease of introduction into transformants, the DNA of the present invention is cDNA
It is preferred that

The DNA of the present invention is particularly preferably human MD-
2 or has the entire nucleotide sequence encoding the amino acid of mouse MD-2. This sequence can be used to produce proteins for applying the biological activity of human MD-2 or mouse MD-2. Human MD-2 or mouse MD
A recombinant DNA molecule in which a DNA having a nucleotide sequence encoding -2 amino acids is connected downstream of an appropriate signal sequence is prepared, and a host cell is transformed with the recombinant DNA molecule. New protein human MD-2 in culture supernatant
Alternatively, a protein having the amino acid sequence of mouse MD-2 can be secreted.

Even if proteins have the same function, diversity may occur in the amino acid sequence or the nucleotide sequence of the DNA encoding the same due to differences between species or individuals.
Therefore, as long as it has a property involved in increasing NF-κB activation through the TLR4 molecule, it encodes human MD-2 or mouse MD-2 having one or more amino acid substitutions, deletions, insertions or additions. DNA is also included in the DNA of the present invention.
Such amino acid substitutions, deletions, insertions or additions may occur simultaneously at a plurality of positions. Here, “plurality” is 2 to 40, preferably 2 to 30, and more preferably 2 to 10.

As one embodiment of the DNA encoding human MD-2 as described above, for example, a DNA having a nucleotide sequence consisting of at least 174 to 605 of the nucleotide sequence shown in SEQ ID NO: 1 in the Sequence Listing DNA that hybridizes under stringent conditions and encodes a protein having a property involved in increasing NF-κB activation through the TLR4 molecule is exemplified. Also,
As one embodiment of the DNA encoding mouse MD-2, for example,
DNA having a base sequence consisting of at least base numbers 98 to 532 among the base sequences described in SEQ ID NO: 3 in the sequence listing
That hybridizes under stringent conditions with DNA
And a DNA encoding a protein having a property involved in increasing NF-κB activation via the TLR4 molecule. The term "stringent conditions" as used herein refers to conditions under which a so-called specific hybrid is formed and a non-specific hybrid is not formed. Although it is difficult to quantify this condition clearly,
As an example, nucleic acids having high homology, for example, 70%
As described above, preferably, DNAs having homology of 80% or more hybridize with each other, and nucleic acids having lower homology do not hybridize with each other.

In the examples described below, the first DNA of the present invention is obtained by synthesizing a gene having homology to the amino acid sequence of mouse MD-1 in the National Center for Biotechnology Information.
It was searched from the xpressed sequence tag (EST) database, identified from ESTs derived from mouse kidney, and prepared from a mouse kidney cDNA library based on its nucleotide sequence. Similarly, the second DNA of the present invention was identified from a human uterus-derived EST by searching a gene database having homology to the amino acid sequence of human MD-1. However, the DNA of the present invention is not limited to those obtained by these methods, and DNA obtained by any method
It may be. That is, it may be chemically synthesized with reference to the nucleotide sequence encoding the amino acid of human MD-2 or mouse MD-2, or may be cloned from an appropriate DNA library.

For chemically synthesizing the DNA of the present invention, for example, there are the following methods. That is, first, human MD-2
Alternatively, DNA having a nucleotide sequence encoding the amino acid of mouse MD-2 is divided into fragments consisting of about 20 bases and synthesized using a DNA chemical synthesizer, and then, if necessary, phosphorylation of the 5 ′ end is performed. The fragments are annealed and ligated to obtain the desired DNA.

The DNA of the present invention can be obtained from a DNA library by screening from an appropriate genomic DNA library or cDNA library by hybridization, immunoscreening using an antibody, or the like. There is a method in which a clone having the above is propagated and cut out therefrom using a restriction enzyme or the like. Screening by the hybridization method is performed by labeling a DNA having the nucleotide sequence encoding the amino acid of human MD-2 or mouse MD-2 or a part thereof with ³² P or the like as a probe, and using a known cDNA library as a probe. Method (Molecular Cloninng, a Laboratory Manual.,
1982, Cold Spring Harbor Laboratory, New York, Mani
atis T.).

The DNA of the present invention can also be prepared by PCR (Polymera) using a genomic DNA library or a cDNA library as a template.
se Chain Reaction). In PCR, sense primers and antisense primers (for example, SEQ ID NOS: 5 and 6, or SEQ ID NOs: 7 and 8) are prepared based on the nucleotide sequence encoding the amino acid of human MD-2 or mouse MD-2, and any DNA live For the rally, a known method (PCR Pr
otocols, a Guide to Molecular and Applications, Aca
demic Press, Michael AI 1990), etc., to obtain the DNA of the present invention.

