WO2004044201A1 - Signaling proteins participating in immunopotentiation by virus-origin double-stranded rna and endotoxin and genes thereof - Google Patents

Abstract

It is intended to provide proteins having an interferon-induced signaling activity and participating in immunopotentiation by a virus-origin double-stranded RNA and an endotoxin, genes thereof, and method of using the same. Namely, DNA’s (SEQ ID NOS:1 and 3) encoding proteins having an interferon-induced signaling activity and participating in immunopotentiation by a virus-origin double-stranded RNA and an endotoxin, and proteins (SEQ ID NOS: 2 and 4) having an interferon-induced signaling activity expressed by these DNA’s. Mutant DNA’s and mutant proteins derived from the above DNA’s and proteins; antibodies specifically binding to the above proteins; nonhuman animals lacking the above genes on chromosome; and a method of judging immunopotentiation ability of cells and a method of screening an interferon-induced signaling substance using such an antibody, e nonhuman animal and a gene probe.

Description

 Description Signaling protein involved in immunostimulation by virus-derived double-stranded RNA and endotoxin and its gene

 The present invention relates to a protein having an interferon-induced signaling activity involved in immunostimulation by virus-derived double-stranded RNA and endotoxin, a gene thereof, and use thereof. Background art

 Living organisms are constantly at risk of invading microorganisms. Host defense against such a crisis is established by the cooperative action of innate immunity and acquired immunity. Antigen-presenting cells such as macrophages and dendritic cells responsible for innate immunity recognize pathogens through a group of membrane proteins called Toll-like receptors (TLRs). The antigen-presenting cells activated by this recognition produce inflammatory cytokines such as IL-12 and TNF and, at the same time, enhance the expression of accessory functional molecules such as CD40. Expression of accessory functional molecules, in cooperation with antigen presenting cells, induces the proliferation of T cells responsible for acquired immunity. In addition, IL-112 induces differentiation into naive T cells and type 1 helper T (Thl) cells that produce in-fu-feron (IFN), thereby establishing cell-mediated immunity. Therefore, the signal via TLR controls the acquired immunity quantitatively and qualitatively.

TLRs are receptors that recognize specific components of microorganisms such as pathogens, but it has been confirmed that there are many families of TLRs. In other words, the TLR receptor family is characterized by each of the components of the microorganism. Recognition of microorganisms by recognizing a constant structure and inducing natural immunoreactivity (A Rev Immunol. 20, 197, 2002; Nat. Immunol 2, 675, 2001; at. Rev. Immunol. 1, 135) , 2001 TL R 4 of the TL R receptor family functions as a receptor for lipopolysaccharide (LPS) derived from Gram-negative bacteria, and TL R 2 functions as a peptide gland derived from Gram-positive bacteria and various other proteins. TLR1 and TLR6 recognize the lipoproteins of triacylated mic mouth plasma by forming a heterodimer with TLR2, respectively, in response to lipoproteins from various fungi. G. Immunol. 169, 10, 2002). TLR 5 recognizes flagellin (f 1 agel 1 in). TLR 3 and TLR 9 are receptors for double-stranded RNA and CpGDNA (microbial D.NA), respectively. TLR 7 has been implicated in the response of antiviral compounds (Nat. Immunol. 3, 196, 2002).

 The TLR family has an extracellular leucine-rich repeat (LRR) region that is homologous to the IL-11 receptor Yuichi (IL-1R) family (Nat. Immunol 2, 675, 2001). It contains a cytoplasmic region of IL_1R and a region with high homology (TIR region). Similar to IL-1R, TLR induces I RAK via the My D88 adapter and induces the activity of TRF6 and ultimately NF-κB. That is, the TLR family recruits IL-1R binding kinase (IRAK) via My D88 which is an adapter protein in the same manner as the above IL_1R, activates TRAF6, and It is known to activate NF-κB 0. Exp. Med. 187, 2097-2101, 1998, Mol. Cell 2, 253-258, 1998, Immunity 11, 115-122, 1999). TLR-mediated production of inflammatory cytokines is completely blocked in MyD88 differentiated cells.

However, recent studies have shown that the signaling pathways through each TLR are different, and therefore produce different biological responses. (Nat. Immunol 2, 675, 2001). In fact, the TLR4 signal contains both My D88-dependent and -independent pathways. The former is essential for the production of cytokines, and the latter is for activation of IRF-3, followed by interferon-beta (IFN-i3) and IFN-inducible (IFN-inducible). ) Gene (J. Immunol. 167, 5887, 2001; Nat. Immunol. 3, 392, 2002; Essential role for TIRAP in activation of the signaling cascade shared by TLR2 and TLR4. Nature in press. 2002) ing. In addition, the MyD-88 independent pathway functionally matures dendritic cells (DCs) (J. Immunol. 166, 5688, 2001). MyD-88 independent pathway has also been observed in the TLR3 signal (Nature 413, 732, 2001).

 Recently, TIRAP / Mal has been discovered as the second adapter molecule hidden in the TIR region (Nat. Immunol., 835, 2001, Nature 413, 78, 2001). In vitro studies suggest that TIR APZM a1 is involved in LPS-induced activation in a My D-88 independent pathway (Nat. Immunol. 2, 835, 2001). . However, studies in TIRAP-deficient mice revealed that TI RAP functions as an adapter in the My D-88-independent signaling pathway via TLR2 and TLR4 (Essential role for TIRAP in act.ivat ion of the signaling cascade shared by TLR2 and TLR4. Nature in press, 2002). These studies indicate that several adapter molecules, including the TIR, are involved in the signaling pathway through the TLR, and that the different uses of these adapters result in specificity of the TLR signal, and in addition, are independent of MyD_88. This suggests that the pathway is mediated by molecules other than TIRAP.

On the other hand, the disclosure of proteins involved in the mechanism that protects against bacterial infection and their genes includes an increase in NF-κB activation via TLR4 molecules. And a gene encoding the protein (Japanese Patent Application Laid-Open No. 2000-226290), and a feedback mechanism for suppressing excessive LPS response due to bacterial infection. It is suggested that mRNA encoded by mRNA selectively generated from the TLR4 gene by splicing and a gene encoding the protein (Japanese Patent Application Laid-Open No. 2002-176696) are suggested. It has been disclosed. However, as described above, the entire contents of proteins involved in the signal pathway through TLR., Ie, genes encoding the proteins, have not been clarified.

An object of the present invention is to provide a protein having an interferon-induced signaling activity involved in immunostimulation by virus-derived double-stranded RNA and endotoxin, a gene thereof, and methods for using them. The present inventors have shown that TLR (Toll-like receptor) family members are receptors that play an essential role in the recognition of pathogen components by creating gene-deficient mice. MyD88 is an adapter with a TIR domain highly homologous to the cytoplasmic region of the TLR family, but from the analysis of gene-deficient mice, induction of inflammatory cytokines via all TLR families. Is essential for the signal transduction. However, in the case of a signal via TLR4 that recognizes lipopolysaccharide, expression of the interferon-inducible gene was induced in MyD88-deficient mice, even though there was no response to inflammatory site force-in. did. Furthermore, although TIRAP was identified as a molecule that specifically binds to TLR4, analysis of gene-deficient mice indicates that TI RAP is essential not only for TLR4 but also for TLR2-mediated production of inflammatory cytokines. It was shown. In TIRAP-deficient mice, TLR4-mediated expression of interferon-inducible genes is also induced. The presence of a missing signal was suggested.

 Since the expression of interferon-inducible genes is also induced by the signal through TLR3 that recognizes the virus-derived double-stranded RNA, MyD88 and TIRAP-independent via TLR4 It was suggested that a common molecule might be involved in the TLR3 and TLR3-mediated signals. Therefore, as a result of searching for a molecule having a TIR domain in the same manner as MyD88 and TIRAP, a gene having a TIR domain (TIR domain containing adapter inducing interferon-beta) was found. This gene has a coding region of 2,136 bp in humans and encodes 712 amino acids. In mouse, it encodes 733 amino acid at 2,199 bp. In both humans and mice, the TIR domain is located in the center of the protein (Fig. 2).

