JPH11123082A - T cell receptor zeta chain protein, refined nucleic acid containing gene encoding the same or part of the gene, and detection of autoimmune disease based upon the protein or nucleic acid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a T cell receptor ζ chain protein useful for treatment for and diagnosis of systemic lupus erythematosus, etc., and applicable to patients suffering from autoimmune diseases. SOLUTION: This T cell receptor ζ chain protein comprises an isolatedly refined amino acid residue of the formula or the substantially same amino acid as this and is used for patients suffering from autoimmune diseases. The other objective nucleic acid refined isolatedly is to contain gene encoding the T cell receptor ζ chain protein or part thereof wherein the part comprises any sequences involved in signal transfer such as tyrosine residue, immune receptor tyrosine dependent activated motif or GTP/GDP bond sequence. Preferably, there are prepared diagnosis kits for autoimmune diseases by using the nucleic acid.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

FIELD OF THE INVENTION The present invention relates to T
The present invention relates to a nucleic acid encoding a ζ-chain protein in a cell receptor.

[0002] The present invention also relates to a method for detecting an autoimmune disease based on the ζ-chain protein or a nucleic acid encoding the same.

[0003]

2. Description of the Related Art Generally, an immune reaction is a reaction in which immune cells recognize an antigen that has entered from a foreign source, generate predetermined antibodies and the like, and these antibodies attack the antigen. That is, immune cells distinguish between self and non-self, and immune tolerance (tolerance) acts on self-antigens to suppress immune responses, while eliciting immune responses against non-self antigens.

[0004] However, when the balance of the immune tolerance is broken, an antibody is also produced against a self-antigen, which causes a problem of attack on a self-antigen such as a self-tissue. Such a phenomenon is called an autoimmune reaction, and various diseases are caused by the autoimmune reaction.

As autoimmune diseases, systemic lupus erythematosus (SLE), dermatomyositis, chronic indirect rheumatism, autoimmune hemolytic anemia, pernicious anemia, etc. are known, and the symptoms are fatal in SLE, dermatomyositis, etc. In some cases.

[0006] As a treatment for these diseases, for example, immunosuppressive therapy is applied to SLE and dermatomyositis which cause the above-mentioned serious symptoms. Also, in rheumatoid arthritis and the like, artificial organs and allogeneic organ transplants are performed because their own organs are completely lost due to autoimmunity. In addition, metabolic enzymes, hormones, and the like that are deficient are administered for those that cause metabolic abnormalities as symptoms. However, these treatments are merely coping treatments, and at present the treatment of fundamental autoimmune diseases has not been attempted.

[0007] At present, research is being conducted to elucidate the causes of these autoimmune diseases in order to aim at the fundamental treatment of these autoimmune diseases.

[0008] In recent years, in SLE, one of the above autoimmune diseases, it is clear that the dysfunction observed in SLE peripheral blood T cells is corrected by directly activating protein kinase C (PKC) by phorbol ester. It has become. From this, it has been pointed out that the cause of the dysfunction may be in the pathway from the T cell receptor (TcR) to PKC.

Usually, an immune response by TcR is induced by recognition of an antigen by TcR and signal transduction into T cells after recognition. FIG. 10 shows the mechanism of immune response induction by T cells.

[0010] Immune responses by T cells include α, β, γ,
Antigen recognition by a T cell receptor (TcR) composed of five subunits of ε and ζ, and signal transduction into cells via the TcR are essential.

Foreign matter invading from outside is presented to T cells together with MHC class I and class II. The antigen presented here binds to the TcR-CD3 molecule, and the MHC molecule binds to the CD4 and CD8 molecules to perform antigen recognition.

[0012] In T cells, antigen recognition via TcR is transmitted as a signal into cells, and early intracellular signal transmission such as intracellular calcium elevation, phospholipid metabolism, and tyrosine phosphorylation occurs. In particular, tyrosine phosphorylation is the biochemical change that occurs earliest after stimulation. The enzymes involved in tyrosine phosphorylation, tyrosine kinases, have been shown to be fyn, ZAP70 associated with CD3 and lck associated with CD4, CD8, IL-2R. These tyrosine kinases phosphorylate and activate various cytoplasmic proteins, allowing signals to reach the nucleus. As a result, new proteins necessary for cell proliferation and the like, for example, various cytokines are synthesized. The protein synthesized here is used to differentiate immune cells such as T cells and B cells,
It promotes proliferation and activates cytotoxic cells such as macrophages to promote antigen destruction.

That is, the above-mentioned studies using phorbol esters suggest that SLE has an abnormality in the signal transduction process by the above-mentioned phosphorylase from this antigen recognition.

[0014]

As described above, in SLE, it is suggested that there is an abnormality in the signal transmission process from antigen recognition, but the cause of this abnormality has not been elucidated. Therefore, as a treatment method for SLE, it has been necessary to rely on immunosuppressive therapy for reducing the symptoms of SLE by reducing the immunity of the patient. However, this immunosuppressive therapy also has the side effect of reducing the aggressiveness against foreign substances that need to be eliminated by an immune reaction, and thus a better treatment is desired.

The inventors of the present invention have conducted intensive studies in order to elucidate the causes of the onset of SLE and to develop a more effective treatment method. Revealed that there is something abnormal.

[0016] More specifically, the present inventors have proposed that human S
Analysis of signal transduction from TcR in LE was performed,
In the signaling pathway from TCR / CD3, ZAP7
It was found that phosphorylation of the ζ chain protein by 0 was reduced. Furthermore, focusing on this point, as a result of analyzing the ζ chain protein of the SLE patient and the DNA encoding the same,
We found that there was a mutation in the tyrosine phosphorylation site of the ζ chain protein. In particular, there are four phosphorylation sites in the ζ chain protein, and it is clear that many mutations are observed in the region containing the tyrosine residue at position 120, which is the most C-terminal side of the ζ chain protein. did.

Accordingly, the present invention aims to provide a protein and a nucleic acid which can be used for the treatment and diagnosis of autoimmune diseases, especially SLE, based on the above findings, and to provide a biochemical detection method of SLE. .

[0018]

Means for Solving the Problems In order to achieve the above object, the present invention relates to an isolated and purified T cell receptor ζ chain protein, which is used for an autoimmune disease patient.

According to the protein of the present invention, normal ζ
It becomes possible to supply chain proteins.

[0020] The nucleic acid of the present invention is a purified nucleic acid containing a gene encoding a T cell receptor ζ chain protein or a part thereof, wherein at least one of the T cell receptor ζ chain protein has It is characterized by including a phosphorylation site.

According to the nucleic acid of the present invention, a gene encoding an abnormal ζ chain protein of a patient is complemented, and the normal ζ chain protein is added to the patient.
It becomes possible to provide a chain protein.

Therefore, the “ζ-chain protein” here means
It refers to normally functioning 意味 chain proteins, especially those that can transmit signals normally during an immune response. In addition, as long as it functions normally, the amino acid sequence of the ζ-chain protein may have a mutation in a region not involved in the function.

Specifically, examples of the normal ζ chain protein include those having the amino acid sequence of SEQ ID NO: 1. However, a substantially identical sequence, ie,
The present invention also includes an amino acid sequence that is partially different from the amino acid sequence described in SEQ ID NO: 1, but normally functions as a ζ chain protein.

