JP3487394B2 - Modified thermostable DNA polymerase and use thereof - Google Patents

Description

【発明の詳細な説明】Detailed Description of the Invention

【０００１】[0001]

【発明の属する技術分野】本発明は改変された耐熱性Ｄ
ＮＡポリメラーゼおよびおよび該酵素を用いた核酸の増
幅方法ならびに該方法に使用する試薬に関する。TECHNICAL FIELD The present invention relates to a modified heat resistance D.
The present invention relates to NA polymerase and a method for amplifying a nucleic acid using the enzyme, and a reagent used in the method.

【０００２】[0002]

【従来の技術】従来から、ポリメラーゼ連鎖反応（ＰＣ
Ｒ）等の核酸を増幅する技術に用いる耐熱性ＤＮＡポリ
メラ−ゼに関する研究が多くなされている。ＰＣＲ反応
に用いられる耐熱性ＤＮＡポリメラ−ゼは、主としてサ
−マス・サ−モフィラス(Thermusthermophilus) 由来の
ＤＮＡポリメラ−ゼ(Tthポリメラ−ゼ) やサ−マス・ア
クアチカス(Thermus aquaticus) 由来のＤＮＡポリメラ
−ゼ(Taqポリメラ−ゼ)などが用いられてきた。また、
超好熱始原菌由来のＤＮＡポリメラーゼ、たとえばパイ
ロコッカス・フリオサス(Pyrococcus furiosus) 由来の
耐熱性ＤＮＡポリメラーゼ(Pfuポリメラーゼ、WO92/096
89、特開平5-328969公報）、サーマス・リトラリス(The
rmococcus litoralis)由来の耐熱性ＤＮＡポリメラ−ゼ
(Tliポリメラーゼ、特開平6-7160号公報）などが知られ
ている。2. Description of the Related Art The polymerase chain reaction (PC
There have been many studies on thermostable DNA polymerases used in techniques for amplifying nucleic acids such as R). Thermostable DNA polymerases used in the PCR reaction are mainly DNA polymerases derived from Thermus thermophilus (Tth polymerase) and DNA polymerases derived from Thermus aquaticus. -Tase (Taq polymerase) and the like have been used. Also,
DNA polymerase derived from hyperthermophilic archaeon, for example, thermostable DNA polymerase derived from Pyrococcus furiosus (Pfu polymerase, WO92 / 096
89, JP-A-5-328969), Thermus litoralis (The
rmococcus litoralis) thermostable DNA polymerase
(Tli polymerase, JP-A-6-7160) and the like are known.

【０００３】さらに、本発明者らは熱安定性やＤＮＡ合
成速度に優れたパイロコッカス(Pyrococcus)ｓｐ．ＫＯ
Ｄ１由来の耐熱性ＤＮＡポリメラーゼ(KODポリメラー
ゼ、特開平7-298879号公報) を見いだした。Further, the present inventors have found that Pyrococcus sp. KO
A thermostable DNA polymerase derived from D1 (KOD polymerase, Japanese Patent Laid-Open No. 7-298879) was found.

【０００４】[0004]

【発明が解決しようとする課題】しかしながら、これら
の耐熱性ＤＮＡポリメラ−ゼは核酸の増幅効率が充分で
ないなどの問題がある。また、超好熱始原菌、例えばパ
イロコッカス(Pyrococcus)ｓｐ．ＫＯＤ１由来のポリメ
ラーゼは３’−５’エキソヌクレアーゼ活性が存在し、
ＰＣＲの反応時間、酵素量、プライマー濃度等の条件が
狭いとの問題がある。したがって、新規な耐熱性ＤＮＡ
ポリメラーゼが待ち望まれていた。However, these thermostable DNA polymerases have problems such as insufficient amplification efficiency of nucleic acids. Also, hyperthermophilic archaeon, such as Pyrococcus sp. The polymerase derived from KOD1 has 3′-5 ′ exonuclease activity,
There is a problem that conditions such as PCR reaction time, amount of enzyme, and primer concentration are narrow. Therefore, a novel thermostable DNA
Polymerase has been awaited.