When luciferase is introduced into huian embryonic kidney cell (293M) together with a luciferase-incorporated luciferase (plasmid) gene under the interferon (IFN-beta) gene promoter, the luciferase activity is expressed when this gene is expressed. Has been enhanced. This result indicates that the protein encoded by the gene is involved in the expression of interferon) 3. The expression of My D88 and TI RAP does not activate the interferon / 3 gene promoter. Therefore, among proteins having a TIR domain, the protein encoded by the gene is interferon. Was specifically involved in the induction signal. Among mutant proteins of the protein encoded by this gene, which is made up of only the TIR domain, dominant negative: a dominant negative that blocks the activation of the interferon / 3 gene promoter by introducing the full-length protein encoded by this gene. Suppress). In cells in which human TLR3 (human TLR3) was expressed in 293 cells, stimulation of the double-stranded RNA activates the gene promoter of interleukin13. This suggests that TLR3-mediated signaling is involved in the induction of interferon] 3. The introduction of the dominant negative (suppressed) form of the protein encoded by this gene, the protein-TIR, blocked the activation of the interferon / 3 gene promoter induced by double-stranded RNA. . Since this block cannot be induced by the TIR domain of My D88 or TI RAP, the protein encoded by this gene is involved in MyD88 or TI RAP-independent signal induction of interferin. It is shown that. The present invention has been completed based on the above findings. That is, the present invention relates to a DNA encoding a protein having an inducible pheron-inducing signaling activity involved in immunostimulation by virus-derived double-stranded RNA and endotoxin, and an inducible DNA expressed by the DNA. It consists of a protein having terferon-inducing signaling activity. The DNA is shown in SEQ ID NO: 1 and SEQ ID NO: 3 in the sequence listing, and the protein is shown in SEQ ID NO: 2 and SEQ ID NO: 4 in the sequence listing. In addition, the present invention includes mutant DNAs and mutant proteins of the DNA and the protein. Furthermore, the present invention provides an antibody that specifically binds to the protein of the present invention, a non-human animal in which the gene of the present invention has been deleted on the chromosome, and the antibody, the non-human animal, and the gene probe of the present invention. And a method for screening for an interferon-induced signal transduction active substance, and the like. Disclosure of the invention

Specifically, the present invention has an interferon-induced signaling activity involved in immunostimulation by virus-derived double-stranded RNA and endotoxin. The interferon-induced signal transduction activity according to claim 1, wherein the DNA encoding the protein (claim 1) and the interferon-induced signal transduction activity are interferon | 3 inducible signal transduction activities. A DNA encoding a protein having the formula (Claim 2), the base sequence shown in SEQ ID NO: 1 or its complementary sequence, and part or all of these sequences. Or the nucleotide sequence shown in SEQ ID NO: 3 in the sequence listing or a complementary sequence thereof and a part or all of these sequences. The DNA according to claim 1 or 2, characterized in that it hybridizes under stringent conditions with the DNA according to claim 2 (claim 4) or the DNA constituting the gene according to claim 3 or 4. Claim 5) The DNA according to claim 1 or 2, wherein the protein having an interferon-inducing signaling activity is the following protein (a) or (b): A protein consisting of the amino acid sequence shown in SEQ ID NO: 2, or (b) an amino acid sequence shown in SEQ ID NO: 2 in the sequence listing in which one or several amino acids have been deleted, substituted or added. And a protein having an interferon-induced signal transducing activity (claim 6) or a protein having an interferon-induced signal transducing activity is a protein of the following (a) or (b): Or the DNA according to 2, that is, (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing; A protein comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence shown, and having an interphenin-induced signal transduction activity (Claim 7); Proteins with interferon-induced signaling activity involved in immunostimulation by RNA and endotoxin (claim Item 8), the protein having an interferon-induced signal transducing activity according to claim 8, wherein the interferon-induced signal transducing activity is an interferin-induced signal transducing activity (Claim 9). 10. The protein according to claim 8 or 9, wherein the protein comprises the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing (claim 10); A protein consisting of an amino acid sequence in which several amino acids have been deleted, substituted or added and having an interferon-inducing signal transduction activity (claim 11); or a protein having the amino acid sequence shown in SEQ ID NO: 4 in the sequence listing. The protein according to claim 8 or claim 9 (claim 12) or the amino acid sequence represented by SEQ ID NO: 4 in the sequence listing. And a protein having an amino acid sequence in which one or several amino acids have been deleted, substituted or added, and having an interferon-inducing signaling activity (Claim 13).

Also, the present invention provides a protein expression recombinant vector having an interfacial protein-inducing signal transduction activity, wherein the DNA according to any one of claims 1 to 7 is incorporated into an expression vector. (Claim 14) or a transformed cell obtained by introducing into a host cell a recombinant vector for expressing a protein having an interface-inducing signal transduction activity according to claim 14 (Claim 14). 15. The antibody according to claim 16, wherein the antibody specifically binds to the protein according to any one of claims 8 to 13 (claim 16), and the antibody is a monoclonal antibody. (Claim 17) and a chromosome-deficient gene function encoding a protein having an interferon-induced signaling activity involved in immunostimulation by the virus-derived double-stranded RN and endotoxin. Non-human animals (claim 1 8) and that the non-human animal, which encodes a protein having an inter one Hue port emissions induced signaling activity of claims 1 to 8, wherein a is a mouse genetically Non-human animals characterized in that their child functions are deficient on the chromosome (Claim 19), and virus-derived double-stranded RNAs and proteins having interphenin-induced signal transduction activity related to immunostimulation by endotoxin A cell that expresses a protein having an activity of inducing signal transduction induced by interferin, which comprises introducing the DNA according to any one of claims 1 to 7 into a cell having a chromosome-deficient gene function. (Claim 20).

Furthermore, the present invention provides a protein having interferon-induced signal transduction activity related to immune activation by endotoxin, comprising a virus or a double-stranded RNA derived from the DNA according to any one of claims 1 to 7 or a part thereof. A virus-derived double-stranded RNA and an endotoxin which detect and determine a gene using the gene detection probe encoding the gene (Claim 21) or the gene detection probe according to Claim 21. Using the method for determining the immunostimulatory function by syn (Section 22) and the antibody according to Claim 16 or 17, an interface involved in immunostimulation by virus-derived double-stranded RNA of cells and endotoxin is used. A virus-derived double-stranded RNA and endotoxin of a cell, characterized by detecting the expression state of a protein having a mouth-inducing signaling activity. The method for determining the immunostimulatory function of the virus (claim 23) and the signal transduction activity induced by the virus-derived double-stranded RNA and endotoxin-induced immunostimulation according to claim 18 A virus derived from a cell, characterized in that a test substance is administered to a non-human animal whose gene function to encode a protein is deficient on a chromosome, and the interferon-inducing activity of the non-human animal is measured and evaluated. A method for screening for an interferon-induced signal transduction active substance involved in immunostimulation by double-stranded RNA and endotoxin (claim 24). BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 is a view showing a human TRIF (TRIF) identified in an example of the present invention. a: Comparison of the amino acid sequence of human TRIF with the amino acid sequence of known smart 0255 TIR, b: Structure of TRIF, My D88, and TIRAP / M a1 In the schematic diagram, c shows the tissue distribution of human TRIF mRNA.

 FIG. 2 is a diagram showing that the amino acid sequences of human TRIF and mouse TRIF were compared in Examples of the present invention. The boxed sequence indicates the TIR domain.

 FIG. 3 is a diagram showing that TR IF induces the activity of I NF-i3 promoter and N-κΒ in Examples of the present invention.

 FIG. 4 is a diagram showing that the suppressed form of TRIF inhibits the activation of a signal transduction pathway via TLR in the example of the present invention.

 FIG. 5 is a photograph showing that the Cho 1 area of Cho Sha 1 to TLR 3 and IRF-3 are recognized in the example of the present invention. In the figures, IP indicates the antibody used for the immunoprecipitation, and IB indicates the antibody used for the immunoplot.

 FIG. 6 is a diagram showing a target deletion of a mutant Trif gene in Examples of the present invention.