The term “gene encoding a ζ chain protein” (hereinafter referred to as ζ chain gene) means the gene encoding a normal ζ chain protein described above, and specifically, the nucleic acid sequence represented by SEQ ID NO: 1. Can be mentioned. Further, the sequence is not limited to the sequence described in SEQ ID NO: 1, and includes a sequence having a nonsense mutation. further,
It includes a gene that differs from the sequence of SEQ ID NO: 1 but encodes a normal ζ-chain protein.

The above “gene” includes genomic DNA, cD
NA and also RNA are included. In addition, not only a structural gene but also a control sequence can be included.

When complementing a gene encoding a patient's abnormal ζ-chain protein, it is desirable to use a full-length ζ-chain gene, but the purpose can be achieved even with a part of the ζ-chain gene. For example, when replacing a region having a patient mutation with a normal sequence as in gene targeting, the full length is not necessarily required. Therefore, the “part of the gene encoding the ζ-chain protein” described here only needs to have at least a length that can complement the region having the patient mutation.

For example, as observed from gene analysis of SLE patients, when it is considered that an autoimmune disease is caused by a mutation in a region involved in signal transduction on the ζ chain protein, the “partial” Can be composed of a region necessary for replacing the mutation.
More specifically, when complementing a deletion mutation from residue 110 to residue 121 of the ζ-chain protein detected from an SLE patient, the portion is at least 110 to 1 to 1
It can be composed of a region containing up to 21 residues.

The “sequences involved in signal transduction” include tyrosine residues and immunoreceptor tyrosine-dependent activation motifs containing the same (ITAM: Immunorecepter T).
yroshine-based Activation Motif) (Romeo et al., Cell 6
8: 889-897 and Irving et al., Cell 64: 891-901) or GT
P / GDP binding sequence (Peter. ME et al., Embo J. 11; 933-94
1) and the like.

The tyrosine residue on the chain and the ITAM are p
It is a substrate for tyrosine kinases such as 56 ^lck , p59 ^fyn and ZAP70, and it is known that signal transduction in the early stage of immunity is performed by phosphorylation of these substrates by tyrosine kinase.

Further, although there are three ITAM domains in the に は chain, the deletion mutation
Part of the ITAM and the GTP /
It corresponds to the GDP binding sequence. The GTP / GDP binding sequence plays an important role in the pathway for activating G proteins via Shc / Grb2 / SOS. Therefore, S
Deletion mutations in LE may prevent this pathway from working, resulting in an autoimmune disease.

The above-mentioned “purified nucleic acid” is mainly used to distinguish it from a nucleic acid naturally present in an individual such as a human. Therefore, the “purified nucleic acid” includes a nucleic acid isolated and purified from an individual such as a human, a synthesized nucleic acid, and a nucleic acid modified in vitro or the like.
Further, the term also includes a nucleic acid introduced into a suitable host.

[0032] The modified nucleic acids include those in which the gene or a part thereof is connected to another sequence, for example, a vector or any nucleic acid fragment.

This vector includes a vector for amplification or a vector for expression of the above gene or a partial sequence thereof. The vector also includes a vector that can self-amplify in prokaryotic or eukaryotic cells, or a shuttle vector that can amplify in both cells. In particular, when applied to gene therapy, it is effective to use vectors developed for gene therapy, such as retrovirus vectors, adenovirus vectors, adeno-associated virus vectors, and HIV virus vectors.

In order to easily treat an autoimmune disease using the above-described nucleic acid or protein, it is necessary to provide a kit for treating an autoimmune disease containing the nucleic acid or protein and necessary reagents and the like. Is preferred. The reagents necessary here are not limited to reagents such as a pharmaceutically acceptable dissolving agent, but may include equipment necessary for administration.

The above-mentioned optional nucleic acid fragment includes a selection gene, a sequence involved in a nuclear localization signal, a linker or a partial sequence of a vector, and the like. The selection gene is drug resistance,
Genes that can be selected using auxotrophy, metabolic abnormalities, and the like as indices. The linker may be, in addition to the nucleic acid containing one or more restriction sites,
And nucleic acids complementary to the primers.

[0036] The nucleic acid of the present invention is a purified nucleic acid comprising a gene encoding a T cell receptor ζ chain protein or a part thereof, the part comprising at least 16 bases, and an autoimmune disease caused by the nucleic acid. Is characterized by being able to detect the presence or absence of the disease.

By detecting a mutation in the ζ chain gene using the above nucleic acid, it is possible to diagnose whether or not the subject has an autoimmune disease. For example, using this nucleic acid, S
By detecting the presence or absence of a mutation in the tyrosine phosphorylation site frequently observed in LE patients, the presence or absence of SLE can be diagnosed. Also, this nucleic acid is
However, the present invention can be effectively used for diagnosis of an autoimmune disease caused by an abnormality in the T cell ζ chain.

In the above description, “gene encoding T cell receptor リ chain protein” includes cDNA of ζ chain gene.
And genomic DNA. These include not only structural genes but also flanking sequences such as regulatory sequences.

The cDNA herein includes, for example, the nucleotide sequence shown in SEQ ID NO: 1 and a sequence substantially identical thereto. Examples of the substantially identical sequence include a nonsense mutation and a sequence encoding the above “normal ζ chain protein”.

In humans, the genomic DNA of the ζ chain gene is the long arm of chromosome 1 of humans 23-24 (1q23-24).
(Jefferson et al., Immu
nogenetics 29: 331), which can be obtained by collecting this. It can also be recovered from those already cloned as a genomic library.

Further, a part of the ζ chain gene can be used as a primer for PCR and a probe for hybridization, which will be described later. In order to ensure specificity as a primer or a probe, it is preferable that a part of the ζ chain gene is composed of at least 16 bases. However, a sequence of 16 bases or less can also be used if the above object can be accurately achieved with a base number of less than this. This partial sequence can be selected according to the purpose. For example, when it is desired to amplify the entire ζ chain gene by PCR, a sequence at both ends of the structural gene or outside thereof is selected as a primer sequence. When it is desired to specifically amplify a region containing a sequence involved in signal transduction, a primer pair complementary to a position sandwiching this region is selected. In addition, this partial sequence can be generated by cutting or synthesizing the above cDNA or genomic DNA.

The term "purified nucleic acid" as used above is mainly used for distinguishing from a naturally occurring in vivo ζ chain gene, and a naturally occurring ζ chain gene is isolated and purified. It means those recovered from the library, those amplified in prokaryotes, and those synthesized further.

Methods for detecting an autoimmune disease using this nucleic acid include DNA- or RNA-based hybridization and PCR (polymerase chain reaction;
merase chain reaction), RFLP method (restriction fragment length polymorphism) and the like.

In the hybridization, the full-length ζ
By comparing the stranded gene with the ζ chain gene of the subject, it is possible to detect whether or not there is a mutation on the 上 chain gene of the subject. In this case, the DN of the ζ chain gene of the subject
If A is used as a sample, abnormalities on the chromosome can be detected. Further, by using the mRNA of the ζ-chain gene of the subject as a sample, it is possible to detect the expression of the 発 現 -chain and abnormality of the し て -chain gene on the chromosome via the mRNA.