As the DNA library used in the above-mentioned various methods, any library can be used as long as it has the DNA of the present invention. A commercially available DNA library can be used, or a known library can be obtained from appropriate cells. Method (Molecular Clo
ning, a Laboratory Manual 2nd ed., 1989, Cold Spri
ng Harbor Laboratory, New York, supra). Cells for obtaining the cDNA library are not particularly limited, but include kidney cells, lymphocytes and B cells obtained from human peripheral blood and spleen cells.
Cell-based cell lines, hybridomas and the like are suitable.
As a cell for obtaining the library, it is preferable to use a cell that highly expresses MD-2. Such cells include Daudi cells.

If the cells as described above are required, they may be activated with an appropriate activator and then a cDNA library may be prepared by the above-mentioned known method.

According to the present invention, DNA encoding human MD-2
And the nucleotide sequence of the DNA encoding mouse MD-2 has been elucidated, so that the sequence of the complementary DNA and the sequence of the RNA can be uniquely determined, so that the RNA corresponding to the DNA of the present invention
Alternatively, DNA having a complementary sequence is also provided.

The novel DNA of the present invention may be single-stranded,
It may combine with DNA or RNA having a sequence complementary thereto to form a double or triple strand.

DNA encoding human MD-2 or mouse MD-2 having amino acid residue substitutions, deletions or insertions can be obtained by ordinary mutation procedures such as a method using a mutagen or a site-directed mutagenesis method. Can be There are various techniques for site-directed mutagenesis (R. Higuchi et al. Recombinant PCR in "PCR Pr.
otocols: A Guide to Methods and Applications "p.17
7, Academic Press, 1990: Sambrook, Fritsch and M
aniatis, "MolecularCloning" Chapter 15 Site-direct
ed Mutagenesis of Cloned DNA, Cold SpringHarbor La
boratory Press, 1989), but any method may be used as long as it is a method for introducing a mutation in a site-specific manner.

Incidentally, as shown in Examples described later, the DN of the present invention
Using DNA encoding A and TLR4, when MD-2 and TLR4 were transiently expressed in human kidney cell line 293T, MD-2 without a cell membrane transit site was detected on the cell membrane surface only in the presence of TLR4, It was not detected in the absence of TLR4. Therefore, MD-2 and TLR4 were expected to be associated on the cell surface.

<2> Protein of the Present Invention The first protein of the present invention is a protein having the protein (human MD-2) of (A) or (B) or a part thereof. Further, the second protein of the present invention is a protein having the protein (mouse MD-2) of (C) or (D) or a part thereof. Here, “part” means any part of the protein of (A) or (B) or the protein of (C) or (D), and “having at least a part” means ( (A) or (B) or (C) or (D) all or part of the N-terminal, the C-terminal, or both, and any one or more amino acids added thereto It means that the amino acid sequence may be defined by the following amino acid sequence.

The protein of the present invention is a protein having a property involved in increasing NF-κB activation through a TLR4 molecule, and a protein containing a part thereof.

Even if proteins have the same function, diversity may occur in the amino acid sequence and the nucleotide sequence of the DNA encoding the same due to differences between species and between individuals.
Further, depending on the position of the amino acid, it can be substituted with another amino acid without affecting the biological activity of the protein. Therefore, as long as it has a property involved in increasing NF-κB activation through the TLR4 molecule, it encodes human MD-2 or mouse MD-2 having one or more amino acid substitutions, deletions, insertions or additions. DNA is also included in the DNA of the present invention. Such amino acid substitutions, deletions, insertions or additions may occur simultaneously at a plurality of positions. Here, “plurality” is 2 to 40, preferably 2 to 30, and more preferably 2 to 10.

[0033] The first protein of the present invention preferably has at least a part of the amino acid sequence shown in SEQ ID NO: 2. Further, the second protein of the present invention preferably has at least a part of the amino acid sequence shown in SEQ ID NO: 4.

Human MD-2 and mouse MD-2 exist as cell surface molecules. Therefore, to obtain these proteins, it is necessary to purify them from lysates of cells and tissues expressing these proteins. By the way, such a protein existing on the cell surface is often present in a culture supernatant of a cell that produces the protein, or in a body fluid such as urine or blood. Generally, protein is purified from cell or tissue lysate or from culture supernatant or urine. The latter is more efficient and yields better. As described above, the novel protein has high utility in production because it is present in the culture supernatant or the like. Therefore, the present invention provides such a protein having at least a part of the novel protein as a preferred form of the protein of the present invention.

The novel protein of the present invention includes both those having a sugar chain and those having no sugar chain. In the amino acid sequences of human MD-2 and mouse MD-2, there are two sites where sugar chains can be added (N-glycosylation sites). Therefore, even if the protein is produced by animal cells, or if it is produced by genetic engineering using eukaryotic cells such as yeast or animal cells as a host, a sugar chain is added. May be On the other hand, when a prokaryotic cell such as Escherichia coli is used as a host to produce a protein by genetic engineering, the protein does not have a sugar chain.

