JP2000157297A - Method for detecting factor of kawasaki disease - Google Patents
Method for detecting factor of kawasaki diseaseInfo
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- JP2000157297A JP2000157297A JP10341661A JP34166198A JP2000157297A JP 2000157297 A JP2000157297 A JP 2000157297A JP 10341661 A JP10341661 A JP 10341661A JP 34166198 A JP34166198 A JP 34166198A JP 2000157297 A JP2000157297 A JP 2000157297A
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Abstract
Description
【0001】[0001]
【発明の属する技術分野】本発明は、川崎病の発症に関
与する川崎病因子の検出法に関する。The present invention relates to a method for detecting a Kawasaki disease factor involved in the development of Kawasaki disease.
【0002】[0002]
【従来技術と発明が解決しようとする課題】川崎病は、
1963年川崎冨作により初めて報告された乳幼児期に
認められる病気であり、近年では、人種毎の発症頻度が
異なるものの世界各地で発症例が報告されている。川崎
病は男児に好発し、その患者年齢としては4才以下が80
〜85%を占めているが、年長者にも発症が認められてい
る。また、冠動脈障害や冠動脈瘤による突然死をきたす
こともある。死亡頻度は、0.3〜0.5%であるが、乳幼児
における心臓疾患の主要原因でもある。また、後遺症と
して冠状動脈瘤及び狭窄を来すことがあり、心筋梗塞の
原因となる。発症初期におけるガンマグロブリン療法等
により冠動脈瘤形成頻度の減少が認められるものの、ガ
ンマグロブリン療法の無効例も多く存在している。川崎
病はその病態が、ブドウ球菌由来の細菌外毒素(TSST−
1:スーパー抗原の一種)による毒素性ショック症候群
(Toxic shock Syndrom: TSS)等に見られる全身性の症
状に類似しており、リンパ球、マクロファージ系の細胞
活性化を初めとした免疫学的変化顕著である(臨床免
疫,26(7): 738-744, 1994)。更には、患者の急性期末
梢血リンパ球においてVβ2及びVβ8陽性T細胞が選
択的に増加しているとの報告もあり(J., Kotozin, B.
L., Jujo, K., Melish, M.E., Glode, M.P., Kohsaka,
T. and Leung, D.Y.M. (1992) Selective expansion of
T cells expressing T-cell receptor variable region
s V beta 2 and V beta8 in Kawasaki disease, Proc N
atl Acad Sci USA 89, 4066; Abe J., Kotozin,B.L., M
eissner, C., Melish, M.E., Masato, T., Fuluton,
D., Romagne, F.,Malissen, B. and Leung, D.Y.M. (19
93) Characterization of T cell repertoire changes
in acute Kawasaki disease, J Exp Med 177,791; Curt
is, N., Zheng, R., Lamb, J.R. and Levin, M.(1995)
Evidence for superantigen mediated process in Kawa
saki disease, Arch Dis Child 72, 308; Donald et. a
l.,1995, J. Immunol. (1995), 155: 5018-5021)、その
発症にはスーパー抗原(SA)の関与が示唆されていた。2. Description of the Related Art Kawasaki disease
This disease was first reported by Tosaku Kawasaki in 1963, and was observed in infancy. In recent years, cases of occurrence have been reported in various parts of the world, although the frequency of occurrence varies depending on the race. Kawasaki disease is common among boys, and the age of the patient is 80 years old or younger.
It accounts for ~ 85%, but has also been found in older people. In addition, sudden death due to coronary artery disorder or coronary aneurysm may occur. Death frequency is 0.3-0.5%, but is also the leading cause of heart disease in infants. Further, as a sequela, coronary aneurysm and stenosis may occur, which causes myocardial infarction. Although the frequency of coronary aneurysm formation is reduced by gamma globulin therapy and the like in the early stage of onset, there are many cases where gamma globulin therapy is ineffective. Kawasaki disease is characterized by a bacterial exotoxin (TSST-
1: a kind of superantigen), similar to systemic symptoms such as Toxic shock Syndrom (TSS), and immunological changes including lymphocyte and macrophage cell activation It is remarkable (clinical immunity, 26 (7): 738-744, 1994). Furthermore, it has been reported that Vβ2 and Vβ8 positive T cells are selectively increased in acute peripheral blood lymphocytes of patients (J., Kotozin, B. et al.
L., Jujo, K., Melish, ME, Glode, MP, Kohsaka,
T. and Leung, DYM (1992) Selective expansion of
T cells expressing T-cell receptor variable region
s V beta 2 and V beta 8 in Kawasaki disease, Proc N
atl Acad Sci USA 89, 4066; Abe J., Kotozin, BL, M
eissner, C., Melish, ME, Masato, T., Fuluton,
D., Romagne, F., Malissen, B. and Leung, DYM (19
93) Characterization of T cell repertoire changes
in acute Kawasaki disease, J Exp Med 177,791; Curt
is, N., Zheng, R., Lamb, JR and Levin, M. (1995)
Evidence for superantigen mediated process in Kawa
saki disease, Arch Dis Child 72, 308; Donald et.a
l., 1995, J. Immunol. (1995), 155: 5018-5021), suggesting the involvement of superantigen (SA) in its onset.
【0003】また、ガンマグロブリン療法が川崎病に奏
効するということも、川崎病の発症にスーパー抗原が関
与することを示唆している。即ち、ガンマグロブリン製
剤中にブドウ球菌由来スーパー抗原に対する抗体が存在
し、この抗SA抗体がSAによるT細胞活性化特異的に抑制
するとの報告(Takei, S., Arora, Y.K. and Walker,M.
(1993) Intravenous immunoglobulin contains specif
ic antibodies inhibitory to activation of T cell b
y staphylococcal toxin superantigens, J.Clin Inves
t, 91, 602-607)がある。しかしながら、その病因は未
だ解明されていない状態である。[0003] The fact that gamma globulin therapy responds to Kawasaki disease also suggests that superantigens are involved in the development of Kawasaki disease. That is, there is a report that an antibody against a staphylococcal superantigen is present in a gamma globulin preparation, and that this anti-SA antibody specifically suppresses T cell activation by SA (Takei, S., Arora, YK and Walker, M., et al.
(1993) Intravenous immunoglobulin contains specif
ic antibodies inhibitory to activation of T cell b
y staphylococcal toxin superantigens, J. Clin Inves
t, 91, 602-607). However, its etiology has not yet been elucidated.
【0004】従来、本症の診断は、1) 5日以上続く発
熱;2) 四肢末端の変化;硬性浮腫、紅斑等;3) 不定形
発疹;4) 両眼球結膜の充血;5) ***、口腔所見;口腔
の紅潮、イチゴ舌、口腔咽頭粘膜のびまん性発赤;6)
急性期における非化膿性頚部リンパ節腫眼からなる臨床
症状の内、5つ以上の症状を伴うことを基準として行わ
れている。しかし、必ずしもこれら主要症状が早期に揃
って出現するとは限らず、特に年長発症の場合には、重
症例が多い上、主要症状の出現に時間がかかることか
ら、早期に的確な診断を行うための基準の確立が強く求
められている。Conventionally, this disease has been diagnosed by: 1) fever lasting 5 days or more; 2) changes in extremities; hard edema, erythema, etc .; 3) irregular rash; 4) conjunctival congestion in both eyes; Oral findings; flushing of the oral cavity, strawberry tongue, diffuse redness of the oropharyngeal mucosa; 6)
Non-suppurative cervical lymphadenopathy in the acute phase is performed on the basis of clinical symptoms involving five or more symptoms. However, these major symptoms do not always appear at an early stage, and especially in the case of older onset, an accurate diagnosis is made early because many severe cases and the appearance of major symptoms take time. There is a strong demand for establishing standards for this.
【0005】[0005]
【課題を解決するための手段】本発明者らは、川崎病の
的確な早期診断を確立するために、鋭意研究を重ねた結
果、川崎病の発症期の患者に、川崎病に特異的な因子の
存在を示唆する、科学的に検出可能な徴候が現れている
ことを見出し、本発明を完成するに至った。即ち、本発
明は、Vβ6.5陽性T細胞数の増加、又はVβ6.5及
びVβ2.1陽性T細胞数の増加を指標とする、川崎病
因子の検出法を提供するものである。「川崎病因子」と
は、川崎病の臨床症状を顕在化させる因子を意味し、何
らかの原因で川崎病に罹患した患者の血中に存在する病
因物質を含む。そのような物質として、例えば、細菌性
スーパー抗原の可能性がある。スーパー抗原の代表例と
して、A群連鎖球菌由来の外毒素(Streptococcal pyog
enic exptoxin C(SPEC))を挙げることができる。Means for Solving the Problems The present inventors have conducted intensive studies in order to establish an accurate early diagnosis of Kawasaki disease. The inventors have found that scientifically detectable signs suggesting the presence of the factor have appeared, and have completed the present invention. That is, the present invention provides a method for detecting Kawasaki disease factor using an increase in the number of Vβ6.5-positive T cells or an increase in the number of Vβ6.5 and Vβ2.1-positive T cells as an index. “Kawasaki disease factor” refers to a factor that manifests the clinical symptoms of Kawasaki disease, and includes an etiological substance present in the blood of a patient suffering from Kawasaki disease for some reason. Such a substance may be, for example, a bacterial superantigen. As a representative example of the superantigen, an exotoxin derived from group A streptococcus (Streptococcal pyog
enic exptoxin C (SPEC)).
【0006】本発明の川崎病因子の検出法を実施するに
は、川崎病を疑われる患者から末梢血を得、TCRV領
域の遺伝子群を増幅し、TCRVサブファミリーの発現
頻度を検出し、Vβ6.5陽性T細胞、又はVβ6.5陽
性T細胞及びVβ2.1陽性T細胞の増加を評価する。
T細胞のVβレパートリーは、当該技術分野で既知の任
意の方法で増幅し、発現を調べることができる。例え
ば、患者から末梢血を得、トータルRNAを抽出し、既
知のアダプター付加PCR(アダプターライゲーション
PCR=AL−PCR)でTCRV領域の遺伝子群を増
幅し、リバース・ドット・ブロット法(Reverse dot bl
otting)により、TCRVサブファミリーに特異的なオ
リゴプローブを用いて、それらサブファミリーの発現状
態を調べ、TCRVβ6.5、又はTCRVβ6.5及び
TCRVβ2.1サブファミリーの発現頻度の有意な増
大(絶対的及び/又は相対的)を比較する。リバース・
ドット・ブロット法に用いるオリゴヌクレオチドプロー
ブは、TCRVβサブファミリーに特異的であり、少な
くとも、TCRVβ2.1及びTCRVβ6.5に対応す
るプローブを含むことを条件として任意である。本発明
の実施に用いることができるオリゴヌクレオチドプロー
ブの一例として、表7〜13に記載のオリゴヌクレオチ
ドプローブが挙げられる。これらは配列番号10及び1
1に記載の塩基配列で示されるオリゴヌクレオチドプロ
ーブ(表10のVB02-1及び表11のVB06-4、それぞれT
CRVβ2.1及びTCRVβ6.5に対応する)と、配
列番号12〜96に記載のオリゴヌクレオチドを含む。
これらオリゴヌクレオチドプローブを用いる場合、最終
的に得られたサブファミリーの発現頻度のデータから、
配列番号10及び11に記載のオリゴヌクレオチドプロ
ーブに対応するTCRVβ6.5、又はTCRVβ6.5
及びTCRVβ2.1サブファミリーの発現頻度が他の
サブファミリーの発現頻度よりも有意に高いことを指標
として、川崎病因子の存在を決定すればよい。To carry out the method for detecting a Kawasaki disease factor of the present invention, peripheral blood is obtained from a patient suspected of having Kawasaki disease, the genes of the TCRV region are amplified, the expression frequency of the TCRV subfamily is detected, and Vβ6 Evaluate the increase of .5 positive T cells or Vβ6.5 positive T cells and Vβ2.1 positive T cells.
The V cell repertoire of T cells can be expanded and examined for expression by any method known in the art. For example, peripheral blood is obtained from a patient, total RNA is extracted, a group of genes in the TCRV region is amplified by a known adapter-added PCR (adapter ligation PCR = AL-PCR), and reverse dot blotting (Reverse dot bl
otting), using oligo probes specific for the TCRV subfamily to examine the expression status of those subfamilies and to significantly increase the frequency of expression of the TCRVβ6.5 or TCRVβ6.5 and TCRVβ2.1 subfamilies (absolute And / or relative). reverse·
The oligonucleotide probe used for the dot blot method is specific to the TCRVβ subfamily, and is optional provided that it contains at least probes corresponding to TCRVβ2.1 and TCRVβ6.5. Examples of the oligonucleotide probes that can be used in the practice of the present invention include the oligonucleotide probes described in Tables 7 to 13. These are SEQ ID NOs: 10 and 1.
The oligonucleotide probe represented by the nucleotide sequence shown in Table 1 (VB02-1 in Table 10 and VB06-4 in Table 11;
CRVβ2.1 and TCRVβ6.5) and the oligonucleotides set forth in SEQ ID NOs: 12-96.
When using these oligonucleotide probes, from the data on the expression frequency of the finally obtained subfamily,
TCRVβ6.5 or TCRVβ6.5 corresponding to the oligonucleotide probes of SEQ ID NOS: 10 and 11.
