WO2000024776A1 - Novel toll-like receptor and gene thereof - Google Patents

Novel toll-like receptor and gene thereof Download PDF

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WO2000024776A1
WO2000024776A1 PCT/JP1999/005917 JP9905917W WO0024776A1 WO 2000024776 A1 WO2000024776 A1 WO 2000024776A1 JP 9905917 W JP9905917 W JP 9905917W WO 0024776 A1 WO0024776 A1 WO 0024776A1
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tlr6
toll
amino acid
receptor
mouse
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PCT/JP1999/005917
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French (fr)
Japanese (ja)
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Science And Technology Corporation Japan
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Akira, Shizuo
Takeuchi, Osamu
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants

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  • the early stages of the immune response to infection require the production of inflammatory cytodynamics from antigen-presenting cells such as macrophages, which induces a specific response by lymphocytes. . However, little is known about the molecular mechanism of this early reaction.
  • the Drosophila Toll gene is known to be involved in the development of the dorsal-ventral axis during development and in the immune response to bacteria and fungi in adults.
  • the Toll gene product is a type I receptor pig and, when combined with the ligand spaetzle, ultimately induces the production of antibacterial and antifungal substances in adults. Its intracellular signaling pathways have also been investigated in detail.
  • Drosophila Toll activates Pelle, a type of serine reonin kinase, through a tube, an adapter molecule, resulting in transcription of the Rel family. Escapes repression by Dorsal i Cactus with homology to the factor, translocates to the nucleus, and induces transcription.
  • the intracellular region of the Toll gene is widely conserved from plants to vertebrates. In mammals, the intracellular region of the IL-11 receptor family is known to have homology. ing.
  • the signaling mechanism from the IL-11 receptor family is the adapter molecule My D88, and the receptor for serine leonin kinase, IL-1
  • the repression of Ic ⁇ is released via the body-associated kinase (IL-1 Receptor-associated kinase (IRAK)), which activates the rel (Rel) family transcription factor NF- ⁇ .
  • IRAK body-associated kinase
  • This is in contrast to the signal transmission of the Drosophila 'Toll' gene and is thought to be evolutionarily conserved (O'Neill LA, et al., J Leukoc Biol. 1998 63 (6): 650 -657.).
  • Toll-related molecules in humans have been identified. It is a once-transmembrane type I receptor. Like the Drosophila Toll gene, the extracellular domain is a leucine repeat repeat, and the intracellular domain is an IL-1 receptor phage. There is a region homologous to the plant. Toll-related molecules in humans have so far been cloned into five families (TLR1-5) (Rock FL, et al., Proc Natl Acad Sci US A. 1998 95 (2): 588-593.).
  • TLR 4 (hToll) has been the most analyzed among them, and it is possible to express activated TLR4 (hToll) in human T cell line Jurkat cells.
  • NF- / cB is activated to induce the production of inflammatory cytokines such as IL-11, IL-6, and IL-8 (Medzhitov R, et Al., Mol Cell. 1998 2 (2) Medzhitov R, et al., Nature. 1997 388 (6640): 394-397.).
  • monocytes the expression of TLR4 gene is induced in response to lipopolysaccharide (LPS) (Muzio M, et al., J Exp Med. 1998 187 (12): 2097- 2101.). Therefore, it is thought that this molecule is involved in the production of inflammatory cytokines at the early stage of the immune response and the induction of so-called adaptive immunity by specific lymphocytes.
  • LPS lipopolysaccharide
  • Tol 1 molecule may form a larger family, elucidate the mechanism of the responding immune system, and investigate various immune system illnesses and the effects of ⁇ disease infection. To establish toxins and other treatments and preventive measures, Toll There is a demand for elucidation of the whole family. Disclosure of the invention
  • the present inventors have attempted to identify a new molecule belonging to this family, and provide a new molecular species that is considered to belong to the Tol (Tol 1) family.
  • the present invention provides TLR6, a novel molecular species of tolfamily related to a transcription factor NF- ⁇ that regulates the expression of various genes involved in an immune response, and a gene encoding the same. I do.
  • the present invention provides an amino acid sequence represented by SEQ ID NO: 2 or 4 in the Sequence Listing or one or more amino acids in the amino acid sequence are deleted and substituted with another amino acid, And / or a toll-like receptor (TLR6) having an amino acid sequence to which one or more other amino acids have been added.
  • TLR6 toll-like receptor
  • the present invention also relates to a protein having a toll-like receptor (TLR6) action.
  • the present invention also relates to a gene having a nucleotide sequence encoding the novel receptor or protein. More specifically, the present invention relates to a gene having a base acid sequence represented by SEQ ID NO: 1 or 3. BRIEF DESCRIPTION OF THE FIGURES
  • FIG. 1 shows the nucleotide sequence and amino acid sequence (one letter code) of human TLR6 of the present invention.
  • FIG. 2 shows the nucleotide sequence and amino acid sequence (one-letter code) of mouse TLR6 of the present invention.
  • FIG. 3 shows a comparison of amino acid sequences in the intracellular region of the human Toll family.
  • FIG. 4 is a photograph in place of a drawing showing the result of Northern blot analysis of mouse TLR6 in each mouse organ. The lower part of the figure shows the RNA content due to /?-Actin.
  • FIG. 5 shows the results of the TLR6 dual luciferase reporter assay of the present invention.
  • the upper row shows the case of 293 cells, and the lower row shows the case of NIH 3T3 cells.
  • FIG. 1 shows the nucleotide sequence and the amino acid sequence (one-letter code).
  • the nucleotide sequence of the obtained human TLR6 is shown in SEQ ID NO: 1 in the sequence listing.
  • the amino acid sequence is shown in SEQ ID NO: 2 in the sequence listing.
  • FIG. 2 shows the nucleotide sequence and the amino acid sequence (single-letter code).
  • the nucleotide sequence of the obtained mouse TLR6 is shown in SEQ ID NO: 3 in the sequence listing.
  • the amino acid sequence is shown in SEQ ID NO: 4 in the sequence listing.
  • the human TLR6 cDNA has an open reading frame of 2,388 bp, and the expected protein consists of 796 amino acid.
  • the mouse TLR6 cDNA was expected to have an oven reading frame of 2,42 lbp and to consist of 807 amino acids.
  • the homology between the human and the mouse was 77.6% at the DNA level and 73.7% at the amino acid level.
  • TLR6 The amino acid sequence of TLR6 is characteristic of a type I membrane protein. There are hydrophobic sites near the N-terminus and the center, which are thought to correspond to the signal peptide and the ⁇ penetration region, respectively.
  • the To11 family is characterized by an extracellular domain, the leucine litulin repeat domain, and a region homologous to the intracellular IL-1 receptor.
  • Human and mouse TLR6 also have a leucine litulin chili beat domain in the extracellular region, and homology within the cell with the IL_1 receptor family and other To11 families. Having.
  • FIG. 3 shows a comparison of amino acid sequences in the intracellular region of the human To11 family.
  • TLR6 of the present invention has the highest homology to TLR1, with 69.1% amino acid levels between human TLR1 and human TLR6. C. Among them, high homology was shown particularly in the intracellular region.
  • a primer was designed so that about 800 bp of the sequence in the N-terminal region was amplified.
  • the position on the chromosome was determined by the FISH method using the cDNA probe of mouse TLR6. This revealed that mouse TLR6 was present on chromosome 5.
  • TLR6 is thought to be on the same chromosome as TLR1, TLR2, and TLR3 that have been reported to date.
  • TLR6 of the present invention activates NF-B.
  • TLR6 The intracellular region of TLR6 has homology to the IL-1 receptor (IL-1R) family, and may have a similar intracellular signaling pathway. Signals from the IL-1R family cause activation of NF-B. Therefore, it was analyzed whether TLR6 was also involved in the activation of NF-B.
  • IL-1R IL-1 receptor
  • Human wild-type TLR6 was inserted into the expression vector pEF-BOS.
  • a tag was applied to the C-terminus with My c as an epitope (pE F—B ES—My c—T L R 6).
  • the cells were transiently transfected with 0.5, 1.0, and 2.0 ⁇ g of these into 2993 cells and NIH3T3 cells using the Lipofxion method and expressed.
  • 2.0 ⁇ g of wild-type MyD88 pEF-BOS-Flag-MyD88
  • 1.0 ⁇ g of NF—B / reporter plasmid and 0.1 g of pRL—SV40 Vector were simultaneously introduced. Cells were harvested 8 hours after transfection, and the luciferase activity was determined using a dual luciferase reporter assay system (Promega) using a luminometer (Lumminometer). Was measured.
  • FIG. 5 shows the case where 293 cells were flowed, and the lower part Indicates the case where NIH 3 T 3 cells were used.
  • the control of MyD888 showed about a 3-fold increase in luciferase activity in 2993 cells and a about 2-fold increase in luciferase activity in NIH3T3 cells.
  • the group in which TLR6 was expressed no increase in activity was observed in any of the cell types.
  • NF-B could not be activated only by expressing human TLR6 alone, which was the only receptor pig.
  • the human and mouse TLR6 of the present invention have a leucine-rich repeat domain at the N-terminal side, and a cell of the IL-11R family at the C-terminal side following the transmembrane portion. It had homology with the inner domain. It showed the highest homology with TLR1 among TLR-related molecules, TLR families.
  • TLR6 of the present invention Analysis of expression of TLR6 of the present invention by RT-PCR reveals expression in immunocompetent tissues such as thymus and spleen, suggesting a role for this molecule in host defense.
  • TLR6 was thought to transmit a signal to NF- / cB due to its intracellular domain being homologous to the intracellular domain of IL_1R. NF-B could not be activated simply by expressing 293- and NIH3T3 cells. This is because (1) the signal is not transmitted into the cell by the expression of the receptor alone, (2) the signal is transmitted through another pathway, not NF-B, (3) It is conceivable that the response differs depending on the type of cell into which the gene is introduced. In the future, it is necessary to produce active TLR6 and analyze its signaling mechanism.
  • Toll gene product is involved in nonspecific immune responses in the early stage of infection and signaling to adaptive immunity by lymphocytes.
  • the biological response to infection is very important, but in severe infections, too strong a response can cause fatal situations, such as endotoxin. This control of biological response is considered to be very useful clinically.
  • a detailed analysis of the function of the To11 family is expected to help elucidate the molecular mechanism of the initial response to inflammation.
  • Example 1 (cloning of cDNA of mouse TLR6)
  • a homologous search of the EST database using the human TLR1 nucleotide sequence revealed a mouse-derived nucleotide sequence (AA177549) highly homologous to the human TLR1 nucleotide sequence.