 FIG. 7 is a diagram showing a defect in poly (I: C) response in TRIF-deficient cells in an example of the present invention.

 FIG. 8 is a view showing a defect in response to LPS in TRIF-deficient cells in an example of the present invention.

FIG. 9 shows the activation of signal cascade in TRIF-deficient and TRIFZMyD88 double knockout cells in the examples of the present invention. FIG. BEST MODE FOR CARRYING OUT THE INVENTION

 The present invention comprises a gene encoding a protein having an interferon-induced signaling activity involved in immunostimulation by virus-derived double-stranded RNA and endotoxin. The gene comprises the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 in the sequence listing or a complementary sequence thereof, and a DNA containing a part or all of these sequences. Examples of the interferon-induced signaling activity of the present invention include inducible signaling activity of interferon) 3.

 Further, the present invention provides a DNA encoding the following protein (a) or (b):

(a) a protein consisting of the amino acid sequence of SEQ ID NO: 2

 (b) In the immunostimulation by virus-derived double-stranded RNA and endotoxin, the amino acid sequence shown in SEQ ID NO: 2 has an amino acid sequence in which one or several amino acids have been deleted, substituted or added. Related proteins include mutant genes consisting of proteins having feron-induced signaling activity. In order to obtain the mutant gene, it can be obtained by appropriately mutating the gene by the known genetic engineering technique based on the gene sequence information in the sequence listing of the present invention.

Furthermore, the present invention provides a method for hybridizing with the gene of the present invention (the DNA of SEQ ID NO: 1 or SEQ ID NO: 3 in the sequence listing) under stringent conditions and inducing interferon involved in immunostimulation by virus-derived double-stranded RNA. Includes DNA that encodes a protein having signal transduction activity, ie, a mutant of an intact gene. To obtain the gene, for example, a DNA probe is prepared from the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 in the sequence listing, and the DNA probe is used to prepare a DNA library. Under high stringency conditions, and those having a machine-accepting channel function can be selected and obtained. Hybridization conditions for obtaining the DNA include, for example, hybridization at 42 ° C., and washing at 42 ° C. with a buffer containing 1 × SSC and 0.1% SDS. More preferably, the hybridization at 65 ° C and the washing treatment at 65 ° C with a buffer containing 0.1 XSSC and 0.1% SDS can be mentioned more preferably.

 The protein of the present invention having an interferon-induced signaling activity involved in immunostimulation by virus-derived double-stranded RNA and endotoxin comprises an amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 in the sequence listing. One or several amino acids are deleted in the protein having an interferon-induced signal transduction activity involved in immunostimulation by virus-derived double-stranded RNA or the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 in the sequence listing. Specific examples include mutant proteins having a function of a protein having an inducible ferron-induced signal transduction activity involved in immunostimulation by a virus derived double-stranded RNA comprising a substituted or added amino acid sequence. it can. These proteins can be prepared by a known method by genetic engineering using the gene of the present invention encoding the protein or by recombination of the sequence based on the DNA sequence information of the present invention. All of the proteins having an interferon-inducing signal transmission activity consisting of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 in the sequence listing obtained in the present invention have a TIR domain in the central part of the protein. . The interferon-induced signaling activity of the present invention includes the induced signaling activity of interferon 3.

Immunostimulation with the double-stranded RNA derived from the virus of the present invention and endotoxin A protein having an interferon-induced signaling activity involved in activity can be produced using the gene of the present invention by a method known in genetic engineering. That is, the gene of the present invention is integrated into an expression vector for expressing a protein having an interface-inducing signal transduction activity of the present invention, and the recombinant vector is introduced into a host cell and transformed. By culturing the cells, the gene can be expressed, and the protein having the interfacial protein-inducing signal transduction activity of the present invention can be produced.

 The expression system used for the genetic engineering production of the protein of the present invention may be any expression system capable of expressing the protein of the present invention in a host cell, such as a chromosome, an episome, and a chromosome. Expression systems derived from viruses, e.g., derived from bacterial plasmid, yeast plasmid, papovavirus such as SV40, vaccinia virus, adenovirus, fowlpox virus, pseudorabies virus, retrovirus derived vector, bacteriophage derived, Vectors derived from transposons and vectors derived from combinations thereof, for example, those derived from plasmids such as cosmid-phagemid and genetic elements of pacteriophage can be mentioned. These expression systems may contain control sequences that regulate expression as well as cause expression.

The host cells used for the genetic engineering production of the protein of the present invention include bacterial prokaryotic cells such as Escherichia coli, Streptomyces, Bacillus subtilis, Streptococcus, and S. cerevisiae, and fungi such as yeast and Aspergillus. Cells, insect cells such as Drosophila S2, Spodoptera Sf9, L cells, CHO cells, COS cells, HeLa cells, C127 cells, BALB / c3T3 cells (dihydrofolate reductase ゃ(Including mutants lacking thymidine kinase etc.), BHK21 cells, HEK293 cells, Bow It can be used by selecting from known host cells such as animal and plant cells such as es melanoma cells and oocytes. In addition, the gene of the present invention can be introduced into a host cell using a method described in a known standard laboratory manual or the like.

 The present invention includes an antibody that specifically binds to the protein of the present invention. Specific examples of the antibody include a monoclonal antibody and a polyclonal antibody. These can be prepared by a conventional method using, as an antigen, a protein having an interferon-induced signaling activity involved in immunostimulation by the virus-derived double-stranded RNA of the present invention. Among them, a monoclonal antibody is more preferable in terms of its specificity. By using such monoclonal antibodies or the like to detect the expression state of the virus-derived double-stranded RNA of the cell and the interferon-induced protein involved in immunostimulation by endotoxin, the expression state of the protein having the signal transduction activity can be detected. The immunostimulatory function of the double-stranded RNA can be determined.

As a detection means used for detecting a protein using the antibody as described above, known means can be used as appropriate. For example, the monoclonal antibody or the like of the present invention, eg, FITC fluorescent substance or the like (Full O receptacle I Ni Société § sulphonate) or tetramethyl port one da Min iso Xia ^{sulfonate, 1 2 5 I, 3 2} P, 3 5 S or ³ or radioisotope such as H, fused alkali phosphatase, Peruokishida Ichize, / 3-galactosidase, or those labeled with enzymes Fikoeri Trindade like or a fluorescent emission proteins such as green fluorescent protein (GFP) By using the fusion protein, the reaction of the antibody can be detected, and the expression of the protein can be detected. Immunological measurement methods include RIA, ELISA, fluorescent antibody, plaque, spot, hemagglutination, octotarony, etc. Can be used.

 The present invention further provides a gene deficient in a gene encoding a protein having an interferon-inducible signal transduction activity involved in immunostimulation by the virus-derived double-stranded RNA of the present invention and endotoxin on a chromosome. Includes knockout non-human animals deficient in function. In order to produce the non-human animal, a gene encoding an amino acid sequence obtained by converting some amino acids to other amino acids in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 in the sequence listing is used. By introducing the gene, it can be carried out by deleting the function of the gene encoding the interfering signal-inducing signaling protein. In addition, in the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 of the animal gene, a portion corresponding to a part of the amino acid sequence is deleted to encode an amino acid-inducible signaling protein. Can be performed by deficient gene function. Specific examples of the non-human animal in the present invention include non-human animals such as birds, egrets, mice, and rats, but mouse is particularly preferable for use in experiments. When producing a non-human animal deficient in the function of the gene encoding the protein of the present invention, the gene may be introduced by a known appropriate method such as construction of a targeting vector. Can be.

 In the present invention, the gene (DNA) of the present invention is added to a cell deficient on a chromosome in a gene functioning a protein having an interferon-induced signal transduction activity involved in immunostimulation by virus-derived double-stranded RNA and endotoxin. By introducing the cell, the cell function can be repaired, and cells expressing a protein having interferon-induced signal transduction activity can be prepared.