Further, any sequence of the above-mentioned ζ chain gene may be selected as the hybridization probe. For example, when a sequence within a mutation sequence or a sequence containing a mutation is selected as a probe, the presence or absence of a mutation can be determined by the presence or absence of a hybridization signal.
When a probe that forms a hybrid with a region other than the mutation site of the fragment containing the mutation site is selected, the mutation can be detected based on a difference in length, a difference in the number of restriction enzyme fragments, and the like. .

More specifically, in some cases of SLE, deletion mutations were detected from about 465 bases to 500 bases of SEQ ID NO: 1. From this, it is possible to diagnose the possibility of SLE by detecting by hybridization that the fragment containing this region is shortened by about 35 bp.

In the PCR method, a region containing a mutation site on the ζ chain gene is amplified, or a sequence having a mutation is specifically amplified, and the mutation is detected by analyzing the amplified fragment. Can be.

The primers used in this PCR method can be designed from the base sequence of the ζ chain gene. Specifically, when a region containing a mutation site is amplified, a position sandwiching the mutation site may be selected as a primer sequence, and when a sequence having a mutation is specifically desired to be amplified, at least One primer is designed to include the mutation site.

For example, in SLE, the region containing the above-mentioned deletion mutation is selectively amplified, and the amplified fragment is shortened by a length corresponding to the deletion as compared with an amplified fragment derived from a normal ζ chain gene. , The deletion mutation can be detected. In addition, since mutations due to single base substitution have also been detected in addition to deletion mutations, in this case, it is also possible to detect the presence or absence of the mutation based on the number of fragments obtained by cutting the amplification product with a restriction enzyme. it can.

The present invention also provides a material and a method capable of biochemically detecting an autoimmune disease, not limited to the above-described genetic detection method.

Specifically, the present invention provides an antibody that recognizes a T cell receptor ζ chain protein as a material capable of biochemically detecting an autoimmune disease. In SLE, which is an autoimmune disease, it has been detected that ζ chain protein expression is reduced and a deletion mutation is present in a part of ζ chain protein. Therefore, if the above antibody is used, SLE can be diagnosed through abnormal expression of the ζ-chain protein. In the diagnosis using this antibody, analysis methods such as Western blotting, ELISA, and immunoprecipitation can be used.

In order to easily carry out the diagnosis of an autoimmune disease using the above-mentioned nucleic acid or the above-mentioned antibody, a kit for diagnosing an autoimmune disease containing the above-mentioned nucleic acid or antibody and necessary reagents is provided. Is preferred.

[0053]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the present invention will be described based on preferred embodiments.

1. Generation of T cell receptor ζ chain gene The ζ chain gene on genomic DNA is
3-24 (Gene 1)
48: 2563-2571, 1992). Therefore, genomic DNA collected from normal human peripheral blood mononuclear cells or the like or a commercially available human genomic library (Clontech)
Labs, Polo Alto, Calif.). These can be selected by hybridization using the nucleic acid of SEQ ID NO: 1 as a probe.

It should be noted that genome D from peripheral blood mononuclear cells
The method for recovering NA is described, for example, in Maniatis et al.
rectangular Cloning, Cold Spri
ngHarbor Laboratory Pres
s, 1989).

The ζ chain gene obtained here can be connected to a vector or the like according to a conventional method and amplified as necessary.

2. CDN of T cell receptor ζ chain gene
Production of A mRNA is collected from peripheral blood mononuclear cells of normal humans and the like, and cDNA is synthesized using this mRNA as a template.

A series of operations from the recovery of mRNA to the synthesis of cDNA can be performed by the method described in the above-mentioned Maniatis et al. Alternatively, it can be easily carried out using a commercially available kit. mRNA
As a kit for recovery, Pharmacia and the like can be used. In addition, as a kit for cDNA synthesis, for example, Amersham and the like can be used.

The base sequence of the ζ-chain cDNA shown in SEQ ID NO: 1 was determined using a Jarkat T cell line (Proc. Nat. Acad. Sci. USA 85: 9709-971).
3) According to this sequence, it can be synthesized using a DNA synthesizer or the like.

3. Autoimmune Disease Diagnosis: Detection of Abnormality in T Cell Receptor ζ Chain Protein in Patients with Autoimmune Disease In autoimmune diseases such as SLE, abnormalities of ζ chain protein, such as decreased expression, ζ chain protein, as compared to healthy individuals Deletion mutation and substitution mutation, and reduced tyrosine phosphorylation. Thus, it has been suggested that certain autoimmune diseases are caused by abnormalities in T cell receptors (especially, ζ chain) resulting in the failure of normal signal transduction. Thus, by comparing the ζ-chain protein of an autoimmune patient with the T cell receptor of a healthy subject,
An autoimmune disease can be detected.

(1) Recovery of T cell protein from autoimmune disease Mononuclear cells are collected from venous blood of healthy individuals and patients with autoimmune disease. These mononuclear cells are separated and collected from venous blood by Ficoll-Hypaque density gradient centrifugation. The collected mononuclear cells are passed through a nylon wool column to obtain a roughly purified T cell solution.

The T cells in this solution are destroyed, and a T cell lysate is recovered. The T cells may be destroyed by either a crushing method using ultrasonic waves or a cell lysis method using a predetermined lysis solution.

(2) Analysis of ζ-chain protein The ζ-chain protein in the above cell lysate is analyzed using an anti-TCRζ-chain antibody. The antibody used here can be either polyclonal or monoclonal.

A polyclonal antibody can be produced according to a conventional method. For example, after mixing the ζ-chain protein with the adjuvant, it is injected subcutaneously or into a footpad of a rabbit or the like. Repeat this operation about three times with a certain period of time, ζ
When the antibody titer to the chain is increased, collect the serum,
This is referred to as a polyclonal antibody.

A monoclonal antibody against the ζ chain is also
It can be generated according to known methods. That is,
Collect spleen cells from mice immunized with ζ chain protein,
It can be produced by fusing it with a hybridoma. In addition, for convenience, a monoclonal antibody against the 、 chain is, for example, TIA: IgG1 (Coulter Immuno
logy, Hialeah, Florida, USA) and 6B1
0.2: Commercial products such as IgG (SantaCruze Biotechnology, Santa Cruz, CA, USA) can also be used.

The analysis of the ζ chain using these antibodies is preferably performed based on the Western blotting method. That is, after fractionating the cell lysate by polyacrylamide gel electrophoresis, the proteins in the gel are transferred to a membrane filter. After blocking the membrane after transfer with bovine serum albumin (BSA) or the like, the membrane is immersed in the buffer containing the anti-chain antibody, washed, and visualized using a labeled secondary antibody or the like. In the analysis of ζ-chain protein using this Western blotting method, changes in the expression amount and size of ζ-protein in a patient can be detected.

For analysis of ζ chain protein, ELISA method,
Immunoprecipitation can also be used. These ELISAs
The method and the immunoprecipitation method can be performed according to a conventional method.
These methods are effective when measuring the expression level of ζ-chain protein in patients. Also, in these methods,
By using a monoclonal antibody that recognizes a specific region of a chain protein, a mutation in the specific region can be specifically detected.

For example, in SLE patients, since deletion of exon 7 of the 検 出 chain gene has been detected, an antibody against the amino acid sequence encoded by this exon 7 (anti-ζ chain exon 7 antibody) is used. , SLE
Exon 7 deletion mutations in patients can be detected.