The presence of the Kawasaki disease factor may be determined by using the expression frequency of the TCRVβ2.1 subfamily and the expression frequency of the other subfamilies significantly higher as an index.
【0007】別法として、Vβ2.1及びVβ6.5に特
異的なプライマーで増幅し、その後、増幅量を定量して
もよい。定量法としては、電気泳動に供し、ハウスキー
ピング遺伝子をインナーコントロールとして定量するこ
とができる。川崎病因子の存在は、TCRVβ鎖におけ
る、Vβ2.1及びVβ6.5を検出し、Vβ2.1とV
β6.5陽性T細胞が、川崎病発症期において発現量の
増加が健常人群コントロールの平均値に比べ有意に高い
場合を陽性、低い場合を陰性とする。その他のファクタ
ー、例えば、T細胞の増加がポリクローナルであるこ
と、各種炎症性サイトカインの増加、従来の症状の発現
も診断に際して補助的に用いることができる。[0007] Alternatively, amplification may be carried out with primers specific for Vβ2.1 and Vβ6.5, and then the amount of amplification may be quantified. As a quantification method, housekeeping genes can be quantified as an inner control by subjecting to electrophoresis. The presence of Kawasaki disease factor detected Vβ2.1 and Vβ6.5 in the TCRVβ chain,
ββ-positive T cells are positive if the increase in the expression level during the Kawasaki disease onset phase is significantly higher than the average value of the control group of healthy individuals, and negative if the increase is low. Other factors such as polyclonal increase in T cells, increase in various inflammatory cytokines, and expression of conventional symptoms can also be used as an aid in diagnosis.
【0008】実施例1 表1に示す川崎病患者22名の治療前(発症期)、治療
後(79日以上経過後)及び発熱を伴う川崎病以外の疾
患群の子供14名、及び健常群の大人10名について末
梢血中のTCRVレパートリーを表2に示すオリゴヌク
レオチドプライマーを用いるアダプター付加PCR法で
増幅し、表7〜13に記載のオリゴヌクレオチドプロー
ブを用いるリバース・ドット・ブロット法でTCRVサ
ブファミリーの発現状態を調べ、TCRVβ6.5及び
TCRVβ2.1サブファミリーの発現頻度を解析し
た。 I. TCRV領域の増幅表1 川崎病因子の検出対象 Example 1 Twenty-two Kawasaki disease patients shown in Table 1 before treatment (onset), after treatment (after 79 days or more), and 14 children of a disease group other than Kawasaki disease accompanied by fever, and a healthy group The TCRV repertoire in the peripheral blood of 10 adults was amplified by an adapter-added PCR method using the oligonucleotide primers shown in Table 2, and was subjected to reverse dot blotting using The expression status of the families was examined, and the expression frequencies of the TCRVβ6.5 and TCRVβ2.1 subfamilies were analyzed. I. Amplification of TCRV region Table 1 Targets for detection of Kawasaki disease factor
【表1】 [Table 1]
【0009】表2 アダプター付加PCR法及びTCR
αβcDNAの増幅に用いるオリゴヌクレオチドプライ
マー Table 2 PCR method with adapter and TCR
Oligonucleotide primers used for amplification of αβ cDNA
【表2】 [Table 2]
【0010】表2のTCRαβcDNAのPCR増幅用
オリゴヌクレオチドプライマーの内、CA1(配列番号
4)、CA2(配列番号5)及びCA3(配列番号6)
は2種類のCα領域の共通部分に、CB1(配列番号
7)、CB2(配列番号8)及びCB3(配列番号9)
は2種類のCβ領域の共通配列上に、それぞれ対応する
よう設定されている。Among the oligonucleotide primers for PCR amplification of TCRαβ cDNA shown in Table 2, CA1 (SEQ ID NO: 4), CA2 (SEQ ID NO: 5) and CA3 (SEQ ID NO: 6)
Indicates that CB1 (SEQ ID NO: 7), CB2 (SEQ ID NO: 8) and CB3 (SEQ ID NO: 9)
Are set on the common sequence of the two types of Cβ regions so as to correspond to each other.
【0011】(1)cDNAの合成 表2に記載のPCRプライマーP10EA(配列番号
1)、P20EA(配列番号2)及びBSL−18(配
列番号3)を用いた。対象から末梢血5−10mlを採取
し、Ficoll-Hypaque (ファルマシア社)を用いてPBM
Cと血清に分けた。Trizol-Reagents(BRL社)を使用し、
業者の指示に従って、PBMCよりトータルRNAを抽
出した。抽出したRNAから、Adaptor−ligation PC
R法(Tsuruta, Y., Iwagami,S., Furue, S., Teraoka,
H., Yoshida, T., Sakata, T. and Suzuki, R. (1993)
Detection of human T cell receptor cDNAs (α、
β、γ、and δ)by ligation of a universal adaptor
to variable region J Immunol Methods 161, 7)によ
り、TCR V領域を含む遺伝子群を増幅した。まず、
抽出されたRNAから、BSL−18プライマー(表2
及び配列番号3)を用いて、1stストランドDNAを合
成し、次いで、2ndストランドDNAを合成し、フェノ
ール抽出し、次いでポリエチレングリコール(PEG)を用
いたDNA沈殿法で余分なBSL−18を除去し、精製
した。(1) Synthesis of cDNA PCR primers P10EA (SEQ ID NO: 1), P20EA (SEQ ID NO: 2) and BSL-18 (SEQ ID NO: 3) described in Table 2 were used. 5-10 ml of peripheral blood is collected from the subject and subjected to PBM using Ficoll-Hypaque (Pharmacia).
C and serum. Using Trizol-Reagents (BRL),
According to the manufacturer's instructions, total RNA was extracted from PBMC. Adapter-ligation PC from extracted RNA
R method (Tsuruta, Y., Iwagami, S., Furue, S., Teraoka,
H., Yoshida, T., Sakata, T. and Suzuki, R. (1993)
Detection of human T cell receptor cDNAs (α,
β, γ, and δ) by ligation of a universal adaptor
The gene group containing the TCR V region was amplified by to variable region J Immunol Methods 161, 7). First,
From the extracted RNA, a BSL-18 primer (Table 2)
And the second strand DNA was synthesized by using SEQ ID NO: 3), then the second strand DNA was synthesized, extracted with phenol, and the extra BSL-18 was removed by a DNA precipitation method using polyethylene glycol (PEG). And purified.
【0012】(2)アダプターの付加 1)P20EA/P10EAアダプターの調製 RNaseを含有しない滅菌水(和光純薬)に溶かした各10
0pmol/μlのP20EAとP10EA(配列番号2及
び1)を、1.5mlスクリューキャップ付き滅菌チュ
ーブ内で1:1の割合で混合し、蓋をして70℃で10分
間加熱し、室温まで徐冷した。得られたP20EA/P
10EA溶液を冷房室に移し10℃以下になった時点で−
70℃にて凍結させ、使用するまで−20℃で保存した。(2) Addition of Adapter 1) Preparation of P20EA / P10EA Adapter Each of 10 adapters dissolved in sterile water (Wako Pure Chemical Industries, Ltd.) containing no RNase.
0 pmol / μl of P20EA and P10EA (SEQ ID NOs: 2 and 1) were mixed at a ratio of 1: 1 in a sterile tube with a 1.5 ml screw cap, heated with a lid at 70 ° C. for 10 minutes, and gradually cooled to room temperature. Cooled down. P20EA / P obtained
Transfer the 10EA solution to the cooling room, and when the temperature drops below 10 ° C-
It was frozen at 70 ° C and stored at -20 ° C until use.
【0013】2)2本鎖cDNAに対するアダプターの
付加 氷上で、2本鎖cDNA溶液(12.5μl)、x5 T4 DNA
Ligase Buffer (BRL;250 mM Tris-HCl (pH 7.6), 50 mM
MgCl2, 5 mM ATP, 5 mM DTT,25%(W/V) PEG8000)(5.0
μl = x1 buffer)、50 pmol/μl P20EA/P10EA アダプ
ター(5.0μl,10μM)、T4 DNA Ligase (BRL) (2.5μ
l)を混合し、合計25.0μlの反応液をピペッティング
及びタッピングで良く攪拌し、4℃でFlash遠心した後、
16℃で20時間靜置した。反応終了後、4℃でFlash遠心し
た。次いで、アダプターを付加した2本鎖cDNAをフ
ェノール抽出で精製し、PEGを用いるDNA沈殿によ
り余分なP20EA/P10EAアダプターを除去し
た。2) Addition of adapter to double-stranded cDNA On ice, double-stranded cDNA solution (12.5 μl), x5 T4 DNA
Ligase Buffer (BRL; 250 mM Tris-HCl (pH 7.6), 50 mM
MgCl 2 , 5 mM ATP, 5 mM DTT, 25% (W / V) PEG8000) (5.0
μl = x1 buffer), 50 pmol / μl P20EA / P10EA adapter (5.0 μl, 10 μM), T4 DNA Ligase (BRL) (2.5 μl
l) was mixed, and a total of 25.0 μl of the reaction solution was thoroughly stirred by pipetting and tapping, followed by Flash centrifugation at 4 ° C.
The mixture was allowed to stand at 16 ° C for 20 hours. After completion of the reaction, the mixture was subjected to Flash centrifugation at 4 ° C. Next, the double-stranded cDNA to which the adapter was added was purified by phenol extraction, and the extra P20EA / P10EA adapter was removed by DNA precipitation using PEG.
【0014】3)TCR遺伝子C領域側に付いたアダプ
ターの切断 上記2)で得た34.0μlのアダプター付加2本鎖cDN
Aに、氷上で、0.1% Triton-X(5.0μl, 0.01%, TAKAR
A),0.1% BSA (5.0μl, 0.01%, TAKARA), x10 buffer H
(5.0 μl, x1 buffer, TAKARA)を混合し、最後に Not
I (約50U/μl, TAKARA)を加えて、合計50.0μl
の混合液をタッピングにより良く混合し、4℃でFlash遠
心を行った後、37℃で2時間靜置した。反応終了後、直
ちに氷上に移した。次いで、NotI処理した2本鎖cDN
Aをフェノール抽出して精製し、PEGを用いるDNA
沈殿法でNotI処理により生じたP20EA/P10EA
アダプターを除去した。得られたDNAを20μlの滅
菌水に懸濁し、‐20℃で保存した。3) Cleavage of the adapter attached to the C region of the TCR gene 34.0 μl of the adapter-added double-stranded cDN obtained in 2) above
A. On ice, 0.1% Triton-X (5.0 μl, 0.01%, TAKAR
A), 0.1% BSA (5.0μl, 0.01%, TAKARA), x10 buffer H
(5.0 μl, x1 buffer, TAKARA).
I (about 50 U / μl, TAKARA) and add 50.0 μl
Was mixed well by tapping, and the mixture was subjected to Flash centrifugation at 4 ° C, and then allowed to stand at 37 ° C for 2 hours. Immediately after the reaction was completed, the mixture was transferred to ice. Then, NotI-treated double-stranded cDN
A is purified by phenol extraction and DNA using PEG
P20EA / P10EA generated by NotI treatment by precipitation method
Adapter was removed. The obtained DNA was suspended in 20 μl of sterile water and stored at −20 ° C.
【0015】(3)PCR PCRは、以下の表に示すプライマーセットを用いて行
った。表3 PCR用プライマーセット(3) PCR PCR was carried out using the primer sets shown in the following table. Table 3 Primer set for PCR
【表3】 [Table 3]
【0016】1)第1回(1st)PCR 上記(2)で最終的に20μlに調製したcDNAを0.
5ml滅菌チューブに1μlずつ分注し、氷上で保管した。
x10 PCR用バッファー(Promega)、25mM MgCl2(Promeg
a)、P20EA/CA1及びP20EA/CB1プライマーセット(50pmol
/μl)、25mM dNTP mix(g)を氷上で解凍し、ボルテック
スで攪拌した後、Flash遠心により溶液を回収した。上
記の試薬を用い、α、β各500μl(10回分)の1stPC
R溶液を氷上で以下の組成に従い調製した。1) First (1st) PCR The cDNA finally prepared in 20 μl in the above (2) was used in 0.1 μl.
1 μl was dispensed into a 5 ml sterile tube and stored on ice.
x10 PCR buffer (Promega), 25mM MgCl 2 (Promeg
a), P20EA / CA1 and P20EA / CB1 primer set (50 pmol
/ μl), 25 mM dNTP mix (g) was thawed on ice, vortexed, and the solution was recovered by Flash centrifugation. Using the above reagents, 500 µl each of α and β (10 times) of 1stPC
An R solution was prepared on ice according to the following composition.
【0017】表4 α、β鎖用1stPCR溶液 Table 4 1st PCR solution for α and β chains
【表4】 混合物を良く攪拌し、Flash遠心したのち、α、β各4
90μl(10回分)の1stPCR溶液を、先にcDNAを
分注しておいたチューブに49μlずつ分注した。残っ
たPCR溶液は、ネガティブコントロールに用いた。
α、β各10本ずつのチューブにミネラルオイル(SIGM
A)約50μlを分注し、合計150μlの混合液を用い
て以下の手順でPCR増幅を行った(変成94℃30秒を1
サイクル、変成94℃1分、アニール55℃、1.5分、伸長7
2℃、2分を20サイクル、Last extension72℃、5分以
下で4℃で保存)。Flash遠心後、-70℃に靜置し、氷結
させた。室温に戻し、ミネラルオイルのみが解凍した時
点で、マイクロピペットによりこれを除去した。[Table 4] Stir the mixture well and centrifuge in Flash, then 4 each for α and β
90 μl (10 times) of the 1st PCR solution was dispensed in 49 μl portions into the tube into which cDNA was previously dispensed. The remaining PCR solution was used for a negative control.