  • PCR Polymerase chain reaction
  • PCR was performed using Taq polymerase (Takara Shuzo), 30 cycles of 94 ° C for 30 seconds, 56 ° C for 30 seconds, and 72 ° C for 30 seconds, and one cycle of 72 ° C for 10 minutes. Kuru went.
  • a portion of this PCR product was electrophoresed in agarose gel, stained with ethidium bromide (Nippon Gene), and amplified with approximately 400 bp of cDNA under UV irradiation. It was confirmed. This band was excised from the gel, purified with a wizard (Wizard) (Promega), and then cloned using a TA cleaning kit (Novagen).
  • pT7blue (Novagen) T vector was used as a vector, and the vector and the above DNA were mixed at a molar ratio of 1: 3. DNA was integrated into the vector at.
  • the T vector incorporating DNA was transfected into E. coli DH5, and L containing 100 ⁇ g / ml of Ambicillin (Sigma) and 200 ⁇ g / ml of X-gal (Nacalai Tesque) One broth (L-Broth) I sprayed on a plate of semi-solid medium, about 12 hours 37.
  • the nucleotide sequence of the imported cDNA fragment was determined using a fluorescent sequencer 377 manufactured by Applied Biosystems.
  • the sequencing sam- ble was prepared using PRIMSM, a Ready Reaction Dye Terminator Cycle Sequencing Kit (Available Biosystems).
  • PRIMSM a Ready Reaction Dye Terminator Cycle Sequencing Kit
  • In a microtube add 10 ⁇ 1 of the reaction stock solution, 2.0 ⁇ 1 of 1.6 pmol / ⁇ l of T7 Promoter Primer and 8.0 ⁇ 1 of 0.1 l ⁇ g / ⁇ l. was added and mixed, and 25 cycles of PCR reaction were performed at 96 ° C for 10 seconds, 50 ° C for 5 seconds, and 6 ° C for 4 minutes.
  • the six clones had a nucleotide sequence corresponding to EST (AA177549).
  • a mouse genomic clone containing this sequence was searched in the mouse 129 s V genome library (Stratagene). 1 0 6 corresponding plaques were plates, emerging plaques Nai Ronfiru terpolymer (colony / plaquescreen, NEN Co.) was transferred to, alkali processes Nai b Nfu Iruta has been transferred (1. 5 MN a C 1 , 0.5 minutes on a filter paper impregnated with 0.5 MNaOH, followed by neutralization treatment (1.5 MNaCl, 0.5 MTris-HClH7.5 filter paper impregnated with it).
  • EST (AA177549) fragment probe labeled with 32 P radioisotope was prepared as follows. That is, the EST (AA1774949) fragment was excised from the vector with the restriction enzymes BamHI and SalI and incorporated into an agarose gel. After electrophoresis, the band was stained with ethidium Mubuchi Mide (Nippon Gene Co., Ltd.), and a band of about 400 bp was cut out from the gel under ultraviolet irradiation and purified with a wizard (Promega). The obtained fragment was labeled using a DNA labeling kit (Megaprime DNA labeling kit: Amersyam).
  • the filter prepared by the above method was applied to a SSC solution with a final concentration of 6 times for each component, a Denhardt's solution with a 5 times concentration, 1% SDS, and a boiling water bath with a concentration of 100 g / m1. After immersion in a hybridization solution containing denatured salmon sperm DNA (Sigma) and shaking at 65 ° C for 1 hour, the probe labeled by the method described above was hybridized. The mixture was shaken at 65 ° C. for 18 hours to perform hybridization.
  • the filter is immersed in SSC solution containing 0.1% SDS at a final concentration of 2 times, washed once at 65 ° C, and then added to the final concentration of 0.1% SDS. It was immersed in a 0.2 times concentration SSC solution and washed once at 65 ° C.
  • the washed filter was subjected to autoradiography at 180 ° C using a sensitizing screen. As a result, the strongly exposed clones were picked up, plaques were replated again, and screened twice by the above-mentioned method to completely separate two clones from a single clone.
  • This phage DNA was digested with the restriction enzymes BamHI alone or with BamHI and Sa1I, and similarly with BamHI alone or with BamHI and Sa1I. It has been incorporated into the Smid p Bluscript KS (+).
  • BamHI alone or with BamHI and Sa1I restriction enzymes BamHI alone or with BamHI and Sa1I
  • This sequence was amplified using PCR (Polymerase chain reaction) using the cDNA library of the 17th mouse embryo as a material.
  • the PCR was performed for 30 cycles at 94 ° C. for 30 seconds, 56 ° C. for 30 seconds, 72 ° C. for 30 seconds, and one cycle for 72 ° C. for 10 minutes.
  • the PCR product was subjected to agarose gel electrophoresis and stained. As a result, a band of about 2 kbp was confirmed. This band was cut out of the gel, subjected to TA cloning as described above, and its nucleotide sequence was determined using a sequencer.The nucleotide sequence corresponding to mouse TLR6 in Fig. 2 was obtained. .
  • FIG. 2 shows the nucleotide sequence and the amino acid sequence (one-letter code).
  • the nucleotide sequence of the obtained mouse TLR6 is shown in SEQ ID NO: 3 in the sequence listing.
  • the amino acid sequence is shown in SEQ ID NO: 4 in the sequence listing.
  • Example 2 (cloning of cDNA of human TLR6)
  • a probe for screening human TLR6 was prepared from the nucleotide sequence obtained in Example 1. Specifically, the nucleotide sequence information of mouse TLR6 is changed to ⁇ to synthesize oligonucleotides, 5 '-cctcgagctgagatagagagcatcttg-3' and
  • a partial base sequence of mouse TLR6 was amplified by PCR. This PCR product was similarly TA-cloned, and the nucleotide sequence was determined by sequencing. It was confirmed that a part of the nucleotide sequence of mouse TLR6 was partially amplified. Using this fragment as a probe, a human cDNA clone containing this sequence was searched in the human placenta cDNA library (CLONTECH). A nylon filter was prepared as described above, and a probe of a mouse TLR6 fragment labeled with a radioactive isotope 32 P was prepared. At this time, Xhol and SalI were used as restriction enzymes.
  • the filter prepared by the method described above was applied to a SSC solution with a final concentration of 6 times the concentration of each component, a Denhardt's solution with a concentration of 5 times, 1% SDS, and a boiling water bath of 10 ° g / m1. After immersion in a hybridization solution containing denatured salmon sperm DNA (Sigma) and shaking at 60 ° C for 1 hour, the probe labeled by the above-mentioned method was hybridized. The solution was shaken at 60 ° C. for 20 hours to perform hybridization.
  • the filter is immersed in SSC solution at a final concentration of 2% each containing 0.1% SDS, washed once at 60 ° C, and then further washed with 0.1% SDS.
  • Autoradiography was performed at C. As a result, the strongly exposed clones were picked up, plaques were replated again, and screened twice by the method described above, and 6 clones of a single clone were completely separated.
  • the phage DNA was purified, digested with the restriction enzyme NotI, and incorporated into pBluescriptKs (+) similarly digested with NotI.
  • the DNA sequences of these clones were analyzed by a sequencer, and the DNA sequences shown in FIG. 1 were determined. As described above, the total length of human TLR6 was determined.
  • Fig. 1 shows the nucleotide sequence and amino acid sequence (single-letter code).
  • the nucleotide sequence of the obtained human TLR6 is shown in SEQ ID NO: 1 in the sequence listing.
  • the amino acid sequence is shown in the Sequence Listing. This is shown in array number ⁇ 2.
  • Example 3 Expression of mouse TLR 6 in each tissue
  • Trizol Trizol
  • PCR was performed using Taq polymerase (Takara Shuzo Co., Ltd.), and was carried out for 25 cycles at 94 ° C, 60 ° C for 30 seconds, and 74 ° C for 60 seconds. Cycled. The PCR product was subjected to 1.5% agarose gel electrophoresis, and the amount of amplification was determined by staining. Mouse TLR6 was expressed in thymus, spleen, ovary and lung.
  • Fig. 4 shows the results.
  • Example 4 (Mouse TLR6 chromosomal location)
  • Human wild-type TLR6 was prepared by using PCR with Myc as a shredding tag at the C-terminus. Synthetic oligonucleotides,
  • the expression vector pEF-BOS-Myc-TLR6 constructed in Example 5 was applied to 2933 cells and NIH3T3 cells using the Lipofection method, respectively. , 2.0 g—transiently expressed.
  • wild-type My D 8 8 a (P EF - - BOS F lag -M y D 8 8) 2. was introduced into 0 ⁇ g- excessive resistance.
  • NF—B / reporter plasmid 1.0 ⁇ g
  • the cells were washed with PBS (Phosphate Buffered saline), and then lysed and recovered with a lysis buffer (Lysis Buffer).
  • PBS Phosphate Buffered saline
  • a lysis buffer Lysis Buffer
  • L-II as a substrate for luciferase enzyme and Stop and Glo as an internal control were added, and immediately after that, luciferase activity was measured using a luminometer.
  • the TLR6 of the present invention shows high homology to TLR1 of the TLR family, which is a Toll-related molecule, has a leucine litulin bite domain at the N-terminal side, and has a transmembrane region. Subsequently, the C-terminal side is a novel toll-like (T01-1 ike) receptor having homology to the intracellular domain of IL_1R. It is also clinically important as a substance involved in the signal transmission system in the early stage of the immune response.

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Abstract

A novel molecular species TLR6 which belongs to the Toll family relating to, for example, a transcription factor NF-'kappa'B regulating the expression of various genes participating in immune response; and a gene encoding the same are provided. A novel Toll-like receptor (TLR6) which is a molecule relating to Toll relating to a transcription factor NF-'kappa'B regulating the expression of various genes participating in immune response; and a gene encoding the same.

Description

新規トル様 （ T o 1 1 _ 1 i k e ) レセプター及びその遺伝子  Novel Toll-like (To11_1ik) receptor and its gene
技術分野 Technical field
本発明は、 新規な トル様 （Toll- like) レセブター、 その遺伝子及び医薬組成物 に関する。 よ り詳細には、 本発明は免疫応答に関わる種々の遺伝子の発現を制御 する転写因子 N F— Bに関連する トル （Toll) 関連分子である新規トル様 （To 1卜 like) レセブター （T L R 6 ) に関す細る。 」  The present invention relates to a novel Toll-like receptor, its gene and a pharmaceutical composition. More specifically, the present invention relates to a novel Toll-like receptor (TLR6) which is a Toll-related molecule related to the transcription factor NF-B that controls the expression of various genes involved in the immune response. ) "
背景技術 Background art
感染に対する免疫応答の初期の段階では、 マクロファージなど抗原提示細胞か らの炎症性サイ ト力イ ンの産生などを必要と し、 これによ り、 リ ンパ球による特 異的反応が誘導される。 しかし、 この初期の反応の分子メカニズムに関しては余 り ょ く解っていない。  The early stages of the immune response to infection require the production of inflammatory cytodynamics from antigen-presenting cells such as macrophages, which induces a specific response by lymphocytes. . However, little is known about the molecular mechanism of this early reaction.