【０００５】[0005]

【課題を解決するための手段】そこで、本発明者らは鋭
意検討した結果、パイロコッカス(Pyrococcus)ｓｐ．Ｋ
ＯＤ１由来のポリメラーゼのＤＮＡの合成速度や熱安定
性を保持したまま、改変前の該酵素に比べて３’−５’
エキソヌクレアーゼ活性を少なくとも５％以下に低下さ
せた改変酵素を作り出すことに成功した。また、ＤＮＡ
合成速度が少なくとも３０塩基／秒であって、ｐＨ８．
８（２５℃での測定値、９５℃にてｐＨを測定すること
は困難である。）にて９５℃、６時間の処理で６０％以
上の残存活性を保持することができる耐熱性ＤＮＡポリ
メラーゼであって、改変前の酵素に比べて、３’−５’
エキソヌクレアーゼ活性が少なくとも５％以下に低下し
た酵素を用いることにより、改変前の酵素を用いるより
も増幅効率が上昇することを見いだし、本発明を完成す
るに到った。Therefore, as a result of diligent studies, the present inventors have found that Pyrococcus sp. K
3'-5 'compared to the enzyme before modification while maintaining the DNA synthesis rate and thermal stability of the OD1-derived polymerase
We have succeeded in producing a modified enzyme that reduces the exonuclease activity to at least 5% or less. Also, DNA
A synthesis rate of at least 30 bases / sec and a pH of 8.
A thermostable DNA polymerase capable of retaining 60% or more residual activity after treatment at 95 ° C for 6 hours at 8 (measured value at 25 ° C, pH measurement at 95 ° C is difficult). And compared with the enzyme before modification, 3'-5 '
The inventors have found that the use of an enzyme having an exonuclease activity reduced to at least 5% or less increases the amplification efficiency as compared with the use of an enzyme before modification, and has completed the present invention.

【０００６】すなわち、本発明は下記理化学的性質を有
する改変された耐熱性ＤＮＡポリメラーゼである。 作用：ＤＮＡ合成活性を有し、改変前の酵素に比べて、
５％以下である３’−５’エキソヌクレアーゼ活性を有
する。 ＤＮＡ合成速度：少なくとも３０塩基／秒 熱安定性：ｐＨ８．８（２５℃ので測定値）にて９５
℃、６時間の処理で６０％以上の残存活性を保持するこ
とができる。That is, the present invention is a modified thermostable DNA polymerase having the following physicochemical properties. Action: It has a DNA synthesis activity and, compared to the enzyme before modification,
It has a 3'-5 'exonuclease activity that is 5% or less. DNA synthesis rate: at least 30 bases / second Thermal stability: 95 at pH 8.8 (measured at 25 ° C)
The residual activity of 60% or more can be retained by the treatment at 6 ° C. for 6 hours.

【０００７】本発明はＤＮＡを鋳型とし、プライマー、
ｄＮＴＰおよび請求項１〜３記載の耐熱性ＤＮＡポリメ
ラーゼを反応させて、プライマーを伸長して、ＤＮＡプ
ライマー伸長物を合成することを特徴とする核酸増幅法
である。The present invention uses DNA as a template, a primer,
A nucleic acid amplification method comprising reacting dNTP with the thermostable DNA polymerase according to any one of claims 1 to 3 to extend a primer to synthesize a DNA primer extension product.

【０００８】さらに、本発明は１方のプライマーが他方
のプライマーのＤＮＡ伸長生成物に相補的である２種の
プライマー、ｄＮＴＰおよび上記耐熱性ＤＮＡポリメラ
ーゼ、カリウムイオン、アンモニウムイオンおよびマグ
ネシウムイオン、ＢＳＡおよび非イオン界面活性剤およ
び緩衝液を含む核酸増幅用試薬である。Furthermore, the present invention provides two primers, one of which is complementary to the DNA extension product of the other primer, dNTP and the thermostable DNA polymerase, potassium ion, ammonium ion and magnesium ion, BSA and A nucleic acid amplification reagent containing a nonionic surfactant and a buffer.