Mineral oil (SIGM) in 10 tubes each for α and β
A) Approximately 50 μl was dispensed, and PCR amplification was performed using a total of 150 μl of the mixed solution according to the following procedure (denaturation was performed at 94 ° C. for 30 seconds for 1 second).
Cycle, denaturation 94 ° C for 1 minute, annealing 55 ° C, 1.5 minutes, extension 7
20 cycles of 2 minutes at 2 ° C, Last extension 72 ° C, stored at 4 ° C for 5 minutes or less). After flash centrifugation, the mixture was allowed to stand at -70 ° C and frozen. After returning to room temperature and only the mineral oil was thawed, it was removed with a micropipette.
【0018】2)第2回(2nd)PCR 上記1)で増幅したPCR産物を、0.5ml滅菌チューブ
に5μlずつ、ネガティブコントロールとして増幅した
産物は2μlを分注し、氷上で保管した。x10 PCR用
バッファー(Promega)、25mM MgCl2(Promega)、P20EA/CA
2及びP20EA/CB2プライマーセット(5pmol/μl)、25mM
dNTP mix(TOYOBO)を氷上で解凍し、ボルテックスで良く
攪拌した後、Flash遠心により溶液を回収した。上記の
試薬を用い、α、β各500μl(10回分)の2ndPCR溶
液を氷上で以下の組成に従い調製した。2) 2nd (2nd) PCR 5 μl of the PCR product amplified in the above 1) was dispensed into a 0.5 ml sterilized tube, and 2 μl of the amplified product was stored as a negative control and stored on ice. x10 PCR buffer (Promega), 25mM MgCl 2 (Promega), P20EA / CA
2 and P20EA / CB2 primer set (5 pmol / μl), 25 mM
The dNTP mix (TOYOBO) was thawed on ice, vortexed well, and the solution was recovered by Flash centrifugation. Using the reagents described above, 500 μl each of α and β (10 times) of a 2nd PCR solution was prepared on ice according to the following composition.
【0019】表5 α、β鎖用2ndPCR溶液 Table 5 2nd PCR solution for α and β chains
【表5】 混合物を良く攪拌し、Flash遠心したのち、α、β各4
50μl(10回分)の2ndPCR溶液を、先にcDNAを
分注しておいたチューブに45μlずつ分注した。残っ
たPCR溶液のうち、18μlをネガティブコントロール
増幅に用いた。α、β各10本ずつのチューブにミネラル
オイル(SIGMA)を分注し、以下の手順でPCR増幅を
行った(変成94℃30秒を1サイクル、変成94℃1分、ア
ニール55℃、1.5分、伸長72℃、2分を20サイクル、La
st extension72℃、5分以下で4℃で保存)。Flash遠心
後、-70℃に靜置し、氷結させた。室温に戻し、ミネラ
ルオイルのみが解凍した時点で、マイクロピペットによ
りこれを除去した。第2回のPCR産物5μlに2μl
の電気泳動用色素を加えた。x1TAE中で定電圧100m
vの条件で、1%アガロース電気泳動を行い、サイズ分
画されたDNAをエチジウムブロマイド染色し、トラン
スイルミネーターを用い、増幅を確認した。[Table 5] Stir the mixture well and centrifuge in Flash, then 4 each for α and β
50 μl (10 times) of the 2nd PCR solution was dispensed in 45 μl portions into the tube into which cDNA was previously dispensed. Of the remaining PCR solution, 18 μl was used for negative control amplification. Mineral oil (SIGMA) was dispensed into 10 tubes each of α and β, and PCR amplification was performed according to the following procedure (1 cycle of denaturation at 94 ° C for 30 seconds, denaturation at 94 ° C for 1 minute, annealing at 55 ° C and 1.5 Min, extension 72 ° C, 20 cycles of 2 min, La
st extension 72 ℃, stored at 4 ℃ for 5 minutes or less). After flash centrifugation, the mixture was allowed to stand at -70 ° C and frozen. After returning to room temperature and only the mineral oil was thawed, it was removed with a micropipette. 2 μl for 5 μl of second PCR product
Was added. Constant voltage 100m in x1 TAE
Under the conditions of v, 1% agarose electrophoresis was performed, the size-fractionated DNA was stained with ethidium bromide, and amplification was confirmed using a transilluminator.
【0020】3)第3回(3rd)PCR(ビオチン付
加) 上記2)で増幅を確認したPCR産物を、0.5ml滅菌チ
ューブに1μlずつ分注し氷上で保管した。x10 PCR
用バッファー(Promega)、25mM MgCl2(Promega)、P20EA/
CA3及びP20EA/CB3プライマーセット(50pmol/μl)、1
0mM dATP, dCTP, dGTP mix, 0.5mMdTTP mix, 0.5mM Bio
tin-21-dUTPを氷上で解凍し、ボルテックスで良く攪拌
した後、Flash遠心により溶液を回収した。上記の試薬
を用い、α、β各1000μl(10回分)の3rdPCR溶液
を氷上で以下の組成に従い調製した。3) Third (3rd) PCR (addition of biotin) The PCR product confirmed to be amplified in the above 2) was dispensed at 1 μl per 0.5 ml sterile tube and stored on ice. x10 PCR
Buffer (Promega), 25 mM MgCl 2 (Promega), P20EA /
CA3 and P20EA / CB3 primer set (50 pmol / μl), 1
0mM dATP, dCTP, dGTP mix, 0.5mM dTTP mix, 0.5mM Bio
Tin-21-dUTP was thawed on ice, vortexed well, and the solution was recovered by Flash centrifugation. Using the reagents described above, 1000 μl (10 times) of 3rd PCR solution for each of α and β was prepared on ice according to the following composition.
【0021】表6 α、β鎖用3rdPCR溶液 Table 6 3rd PCR solution for α and β chains
【表6】 混合物を良く攪拌し、Flash遠心したのち、α、β各1
000μl(10回分)の3rdPCR溶液を、先に2ndcD
NAを分注しておいたチューブに100μlずつ分注し
た。残ったPCRR溶液のうち、20μlをネガティブコ
ントロール増幅に用いた。α、β各10本ずつのチューブ
にミネラルオイル(SIGMA)を分注し、以下の手順でP
CR増幅を行った(変成94℃30秒を1サイクル、変成94
℃1分、アニール55℃、2分、伸長72℃、2.5分を20
サイクル、Last extension72℃、5分以下で4℃で保
存)。Flash遠心後、-70℃に靜置し、氷結させた。室温
に戻し、ミネラルオイルのみが解凍した時点で、マイク
ロピペットによりこれを除去した。第3回のPCR産物
5μlに2μlの電気泳動用色素を加えた。x1TAE
中で定電圧100mvの条件で、1%アガロース電気泳動
を行い、サイズ分画されたDNAをエチジウムブロマイ
ド染色し、トランスイルミネーターを用い、増幅を確認
した。[Table 6] Stir the mixture well, flash centrifuge, then each of α and β
2,000 μl (10 times) of the 3rd PCR solution was added to the 2ndcD
100 μl was dispensed into tubes into which NA had been dispensed. Of the remaining PCRR solution, 20 μl was used for negative control amplification. Dispense mineral oil (SIGMA) into 10 tubes each of α and β and follow the procedure below.
CR amplification was performed (1 cycle of denaturation 94 ° C for 30 seconds, denaturation 94
℃ 1 minute, annealing 55 ℃, 2 minutes, extension 72 ℃, 2.5 minutes 20
Cycle, Last extension 72 ° C, stored at 4 ° C for less than 5 minutes). After flash centrifugation, the mixture was allowed to stand at -70 ° C and frozen. After returning to room temperature and only the mineral oil was thawed, it was removed with a micropipette. To 5 μl of the third PCR product, 2 μl of electrophoresis dye was added. x1 TAE
In the medium, 1% agarose electrophoresis was performed under the condition of a constant voltage of 100 mv, and the size-fractionated DNA was stained with ethidium bromide, and amplification was confirmed using a transilluminator.
【0022】II. Reverse dot blotting Reverse dot blottingは、以下の表7〜13に記載の、
各TCRVサブファミリーに特異的に対応する20bp前
後のオリゴヌクレオチドプローブを用い、常法に従って
行った。表7〜9には、TCRVα及びCαのための一
本鎖オリゴヌクレオチドプローブ(SSOP)、表10
から13には、TCRVβ及びCβのための一本鎖オリ
ゴヌクレオチドプローブ(SSOP)が記載されてい
る。表7〜9 Reverse dot blotting用TCRVα及び
CαのためのSSOPII. Reverse dot blotting Reverse dot blotting is described in Tables 7 to 13 below.
This was carried out according to a conventional method using an oligonucleotide probe of about 20 bp specifically corresponding to each TCRV subfamily. Tables 7-9 show single stranded oligonucleotide probes (SSOP) for TCRVα and Cα, Table 10
To 13 describe single-stranded oligonucleotide probes (SSOPs) for TCRVβ and Cβ. Tables 7-9 SSOPs for TCRVα and Cα for Reverse dot blotting
【表7】 [Table 7]
【表8】 [Table 8]
【表9】 [Table 9]
【0023】表10〜13 Reverse dot blotting用T
CRVβ及びCβのためのSSOP Tables 10-13 T for reverse dot blotting
SSOP for CRVβ and Cβ
【表10】 [Table 10]
【表11】 [Table 11]
【表12】 [Table 12]
【表13】 [Table 13]
【0024】表7〜13に記載のオリゴヌクレオチドを
合成し、poly-T-tailを付加し、標識し、供給者の指示
に従ってHybond Nfp メンブレン(アマーシャム社)に固
相化した。次いで、上記I.に記載のアダプター付加P
CRで増幅した遺伝子群を、固相化したメンブレン上の
オリゴマーに37℃で1時間、指定のバッファー中でハ
イブリダイズさせ、標識に用いたビオチンと、アルカリ
フォスファターゼ標識したアヴィジンによるAMPDD
の発光量の強度に基づいて検出することにより、同定、
定量した。なお、TCRVβ2.1及びVβ6.5に特異
的なオリゴプローブVB02−1及びVB06−4の塩
基配列は配列番号10及び11に示されている。Oligonucleotides shown in Tables 7 to 13 were synthesized, added with poly-T-tail, labeled, and immobilized on Hybond Nfp membrane (Amersham) according to the supplier's instructions. Next, the adapter-added P described in I. above.
The genes amplified by CR were hybridized with the oligomer on the immobilized membrane at 37 ° C. for 1 hour in a designated buffer, and the biotin used for labeling and the AMPDD with alkaline phosphatase-labeled avidin were used.
Identification by detecting based on the intensity of the luminescence amount of
Quantified. The nucleotide sequences of oligo probes VB02-1 and VB06-4 specific to TCRVβ2.1 and Vβ6.5 are shown in SEQ ID NOS: 10 and 11, respectively.
【0025】川崎病患者22名の治療前(発症期)、γ-
グロブリン投与時及び回復期における測定結果を図1及
び2に示す。さらに、川崎病患者22名の治療前(発症
期)と回復期、発熱を伴う川崎病以外の疾患群の子供1
4名、及び健常大人10名についてTCRVβ鎖のVβ
2.1及びVβ6.5の発現頻度を図3に示す。図1及び
2において、横軸のVA及びVBは、表7〜13に記載
の、Reverse dot blottingに用いたオリゴプローブの番
号を表し、縦軸は各サブファミリーの発現率(%)を示
す。なお、図1における1D,2D,3Dは、それぞ
れ、表2におけるVD1,VD2,VD3に相当し、V
B02−1はVβ2.1を、VB06−4はVβ6.5を
表す。図1および2から、川崎病発症期にはVβ鎖のV
β2.1及びVβ6.5が高頻度で発現していることが分
かる また、図3からTCRVβ鎖については、川崎病発症期
の患者の場合、健常人コントロール及び川崎病回復期の
患者に比べて、Vβ2.1(P<0.05)及びVβ6.5(P<
0.05)の使用頻度が有意に高いこと、他の疾患群と比較
した場合、Vβ6.5(P<0.01)の使用頻度が有意に高
いことが分かる。以上の結果は、川崎病の発症に関与す
る因子は、Vβ6.5陽性T細胞、又はVβ6.5陽性及
びVβ2.1陽性T細胞の増加をもたらすこと、即ち、
これらT細胞の増加に基づいて検出することが可能であ
ることを示している。Before treatment (onset) of 22 Kawasaki disease patients, γ-
The measurement results at the time of globulin administration and during the recovery period are shown in FIGS. In addition, 22 children with Kawasaki disease before treatment (onset period), convalescent period, and children with disease other than Kawasaki disease with fever.
VCR of TCRVβ chain for 4 and 10 healthy adults
The expression frequencies of 2.1 and Vβ6.5 are shown in FIG. In FIGS. 1 and 2, VA and VB on the horizontal axis represent the numbers of the oligo probes used for Reverse dot blotting described in Tables 7 to 13, and the vertical axis represents the expression rate (%) of each subfamily. 1D, 2D, and 3D in FIG. 1 correspond to VD1, VD2, and VD3 in Table 2, respectively.