ショ ウジヨウバエの トル （Toll) 遺伝子は、 発生段階においては背腹軸の決定、 また成体では細菌、 真菌に対する免疫応答に関与する事が知られている。 トル (Toll) 遺伝子産物は、 I型レセブタ一であり リ ガン ドスパチユ レ （Spaetzle) が結合すると、 成体では最終的に抗細菌、 抗真菌物質の産生を誘導する。 その細 胞内情報伝達経路も詳しく調べられている。 ショ ウジヨ ウバエ ' トル （Toll) は アダプタ一分子であるチューブ （ Tube ) を介しセ リ ンス レオニンキナーゼの一種 であるペレ （Pelle) を活性化し、 その結果、 レル （Rel) ファ ミ リ一の転写因子 と相同性を持つ ドルサル （Dorsal) i カクタス （Cactus) による抑制を離れ、 核に移行し、 転写を誘導する。  The Drosophila Toll gene is known to be involved in the development of the dorsal-ventral axis during development and in the immune response to bacteria and fungi in adults. The Toll gene product is a type I receptor pig and, when combined with the ligand spaetzle, ultimately induces the production of antibacterial and antifungal substances in adults. Its intracellular signaling pathways have also been investigated in detail. Drosophila Toll activates Pelle, a type of serine reonin kinase, through a tube, an adapter molecule, resulting in transcription of the Rel family. Escapes repression by Dorsal i Cactus with homology to the factor, translocates to the nucleus, and induces transcription.
トル （Toll) 遺伝子の細胞内領域は、 広 く植物から脊椎動物に至るまで保存さ れており、 哺乳類では、 I L一 1 レセブターフ ァ ミ リ一の細胞内領域が相同性を 持つことが知られている。 I L一 1 レセブターファ ミ リーからのシグナル伝達機 構はアダプター分子 My D 8 8、 セ リ ンス レオニンキナーゼである I L— 1受容 体会合キナーゼ ( I L - 1 Receptor-associated kinase ( I R A K ) ) を介し、 I c Βによる抑制が解除されるこ とによ り、 レル （Rel) ファ ミ リー転写因子 N F - κ を活性化させる。 これはショ ウジヨ ウバエ ' トル （Toll) 遺伝子のシグナ ル伝達と対比され、 進化的に保存されたものと考えられる （O'Neill LA, et al., J Leukoc Biol . 1998 63(6): 650-657. ) 。 The intracellular region of the Toll gene is widely conserved from plants to vertebrates. In mammals, the intracellular region of the IL-11 receptor family is known to have homology. ing. The signaling mechanism from the IL-11 receptor family is the adapter molecule My D88, and the receptor for serine leonin kinase, IL-1 The repression of IcΒ is released via the body-associated kinase (IL-1 Receptor-associated kinase (IRAK)), which activates the rel (Rel) family transcription factor NF-κ. This is in contrast to the signal transmission of the Drosophila 'Toll' gene and is thought to be evolutionarily conserved (O'Neill LA, et al., J Leukoc Biol. 1998 63 (6): 650 -657.).
最近、 ヒ トにおける トル （Toll) 関連分子が同定された。 1 回膜貫通型の I型 レセプターであ り、 ショ ウジヨ ウバエの トル （Toll) 遗伝子と同じように細胞外 領域にはロイ シンリ ツチ リ ピー ト、 細胞内領域には I L— 1 レセブターフア ミ リ 一と相同性のある領域が存在する。 ヒ トにおける トル （Toll) 関連分子は、 現在 までに 5つのファ ミ リー （ T L R 1 ~ 5 ) がク ロ一ニングされている （Rock FL, et al. , Proc Natl Acad Sci U S A. 1998 95(2) : 588-593. ) 。  Recently, Toll-related molecules in humans have been identified. It is a once-transmembrane type I receptor. Like the Drosophila Toll gene, the extracellular domain is a leucine repeat repeat, and the intracellular domain is an IL-1 receptor phage. There is a region homologous to the plant. Toll-related molecules in humans have so far been cloned into five families (TLR1-5) (Rock FL, et al., Proc Natl Acad Sci US A. 1998 95 (2): 588-593.).
T L R 4 ( h T o l l ) は中でも最も解析が進んでおり、 活性化型の T L R 4 ( h T o 1 1 ) をヒ ト T細胞株であるジャーカツ ト （Jurkat) 細胞に発現させる こ とによ り、 N F— / c Bが活性化され I L一 1、 I L— 6、 I L— 8など炎症性 サイ トカイ ンの産生が誘導される （Medzhitov R, et Al., Mol Cell. 1998 2 (2): 253-258. ； Medzhitov R, et al. , Nature. 1997 388(6640) : 394-397.) 。 単球ではリポポリサッカライ ド （Lipopolysaccharide ( L P S ) ) に反応して、 T L R 4の遺伝子の発現が誘導される （Muzio M， et al . , J Exp Med. 1998 18 7( 12): 2097-2101. ) 。 このため、 この分子が免疫応答初期の段階での炎症性サイ トカイ ンの産生や、 特異的リ ンパ球によるいわゆる適応免疫の誘導に関わってい ることが考えられる。  TLR 4 (hToll) has been the most analyzed among them, and it is possible to express activated TLR4 (hToll) in human T cell line Jurkat cells. NF- / cB is activated to induce the production of inflammatory cytokines such as IL-11, IL-6, and IL-8 (Medzhitov R, et Al., Mol Cell. 1998 2 (2) Medzhitov R, et al., Nature. 1997 388 (6640): 394-397.). In monocytes, the expression of TLR4 gene is induced in response to lipopolysaccharide (LPS) (Muzio M, et al., J Exp Med. 1998 187 (12): 2097- 2101.). Therefore, it is thought that this molecule is involved in the production of inflammatory cytokines at the early stage of the immune response and the induction of so-called adaptive immunity by specific lymphocytes.
他の トル （Toll) ファ ミ リーのいくつかにおいても免疫初期応答や、 発生への 関与が示唆されているが （Chaudhary PM, et al. , Blood. 1998 91( 11 ): 4020-4 027. ； Ruey-Bing Yang, et al., Nature. 1998 395: 284-288. ) 、 生体内の機能 に関しては不明な点が多い。 感染に対する生体防御初期の反応をコ ン 卜 ロールす る分子メカニズムの解析は最近始まったばかりである。  Some other Toll families have also been implicated in early immune response and development (Chaudhary PM, et al., Blood. 1998 91 (11): 4020-4027. Ruey-Bing Yang, et al., Nature. 1998 395: 284-288.) There are many unclear points about functions in living organisms. Analysis of molecular mechanisms that control the initial response of host defense to infection has only recently begun.
また、 トル （ Tol 1 ) 分子はさらに大きなファ ミ リーを形成している可能性が考 えられ、 応答免疫系の機構を解明し、 各種の免疫系疾忠や、 ^症感染症時のェン ド トキシンショ ックなどの治療、 予防法を確立してゆく ためにも トル （Toll) フ ァ ミ リーの全貌の解明が要望されている。 発明の開示 In addition, it is thought that the Tol 1 molecule may form a larger family, elucidate the mechanism of the responding immune system, and investigate various immune system illnesses and the effects of ^ disease infection. To establish toxins and other treatments and preventive measures, Toll There is a demand for elucidation of the whole family. Disclosure of the invention
本発明者らは、 このフ ァ ミ リーに属する新しい分子の同定を試み、 トル （Tol 1) フア ミ リーに属する と考えられる新たな分子種を提供する。  The present inventors have attempted to identify a new molecule belonging to this family, and provide a new molecular species that is considered to belong to the Tol (Tol 1) family.
本発明は、 免疫応答に関わる種々の遺伝子の発現を制御する転写因子 N F - κ Βなどに関連する トルファ ミ リ一の新規な分子種である T L R 6、 及び、 それを コー ドする遺伝子を提供する。  The present invention provides TLR6, a novel molecular species of tolfamily related to a transcription factor NF-κΒ that regulates the expression of various genes involved in an immune response, and a gene encoding the same. I do.
本発明は、 配列表の配列番号 2若しくは 4で表されるアミ ノ酸配列又は当該ァ ミ ノ酸配列中の 1個以上のアミ ノ酸が欠失し、 他のアミ ノ酸で置換され、 及び/ 又は、 1個以上の他のア ミ ノ酸が付加されたア ミ ノ酸配列を有する トル様レセプ ター （ T L R 6 ) に関する。 また、 本発明は、 トル様レセプター （ T L R 6 ) 作 用を有する蛋白質に関する。  The present invention provides an amino acid sequence represented by SEQ ID NO: 2 or 4 in the Sequence Listing or one or more amino acids in the amino acid sequence are deleted and substituted with another amino acid, And / or a toll-like receptor (TLR6) having an amino acid sequence to which one or more other amino acids have been added. The present invention also relates to a protein having a toll-like receptor (TLR6) action.
また、 本発明は、 前記の新規なレセプター又は蛋白質をコー ドする塩基配列を 有する遺伝子に関する。 よ り詳細には、 本発明は、 配列番号 1 又は 3で表される 塩基酸配列を有する遺伝子に関する。 図面の簡単な説明  The present invention also relates to a gene having a nucleotide sequence encoding the novel receptor or protein. More specifically, the present invention relates to a gene having a base acid sequence represented by SEQ ID NO: 1 or 3. BRIEF DESCRIPTION OF THE FIGURES
第 1 図は、 本発明のヒ ト T L R 6の塩基配列及びァミ ノ酸配列 （ 1文字コー ド） を示す。  FIG. 1 shows the nucleotide sequence and amino acid sequence (one letter code) of human TLR6 of the present invention.
第 2図は、 本発明のマウス T L R 6の塩基配列及びア ミ ノ酸配列 （ 1 文字コー ド） を示す。  FIG. 2 shows the nucleotide sequence and amino acid sequence (one-letter code) of mouse TLR6 of the present invention.
