WO2005040380A1

WO2005040380A1 - Rodent immune response regulatory proteins

Info

Publication number: WO2005040380A1
Application number: PCT/JP2004/016376
Authority: WO
Inventors: Saburo Saito; Kinya Nagata; Kazuyuki Ogawa; Kunou Suzuki; Ryutaro Oba; Nobutake Akiyama
Original assignee: Bml, Inc.
Priority date: 2003-10-29
Filing date: 2004-10-28
Publication date: 2005-05-06

Abstract

It is intended to provide a rodent immune response regulatory protein having an amino acid sequence represented by SEQ ID NO:2 or 4; an immune response regulatory gene encoding this protein; and a method of constructing an arbitrary antibody in a rodent with the use of the same. By immunizing an experimental animal with the above-described protein and a fused protein thereof, immunization can be effectively performed and various polyclonal and monoclonal antibodies can be conveniently obtained. Since immunization can be efficiently carried out in experimental animals, model mice of immunopathy such as auto immune diseases can be efficiently constructed.

Description

Description: Immune response regulatory proteins in rodents

TECHNICAL FIELD The invention of this application relates to a novel immune response regulatory gene and an immune response regulatory protein that regulate an immune response.

The W immune system plays an essential role in host defense. Activation of the immune system is regulated by a variety of trace proteins secreted between cells, intracellularly or extracellularly. Immune cells work through these molecules in host defense while maintaining homeostasis. On the other hand, disruption of the immune system not only causes serious infections, but also causes various diseases such as autoimmune diseases, allergies, immunodeficiency and cancer. Therefore, elucidation of trace protein substances that regulate immunity will not only contribute to the defense of the immune system in vivo, but will also be useful in overcoming various immune disorders. Proteins involved in the regulation of the immune response include, for example, inuichi leukin 1-15, lipid A, phospholipase A2, endotoxin, staphylococcal enterotoxin B and other toxins, influenza I type, interferon type II , Tumor necrosis factor, transforming growth factor-J3 ("TGF- / 3"), lymphotoxin, migration inhibitor, granulocyte macrophage colony stimulating factor (CSF), monocyte macrophage CSF, granulocyte CSF, vascular epithelium Growth factors, angiotensin, transforming growth factor, heat shock protein, sugar group of blood group, Rh factor, fibroblast growth factor and the like are known. In addition, a labeled delivery system in which these immune response modifiers are bound to a metal colloid or the like, and a method for enhancing an immune response and a method for producing a monoclonal antibody using the same have been proposed (Japanese Patent Application Publication No. 2002-503639). Gazette).

DISCLOSURE OF THE INVENTION Trace proteins that regulate immune responses have the potential as target proteins for the development of low-molecular-weight drugs related to carcinogenesis and immune disorders, and it is desirable to obtain as many immune-related proteins as possible. It is rare. In addition, proteins having excellent functions of promoting an immune response are useful for producing useful antibodies in animals. Furthermore, overexpressed or deficient animals of the protein are expected to greatly contribute to the establishment of therapeutic methods and the development of pharmaceuticals as various disease model animals. The invention of this application has been made in view of the above circumstances, and has as its object to provide a novel rodent immune response regulatory protein and its gene, and an invention utilizing them. . This application provides the following inventions to solve the above-mentioned problems. That is, the first invention is an isolated rodent gene, wherein the amino acid sequence of any of SEQ ID NO: 2 or 4 or one or more amino acid residues of SEQ ID NO: 2 or 4 is It is an immune response regulatory gene encoding an immune response regulatory protein having a sequence deleted, added or substituted with another amino acid residue. In one embodiment of the immune response regulating gene of the first invention, cDNA synthesized from the transcript mRNA is a gene having the nucleotide sequence of SEQ ID NO: 1 or 3, respectively. A second invention is a polynucleotide purified from the genomic DNA, mRNA, cDNA or their complementary sequence of the immune response regulatory gene of the first invention. A third invention is an oligonucleotide probe that hybridizes under stringent conditions with the immune response regulator gene of the first invention or the purified polynucleotide of the second invention. A fourth invention is a primer set for PCR-amplifying the immune response regulator gene of the first invention or the purified polynucleotide of the second invention. A fifth invention is a recombinant vector having the purified polynucleotide of the second invention. A sixth invention is a transformed cell obtained by the recombinant vector according to the fifth invention. A seventh invention is a purified rodent protein, wherein the amino acid sequence of any one of SEQ ID NO: 2 or 4 or one or more amino acid residues in SEQ ID NO: 2 or 4 are deleted or added. Or an immune response regulatory protein having a sequence substituted with another amino acid residue. One embodiment of the immune response regulating protein of the seventh invention is a protein that is an expression product of the immune response regulating gene of the first invention or the polynucleotide of the second invention. The eighth aspect is a purified or synthesized peptide comprising a part of the immune response regulating protein of the seventh aspect. One embodiment of the peptide of the eighth invention is a peptide consisting of 10 or more consecutive amino acid sequences of amino acids 1-163 in SEQ ID NO: 2, or a continuous peptide of amino acids 1-162 in SEQ ID NO: 4. A peptide having the above amino acid sequence. The A ninth invention is an antibody that specifically recognizes the immune response control protein of the seventh invention. The 10th invention is a cell or animal in which the immune response regulatory protein of the 7th invention is overexpressed. The eleventh invention is a cell or animal in which the function of the immune response regulatory gene of the first invention is defective. A twelfth invention is a method for producing an antibody against an arbitrary protein in a rodent, the method comprising administering an antigenic substance to the animal according to the tenth invention, and isolating the antibody from the animal. It is a method characterized by including a process. In the present invention, the term "polynucleotide" refers to a phosphate ester of a nucleoside having a 3-N-glycosidic bond (ATP, GTP, CTP, UTP; or dATP, dGTP, dCTP, dTTP). ) Refers to a molecule with 100 or more bonds, and “oligonucleotide” refers to a molecule with 2-99 bonds. "Protein" and "peptide" refer to a molecule composed of a plurality of amino acid residues linked together by amide bonds (peptide bonds) or unnatural residue linkages fc. Further, in the present invention, “deletion or addition of one or more amino acid residues or substitution with another amino acid residue” refers to a range in which the function of the immune response regulatory protein of the present invention is not practically changed. In the above, it means that 30% or less, preferably 20% or less, more preferably 10% or less of amino acid residues in the entire amino acid sequence are added, deleted or substituted. Other terms and concepts in each invention of this application are defined in detail in the description of the embodiments of the invention and examples. Also, various techniques used to implement the present invention. The technique can be easily and reliably performed by those skilled in the art based on known literature and the like, except for the technique whose source is clearly indicated. For example, genetic engineering and molecular biology fe Sambrook and Maniatis, in Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989; Ausubel, FM et al., Current Protocols in Molecular Biology, John Wiley & Sons , New York, NY, 1995. Further, the terms in the present invention are basically based on the IUPAC-IUB Commission on Biochemical Nomenclature or based on the meanings of terms commonly used in the art.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing the results of testing the effect of m-UNK on promoting B cell proliferation. FIG. 2 is a graph showing the results of testing the effect of m-UNK on the immunization of mice with ovalbumin (OVA). FIG. 3 shows the results of detection of the m-UNK protein by the eight-muster polyclonal antibody. FIG. 4 is a graph showing the results of confirming the effect of m-UNK on the anti-sheep erythrocyte antibody production ability in mouse spleen cells.