B02-1 represents Vβ2.1 and VB06-4 represents Vβ6.5. From FIGS. 1 and 2, the Vβ chain V
It can be seen that β2.1 and Vβ6.5 are expressed at a high frequency. From FIG. 3, the TCRVβ chain is more evident in patients with Kawasaki disease onset than in healthy control and patients with Kawasaki disease recovery. , Vβ2.1 (P <0.05) and Vβ6.5 (P <0.05)
0.05) is significantly higher, and when compared to other disease groups, Vβ6.5 (P <0.01) is significantly higher. The above results indicate that the factor involved in the development of Kawasaki disease is an increase in Vβ6.5-positive T cells or Vβ6.5-positive and Vβ2.1-positive T cells,
This indicates that detection can be performed based on the increase in these T cells.
【0026】III.CDR3領域の塩基配列の決定 治療前の発症期において、治療後の回復期より特に高い
割合でVβ2.1及びVβ6.5が発現しているサンプル
について、CDR3領域(可変領域)の塩基配列を決定
した。即ち、アダプター付加PCRで増幅した産物を、
それぞれ、Vβ2.1及びVβ6.5のための、VB02
−1(表10)とCB3(表2)若しくはVB06−4
(表11)とCB3(表2)で、アダプター付加PCR
と同じ条件下でPCR増幅し、アガロース電気泳動を行
った。ゲルから常法通り目的のDNAを抽出した後、T
Aクローニング法(Holton, T.A. and Ghanem, M.W. (1
991) A simple and efficient method for direct clon
ing of PCR products using ddT-tailed vectors Nucle
ic Acids Res 19, 1156)に従い、Vβ2.1及びVβ6.
5の配列をもつPCR産物を、既知のベクター、pGE
MTのクローニングサイトに導入した。遺伝子導入され
たベクターを、電気穿孔法により、DH10B由来の
E.coli K-12株に導入し、培養した。それぞれの形質転
換された大腸菌より抽出されたプラスミドは、T7DN
Aポリメラーゼと、VB02−1若しくはVB06−4
とCB3プライマーのセットを用い、サンガー法(Sange
r ら、1987)によって塩基配列を決定した。該塩基配列
から予想されるアミノ酸配列を表14及び表15、16
に示す。III. Determination of Nucleotide Sequence of CDR3 Region The nucleotide sequence of the CDR3 region (variable region) was determined for a sample in which Vβ2.1 and Vβ6.5 were expressed at a particularly high ratio during the onset period before treatment and during the recovery period after treatment. did. That is, the product amplified by the adapter-added PCR is
VB02 for Vβ2.1 and Vβ6.5, respectively.
-1 (Table 10) and CB3 (Table 2) or VB06-4
(Table 11) and CB3 (Table 2)
PCR was performed under the same conditions as described above, and agarose electrophoresis was performed. After extracting the target DNA from the gel as usual,
A cloning method (Holton, TA and Ghanem, MW (1
991) A simple and efficient method for direct clon
ing of PCR products using ddT-tailed vectors Nucle
ic Acids Res 19, 1156), Vβ2.1 and Vβ6.
The PCR product having a sequence of 5
It was introduced into the cloning site of MT. The transfected vector was introduced into DH10B-derived E. coli K-12 strain by electroporation and cultured. Plasmids extracted from each transformed E. coli were T7DN
A polymerase and VB02-1 or VB06-4
And Sanger method (Sange method)
r et al., 1987). Table 14 and Tables 15 and 16 show the amino acid sequence deduced from the nucleotide sequence.
Shown in
【0027】表14 川崎病発症期の患者から得たVβ
2.1−陽性cDNAクローンのCDR3のアミノ酸配
列(n=2、クローン数10又は11) Table 14 Vβ obtained from patients at the onset of Kawasaki disease
2.1-Amino acid sequence of CDR3 of positive cDNA clone (n = 2, number of clones 10 or 11)
【表14】 表14に記載の21クローンのアミノ酸配列は、上から
順次、配列番号97〜配列番号117のアミノ酸配列
に、それぞれ対応している。[Table 14] The amino acid sequences of the 21 clones shown in Table 14 correspond to the amino acid sequences of SEQ ID NO: 97 to SEQ ID NO: 117, respectively, from the top.
【0028】表15〜16 川崎病発症期の患者から得
たVβ6.5−陽性cDNAクローンのCDR3のアミ
ノ酸配列(n=4、クローン数10〜12) Table 15-16 Amino acid sequence of CDR3 of Vβ6.5-positive cDNA clone obtained from Kawasaki disease onset patient (n = 4, number of clones 10-12)
【表15】 [Table 15]
【表16】 表15に記載の20クローンのアミノ酸配列は、配列番
号118〜137、表16に記載の23クローンのアミ
ノ酸配列は、配列番号138〜160に記載のアミノ酸
配列に、それぞれ対応している。表14〜16から明ら
かなように、同一のクローンは存在していない。これ
は、川崎病発症期におけるVβ2.1、Vβ6.5ポジテ
ィブT細胞の発現量増加が、ポリクローナルであること
を示している。[Table 16] The amino acid sequences of the 20 clones shown in Table 15 correspond to SEQ ID NOs: 118 to 137, and the amino acid sequences of the 23 clones shown in Table 16 correspond to the amino acid sequences shown in SEQ ID NOs: 138 to 160, respectively. As is clear from Tables 14 to 16, no identical clone exists. This indicates that the increase in the expression level of Vβ2.1 and Vβ6.5 positive T cells in the onset stage of Kawasaki disease is polyclonal.
【0029】IV.各種サイトカインの測定 川崎病(発症期及び回復期)及び他の疾患群の血清につ
いて、Quantikine immunoassay kit (R & D system
社)、Cytoscreen immunoassay kit (BioSouce Internat
ional 社)、Human Interferon Gamma ELISA test kit(B
RL社)を用い、IL−1β、IL−2、IL−6、IL
−8、IL−10、IFNγ、G−CSF、TNF−α
量を測定した。結果を表17に示す。表17 川崎病の発症期及び回復期、及び他の疾患の患
者の、血清サイトカイン濃度IV. Measurement of various cytokines Serum from Kawasaki disease (onset and recovery) and other disease groups was analyzed using the Quantikine immunoassay kit (R & D system).
), Cytoscreen immunoassay kit (BioSouce Internat
ional), Human Interferon Gamma ELISA test kit (B
RL), IL-1β, IL-2, IL-6, IL
-8, IL-10, IFNγ, G-CSF, TNF-α
The amount was measured. The results are shown in Table 17. Table 17 Serum cytokine levels in patients with Kawasaki disease onset and recovery, and other diseases
【表17】 注:a)検出可能なサイトカイン濃度以下の血清濃度を
ゼロとして、平均値を算出した。表から、各種炎症性の
サイトカイン産生もTCR Vβ2.1やVβ6.5同様、
川崎病回復期に比べ発症期において高い傾向が見られ
る。また、他の疾患群と比較した場合も、多くのサイト
カインが、その発症期で高い傾向にあることが分かる。
この結果は、末梢血中でT細胞が増加していることを示
している。以上の結果は、川崎病の発症には、スーパー
抗原が関与していること、さらには、A群連鎖球菌由来
の外毒素 (SPEC)の関与を示唆するものである。[Table 17] Notes: a) The mean value was calculated with the serum concentration below the detectable cytokine concentration as zero. From the table, the production of various inflammatory cytokines was similar to that of TCR Vβ2.1 and Vβ6.5.
A higher tendency is seen in the onset period than in the Kawasaki disease recovery period. Also, when compared with other disease groups, it can be seen that many cytokines tend to be high in the onset period.
This result indicates that T cells are increased in peripheral blood. The above results suggest that superantigens are involved in the development of Kawasaki disease, and that exotoxins (SPEC) derived from group A streptococci are involved.
【0030】[0030]
【産業上の有用性】原因が未解明であり診断の確定に時
間を要するために、重篤な症状に至る例が多い川崎病に
関連する因子を、その発症期に科学的な方法で検出する
手段を提供することにより、川崎病の早期診断、早期治
療を可能にすることができる。[Industrial usefulness] Factors related to Kawasaki disease that often lead to serious symptoms are detected by scientific methods during the onset period because the cause is unknown and it takes time to confirm the diagnosis. By providing means for performing the above, early diagnosis and early treatment of Kawasaki disease can be made possible.
【0031】[0031]
【配列表】 <110> 塩野義製薬株式会社 Shionogi & Co., Ltd. <120> 川崎病因子の検出法 <130> 155380 <160> 160 <210> 1 <211> 10 <212> DNA <213> Artificial Sequence <400> 1 gggaattcgg 10 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <400> 2 taatacgact ccgaattccc 20 <210> 3 <211> 45 <212> DNA <213> Artificial Sequence <400> 3 gactagtcaa agcggccgcg agctcttttt tttttttttt ttttt 45 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <400> tgttgaaggc gtttgcacat gca 23 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <400> 5 gtccatagac ctcatgtcta gca 3 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <400> 6 actttgtgac acatttgttt gag 23 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <400> 7 gaactggact tgacagcgga agt 23 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <400> 8 aggcagtatc tggagtcatt gag 23 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <400> 9 actgtgcacc tccttcccat tca 23 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <400> 10 tcatcaacca tgcaagcctg a 21 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <400> 11 aaggctgctc agtgatcggt 20 <210> 12 <211> 23 <212> DNA <213> Artificial Sequence <400> 12 gaaacaagtt ggtggtcata tta 23 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <400> 13 aaaggagaag cgatcggtaa c 21 <210> 14 <211> 23 <212> DNA <213> Artificial Sequence <400> 14 gacactgtat attcaaatcc aga 23 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <400> 15 atcaagggct ttgaggctga a 21 <210> 16 <211> 22 <212> DNA <213> Artificial Sequence <400> 16 atcaacggtt ttgaggctga at 22 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <400> 17 gcattaaagg ctttgaggct g 21 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <400> 18 catcacggtt ttgaggctga a 21 <210> 19 <211> 21 <212> DNA <213> Artificial Sequence <400> 19 acagctcaat aaagccagcc a 21 <210> 20 <211> 21 <212> DNA <213> Artificial Sequence <400> 20 cacaggtcga taaatccagc a 21 <210> 21 <211> 21 <212> DNA <213> Artificial Sequence <400> 21 agagccagcc agtatatttc c 21 <210> 22 <211> 21 <212> DNA <213> Artificial Sequence <400> 22 gtggaagatt aagagtcacg c 21 <210> 23 <211> 21 <212> DNA <213> Artificial Sequence <400> 23 aacaacagaa tggcctctct g 21 <210> 24 <211> 21 <212> DNA <213> Artificial Sequence <400> 24 cctctctgat catcacagaa g 21 <210> 25 <211> 21 <212> DNA <213> Artificial Sequence <400> 25 ctgaaggtca cctttgatac c 21 <210> 26 <211> 21 <212> DNA <213> Artificial Sequence <400> 26 tgacaccagt gatccaagtt a 21 <210> 27 <211> 22 <212> DNA <213> Artificial Sequence <400> 27 attccttagt cgctctgata gt 22 <210> 28 <211> 21 <212> DNA <213> Artificial Sequence <400> 28 ttccttagtc ggtctaaagg g 21 <210> 29 <211> 21 <212> DNA <213> Artificial Sequence <400> 29 tgggcgaaaa gaaagaccaa c 21 <210> 30 <211> 21 <212> DNA <213> Artificial Sequence <400> 30 ggccaaagag tcaccgtttt a 21 <210> 31 <211> 21 <212> DNA <213> Artificial Sequence <400> 31 catcaaaggc ttcactgctg a 21 <210> 32 <211> 21 <212> DNA <213> Artificial Sequence <400> 32 gtggagaagt gaagaagctg a 21 <210> 33 <211> 21 <212> DNA <213> Artificial Sequence <400> 33 agggacgata caacatgacc t 21 <210> 34 <211> 21 <212> DNA <213> Artificial Sequence <400> 34 gtcggtattc ttggaacttc c 21 <210> 35 <211> 21 <212> DNA <213> Artificial Sequence <400> 35 aagattaagc gccacgactg t 21 <210> 36 <211> 21 <212> DNA <213> Artificial Sequence <400> 36 cgtttctctg tgaacttcca g 21 <210> 37 <211> 21 <212> DNA <213> Artificial Sequence <400> 37 gaccaaagac tcactgttct a 21 <210> 38 <211> 21 <212> DNA <213> Artificial Sequence <400> 38 cagctatggc tttgaagctg a 21 <210> 39 <211> 22 <212> DNA <213> Artificial Sequence <400> 39 gaaggaagat tcacaatctc ct 22 <210> 40 <211> 21 <212> DNA <213> Artificial Sequence <400> 40 ggacgatata gtgccactct t 21 <210> 41 <211> 22 <212> DNA <213> Artificial Sequence <400> 41 gcatggaaga ttaattgcca ca 22 <210> 42 <211> 23 <212> DNA <213> Artificial Sequence <400> 42 gactatacta acagcatgtt tga 23 <210> 43 <211> 21<212> DNA <213> Artificial Sequence <400> 43 aacaaaagtg ccaagcacct c 21 <210> 44 <211> 21 <212> DNA <213> Artificial Sequence <400> 44 tgacaaggga agcaacaaag g 21 <210> 45 <211> 21 <212> DNA <213> Artificial Sequence <400> 45 aagtggaaga cttaatgccc c 21 <210> 46 <211> 21 <212> DNA <213> Artificial Sequence <400> 46 caactctgga tgcagacaca a 21 <210> 47 <211> 21 <212> DNA <213> Artificial Sequence <400> 47 aagactgact gctcagtttg g 21 <210> 48 <211> 22 <212> DNA <213> Artificial Sequence <400> 48 agataactgc caagttggat ga 22 <210> 49 <211> 21 <212> DNA <213> Artificial Sequence <400> 49 ggacgattaa tggcctcact t 21 <210> 50 <211> 22 <212> DNA <213> Artificial Sequence <400> 50 gtcaggaaga ctaagtagca ta 22 <210> 51 <211> 20 <212> DNA <213> Artificial Sequence <400> 51 ggagaacaga tgcgtcgtga 20 <210> 52 <211> 21 <212> DNA <213> Artificial Sequence <400> 52 tggagaacag aagggtcatg a 21 <210> 53 <211> 22 <212> DNA <213> Artificial Sequence <400> 53 tgaagaagca gaaaagactg ac 22 <210> 54 <211> 21 <212> DNA <213> Artificial