第 3図は、 ヒ ト Tollフア ミ リーの細胞内領域のア ミ ノ酸配列の比較を示す。 第 4図は、 マウスの各臓器におけるマウス T L R 6のノザンブロ ヅ ト解析に結 果を示す図面に代わる写真である。 図の下段は /?—ァクチンによる R NA量を示 している。  FIG. 3 shows a comparison of amino acid sequences in the intracellular region of the human Toll family. FIG. 4 is a photograph in place of a drawing showing the result of Northern blot analysis of mouse TLR6 in each mouse organ. The lower part of the figure shows the RNA content due to /?-Actin.
第 5図は、 本発明の T L R 6のデュアルルシフェラ一ゼレポーターアツセィの 結果を示す。 上段は 2 9 3細胞の場合を、 下段は N I H 3 T 3細胞の場合をそれ それ示す。 発明を実施するための最良の形態 FIG. 5 shows the results of the TLR6 dual luciferase reporter assay of the present invention. The upper row shows the case of 293 cells, and the lower row shows the case of NIH 3T3 cells. BEST MODE FOR CARRYING OUT THE INVENTION
まず、 本発明の T L R 6の c D NAクローニングについて説明する。  First, the cDNA cloning of TLR6 of the present invention will be described.
マウス T L R 1の細胞質内領域をプローブと してマウスゲノムライ ブラ リーを スク リ ーニングした。 その結果、 T L R 1以外に T L R 1 と有意な相同性を示す ク ローンが得られた。 この断片をプローブと してヒ ト胎盤 c D NAライ ブラ リー をスク リ一ニングしヒ ト c D N Aの全長を得た。 その塩基配列及びァミ ノ酸配列 ( 1文字コー ド） を第 1図に示す。  The mouse genomic library was screened using the cytoplasmic region of mouse TLR1 as a probe. As a result, a clone showing significant homology with TLR1 other than TLR1 was obtained. Using this fragment as a probe, a human placenta cDNA library was screened to obtain the full length human cDNA. FIG. 1 shows the nucleotide sequence and the amino acid sequence (one-letter code).
得られたヒ ト T L R 6の塩基配列を配列表の配列番号 1 に示す。 そのア ミ ノ酸 配列を配列表の配列番号 2に示す。  The nucleotide sequence of the obtained human TLR6 is shown in SEQ ID NO: 1 in the sequence listing. The amino acid sequence is shown in SEQ ID NO: 2 in the sequence listing.
また、 マウスに関しても全長の塩基配列を決定した。 その塩基配列及びアミ ノ 酸配列 （ 1文字コー ド） を第 2図に示す。  The full-length nucleotide sequence of the mouse was also determined. FIG. 2 shows the nucleotide sequence and the amino acid sequence (single-letter code).
得られたマウス T L R 6の塩基配列を配列表の配列番号 3に示す。 そのア ミ ノ 酸配列を配列表の配列番号 4に示す。  The nucleotide sequence of the obtained mouse TLR6 is shown in SEQ ID NO: 3 in the sequence listing. The amino acid sequence is shown in SEQ ID NO: 4 in the sequence listing.
ヒ ト T L R 6の c D NAは 2 , 3 8 8 b pのオープン リーディ ングフ レーム (open reading frame) を持ち、 予想されるタンパク質は 7 9 6ア ミノ酸よ りな る。 マウス T L R 6の c D NAは 2 , 4 2 l b pのオーブン リーディ ングフ レー ム （open reading frame) を持ち、 8 0 7アミ ノ酸からなると予想された。 ヒ 卜 とマウスの間での相同性は D N Aレベルで 7 7 . 6 %、 ア ミ ノ酸レベルで 7 3. 7 %であった。  The human TLR6 cDNA has an open reading frame of 2,388 bp, and the expected protein consists of 796 amino acid. The mouse TLR6 cDNA was expected to have an oven reading frame of 2,42 lbp and to consist of 807 amino acids. The homology between the human and the mouse was 77.6% at the DNA level and 73.7% at the amino acid level.
T L R 6のア ミ ノ酸配列は I型膜タ ンパクの特徴を持つ。 N末端と中心付近に 疎水性の部位があり、 それぞれシグナルべプチ ド及び胶貫通領域に対応する と思 われる。  The amino acid sequence of TLR6 is characteristic of a type I membrane protein. There are hydrophobic sites near the N-terminus and the center, which are thought to correspond to the signal peptide and the 胶 penetration region, respectively.
ショ ウジヨ ウバエから哺乳類まで T o 1 1フア ミ リーは細胞外領域のロイ シン リ ツチリ ピー ト ドメイ ンと、 細胞内の I L— 1 レセプターと相同性を持つ領域に よ り特徴付けられる。 ヒ ト、 マウス T L R 6も同様に細胞外領域にロイ シン リ ツ チリ ビー ト ドメ イ ンを持ち、 細胞内は I L _ 1 レセプターフア ミ リーや他の T o 1 1ファ ミ リ一と相同性を有する。  From Drosophila to mammals, the To11 family is characterized by an extracellular domain, the leucine litulin repeat domain, and a region homologous to the intracellular IL-1 receptor. Human and mouse TLR6 also have a leucine litulin chili beat domain in the extracellular region, and homology within the cell with the IL_1 receptor family and other To11 families. Having.
第 3図にヒ ト T o 1 1 ファ ミ リーの細胞内領域のア ミ ノ酸配列の比較を示す。 T L Rファ ミ リーの中では、 本発明の T L R 6は T L R 1 と最も高い相同性を有 し、 ヒ ト T L R 1 とヒ ト T L R 6の間ではア ミ ノ酸レベルで 6 9 . 1 %であった c この中でも特に細胞内領域で高い相同性を示した。 FIG. 3 shows a comparison of amino acid sequences in the intracellular region of the human To11 family. Among the TLR families, TLR6 of the present invention has the highest homology to TLR1, with 69.1% amino acid levels between human TLR1 and human TLR6. C. Among them, high homology was shown particularly in the intracellular region.
T L R 6の各組織における発現を調べるために、 マウスの各組織よ り R N Aを 抽出し、 R T— P C Rによる解析を行った。 N末端領域の約 8 0 0 b pの配列が 増幅されるようにプライマ一 （Primer) を設計した。 マウス T L R 6は胸腺、 脾 臓、 卵巣、 肺において発現がみられた。 （第 4図参照）  To examine the expression of TLR6 in each tissue, RNA was extracted from each tissue of the mouse and analyzed by RT-PCR. A primer was designed so that about 800 bp of the sequence in the N-terminal region was amplified. Mouse TLR6 was expressed in thymus, spleen, ovary and lung. (See Fig. 4)
マウス T L R 6の c D NAプローブを用いて F I S H法によ り、 染色体上の位 置を決定した。 これによ りマウス T L R 6は、 5番染色体に存在するこ とが明ら かとなつた。  The position on the chromosome was determined by the FISH method using the cDNA probe of mouse TLR6. This revealed that mouse TLR6 was present on chromosome 5.
ヒ トではこの部位は 4番染色体に相当 し、 T L R 6は現在までに報告されてい る T L R 1、 T L R 2、 T L R 3 と同じ染色体上に存在する と考えられる。  In humans, this site corresponds to chromosome 4, and TLR6 is thought to be on the same chromosome as TLR1, TLR2, and TLR3 that have been reported to date.
次に、 本発明の T L R 6が N F— Bを活性化するか否かを検討した。  Next, it was examined whether or not TLR6 of the present invention activates NF-B.
T L R 6の細胞内領域は I L— 1 レセブター （ I L— 1 R) フ ァ ミ リーと相同 性を持ち、 同様の細胞内シグナル伝達系路を持つ可能性が考えられる。 I L— 1 Rファ ミ リーからのシグナルは N F— Bの活性化を引き起こす。 このため、 T L R 6が同様に N F— Bの活性化に関わるかどうか解析した。  The intracellular region of TLR6 has homology to the IL-1 receptor (IL-1R) family, and may have a similar intracellular signaling pathway. Signals from the IL-1R family cause activation of NF-B. Therefore, it was analyzed whether TLR6 was also involved in the activation of NF-B.
ヒ ト野生型 T L R 6を、 発現べクタ一 p E F— B 0 Sへ挿入した。 このとき C 末端に My cをェビ ト一プと してタグを施した （p E F— B〇 S— My c— T L R 6 ) 。 これを 2 9 3細胞、 および N I H 3 T 3細胞に、 リポフ エクシヨ ン法を 用いてそれそれ 0. 5、 1 . 0、 2. 0〃 gを一過性的に導入し発現させた。 ま た対照と して、 野生型 My D 8 8 ( p E F - B O S - F l a g-M yD 8 8 ) を 2. 0〃 g—過性的に導入した。 この時、 N F— B /レポーターブラス ミ ド ( reporter plasmid) 1 . 0〃 g、 p R L— S V 4 0ベクタ一 (Vector) 0. 1 gも同時に導入した。 導入 4 8時間後細胞を回収し、 ルシフ ェラーゼ （lucife rase) 活性をデュアルルシフェラ一ゼレポ一ターァヅセィ系 （Dua卜 Luciferase Reporter Assay System) ( P r o m e g a社) を用いてルミ ノ メ一ター ( Lummi nometer ) で測定した。  Human wild-type TLR6 was inserted into the expression vector pEF-BOS. At this time, a tag was applied to the C-terminus with My c as an epitope (pE F—B ES—My c—T L R 6). The cells were transiently transfected with 0.5, 1.0, and 2.0 μg of these into 2993 cells and NIH3T3 cells using the Lipofxion method and expressed. As a control, 2.0 μg of wild-type MyD88 (pEF-BOS-Flag-MyD88) was transiently introduced. At this time, 1.0 μg of NF—B / reporter plasmid and 0.1 g of pRL—SV40 Vector were simultaneously introduced. Cells were harvested 8 hours after transfection, and the luciferase activity was determined using a dual luciferase reporter assay system (Promega) using a luminometer (Lumminometer). Was measured.
結果を第 5図に示す。 第 5図の上段は、 2 9 3細胞を川いた場合を示し、 下段 は N I H 3 T 3細胞を用いた場合を示す。 The results are shown in FIG. The upper part of Fig. 5 shows the case where 293 cells were flowed, and the lower part Indicates the case where NIH 3 T 3 cells were used.
第 5図に示すように、 対照と した M y D 8 8では 2 9 3細胞で約 3倍、 N I H 3 T 3細胞で約 2倍のルシフ ェラーゼ （ luciferase) 活性の増加が認められた。 それに対し、 T L R 6を発現させた群ではどち らの細胞種においても活性の増加 は認めなかった。  As shown in FIG. 5, the control of MyD888 showed about a 3-fold increase in luciferase activity in 2993 cells and a about 2-fold increase in luciferase activity in NIH3T3 cells. On the other hand, in the group in which TLR6 was expressed, no increase in activity was observed in any of the cell types.