The novel protein of the present invention may be produced by any method. For example, it may be chemically synthesized using a peptide synthesizer, or may be purified from human, mouse or any other biological tissue, cell, or body fluid. Body fluids include blood and urine. As the cells, cells that produce the novel protein of the present invention can be appropriately selected and used. Examples include thymocytes, spleen cells, lymphoid cells, and cell lines thereof. Then, the protein is purified from the lysate or culture supernatant of these cells. Purification can be performed by a combination of concentration, various types of chromatography, salting out, and the like.

However, in view of its purity, the novel protein is preferably produced by genetic engineering, that is, a recombinant protein. To produce the protein by genetic engineering, the DN of the present invention is used.
A, or using the following recombinant DNA containing this DNA,
An appropriate host cell may be transformed, the resulting transformant may be cultured, and the protein of the present invention may be recovered and purified from the culture.

According to recent techniques, proteins can be bound to polymers such as polyethylene glycol, dextran, pyran copolymer, polylysine, and styrene-maleic acid copolymer; natural polymers such as polysaccharides and proteins; and physiologically active substances such as hormones. It can be combined with inorganic substances such as magnetite. In the present invention, such modifications are also possible by combining known methods.
Therefore, the proteins of the present invention include those modified as described above.

When the novel protein of the present invention is used as a pharmaceutical, the pharmaceutical composition is produced according to the usual method for producing a pharmaceutical composition containing the protein as an active ingredient. As an active ingredient of the pharmaceutical composition, among the novel proteins of the present invention, antigens against humans have the lowest antigenicity,
It is preferable to use a protein having the amino acid sequence of human MD-2 or at least a part thereof.

<3> Recombinant DNA of the Present Invention The recombinant DNA of the present invention includes the DNA of the present invention. The recombinant DNA of the present invention may be in any form, such as circular or linear. The recombinant DNA of the present invention may be used for any purpose. For example, it may be used for amplifying the DNA of the present invention to obtain a large amount, or may be used for producing the protein of the present invention.

The recombinant DNA of the present invention comprises, in addition to the DNA of the present invention,
If necessary, it may have another base sequence. Other base sequences include enhancer sequences, promoter sequences,
Ribosome binding site, a sequence used for the purpose of amplifying the copy number, a nucleotide sequence encoding a signal peptide,
It refers to a base sequence encoding another polypeptide, a polyA addition sequence, a splicing sequence, a replication initiation sequence, a base sequence of a gene serving as a selection marker, and the like. The necessity of these nucleotide sequences is determined depending on the purpose of use of the recombinant DNA molecule, and the recombinant DNA of the present invention has at least a replication origin and a marker gene in addition to the DNA of the present invention. Is preferred. Examples of the marker gene include an ampicillin resistance gene, a kanamycin resistance gene, a neomycin resistance gene, a thymidine kinase gene and the like.

The recombinant DNA of the present invention is preferably capable of transforming Escherichia coli so as to express the protein of the present invention, human MD-2 or mouse MD-2. That is, it is preferable to have at least a promoter sequence that functions in Escherichia coli in addition to an E. coli replication origin and a marker gene. In addition, it is preferable to have at least a sequence encoding a signal peptide.

The recombinant DNA may be one that can transform a eukaryotic cell such as a yeast, an insect cell, or an animal cell so as to express the protein of the present invention. Such a recombinant DNA preferably has at least a polyA addition sequence in addition to the marker gene. In addition to these, at least a promoter of alcohol oxidase (AOX) 1 that functions in yeast, or a polyhedrin promoter that functions in insect cells, or an SV40, CMV, SRα, or FE1α promoter that functions in animal cells. .

The recombinant DNA of the present invention can be obtained by ligating the DNA of the present invention to another DNA fragment having an arbitrary nucleotide sequence, or inserting the DNA into an arbitrary vector.
Methods for inserting DNA into a vector are known. (Molecula
r Cloning, a Laboratory Manual 2nd ed., 1989, Cold
(See Spring Harbor Laboratory, New York.) That is, the DNA and the vector may be digested with appropriate restriction enzymes, and the obtained fragments may be ligated using DNA ligase. The vector may be any vector such as a plasmid vector, a phage vector, and a virus vector.

<4> Transformant of the Present Invention The transformant of the present invention has been transformed with the DNA of the present invention or the recombinant DNA. As a method for introducing the DNA or the recombinant DNA of the present invention into host cells, known methods such as an electroporation method, a protoplast method, an alkali metal method, a calcium phosphate method, a DEAE dextran method, a microinjection method, and a method using a virus (experiments) Extraordinary Medicine, Genetic Engineering Handbook, 1991, Youtosha), but any method may be used. The transformant of the present invention can also be used for the purpose of producing the DNA of the present invention in large quantities. Further, the DN of the present invention
When A is integrated downstream of an appropriate promoter in the host cell, the transformant produces the novel protein of the present invention. Therefore, such a transformant can be used for the purpose of producing a protein having an amino acid sequence encoded by the novel DNA of the present invention.