Sequence <400> 54 ctgtattcag ctggggaaga a 21 <210> 55 <211> 22 <212> DNA <213> Artificial Sequence <400> 55 tcagagagag acaatggaaa ac 22 <210> 56 <211> 20 <212> DNA <213> Artificial Sequence <400> 56 tatccagaac cctgaccctg 20 <210> 57 <211> 21 <212> DNA <213> Artificial Sequence <400> 57 tgtaccagct gagagactct a 21 <210> 58 <211> 21 <212> DNA <213> Artificial Sequence <400> 58 acaacagttc cctgacttgc a 21 <210> 59 <211> 21 <212> DNA <213> Artificial Sequence <400> 59 ccatcaacca tccaaacctg a 21 <210> 60 <211> 21 <212> DNA <213> Artificial Sequence <400> 60 gtctctagag agaagaagga g 21 <210> 61 <211> 21 <212> DNA <213> Artificial Sequence <400> 61 ttgacaagtt tcccatcagc c 21 <210> 62 <211> 21 <212> DNA <213> Artificial Sequence <400> 62 cttcagtgag acacagagaa a 21 <210> 63 <211> 22 <212> DNA <213> Artificial Sequence <400> 63 attatgagga ggaagagaga ca 22 <210> 64 <211> 23 <212> DNA <213> Artificial Sequence <400> 64 tattatgaga aagaagagag agg 23 <210> 65 <211> 21 <212> DNA <213> Artificial Sequence <400> 65 tgacgagggt gaagagagaa a 21 <210> 66 <211> 22 <212> DNA <213> Artificial Sequence <400> 66 attataggga ggaagagaat gg 22 <210> 67 <211> 21 <212> DNA <213> Artificial Sequence <400> 67 acgatcggtt ctttgcagtc a 21 <210> 68 <211> 21 <212> DNA <213> Artificial Sequence <400> 68 tcaactagac aaatcggggc t 21 <210> 69 <211> 21 <212> DNA <213> Artificial Sequence <400> 69 ccaacaagac aaatcagggc t 21 <210> 70 <211> 20 <212> DNA <213> Artificial Sequence <400> 70 tcgcttctct gcagagagga 20 <210> 71 <211> 21 <212> DNA <213> Artificial Sequence <400> 71 aactggaaaa atcagggctg c 21 <210> 72 <211> 20<212> DNA <213> Artificial Sequence <400> 72 ctgaatgccc caacagctct 20 <210> 73 <211> 23 <212> DNA <213> Artificial Sequence <400> 73 tttactttaa caacaacgtt ccg 23 <210> 74 <211> 20 <212> DNA <213> Artificial Sequence <400> 74 aaggactgga gttgctggct 20 <210> 75 <211> 22 <212> DNA <213> Artificial Sequence <400> 75 aaacagttcc aaatcgcttc tc 22 <210> 76 <211> 22 <212> DNA <213> Artificial Sequence <400> 76 ccaaaaactc atcctgtacc tt 22 <210> 77 <211> 22 <212> DNA <213> Artificial Sequence <400> 77 tcaacagtct ccagaataag ga 22 <210> 78 <211> 21 <212> DNA <213> Artificial Sequence <400> 78 caaaggagaa gtctcagatg g 21 <210> 79 <211> 21 <212> DNA <213> Artificial Sequence <400> 79 caatggctac aatgtctcca g 21 <210> 80 <211> 20 <212> DNA <213> Artificial Sequence <400> 80 ggtccctgat ggctacaatg 20 <210> 81 <211> 21 <212> DNA <213> Artificial Sequence <400> 81 tgatggttat agtgtctcca g 21 <210> 82 <211> 20 <212> DNA <213> Artificial Sequence <400> 82 ccgaatggct acaacgtctc 20 <210> 83 <211> 21 <212> DNA <213> Artificial Sequence <400> 83 gtctctcgaa aagagaagag g 21 <210> 84 <211> 20 <212> DNA <213> Artificial Sequence <400> 84 agtgtctctc gacaggcaca 20 <210> 85 <211> 21 <212> DNA <213> Artificial Sequence <400> 85 gtgaaagagt ctaaacagga t 21 <210> 86 <211> 23 <212> DNA <213> Artificial Sequence <400> 86 cacagatagt aaatgacttt cag 23 <210> 87 <211> 21 <212> DNA <213> Artificial Sequence <400> 87 gatgagtcag gaatgccaaa g 21 <210> 88 <211> 20 <212> DNA <213> Artificial Sequence <400> 88 ctcaatgccc caagaacgca 20 <210> 89 <211> 20 <212> DNA <213> Artificial Sequence <400> 89 tctgaggtgc cccagaatct 20 <210> 90 <211> 20 <212> DNA <213> Artificial Sequence <400> 90 tctgcagaga ggctcaaagg 20 <210> 91 <211> 23 <212> DNA <213> Artificial Sequence <400> 91 ttcagtgact atcattctga act 23 <210> 92 <211> 21 <212> DNA <213> Artificial Sequence <400> 92 attcttgggg cagaaagtcg a 21 <210> 93 <211> 19 <212> DNA <213> Artificial Sequence <400> 93 ccaggaggcc gaacacttc 19 <210> 94 <211> 21 <212> DNA <213> Artificial Sequence <400> 94 aacaggtatg cccaaggaaa g 21 <210> 95 <211> 19 <212> DNA <213> Artificial Sequence <400> 95 cacccgaggt cgctgtgtt 19 <210> 96 <211> 21 <212> DNA <213> Artificial Sequence <400> 96 gctgtgtttg agccatcaga a 21 <210> 97 <211> 27 <212> PRT <213> Homo sapiens <400> 97 Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala Arg Val Gly Asn Gly Tyr 1 5 10 15 Thr Phe Gly Ser Gly Thr Arg Leu Thr Val Val 20 25 <210> 98 <211> 30 <212> PRT <213> Homo sapiens <400> 98 Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala Arg Gly Asp Glu Ala Ile 1 5 10 15 Thr Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr Val Leu 20 25 30 <210> 99 <211> 31 <212> PRT <213> Homo sapiens <400> 99 Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala Arg Asp Pro Thr Gly Arg 1 5 10 15 His Asn Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr Val Leu 20 25 30 <210> 100 <211> 29 <212> PRT <213> Homo sapiens <400> 100 Glu Asp Ser Ser Phe Tyr Ile Cys Arg Pro Ser Ser Gly Ser Tyr Asn 1 5 10 15 Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr Val Leu 20 25 <210> 101 <211> 34 <212> PRT <213> Homo sapiens <400> 101 Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala Arg Gly Leu Ala Gly Thr 1 5 10 15 Ser Ser Ser Tyr Asn Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr 20 25 30 Val Leu <210> 102 <211> 34 <212> PRT <213> Homo sapiens <400> 102 Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala Ile Gly Thr Thr Ser Gly 1 5 10 15 Ser His Arg Thr Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr 20 25 30 Val Leu <210> 103 <211> 26 <212> PRT <213> Homo sapiens <400> 103 Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala Ser Gly Glu Thr Gln Tyr 1 5 10 15 Phe Gly Pro Gly Thr Arg Leu Leu Val Leu 20 25 <210> 104 <211> 30 <212> PRT <213> Homo sapiens <400> 104 Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala Pro Ser Gly Arg Ala Gln 1 5 10 15 Glu Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu Leu Val Leu 20 25 30 <210> 105 <211> 28 <212> PRT <213> Homo sapiens <400> 105 Glu Asp Ser Ser Phe Tyr Ile Cys Ser Val Gly Gly Gln Ser Tyr Glu 1 5 10 15 Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Thr 20 25 <210> 106 <211> 28 <212> PRT <213> Homo sapiens <400> 106 Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala Ala Gln Asp Val Tyr Glu 1 5 10 15 Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Thr 20 25 <210> 107 <211> 29 <212> PRT <213> Homo sapiens <400> 107 Glu Asp Ser Ser Phe Tyr Ile Cys Ser Asp Thr Arg Gly Met Asn Thr 1 5 10 15 Glu Ala Phe Phe Gly Gln Gly Thr Arg Leu Thr Val Val 20 25 <210> 108 <211> 31 <212> PRT <213> Homo sapiens <400> 108 Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala Arg Gly Ala Asp Arg Tyr 1 5 10 15 Asn Thr Glu Ala Phe Phe Gly Gln Gly Thr Arg Leu Thr Val Val 20 25 30 <210> 109 <211> 29 <212> PRT <213> Homo sapiens <400> 109 Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala Arg Glu Thr Ser Asn Thr 1 5 10 15 Glu Ala Phe Phe Gly Gln Gly Thr Arg Leu Thr Val Val 20 25 <210> 110 <211> 28 <212> PRT <213> Homo sapiens <400> 110 Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala Arg Phe Ser Ser Gly Gly 1 5 10 15 Tyr Thr Phe Gly Ser Gly Thr Arg Leu Thr Val Val 20 25 <210> 111 <211> 30 <212> PRT <213> Homo sapiens <400> 111 Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala Asn Leu Ala Gly Val Gly 1 5 10 15 Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Leu 20 25 30 <210> 112 <211> 30 <212> PRT <213> Homo sapiens <400> 112 Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala Arg Val Ser Gly Arg Thr 1 5 10 15 Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Leu 20 25 30 <210> 113 <211> 31 <212> PRT <213> Homo sapiens <400> 113 Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala Tyr Thr Ser Gly Arg Ser 1 5 10 15 Lys Asn Ile Gln Tyr Phe Gly Ala Gly Thr Arg Leu Ser Val Leu 20 25 30 <210> 114 <211> 29 <212> PRT <213> Homo sapiens <400> 114 Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala Arg Gly Leu Ala Val Glu 1 5 10 15 Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu Leu Val Leu 20 25 <210> 115 <211> 31 <212> PRT <213> Homo sapiens <400> 115 Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala Arg Pro Ile Val Gly Gly 1 5 10 15 Ser His Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Thr 20 25 30 <210> 116 <211> 30 <212> PRT <213> Homo sapiens <400> 116 Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala Arg Gly Pro Gly Thr Ser 1 5 10 15 Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Thr 20 25 30 <210> 117 <211> 30 <212> PRT <213> Homo sapiens <400> 117 Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala Thr Ser Ser Gly Ser Ser 1 5 10 15 Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Thr 20 25 30 <210> 118 <211> 29 <212> PRT <213> Homo sapiens <400> 118 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Glu Asp Val Gly Thr 1 5 10 15 Glu Ala Phe Phe Gly Gln Gly Thr Arg Leu Thr Val Val 20 25 <210> 119 <211> 32 <212> PRT <213> Homo sapiens <400> 119 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Pro Thr Ser Gly Gly 1 5 10 15 Ala Trp Asn Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr Val Leu 20 25 30 <210> 120 <211> 29 <212> PRT <213> Homo sapiens <400> 120 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ile Lys Thr Asp Pro Arg Arg 1 5 10 15 Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr Val Leu 20 25 <210> 121 <211> 28 <212> PRT <213> Homo sapiens <400> 121 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Arg Ser Gly Gly Gly Glu 1 5 10 15 Leu Phe Phe Gly Glu Gly Ser Arg Leu Thr Val Leu 20 25 <210> 122 <211> 33 <212> PRT <213> Homo sapiens <400> 122 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Leu Ala Ala Gln Gly 1 5 10 15 Gln Gly Lys Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val 20 25 30 Leu <210> 123 <211> 31 <212> PRT <213> Homo sapiens <400> 123 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Arg Leu Ala Gly Ala Phe 1 5 10 15 Thr Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Leu 20 25 30 <210> 124 <211> 28 <212> PRT <213> Homo sapiens <400> 124 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Phe Ile Thr Asp Thr 1 5 10 15 Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Leu 20 25 <210> 125 <211> 32 <212> PRT <213> Homo sapiens <400> 125 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Leu Gly Gly Gly Gly 1 5 10 15 Arg Val Ala Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Thr 20 25 30 <210> 126 <211> 31 <212> PRT <213> Homo sapiens <400> 126 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Leu Ala Gly Gln Glu 1 5 10 15 Asn Asn Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr Val Leu 20 25 30 <210> 127 <211> 32 <212> PRT <213> Homo sapiens <400> 127 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Leu Arg Ala Ser Gly 1 5 10 15 Arg Ser Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Thr 20 25 30 <210> 128 <211> 27 <212> PRT <213> Homo sapiens <400> 128 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Pro Glu Leu Gly Phe 1 5 10 15 Thr Phe Gly Ser Gly Thr Arg Leu Thr Val Val 20 25 <210> 129 <211> 29 <212> PRT <213> Homo sapiens <400> 129 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Leu Phe Gly Gly Glu 1 5 10 15 Lys Leu Phe Phe Gly Ser Gly Thr Gln Leu Ser Val Leu 20 25 <210> 130 <211> 29 <212> PRT <213> Homo sapiens <400> 130 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Leu Gly Arg Asp Gln 1 5 10 15 Pro Gln His Phe Gly Asp Gly Thr Arg Leu Ser Ile Leu 20 25 <210> 131 <211> 30 <212> PRT <213> Homo sapiens <400> 131 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Pro Gln Ser Gly His Ala 1 5 10 15 Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Leu 