【０００９】[0009]

【発明の実施態様】本発明において、ＤＮＡ合成活性と
は鋳型ＤＮＡにアニールされたオリゴヌクレオチドまた
はポリヌクレオチドの３’−ヒドロキシル基にデオキシ
リボヌクレオシド５’−トリホスフェートのα−ホスフ
ェートを共有結合せしめることにより、デオキシリボ核
酸にデオキシリボヌクレオシド５’−モノホスフェート
を鋳型依存的に導入する反応を触媒する活性をいう。BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, the DNA synthesis activity means the covalent bond of α-phosphate of deoxyribonucleoside 5′-triphosphate to the 3′-hydroxyl group of an oligonucleotide or a polynucleotide annealed to a template DNA. , Refers to the activity of catalyzing the reaction of introducing deoxyribonucleoside 5′-monophosphate into deoxyribonucleic acid in a template-dependent manner.

【００１０】その活性測定法は、酵素活性が高い場合に
は、保存緩衝液でサンプルを希釈して測定を行う。本発
明では、下記Ａ液２５μｌ、Ｂ液およびＣ液各５μｌお
よび滅菌水１０μｌをエッペンドルフチューブに加えて
攪拌混合した後、上記酵素液５μｌを加えて７５℃で１
０分間反応する。その後、氷冷し、Ｅ液５０μｌ、Ｄ液
１００μｌを加えて、攪拌後、さらに１０分間氷冷す
る。この液をガラスフィルター（ワットマンＧＦ／Ｃフ
ィルター）で濾過し、Ｄ液及びエタノールで充分洗浄
し、フィルターの放射活性を液体シンチレーションカウ
ンター（パッカード社製）で計測し、鋳型ＤＮＡへのヌ
クレオチドの取り込みを測定する。酵素活性の１単位は
この条件下で３０分あたり１０ｎモルのヌクレオチドを
酸不溶性画分に取り込む酵素量とする。 Ａ： ４０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．５） １６ｍＭ 塩化マグネシウム １５ｍＭ ジチオスレイトール １００μｇ／ｍｌ ＢＳＡ Ｂ： ２μｇ／μｌ 活性化仔牛胸腺ＤＮＡ Ｃ： １．５ｍＭ ｄＮＴＰ（２５０cpm/pmol〔 ³Ｈ〕ｄＴＴＰ） Ｄ： ２０％ トリクロロ酢酸（２ｍＭピロリン酸ナトリウム） Ｅ： １μｇ／μｌ キャリアーＤＮＡIn the activity measuring method, when the enzyme activity is high, the sample is diluted with a storage buffer for measurement. In the present invention, the following solution A (25 μl), solution B and solution C (5 μl) and sterilized water (10 μl) were added to an Eppendorf tube and mixed by stirring, and then the above enzyme solution (5 μl) was added to the solution at 75 ° C. for 1 hour.
React for 0 minutes. Then, the mixture is ice-cooled, 50 μl of E liquid and 100 μl of D liquid are added, and the mixture is stirred and ice-cooled for 10 minutes. This solution was filtered through a glass filter (Whatman GF / C filter), thoroughly washed with solution D and ethanol, and the radioactivity of the filter was measured with a liquid scintillation counter (made by Packard) to incorporate nucleotides into the template DNA. taking measurement. One unit of the enzyme activity is the amount of the enzyme that incorporates 10 nmol of nucleotide into the acid-insoluble fraction per 30 minutes under this condition. A: 40 mM Tris-HCl (pH 7.5) 16 mM magnesium chloride 15 mM dithiothreitol 100 μg / ml BSA B: 2 μg / μl activated calf thymus DNA C: 1.5 mM dNTP (250 cpm / pmol [ ³ H] dTTP) D: 20% trichloroacetic acid (2 mM sodium pyrophosphate) E: 1 μg / μl carrier DNA