BEST MODE FOR CARRYING OUT THE INVENTION The immune response regulator gene of the present invention is a genomic gene encoding a rodent immune response regulator protein (hereinafter sometimes referred to as “UNK:”). Target Contains a mouse gene encoding a mouse immune response regulatory protein having the amino acid sequence of SEQ ID NO: 2 (hereinafter sometimes referred to as “M-UNK”); and a rat immune response regulatory protein having the amino acid sequence of SEQ ID NO: 4. (Hereinafter sometimes referred to as “R-UNK:”). The cDNA of the M-UNK gene has the nucleotide sequence of SEQ ID NO: 1, and the cDNA of the R-UNK gene has the nucleotide sequence of SEQ ID NO: 3.

The UNK gene can be isolated by screening using a mouse or rat genomic cDMA library using the oligonucleotide probes provided by this invention, respectively. As the probe, for example, a partial sequence (15 bp or more) of the purified polynucleotide (eg, cDNA) provided by the present invention or its complementary sequence can be used. Screening with a probe can be performed under stringent end conditions that allow specific hybridization between the genomic DNA and the probe. Stringent conditions are defined by the salt concentration in the hybridization and washing steps, the concentration of organic solvents (such as formaldehyde), temperature conditions, and the like. For example, the conditions disclosed in U.S. Patent No. 6,100,037 can be employed. The probe can be labeled by a radioisotope (RI) method or a non-RI method, but it is preferable to use a non-RI method. Examples of the non-RI method include a fluorescent labeling method, a biotin labeling method, and a chemiluminescent method, and it is preferable to use a fluorescent labeling method. As the fluorescent substance, a substance capable of binding to the base moiety of the oligonucleotide can be appropriately selected and used. Examples of the fluorescent substance include a cyanine dye (for example, Cy3 and Cy5 of the Cy DyeTM series), a monodamine 6G reagent, -N2-Acetylaminofluorene (AAF), AAIF (iodine derivative of AAF) and the like can be used.