20 25 30 <210> 132 <211> 28 <212> PRT <213> Homo sapiens <400> 132 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Leu Phe Thr Asp Thr 1 5 10 15 Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Leu 20 25 <210> 133 <211> 26 <212> PRT <213> Homo sapiens <400> 133 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Ser Pro Pro Gln Tyr 1 5 10 15 Phe Gly Pro Gly Thr Arg Leu Leu Val Leu 20 25 <210> 134 <211> 27 <212> PRT <213> Homo sapiens <400> 134 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Phe Lys Trp Thr Gln 1 5 10 15 Tyr Phe Gly Pro Gly Thr Arg Leu Leu Val Leu 20 25 <210> 135 <211> 27 <212> PRT <213> Homo sapiens <400> 135 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Arg Glu Gly Gly Gln 1 5 10 15 Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Thr 20 25 <210> 136 <211> 31 <212> PRT <213> Homo sapiens <400> 136 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Phe Gly Ser Leu Ser 1 5 10 15 Ser Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Thr 20 25 30 <210> 137 <211> 30 <212> PRT <213> Homo sapiens <400> 137 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Pro Ala Gln Gly Ala 1 5 10 15 Ala Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Thr 20 25 30 <210> 138 <211> 30 <212> PRT <213> Homo sapiens <400> 138 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Gly Ser Leu Val Asp 1 5 10 15 Gln Pro Gln His Phe Gly Asp Gly Thr Arg Leu Ser Ile Leu 20 25 30 <210> 139 <211> 30 <212> PRT <213> Homo sapiens <400> 139 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Gln Gln Gly Gly Gly 1 5 10 15 Asn Thr Ile Tyr Phe Gly Glu Gly Ser Trp Leu Thr Val Val 20 25 30 <210> 140 <211> 31 <212> PRT <213> Homo sapiens <400> 140 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Leu Gly Ser Leu Val 1 5 10 15 Asp Gln Pro Gln His Phe Gly Asp Gly Thr Arg Leu Ser Ile Leu 20 25 30 <210> 141 <211> 30 <212> PRT <213> Homo sapiens <400> 141 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Leu Ile Gly Ala Ala 1 5 10 15 His Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr Val Leu 20 25 30 <210> 142 <211> 27 <212> PRT <213> Homo sapiens <400> 142 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Leu Asp Glu Arg Glu 1 5 10 15 Phe Phe Gly Pro Gly Thr Arg Leu Thr Val Leu 20 25 <210> 143 <211> 31 <212> PRT <213> Homo sapiens <400> 143 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Leu Gly Ser Thr Arg 1 5 10 15 Gly Asp Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr Val Leu 20 25 30 <210> 144 <211> 31 <212> PRT <213> Homo sapiens <400> 144 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Gln Gly Gly Gly Gly 1 5 10 15 Thr Gly Glu Leu Phe Phe Gly Glu Gly Ser Arg Leu Thr Val Leu 20 25 30 <210> 145 <211> 33 <212> PRT <213> Homo sapiens <400> 145 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Leu Ala Leu Val Arg 1 5 10 15 Asp Tyr Ser Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val 20 25 30 Thr <210> 146 <211> 30 <212> PRT <213> Homo sapiens <400> 146 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Leu Thr Gly Gly Gly 1 5 10 15 Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Leu 20 25 30 <210> 147 <211> 30 <212> PRT <213> Homo sapiens <400> 147 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Leu Thr Thr Asp Ala 1 5 10 15 Glu Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu Leu Val Leu 20 25 30 <210> 148 <211> 31 <212> PRT <213> Homo sapiens <400> 148 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Ala Ile Ala Gly Ala 1 5 10 15 Tyr Asn Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr Val Leu 20 25 30 <210> 149 <211> 30 <212> PRT <213> Homo sapiens <400> 149 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Ala Gly Gln Gly Ser 1 5 10 15 Thr Glu Ala Phe Phe Gly Gln Gly Thr Arg Leu Thr Val Val 20 25 30 <210> 150 <211> 31 <212> PRT <213> Homo sapiens <400> 150 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Ser Leu Val Gly Gly 1 5 10 15 Arg Gly Glu Ala Phe Phe Gly Gln Gly Thr Arg Leu Thr Val Val 20 25 30 <210> 151 <211> 30 <212> PRT <213> Homo sapiens <400> 151 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Phe Leu Gly Gln Asn 1 5 10 15 Tyr Gly Tyr Thr Phe Gly Ser Gly Thr Arg Leu Thr Val Val 20 25 30 <210> 152 <211> 27 <212> PRT <213> Homo sapiens <400> 152 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Leu Val Asp Thr Ile 1 5 10 15 Tyr Phe Gly Glu Gly Ser Trp Leu Thr Val Val 20 25 <210> 153 <211> 32 <212> PRT <213> Homo sapiens <400> 153 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Pro Gly Thr Ser Gly 1 5 10 15 Ser Tyr Asn Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr Val Leu 20 25 30 <210> 154 <211> 31 <212> PRT <213> Homo sapiens <400> 154 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Phe Gly Arg Gly Asn 1 5 10 15 Thr Gly Glu Leu Phe Phe Gly Glu Gly Ser Arg Leu Thr Val Leu 20 25 30 <210> 155 <211> 33 <212> PRT <213> Homo sapiens <400> 155 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Leu Ala Ile Arg Gly 1 5 10 15 Ala Ser Thr Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val 20 25 30 Leu <210> 156 <211> 31 <212> PRT <213> Homo sapiens <400> 156 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Leu Gly Thr Ser Gly 1 5 10 15 Ala Glu Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu Leu Val Leu 20 25 30 <210> 157 <211> 31 <212> PRT <213> Homo sapiens <400> 157 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Leu Gly Val Thr Pro 1 5 10 15 Gln Glu Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu Leu Val Leu 20 25 30 <210> 158 <211> 36 <212> PRT <213> Homo sapiens <400> 158 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Ile Pro Arg Leu Gly 1 5 10 15 Gln Gly Ala Ser Gly Ala Asn Val Leu Thr Phe Gly Ala Gly Ser Arg 20 25 30 Leu Thr Val Leu 35 <210> 159 <211> 30 <212> PRT <213> Homo sapiens <400> 159 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Leu Gly Thr Gly Gly 1 5 10 15 Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Thr 20 25 30 <210> 160 <211> 32 <212> PRT <213> Homo sapiens <400> 160 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Ser Ile Pro Gly Leu 1 5 10 15 Arg Asn Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Thr 20 25 30[Sequence List] <110> Shionogi & Co., Ltd. <120> Detection method of Kawasaki disease factor <130> 155 380 <160> 160 <210> 1 <211> 10 <212> DNA <213 > Artificial Sequence <400> 1 gggaattcgg 10 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <400> 2 taatacgact ccgaattccc 20 <210> 3 <211> 45 <212> DNA <213> Artificial Sequence <400> 3 gactagtcaa agcggccgcg agctcttttt tttttttttt ttttt 45 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <400> tgttgaaggc gtttgcacat gca 23 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <400> 5 gtccatagac ctcatgtcta gca 3 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <400> 6 actttgtgac acatttgttt gag 23 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <400> 7 gaactggact tgacagcgga agt 23 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <400> 8 aggcagtatc tggagtcatt gag 23 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <400> 9 actgtgcacc tccttcccat tca 23 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <400> 10 tcatcaacca tgcaagcctg a 21 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <400> 11 aaggctgctc agtgatcggt 20 <210> 12 <211> 23 <212> DNA <213> Artificial Sequence <400> 12 gaaacaagtt ggtggtcata tta 23 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <400> 13 aaaggagaag cgatcggtaa c 21 <210> 14 <211> 23 <212> DNA <213> Artificial Sequence <400> 14 gacactgtat attcaaatcc aga 23 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <400> 15 atcaagggct ttgaggctga a 21 <210> 16 <211> 22 <212> DNA <213> Artificial Sequence <400> 16 atcaacggtt ttgaggctga at 22 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <400> 17 gcattaaagg ctttgaggct g 21 <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <400> 18 catcacggtt ttgaggctga a 21 <210> 19 <211> 21 <212> DNA <213> Artificial Sequence <400> 19 acagctcaat aaagccagcc a 21 <210> 20 <211> 21 <212> DNA <213> Artificial Sequence <400> 20 cacaggtcga taaatccagc a 21 <210> 21 <211> 21 <212> DNA <213> Artificial Sequence <400> 21 agag ccagcc agtatatttc c 21 <210> 22 <211> 21 <212> DNA <213> Artificial Sequence <400> 22 gtggaagatt aagagtcacg c 21 <210> 23 <211> 21 <212> DNA <213> Artificial Sequence <400> 23 aacaacagaa tggcctctct g 21 <210> 24 <211> 21 <212> DNA <213> Artificial Sequence <400> 24 cctctctgat catcacagaa g 21 <210> 25 <211> 21 <212> DNA <213> Artificial Sequence <400> 25 ctgaaggtca cctttgatac c 21 <210> 26 <211> 21 <212> DNA <213> Artificial Sequence <400> 26 tgacaccagt gatccaagtt a 21 <210> 27 <211> 22 <212> DNA <213> Artificial Sequence <400> 27 attccttagt cgctctgata gt 22 <210> 28 <211> 21 <212> DNA <213> Artificial Sequence <400> 28 ttccttagtc ggtctaaagg g 21 <210> 29 <211> 21 <212> DNA <213> Artificial Sequence <400> 29 tgggcgaaaa gaaagaccaa c 21 <210> 30 <211> 21 <212> DNA <213> Artificial Sequence <400> 30 ggccaaagag tcaccgtttt a 21 <210> 31 <211> 21 <212> DNA <213> Artificial Sequence <400> 31 catcaaaggc ttcactgctg a 21 <210> 32 <211> 21 <212> DNA <213> Artificial Sequence <400> 32 gtggagaagt ga agaagctg a 21 <210> 33 <211> 21 <212> DNA <213> Artificial Sequence <400> 33 agggacgata caacatgacc t 21 <210> 34 <211> 21 <212> DNA <213> Artificial Sequence <400> 34 gtcggtattc ttggaacttc c 21 <210> 35 <211> 21 <212> DNA <213> Artificial Sequence <400> 35 aagattaagc gccacgactg t 21 <210> 36 <211> 21 <212> DNA <213> Artificial Sequence <400> 36 cgtttctctg tgaacttcca g 21 <210> 37 <211> 21 <212> DNA <213> Artificial Sequence <400> 37 gaccaaagac tcactgttct a 21 <210> 38 <211> 21 <212> DNA <213> Artificial Sequence <400> 38 cagctatggc tttgaagctg a 21 <210> 39 <211> 22 <212> DNA <213> Artificial Sequence <400> 39 gaaggaagat tcacaatctc ct 22 <210> 40 <211> 21 <212> DNA <213> Artificial Sequence <400> 40 ggacgatata gtgccactct t 21 <210> 41 <211> 22 <212> DNA <213> Artificial Sequence <400> 41 gcatggaaga ttaattgcca ca 22 <210> 42 <211> 23 <212> DNA <213> Artificial Sequence <400> 42 gactatacta acagcatgtt tga 23 <210> 43 <211> 21 <212> DNA <213> Artificial Sequence <400> 43 aacaaaagtg ccaagcacc tc 21 <210> 44 <211> 21 <212> DNA <213> Artificial Sequence <400> 44 tgacaaggga agcaacaaag g 21 <210> 45 <211> 21 <212> DNA <213> Artificial Sequence <400> 45 aagtggaaga cttaatgccc c 21 <210> 46 <211> 21 <212> DNA <213> Artificial Sequence <400> 46 caactctgga tgcagacaca a 21 <210> 47 <211> 21 <212> DNA <213> Artificial Sequence <400> 47 aagactgact gctcagtttg g 21 <210> 48 <211> 22 <212> DNA <213> Artificial Sequence <400> 48 agataactgc caagttggat ga 22 <210> 49 <211> 21 <212> DNA <213> Artificial Sequence <400> 49 ggacgattaa tggcctcact t 21 <210> 50 <211> 22 <212> DNA <213> Artificial Sequence <400> 50 gtcaggaaga ctaagtagca ta 22 <210> 51 <211> 20 <212> DNA <213> Artificial Sequence <400> 51 ggagaacaga tgcgtcgtga 20 <210> 52 <211> 21 <212> DNA <213> Artificial Sequence <400> 52 tggagaacag aagggtcatg a 21 <210> 53 <211> 22 <212> DNA <213> Artificial Sequence <400> 53 tgaagaagca gaaaagactg ac 22 <210> 54 <211> 21 <212> DNA <213> Artificial Sequence <400> 54 ctgtattcag ctggggaaga a 21 <2 10> 55 <211> 22 <212> DNA <213> Artificial Sequence <400> 55 tcagagagag acaatggaaa ac 