Further, the gene (DNA) of the present invention or a DNA sequence comprising The gene is detected using a double-stranded RNA derived from virus and a protein having an interferon-induced signaling activity involved in immunostimulation by endotoxin as a gene detection probe, and the gene is detected and compared with the DNA sequence in the sample. By comparing with the DNA sequence of SEQ ID NO: 1 or SEQ ID NO: 3 in the column list, the immunostimulatory function by the virus-derived double-stranded RNA and endotoxin can be determined. By this determination, it is possible to diagnose a disease associated with the function or expression of a protein having an inducible protein-inducing signaling activity involved in immunostimulation by virus-derived double-stranded RNA.

 Further, as described above, the antibody of the present invention is used for detecting the expression state of a virus-derived double-stranded RNA of a cell and a protein having an interferon-induced signal transduction activity involved in endotoxin-induced immune activity. Can be used to determine the immunostimulatory function of the virus-derived double-stranded RNA and endotoxin. By this determination, it is possible to diagnose a disease relating to the function or expression of a protein having an interferon-induced signal transduction activity involved in immunostimulation by virus-derived double-stranded RNA and endotoxin.

 The probe for gene detection encoding the protein having the interferon-inducing signal transduction activity of the present invention and the antibody of the present invention include a double-stranded RNA derived from a virus and a protein having an interferon-inducing signal transduction activity involved in immunostimulation by endotoxin. It can be commercialized as a kit for diagnosis of diseases related to function or expression.

The present invention provides a non-human animal having a chromosome-deficient gene function that encodes a protein having an interferon-induced signaling activity involved in immunostimulation by the virus-derived double-stranded RNA and endotoxin of the present invention. Immunostimulation by virus-derived double-stranded RNA and endotoxin Screening of interferon-induced signal transduction active substances related to the above. In screening for the interferon-inducing signal transducing substance, a test substance is administered to a non-human animal, and the non-human animal, for example, an interferon-inducing activity such as inferon feron 3 is measured and evaluated. Can be performed. The interferon-induced signal transducing substance is used for the treatment of diseases related to the function or expression of protein having interferon-induced signal transducing activity related to immunostimulation by virus-derived double-stranded RNA and endotoxin. Can be used. Hereinafter, the present invention will be described more specifically with reference to examples, but the technical scope of the present invention is not limited to these examples. Example 1

 In this example, in order to further elucidate the signaling pathway via the TLR, adapter molecules containing a TIR region other than MyD88 and TI RAP were searched, and TRIF (interferon 1 / We identified a new adapter molecule named an adapter containing a TIR region that induces 3 (interferon-beta). According to this experiment, the TR IF is not MyD88 or TI RAP, but the dominant negative (suppressed) form of IFN-] 3 and the dominant negative (suppressed) form of TR IF. By inhibiting the TLR3 reaction mediated by TLR3, it was confirmed that this novel adapter has a specific role in the TLR3 signal. [Identification of genes and elucidation of functions]

Materials and methods (Immunoprecipitation and immunoplot)

Into 293 cells, a plasmid containing the DNA fragment indicated in the expression vector was transfected and transiently expressed. 0.15% NP-40, 20 mM Tris-HCL (pH 7.5), 150 mM NaCl, 1 mM EDTA, 10% glycerol, 10 mM j3-glyceroline salt, 1 mM N a ₃ V_〇 _4, and protease Ichize inhibitors: were dissolved in lysis buffer one containing (mixture Roche Diagnostics Corporation). The cell lysate was pre-purified with Protein G-sepharose (Amersham) for 1 hour, then 2 g of anti-F1ag M2 antibody (Sigma) and 2 g of anti-MycPL14 Immunoprecipitation was performed with an antibody (MBL) or lg anti-human IRF-3 antibody (SantaCruz) and Protein G-sepharose (Amersham Chito) for 2 hours.

 The immunoprecipitate was washed in a lysate, dissolved in an SDS sample buffer, separated by SDS-PAGE, and transferred to a polyvinylidene fluoride membrane (BIO-RAD). F1 ag-tagged (Flag-tagged) protein or Myc-tagged (Myc-tagged) Protein is HRP-labeled anti-F1ag M2 antibody or HRP-labeled anti-Myc9E10 Each was reacted with an antibody (manufactured by SantaCruz). Endogenous IRF-3 was reacted with anti-human IRF-3 antibody and HRP-labeled anti-Peacock IgG antibody (Amersham), and the band recognized by the antibody was detected by the ECL system ( PerkmElmer Life Sciences ft).

[Experimental results and discussion]

 (Identification of TRIF)

Predict that MyD88 and molecules other than TIRAP in the TIR region are involved in TLR-mediated signaling pathways, and Expressed sequence tag (EST) Searched on a database overnight to identify a new human cDNA clone (accession number: BC 009860, sequence number 1) and named it TRIF did. This gene was very similar to the TIR region registered in NCBI (smalt 0 255 TIR) (Fig. 1a). Using this fragment as a probe, the full-length cDNA of this gene was identified. Compared to MyD 88 (encoding 296 amino acids) and TI RA P / M a1 (235 amino acids), this gene has a long open reading frame of 213 bp, (Sequence number 2) (Figure 1b).

 This cDNA encoding the human TRIF amino acid sequence has 55% homology with a mouse TRIF cDNA clone of unknown function (accession number: XM110244, SEQ ID NO: 3). Indicated. This gene product was designated as TRIF, ie, a TIR region containing an adapter that induces IFN-i3 (FIG. 2).

The TIR region of TRIF was located on the C-terminal side of TRIF protein. In addition, a proline residue conserved in the TLR and essential for TLR-mediated signal activity was observed (Fig. 1a) (Science 282, 2085, 1998; J. Immunol. 162, 374, 1999; Nature). 401, 811, 1999). TRIF expression was analyzed by Northern hybridization using total RNA extracted from human tissues. As a probe, a 6 7 8-1 48 1 th c DNA fragment of human ^{TRIF c DNA, [32 P]} - labeled with d CTP in megaprime DNA la Beringuki' preparative (Amersham Corp.), human MTN b1 ot (Clontech) ExpressHyb solution (Clontech) 10 ml after hybridization at 65 ° C for 12 hours, 2 XSSC, 0.1% SDS at 65 ° C for 30 hours The plate was washed with 0.2 XSSC containing 0.1% SDS at 65 ° C for 30 minutes and developed with BioMaxMS film (manufactured by KODAK). That As a result, it was observed in many tissues such as brain, lung, heart, liver, spleen, kidney, and skeletal muscle, and was particularly highly expressed in liver (Fig. 1c).

(IFN_i3 promoter and NF-κB-inducing activity by TRIF) It was shown that forced expression of MyD88 and TIRAP induces NF-κB activity in 293 cells. Analysis was performed by measuring the luciferase activity of the NF_κB responsive promoter (SEQ ID NO: 5). (Fig. 3a, left). TRIF expression induced NF-κB activation, albeit at a lower level compared to MyD88 or TIRAP-mediated induction. LPS (TLR 4 ligand) and double-stranded RNA (dsRNA) (TLR 3 ligand) induced the expression of IFN- / 3 independently of My D88 (Nat. Immunol. 3, 392, 2002, J. Immunol. 162, 374, 2002, Nature 413, 732, 2001). Next, the activity of mouse IFN-j8 promoter overnight (SEQ ID NO: 6) was observed using the Lucifera zelepo overnight gene (Fig. 3a, right). No promoter activity was observed when My D88 or TIRAP expression induced with the reporter plasmid was induced. However, expression of TRIF dramatically induced promoter activity of IFN-; 8.

In order to identify regions essential for promoter activity in the TRIF protein, several types of truncated TRIFs were created. The C-terminal half of the D1 region and the N-terminal half (1st to 54th amino acids) of D11F are the amino acids from the AC, TIR region and the C-terminal half of TRIF (380th to the C-terminal end) ) Included are ΔΝ, and those composed only of TIR region are ANAC (Fig. 3b). When co-transfection of NF-κΒ responsive luciferase lysate with 293 cells in 293 cells, either ΔC or ΔΝ has reduced activity compared to full-length TRIF-mediated induction. Induced luciferase activity (Fig. 3c, left). In the case of the IFN- / 3 promoter overnight luciferase gene, AC exhibited a promoter activity similar to that induced by full-length TRIF, but was not observed in ΔΝ (Fig. 3c, right). In ANAC, no luciferase repo overnight promoter activity was observed. These results indicate that the distinct region of TRIF is responsible for the activation of the following two types of promoters: a part of the N-terminus of TRIF is essential for the activity of the IFN-3 promoter It was suggested that both N-terminal and C-terminal parts of TRIF are involved in NF-κ activation.