The UNK gene can also be amplified by a PCR (Polymerase Chain Reaction) method using rat genomic DNA or mouse genomic DNA as a 踌 type using the primer set provided by the present invention. The primer set can be prepared by combining two or more of partial sequences (15 bp or more) selected from the purified polynucleotide (cDNA) provided by the present invention. In addition, the upstream of the gene was determined by the RACE method using one primer on the fifth side of the cDNA. It is also possible to PCR amplify the downstream part of the gene by the RACE method using one primer on the three sides of the cDNA. In addition, the following points can be pointed out as points to keep in mind when designing primers. The size (number of bases) of the primer is 15-40 bases, preferably 15-30 bases, in view of satisfying the specific annealing with type I DNA. However, when performing LA (long and accurate) PCR, at least 30 bases are efficient. Avoid the complementary sequence between one primer or one pair (two) consisting of the sense strand (5 'end) and the antisense strand (3, end) so that they do not anneal to each other. In addition, the GC content should be about 50% to ensure stable binding to type I DNA, so that GC-rich or AT-rich is not unevenly distributed in the primers. Since the annealing temperature depends on the melting temperature (Tm), primers with a Tm value of 55-65 and similarity to each other should be selected to obtain highly specific PCR products. It is also necessary to take care to adjust the final concentration of primer used in PCR to be about 0.1 to about l_iM. In addition, commercially available software for primer design, for example, OligoTM (manufactured by National Bioscience Inc. (USA)), GENETYX (manufactured by Softea Development Co., Ltd. (Japan)) and the like can also be used. The full-length genomic gene obtained in this manner is usually used, for example, by PCR, NASBN (Nucleic acid sequence based amplification), TMA (transcription-mediated amplification), and SDA (Strand Displacement Amplification). It can also be amplified by a gene amplification method. The UNK genomic gene can be used to prepare purified polynucleotides (DNA fragments and RNA fragments) from mRNA transcribed by this gene and cDNA synthesized from mRNA. For example, cDNA can be synthesized as type III using poly (A) + RNA extracted from rodent cells such as mouse and rat. The rodent cells may be cells extracted from animals or cultured cells. cDNA can be synthesized using a known method (Mol. Cell. Biol. 2, 167-170, 1982; J. Gene 25, 263-269, 1983; Gene, 150, 243-250, 1994). Or according to the invention The target cDNA can also be synthesized using the set of primers provided by the above-mentioned method and the RT-PCR method in which mRNA isolated from a rodent cell is type II. Alternatively, the target cDNA can also be synthesized by synthesizing partial sequences using a DNA oligo synthesizer and joining them using enzymatic and subcloning techniques. The cDNA prepared in this manner specifically has the nucleotide sequences of SEQ ID NOS: 1 and 3. These polynucleotides can be used for genetically engineered production of UNK proteins. These polynucleotides can also be used as genetic material (transgene) for overexpressing UNK protein in animals. When the UNK protein is produced by genetic engineering or used as a transgene, it does not need to be the full length of SEQ ID NOS: 1 and 3, respectively. For example, at least the respective protein coding regions (CDS) constitute Polynucleotide sequence. In addition, when transfecting a gene to improve the animal's antigenic wisdom for the antigen of interest, for example, a sequence consisting of more than 60 consecutive nucleotides of the 1st to 489th nucleotides of SEQ ID NO: 1 or the first nucleotide of SEQ ID NO: 3 Oligonucleotides or polynucleotides each containing more than 60 consecutive 527 bases can also be used. Such oligonucleotides or polynucleotides can be made, for example, by cleaving cDNA with an appropriate restriction enzyme, or can be prepared by literature (eg, Carruthers (1982) Cold Spring Harbor Symp. Quant. Biol. 47:41 1-418). ; Adams (1983) J. Am. Chem. Soc. 105: 661; Belousov (1997) Nucleic Acid Res. 25: 3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19: 373-380; Blommers ( 1994) Biochemistry 33: 7886--7896; Narang (1979) Meth. Enzymol. 68:90; Brown (1979) Meth. Enzymol. 68: 109; Beaucage (1981) Tetra. Lett. 22: 1859; U.S. Pat. No. 458,066), and can be synthesized in vitro by a well-known chemical synthesis technique. The recombination vector of the present invention is a cloning vector or an expression vector, and an appropriate one is used depending on the kind of the polynucleotide as the insert and the purpose of use. For example, when producing UNK protein using cDNA or its ORF region as an insert, expression vectors for in vitro transcription, E. coli, Speech vectors suitable for prokaryotic cells such as bacteria, eukaryotic cells such as yeast cells, insect cells, and mammalian cells can also be used. In the case of genomic DNA insert of UNK gene, BAC (Bacterial Artificial Chromosome) vector or cosmid vector can also be used. As the transformed cell of the present invention, for example, when a large amount of UNK protein is produced, prokaryotic cells such as Escherichia coli and Bacillus subtilis, and eukaryotic cells such as yeast, insect cells, and mammalian cells can be used. . These transformed cells can be prepared by introducing a recombinant vector into the cells by a known method such as an electroporation method, a calcium phosphate method, a ribosome method, and a DEAE dextran method. The UNK protein of the present invention can be isolated from a rodent organ or a cell line, a method for preparing a peptide by chemical synthesis based on the amino acid sequences of SEQ ID NOS: 2 and 4, or the purification provided by the present invention. It can be obtained by a method using a polynucleotide (cDNA or a translation region thereof) by recombinant DNA technology, but a method obtained by recombinant DNA technology is preferably used. For example, a protein can be expressed in vitro by preparing RNA from a vector having the above-mentioned polynucleotide by in vitro transcription and performing in vitro translation using this as a type III. If the polynucleotide is recombined into an appropriate expression vector by a known method, the polynucleotide is encoded in prokaryotic cells such as Escherichia coli and Bacillus subtilis, and eukaryotic cells such as yeast, insect cells, and mammalian cells. You can express a large amount of protein.