この結果よ り、 レセブタ一であるヒ ト T L R 6を単独に発現させるこ とのみで は N F— Bを活性化することはできないと考えられた。  From these results, it was considered that NF-B could not be activated only by expressing human TLR6 alone, which was the only receptor pig.
このように、 本発明のヒ ト及びマウス T L R 6は、 N末端側にロイ シンリ ッチ リ ピー ト ドメイ ンを持ち、 膜貫通部分に続いて C末端側は I L一 1 Rファ ミ リー の細胞内 ドメイ ンと相同性を有した。 T o l l関連分子である T L Rフア ミ リー の中では T L R 1 と最も高い相同性を示した。  Thus, the human and mouse TLR6 of the present invention have a leucine-rich repeat domain at the N-terminal side, and a cell of the IL-11R family at the C-terminal side following the transmembrane portion. It had homology with the inner domain. It showed the highest homology with TLR1 among TLR-related molecules, TLR families.
本発明の T L R 6は R T— P C Rによる発現の解析にて、 胸腺、 脾臓といった 免疫担当組織での発現が確認され、 生体防御におけるこの分子の役割が示唆され る。  Analysis of expression of TLR6 of the present invention by RT-PCR reveals expression in immunocompetent tissues such as thymus and spleen, suggesting a role for this molecule in host defense.
T L R 6はその細胞内領域は I L _ 1 Rの細胞内 ドメイ ンと相同性を持つこと よ り N F— / c Bにシグナルを伝えるこ とが考えられたが、 本発明の T L R 6の全 長を 2 9 3細胞、 および N I H 3 T 3細胞に発現させることのみでは、 N F— Bを活性化させるこ とは出来なかった。 この原因と しては、 （ 1 ) レセプターの みの発現では細胞内にシグナルが伝達されない、 （ 2 ) N F— Bではな く、 他 の経路を介してシグナル伝達を行っている、 （ 3 ) 遺伝子導入する細胞の種類に よって応答性が異なるこ となどが考えられる。 今後は、 活性型 T L R 6を作製し、 そのシグナル伝達機構を解析することが求められる。  TLR6 was thought to transmit a signal to NF- / cB due to its intracellular domain being homologous to the intracellular domain of IL_1R. NF-B could not be activated simply by expressing 293- and NIH3T3 cells. This is because (1) the signal is not transmitted into the cell by the expression of the receptor alone, (2) the signal is transmitted through another pathway, not NF-B, (3) It is conceivable that the response differs depending on the type of cell into which the gene is introduced. In the future, it is necessary to produce active TLR6 and analyze its signaling mechanism.
T o l l遺伝子産物は感染初期の非特異的免疫応答、 リ ンパ球による適応免疫 への情報伝達に関与している事が考えられる。 感染に対する生体反応は非常に重 要であるが、 重症感染症においては、 その反応が強すぎると例えばエン ド トキシ ンショ ックのように致死的な状況が引き起こされる。 この生体反応のコ ン ト ロー ルは臨床的にも非常に有益である と考えられる。 T o 1 1フ ァ ミ リ ーの機能の詳 細な解析は、 炎症初期反応の分子メ力二ズムの解明に役立つことが期待される。 実施例 It is thought that the Toll gene product is involved in nonspecific immune responses in the early stage of infection and signaling to adaptive immunity by lymphocytes. The biological response to infection is very important, but in severe infections, too strong a response can cause fatal situations, such as endotoxin. This control of biological response is considered to be very useful clinically. A detailed analysis of the function of the To11 family is expected to help elucidate the molecular mechanism of the initial response to inflammation. Example
次に実施例によ り本発明をよ り具体的に説明するが、 本発明はこれらの実施例. に限定されるものではない。 実施例 1 (マウ ス T L R 6の c D NAのク ロ一ニング）  Next, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples. Example 1 (cloning of cDNA of mouse TLR6)
ヒ ト T L R 1 の塩基配列を用いて、 E S Tデータベースをホモロ ジ一検索した ところ、 ヒ ト T L R 1の塩基配列と相同性の高いマウス由来の塩基配列 （AA 1 7 7 5 4 9 ) が発見された。  A homologous search of the EST database using the human TLR1 nucleotide sequence revealed a mouse-derived nucleotide sequence (AA177549) highly homologous to the human TLR1 nucleotide sequence. Was.
E S T (A A 1 7 7 5 4 9 ) の塩基配列情報を基に合成オリゴヌ クレオチ ド、 5' -ttgcctccatgagaggaa-3'と、  Based on the base sequence information of EST (AA177549), a synthetic oligonucleotide, 5'-ttgcctccatgagaggaa-3 ',
5' -ggatgctcttaggtttgc-3' 5 '-ggatgctcttaggtttgc-3'
を用いマウス脳の c D N Aライブラ リ一を材料に して P C R (Polimerase chain reaction) を用いて E S T ( A A 1 7 7 5 4 9 ) の配列を增幅した。 P C Rは T a qポリメラーゼ （宝酒造社） を用い、 9 4 °C 3 0秒、 5 6 °C 3 0秒、 7 2 °C 3 0秒を 3 0サイ クル 7 2 °C 1 0分を 1サイ クル行った。 この P C R産物の一部を ァガロースゲル中で電気泳動し、 ェチジゥムブロマイ ド （日本ジーン社） にて染 色後、 紫外線照射下で、 約 4 0 0 b pの c D N Aが増幅されていることを確認し た。 このバン ドをゲルから切り出 し、 ウイザー ド （W i z a r d ) ( P r o m e g a社） で精製後、 TAクロ一ニングキヅ ト （N o v a g e n社） を用いクロ一 ニングした。  Using the DNA library of the mouse brain as a material, the sequence of EST (AA177549) was amplified using PCR (Polymerase chain reaction). PCR was performed using Taq polymerase (Takara Shuzo), 30 cycles of 94 ° C for 30 seconds, 56 ° C for 30 seconds, and 72 ° C for 30 seconds, and one cycle of 72 ° C for 10 minutes. Kuru went. A portion of this PCR product was electrophoresed in agarose gel, stained with ethidium bromide (Nippon Gene), and amplified with approximately 400 bp of cDNA under UV irradiation. It was confirmed. This band was excised from the gel, purified with a wizard (Wizard) (Promega), and then cloned using a TA cleaning kit (Novagen).
すなわち、 ベクターと して p T 7 b l u e (N o v a g e n社） Tベクタ一を 用い、 ベクターと先の D NAをそのモル比が 1 ： 3 となるように混合し、 ライゲ ーシヨ ンキッ ト （宝酒造社） にてベクタ一に D N Aを組み込んだ。 D NAを組み 込んだ Tベクターを大腸菌 D H 5 ひに遺伝子導入し、 アンビシリ ン （シグマ社） を 5 0〃 g/m l , X— g a l (ナカライテスク社） 2 0 0〃 g/m lを含む L 一ブロース （ L一 B r o t h ) 半固形培地のプレー トに I涛き、 1 2時間程度 3 7 。(：に放置し、 現れてきた ΰいコロニーを無作為選択し、 冋濃度のアンビシリ ンを 含む L一ブロース液体培地 2 m lに植え付け、 8時問程度 3 7 °Cで振盪培養し、 菌体を回収し、 ウイザー ド ミ ニプレ ップ （ P r o m e g a社） を用いてプラス ミ ドを分離し、 このプラス ミ ドを制限酵素 B a m H I と S a 1 Iにて消化して、 約 4 0 0 b pのバン ドが切り出されて来るこ とで P C R産物が組み込まれているこ. とを確認し、 確認されたクローンについて組み込まれている c D N Aの塩基配列 決定を行つた。 That is, pT7blue (Novagen) T vector was used as a vector, and the vector and the above DNA were mixed at a molar ratio of 1: 3. DNA was integrated into the vector at. The T vector incorporating DNA was transfected into E. coli DH5, and L containing 100 μg / ml of Ambicillin (Sigma) and 200 μg / ml of X-gal (Nacalai Tesque) One broth (L-Broth) I sprayed on a plate of semi-solid medium, about 12 hours 37. (: Left), randomly select the emergent blue colonies, inoculate them in 2 ml of L-broth liquid medium containing low-concentration ambisilin, and culture with shaking at 37 ° C for about 8 hours. And collected using Wizard Mini Prep (Promega). This plasmid is digested with the restriction enzymes BamHI and Sa1I, and a band of about 400 bp is cut out to confirm that the PCR product has been incorporated. Was confirmed, and the nucleotide sequence of the cDNA incorporated in the confirmed clone was determined.
揷入 c D N A断片の塩基配列の決定は、 アプライ ドバイォシステムズ （Applie d Biosystems) 社製の蛍光シークェンサ一 3 7 7を用いて実施した。 シークェン スサンブルの調製は P R I S M, レディ リアクショ ンダイターミネータ一法サイ クノレシークェンシングキッ 卜 (Ready Reaction Dye Terminator Cycle Sequenci ng Kit) (アブライ ドバイオシステムズ社） を用いて行った。 マイ クロチューブ に 1 0〃 1の反応ス ト ック液 2. 0〃 1の 1 . 6 p m o l /〃 lの T 7プロモ一 ターブライ マー及び 8. 0〃 1の 0. l〃 g/〃 lのシークェンス用錶型 D NA を加えて混合し、 9 6 °C 1 0秒、 5 0 °C 5秒、 6 ◦ °C 4分をサイ クルとする P C R反応を 2 5サイ クル行つた。 反応後、 2〃 1の 3 M酢酸ナ ト リ ウム （ p H 5. 2 ) および 5 0〃 1のエタノールを加え攪拌後室温で 1 5分放置し、 1 4 0 0 0 r p mにて 1 5分の遠心を行い沈殿を回収した。 沈殿を 7 0 %エタノールで洗浄 後、 真空下に 2分間静置して乾燥させ、 シークェンス用サンプルと した。 シーク エンスサンプルは 6〃 1の E D T Aを含むホルムア ミ ドに溶解して 9 0 °C 2分変 性後、 氷中で冷却してシークェンスに供した。  The nucleotide sequence of the imported cDNA fragment was determined using a fluorescent sequencer 377 manufactured by Applied Biosystems. The sequencing sam- ble was prepared using PRIMSM, a Ready Reaction Dye Terminator Cycle Sequencing Kit (Available Biosystems). In a microtube, add 10〃1 of the reaction stock solution, 2.0〃1 of 1.6 pmol / 〃l of T7 Promoter Primer and 8.0〃1 of 0.1 l〃g / 〃l. Was added and mixed, and 25 cycles of PCR reaction were performed at 96 ° C for 10 seconds, 50 ° C for 5 seconds, and 6 ° C for 4 minutes. After the reaction, 2〃1 of 3 M sodium acetate (pH 5.2) and 50〃1 of ethanol were added, and the mixture was stirred and left at room temperature for 15 minutes. The precipitate was collected by centrifugation for 1 minute. After the precipitate was washed with 70% ethanol, it was left standing under vacuum for 2 minutes and dried to obtain a sample for sequence. The sequence samples were dissolved in formamide containing 61〃EDTA, changed at 90 ° C for 2 minutes, cooled in ice, and subjected to a sequence.