(Effect of T RIF suppression on signaling pathway via TLR)

As in the case of My D88 and TIRAP, the TIR region of TRIF (expression of ΝΔΔΟ) acted as a dominant inhibitor.NF-κB and IFN-] 3 induced by full-length TRIF Activation of the promoter was significantly suppressed by the expression of TRIF ANA C. (Fig. 4a) Using TRIF ANAC, we analyzed whether TRIF is a TLR-dependent signaling pathway. Expression of TLR4 ZMD-2 in E. coli enabled the cells to activate the NF-κΒ reporter in response to LPS TRI-ANAC co-expression was dependent on TLR4-dependent NF-κB In addition, TLR_2 and TLR_7-dependent activation of NF-κB was inhibited by the expression of TRI FANA C (Fig. 4b). (Fig. 4c, d) Forced expression of MyD88 and TIRAP resulted in ligand-independent activation of NF-κΒ. The co-expression of IF ΔΝΔC markedly inhibited the activation of NF-κB via My D88 and TI RAP (FIGS. 4e, f). 8 8 and downstream of TI RAP Suggested that it is involved in many TLR-mediated signaling pathways. Compared to other TLR members, TLR3 is even more dominant than other inflammatory cytokines such as IL-12 and TNF-a (Biochem. Biophys. Res. Comraun. 293, 1364, 2002). Via a unique signaling pathway that induces IFN- / 3. 100 ng of repo overnight plasmid was transfected to 293 cells stably expressing TLR3 using 1 ipo feet amine 2000 (manufactured by Invitrogen) for 24 hours. After 5

Stimulation with 0 g / m 1 poly (I: C) for 8 hours activated both the NF-κB and IFN-β promoters (Fig. 4e, suppression of fMyD88 and TIRAP). Co-expression did not inhibit the activity of IFN-promoter-1 and NF-κB-responsive ELAM-1 promoter-1 in a poly (I: C) -dependent manner. My D— 8 8 indicates that it is composed of independent pathways (Fig. 4, e, f TR

We focused on the role of TRIF in the TLR3 signal, since forced expression of 1F predominantly activated the IFN / 3 promoter.

As a clear control for the suppressed forms of MyD88 and TIRAP, TRI FANAC is a poly (I: C) -dependent activity of both promoters in 293 cells stably expressing TLR3 Was inhibited. These results indicate that TRIF is involved in My D88-independent activation of the TLR3 signal.

(Recognition of TLR3 and IRF-3 by TRIF)

It was analyzed whether TRIF recognizes TLR3 or TLR2. Human renal cells 293 cells contain 7 ^ g of Flag-TLR2 (FLR-tagged TLR2: Flag-gged TLR2) or F1ag-TLR3 (FLRag-tagged TLR3 : Flag-tagged TLR3) and 3 8 1 ^ -TRIF (TR IF Myc-tagged: Myc-tagged TRIF) was transfected and transiently expressed. In the mo ck (control) group, an empty vector subjected to 10 zg transfection was used. 36 hours after the transfection, the cells were treated with 0.15% NP—40, 20 mM MT ris—HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 10% glycerol, l Omm I3- glycerin port phosphate, 1 m MN a ₃ V_〇 _4, and protease Ichize inhibitor (mixture Roche Diagnostics Corp.) were dissolved in lysis buffer one containing.

 The cell lysate was pre-purified for 1 hour using Protein G-sepharose (Amersham), and then 2 g of anti-F1 ag M2 antibody (anti-Flag M2 antibody: Sigma), g anti-Myc PL14 antibody (manufactured by MBL), 1 g anti-human IRF-3 antibody (anti-human IRF-3 antibody: manufactured by SantaCruz) and Protein G-sepharose ( (Amersham) for 2 hours (Fig. 5a). The immunoprecipitate is washed with a lysis buffer, dissolved in an SDS sample buffer, separated by SDS-PAGE according to molecular weight, and applied to a polyvinylidene fluoride (PVDF) membrane (BIO-RAD). Moved. The Flag-Tagged protein and Myc-Tagged protein were respectively reacted with an HRP-labeled anti-FlagM2 antibody or an HRP-labeled anti-Myc9E10 antibody (manufactured by SantaCruz). Endogenous IRF_3 is reacted with an anti-human IRF-3 antibody and an HRP-labeled anti-Egret IgG antibody (Amersham), and the band recognized by the antibody is subjected to an ECL system (PerkinElmer Life Sciences). Detected by

 As previously reported, the TIR region of MyD88 and TIRAP is essential for interaction with TLR. Therefore, the TIR region of TRIF

We analyzed whether C) recognized TLR3. F lag — TL R 3 An empty vector or My c — TRIF (ΔΝΔΟ expression vector (10 g)) was transfected into the stably expressed 293 cells and transiently expressed. After immunoprecipitation of FANAC with F1ag_TLR3, an immunoplot was performed in the same manner as above, and as a result, TRIF recognized TLR3 via the TIR region (Fig. 5b). This suggests that LPS stimulation activates IRF-3 through induction of IFN-3 in response to poly (I: C) .poly (I: C) (J. Biol. Chem. 274, 35535, 1999, FEBS Lett. 517, 251, 2001) _o Furthermore, we analyzed whether TRIF recognizes endogenous IRF-3. Transfection of GST or F1ag-TRIF expression vector and transient expression Transfection 24 hours after cell lysis, whole cell lysate After immunoprecipitation with 1 ag antibody or anti-human IRF-3 antibody, immuno-plotting was performed using anti-F1 ag antibody in the same manner as above, and to detect endogenous IRF-3. Then, whole cell lysate (WCL) was immunized with an anti-IRF-3 antibody, and as a result, TRIF was recognized by IRF-3. It is suggested that they interact (Fig. 5c).

In this experiment, a novel adapter molecule containing a TIR region was identified, named TRIF, and analyzed. Overexpression of TRIF activated the IFN- / 3 gene promoter as well as the NF-κB responsive promoter of the ELAM-1 gene. Unlike the known adabu molecules My D88 and TIRAP, TRIF activated the IFN-; 3 promoter predominantly as compared to the NF-κB responsive promoter. Pay attention What should be noted is that the suppressed form of TRIF completely inhibited TLR3-mediated signaling, but not MyD88 or TI RAP. This indicates a specific role of TRIF in TLR3 signal. In addition, the repressed form of TRIF inhibits TNF2, TLR4, or TLR7-mediated NF-κB activity, and may play a role in other TLR signaling pathways. Is suggested. Even though the functional role of TRIF in each TLR response was observed during the development of knockout mice, TRIF predominantly activated the IFN-3 promoter and TRIF activated IRF. Recognition of —3 indicates that TRIF is involved in the My D88-independent pathway of TLR 3 signaling. Example 2

 Stimulation of Toll-like receptor (TLR) leads to a common My D88-dependent pathway that leads to the production of inflammatory site force-in, and the generation of IFN—, a unique My D88-specific signal for TLR3 and TLR4 signals. Triggers activation of the D88-independent pathway. In the present example, the present inventors constructed a TIF-domain-specific adapter, disrupting a gene encoding TRIF, producing a TRIF-deficient mouse, and playing a role of TRIF in the signal transduction pathway via TLR. Was further clarified.

That is, TRIF-deficient mice were defective in expression of IFN-) 3 and activation of IRF-3 through TLR3 and TLR4. In addition, the production of inflammatory cytokines via TLR4 was abolished in TRIF-deficient macrophages. In mice deficient in both My D88 and TRIF, activation of NF-κB in response to TLR4 stimulation was completely abolished. These results indicate that TRIF was not mediated by TLR3 and TLR4. Proven to be essential for the Gnar route.