When the UNK protein is produced by expressing DNA by in vitro translation, the above-mentioned polynucleotide is inserted into a vector having an RNA polymerase promoter to prepare a recombinant vector. The RNA polymerase promoter that can produce UNK protein in vitro when added to an in vitro translation system such as a heron reticulocyte lysate or a wheat germ extract containing RNA polymerase corresponding to T3, T7, and SP6 And the like. Examples of vectors containing these RNA polymerase promoters include pKAl, pCDM8, pT3 / T718, pT7 / 319, and pBluescriptll. When expressing the DNA of a UNK protein in a microorganism such as Escherichia coli, an expression vector having an origin, a promoter, a ribosome binding site, a DNA cloning site, a sunset and a mineral sequence that can be replicated in the microorganism, etc. First, an expression vector is prepared by recombining the above-mentioned polynucleotide, a host cell is transformed with the expression vector, and the resulting transformant is cultured, whereby the protein encoded by the polynucleotide is obtained. Can be mass-produced in microorganisms. At this time, a protein fragment containing an arbitrary region can also be obtained by adding a start codon and a stop codon before and after the arbitrary translation region and expressing it. Alternatively, it can be expressed as a fusion protein with another protein. By cleaving this fusion protein with an appropriate protease, it is possible to obtain only the protein portion encoded by the polynucleotide. Examples of expression vectors for Escherichia coli include a pUC system, a BluescriptII, pET expression system, and a PGEX expression system.

When the UNK protein is produced by expressing DNA in a eukaryotic cell, the polynucleotide is converted into an eukaryotic expression vector having a promoter, a splicing region, a poly (A) addition site, and the like. If inserted and made into a recombinant vector and introduced into eukaryotic cells, UNK protein can be produced in eukaryotic cells. Examples of expression vectors include pKAl, pCDM8, pSV3, pSVL, pBK-CMV, pBK-RSV, EBV vector, pRS, and pYE82. When pIND / V5-His, pFLAG-CMV-2, pEGFP-Nl, pEGFP-Cl, etc. are used as expression vectors, they can be expressed as fusion proteins with various tags such as His tag, FLAG tag, and GFP. I can come out. As eukaryotic cells, monkey kidney cells COS-7, Chinese hamster ovary cells, cultured mammalian cells such as CHO, budding yeast, fission yeast, silkworm cells, African frog egg cells and the like are generally used. Any eukaryotic cell that can express it can be used. To introduce the expression vector into eukaryotic cells, known methods such as an electroporation method, a calcium phosphate method, a ribosome method, and a DEAE dextran method can be used.