6個のクローンについて塩基配列決定を行ったところ、 6個のク ローンが E S T ( A A 1 7 7 5 4 9 ) に対応する塩基配列を有していた。  When the nucleotide sequence of six clones was determined, the six clones had a nucleotide sequence corresponding to EST (AA177549).
次に上記クローンをプローブと して、 マウス 1 2 9 s Vゲノムライブラ リ一 ( S t r a t a g e n e ) にてこの配列を含むマウスゲノ ムクローンの検索を行 つた。 1 06個相当のプラークをプレー ト し、 出現したプラークをナイ ロンフィル ター ( c o l o n y/p l a q u e s c r e e n , N E N社） に転写し、 転写 したナイ ロ ンフ ィルターをアルカ リ処理 （ 1 . 5 M N a C 1 , 0 . 5 M N a 0 Hを染み込ませた濾紙上に 5分間放置） し、 次いで中和処理 （ 1 . 5 M N a C 1 , 0. 5 M T r i s - H C l H 7. 5を染み込ませた濾紙上に 5分間 放置） を 2回行い次に 2 X S S C中で 5分問洗、净し風乾した。 このフィルターを 用いて放射性同位元素32 Pにて標識された E S T ( A A 1 7 7 5 4 9 ) 断片をブ ローブに してハイ ブリダィゼ一シヨ ンを行った。 Next, using the above-mentioned clone as a probe, a mouse genomic clone containing this sequence was searched in the mouse 129 s V genome library (Stratagene). 1 0 6 corresponding plaques were plates, emerging plaques Nai Ronfiru terpolymer (colony / plaquescreen, NEN Co.) was transferred to, alkali processes Nai b Nfu Iruta has been transferred (1. 5 MN a C 1 , 0.5 minutes on a filter paper impregnated with 0.5 MNaOH, followed by neutralization treatment (1.5 MNaCl, 0.5 MTris-HClH7.5 filter paper impregnated with it). (Left for 5 minutes) twice, then washed in 2 XSSC for 5 minutes, and then air-dried. Using this filter, EST (AA177549) fragments labeled with 32 P radioisotope were blocked. Hybridization was performed as a robe.
放射性同位元素32 Pにて標識された E S T ( A A 1 7 7 5 4 9 ) 断片プローブ は以下のよ う に して作製した。 すなわち、 E S T ( A A 1 7 7 5 4 9 ) の断片の 組み込まれた Tベクターよ り、 制限酵素 B am H I と S a l Iにてベクターよ り 切り出し、 ァガロースゲル中で？1気泳動し、 ェチジゥムブ口マイ ド （日本ジーン 社） にて染色後、 紫外線照射下で、 約 4 0 0 b pのバン ドをゲルから切り出し、 ウイザー ド （P r o m e g a社） で精製した。 得られた、 断片を D NAラベリ ン グキッ ト （Megaprime DNA labeling kit： A m e r s y a m社） を用いて標識し た。 すなわち、 D NA 5 0 n gにブライマ一液 5〃 1及び脱イオン水を加えて 3 3〃 1 と し 9 6 °C熱変性を 5分行い、 その後 5 X緩衝液 1 0〃 1、 （ ひ — 32 P ) d C T P 5 1 及びク レノ ウ （ K l e n o w) 酵素 2〃 1を加えて 3 7 °C 1 0 分間水浴し、 放射性標識した E S T (A A 1 7 7 5 4 9 ) の断片を合成した。 更 にその後、 セフ アデヅクスカラムで (ProbeQuant G-50Micro Columns： ファ レマ シァ社） で精製し、 5分間 9 6 °C煮沸した後氷冷した。 An EST (AA177549) fragment probe labeled with 32 P radioisotope was prepared as follows. That is, the EST (AA1774949) fragment was excised from the vector with the restriction enzymes BamHI and SalI and incorporated into an agarose gel. After electrophoresis, the band was stained with ethidium Mubuchi Mide (Nippon Gene Co., Ltd.), and a band of about 400 bp was cut out from the gel under ultraviolet irradiation and purified with a wizard (Promega). The obtained fragment was labeled using a DNA labeling kit (Megaprime DNA labeling kit: Amersyam). That is, 50 ng of DNA was added with 5〃1 of the primer solution and deionized water to obtain 33〃1, heat-denatured at 96 ° C for 5 minutes, and then 5 5 buffer solution was added to 10〃1 、 — 32 P) d CTP51 and Klenow enzyme (2〃1) are added and the mixture is water-bathed at 37 ° C for 10 minutes to synthesize radiolabeled EST (AA177549) fragments. did. Thereafter, purification was carried out using a Sephadex column (ProbeQuant G-50Micro Columns: Pharmacia), and the mixture was boiled at 96 ° C for 5 minutes and cooled with ice.
前述の方法にて作成したフィルターを、 各々の成分の最終濃度が 6倍濃度の S S C溶液 5倍濃度のデンハル ト液、 1 % S D S、 及び 1 0 0〃 g/m 1の煮沸水 浴によ り変性したサケ精子 D N A ( S i g m a社） を含むハイ ブリ ダィゼ一ショ ン液中に浸し、 6 5 °Cにて 1時間振盪した後、 前述の方法で標識されたプローブ をハイ ブリ ダィゼ一シヨ ン液中に添加し、 6 5 °Cにて 1 8時間振盪し、 ハイ プリ ダイゼーショ ンを行った。  The filter prepared by the above method was applied to a SSC solution with a final concentration of 6 times for each component, a Denhardt's solution with a 5 times concentration, 1% SDS, and a boiling water bath with a concentration of 100 g / m1. After immersion in a hybridization solution containing denatured salmon sperm DNA (Sigma) and shaking at 65 ° C for 1 hour, the probe labeled by the method described above was hybridized. The mixture was shaken at 65 ° C. for 18 hours to perform hybridization.
次にフ ィ ルターを 0. 1 % S D Sを含む各々の最終濃度が 2倍濃度の S S C溶 液に浸し、 6 5 °C 1回洗浄後、 さ らに 0. 1 % S D Sを含む各々の最終濃度が 0. 2倍濃度の S S C溶液に浸し、 6 5 °C 1回洗浄した。 洗浄の終了 したフ ィ ルター を増感スク リーンを使用 して、 一 8 0 °Cでオー トラジオグラフィーをおこなった。 その結果、 強く露光された部分のクローンを拾い、 再度プラークを蒔き直し前述 の方法にて 2回スク リ一ニングを行い、 完全に単独のク ローンを 2 クローン分離 した。  Next, the filter is immersed in SSC solution containing 0.1% SDS at a final concentration of 2 times, washed once at 65 ° C, and then added to the final concentration of 0.1% SDS. It was immersed in a 0.2 times concentration SSC solution and washed once at 65 ° C. The washed filter was subjected to autoradiography at 180 ° C using a sensitizing screen. As a result, the strongly exposed clones were picked up, plaques were replated again, and screened twice by the above-mentioned method to completely separate two clones from a single clone.
「モレキュラークローニング実験室マニュアル （Molecular Cloning A labora tory manua丄 j 」 , ( 1989，Cold Spring Harbor Laboratory Press) の 法に従！^ これらのク ローンのファージを約 1 09p f u (plaque forming unit) 調整し、 ウイザ一 ドラムダブレ ツブス （wizard lambda preps) ( P r o m e g a社） を用 いてフ ァージ D NAを精製した。 According to the method of "Molecular Cloning A labora tory manua 丄 j", (1989, Cold Spring Harbor Laboratory Press)! ^ Phage of these clones about 1 0 9 pfu (plaque forming unit ) adjusted to purify the use Itefu Aji D NA the Uiza one Doramudabure crush (wizard lambda preps) (P romega Inc.).
このフ ァージ D N Aを制限酵素 B a mH I単独も しく は B a mH I と S a 1 I で消化し、 同様に B a m H I単独も し くは B a m H I と S a 1 Iで消化したブラ ス ミ ド p B l u e s c r i p t K S ( + ) に組み込んだ。 これらのク ロ一ンを シークェンサ一によ り解析したところ、 1 クローンよ り E S T ( A A 1 7 7 5 4 9 ) と完全に一致する塩基配列が得られたが、 もう 1 クローンからは E S T ( A A 1 7 7 5 4 9 ) と 8 2 %の相同性を持つ D N A配列が得られた。  This phage DNA was digested with the restriction enzymes BamHI alone or with BamHI and Sa1I, and similarly with BamHI alone or with BamHI and Sa1I. It has been incorporated into the Smid p Bluscript KS (+). When these clones were analyzed by a sequencer, a nucleotide sequence completely identical to EST (AA177549) was obtained from one clone, but EST ( A DNA having a homology of 82% with AA177549) was obtained.
この E S T ( A A 1 7 7 5 4 9 ) と 8 2 %の相同性を持つク ローンの塩基配列 をその前後にわたって解析すると、 第 2図と同じ塩基配列を得た。 この塩基配列 を基に合成オリ ゴヌ ク レオチ ド、  When the base sequence of the clone having 82% homology with this EST (AA177549) was analyzed before and after that, the same base sequence as in FIG. 2 was obtained. Based on this base sequence, a synthetic oligonucleotide,
5， -ctgtgaagaatggtaaagtccctctgg- J' と、 5, -ctgtgaagaatggtaaagtccctctgg- J '
5' -ccactcactctggatgaagt-3' 5 '-ccactcactctggatgaagt-3'
を用いマウス胎児 1 7日の c D NAライブラ リ一を材料に して P C R (Polimera se chain reaction) を用いてこの配列を増幅した。 P C Rは、 9 4°C 3 0秒、 5 6 °C 3 0秒、 7 2 °C 3 0秒を 3 0サイ クル 7 2 °C 1 0分を 1サイ クル行った。 P C R産物をァガロースゲル電気泳動し、 染色した結果、 約 2 k b pのバン ドが確 認された。 このバン ドをゲルよ り切り 出し、 前述のように T Aク ロ一ニングを行 い、 その塩基配列をシークェンサ一を用いて決定したところ第 2図のマウス T L R 6に相当する塩基配列を得た。 This sequence was amplified using PCR (Polymerase chain reaction) using the cDNA library of the 17th mouse embryo as a material. The PCR was performed for 30 cycles at 94 ° C. for 30 seconds, 56 ° C. for 30 seconds, 72 ° C. for 30 seconds, and one cycle for 72 ° C. for 10 minutes. The PCR product was subjected to agarose gel electrophoresis and stained. As a result, a band of about 2 kbp was confirmed. This band was cut out of the gel, subjected to TA cloning as described above, and its nucleotide sequence was determined using a sequencer.The nucleotide sequence corresponding to mouse TLR6 in Fig. 2 was obtained. .