[Creation and function of TRIF-deficient mice]

Materials and methods

 (Generation of TRIF-deficient mice)

 From the genomic DNA of ES cells (E14.1), the Trif gene was isolated by PCR using Takara LA Taq ™ (TaKaRa). A targeting vector was constructed by replacing the 2.1 Kb fragment encoding the entire TRIF〇RF with the neomycin resistance gene cassette (neo). For a negative selection, a simple viral Will thymidine kinase driven by the PGK promoter was introduced into the genomic fragment (Fig. 1a). After inserting the targeting vector into ES cells, G418 and gancyclovir double resistant colonies were selected and screened by the PCR method and the Southern blot method. Homologous recombinants were injected into C57BL / 6 female mice and heterozygous F1 offspring were crossed over to produce TRIF-deficient mice. TRIF-deficient mice and wild-type littermates that intercrossed them were used in the experiments.

(Antibody)

R-484 was provided by Japan Energy, Inc., Pharmaceutical Biotechnology Laboratory. CpG oligodeoxynucleotides were prepared according to the literature (14). LPS from Salmonella endotoxin R e595 was prepared by a phenol-chloroform petroleum ether extraction process. PGN and poly (I: C) from S. aureus (Staphylococcus aureus) were purchased from Fluka and Amersham, respectively. Anti-phospho JNK antibody and anti-ERK 1/2 antibody were purchased from Cell Signaling. Anti-JNK1 antibody is from Santa Cruz Purchased from the company. Polyclonal anti-TRIF antibodies against amino acids 672-684 or 718-732 of TRIF mice were generated for immunoprecipitation or immunization plots, respectively. Polyclonal anti-IRF3 antibodies were generated against amino acids 1311-14 of IFR-3 mice. IgMi bodies subjected to FACS analysis were purchased from Jackson ImmunoResearch Laboratory.

(Electrophoretic mobility shift method)

Fetal fibroblasts and lung fibroblasts (1 × 10 ⁶ ) were converted to 10 g / m 1 LPS, 50 / X gZm 1 poly (I: C) and 10 ng gZm 1 TNF—Q ; Stimulated for the indicated time. Nuclear extracts were purified from cells, incubated with specific probes for the NF-κB DNA binding site, electrophoresed, and visualized by autoradiography according to the literature (Immunity 9: 143, 1998).

(Measurement of proinflammatory cytokine concentration)

Peritoneal macrophages induced by thiodaricholic acid were cultured in 96-well plates at the indicated concentrations of PGN, LPS, R-848 or CpG DNA (5 × 10 ⁴ cells per well). TNF- «, IL-6 and IL-12p40 produced by peritoneal macrophages were measured by ELISA according to the manufacturer's instructions (Genzyme).

(B cell proliferation analysis)

In 96 well plate, poly (I: C), LP S , at the indicated concentrations of R- 848 or Cp GD NA, and cultured for 24 hours spleen cells (1 X 1 0 ^5). 1 Microcurie [ ³ H] thymidine was pulsed for the last 12 hours [ ³ H] intake was measured with a scintillation counter (Packard).

(Flow cytometry analysis)

 Two hundred thousand spleen cells were cultured with 50 g / ml poly (I: C), 10 g / ml LPS or 10 g / ml anti-IgM antibody. After 36 hours of culture, cells were harvested and stained with a PE-conjugated anti-B220 antibody and a biotin-conjugated anti-CD69, CD86, I-Ab antibody, followed by streptavidin FITC. The stained cells were analyzed on a FAC S Calibur using CellQuest software (Becton Dickison).

(Western blot analysis)

Lung fibroblasts (1 X 1 0 ^6), in 1 0 g / m 1 of LPS, stimulated indicated times. Next, 1.0% Nonidet-P40 150 mM NaC1, 20 mM Tris-C1 (pH 7.5), ImM EDTA and a cocktail of the protease inhibitor ( The cells were lysed in a lysis buffer containing Roche). Cell lysates were degraded by SDS-PAGE (polyacrylamide gel electrophoresis) and transferred onto PVDF cell membranes (BioRad). Cell membranes were plotted with anti-phospho JNK antibody or anti-JNK1 antibody and visualized with an enhanced chemiluminescence system (NEN Life Science Product).

(Preparation of lung fibroblasts)

Mouse lungs were excised, washed in PBS, cut into small pieces, vortexed and enzymatically digested at 37 ° C for 30 minutes. Digestion buffer (10 ml / lung) is a 0.25% trypsin solution containing 400 nM EDTA. The resulting cell Cold complete DMEM medium was added to the suspension. After centrifugation (110 rpm, 5 minutes), the pellet was resuspended in complete medium and then cultured in a dish. Ten days after resection, lung fibroblasts were used for each experiment. (Native- P AGE analysis: non-reducing polyacrylamide gel electrophoresis) lung fibroblasts (1 X 1 0 ⁶⁾ and peritoneal macrophages (5 X 1 0 ^6), respectively 5 0 g / m 1 of poly (I : C) and stimulated with 1 gZml LPS for the indicated times and then dissolved. native—Cell lysate (62.5 mM Tris—Cl, pH 6.8, 15% glycerol and 1% deoxycholate) in PAGE sample buffer was isolated with native—PAGE. After that, immunization was performed with an anti-IRF-3 antibody according to the description in the literature (Int. Immunol. 14, 783, 2002).

[Experimental results and discussion]

Toll-like receptor (TLR) recognizes the specific pattern of the constituents of microorganisms, and adapts adapters having Tol 1 ZIL-11 receptor (TIR) domain such as MyD88, TIRAP and TRIF. Induces an innate immune response through the activation of the signaling pathway via (Annu. Rev. Immunol. 0: 197, 2002; Annu. Rev. Immunol. 21: 335, 2003). MyD88 is an adapter common to all TLRs, but TI RAP is specifically involved in TLR2 and TLR4-mediated signaling pathways (Nat. Immunol. 2, 675, 200K Nature 420, 324, 2002, Nature 420, 329, 2002). In addition to the common MyD88-dependent pathway, TLR3 and TLR4 use a MyD88-independent pathway that leads to IRF-3 activation and IFN-3 induction (J. Immunol. 167, 5887, 2001, Int. Immunol. 14, 1225, 2002, Immunity 17, 251, 2002). Has a TIR domain that induces IFN-3 U. Immunol. 169, 6668, 2002> Nat. Immunol. 4 (TRIF) identified as a third adapter that activates the IFN-promoter and has a TIR domain, which is associated with TLR3. , 161, 2003).

 To assess the physiological role of TRIF, we performed homologous recombination in embryonic stem (ES) cells to generate mice lacking TRIF. The mouse Trif gene is composed of one exon. The present inventors constructed a targeting vector in which one entire exon was replaced with a neomycin resistance gene (neo) (FIG. 6A). A correctly targeted ES cell clone was used to generate a mouse with a mutant allele (Figure 6B). Homozygous mutant mice that broke the Trif gene were born at the expected Mendelian ratio and developed well. Northern blot analysis suggested that peritoneal macrophages from mutant mice did not express TRIF mRNA (FIG. 6C). Immunoblot analysis of lung fibroblasts confirmed that expression of the TRIF protein was lost when the Trif gene was disrupted (Fig. 6D).

Early in vitro studies suggested that TRIF was involved in the induction of 11 ^-via shinguru3 (j. Immunol. 169: 6668, 2002; Nat. Immunol. 4: 161, 2003). Therefore, the present inventor first considered that IFN-i3 and several IFN-inducible genes such as RANTES, IP-10, and MCP-1 induced by poly (I: C) stimulation in peritoneal macrophages. mRNA was analyzed (Fig. 7A). In macrophages of TRIF ~ ^/ one mouse, expression of IFN-) 3 and IFN-inducible genes in response to poly (I: C) was defective. This was consistent with the results seen in the TLR3-/-mouse (Nature 413, 732, 2001). In addition, the spleen cells of TRIF- — mice showed a marked proliferation defect in response to poly (I: C). However, TLR9 ligand C Responded (Fig. 7B). TR IF- -B cells had a marked defect in poly (I: C) -induced increased expression of CD69, CD86 and MHC class II on the cell surface, but did not have anti-IgM There was no impairment in Ab antibody induction (Fig. 7C). Thus, TRIF-z-mouse and TLR3-z-mouse had defective responses to poly (I: C), suggesting that TRIF is essential for the signaling pathway through TLR3.