When the recombinant DNA of the present invention is used for transformation, the vector used for preparing the recombinant DNA is as follows:
It is necessary to select one suitable for the host cell. at the same time,
The promoter, the base sequence encoding the signal peptide, the marker gene and the like contained in the recombinant DNA need to be suitable for the host cell. (See Special Issue on Experimental Medicine, Genetic Engineering Handbook, 1991)

The transformant of the present invention may be either a prokaryotic cell or a eukaryotic cell. Representative examples of prokaryotic cells include Escherichia coli and Bacillus subtilis. Representative examples of eukaryotic cells include mammalian cells such as CHO cells, Hela cells, COS cells, and Namalwa cells, as well as insect cells such as Sf cells and yeast. However, the transformant of the present invention is preferably one obtained by transforming Escherichia coli, mammalian cells, or yeast. Among animal cells, dhfr-deficient cells of CHO cells capable of increasing the copy number of a gene are preferable. As for yeast, a yeast belonging to the genus Pichia is preferable in that the secretory expression of a foreign protein is large.

The transformant of the present invention can be used for the purpose of obtaining a large amount of the DNA of the present invention or for producing the protein of the present invention. The transformant may be used for any purpose, but preferably, the transformant may produce the protein of the present invention, and more preferably, the protein of the present invention may be used as a medium. The secretion is good.

In order to obtain a transformant producing the protein of the present invention, the recombinant DN of the present invention to be introduced into a host cell is used.
It is necessary that A contains at least a promoter sequence that can function in the host cell. If the host cell is a prokaryotic cell such as Escherichia coli, the recombinant DNA
An origin of replication must be included in addition to the promoter sequence of the molecule. When the host cell is a eukaryotic cell, it must contain a polyA addition site and a replication origin in addition to the promoter sequence of the recombinant DNA molecule.
In any case, it is preferable that the recombinant DNA molecule used contains the marker gene described above. In addition, in order for the transformant to express and secrete the protein of the present invention, the recombinant DNA used for the transformation contains, in addition to the sequence necessary for the production described above, 5 ′ of the DNA of the present invention.
It is necessary to have a base sequence encoding a signal peptide at the terminal. Culturing the transformant of the present invention,
The protein of the present invention can be produced by collecting and purifying the protein of the present invention from the culture. In the production method, first, the transformant of the present invention is cultured, and if necessary, gene amplification and expression induction are performed. Next, the cultures are collected, and the proteins of the present invention are purified by using them as materials, performing operations such as concentration, solubilization, dialysis, and various types of chromatography as necessary. The transformant used in the production method may be any cell transformed. However, preferably, the transformant is preferably a mammalian cell such as a CHO cell or a cell selected from yeast or Escherichia coli as a host.

[0050] The transformant can be cultured by a general method. (Microbial Experiment Method, Tokyo Chemical Doujin,
1992) The method and necessity of gene amplification and expression induction vary depending on the type of host cell and the promoter used. For example, dh as host
When fr-deficient CHO cells are used, the gene can be amplified by using a vector having dhfr as the vector and using methotrexate (MTX).

In the production method of the present invention, the culture is
Culture supernatant or cell lysate. That is, when the transformant secretes the protein extracellularly, the protein of the present invention is recovered and purified using the culture supernatant as a material. On the other hand, when the protein accumulates in cells, lysozyme, detergent, freeze-thaw,
After disrupting the cells using a means such as pressurization, the supernatant is collected by centrifugation, and after removing insoluble cell fragments and the like by filtration or the like, the protein of the present invention is purified using the material as a material.

The method of purifying the protein of the present invention from the culture can be carried out by appropriately selecting an appropriate method from those usually used for protein purification.
That is, various kinds of chromatography such as salting-out method, ultrafiltration method, isoelectric point precipitation method, gel filtration method, electrophoresis method, ion exchange chromatography, hydrophobic chromatography, antibody chromatography, chromatofocusing method, adsorption chromatography An appropriate method may be appropriately selected from commonly used methods such as chromatography and reverse phase chromatography, and if necessary, purification may be performed in an appropriate order using an HPLC system or the like.

In the production method, the protein of the present invention may be produced in a transformant as a fusion protein with another protein. For example, a method of connecting a DNA encoding a protein of interest to the downstream of a DNA encoding β-galactosidase of Escherichia coli and expressing the protein of interest as a fusion protein with β-galactosidase is generally expected because a high production amount can be expected. This is the way it is done. When the novel protein is expressed as a fusion protein with another protein, in any step of the purification process, the fusion protein is treated with a chemical substance such as bromocyan or an enzyme such as a protease to cut out the novel protein. Operation is required.

When the transformant to be used is Escherichia coli, and when the novel protein is produced as an inclusion body which is an insolubilized protein, during purification, the inclusion body is solubilized, denatured, and reconstituted. The operation of folding may be performed at an appropriate purification step.