After expressing the UN protein in prokaryotic or eukaryotic cells, isolation and purification of the target protein from the culture can be performed by a combination of known separation procedures. example For example, treatment with a denaturant such as urea or a surfactant, sonication, enzyme digestion, salting-out, solvent precipitation, dialysis, centrifugation, ultrafiltration, gel filtration, SDS-PAGE, isoelectric focusing, Examples thereof include ion exchange chromatography, hydrophobic chromatography, affinity chromatography, and reverse phase chromatography. In addition, the recombinant UNK protein obtained by the above method includes a fusion protein with any other protein. For example, a fusion protein with glutathione-S-transferase (GST) recording color fluorescent protein (GFP) can be exemplified. Further, the protein of the invention may be subjected to various modifications in cells after being translated. Therefore, modified proteins are also included in the scope of the protein of the present invention. Examples of such post-translational modifications include elimination of N-terminal methionine, N-terminal acetylation, addition of sugar chains, limited degradation by intracellular protease, mistrailization, isoprenylation, phosphorylation and the like. The peptides of these inventions are peptide fragments consisting of a part of the UNK protein. That is, it is a peptide having a continuous 10 amino acid sequence, 11 to 30 amino acid sequence, 31 to 60 amino acid sequence, 61 to 110 amino acid sequence, and 11 to 160 amino acid sequence in SEQ ID NOs: 2 and 4. More specifically, a peptide consisting of a continuous amino acid sequence of amino acids 1 to 163 in SEQ ID NO: 2 or 10 or a continuous amino acid sequence of amino acids 1 to 162 in SEQ ID NO: 4 It is a peptide. That is, as shown in Examples described later, each UNK protein has an antigen recognition increasing activity on the C-terminal side. Specifically, M-UNK has an activity of increasing antigen recognition at amino acid residues 1-163 of SEQ ID NO: 2 and R-UNK has amino acid residues 1-162 of SEQ ID NO: 4 (FIG. 2). ). Therefore, peptides prepared from these regions can be used as a raw material for improving antigen recognition in animals, for example, by fusing antigens, and also used to induce tolerance in animals by overdose, etc. You can do it. These peptides can be prepared by cleaving the UNK protein with any restriction enzyme. Also, based on the amino acid sequence of SEQ ID NO: 2 or 4, Known peptide synthesis methods (Merrifield, RBJ Solid phase peptide synthesis I. The synthesis of tetrapeptide. J. Amer. Chem. Soc. 85, 2149-2154, 1963; Fmoc Solid Phase Peptide Synthesis. A Practical Approach. Chan , WC and White, PD, Oxford University Press, 2000). Further, these peptides may be composed of a residue linkage other than a natural amide bond. Residue linkages other than the natural amide bond include, for example, daltaraldehyde, N-hydroxysuccinimide ester, bifunctional maleimide, Ν, Ν, -dicyclohexylcarpoimide (DCC), or Ν, Ν, Examples include chemical bonding or coupling means such as -diisopropyl carpoimide (DIC). Furthermore, the linking group can be a substitute for a peptide bond, for example, ketomethylene (e.g., - C (= θ) -CH 2 - for - C (= 0) -NH-) , aminomethylene (CH ₂ - NH), ethylene, Orefin (CH = CH), ether (CH2-0), Chioete Le (CH ₂ -S), tetrazole (CN ₄ -), thiazole Ichiru, retro amide, including Chioami de or esters, (e.g., Spatola (1983 ) in Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp 267-357, "Peptide Backbone Modifications," Marcell Dekker, NY). The antibody of the present invention is the aforementioned purified rodent polyclonal antibody or monoclonal antibody. Specifically, it is an antibody prepared using each purified UNK protein or its partial peptide as an antigen. The antibodies of the present invention include whole molecules capable of binding to the epitope of each UNK protein, Fab, F (ab ') ₂ , Fv fragments, etc. For example, in the case of a polyclonal antibody, the antibody can be obtained from serum after immunizing an animal using the UNK protein or peptide as an antigen. As animals, mice, rats, hamsters, egrets, goats, sheep, geese, geese, bush, dogs, cats, monkeys, birds, etc. are used. The immunization of animals with antigenic peptides can be performed by known methods (for example, Shigeru Muramatsu, et al., Experimental Biology Lecture 14, Immunobiology, Maruzen Co., Ltd., 1985, Japanese Biochemical Society, Ed. Immune Biochemistry Research, Tokyo Chemical Dojin, 1986, The Japanese Biochemical Society, Ed., New Chemistry Laboratory 12, Molecular Immunology III, Antigen · antibody · complement, the method described in Tokyo Chemical Dojin, 1992, etc.). For example, as a general method, immunization can be performed by injecting an antigenic peptide intraperitoneally or subcutaneously into a mammal. Further, the antigen peptide may be administered together with adipant. Adjuvants include, for example, Freund's complete (or incomplete) adjuvant, Ribi adjuvant, pertussis vaccine, BCG, lipid A, ribosomes, aluminum hydroxide, silica, and the like. Furthermore, a recombinant plasmid vector (naked DNA, DNA peptide) is directly injected into mammalian skeletal muscle, skin, or myocardium, and a protein or polypeptide expressing the gene incorporated in the plasmid vector is used as an antigen for immunization. (Science, 247: 1465-1468, 1990, etc.). An antiserum containing a polyclonal antibody can be prepared from blood collected from an immunized animal after breeding the animal for a predetermined period of time. Monoclonal antibodies can be prepared using known monoclonal antibody production methods (“Monoclonal Antibodies”, by Kamei Nagamune and Hiroshi Terada, Hirokawa Shoten, 1990; “Monoclonal Antibody” James W. Godmg, third edition, Academic Press, 1996; Monoclonal Antibody Production Techniques and Applications, pp. 79-97, Marcel Dekker, Inc., New York, 1987, etc.). The antibodies of the present invention also include those conjugated with various labels (enzymes, radioisotopes, fluorescent dyes, etc.). These antibodies can be used, for example, to detect the expression of the UNK gene, or to administer to experimental animals to neutralize endogenous UNK and create an experimental model of the above immune response. Next, the cell or animal of the tenth invention in which the UNK protein is excessively expressed will be described. This animal or cell is, for example, a method of injecting an UNK protein solution into an animal; a metal colloid and an UNK protein as described in Patent Document 1 A method of administering to an animal by binding to the animal; An active UNK gene (that is, a purified polynucleotide linked to a high-expression promoter, etc.) is administered to an animal according to a gene therapy method using a retrovirus vector or an adenovirus vector. Method for introduction; Preparation using the calcium phosphate method, a method using ribosomes or erythrocyte ghosts, an electoral portation method, a method for introducing the UNK gene into cells by a microinjection method using a glass pipe, etc. Can be. Furthermore, these animals and cells can be prepared according to a known transgenic animal preparation method (eg, ', Proc. Natl. Acad. Scl. USA 77; 7380-7384, 1980). That is, an activated UNK gene is introduced into a totipotent cell of a non-human animal (preferably a mouse or a lad), the cell is developed into an individual, and an individual having the somatic cell genome into which the UNK gene is integrated is used. By selecting, a transgenic animal of interest can be produced. Considering the yield of transgenic animals and the efficiency of transgene transfer to the next generation, the method of gene transfer into totipotent cells is a physical injection (microinjection) method of foreign gene DNA. Is optimal. The fertilized egg into which the gene has been injected is then transplanted into the fetal oviduct of the foster parent, and the animals that have developed and born are placed in foster parents and reared, and then DNA is extracted from a part of the body (tail tip, etc.). By confirming the presence of the introduced foreign gene by Southern blot analysis or PCR assay, a primary heterozygous animal in which the foreign gene was introduced into one of the diploid chromosomes was obtained. Homozygous animals are obtained by crossing zygotes. The transgenic animal of the present invention includes a primary heterozygous animal, a homozygous animal obtained by crossing heterozygotes, their progeny, or their fetuses. The eleventh invention is a cell or animal in which the function of the immune response regulatory gene of the first invention is defective. “Functional deficiency” in this case means, for example, that the UNK gene product, UNK protein, is not functioning normally in the immune response system. Therefore, the cells or animals of the eleventh invention are immunodeficient cells or immunodeficient animals in which the immune response is not functioning normally. One embodiment of such a cell or animal is a cell or animal that does not produce UNK protein due to the absence of the UNK gene coding region in the chromosomal genome. In another embodiment, one or more nucleotides in the coding region of the UNK gene are deleted, added, or substituted with other nucleotides (genetic mutation) so that they do not produce the UNK protein or have a mutant form. A cell or animal that produces UNK protein. Yet another embodiment is a cell or animal that does not express the UNK gene due to deletion or partial mutation of the expression control region of the UNK gene, and thus does not produce UNK protein. In still another embodiment, a ribozyme that cleaves a sense strand, antisense strand, sense / antisense double strand (siRNA), RNAi, and UNK mRNA for a transcript (mRNA) from the UNK gene is expressed, and UNK is expressed from the mRNA. A cell or animal in which no protein is synthesized. Cells expressing the sense strand, antisense strand, sense 'antisense duplex (siRNA), and RNAi lipozyme can be prepared by introducing a DNA fragment encoding them into cells, but the UNK gene It is preferable to introduce a deletion mutation into the coding region and / or the expression control region of the UNK gene in order to ensure that the gene is defective. Such gene-specific mutagenesis can be performed by a known target gene recombination method (Gene Yuichi targeting method: Science 244: 1288-1292, 1989). Then, by generating a mutagenized totipotent cell (ES cell) in an individual, an animal (knockout animal) in which the function of UNK protein is deficient in all somatic cells can be obtained. A twelfth invention is a method for producing an antibody against an arbitrary protein in a rodent, comprising the steps of: administering an antigen to the animal of the tenth invention; and isolating the antibody from the animal. Is included. Specifically, the same means as in the production of an animal of the tenth invention (for example, a method of administering an UNK protein to an animal or a method of introducing an UNK gene into an animal in accordance with a method of gene therapy) can be used in an animal. Increase the activity of UNK protein and administer any antigenic substance to animals. Since the immune response of animals is activated by administration and high expression of UNK protein, antibodies can be efficiently produced. In another aspect of the twelfth invention, an antibody is produced using a transgenic animal having an activated UNK gene in somatic cells. It is a method to accomplish. This animal overexpresses the UNK protein due to high expression of the introduced UNK gene, and has a high immune response ability, so that it efficiently produces antibodies. The antibody is a polyclonal antibody or a monoclonal antibody, and can be prepared by the same procedure as the antibody of the ninth invention.