その塩基配列及びアミ ノ酸配列 （ 1文字コー ド） を第 2図に示す。 得られたマ ウス T L R 6の塩基配列を配列表の配列番号 3に示す。 そのアミ ノ酸配列を配列 表の配列番号 4に示す。 実施例 2 (ヒ ト T L R 6の c D NAのク ロ一ニング）  FIG. 2 shows the nucleotide sequence and the amino acid sequence (one-letter code). The nucleotide sequence of the obtained mouse TLR6 is shown in SEQ ID NO: 3 in the sequence listing. The amino acid sequence is shown in SEQ ID NO: 4 in the sequence listing. Example 2 (cloning of cDNA of human TLR6)
実施例 1で得られた塩基配列よ り ヒ ト T L R 6をス ク リ一二ングするためのブ ローブを作製した。 具体的にはマウス T L R 6の塩基配列情 ¾を^に合成ォ リゴ ヌ ク レオチ ド、 5' -cctcgagctgagatagagagcatcttg-3' と、 A probe for screening human TLR6 was prepared from the nucleotide sequence obtained in Example 1. Specifically, the nucleotide sequence information of mouse TLR6 is changed to ^ to synthesize oligonucleotides, 5 '-cctcgagctgagatagagagcatcttg-3' and
5， -ggtcgacaatagtcggaagcatgaccc-3' 5, -ggtcgacaatagtcggaagcatgaccc-3 '
を作製し、 マウス胎児 1 7 日の c D NAライ ブラ リ一を材料に して P C Rにてマ ウス T L R 6の一部の塩基配列を増幅した。 この P C R産物を同様に TAクロー ニングし、 塩基配列をシークェンスによ り決定し、 マウス T L R 6の一部の塩基 配列の一部が増幅されていることを確認した。 この断片をプローブと してヒ ト胎 盤 c D NAライ ブラ リ一 （ C L O N T E C H社） にて、 この配列を含むヒ ト c D N Aクローンの検索を行った。 前述のようにナイ ロ ンフ ィルタ一を作製し、 放射 性同位元素32 Pにて標識されたマウス T L R 6断片のプローブの作製を行った。 この際、 制限酵素と して X h o l並びに S a l Iを使用 した。 Using the cDNA library of the 17th mouse embryo, a partial base sequence of mouse TLR6 was amplified by PCR. This PCR product was similarly TA-cloned, and the nucleotide sequence was determined by sequencing. It was confirmed that a part of the nucleotide sequence of mouse TLR6 was partially amplified. Using this fragment as a probe, a human cDNA clone containing this sequence was searched in the human placenta cDNA library (CLONTECH). A nylon filter was prepared as described above, and a probe of a mouse TLR6 fragment labeled with a radioactive isotope 32 P was prepared. At this time, Xhol and SalI were used as restriction enzymes.
前述の方法にて作成したフィ ルターを、 各々の成分の最終濃度が 6倍濃度の S S C溶液 5倍濃度のデンハル ト液、 1 % S D S、 及び 1 0 ◦〃 g/m 1の煮沸水浴 によ り変性したサケ精子 D N A ( S i gm a社） を含むハイ ブリダィゼ一シヨン 液中に浸し、 6 0 °Cにて 1時間振盪した後、 前述の方法で標識されたプローブを ハイブリダィゼーシヨ ン液中に添加し、 6 0 °Cにて 2 0時間振盪し、 ハイブリダ ィゼーショ ンを行った。 The filter prepared by the method described above was applied to a SSC solution with a final concentration of 6 times the concentration of each component, a Denhardt's solution with a concentration of 5 times, 1% SDS, and a boiling water bath of 10 ° g / m1. After immersion in a hybridization solution containing denatured salmon sperm DNA (Sigma) and shaking at 60 ° C for 1 hour, the probe labeled by the above-mentioned method was hybridized. The solution was shaken at 60 ° C. for 20 hours to perform hybridization.
次にフィ ルターを 0. 1 % S D Sを含む各々の最終濃度が 2倍濃度の S S C溶 液に浸し、 6 0 °C 1回洗浄後、 さ らに 0. 1 % S D Sを含む各々の最終濃度が 0. 2倍濃度の S S C溶液に浸し、 6 0 °C 1回洗浄した。 洗浄の終了 したフィルター を增感スク リーンを使用 して、 一 8 0。Cでオー ト ラジオグラフィ一をおこなった。 その結果、 強く露光された部分のクローンを拾い、 再度プラークを蒔き直し前述 の方法にて 2回スク リーニングを行い、 完全に単独のク ローンを 6クローン分離 した。  Next, the filter is immersed in SSC solution at a final concentration of 2% each containing 0.1% SDS, washed once at 60 ° C, and then further washed with 0.1% SDS. Was immersed in a 0.2-fold concentration of SSC solution and washed once at 60 ° C. After washing, use a sensitive screen to remove the filter. Autoradiography was performed at C. As a result, the strongly exposed clones were picked up, plaques were replated again, and screened twice by the method described above, and 6 clones of a single clone were completely separated.
ファージ D N Aを精製、 制限酵素 N o t Iで消化し、 同様に N o t Iで消化し た p B l u e s c r i p t K S ( + ) に組み込んだ。 これらのク ローンの D N A配列をシークェンサ一によ り解析し、 第 1図にある D N A配列を決定した。 以上のように してヒ ト T L R 6の全長を決定した。  The phage DNA was purified, digested with the restriction enzyme NotI, and incorporated into pBluescriptKs (+) similarly digested with NotI. The DNA sequences of these clones were analyzed by a sequencer, and the DNA sequences shown in FIG. 1 were determined. As described above, the total length of human TLR6 was determined.
その塩基配列及びアミ ノ酸配列 （ 1文字コー ド） を第 1図に示す。 得られたヒ ト T L R 6の塩基配列を配列表の配列番号 1に示す。 そのア ミ ノ酸配列を配列表 の配列番^ 2に示す。 実施例 3 (マウス T L R 6の各組織における発現） Fig. 1 shows the nucleotide sequence and amino acid sequence (single-letter code). The nucleotide sequence of the obtained human TLR6 is shown in SEQ ID NO: 1 in the sequence listing. The amino acid sequence is shown in the Sequence Listing. This is shown in array number ^ 2. Example 3 (Expression of mouse TLR 6 in each tissue)
T L R 6の各組織における発現を調べるために、 マウスの各組織よ り mR NA を抽出し、 R T— P C Rによる解析を行った。 具体的には 4週齡のマウス （ c 5 7 B L/ 6 ) を屠殺し、 胸腺、 脾臓、 肝臓、 卵巣、 腎臓、 心臓、 肺を採取した。 これにそれぞれ l m lの ト リ ゾール （ T r i z o l ) ( G i b c o社） を加え、 ホモジナイザーでホモジナイズした。 これに、 2 0 0 1のクロ口ホルム （ナカ ライテスク社） を加え 1 2 0 0 0 r p m l 5分遠心後その上清にイ ソプロパノ一 ル （ナカライテスク社） を加え転倒混和後 1 0 0 0 0 r p m 5分間遠心し、 その 沈殿を 7 0 %エタ ノール （ナカライテスク社） にて洗浄した。 その後沈殿を蒸留 水で溶解した。  To examine the expression of TLR6 in each tissue, mRNA was extracted from each mouse tissue and analyzed by RT-PCR. Specifically, a 4-week-old mouse (c57BL / 6) was sacrificed, and the thymus, spleen, liver, ovary, kidney, heart, and lung were collected. To this, lml of Trizol (Trizol) (Gibco) was added, and homogenized with a homogenizer. To this, add 200 ml of Cloguchi Holm (Nacalai Tesque), centrifuge at 1200 rpm for 5 minutes, add isopropanol (Nacalai Tesque) to the supernatant, mix by inversion, and then add 100 ml. After centrifugation at 0 rpm for 5 minutes, the precipitate was washed with 70% ethanol (Nacalai Tesque). Thereafter, the precipitate was dissolved with distilled water.
この、 mR NAのうち l〃 gを取り出し、 D N a s e l (G I B C O) 1〃 1、 緩衝液 1 1を加え、 室温で 1 5分反応を行い、 その後、 スーパ一スク リ プト II (SUPERSCRIPTII) ( G I B C O社） 逆転写酵素キッ トを用いてファース トス トラ ン ド （ 1 s t s t r a n d ) c D N A合成を行つた。 具体的には D N a s e I 処理後の m RNAにランダムへキサマー l z lを加え 7 0 °C 1 0分保持し、 これ にキッ ト添え付けの緩衝液、 M g C l 2、 1 0 mMd N T P、 0. 1 MD T T、 逆 転写酵素を加え 2 5 °C 1 0分、 4 2 °C 5 0分、 7 0 °C 1 5分の反応を行った。 こ の反応後のフ ァース トス トラン ド （ 1 s t s t r a n d ) c D NAを錶型にし て P C Rを行った。 マウス T L R 6の N末端領域の約 8 0 0 b pの配列が増幅さ れるようにプライマ一を設計した。 合成オ リ ゴヌク レオチ ド、 From this mRNA, l〃 g was taken out, DN asel (GIBCO) 1〃1 and buffer solution 11 were added, and the mixture was reacted at room temperature for 15 minutes.After that, Superscript II (SUPERSCRIPTII) (GIBCO First-strand cDNA synthesis was performed using a reverse transcriptase kit. Specifically random to the added holding 7 0 ° C 1 0 minute hexamers lzl to m RNA after DN ase I treatment, to which kit accompanied with the buffer, M g C l 2, 1 0 mMd NTP, 0.1 MDTT and reverse transcriptase were added, and the reaction was performed at 25 ° C for 10 minutes, at 42 ° C for 50 minutes, and at 70 ° C for 15 minutes. After this reaction, PCR was performed using the first strand (1 ststrand) cDNA as type II. The primer was designed to amplify a sequence of about 800 bp in the N-terminal region of mouse TLR6. Synthetic oligonucleotides,
5' -cttaatagtcggaagcatgaccccg-3'と、 5'-cttaatagtcggaagcatgaccccg-3 ',
5' -aaggttggacctctggtgagttctg-3" 5 '-aaggttggacctctggtgagttctg-3 "
を合成しブライマーと した。 対照と してどの組織でも冋じょうに発現していると 考えられる /?ーァクチンを P C Rに使用した。 5—ァクチンを増幅するプライマ 一は、 Was synthesized into a primer. As a control, / actin, which is considered to be highly expressed in any tissue, was used for PCR. Primers that amplify 5-actin
5' -ctatgtgggtgacgaggcccagag-3'とゝ  5 '-ctatgtgggtgacgaggcccagag-3' and ゝ
5' -gggtacatggtggtaccaccagac-3' を用いた。 P C Rは T a qポ リ メ ラーゼ （宝酒造社） を用い、 9 4 3 0秒、 6 0 °C 3 0秒、 7 4 °C 6 0秒を 2 5サイ クル 7 4 °C Γ◦分を 1サイ クル行った。 P C R産物を 1 . 5 %ァガロースゲル電気泳動し、 染色によ り、 増幅量を見た。 マ ウス T L R 6は胸腺、 脾臓、 卵巣、 肺において発現がみられた。 5 '-gggtacatggtggtaccaccagac-3' Was used. PCR was performed using Taq polymerase (Takara Shuzo Co., Ltd.), and was carried out for 25 cycles at 94 ° C, 60 ° C for 30 seconds, and 74 ° C for 60 seconds. Cycled. The PCR product was subjected to 1.5% agarose gel electrophoresis, and the amount of amplification was determined by staining. Mouse TLR6 was expressed in thymus, spleen, ovary and lung.