【００１１】本発明において、３’−５’エキソヌクレ
アーゼ活性とは、ＤＮＡの３’末端領域を切除し、５’
−モノヌクレオチドを遊離する活性をいう。その活性測
定法は、５０μｌの反応液（120mM Tris-HCl(pH8.8 at
25℃), 10mM KCl, 6mM 硫酸アンモニウム，1mM MgCl₂,
0.1% Triton X-100, 0.001% BSA,5 μg トリチウムラ
ベルされた大腸菌ＤＮＡ）を１．５ｍｌのエッペンチュ
ーブにに分注し、ＤＮＡポリメラーゼを加える。７５℃
で１０分間反応させた後、氷冷によって反応を停止し、
次にキャリアーとして、０．１％のＢＳＡを５０μｌ加
え、さらに１０％のトリクロロ酢酸、２％ピロリン酸ナ
トリウム溶液を１００μｌ加え混合する。氷上で１５分
放置した後、１２，０００回転で１０分間遠心し沈殿を
分離する。上清１００μｌの放射活性を液体シンチレー
ションカウンター（パッカード社製）で計測し、酸可溶
性画分に遊離したヌクレオチド量を測定する。In the present invention, the 3'-5 'exonuclease activity means that the 3'terminal region of DNA is excised and 5'.
-Refers to the activity of releasing mononucleotides. The activity is measured by 50 μl of reaction solution (120 mM Tris-HCl (pH 8.8 at
25 ℃), 10mM KCl, 6mM ammonium sulfate, 1mM MgCl ₂ ,
0.1% Triton X-100, 0.001% BSA, 5 μg tritium-labeled E. coli DNA) is dispensed into a 1.5 ml Eppendorf tube, and a DNA polymerase is added. 75 ° C
After reacting for 10 minutes, stop the reaction by cooling with ice,
Next, 50 μl of 0.1% BSA is added as a carrier, and further 100 μl of 10% trichloroacetic acid and 2% sodium pyrophosphate solution is added and mixed. After leaving it on ice for 15 minutes, it is centrifuged at 12,000 rpm for 10 minutes to separate the precipitate. The radioactivity of 100 μl of the supernatant is measured with a liquid scintillation counter (manufactured by Packard) to measure the amount of nucleotides released in the acid-soluble fraction.

【００１２】本発明において、ＤＮＡ合成速度とは、単
位時間当たりのＤＮＡの合成数をいう。その測定法はＤ
ＮＡポリメラーゼの反応液(20mM Tris-HCl(pH7.5), 8mM
塩化マグネシウム、7.5mM ジチオスレイトール、100 μ
g/ml BSA, 0.1mM dNTP, 0.2μCi [α-³²P]dCTP)を、プ
ライマーをアニーリングさせたＭ１３ｍｐ１８１本鎖Ｄ
ＮＡと７５℃で反応させる。反応停止は等量の反応停止
液（50mM 水酸化ナトリウム、10mM EDTA, 5% フィコー
ル、0.05% ブロモフェノールブルー) を加えることによ
り行う。上記反応にて合成されたＤＮＡをアルカリアガ
ロースゲル電気泳動にて分画した後、ゲルを乾燥させオ
ートラジオグラフィーを行う。ＤＮＡサイズマーカーと
してはラベルしたλ/HindIIIを用いる。このマーカーの
バンドを指標として合成されたＤＮＡのサイズを測定す
ることによって、ＤＮＡ合成速度を求める。In the present invention, the DNA synthesis rate means the number of DNAs synthesized per unit time. The measurement method is D
NA polymerase reaction solution (20 mM Tris-HCl (pH 7.5), 8 mM
Magnesium chloride, 7.5 mM dithiothreitol, 100 μ
g / ml BSA, 0.1 mM dNTP, 0.2 μCi [α- ³² P] dCTP) was added to the primer-annealed M13mp181 single-stranded D
React with NA at 75 ° C. Stop the reaction by adding an equal volume of the reaction stop solution (50 mM sodium hydroxide, 10 mM EDTA, 5% Ficoll, 0.05% bromophenol blue). The DNA synthesized in the above reaction is fractionated by alkaline agarose gel electrophoresis, then the gel is dried and autoradiography is performed. Labeled λ / HindIII is used as a DNA size marker. The rate of DNA synthesis is determined by measuring the size of the synthesized DNA using the band of this marker as an index.