<5> Utilization of DNA and Protein of the Present Invention The protein of the present invention and DNA encoding the same are used as immunostimulants or therapeutic and diagnostic agents for autoimmune diseases, allergic diseases, asthma, and atopic dermatitis. Can be used.

The DNA of the present invention is labeled with an enzyme such as horseradish peroxidase (HRP), a radioisotope, a fluorescent substance, a chemiluminescent substance, etc., and used to examine the expression status of MD-2 in tissues. it can. Using the DNA, the expression level of MD-2 in cells can be confirmed,
Cells and cell culture conditions suitable for the production of MD-2 can be determined. In addition, the DNA of the present invention can be used as an antisense DNA against the DNA, for example, sepsis, inflammation,
It can be used as a therapeutic agent for diseases such as autoimmune diseases, allergic diseases, asthma, and atopic dermatitis.

The protein of the present invention can be used for producing an antibody used for detecting the protein, and the antibody can be used in the fields of reagents and diagnostics. That is, the protein of the present invention and antibodies thereto can be provided as therapeutic and diagnostic agents for diseases such as sepsis, inflammation, autoimmune disease, allergic disease, asthma, and atopic dermatitis.

[0058]

EXAMPLES The present invention will be described in more detail with reference to the following examples, which are merely examples and the present invention is not limited thereto. Abbreviations used in the following description are based on commonly used abbreviations in the art.

(Example 1) MD-2 cDNA of mouse and human
And analysis of the gene encoding a protein homologous to the amino acid sequence of mouse MD-1 was obtained from the National Center for Biotechnol
It was searched in the expressed sequence tag (EST) database of ogy Information. As a result, mouse kidney-derived EST (Genbank accesion numb) having homology to mouse MD-1
er: AA109204). Referring to the nucleotide sequence of the EST clone, 3'RA
CE Method (Experimental Medicine Separate Volume New Genetic Engineering Handbook, 1996
(Yodosha), and cloned the full-length cDNA. That is, total RNA was prepared from mouse kidney, and 3'R
CDNA was synthesized using an ACE adapter primer (Gibco, 10542-017), and PCR was performed using this cDNA as a template. Oligonucleotides having the nucleotide sequences shown in SEQ ID NOS: 5 and 6 were used as PCR primers. Insert the insert DNA of the cDNA clone obtained by PCR into Bluescr
It was subcloned into ipt II (Stratagene), and its entire nucleotide sequence was sequenced using a DNA sequencer (Amersham Pharmacia Biote).
ch) and Thermo Seaquence cycle sequencing kit (Am
ersham Pharmacia Biotech).

The determined nucleotide sequence and the amino acid sequence that can be encoded by the nucleotide sequence are shown in SEQ ID NO: 3 in the sequence listing.
SEQ ID NO: 4 shows only the amino acid sequence. This mouse
The cDNA consisted of 639 bases, the longest open reading frame was 480 bases and encoded 160 amino acids. In this amino acid sequence, one site rich in hydrophobic amino acids (amino acid numbers -1 to -15) was present at the amino terminal portion. This hydrophobic region corresponds to the signal sequence, and it was assumed that MD-2 was a secretory protein. The predicted putative mature protein is at amino acid 145
Consisted of pieces.

On the other hand, a gene encoding a protein having homology to the amino acid sequence of human MD-1 was searched in the above database to obtain a base sequence of an EST clone derived from human uterus (Genbank accesion number: AA099571). . The EST
Genome Systems Inc. (St Louis, MO, USA)
And the nucleotide sequence was determined in the same manner as described above. This EST clone contained the complete coding region for human MD-2.

The determined nucleotide sequence and the amino acid sequence that can be encoded by the nucleotide sequence are shown in SEQ ID NO: 1 in the Sequence Listing.
SEQ ID NO: 2 shows only the amino acid sequence. This human cD
NA consisted of 645 bases and the longest open reading frame encoded 160 amino acids. This amino acid sequence also had a hydrophobic site at the amino terminus (amino acids -1 to -16), and no other hydrophobic site was recognized. Therefore, it was presumed to be a secretory protein. The predicted site of the mature protein consisted of 144 amino acids.

A new molecule having a high homology to MD-1 obtained as described above was named MD-2. When the amino acid sequences of these proteins and MD-2 were analyzed by the BLAST program, the homology of the mature proteins of human MD-2 and mouse MD-2 was 64%, that of human MD-2 and human MD-1 (particularly Nippon 10-2864
The homology with (70) was 23%.

(Example 2) Northern hybridization of human TLR4 and human MD-2 In Example 1, since the homology between human MD-2 and human MD-1 was confirmed, mutual interaction between human MD-2 and TLRs was confirmed. The effect was investigated. First, TL with the highest homology between human RP105 and extracellular LRR
We focused on R4. Various human lymphocyte and monocyte cell lines (Na
lm-6, Ramos, Daudi, RPMI8866, CEM, Molt4, U937, K5
RNA (20 μg / lane each) extracted from 62, THP-1) was subjected to agarose gel electrophoresis, and a nylon membrane (Hybond N
+, Amersham Japan). Hybridization was performed on the membrane using probes for MD-2 and TLR4. As a control, the above membrane was rehybridized using a probe for the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene. As a result, the lymphocyte and monocyte cell lines expressing TLR4 were found to be MD-2 mRNA positive.