EXAMPLES Next, the present invention will be described in more detail and specifically with reference to examples, but the present invention is not limited to these examples. The basic operation and enzymatic reaction for DNA recombination were in accordance with the literature ("Molecular Cloning, Laboratory Manual", Cold Spring Harbor Laboratory, 1989). Unless otherwise specified, restriction enzymes and various modifying enzymes were manufactured by Invitrogen. The buffer composition and reaction conditions for each enzyme reaction were in accordance with the attached instructions.

Example

Using a 30-mer sense primer (oligonucleotide of SEQ ID NO: 5) and a 30-mer antisense primer (oligonucleotide of SEQ ID NO: 6), a cDNA prepared from mRNA extracted from mouse spleen was amplified by PCR as type III. . The PCR product was blunted with KOD polymerase (manufactured by Toyobo), inserted into the EcoRV site of pZero-2.0, and transformed into host Escherichia coli DH-5a FT (manufactured by Invitorgen). The nucleotide sequence of the obtained clone was determined, and a clone in which an amplification error did not occur during the reverse transcription reaction and the PCR reaction was selected. The resulting clone, m-UNK, has a total length of 578 bp and an open reading frame of 489 bp.

(ORF) had a structure consisting of a 53 bp 3 ′ untranslated region (SEQ ID NO: 1).

The ORF encoded a protein consisting of 163 amino acid residues, and had a characteristic signal sequence of a secreted protein. (SEQ ID NO: 2). Example 2

Using a 30-mer sense primer (oligonucleotide of SEQ ID NO: 7) and a 30-mer antisense primer (oligonucleotide of SEQ ID NO: 8), the cDNA prepared from the mRNA extracted from the rat spleen was amplified by PCR as type III. . The PCR product was blunted with KOD polymerase (manufactured by Toyobo), inserted into the EcoRV site of pZero-2.0, and transformed into host E. coli DH-5a FT (manufactured by Invitorgen). The nucleotide sequence of the obtained clone was determined, and a clone in which no amplification error occurred during the reverse transcription reaction and the PCR reaction was selected. The obtained clone RUNK had a total length of 527 bp, and had a structure of an open reading frame (ORF) of 483 bp, a structure of 44 bp and 3 untranslated regions (SEQ ID NO: 3). The ORF encoded a protein consisting of 162 amino acid residues, and had a characteristic signal sequence of a secreted protein. (SEQ ID NO: 4).

Example 3 The translation region was amplified by PCR using the m-UNK cDNA specified in Example 1 as type III.

As a PCR primer, a 30-mer sense primer (SEQ ID NO: 9) to which a BamHI site was added and a 30-mer antisense primer (SEQ ID NO: 10) to which an Apal site was added were used.