【００１３】本発明において、熱安定性とは、ｐＨ８．
８（２５℃での測定値）にて９５℃、６時間の処理での
残存活性を意味する。In the present invention, the thermal stability means a pH of 8.
8 (measured value at 25 ° C.) means residual activity after treatment at 95 ° C. for 6 hours.

【００１４】本発明の一実施態様は下記理化学的性質を
有する改変された耐熱性ＤＮＡポリメラーゼである。 作用：ＤＮＡ合成活性を有し、改変前の酵素に比べて、
５％以下である３’−５’エキソヌクレアーゼ活性を有
する。 ＤＮＡ合成速度：少なくとも３０塩基／秒 熱安定性：ｐＨ８．８（２５℃での測定値）にて９５
℃、６時間の処理で６０％以上の残存活性を保持するこ
とができる。 至適温度：約７５℃ 分子量：８８〜９０ＫＤａ アミノ酸配列：配列番号２に記載のアミノ酸配列の第１
４１、１４３、２１０および３１１番目のアミノ酸の少
なくとも１つを他のアミノ酸に置換したアミノ酸配列One embodiment of the present invention is a modified thermostable DNA polymerase having the following physicochemical properties. Action: It has a DNA synthesis activity and, compared to the enzyme before modification,
It has a 3'-5 'exonuclease activity that is 5% or less. DNA synthesis rate: at least 30 bases / sec Thermal stability: 95 at pH 8.8 (measured value at 25 ° C)
The residual activity of 60% or more can be retained by the treatment at 6 ° C. for 6 hours. Optimum temperature: about 75 ° C. Molecular weight: 88-90 KDa Amino acid sequence: First of the amino acid sequences set forth in SEQ ID NO: 2
Amino acid sequence in which at least one of the 41st, 143rd, 210th and 311th amino acids has been replaced with another amino acid

【００１５】また、本発明の別な実施態様は下記理化学
的性質を有する改変された耐熱性ＤＮＡポリメラーゼで
ある。 作用：ＤＮＡ合成活性を有し、３’−５’エキソヌクレ
アーゼ活性を有しない。 ＤＮＡ合成速度：少なくとも３０塩基／秒 熱安定性：ｐＨ８．８（２５℃での測定値）にて９５
℃、６時間の処理で６０％以上の残存活性を保持するこ
とができる。 至適温度：約７５℃ 分子量：８８〜９０ＫＤａ アミノ酸配列：配列番号２に記載のアミノ酸配列の第１
４１、１４３、２１０および３１１番目のアミノ酸の少
なくとも１つを他のアミノ酸に置換したアミノ酸配列Another embodiment of the present invention is a modified thermostable DNA polymerase having the following physicochemical properties. Action: It has a DNA synthesis activity and does not have a 3′-5 ′ exonuclease activity. DNA synthesis rate: at least 30 bases / sec Thermal stability: 95 at pH 8.8 (measured value at 25 ° C)
The residual activity of 60% or more can be retained by the treatment at 6 ° C. for 6 hours. Optimum temperature: about 75 ° C. Molecular weight: 88-90 KDa Amino acid sequence: First of the amino acid sequences set forth in SEQ ID NO: 2
Amino acid sequence in which at least one of the 41st, 143rd, 210th and 311th amino acids has been replaced with another amino acid