Example 3 Analysis of the Role of MD-2 in TLR4-Dependent Signaling The human kidney cell line 293T was transformed into a vector having the TLR4 gene (Rusl
an Medzhitov, et al., Nature, 388, 394-397, 1997)
To obtain a cell line stably expressing TLR4. A reporter plasmid for measuring NF-κB (Fujita, T. et al., Genes Dev., 7: 1354-
1363, 1993), and a vector carrying a gene encoding β-galactosidase. The reporter plasmid is NF-
It has a κB gene and a luciferase gene whose transcription is regulated by its gene product, NF-κB. Therefore,
NF-κB activity directly correlates with luciferase activity. Relative NF-κB activity is calculated by dividing relative luciferase activity by β-galactosidase activity.

The above cells were further transfected with a recombinant vector having a gene encoding MD-2, and the relative NF-κB activities were compared. The recombinant vector is obtained by cutting the MD-2 cDNA shown in SEQ ID NO: 1 with restriction enzymes XhoI / BamHI and preparing the vector pEFBOS (Mizushima S., Nagata S, Nucleic
Acids Res., 18, 5322, 1990). The results are shown in FIG. As is evident from the results, transfection of cells with a vector having a gene encoding MD-2 significantly increased NF-κB activation by signaling through ligand-nonspecific TLR4.

[0067]

Industrial Applicability According to the present invention, there are provided human and mouse MD-2 molecules having the property of increasing NF-κB activation via ligand-nonspecific TLR4, and DNAs encoding them.

[0068]

[Sequence List] SEQUENCE LISTING <110> Mitsubishi Chemical Corporation <120> Novel protein that controls defense response to bacteria and DNA encoding it <130> J03192 <141> 1999-03-18 <160> 6 < 170> PatentIn Ver. 2.0

<210> 1 <211> 645 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (126) .. (605) <220> <221> sig_peptide <222> (126 ) .. (173) <220> <221> mat_peptide <222> (174) .. (605) <220> <221> misc_feature <222> (641) <223> r = g or a <400> 1 ggcgggccgc tcccacttcg gcacgagggg cacgaggtaa atcttttctg cttactgaaa 60 aggaagagtc tgatgattag ttactgatcc tctttgcatt tgtaaagctt tggagatatt 120 gaatc atg tta cca ttt ctg ttt ttt tcc acc ctg ttt Tct Pt Tt Pt Pt Met t Tt Pt Pt Tt Pt Pt Mt 5 act gaa gct cag aag cag tat tgg gtc tgc aac tca tcc gat gca agt 218 Thr Glu Ala Gln Lys Gln Tyr Trp Val Cys Asn Ser Ser Asp Ala Ser -1 1 5 10 15 att tca tac acc tac tgt gat aaa atg caa tac cca att tca att aat 266 Ile Ser Tyr Thr Tyr Cys Asp Lys Met Gln Tyr Pro Ile Ser Ile Asn 20 25 30 gtt aac ccc tgt ata gaa ttg aaa gga tcc aaa gga tta ttg cac att 314 Val Asn Pro Cys Ile Glu Leu Lys Gly Ser Lys Gly Leu Leu His Ile 35 40 45 ttc tac att cca agg aga gat tta aag caa tta ta t ttc aat ctc tat 362 Phe Tyr Ile Pro Arg Arg Asp Leu Lys Gln Leu Tyr Phe Asn Leu Tyr 50 55 60 ata act gtc aac acc atg aat ctt cca aag cgc aaa gaa gtt att tgc 410 Ile Thr Val Asn Thr Met Asn Leu Pro Lys Arg Lys Glu Val Ile Cys 65 70 75 cga gga tct gat gac gat tac tct ttt tgc aga gct ctg aag gga gag 458 Arg Gly Ser Asp Asp Asp Tyr Ser Phe Cys Arg Ala Leu Lys Gly Glu 80 85 90 95 act gtg aat aca aca ata tca ttc tcc ttc aag gga ata aaa ttt tct 506 Thr Val Asn Thr Thr Ile Ser Phe Ser Phe Lys Gly Ile Lys Phe Ser 100 105 110 aag gga aaa tac aaa tgt gtt gtt gaa gct att tct ggg agc cca gaa 554 Lys Gly Lys Tyr Lys Cys Val Val Glu Ala Ile Ser Gly Ser Pro Glu 115 120 125 gaa atg ctc ttt tgc ttg gag ttt gtc atc cta cac caa cct aat tca 602 Glu Met Leu Phe Cys Leu Glu Phe Val Ile Leu His Gln Pro Asn Ser 130 135 140 aat tagaataaat tgagtattta aaaaaaaaaa aaraaaaaaa 645 Asn