(3) Detection of Abnormality in Tyrosine Phosphorylation of ζ Chain Protein Tyrosine phosphorylation of ζ chain protein can be measured by Western blotting or the like using an antibody against phosphorylated tyrosine. For example, the measurement can be performed by the following operation.

T cells in peripheral blood are suspended in a cell culture,
A mouse anti-CD3 antibody, a mouse anti-CD4 antibody or a mixed antibody thereof is added to treat the T cell surface molecule. After treatment,
Crosslink with anti-mouse antibody. The cells are solubilized in a predetermined solution, fractionated by polyacryl electrophoresis, and transferred to a membrane filter. The membrane after the transfer is blotted using an anti-tyrosine phosphorylated monoclonal antibody, and visualized using a labeled secondary antibody or the like. The degree of phosphorylation can be measured based on the intensity of the signal at each tyrosine residue.

4. Autoimmune Disease Diagnosis: Detection of Abnormality on the ζ-chain Gene in Patients with Autoimmune Disease An abnormality on the ζ-chain gene was detected in patients with autoimmune diseases such as SLE patients. Therefore, an autoimmune disease, in particular, SLE can be suitably detected through detection of an abnormality in the ζ chain gene in the subject.

(1) Detection of mutation of ζ-chain gene on chromosome based on ζ-chain mRNA of subject The mRNA of the subject is obtained by using the same method or kit as described in the above-mentioned cDNA generation, and using the same method or kit. Recover the mRNA from the sphere. If necessary, collect
CDNA is synthesized by the method described above using RNA as a template.

Using mRNA or cDNA as a template, P
Perform CR to amplify the ζ chain gene of the subject. This PCR
Can be synthesized, for example, based on the sequence described in SEQ ID NO: 1. This PCR primer is composed of at least 16 bases or more, is preferably a GC-rich region, and has a double-stranded structure such as a palindromic structure.
A sequence that does not form the next structure is selected. For example, a suitable primer pair used herein includes 5′-TCAGCCTCTGCCTCC
CAGCCTCTTTCT-3 '(SEQ ID NO: 2) and 5'-ATGCTTCATCCTGT
GTCTCATAATCTG-3 ′ (SEQ ID NO: 3). These primers can complement the flanking regions on both sides of the ζ-chain structural gene, ie, the positions sandwiching the structural gene, and amplify the entire length of the ζ-chain structural gene. Therefore, this primer is suitable for searching the entire region of the 遺 伝 子 -chain structural gene. In addition to these primers, a primer that specifically amplifies a specific region of the ζ chain, for example, a region involved in signal transduction (ITAM, GTP / GDP binding sequence, etc.) can also be used.

The PCR-amplified fragment is cleaved directly or with a restriction enzyme, fractionated by electrophoresis, and the electrophoresis patterns of the amplified fragment of a healthy subject and the amplified fragment of a subject are compared to determine the presence or absence or the length of a predetermined fragment. Detect differences.

In order to analyze the details of the mutation,
Preferably, a determination of the sequence is made. When performing this sequence determination, a known sequence determination method such as the dideoxy method or the Sanger method can be used. In addition, a mutant sequence can be easily determined by using a commercially available sequence determination kit or an automatic sequencer.

(2) Detection of Mutation of the ζ-chain Gene Directly on the Chromosome The chromosome of the subject can be obtained, for example, from the whole DN cells by the method described in the above-mentioned document of Maniatis et al.
Collect as A.

The recovered total DNA is used to detect the presence or absence of a mutation after amplifying the ζ chain gene as a template for the PCR. Further, the mutation may be detected by a restriction enzyme fragment length polymorphism (RFLP) test method. That is, after cutting the entire DNA with an appropriate restriction enzyme, the restriction enzyme fragment is separated by electrophoresis, and the mutation can be detected based on the polymorphism of the migration pattern of the restriction enzyme fragment. Other analysis methods can be performed in the same manner as in the case of using cDNA.

5. Applications: Treatment of autoimmune diseases Autoimmune diseases, such as SLE, are thought to involve the inhibition of normal signaling by mutations in the patient's ζ chain gene. Therefore, it is expected that a normal immune response can be restored by introducing the above-mentioned normal 正常 chain gene or normal ζ chain protein into a patient.

(1) Administration of ζ-chain gene to patient: gene therapy using ζ-chain gene In gene therapy using ζ-chain gene, the mutant ζ-chain gene on the chromosome is replaced with the normal ζ-chain gene described above. And a method of introducing a transgene into any position of the chromosome.

When replacing the mutant ζ-chain gene on the chromosome with the above-mentioned normal ζ-chain gene, for example, a normal ζ-chain gene is introduced into bone marrow cells collected from a patient using a gene transfer method. After gene transfer, the ζ chain locus is screened by PCR or the like. In this screening,
Select cells in which the ζ-chain locus has been replaced with a normal ζ-chain gene. By returning the selected cells to the patient's bone marrow again, a normal ζ chain gene can be supplied to the patient.

The ζ chain gene used for the above-mentioned introduction may be linear or circular, and may have a vector sequence or a selection marker gene in the flanking sequence.

The gene introduction method may be any of microinjection method, microporation method, calcium phosphate method, and liposome method (Mich
aelKriegler, Gene Transfer and Expression, Stockton
Press).

On the other hand, when a normal ζ-chain gene is introduced as a transgene, the ζ-chain gene can be introduced into a patient's bone marrow cells or the like as a fragment or in a state of being connected to an appropriate vector in the same manner as described above. it can. After introduction, the cells of interest are selected and returned to the patient's bone marrow.

As this vector, a retrovirus vector, an HIV virus vector or the like which has been developed for gene therapy can be suitably used. By utilizing the ability of these viruses to infect human cells and to integrate into chromosomes using reverse transcriptase, the desired normal ζ chain gene can be introduced into patients. These introduction methods can be executed based on the above-mentioned document of Michael Kriegler.

[0085] As a vector, an adenovirus vector developed for gene therapy can also be used. Adenoviruses are known to exist in the nucleus separately from chromosomes. Therefore, by using an adenovirus vector, the ζ chain gene can be retained in the cell without introducing a foreign gene into the chromosome of the patient.

The ζ-chain gene thus introduced is expressed in cells and produces a normal ζ-chain protein.

(2) Administration of ζ-chain protein to patient: Treatment with ζ-chain protein By directly administering 自己 -chain protein to a patient, it can be expected that an autoimmune disease can be alleviated or treated.

The ζ-chain protein can be produced in vitro by connecting the ζ-chain gene to any expression vector. This protein can also be modified in sugar chain structure and the like in order to increase the absorption rate in humans. In addition, upon administration to a patient, a pharmaceutically acceptable excipient,
Additives and the like can be mixed.

[0089]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below based on embodiments.
Here, systemic lupus erythematosus (SLE) is described in detail as an autoimmune disease associated with abnormalities in the ζ chain.

Example 1 T cell Tyrosine Phosphorylation Abnormality in Patients An analysis was performed on the T cell receptor subunits of 21 SLE patients satisfying the ARC classification criteria. In this analysis, 12 healthy subjects and 7 scleroderma were used as controls.