その結果を第 4図に示す。 実施例 4 (マウス T L R 6の染色体上の位置）  Fig. 4 shows the results. Example 4 (Mouse TLR6 chromosomal location)
マウス T L R 6の c D NAプローブを用いて F I S H法によ り、 染色体上の位 置を決定した。 これによ りマウス T L R 6は 5番染色体に存在することが明らか となった。 実施例 5 (ヒ ト T L R 6発現べクタ一の構築）  The position on the chromosome was determined by the FISH method using the cDNA probe of mouse TLR6. This revealed that mouse TLR6 was present on chromosome 5. Example 5 (Construction of human TLR6 expression vector)
C末端に My cをェビ ト一ブと してタグを施したヒ ト野生型 T L R 6を P C R を用いて作製した。 合成オリ ゴヌ ク レオチ ド、  Human wild-type TLR6 was prepared by using PCR with Myc as a shredding tag at the C-terminus. Synthetic oligonucleotides,
5' -gtcgaccaccatgaccaaagacaaagaacc- J'と、 5'-gtcgaccaccatgaccaaagacaaagaacc-J '
5， -gtcgactatctcaacaagtcctctctgaaattaacttttgctcagatttcacatcattgttttc-3' を用い、 ヒ ト T L R 6 c D NAを鍩型に P C Rを行いその産物を T Aクロク ロ一 ニングした （M y c— T L R 6 ) 。  5, -gtcgactatctcaacaagtcctctctgaaattaacttttgctcagatttcacatcattgttttc-3 'was used to perform PCR on human TLR6c DNA in type II, and the product was subjected to TA cloning (Myc-TLR6).
C末端に My cをェピ トーブと してタグを施したヒ ト野生型 T L R 6を S a l Iで消化し、 同様に S a l Iで消化した発現べクタ一 p E F _ B O Sへ挿入し、 発現べクタ一 p E F— B O S— M y c— T L R 6を構築した。 実施例 6 (T L R 6の N F— B活性化の有無の解析）  Human wild-type TLR6, tagged with Myc at the C-terminus as an epitope, was digested with SalI, and similarly inserted into the expression vector pEF_BOS digested with SalI. An expression vector pEF-BOS-Myc-TLR6 was constructed. Example 6 (Analysis of presence or absence of NF-B activation of TLR6)
実施例 5で構築した発現べクタ一 p E F— B O S— M y c— T L R 6を 2 9 3 細胞、 及び、 N I H 3 T 3細胞に、 リポフエクシヨ ン法を用いてそれそれ◦ . 5、 1 . 0、 2. 0 g—過性に導入し発現させた。  The expression vector pEF-BOS-Myc-TLR6 constructed in Example 5 was applied to 2933 cells and NIH3T3 cells using the Lipofection method, respectively. , 2.0 g—transiently expressed.
また、 対照と して、 野生型 My D 8 8 ( P E F - B O S - F l a g -M y D 8 8 ) を 2. 0〃 g—過性に導入した。 この時、 N F— B /レポ一タ一プラス ミ ド （reporter plasmid) 1 . 0〃 g、 p R L— S V 4 0ベクタ一 （Vector) 0. 1 〃 gも同時に導入した。 導入 4 8時間後細胞を回収し、 ルシフェラーゼ （ luci f erase) 活性をデュアルルシフェラーゼレポーターアツセィ系 （ Dua卜 Luci f eras . e Reporter Assay System) ( P r o m e g a社) を用いてルミ ノ メ一ター ( LUM minometer) で測定した。 Further, as a control, wild-type My D 8 8 a (P EF - - BOS F lag -M y D 8 8) 2. was introduced into 0〃 g- excessive resistance. At this time, NF—B / reporter plasmid 1.0〃g, p RL—SV 40 Vector 0. 1 〃g was also introduced at the same time. 48 hours after transfection, the cells were collected, and the luciferase (luci f erase) activity was determined using a dual luciferase reporter assay system (Du Luc Luceras. E Reporter Assay System) (Promega) (Promega). LUM minometer).
具体的には細胞を P B S (Phosphate Buffered saline) にて洗浄後、 溶解緩衝 液 （Lysis Buffer) にて溶解、 回収した。 これにルシフヱラ一ゼ酵素の基質と し て L一 IIまた、 内部の対照と してス ト ップアン ドグロ一 (Stop and Glo) を加え 直後にそれそれルシフェラ一ゼ活性をルミ ノメーターで測定した。  Specifically, the cells were washed with PBS (Phosphate Buffered saline), and then lysed and recovered with a lysis buffer (Lysis Buffer). To this, L-II as a substrate for luciferase enzyme and Stop and Glo as an internal control were added, and immediately after that, luciferase activity was measured using a luminometer.
結果を第 5図に示す。 産業上の利用可能性  The results are shown in FIG. Industrial applicability
本発明の T L R 6は、 トル （T o l l ) 関連分子である T L Rファ ミ リ一の T L R 1 と高い相同性を示し、 N末端側にロイ シン リ ツチリ ビ一 ト ドメイ ンを持ち、 膜貫通部分に続いて C末端側は I L _ 1 Rの細胞内 ドメイ ンと相同性を有する新 規な トル様 （ T o 1 1— 1 i k e ) レセプターである。 免疫応答初期段階のシグ ナル伝達系に関与する物質と して、 臨床的にも重要なものである。  The TLR6 of the present invention shows high homology to TLR1 of the TLR family, which is a Toll-related molecule, has a leucine litulin bite domain at the N-terminal side, and has a transmembrane region. Subsequently, the C-terminal side is a novel toll-like (T01-1 ike) receptor having homology to the intracellular domain of IL_1R. It is also clinically important as a substance involved in the signal transmission system in the early stage of the immune response.

Claims

請 求 の 範 囲 The scope of the claims
1 . 配列番号 2若し く は 4で表されるアミ ノ酸配列又は当該ア ミ ノ酸配列中の 1 個以上のア ミ ノ酸が欠失し、 他のアミ ノ酸で置換され、 及び/又は、 1個以上の 他のアミ ノ酸が付加されたア ミ ノ酸配列を有する トル様レセプター。 1. the amino acid sequence represented by SEQ ID NO: 2 or 4 or one or more amino acids in the amino acid sequence is deleted and replaced with another amino acid; and And / or a toll-like receptor having an amino acid sequence to which one or more other amino acids have been added.
2 . 配列番号 2若し くは 4で表されるアミ ノ酸配列又は当該ア ミ ノ酸配列中の 1 個以上のア ミ ノ酸が欠失し、 他のアミ ノ酸で置換され、 及び/又は、 1個以上の 他のア ミ ノ酸が付加されたアミ ノ酸配列を有し、 トル様レセプター作用を有する 蛋白質。  2. The amino acid sequence represented by SEQ ID NO: 2 or 4 or one or more amino acids in the amino acid sequence is deleted and replaced with another amino acid, and And / or a protein having an amino acid sequence to which one or more other amino acids are added and having a toll-like receptor action.
3 . 請求の範囲第 1項に記載のレセプターをコー ドする塩基配列を有する遺伝子,  3. A gene having a nucleotide sequence encoding the receptor according to claim 1,
4 . 塩基配列が、 配列番号 1 又は 3で表される塩基酸配列を有する請求の範囲第 3項に記載の遺伝子。 4. The gene according to claim 3, wherein the base sequence has a base acid sequence represented by SEQ ID NO: 1 or 3.
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US7230078B2 (en) 2001-09-17 2007-06-12 Nestec S.A. Soluble toll-like receptor
US9157914B2 (en) 2006-01-10 2015-10-13 Colgate-Palmolive Company Methods of modulating cell surface receptors to prevent or reduce inflammation
US8546324B2 (en) 2008-09-22 2013-10-01 Cedars-Sinai Medical Center Short-form human MD-2 as a negative regulator of toll-like receptor 4 signaling
US9512196B2 (en) 2008-09-22 2016-12-06 Cedars-Sinai Medical Center Short-form human MD-2 as a negative regulator of toll-like receptor 4 signaling
US20160184426A9 (en) * 2010-07-19 2016-06-30 Yeda Research And Development Co., Ltd. Toll-Like Receptor 4 (Tlr-4) Agonist Peptides For Modulating Tlr-4 Mediated Immune Response
US9890202B2 (en) 2010-07-19 2018-02-13 Yeda Research And Development Co. Ltd. Peptides based on the transmembrane domain of a toll-like receptor (TLR) for treatment of TLR-mediated diseases
CN102533775A (en) * 2012-01-09 2012-07-04 天津师范大学 Full-length complementary deoxyribonucleic acid (cDNA) sequence of Paralichthys olivaceus pattern recognition Toll-like receptor TLR21 and applications thereof
CN102533775B (en) * 2012-01-09 2014-06-25 天津师范大学 Full-length complementary deoxyribonucleic acid (cDNA) sequence of Paralichthys olivaceus pattern recognition Toll-like receptor TLR21 and applications thereof
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