22 <210> 56 <211> 20 <212> DNA <213> Artificial Sequence <400> 56 tatccagaac cctgaccctg 20 <210 > 57 <211> 21 <212> DNA <213> Artificial Sequence <400> 57 tgtaccagct gagagactct a 21 <210> 58 <211> 21 <212> DNA <213> Artificial Sequence <400> 58 acaacagttc cctgacttgc a 21 <210 > 59 <211> 21 <212> DNA <213> Artificial Sequence <400> 59 ccatcaacca tccaaacctg a 21 <210> 60 <211> 21 <212> DNA <213> Artificial Sequence <400> 60 gtctctagag agaagaagga g 21 <210 > 61 <211> 21 <212> DNA <213> Artificial Sequence <400> 61 ttgacaagtt tcccatcagc c 21 <210> 62 <211> 21 <212> DNA <213> Artificial Sequence <400> 62 cttcagtgag acacagagaa a 21 <210 > 63 <211> 22 <212> DNA <213> Artificial Sequence <400> 63 attatgagga ggaagagaga ca 22 <210> 64 <211> 23 <212> DNA <213> Artificial Sequence <400> 64 tattatgaga aagaagagag agg 23 <210 > 65 <211> 21 <212> DNA <213> Artificial Sequence <400> 65 tgacgagggt gaagagagaa a 21 <210> 66 < 211> 22 <212> DNA <213> Artificial Sequence <400> 66 attataggga ggaagagaat gg 22 <210> 67 <211> 21 <212> DNA <213> Artificial Sequence <400> 67 acgatcggtt ctttgcagtc a 21 <210> 68 < 211> 21 <212> DNA <213> Artificial Sequence <400> 68 tcaactagac aaatcggggc t 21 <210> 69 <211> 21 <212> DNA <213> Artificial Sequence <400> 69 ccaacaagac aaatcagggc t 21 <210> 70 < 211> 20 <212> DNA <213> Artificial Sequence <400> 70 tcgcttctct gcagagagga 20 <210> 71 <211> 21 <212> DNA <213> Artificial Sequence <400> 71 aactggaaaa atcagggctg c 21 <210> 72 <211 > 20 <212> DNA <213> Artificial Sequence <400> 72 ctgaatgccc caacagctct 20 <210> 73 <211> 23 <212> DNA <213> Artificial Sequence <400> 73 tttactttaa caacaacgtt ccg 23 <210> 74 <211> 20 <212> DNA <213> Artificial Sequence <400> 74 aaggactgga gttgctggct 20 <210> 75 <211> 22 <212> DNA <213> Artificial Sequence <400> 75 aaacagttcc aaatcgcttc tc 22 <210> 76 <211> 22 <212> DNA <213> Artificial Sequence <400> 76 ccaaaaactc atcctgtacc tt 22 <210> 77 <211> 22 <212 > DNA <213> Artificial Sequence <400> 77 tcaacagtct ccagaataag ga 22 <210> 78 <211> 21 <212> DNA <213> Artificial Sequence <400> 78 caaaggagaa gtctcagatg g 21 <210> 79 <211> 21 <212 > DNA <213> Artificial Sequence <400> 79 caatggctac aatgtctcca g 21 <210> 80 <211> 20 <212> DNA <213> Artificial Sequence <400> 80 ggtccctgat ggctacaatg 20 <210> 81 <211> 21 <212> DNA <213> Artificial Sequence <400> 81 tgatggttat agtgtctcca g 21 <210> 82 <211> 20 <212> DNA <213> Artificial Sequence <400> 82 ccgaatggct acaacgtctc 20 <210> 83 <211> 21 <212> DNA <213> Artificial Sequence <400> 83 gtctctcgaa aagagaagag g 21 <210> 84 <211> 20 <212> DNA <213> Artificial Sequence <400> 84 agtgtctctc gacaggcaca 20 <210> 85 <211> 21 <212> DNA < 213> Artificial Sequence <400> 85 gtgaaagagt ctaaacaggat 21 <210> 86 <211> 23 <212> DNA <213> Artificial Sequence <400> 86 cacagatagt aaatgacttt cag 23 <210> 87 <211> 21 <212> DNA < 213> Artificial Sequence <400> 87 gatgagtcag gaatgccaaa g 21 <210> 88 <211> 20 <212> DNA <213> A rtificial Sequence <400> 88 ctcaatgccc caagaacgca 20 <210> 89 <211> 20 <212> DNA <213> Artificial Sequence <400> 89 tctgaggtgc cccagaatct 20 <210> 90 <211> 20 <212> DNA <213> Artificial Sequence <400> 90 tctgcagaga ggctcaaagg 20 <210> 91 <211> 23 <212> DNA <213> Artificial Sequence <400> 91 ttcagtgact atcattctga act 23 <210> 92 <211> 21 <212> DNA <213> Artificial Sequence < 400> 92 attcttgggg cagaaagtcg a 21 <210> 93 <211> 19 <212> DNA <213> Artificial Sequence <400> 93 ccaggaggcc gaacacttc 19 <210> 94 <211> 21 <212> DNA <213> Artificial Sequence <400 > 94 aacaggtatg cccaaggaaa g 21 <210> 95 <211> 19 <212> DNA <213> Artificial Sequence <400> 95 cacccgaggt cgctgtgtt 19 <210> 96 <211> 21 <212> DNA <213> Artificial Sequence <400> 96 gctgtgtttg agccatcaga a 21 <210> 97 <211> 27 <212> PRT <213> Homo sapiens <400> 97 Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala Arg Val Gly Asn Gly Tyr 1 5 10 15 Thr Phe Gly Ser Gly Thr Arg Leu Thr Val Val 20 25 <210> 98 <211> 30 <212> PRT <213> Homo sapiens <400> 98 Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala Arg Gly Asp Glu Ala Ile 1 5 10 15 Thr Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr Val Leu 20 25 30 <210> 99 <211> 31 <212> PRT < 213> Homo sapiens <400> 99 Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala Arg Asp Pro Thr Gly Arg 1 5 10 15 His Asn Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr Val Leu 20 25 30 <210> 100 <211> 29 <212> PRT <213> Homo sapiens <400> 100 Glu Asp Ser Ser Phe Tyr Ile Cys Arg Pro Ser Ser Gly Ser Tyr Asn 1 5 10 15 Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr Val Leu 20 25 <210> 101 <211> 34 <212> PRT <213> Homo sapiens <400> 101 Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala Arg Gly Leu Ala Gly Thr 1 5 10 15 Ser Ser Ser Tyr Asn Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr 20 25 30 Val Leu <210> 102 <211> 34 <212> PRT <213> Homo sapiens <400> 102 Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala Ile Gly Thr Thr Ser Gly 1 5 10 15 Ser His Arg Thr Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr 20 25 30 Val Leu <210> 103 <211> 26 <212> PRT <213> Homo sapie ns <400> 103 Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala Ser Gly Glu Thr Gln Tyr 1 5 10 15 Phe Gly Pro Gly Thr Arg Leu Leu Val Leu 20 25 <210> 104 <211> 30 <212> PRT < 213> Homo sapiens <400> 104 Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala Pro Ser Gly Arg Ala Gln 1 5 10 15 Glu Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu Leu Val Leu 20 25 30 <210> 105 < 211> 28 <212> PRT <213> Homo sapiens <400> 105 Glu Asp Ser Ser Phe Tyr Ile Cys Ser Val Gly Gly Gln Ser Tyr Glu 1 5 10 15 Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Thr 20 25 <210> 106 <211> 28 <212> PRT <213> Homo sapiens <400> 106 Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala Ala Gln Asp Val Tyr Glu 1 5 10 15 Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Thr 20 25 <210> 107 <211> 29 <212> PRT <213> Homo sapiens <400> 107 Glu Asp Ser Ser Phe Tyr Ile Cys Ser Asp Thr Arg Gly Met Asn Thr 1 5 10 15 Glu Ala Phe Phe Gly Gln Gly Thr Arg Leu Thr Val Val 20 25 <210> 108 <211> 31 <212> PRT <213> Homo sapiens <400> 108 Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala Arg Gly Al a Asp Arg Tyr 1 5 10 15 Asn Thr Glu Ala Phe Phe Gly Gln Gly Thr Arg Leu Thr Val Val 20 25 30 <210> 109 <211> 29 <212> PRT <213> Homo sapiens <400> 109 Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala Arg Glu Thr Ser Asn Thr 1 5 10 15 Glu Ala Phe Phe Gly Gln Gly Thr Arg Leu Thr Val Val 20 25 <210> 110 <211> 28 <212> PRT <213> Homo sapiens < 400> 110 Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala Arg Phe Ser Ser Gly Gly 1 5 10 15 Tyr Thr Phe Gly Ser Gly Thr Arg Leu Thr Val Val 20 25 <210> 111 <211> 30 <212> PRT < 213> Homo sapiens <400> 111 Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala Asn Leu Ala Gly Val Gly 1 5 10 15 Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Leu 20 25 30 <210> 112 < 211> 30 <212> PRT <213> Homo sapiens <400> 112 Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala Arg Val Ser Gly Arg Thr 1 5 10 15 Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Leu 20 25 30 <210> 113 <211> 31 <212> PRT <213> Homo sapiens <400> 113 Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala Tyr Thr Ser Gly Arg Ser 1 5 10 15 Lys A sn Ile Gln Tyr Phe Gly Ala Gly Thr Arg Leu Ser Val Leu 20 25 30 <210> 114 <211> 29 <212> PRT <213> Homo sapiens <400> 114 Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala Arg Gly Leu Ala Val Glu 1 5 10 15 Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu Leu Val Leu 20 25 <210> 115 <211> 31 <212> PRT <213> Homo sapiens <400> 115 Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala Arg Pro Ile Val Gly Gly 1 5 10 15 Ser His Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Thr 20 25 30 <210> 116 <211> 30 <212> PRT <213> Homo sapiens < 400> 116 Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala Arg Gly Pro Gly Thr Ser 1 5 10 15 Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Thr 20 25 30 <210> 117 <211> 30 <212 > PRT <213> Homo sapiens <400> 117 Glu Asp Ser Ser Phe Tyr Ile Cys Ser Ala Thr Ser Ser Gly Ser Ser 1 5 10 15 Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Thr 20 25 30 <210 > 118 <211> 29 <212> PRT <213> Homo sapiens <400> 118 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Glu Asp Val Gly Thr 1 5 10 15 Glu Ala Phe Phe Gly Gln Gly Thr Arg Leu Thr Val Val 20 25 <210> 119 <211> 32 <212> PRT <213> Homo sapiens <400> 119 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Pro Thr Ser Gly Gly 1 5 10 15 Ala Trp Asn Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr Val Leu 20 25 30 <210> 120 <211> 29 <212> PRT <213> Homo sapiens <400> 120 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ile Lys Thr Asp Pro Arg Arg 1 5 10 15 Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr Val Leu 20 25 <210> 121 <211> 28 <212> PRT <213> Homo sapiens <400> 121 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Arg Ser Gly Gly Gly Glu 1 5 10 15 Leu Phe Phe Gly Glu Gly Ser Arg Leu Thr Val Leu 20 25 <210> 122 <211> 33 <212> PRT <213> Homo sapiens <400 > 122 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Leu Ala Ala Gln Gly 1 5 10 15 Gln Gly Lys Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val 20 25 30 Leu <210> 123 <211> 31 <212> PRT <213> Homo sapiens <400> 123 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Arg Leu Ala Gly Ala Phe 1 5 10 15 Thr Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Leu 20 25 30 <210> 124 <211> 28 <212> PRT <213> Homo sapiens <400> 124 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Phe Ile Thr Asp Thr 1 5 10 15 Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Leu 20 25 <210> 125 <211> 32 <212> PRT <213> Homo sapiens <400> 125 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Leu Gly Gly Gly Gly 1 5 10 15 Arg Val Ala Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Thr 20 25 30 <210> 126 <211> 31 <212> PRT <213> Homo sapiens <400> 126 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Leu Ala Gly Gln Glu 1 5 10 15 Asn Asn Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr Val Leu 20 25 30 <210> 127 <211> 32 <212> PRT <213> Homo sapiens < 400> 127 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Leu Arg Ala Ser Gly 1 5 10 15 Arg Ser Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Thr 20 25 30 <210> 128 <211> 27 <212> PRT <213> Homo sapiens <400> 128 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Pro Glu Leu Gly Phe 1 5 10 15 Thr Phe Gly Ser Gly Thr Arg Leu Thr Val V al 20 25 <210> 129 <211> 29 <212> PRT <213> Homo sapiens <400> 129 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Leu Phe Gly Gly Glu 1 5 10 15 Lys Leu Phe Phe Gly Ser Gly Thr Gln Leu Ser Val Leu 20 25 <210> 130 <211> 29 <212> PRT <213> Homo sapiens <400> 130 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Leu Gly Arg Asp Gln 1 5 10 15 Pro Gln His Phe Gly Asp Gly Thr Arg Leu Ser Ile Leu 20 25 <210> 131 <211> 30 <212> PRT <213> Homo sapiens <400> 131 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Pro Gln Ser Gly His Ala 1 5 10 15 Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Leu 20 25 30 <210> 132 <211> 28 <212> PRT <213> Homo sapiens <400> 132 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Leu