The venous blood (2
0 ml) was collected and heparin was added. Then Fico
ll-Hypaque (15 ml) was added, and 3000 rpm, 1
Centrifugation was performed for 5 minutes, and peripheral blood mononuclear cells were separated by a density gradient. The cell suspension containing peripheral blood mononuclear cells was passed through a nylon wool column to separate a cell solution containing T cells at a high density. A part of the cell solution obtained here was used for anti-CD3 (UC
THI: IgG1), and the proportion of T cells in the cell fluid was determined. As a result, 92% or more of the cells in the cell solution bound to the anti-CD3 antibody, indicating that the cell solution contained 92% or more T cells.

[0092] The T cells obtained here were transformed into RPMI1640.
The cells were suspended in a medium, and a part of them was suspended in an anti-CD3 antibody (UCTH-
1) Anti-CD4 antibody (T4D11: IgG1) and an antibody obtained by mixing them were added, and incubated on ice for 30 minutes.

After the incubation, the cells were washed, a rabbit anti-mouse polyclonal antibody was added, and the mixture was incubated at 37 ° C. for 5 minutes. After washing the cells three times with PBS, the cell lysate containing 1 mM Na ₃ VO ₄
mM Tris-HCl pH 8.0, 150 mM NaCl,
1% Nonidet-P40, 10 mM EDTA,
1 mM sodium orthovanadate, 1 mM PMS
F, 10 μg / ml aprotinin and 10 μg / ml leupeptin) were added and incubated at 4 ° C. for 15 minutes to lyse the cells.

Electrophoresis buffer (Bio-Rad Laboratories) containing 2-mercaptoethanol in each cell lysate
Was mixed and then boiled for 5 minutes to prepare an electrophoresis sample. Each electrophoresis sample was fractionated by electrophoresis using a 4-20% polyacrylamide gradient gel. After fractionation, the electrophoresis gel was transferred to a transfer buffer (25
(trim HCl, 192 mM glycine and 20% methanol) for 10 minutes to equilibrate. Thereafter, the protein in the gel was converted to a PVDF membrane (Millipore, Be
dford, Mass.).

The membrane after the transcription was blocked by incubating with Block Ace (Dainippon Pharmaceutical Co., Ltd.) at 37 ° C. for 1 hour, and then HRP-labeled anti-phosphotyrosine monoclonal antibody (RC-20, Transduction Laboratories, Inc.
(Kentucky) and incubated. For detection of the signal, an ECL X-ray film (Amersham) was exposed using an ECL chemiluminescent solution (Amersham). The result is shown in FIG.

As shown in FIG. 1, compared to healthy individuals, SLE-derived T cells induced imbalance in tyrosine phosphorylation of anti-CD3 alone and anti-CD4 alone with respect to the mixed antibody of anti-CD3 and anti-CD4. Was. That is, the SLE-derived T
In the cells, the bands shown as p56 and p70 show only a slight decrease in signal intensity as compared with healthy individuals, but for p18 and p100, almost no increase in signal was observed by stimulation with the antibody. Was. This indicated that tyrosine phosphorylation of p56 and p70 was significantly reduced in SLE.

[0097] Such a decrease in tyrosine phosphorylation is due to SL
It was observed in 15 (71%) of the E21 cases. On the other hand, in scleroderma, normal tyrosine phosphorylation similar to that in healthy subjects was observed.

Since this p18 protein is known to be a tyrosine phosphorylated TCRζ chain (Samelson et al., Cell 46: 1083-1090), the tyrosine phosphorylation of the ζ chain protein was examined. .

Example 2 Abnormal Tyrosine Phosphorylation of ζ Chain Protein in SLE Patient p56 is a tyrosine kinase p of the src family
A potential of 56 ^lck was expected. Therefore, SLE and T cell lysates of healthy individuals were analyzed by immunoprecipitation using an anti-56 ^lck antibody. Specifically, an anti-p56 ^lck antibody (10 μl) was added to T cell lysate (500 μl).
And protein G beads (Pharmacia) (40 μl)
Was added and incubated for 2 hours. The sample,
After electrophoresis by SDS-PAGE, the DNA was transferred to a PVDF membrane and subjected to immunoblotting. In addition, the sample amount was standardized based on the signal intensity of the immunoglobulin L chain.

FIG. 2B shows the result. FIG. 2 (B)
Here, SLE HE, KS, and SK indicate SLE patients. SSc indicates a scleroderma patient sample. S
P56 in the samples of the LE group and the control group was anti-p56 ^lck
Reacted with antibody. Furthermore, from the immunoprecipitation reaction with the anti-p56 ^lck antibody, the tyrosine phosphorylation of p56 is significantly increased in SLE T cells, particularly in active patients whose symptoms are remarkable, as compared with T cells of healthy individuals. It was shown that.

Example 3 Analysis of tyrosine phosphorylation of p18ζ chain in SLE Based on the above results, the structure of ζ chain in SLE was further analyzed.

The above cell lysate was added to the T cells,
The cells were lysed by incubation at 4 ° C. for 15 minutes.
After the dissolution, centrifugation was performed at 15,000 rpm for 5 minutes, and the supernatant was recovered.

On the other hand, the cell lysate was replaced with two kinds of anti-TCRζ
Chain monoclonal antibody, TIA-2: IgG1 and 6B
10.2: IgG1 and Protein G Sepharose beads were mixed and incubated at 4 ° C. for 4 hours or overnight. After washing the beads three times with the above cell lysate,
An electrophoresis buffer containing mercaptoethanol (5%) was added. The bead suspension was boiled for 5 minutes to obtain an electrophoresis sample. The electrophoresis sample was injected on a 15% polyacrylamide gel, and electrophoresis was performed. After electrophoresis, the gel was transferred to a transfer buffer (25 mM Tris-HCl, 192
(mM glycine and 20% methanol) for 10 minutes to equilibrate. Thereafter, the protein fractionated in the gel was electrically transferred to a PVDF membrane. The membrane was immersed in a blocking reagent (Blockace (registered trademark), Dainippon Pharmaceutical) at 37 ° C., and then incubated with an anti-phosphotyrosine monoclonal antibody for 1 hour at room temperature. The membrane was incubated with horseradish peroxidase-labeled anti-mouse IgG and the signal was then applied to the chemiluminescent agent (Chemiluminescenc).
e-enhancing reagents, Amersham International). Finally, the membrane was visualized on ECL X-ray film. The results are shown in FIG. In FIG. 3B, HC is immunoglobulin H.
Strands were indicated and the sample intensity was normalized by this band intensity.

As shown in FIG. 2 (A), in the control healthy human, the ζ-chain protein was migrated as a single band in the sample not subjected to the antibody stimulation, while the tyrosine phosphorylation was enhanced by the antibody stimulation. In the one that let me
It migrated as a broad band around 8 to 23 KD.

In the SLE patient (SK), an electrophoresis pattern similar to that of a healthy control was obtained. However, in case HE, the ζ chain did not enhance tyrosine phosphorylation by antibody stimulation, and migrated as a single band regardless of stimulation. In case KS, no phosphorylated ζ chain band was detected. Such defective tyrosine phosphorylation was detected in 15 of 21 SLE cases.

[Example 4] Abnormal expression of ζ-chain protein in SLE Next, the expression of ζ-chain protein in SLE patients was analyzed.

The procedure was performed in the same manner as in Example 2. However, an anti-chain monoclonal antibody was used as an antibody in the immunoprecipitation reaction. The result is shown in FIG.