The PCR product was blunted with KOD polymerase (manufactured by Toyobo), inserted into the EcoRV site of pZero-2.0, and transformed into host Escherichia coli DH-5a FT (manufactured by Invitorgen). The nucleotide sequence of the obtained clone was determined, and a clone having the desired structure was selected without generating amplification errors during the PCR reaction. From this plasmid, BamHI to Apal fragment was inserted between BamHI and Apal of pcDNA3.1-myc-his- (B) + and transformed into host Escherichia coli DH-5 o! FT (manufactured by Invitorgen). . Check the nucleotide sequence and confirm that the Myc tag and His protein are fused to the C-terminal of the ORF of each cDNA. Clones that were expressed as quality were selected. This clone was cultured in LB medium at 37 for 16 hours to purify the expression plasmid DNA (m-UNK-MH / pcDNA3.1-myc-his- (B) +).

Example 4 The cDNA of m-UNK specified in Example 1 was digested with EcoRI and Notl in the multi-cloning site with restriction enzymes.After electrophoresis, the target band was cut out, and pcDNA3-A- ( After introduction between the EcoRI and Notl sites of (+), host Escherichia coli DH-5a FT (Invitrogen) was transformed. The nucleotide sequence was confirmed, and a clone having a structure capable of expressing m-UNK was selected by cytomegalovirus promoter. This clone was cultured in LB medium at 37 for 16 hours, and the expression plasmid DNA (m-UNK / pcDNA3- (A) +) was purified.

Using this plasmid as type I, PCR was performed by PCR using a 45-mer sense primer (oligonucleotide of SEQ ID NO: 11) to which an Xbal site was added and an 18-mer antisense primer (oligonucleotide of SEQ ID NO: 12). Amplified. The PCR product was blunted with KOD polymerase (manufactured by Toyobo), inserted into the EcoRV site of pZero-2.0, and transformed into host E. coli DH-5a FT (manufactured by Invitorgen). The nucleotide sequence was confirmed, and a clone having the desired structure was selected. This clone was cultured in LB medium at 37 for 16 hours, and the expression plasmid DNA was purified. The cDNA fragment between the Xbal site and the Pmel site of this plasmid was digested with restriction enzymes, purified, and inserted between the Xbal site and the EcoRV site of an E. coli expression vector (PET30-A Novagen). ', Host E. coli DH-5 FT

(Manufactured by Invitorgen). The nucleotide sequence was confirmed, and a clone having the desired structure was selected. This clone was cultured in LB medium at 37 for 16 hours, and the expressed plasmid DNA was purified.

This plasmid was transformed into host E. coli BL21-Codon Plus (manufactured by Stratagene), cultured in SOB medium containing kanamycin, and the protein was induced by IPTG, and the expression of m-UNK protein was induced in the host. . 8 M urea after recovery of E. coli The cells were suspended in a PBS buffer solution, freeze-thawed, and disrupted by ultrasonication. The supernatant was separated from the crushed liquid by centrifugation, the purified liquid containing _c- urea purified by NTA-resin (Qiagen) was separated by gel filtration, and the fraction containing m-UNK protein was collected. Dialysis and centrifugation to remove insolubles.

The protein from which the obtained signal sequence was deleted was used as m-UNK-myc-his protein in the subsequent experiments.

Example 5 The expression plasmid DNA (m-UNK / pcDNA3- (A) +) obtained in Example 4 was injected into the muscle of an Armenian hamster for immunization, and two weeks later, the m-UN obtained in Example 4 was used. After immunization with the UNK-myc-his protein, and two weeks later, the expression plasmid DNA (m-UNK / pcDNA3- (A) +) was injected into the muscle for immunization. Two weeks after the final immunization, blood was collected from Hams by the usual method to purify the polyclonal antibody and to prepare a monoclonal antibody from spleen cells.

Using the obtained polyclonal antibody, m-UNK-myc-his protein was detected, and it was confirmed that the antibody was produced. (Figure 3)