【００１６】本発明の改変前の耐熱性ＤＮＡポリメラー
ゼは、鹿児島県子宝島にて単離した超好熱始菌の１種で
あるパイロコッカス(Pyrococcus)ｓｐ．ＫＯＤ由来の酵
素である。該酵素を生産するＫＯＤの菌学的性質は、特
開平7-298879号公報に記載される。該酵素は上記菌株を
培養して生産される。該酵素は下記理化学的性質を有す
る。 作用：ＤＮＡ合成活性を有し、３’−５’エキソヌクレ
アーゼ活性を有する。 ＤＮＡ合成速度：少なくとも１２０塩基／秒 熱安定性：ｐＨ８．８（２５℃での測定値）にて９５
℃、６時間の処理で６０％以上の残存活性を保持するこ
とができる。 至適温度：約７５℃ 分子量：８８〜９０ＫＤａ アミノ酸配列：配列番号２に記載のアミノ酸配列The thermostable DNA polymerase of the present invention before modification is Pyrococcus sp., Which is one of the hyperthermophilic archaeon isolated in Kobojima, Kagoshima Prefecture. It is an enzyme derived from KOD. The mycological properties of KOD producing the enzyme are described in JP-A-7-298879. The enzyme is produced by culturing the above strain. The enzyme has the following physicochemical properties. Action: It has DNA synthesis activity and has 3′-5 ′ exonuclease activity. DNA synthesis rate: at least 120 bases / sec Thermal stability: 95 at pH 8.8 (measured value at 25 ° C)
The residual activity of 60% or more can be retained by the treatment at 6 ° C. for 6 hours. Optimum temperature: about 75 ° C. Molecular weight: 88-90 KDa Amino acid sequence: amino acid sequence set forth in SEQ ID NO: 2

【００１７】また、本発明の好ましい耐熱性ＤＮＡポリ
メラーゼとしては、アミノ酸配列が配列番号２に記載の
アミノ酸配列の第１４１、１４３、２１０および３１１
番目のアミノ酸の少なくとも１つを他のアミノ酸に置換
したアミノ酸配列を有する。その一例としては、配列番
号２の１４１番目のアスパラギン酸をアラニンに置換し
た酵素、配列番号２の１４３番目のグルタミン酸をアラ
ニンに置換した酵素、配列番号２の１４１番目のアスパ
ラギン酸と１４３番目のグルタミン酸をアラニンに置換
した酵素、配列番号２の２１０番目のアスパラギンをア
スパラギン酸に置換した酵素、配列番号２の３１１番目
のチロシンをフェニルアラニンに置換した酵素などがあ
げられる。Further, as a preferable thermostable DNA polymerase of the present invention, the amino acid sequence is the 141st, 143rd, 210th and 311th amino acid sequence of SEQ ID NO: 2.
It has an amino acid sequence in which at least one of the th amino acids has been replaced with another amino acid. Examples thereof include an enzyme in which the 141st aspartic acid of SEQ ID NO: 2 is replaced with alanine, an enzyme in which the 143rd glutamic acid of SEQ ID NO: 2 is replaced with alanine, and the 141st aspartic acid and 143th glutamic acid of SEQ ID NO: 2. And an enzyme in which the 210th asparagine of SEQ ID NO: 2 is replaced with aspartic acid, an enzyme in which the 311th tyrosine of SEQ ID NO: 2 is replaced with phenylalanine, and the like.

【００１８】これらの改変された酵素を製造する方法と
しては、天然型ＫＯＤポリメラーゼをコードする遺伝子
に変異を導入して、蛋白工学的手法により、天然型ＫＯ
Ｄポリメラーゼに比べて３’−５’エキソヌクレアーゼ
活性が低下した新規な酵素を製造する方法がある。As a method for producing these modified enzymes, a mutation is introduced into a gene encoding a natural KOD polymerase and the natural KO is subjected to a protein engineering technique.
There is a method for producing a novel enzyme having a decreased 3′-5 ′ exonuclease activity as compared with D polymerase.

【００１９】変異を導入するためのＫＯＤポリメラーゼ
をコードする遺伝子は特に限定されないが、本発明の一
実施態様は、パイロコッカス(Pyrococcous) ｓｐ．ＫＯ
Ｄ由来の配列表・配列番号３に記載の遺伝子を用いた。The gene encoding the KOD polymerase for introducing the mutation is not particularly limited, but one embodiment of the present invention is a Pyrococcous sp. KO
The gene described in Sequence Listing / SEQ ID NO: 3 derived from D was used.