As shown in FIG. 2 (B), in SK among SLE cases, a single band of the ζ chain protein was detected at the position of 18 KD as in the healthy person. Although not shown in the figure, a single band of ζ-chain protein was detected in scleroderma patients. On the other hand, in HE and KS in which the degree of tyrosine phosphorylation was low or not detected at all in Example 2 above,
The ζ chain band could not be detected.

On the other hand, as shown in the results of FIG. 3B described later, the expression of p56 ^lck protein, which is one of the important enzymes in tyrosine phosphorylation of the 、 chain, was determined in healthy individuals, scleroderma, SLE No differences were seen between the patients. Therefore, it was suggested that the deficiency in expression of the ζ chain in SLE patients was not due to altered regulation of protein expression in T cells.

Next, the difference in the expression level between the control group and healthy subjects was quantified. Specifically, the above-mentioned immunoprecipitation reaction was quantified using a densitometer for each sample of 21 cases of SLE, 13 cases of healthy subjects, and 14 cases of scleroderma. The measured values quantified here were averaged for each control group and each SLE group. FIG. 4 shows the results.

As shown in FIG. 4, the expression level of p56 is not different between the healthy group, the scleroderma group and the SLE group, while the expression level of the ζ-chain protein is higher than that of the other groups. S
The LE group was shown to be significantly reduced.

In order to analyze whether or not the results obtained above were due to changes or deletions in the recognition site of the antibody (TIA-2) on the ζ chain protein,
Anti-chain antibody with other position as recognition site (6B10.2)
And the same experiment as above was performed. In addition, this 6B1
The 0.2 antibody recognizes 36 to 54 amino acid residues of the ζ chain. The experimental results are shown in FIG.

[0113] As shown in FIG.
In K cases, the results using the TIA-2 antibody and the 6B10.2 antibody showed that a single band was similarly detected at the position of 16 KD.

On the other hand, in KS cases, TIA-2 and 6B
10.2 No bands were detected that reacted with both antibodies.

In the case of HE, a band reacting in the same manner as described above was not detected with the TIA-2 antibody.
For the B10.2 antibody, a new band of 14 KD was detected in addition to 16 KD derived from the complete ζ chain protein. Such a mutant ζ-chain protein has been identified by several other SLEs.
It was also detected in cases.

Next, in order to elucidate the cause of the mutant ζ-chain protein and the deficient ζ-chain protein, the Ｅ-chain gene of the SLE patient was analyzed.

Example 5 Gene Analysis of Altered 変 化 Chain from SLE Patient The 解析 chain gene of SLE was analyzed based on human ζ chain cDNA (SEQ ID NO: 1) (Weissman et al., Science 239: 1018-1020). RT-PCR was performed using the selected primers.

Specifically, as an upstream primer,
33 to 59 bases of SEQ ID NO: 1 (5′-TCAGCCTCTGCCTCCCAG
CCTCTTTCT-3 ') (SEQ ID NO: 2) was selected. The downstream primers include 612 to 638 of SEQ ID NO: 1.
The base (5'-ATGCTTCATCCTGTGTCTCATAATCTG-3 ') (SEQ ID NO: 3) was selected. These primers are in the untranslated region of the cDNA, and human
The full length of the coding region of the cRζ chain will be amplified.

The mRNA was obtained from peripheral blood T cells derived from healthy individuals and SLE using a kit (mRNA isolation kit, Pharmacia).
(LKB Biotechnology). cDNA
Can be performed by reverse transcription using a commercially available kit (Clontech Lab.) With mRNA as a template.

More specifically, the above crude extract (5 μl) was mixed with a PCR reaction solution (40 mM Tricine-KOH (pH 9.
2), 15 mM KOAc, 3.5 mM Mg (OA
c) ₂ , 75 μg / ml bovine serum albumin, 100 μmol each of upstream and downstream primers, 0.2 μm
50 μl using mMdNTP and Taq DNA polymerase (1 unit) (Promega)
And The amplification reaction was performed using a thermal cycler,
The denaturation step at 5 ° C. for 30 seconds and the extension step at 68 ° C. for 180 seconds were repeated 35 times.

The obtained amplification product (10 μl) was treated with 1.5%
Electrophoresis was performed using an agarose gel. The gel after electrophoresis was immersed in a buffer solution containing ethidium bromide, and irradiated with ultraviolet rays to visualize the electrophoresis pattern. FIG. 6 shows the result.

Among the healthy subjects and SLE, the expected band of 605 bp was detected in the sample of SK case, but the band shorter than 605 bp was detected in the sample of KS and HE case.

From the results, it was found that ζ chain c in KS and HE cases
It was expected that a deletion mutation was present on DNA, ie, mRNA. To analyze the details of this deletion mutation, K
The amplification products derived from S and HE were cloned and sequenced.

Example 6 Determination of SLE cDNA Sequence The amplified fragment obtained in Example 5 was used for Sure Clone L
It was connected to the pUC18 vector using the Ligation Kit (Pharmacia). This reaction solution was transformed into Escherichia coli DH5α strain, and the connection product was recovered. The ligation product collected here was used as the material for the next sequencing. Sequencing is automatic DN
This was performed using an A sequencer (Pharmacia). FIG. 7 shows healthy individuals and SLE (SK, HE, K
S) shows a part of the ladder pattern of the sample sequence.

The sequence of each cDNA from the five healthy individuals in the control group was identical to the previously reported cDNA sequence from the Jarkat T cell line. The cDNAs of the three cases of SLE were also consistent with the cDNA sequence derived from the Jarkat T cells (Proc. Natl. Acad. Sci. USA 85: 9709-9713). In addition, the ζ chain cDNA sequences of these healthy persons are shown in SEQ ID NO: 1.

On the other hand, c of HE and KS cases among SLE
In the DNA sequence, a partially deleted region was present as shown by the solid line in FIG. This deleted region was a region of 468 to 503 bases of SEQ ID NO: 1 as compared with the sequence derived from a healthy subject.

The deletion mutation corresponding to this deletion site
It corresponds to 110-121 amino residues on the chain protein.
This amino acid region corresponds to exon 7 of the reported human TcRζ chain genomic DNA (Jensen et al., J Immunol 148: 2563-2571).

Example 7 Analysis of Mutant ζ Chain Protein Using Anti-Exon 7 Antibody Further, to confirm the results of Example 6, exon 7 was deleted from the 14 KD protein obtained from the HE case. Whether the mutant 変 異 -chain protein is
The analysis was performed by immunoprecipitation using an antibody against the region of 7 to 121 residues. FIG. 8 shows the result. The immunoprecipitation reaction was performed by the method described above.

As shown in FIG. 8, the anti-exon 7 antibody
It reacted with the 16 KD wild-type の chain protein in healthy individuals and HE cases of SLE. On the other hand, the 14 KD mutant ζ-chain protein in the HE case has an antibody (6
B10.2), but no immunoprecipitation occurred with the anti-exon antibody.

Therefore, as schematically shown in FIG.
The 14KD protein observed in the case was found to be a deletion mutant of exon 7.

[Example 8] Analysis of mutation site of ζ chain in SLE In the same manner as above, mutation in the sequence of the ζ chain gene of SLE was analyzed. Table 1 shows the results.