<210> 2 <211> 160 <212> PRT <213> Homo sapiens <400> 2 Met Leu Pro Phe Leu Phe Phe Ser Thr Leu Phe Ser Ser Ile Phe Thr -15 -10 -5 -1 Glu Ala Gln Lys Gln Tyr Trp Val Cys Asn Ser Ser Asp Ala Ser Ile 1 5 10 15 Ser Tyr Thr Tyr Cys Asp Lys Met Gln Tyr Pro Ile Ser Ile Asn Val 20 25 30 Asn Pro Cys Ile Glu Leu Lys Gly Ser Lys Gly Leu Leu His Ile Phe 35 40 45 Tyr Ile Pro Arg Arg Asp Leu Lys Gln Leu Tyr Phe Asn Leu Tyr Ile 50 55 60 Thr Val Asn Thr Met Asn Leu Pro Lys Arg Lys Glu Val Ile Cys Arg 65 70 75 80 Gly Ser Asp Asp Asp Tyr Ser Phe Cys Arg Ala Leu Lys Gly Glu Thr 85 90 95 Val Asn Thr Thr Ile Ser Phe Ser Phe Lys Gly Ile Lys Phe Ser Lys 100 105 110 Gly Lys Tyr Lys Cys Val Val Glu Ala Ile Ser Gly Ser Pro Glu Glu 115 120 125 Met Leu Phe Cys Leu Glu Phe Val Ile Leu His Gln Pro Asn Ser Asn 130 135 140

<210> 3 <211> 639 <212> DNA <213> Mus musculus <220> <221> CDS <222> (53) .. (532) <220> <221> sig_peptide <222> (53 ) .. (97) <220> <221> mat_peptide <222> (98) .. (532) <220> <221> misc_feature <222> (611) .. (615) <223> n = a or c or g or t <220> <221> misc_feature <222> (636) <223> b = g or c or t <400> 3 gtcgagtccg atggtcttcc tggcgagttt aaagtatcgg agatattaaa tc atg ttg 58 Met Leu -15 cca ttt att ctc ttt tcg acg ctg ctt tct ccc ata ttg act gaa tct 106 Pro Phe Ile Leu Phe Ser Thr Leu Leu Ser Pro Ile Leu Thr Glu Ser -10 -5 -1 1 gag aag caa cag tgg ttc tgc aac tcc tcc gat gca att att tcc tac 154 Glu Lys Gln Gln Trp Phe Cys Asn Ser Ser Asp Ala Ile Ile Ser Tyr 5 10 15 agt tat tgt gat cac ttg aaa ttc cct att tca att agt tct gaa ccc 202 Ser Tyr Cys Asp His Leu Lys Phe Pro Ile Ser Ile Ser Ser Glu Pro 20 25 30 35 tgc ata aga ctg agg gga acc aat gga ttt gtg cat gtt gag ttc att 250 Cys Ile Arg Leu Arg Gly Thr Asn Gly Phe Val His Val Glu Phe Ile 40 45 50 cca aga gga aac tta aaa tat tta t at ttc aac cta ttc atc agt gtc 298 Pro Arg Gly Asn Leu Lys Tyr Leu Tyr Phe Asn Leu Phe Ile Ser Val 55 60 65 aac tcc ata gag ttg ccg aag cgt aag gaa gtt ctg tgc cat gga cat 346 Asn Ser Ile Glu Pro Lys Arg Lys Glu Val Leu Cys His Gly His 70 75 80 gat gat gac tat tct ttt tgc aga gct ctg aaa gga gag act gtg aat 394 Asp Asp Asp Tyr Ser Phe Cys Arg Ala Leu Lys Gly Glu Thr Val Asn 85 90 95 aca tca ata cca ttc tct ttc gag gga ata cta ttt cct aag ggc cat 442 Thr Ser Ile Pro Phe Ser Phe Glu Gly Ile Leu Phe Pro Lys Gly His 100 105 110 115 tac aga tgt gtt gca gaa gct att gct ggg gat act gaa gaa aag ctc 490 Tyr Arg Cys Val Ala Glu Ala Ile Ala Gly Asp Thr Glu Glu Lys Leu 120 125 130 ttc tgt ttg aat ttc acc atc att cac cgc cgt gat gtc aat 532 Phe Cys Leu Asn Phe Thr Ile Asle Arg Arg Val Asn 135 140 145 tagaatatgc tgaatacaca cacacacaca cacacacaca cacatatgta tatatatatt 592 tttttaccca aaaaaaaann nnnaaagtac tagtcgacgc gtgbcca 639