Example 6 Plasmid m-UNK / pcDNA3- (A) + was introduced into monkey kidney cells COS-7 by calcium phosphate method, and after 24 hours, the culture solution was changed to one containing no serum components. After culturing for 48 hours at 37, the cell supernatant and cells were collected separately, and the protein components were separated by SDS-PAGE, and then m-UNK was detected by Western blotting using a polyclonal antibody. It was confirmed that m-UNK was correctly expressed, processed, and released into the supernatant. Example 7 Using the baculovirus expression plasmid pAC-GP67-B (manufactured by Invitrogen) as type III, the sense primer 1 (SEQ ID NO: 13) and the antisense primer 1 containing the sequence of m-UNK (SEQ ID NO: 14) PCR was performed to amplify the signal sequence of paculovirus. Plasmid DNA (m-UNK / pcDNA3- (A) +) is a 铸 type, which is a complementary strand of SEQ ID NO: 14 including a partial baculovirus signal sequence, and downstream of the cleavage site of the m-UNK signal sequence PCR was performed using a primer (SEQ ID NO: 15) containing the above sequence and an antisense primer (SEQ ID NO: 12) to amplify a partial cDNA of m-UNK. Using this cDNA and the DNA of the signal sequence of the paculovirus as described above as a 铸 type, PCR was performed using a sense primer (SEQ ID NO: 13) and an antisense primer (SEQ ID NO: 12) to obtain a baculovirus signal sequence. Amplified DNA (SEQ ID NO: 15) fused with the sequence downstream of the cleavage point of the m-UNK and m-UNK signal sequences, blunted with KOD polymerase (manufactured by Toyobo), and inserted into the EcoRV site of pZero-2.0 Then, the host Escherichia coli DH-5 o! FT (Invitorgen) was transformed. A clone having the desired structure was selected, and the plasmid was purified. This plasmid was digested with Spel and Notl, and the obtained fragment was inserted between Spel and Notl of the Bacchus virus expression plasmid pAC-GP67-B and inserted into the host E. coli DH-50! FT (Invitorgen). Transformation was performed. A clone having the desired structure was selected, and the plasmid was purified. Using the obtained plasmid, a recombinant baculovirus expressing the m-UNK protein was prepared according to the protocol of Pharmingen, the m-UNK protein was released into a protein-free medium, and the insect cells were collected. The protein is dissolved from the supernatant and cells by SDS, and Western blotting is performed to confirm that the m-UNK protein is expressed and released, and to electrophoretically confirm that the sugar chain is modified. confirmed.

Example 8 B cells were fractionated from a spleen cell of BAJLB / c mouse using a column, and m-UNK protein When cultured in the presence of quality, as compared to the absence and _c growth was observed in the dominant B cell, LPS non-responder mice in which C3H / similar proliferative activity in B cells from HeJ The results showed that this reaction was not due to LPS but was induced by H1-UNK protein (FIG. 1).

Example 9 To examine the effect of m-UNK protein on the immune response, BALB / c mice were intraperitoneally administered with ovalbumin (OVA) alone or a mixture of OVA and m-UNK protein. Two weeks after the administration, the serum antibody titer to OVA of each mouse was measured, and a significant increase in the serum antibody titer to OVA was observed in the group administered with the mixture of OVA and m-UNK protein compared to the group administered with OVA alone. (Figure 2).

Example 10 Mouse spleen cells and sheep erythrocytes were cultured in the presence and absence of m-UNK for 4 days, and antibody production against sheep erythrocytes was analyzed by measuring the number of hemolytic plaque forming cells (PFCs) See "Selected Methods in cellular Immunology", edited by Barbara B. Mishell and Stanley M. Shiigi, 1980, WH Freeman and Company, p. 86-89).

The results are shown in Figure 4. In the presence of sheep erythrocytes, the number of hemolytic plaques was increased in the presence of m-UNK compared to the control vector pET30, indicating that antibody production was enhanced. . On the other hand, in the absence of sheep erythrocytes, the number of hemolytic spots did not increase even in the presence of m-UNK, indicating no specific antibody production.

From the above results, it was confirmed that UNK enhances the production of antigen-specific antibodies in vitro.

Claims

The scope of the claims

1. an isolated rodent gene, wherein the amino acid sequence of either SEQ ID NO: 2 or 4 or one or more amino acid residues in SEQ ID NO: 2 or 4 are deleted, added or other amino acids An immune response regulatory gene encoding an immune response regulatory protein having a sequence substituted for a residue.

2. The immune response regulatory gene according to claim 1, wherein the cDNA synthesized from the transcript mRNA has the nucleotide sequence of SEQ ID NO: 1 or 3, respectively.

3. A polynucleotide purified from genomic DNA, mRNA, cDNA, or a sequence complementary thereto, of the immune response regulatory gene of claim 1.

4. An oligonucleotide probe that hybridizes under stringent conditions with the immune response regulatory gene of claim 1 or the purified polynucleotide of claim 3. '

5. A primer set for PCR-amplifying the immune response control gene of claim 1 or the purified polynucleotide of claim 3.

6. A recombinant vector carrying the purified polynucleotide of claim 3.

7. A transformed cell obtained by the recombinant vector according to claim 6.

8. Purified rodent protein, wherein the amino acid sequence of either SEQ ID NO: 2 or 4 or one or more amino acid residues in SEQ ID NO: 2 or 4 are deleted, added or other amino acid residues Immune Response Regulatory Protein Having a Substituted Sequence <

9. The immune response regulatory protein of claim 8, which is an expression product of the immune response regulatory gene of claim 1 or the polynucleotide of claim 3.

10. A purified or synthesized peptide comprising a part of the immune response regulatory protein of claim 8.

1 1. The peptide according to claim 10, comprising 10 or more consecutive amino acid sequences of amino acids 1-163 in SEQ ID NO: 2.

12. The peptide according to claim 10, comprising a continuous 10 or more amino acid sequence of amino acids 1-162 in SEQ ID NO: 4.

13. An antibody that specifically recognizes the immune response regulatory protein of claim 8.

14. A cell or animal in which the immune response modulator protein of claim 8 is overexpressed.

15. A cell or animal in which the immune response regulatory gene according to claim 1 is defective.

16. A method for producing an antibody against a desired protein in a rodent. The method according to claim 14, comprising administering an antigenic substance to the animal, and isolating the antibody from the animal. The method characterized by the above.