[0132]

[Table 1] As shown in Table 1, in addition to the deletion of exon 7, other mutations were detected. Specifically, in the case of Tak, “A” of 484 bases was mutated to “G”, whereby the amino acid at position 116 was mutated from histidine (H) to arginine (R).

As for Ka, two substitution mutations were detected.
On the other hand, “A” of 507 bases is mutated to “G” and 124
Serine (S) at the position was replaced with glycine (G).
On the other hand, “T” of 553 bases is mutated to “C” and 139 bases are mutated.
Leucine (L) at position was replaced by proline (P). In the No case, the 538 base "T" was mutated to "C" and the leucine (L) at position 134 was mutated to proline (P). In the Sk case, 378 bases “A” was mutated to “T”, and arginine (R) at position 81 was replaced with tryptophan (W). In the Ya case, the 154 base "T" was mutated to "C", and the leucine (L) at position 6 was replaced with proline (P). In the Wat case, the 197 base “T” was mutated to “C” and the tyrosine (Y) at position 21 was changed.
Was replaced with histidine (H).

The mutation sites detected among these cases, including the exon 7 deletion mutation described above, were different, but most of these mutation sites were related to sites involved in signal transduction. For example, an ITAM containing a tyrosine residue and a leucine residue (Immunorecepter Tyrosine-based Actam)
tivation Motif) is present at three places on the ζ chain. Of these, the third ITAM partially overlaps Exon 7. In addition, on Exon 7, GTP besides ITAM
/ GDP binding site is present. That is, it has been reported that ZAP70, actin, PI-3K and Shc bind to the above mutations in ITAM (Bu et al., Pro.
c. Natl. Acad. Aci. USA 92: 6693) in which leucine was replaced by another amino acid, and one in which this tyrosine was replaced by another amino acid.
Exon 7 deletion mutants are accompanied not only by mutations in ITAM but also by deletion of GTP / GDP binding sites.

[0135] Although the exact mechanism in the onset of SLE is unknown, the above results indicate that mutations at the sites involved in these signal transductions result in the above-mentioned enzymes, ZAP70,
It was strongly suggested that abnormalities in the regulation of actin, PI-3K, and Shc are related.

In some SLEs, no mutation was detected in the ζ chain gene. In such a case, the other ζ chain [Example 9] Mutation of the gene on the ζ chain was detected in SLE patients. Therefore, the present invention can be applied to a method for genetically or immunologically diagnosing SLE.

In the genetic diagnosis, as described above, cDNA is synthesized based on the mRNA recovered from the subject, the sequence is determined, and the sequence is compared with a normal ζ chain gene. By this comparison,
If the mutation is detected, it can be diagnosed that the possibility of SLE is high.

In addition, according to the above results, most of the mutations
Since mutations exist in the AM region and the GTP / GDP binding region, mutations in the ITAM region and the GTP / GDP binding region can be diagnosed preferentially. In this case,
Based on the nucleotide sequence of SEQ ID NO: 1, an ITAM region, GTP
By performing PCR using PCR primers capable of selectively amplifying the / GDP binding region, these regions can be selectively diagnosed. In addition, mutations in these regions can also be searched for by hybridization using probes that form hybrids with these ITAM regions and GTP / GDP binding regions.

As an immunological diagnostic method, in addition to the anti-chain antibody used in the above example, an antibody that recognizes an ITAM region and a GTP / GDP binding region on the chain is used.
Mutations on the strand can also be detected.

The mutation detected here can be treated or ameliorated by gene therapy.

When performing gene therapy, it is conceivable to replace the mutant ζ chain gene of the patient with a normal gene by homologous recombination.

It is also conceivable that a normal ζ-chain protein is supplied by integrating a normal ζ-chain gene into a chromosome using a retrovirus or the like, or is introduced as an intranuclear extrachromosomal DNA using an adenovirus. . In this case, if it is necessary to exclude the mutant type ζ-chain protein produced from the mutant ζ-chain gene,
Antisense RNA complementary to A can also be introduced.

In addition to the method using a gene, a normal ζ-chain protein can be directly administered to a patient. In addition, the protein used here can also be modified in order to improve the transferability to a target organ or cell.

Although the embodiment has been described focusing on SLE, the present invention is not limited to SLE and can be suitably applied to autoimmune diseases caused by mutations in other TcRζ chain genes.

[0145]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 1472 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence characteristics Characteristic symbol: CDS Location: 75. . 566 Method for determining characteristics: S Symbol indicating characteristic: mat peptide Location: 138. . 563 Method of Characterization: S Sequence CTTTTCTCCT AACCGTCCCG GCCACCGCTG CCTCAGCCTC TGCCTCCCAG CCTCTTTCTG 60 AGGGAAAGGA CAAG ATG AAG TGG AAG GCG CTT TTC ACC GCG GCC ATC CTG 110 Met Lys Trp Lys Ala Leu Phe Thr Ala Ala Ale CAle TTG CCG ATT ACA GAG GCA CAG AGC TTT GGC CTG CTG GAT 158 Gln Ala Gln Leu Pro Ile Thr Glu Ala Gln Ser Phe Gly Leu Leu Asp 1 CCC AAA CTC TGC TAC CTG CTG GAT GGA ATC CTC TTC ATC TAT GGT GTC 206 Pro Lys Leu Cys Tyr Leu Leu Asp Gly Ile Leu Phe Ile Tyr Gly Val 10 20 ATT CTC ACT GCC TTG TTC CTG AGA GTG AAG TTC AGC AGG AGC GCA GAG 254 Ile Leu Thr Ala Leu Phe Leu Arg Val Lys Phe Ser Arg Ser Ala Glu 30 CCC CCC GCG TAC CAG CAG GGC CAG AAC CAG CTC TAT AAC GAG CTC AAT 302 Pro Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn 40 50 CTA GGA CGA AGA GAG GAG TAC GAT GTT TTG GAC AAG AGA CGT GGC CGG 350 Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg 60 70 GAC CCT GAG ATG GGG GGA AAG CCG AGA AGG AAG AAC CCT CAG GAA GGC 398 Asp P ro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly 80 CTG TAC AAT GAA CTG CAG AAA GAT AAG ATG GCG GAG GCC TAC AGT GAG 446 Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu 90 100 ATT GGG ATG AAA GGC GAG CGC CGG AGG GGC AAG GGG CAC GAT GGC CTT 494 Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu 110 TAC CAG GGT CTC AGT ACA GCC ACC AAG GAC ACC TAC GAC GCC CTT CAC 542 Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His 120 130 ATG CAG GCC CTG CCC CCT CGC TAA CAGCCAGGGG ATTTCACCAC TCAAAGGCCA 596 Met Gln Ala Leu Pro Pro Arg 140 GACCTGCAGA CGCCCAGATT ATGAGACACA GGATGAAGCA TTTACAACCC GGTTCACTCT 656 TCTCAGCCAC TGAAGTATTC CCCTTTATGT ACAGGATGCT TTGGTTATAT TTAGCTCCAA 716 ACCTTCACAC ACAGACTGTT GTCCCTGCAC TCTTTAAGGG AGTGTACTCC CAGGGCTTAC 776 GGCCCTGCCT TGGGCCCTCT GGTTTGCCGG TGGTGCAGGT AGACCTGTCT CCTGGCGGTT 836 CCTCGTTCTC CCTGGGAGGC GGGCGCACTG CCTCTCACAG CTGAGTTGTT GAGTCTGTTT 896 TGTAAAGTCC CCAGAGAAAG CGCAGATGCT AGCACATGCC CTAATGTCTG TATCACTCTG 956 TG TCTGAGTG GCTTCACTCC TGCTGTAAAT TTGGCTTCTG TTGTCACCTT CACCTCCTTT 1016 CAAGGTAACT GTACTGGGCC ATGTTGTGCC TCCCTGGTGA GAGGGCCGGG CAGAGGGGCA 1076 GATGGAAAGG AGCCTAGGCC AGGTGCAACC AGGGAGCTGC AGGGGCATGG GAAGGTGGGC 1136 GGGCAGGGGA GGGTCAGCCA GGGCCTGCGA GGGCAGCGGG AGCCTCCCTG CCTCAGGCCT 1296 CTGTGCCGCA CCATTGAACT GTACCATGTG CTACAGGGGC CAGAAGATGA ACAGACTGAC 1356 CTTGATGAGC TGTGCACAAA GTGGCATAAA AAACAGTGTG GTTACACAGT GTGAATAAAG 1316 TGCTGCGGAG CAAGAGGAGG CCGTTGATTC ACTTCACGCT TTCAGCGAAT GACAAAATCA 1376 TCTTTGTGAA GGCCTCGCAG GAAGACGCAA CACATGGGAC CTATAACTGC CCAGCGGACA 1436 GTGGCAGGAC AGGAAAAACC CGTCAATGTA CTAGGG 1472 SEQ ID NO: 2 Sequence length: 27 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA sequence TCAGCCTCTGCTTCCCCCCC Number: 3 Sequence length: 27 Sequence type: Nucleic acid Number of strands: Single strand Topology: Linear Sequence type: Synthetic DNA sequence ATGCTTCATC CTGTGTCTCA TAATCTG 27