 In addition to the TLR 3 ligand, the TLR 4 ligand LPS has been shown to induce IFN-jS and IFN-inducible gene expression in a My D88-independent manner (J. Immunol. 167, 5887, 2001; Int. Immunol. 14, 1225, 2002, I Unity 17, 251, 2002). The present inventors analyzed mRNA of IFN-inducible genes such as RANTES, IP-10 and MCP-1 induced by LPS stimulation in fetal fibroblasts (FIG. 8A). In one TRIF-cell, LPS-induced IFN-inducible gene expression was significantly reduced. Thus, TRIF-no-mice were defective in My D88-independent response to LPS.

 Next, the production of inflammatory cytokines in response to several TLR ligands induced in a My D88-dependent manner was analyzed (FIG. 8B). Both wild-type and TRIF-no macrophages respond to TLR2-ligand peptide glycans, TLR7-ligand R-848 and TLR9-ligand CpG DNA at similar levels of IL- 1 2 p 40 was generated.

However, LPS-induced production of TNF-<<, IL_6, and IL-12p40 was abolished in TRIF-no-macrophages (FIGS. 8B, C). In addition, splenocytes from TRIF-no-mouse showed a significant defect in proliferation in response to LPS, despite the normal proliferation in response to R-848 (Figure 8D). . Furthermore, LPS-induced increases in CD69 and CD89 expression were induced by anti-IgM Ab antibodies in TRIF _ / _ B cells. Despite normal expression, the expression was significantly reduced (Fig. 8E). Thus, TRIF-no mice showed a defective response to LPS through the MyD88-dependent and MyD88-independent pathways.

 U. Immunol. 167, 5887, 2001; Int. Immunol. 14, 1225, 2002. IRF-3 transcribed genes were reported to be activated in the MyD 88-independent pathway through TLR 3 and TLR 4. , Immunity 17, 251, 2002). Indeed, native-PAGE analysis (non-reducing polyacrylamide gel electrophoresis) showed that poly (I: C) and LPS-induced, IRF-3 dimerization in wild-type lung fibroblasts and peritoneal macrophages, respectively. This indicated that the body had formed (Figs. 9A, B). However, no poly (I: C) or LPS-induced dimer of IRF-3 was formed in TRIF-cells. This observation indicates that TRIF is essential for activation of the MyD88-independent pathway in TLR3 and TLR4 signaling. In addition to IRF-13, NF-κB was activated in wild-type lung fibroblasts in response to poly (I: C) (FIG. 9C). TL R3 _ — and TR IF — In a single cell, poly (I: C) -induced NF—KB activation is significantly reduced, and TR IF plays an important role in TL R3 mediated signaling pathways Was suggested. In contrast, LPS stimulation led to almost normal activation of NF-κB and MAP kinase JNK, even in TRIF-no-fetal fibroblasts (FIG. 9D). In TLR4 signaling, the MyD88-dependent pathway leads to early phase activation of NF-κB and MAP kinase, whereas the MyD88-independent pathway leads to MyD88 —No—leads to the late phase activation of NF-κB and MAP kinase, as evidenced by delayed intracellular NF- □ B and MAP kinase activation (J. Immunol. 167, 5887, 2001, Immunity 11, 115, 1999).

Therefore, the present inventors proposed that TRIF-dependent delayed NF-κB and JN Assuming that the K activation defect was masked by My D88-dependent early activation of TRI'F-z mice, create mice deficient in both TRIF and MyD88 did. In IFN / MyD88 double knockout mouse fetal fibroblasts, LPS-induced activation of NF-κB and JNK was completely abolished (FIG. 9E). Furthermore, LPS induction of IFN-inducible genes such as IP-10, MCP-1 and RANTES was not observed at all in TRIF / MyD88 double knockout cells (FIG. 9F). :). These observations clearly demonstrated that TRIF is essential for TLR4-mediated activation of the My D88-independent pathway. We report the physiological function of TRIF as revealed by analysis of TRI mice. TR IF-Z mice had a marked defect in the My D88-independent response induced by TLR3 and TLR4 ligand. TRIF is an essential adapter for TLR3 signaling in the TLR3 signaling pathway, as all poly (I: C) -induced responses have been abolished in TRI mice. In TLR4 stimulation, TRIF—mice showed normal LPS-induced My D88-dependent NF_κB and MAP kinase activation, but LPS-induced production of inflammatory cytokines Had defects. My D88 and TI RAP have been shown to play important roles in the TLR4 signaling pathway. Our observations indicate that TRIF is also involved in the My D88-dependent pathway, probably through the formation of a large complex between TLR4 and an adapter with these three (or more) TIR domains. Suggested that Although activation of the IFN- / 3 promoter involved only the N-terminal portion, NF-κB activation was shown to induce both the N- and C-terminal portions of TRIF (J. Immunol. 169, 6668, 2002). Therefore, the C-terminal part of TRIF is involved in the My D88-dependent pathway of TLR4 signaling. May be required in some cases. The present inventors have identified an essential adapter that regulates the MyD88-independent pathway, which is thought to play an important role in viral infection. A detailed analysis of TRIF − / − mice regarding the involvement of TRIF in viral infection provides new insights into the relationship between TLR and virus recognition.

[Explanation of drawings in Example 2]

 FIG. 6 is a diagram showing the target deletion of the mutant Trif gene.

(A) Trif gene, evening getter vector, and predicted defective gene structure. Solid boxes indicate coded exons. Restriction enzymes: H, Hinc II.

 (B) Southern blot analysis of offspring born from heterozygotes crossing overnight. Genomic DNA was extracted from the mouse tail, digested with Hinc II, electrophoresed, and hybridized with the radiolabeled probe indicated at a. Southern blot analysis showed a single 3.8 kb band for wild-type (+ / +), a 6.1 kb band for homozygotes (—Z_), and a heterozygous (+/—) band. Now both have arisen.

 (C) Northern plot analysis of fetal fibroblasts. Total RNA (10 g) extracted from fetal fibroblasts was electrophoresed, transferred to a nylon cell membrane, and hybridized using the TRIF fragment as a probe. The same cell membrane was again hybridized with the; 8-actin probe.

 (D) Endogenous expression of TR IF. Cell lysates prepared from fetal fibroblasts were immunoprecipitated with an anti-TRIF antibody. Next, the immunoprecipitate was immunized with another anti-TRIF antibody. To examine protein expression, the same lysate was plotted with an antibody against ERK1Z2.

Fig. 7 shows the defective poly (I: C) response in TRIF-deficient cells. FIG.

 (A) Peritoneal macrophages were stimulated with 50 g / ml poly (I: C) for the indicated times. Total RNA (5 g) was extracted and Northern plot analysis was performed to examine the expression of IFN- / 3, IP-10, RANTES and MCP-1. The same cell membrane was re-hybridized with a 3-actin probe.

(B) Proliferation of splenocytes stimulated with poly (I: C) or CpG DNA. Splenocytes were cultured for 24 hours at the indicated concentrations of poly (I: C) or CpG DNA. ^[3 H] were late 1 2 hour pulse thymidine (1 C i).

[ ³ H] thymidine incorporation was measured with a scintillation counter (Packard).

(C) Spleen B220 + cells were cultured with 50 g / ml poly (I: C) or 10 g / ml anti-IgM antibody. After 3 6 hours of culture, cells were harvested, with Piochin conjugated anti CD 6 9 antibody or an anti-CD 8 6 antibody, or stained with streptavidin-PE Following anti-I one A ^b antibody. Cells stained with FAC S Cal ibur were analyzed using Cell Quest software.

 FIG. 8 is a view showing a defective response to LPS in TRIF-deficient cells.