<210> 4 <211> 160 <212> PRT <213> Mus musculus <400> 4 Met Leu Pro Phe Ile Leu Phe Ser Thr Leu Leu Ser Pro Ile Leu Thr -15 -10 -5 -1 1 Glu Ser Glu Lys Gln Gln Trp Phe Cys Asn Ser Serp Ala Ile Ile 5 10 15 Ser Tyr Ser Tyr Cys Asp His Leu Lys Phe Pro Ile Ser Ile Ser Ser 20 25 30 Glu Pro Cys Ile Arg Leu Arg Gly Thr Asn Gly Phe Val His Val Glu 35 40 45 Phe Ile Pro Arg Gly Asn Leu Lys Tyr Leu Tyr Phe Asn Leu Phe Ile 50 55 60 65 Ser Val Asn Ser Ile Glu Leu Pro Lys Arg Lys Glu Val Leu Cys His 70 75 80 Gly His Asp Asp Asp Tyr Ser Phe Cys Arg Ala Leu Lys Gly Glu Thr 85 90 95 Val Asn Thr Ser Ile Pro Phe Ser Phe Glu Gly Ile Leu Phe Pro Lys 100 105 110 Gly His Tyr Arg Cys Val Ala Glu Ala Ile Ala Gly Asp Thr Glu Glu 115 120 125 Lys Leu Phe Cys Leu Asn Phe Thr Ile Ile His Arg Arg Asp Val Asn 130 135 140 145

<210> 5 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for amplifying murine MD-2 cDNA <400> 5 gatgaattcc gatggtcttc ctggcgag 28

<210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for amplifying murine MD-2 cDNA <400> 6 ggccacgcgt cgactagtac 20

[Brief description of the drawings]

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the activation of signaling through a ligand non-specific TLR4 of MD-2.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) (C12P 21/02 C12R 1:91) F term (Reference) 4B024 AA01 AA11 BA21 CA04 DA03 4B063 QA01 QA08 QQ08 QQ22 QQ35 QQ79 QS28 QS38 QX02 4B064 AG02 CA10 CA19 CC24 DA01 DA13 4B065 AA91X AA92X AA92Y AA93X AA93Y AA94Y AB01 BA02 CA24 CA44 CA46 4H045 AA10 AA50 BA10 CA40 DA01 EA22 FA74

Claims (8)

[Claims] 1. A protein having the following human protein (A) or (B) or a part thereof. (A) a protein having the amino acid sequence of SEQ ID NO: 2; (B) the amino acid sequence represented by SEQ ID NO: 2 having an amino acid sequence having one or more amino acid substitutions, deletions, insertions or additions, and NF-mediated through a TLR4 molecule;
A protein having properties involved in increasing κB activation. 2. A protein having the following mouse protein (C) or (D) or a part thereof. (C) a protein having the amino acid sequence of SEQ ID NO: 4; (D) the amino acid sequence represented by SEQ ID NO: 4 having an amino acid sequence having one or more amino acid substitutions, deletions, insertions or additions, and NF-mediated through a TLR4 molecule;
A protein having properties involved in increasing κB activation. 3. A DNA comprising a DNA encoding a human protein of the following (A) or (B) or a D having at least a part thereof:
NA. (A) a protein having the amino acid sequence of SEQ ID NO: 2; (B) the amino acid sequence shown in SEQ ID NO: 2 having an amino acid sequence having one or more amino acid substitutions, deletions, insertions or additions, and NF-mediated through a TLR4 molecule;
A protein having properties involved in increasing κB activation. 4. The DNA according to claim 3, which is a DNA represented by the following (a) or (b): (A) DNA containing at least the base sequence consisting of base numbers 174 to 605 in the base sequence described in SEQ ID NO: 1 in the sequence listing; (b) At least base number in the base sequence described in SEQ ID NO: 1 in the sequence listing Hybridizes with a base sequence consisting of 174 to 605 under stringent conditions, and
DNA encoding a human protein having properties involved in increasing NF-κB activation via the R4 molecule. 5. A DNA encoding the following mouse protein (C) or (D) or a DNA having at least a part thereof. (C) a protein having the amino acid sequence of SEQ ID NO: 4; (D) the amino acid sequence shown in SEQ ID NO: 4 which has an amino acid sequence having one or more amino acid substitutions, deletions, insertions or additions, and NF via a TLR4 molecule
-A protein having properties involved in increasing KB activation. 6. The DNA according to claim 5, which is a DNA shown in (c) or (d) below. (A) DNA containing at least the base sequence consisting of base numbers 98 to 532 in the base sequence described in SEQ ID NO: 3 in the sequence listing; (b) DNA containing at least base number in the base sequence described in SEQ ID NO: 3 in the sequence listing Hybridizes with a base sequence consisting of 98 to 532 under stringent conditions, and
DNA encoding a mouse protein having properties involved in increasing NF-κB activation through four molecules. 7. D according to any one of claims 3 to 6,
A recombinant DNA comprising NA. 8. D according to any one of claims 3 to 6,
A transformant transformed with NA or the recombinant DNA according to claim 7.