[0146]

As described above, according to the present invention, T cells
SLE can be diagnosed through detection of a mutation in a chain gene, and treatment of SLE can be expected by complementing the detected mutation with a normal ζ-chain gene or ζ-chain protein. .

[Brief description of the drawings]

FIG. 1 shows a pattern of a crude extract of T cells recovered from a healthy individual and SLE after stimulation with an anti-CD3 or / and an anti-CD4 antibody, separated by electrophoresis, and subjected to immunoprecipitation with an anti-phosphotyrosine antibody. It is a photograph shown.

FIG. 2 is a set of photographs showing the results of examining the effect of normal cells and SLE on T cell シ ン chain tyrosine phosphorylation after antibody stimulation, and (A) shows the results of electrophoresis of each T cell crude extract.
It is a photograph which shows the pattern at the time of performing immunoprecipitation with an anti-phosphotyrosine antibody, and (B) is a photograph which shows the pattern at the time of performing immunoprecipitation with an anti-chain antibody.

FIG. 3 is a photograph showing a pattern obtained by performing an immunoprecipitation with (A) an anti-ｐ chain antibody and (B) an anti-p56 antibody after electrophoresis of a crude extract of T cells of a healthy individual and SLE.

FIG. 4 is a graph showing the results obtained by immunoprecipitation using an anti-ζ chain antibody or an anti-p56 antibody, and quantifying the obtained signal with a densitometer.

FIG. 5 shows different recognition sites in the ζ chain protein.
4 is a photograph showing a pattern when immunoprecipitation of ζ chain protein was performed using two antibodies.

FIG. 6 is a photograph showing the results of electrophoresis of ζ chain cDNA of a healthy individual and SLE.

FIG. 7 is a diagram showing deletion sites of SLE (HE, KS sequence) based on an electrophoresis pattern (sequence ladder) at the time of sequencing.

FIG. 8 is a photograph showing the results of electrophoresis of SLE ζ chain protein and immunoprecipitation using an anti ζ chain exon 7 antibody.

FIG. 9 is a diagram showing a region involved in signal transduction on the ζ chain protein and schematically showing a detected exon 7 deletion mutant.

FIG. 10 is a diagram showing a regulatory mechanism of T cell signaling in an immune reaction.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI C07K 16/28 G01N 33/53 D G01N 33/53 33/564 Z 33/564 A61K 37/02

Claims (19)

[Claims] 1. An isolated and purified T cell receptor II chain protein, which is used for an autoimmune disease patient. 2. An isolated and purified nucleic acid comprising a gene encoding a T cell receptor ζ chain protein or a part thereof, wherein the part includes a sequence involved in signal transduction. Nucleic acids 3. The nucleic acid according to claim 2, wherein the T cell receptor ζ chain protein comprises the amino acid residue of SEQ ID NO: 1 or an amino acid sequence substantially identical thereto. 4. The nucleic acid according to claim 2, wherein the sequence involved in signal transduction is a tyrosine residue, an immunoreceptor tyrosine-dependent activation motif, or a GTP / GDP binding sequence. 5. The method according to claim 1, wherein the portion includes 11
5. It contains 0 to 121 residues.
The nucleic acid according to any one of the above. 6. The gene encoding the T cell receptor ζ chain protein or a part thereof is connected to a vector or any nucleic acid fragment.
6. The nucleic acid according to any one of 5. 7. The nucleic acid according to claim 1, which is used for an autoimmune disease patient. 8. A purified nucleic acid comprising a gene encoding a T cell receptor ζ chain protein or a part thereof, wherein the part has at least 16 bases, and the presence or absence of an autoimmune disease is detected by the nucleic acid. A nucleic acid characterized in that it can 9. The nucleic acid according to claim 8, wherein the nucleic acid contains a regulatory sequence of a gene encoding a T cell receptor. 10. The nucleic acid according to claim 8, wherein the gene encoding the T cell receptor リ chain protein has the nucleotide sequence of SEQ ID NO: 1. 11. The nucleic acid according to claim 8, wherein the autoimmune disease is systemic erythematosus. 12. An antibody for detecting an autoimmune disease, wherein the antibody immunoreacts with a T cell receptor ζ chain protein. 13. The antibody according to claim 12, wherein the recognition site is exon 7 of a T cell receptor ζ chain protein. 14. A method for detecting an autoimmune disease based on a T cell receptor ζ chain protein or a gene encoding the same, comprising: mutating the length of the gene or the amino acid sequence constituting the ζ chain protein, or A method for detecting an autoimmune disease, wherein a mutation in a gene sequence to be encoded is used as an index. 15. The method for detecting an autoimmune disease according to claim 14, wherein a mutation is detected based on an amino acid sequence involved in signal transduction on the T cell receptor ζ chain protein or a nucleic acid sequence encoding the same. . 16. The nucleotide sequence from 110 to 121 according to SEQ ID NO: 1.
15. The method for detecting an autoimmune disease according to claim 14, wherein the mutation of the amino acid sequence is detected based on the amino acid sequence of the residue or the nucleic acid sequence encoding the residue. 17. The method for detecting an autoimmune disease according to claim 14, wherein the autoimmune disease is systemic lupus erythematosus. 18. A kit for diagnosing an autoimmune disease, comprising the nucleic acid according to claim 8 or the antibody according to claim 13. 19. A kit for treating an autoimmune disease, comprising the ζ-chain protein according to claim 1 or the nucleic acid according to any one of claims 2 to 8.