 (A) Fetal fibroblasts were stimulated with 10 gZm1 LPS for the indicated times. Total RNA (10 g) was extracted and Northern blot analysis was performed to determine the expression of IP-10, RANTES and MCP-1. The same tissue membrane was rehybridized with the] 3-actin probe.

(B) Peritoneal macrophages from TRIF-deficient mice or wild-type mice were made unstimulated, or 10 gZm1 of peptidoglycan (PGN;), 100 nggZml of LPS. , Ι Ο Μ ηΜ R— 8 4 8, 3 Stimulation was performed with 1 M CpGDNA in the presence of 0 ng Zm1 IFN-A. Supernatants were collected 24 hours later for IL-112p40 analysis by ELISA. The indicated value is three times the average soil standard deviation.

 (C) Peritoneal macrophages were stimulated for 24 hours at the indicated LPS concentration in the presence of 30 ng / m1 IFN-r for 24 hours, and the supernatant was subjected to ELISA,

TNF-, IL-6 and IL-12p40 were measured. The displayed value is three times the average standard deviation.

(D)? Proliferation of splenocytes stimulated with 3 or 〇 0 DNA. Splenocytes were cultured at the indicated concentrations of LPS or CpG DNA for 24 hours. ^[3 H] were late 1 2 hour pulse thymidine (1 C i). [ ³ H] thymidine incorporation was measured with a scintillation counter (Packard).

(E) Splenocytes B2220 + were cultured with 10 g / ml LPS or 10 g / ml anti-IgM antibody. At the end of the 36 hour culture, cells were harvested and stained with a biotin-conjugated anti-CD69 or CD86 antibody, followed by streptavidin PE. Stained cells were analyzed on a FAC S Calibur using Cell Quest software.

 FIG. 9 is a diagram showing the activation (A and B) of the signal cascade in TRIF-deficient and TRIF / MyD88 double knockout cells.

Lung fibroblasts (A) or peritoneal macrophages (B) were treated with (A) 50 u § / ml poly (I: C) or (B) 1 PL g / m 1 LPS for the indicated times. Stimulated. Cell lysates were prepared and subjected to native-PAGE.単 量 体 IRF-3 monomer (arrow) or dimer (arrowhead) was detected by the Estanblot method.

((: And 13) Fibroblasts were stimulated with 50 g / ml poly (I: C) and (D) 10 g / ml LPS LPS for the indicated times. Tones NF-κ DNA binding activation was measured by EMSA using an NF-κΒ specific probe. Arrows and asterisks indicate the induced NF-κΒ complex and the non-specific band, respectively ('top panel). JNK activation of LPS-stimulated cells was measured by Western blot using an anti-phospho JΝΚ-specific antibody against the cell extract (lower panel). (F) Fetal fibroblasts of wild type and TRIF / My D88 DKII mice were stimulated with 10 gZml LPS for the indicated times. Total RNA (10 g) was extracted and analyzed by Northern blot to examine the expression of IP-10, RANTES and MCP-1. The same cell membrane was rehybridized with a] 3-actin probe. Industrial applicability

 According to the present invention, a protein having an interferon-induced signal transduction activity involved in immunostimulation by a virus-derived double-stranded RNA and endotoxin, which is involved in a host defense mechanism against invasion of a microorganism by an organism, and a protein encoding the protein Thus, the present invention can not only greatly contribute to elucidation of the molecular mechanism of induction of interface by virus infection, but also to the immunostimulatory mechanism of the virus-derived double-stranded RNA. Diagnosis of related diseases—can provide a powerful means for creating therapeutic agents for treating the diseases.

Claims

The scope of the claims 1. A double-stranded RNA derived from a virus and a DNA encoding a protein having an interferon-induced signaling activity involved in immunostimulation by endotoxin.  2. The DNA encoding the protein having an interferon-induced signaling activity according to claim 1, wherein the interferon-induced signaling activity is an interferon / 3-induced signaling activity. 3. The DNA of claim 1 or 2, wherein the DNA comprises the base sequence shown in SEQ ID NO: 1 in the sequence listing or its complementary sequence, and a part or all of these sequences.  4. The DNA according to claim 1 or 2, comprising a base sequence shown in SEQ ID NO: 3 in the sequence listing or a sequence complementary thereto and a part or all of these sequences.  5. The DNA according to claim 1 or 2, which hybridizes with the DNA constituting the gene according to claim 3 or 4 under stringent conditions.  6. The DNA according to claim 1 or 2, wherein the protein having an interferon-induced signaling activity is the following protein (a) or (b).  (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 in the sequence listing (b) In the amino acid sequence shown in SEQ ID NO: 2 in the sequence listing, one or several amino acids are deleted, substituted or added, and the amino acid sequence has an interfacial protein-induced signal transduction activity. Protein with 7. The method according to claim 1, wherein the protein having an interferon-induced signal transduction activity is a protein of the following (a) or (b): Is the DNA described in 2.  (a) a protein consisting of the amino acid sequence represented by SEQ ID NO: 4 in the sequence listing (b) a protein comprising an amino acid sequence shown in SEQ ID NO: 4 in which one or several amino acids are deleted, substituted or added, and having an interferon-induced signal transduction activity 8. A protein having an inducible pheron-induced signaling activity involved in immunostimulation by virus-derived double-stranded RNA and endotoxin.  9. The protein having an interferon-induced signaling activity according to claim 8, wherein the interferon-induced signaling activity is an interferon) 3 -induced signaling activity.  10. The protein according to claim 8 or 9, comprising an amino acid sequence represented by SEQ ID NO: 2 in the sequence listing.  11. A protein having an amino acid sequence shown in SEQ ID NO: 2 in which one or several amino acids are deleted, substituted or added, and having an interferon-induced signal transduction activity. 12. The protein according to claim 8 or 9, comprising an amino acid sequence represented by SEQ ID NO: 4 in the sequence listing.  13. A protein comprising an amino acid sequence in which one or several amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 4 in the sequence listing, and which has an interferon-induced signal transduction activity. 14. A recombinant protein expression vector having interferon-induced signal transduction activity, wherein the DNA according to any one of claims 1 to 7 is incorporated into an expression vector. 15. A transformed cell, wherein the recombinant vector for expressing a protein having the interferon-induced signal transducing activity according to claim 14 has been introduced into a host cell. 16. A body that specifically binds to the protein according to any one of claims 8 to 13.  17. The antibody according to claim 16, wherein the antibody is a monoclonal antibody.  18. A non-human animal characterized by a chromosomal deficiency in a gene function that encodes a protein having an interfacial protein-inducing signaling activity involved in immunostimulation by virus-derived double-stranded RNA and endotoxin. 19. The non-human animal is a mouse, wherein the gene function of encoding a protein having an interferon-induced signaling activity according to claim 18 is deficient on a chromosome. Non-human animals.  20. In a cell having a chromosome-deficient gene function that encodes a protein having an inferon-feron-induced signaling activity involved in immune activation by virus-derived double-stranded RNA and endotoxin. A method for preparing a cell that expresses a protein having an interferon-induced signaling activity, comprising introducing the DNA according to any one of the above.  21. A protein having the interferon-induced signal transduction activity involved in the activation of immunity by virus-derived double-stranded RNA and endotoxin, comprising a DNA or a portion thereof according to any one of claims 1 to 7. Probe for detecting genes encoding proteins. 22. A method for determining the immunostimulatory function of a cell-derived double-stranded RNA RNA and endotoxin, comprising detecting and determining a gene using the probe for gene detection according to claim 21. 23. Using the antibody according to claim 16 or 17 to detect the expression state of a virus-derived double-stranded RNA of a cell and a protein having an interface-induced signaling activity involved in immunostimulation by endotoxin. It is characterized by double-stranded RNA and endotoxin from virus of cells. Method for determining the immunostimulatory function.  24. A non-human animal deficient on the chromosome in the function of a gene encoding a protein having an interferon-induced signaling activity involved in immune activation by endotoxin and the virus-derived double-stranded RNA according to claim 18. A test substance, and measuring and evaluating the interferon-inducing activity of the non-human animal. Interferon-inducing signal related to immunostimulation by double-stranded RNA and endotoxin of a cell virus. A method for screening a transmission active substance.