Phe Thr Asp Thr 1 5 10 15 Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Leu 20 25 <210> 133 <211> 26 <212> PRT <213> Homo sapiens <400> 133 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Ser Pro Pro Gln Tyr 1 5 10 15 Phe Gly Pro Gly Thr Arg Leu Leu Val Leu 20 25 <210> 134 <211> 27 <212> PRT <213> Homo sapiens <4 00> 134 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Phe Lys Trp Thr Gln 1 5 10 15 Tyr Phe Gly Pro Gly Thr Arg Leu Leu Val Leu 20 25 <210> 135 <211> 27 <212> PRT <213 > Homo sapiens <400> 135 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Arg Glu Gly Gly Gln 1 5 10 15 Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Thr 20 25 <210> 136 <211> 31 <212 > PRT <213> Homo sapiens <400> 136 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Phe Gly Ser Leu Ser 1 5 10 15 Ser Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Thr 20 25 30 < 210> 137 <211> 30 <212> PRT <213> Homo sapiens <400> 137 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Pro Ala Gln Gly Ala 1 5 10 15 Ala Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Thr 20 25 30 <210> 138 <211> 30 <212> PRT <213> Homo sapiens <400> 138 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Gly Ser Leu Val Asp 1 5 10 15 Gln Pro Gln His Phe Gly Asp Gly Thr Arg Leu Ser Ile Leu 20 25 30 <210> 139 <211> 30 <212> PRT <213> Homo sapiens <400> 139 Gly Asp Ser Ala Met Tyr Leu Cys A la Ser Ser Gln Gln Gly Gly Gly 1 5 10 15 Asn Thr Ile Tyr Phe Gly Glu Gly Ser Trp Leu Thr Val Val 20 25 30 <210> 140 <211> 31 <212> PRT <213> Homo sapiens <400> 140 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Leu Gly Ser Leu Val 1 5 10 15 Asp Gln Pro Gln His Phe Gly Asp Gly Thr Arg Leu Ser Ile Leu 20 25 30 <210> 141 <211> 30 <212> PRT <213> Homo sapiens <400> 141 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Leu Ile Gly Ala Ala 1 5 10 15 His Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr Val Leu 20 25 30 <210> 142 <211> 27 <212> PRT <213> Homo sapiens <400> 142 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Leu Asp Glu Arg Glu 1 5 10 15 Phe Phe Gly Pro Gly Thr Arg Leu Thr Val Leu 20 25 <210> 143 <211> 31 <212> PRT <213> Homo sapiens <400> 143 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Leu Gly Ser Thr Arg 1 5 10 15 Gly Asp Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr Val Leu 20 25 30 <210> 144 <211> 31 <212> PRT <213> Homo sapiens <400> 144 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Gln Gly Gly Gly Gly 1 5 10 15 Thr Gly Glu Leu Phe Phe Gly Glu Gly Ser Arg Leu Thr Val Leu 20 25 30 <210> 145 <211> 33 <212> PRT <213> Homo sapiens <400> 145 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Leu Ala Leu Val Arg 1 5 10 15 Asp Tyr Ser Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val 20 25 30 Thr <210> 146 <211> 30 <212> PRT <213> Homo sapiens <400> 146 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Leu Thr Gly Gly Gly 1 5 10 15 Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Leu 20 25 30 <210> 147 <211> 30 <212> PRT <213> Homo sapiens <400> 147 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Leu Thr Thr Asp Ala 1 5 10 15 Glu Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu Leu Val Leu 20 25 30 <210> 148 <211> 31 <212> PRT <213> Homo sapiens <400> 148 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Ala Ile Ala Gly Ala 1 5 10 15 Tyr Asn Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr Val Leu 20 25 30 <210> 149 <211> 30 <212> PRT <213> Homo sapiens <400> 149 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Ala Gl y Gln Gly Ser 1 5 10 15 Thr Glu Ala Phe Phe Gly Gln Gly Thr Arg Leu Thr Val Val 20 25 30 <210> 150 <211> 31 <212> PRT <213> Homo sapiens <400> 150 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Ser Leu Val Gly Gly 1 5 10 15 Arg Gly Glu Ala Phe Phe Gly Gln Gly Thr Arg Leu Thr Val Val 20 25 30 <210> 151 <211> 30 <212> PRT <213> Homo sapiens <400> 151 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Phe Leu Gly Gln Asn 1 5 10 15 Tyr Gly Tyr Thr Phe Gly Ser Gly Thr Arg Leu Thr Val Val 20 25 30 <210> 152 <211> 27 <212> PRT <213> Homo sapiens <400> 152 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Leu Val Asp Thr Ile 1 5 10 15 Tyr Phe Gly Glu Gly Ser Trp Leu Thr Val Val 20 25 <210> 153 <211> 32 <212> PRT <213> Homo sapiens <400> 153 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Pro Gly Thr Ser Gly 1 5 10 15 Ser Tyr Asn Glu Gln Phe Phe Gly Pro Gly Thr Arg Leu Thr Val Leu 20 25 30 <210> 154 <211> 31 <212> PRT <213> Homo sapiens <400> 154 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Phe Gly Arg Gly Asn 1 5 10 15 Thr Gly Glu Leu Phe Phe Gly Glu Gly Ser Arg Leu Thr Val Leu 20 25 30 <210> 155 <211> 33 <212> PRT <213> Homo sapiens <400> 155 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Leu Ala Ile Arg Gly 1 5 10 15 Ala Ser Thr Asp Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val 20 25 30 Leu <210> 156 <211> 31 <212> PRT <213> Homo sapiens <400 > 156 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Leu Gly Thr Ser Gly 1 5 10 15 Ala Glu Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu Leu Val Leu 20 25 30 <210> 157 <211> 31 <212 > PRT <213> Homo sapiens <400> 157 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Leu Gly Val Thr Pro 1 5 10 15 Gln Glu Thr Gln Tyr Phe Gly Pro Gly Thr Arg Leu Leu Val Leu 20 25 30 < 210> 158 <211> 36 <212> PRT <213> Homo sapiens <400> 158 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Ile Pro Arg Leu Gly 1 5 10 15 Gln Gly Ala Ser Gly Ala Asn Val Leu Thr Phe Gly Ala Gly Ser Arg 20 25 30 Leu Thr Val Leu 35 <210> 159 <211> 30 <212> PRT <213> Homo sapiens <400> 159 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Leu Gly Thr Gly Gly 1 5 10 15 Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Thr 20 25 30 <210> 160 <211> 32 <212> PRT <213> Homo sapiens <400> 160 Gly Asp Ser Ala Met Tyr Leu Cys Ala Ser Ser Ser Ile Pro Gly Leu 1 5 10 15 Arg Asn Tyr Glu Gln Tyr Phe Gly Pro Gly Thr Arg Leu Thr Val Thr 20 25 30
【図1】 川崎病患者(22名の)発症期、γグロブリ
ン投与期、及び回復期の末梢血におけるVα鎖サブファ
ミリーの使用頻度(%)を示すグラフ。FIG. 1 is a graph showing the frequency (%) of use of the Vα chain subfamily in peripheral blood of Kawasaki disease patients (22 patients) during onset, γ globulin administration, and recovery.
【図2】 川崎病患者(22名の)発症期、γグロブリ
ン投与期、及び回復期の末梢血におけるVβ鎖サブファ
ミリーの使用頻度(%)を示すグラフ。FIG. 2 is a graph showing the frequency of use (%) of the Vβ chain subfamily in peripheral blood of Kawasaki disease patients (22 patients) during onset, γ globulin administration, and recovery.
【図3】 川崎病患者22名の治療前と回復期、発熱を
伴う川崎病以外の疾患群の子供14名、及び健常な大人
10名についてTCRVβ鎖のVβ2.1及びVβ6.5
の発現頻度を示すグラフ。FIG. 3: Vβ2.1 and Vβ6.5 of TCRVβ chain in 22 children with Kawasaki disease before and after treatment, 14 children in a group of diseases other than Kawasaki disease with fever, and 10 healthy adults.
Is a graph showing the expression frequency.
───────────────────────────────────────────────────── フロントページの続き Fターム(参考) 2G045 AA02 AA25 AA35 CA18 CA25 DA13 FB01 FB02 GA01 4B024 AA01 AA11 CA04 CA09 CA11 DA03 EA04 HA11 4B063 QA01 QA19 QQ03 QQ52 QR31 QR32 QR55 QR62 QR84 QS02 QS16 QS25 QX01 QX02 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 2G045 AA02 AA25 AA35 CA18 CA25 DA13 FB01 FB02 GA01 4B024 AA01 AA11 CA04 CA09 CA11 DA03 EA04 HA11 4B063 QA01 QA19 QQ03 QQ52 QR31 QR32 QR55 QR62 QR84 QS02 QS16 QQSQ
Claims (4)
β6.5及びVβ2.1陽性T細胞数の増加を指標とする
川崎病因子の検出法。1. An increase in the number of Vβ6.5-positive T cells, or
A method for detecting Kawasaki disease factor using an increase in the number of β6.5 and Vβ2.1 positive T cells as an index.
RV領域の遺伝子群を増幅し、TCRVサブファミリー
の発現頻度を検出し、TCRVβ6.5及びTCRVβ
2.1サブファミリーの発現頻度の有意な増大を指標と
する請求項1記載の検出法。2. TC in peripheral blood of a patient suspected of Kawasaki disease
The genes in the RV region were amplified, the frequency of expression of the TCRV subfamily was detected, and TCRVβ6.5 and TCRVβ
2. The detection method according to claim 1, wherein a significant increase in the expression frequency of the 2.1 subfamily is used as an index.
RV領域遺伝子群をアダプター付加PCR法で増幅し、
少なくとも配列番号10及び11に記載の塩基配列で示
されるオリゴヌクレオチドプローブを含むTCRVサブ
ファミリーに特異的なオリゴヌクレオチドプローブを用
いるリバース・ドット・ブロット法により、TCRVβ
サブファミリーの発現頻度を検出し、TCRVβ6.5
サブファミリー、又はTCRVβ6.5及びTCRVβ
2.1サブファミリーの発現頻度が、他のサブファミリ
ーの発現頻度よりも有意に高いことを指標とする請求項
1又は2記載の方法。3. TC in peripheral blood of a patient suspected of Kawasaki disease
The RV region genes are amplified by an adapter-added PCR method,
By reverse dot blotting using an oligonucleotide probe specific to the TCRV subfamily containing at least the oligonucleotide probes represented by the nucleotide sequences of SEQ ID NOS: 10 and 11, TCRVβ
The expression frequency of the subfamily was detected, and TCRVβ6.5 was detected.
Subfamily, or TCRVβ6.5 and TCRVβ
3. The method according to claim 1 or 2, wherein the expression frequency of the 2.1 subfamily is significantly higher than the expression frequency of the other subfamilies.
β6.5陽性cDNAクローンのCDR3領域の塩基配
列を決定し、TCRVβ2.1及びTCRVβ6.5陽性
T細胞数の増加がポリクローナルであることを確認する
ことをも含む請求項1〜3のいずれかに記載の方法。4. The method of claim 1, further comprising the steps of:
4. The method according to claim 1, further comprising determining the nucleotide sequence of the CDR3 region of the β6.5-positive cDNA clone, and confirming that the increase in the number of TCRVβ2.1 and TCRVβ6.5-positive T cells is polyclonal. The described method.
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JP10341661A JP2000157297A (en) | 1998-12-01 | 1998-12-01 | Method for detecting factor of kawasaki disease |
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Family
ID=18347821
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Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1517995A2 (en) * | 2002-07-03 | 2005-03-30 | Institute For Scientific Research, Inc. | Compositions and methods for the detection of human t cell receptor variable family gene expression |
JP2007205842A (en) * | 2006-02-01 | 2007-08-16 | Kobeshi Chiiki Iryo Shinko Zaidan | Kawasaki disease determination method and kit for the same |
WO2009019657A3 (en) * | 2007-08-06 | 2009-08-06 | Univ Cattolica Sacro Cuore | Means for diagnosis and treatment of rheumatoid arthritis |
EP2281065A4 (en) * | 2008-04-16 | 2011-08-31 | Hudsonalpha Inst For Biotechnology | Method for evaluating and comparing immunorepertoires |
-
1998
- 1998-12-01 JP JP10341661A patent/JP2000157297A/en active Pending
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1517995A2 (en) * | 2002-07-03 | 2005-03-30 | Institute For Scientific Research, Inc. | Compositions and methods for the detection of human t cell receptor variable family gene expression |
EP1517995A4 (en) * | 2002-07-03 | 2007-07-11 | Inst Scient Res Inc | Compositions and methods for the detection of human t cell receptor variable family gene expression |
JP2007205842A (en) * | 2006-02-01 | 2007-08-16 | Kobeshi Chiiki Iryo Shinko Zaidan | Kawasaki disease determination method and kit for the same |
WO2009019657A3 (en) * | 2007-08-06 | 2009-08-06 | Univ Cattolica Sacro Cuore | Means for diagnosis and treatment of rheumatoid arthritis |
EP2281065A4 (en) * | 2008-04-16 | 2011-08-31 | Hudsonalpha Inst For Biotechnology | Method for evaluating and comparing immunorepertoires |
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