【００２０】本発明の別な実施態様は、配列番号１に記
載されたアミノ酸配列をコードする遺伝子に変異を導入
して、天然型ＫＯＤポリメラーゼに比べて３’−５’エ
キソヌクレアーゼ活性が低下した新規な酵素を製造す
る。In another embodiment of the present invention, a mutation is introduced into the gene encoding the amino acid sequence set forth in SEQ ID NO: 1 so that the 3'-5 'exonuclease activity is reduced as compared to the natural KOD polymerase. Produce a new enzyme.

【００２１】天然型ＫＯＤポリメラーゼ遺伝子に変異を
導入する方法は、既知のいかなる方法でも用いることが
できる。例えば天然型ＫＯＤポリメラーゼ遺伝子ＤＮＡ
と変異源となる薬剤を接触させる方法や紫外線照射によ
る方法などから、蛋白工学的な手法、例えばＰＣＲ法や
部位特異的変異などの方法を用いることができる。ま
た、遺伝子修復機構が欠損されたため、高頻度に遺伝子
に変異が起こる大腸菌を用いた in vivoでの変異の導入
も可能である。本発明で使用したカメレオン site-dire
cted mutagenesisキット（ストラタジーン社製）とは、
(1) 目的とする遺伝子を挿入したプラスミドを変性さ
せ、該プラスミドに変異プライマーと選択プライマーと
アニーリングさせる。(2) 次にＤＮＡポリメラーゼでＤ
ＮＡ合成を行った後、ライゲースにてライゲーション反
応を行う。(3) 選択プライマー中に存在しないが、鋳型
となるプラスミドに存在する制限酵素でプラスミドを切
断し、変異の挿入されていないＤＮＡを切断する。(4)
次に残されたプラスミドで大腸菌を形質転換する。(5)
形質転換体から変異プラスミドを調製し、(3),(4) を繰
り返し、目的とする変異の挿入されたプラスミドを得る
方法である。As a method for introducing a mutation into the natural KOD polymerase gene, any known method can be used. For example, natural KOD polymerase gene DNA
A method of protein engineering, such as a PCR method or a site-directed mutagenesis method can be used, such as a method of contacting a drug as a mutation source or a method of ultraviolet irradiation. In addition, since the gene repair mechanism has been deleted, it is possible to introduce a mutation in vivo using Escherichia coli in which a gene mutation frequently occurs. Chameleon site-dire used in the present invention
What is the cted mutagenesis kit (manufactured by Stratagene)?
(1) A plasmid in which the gene of interest is inserted is denatured, and the plasmid is annealed with a mutant primer and a selective primer. (2) Then D with DNA polymerase
After NA synthesis is performed, a ligation reaction is performed with a ligase. (3) Cleave the plasmid with a restriction enzyme that is not present in the selection primer but is present in the plasmid serving as a template to cleave DNA having no mutation inserted therein. (Four)
The remaining plasmid is then transformed into E. coli. (Five)
This is a method in which a mutant plasmid is prepared from the transformant and (3) and (4) are repeated to obtain a plasmid having the desired mutation inserted therein.

【００２２】上記のようにして得られた改変ポリメラー
ゼ遺伝子を、例えば大腸菌に形質転換した後、アンピシ
リン等の薬剤を含む寒天培地に塗布し、コロニーを形成
させる。コロニーを栄養培地、例えばＬＢ培地や２×Ｙ
Ｔ培地に接種し、３７℃で１２〜２０時間培養した後、
菌体を破砕して粗酵素液を抽出する。菌体を破砕する方
法は、公知のいかなる手法を用いてもよく、例えば超音
波処理やガラスビール破砕のような物理的破砕法やリゾ
チームのような溶菌酵素を用いることができる。この粗
酵素を熱処理、例えば８０℃、３０分間処理し、宿主由
来のポリメラーゼを失活させ、ＤＮＡポリメラーゼ活性
を測定する。次に３’−５’エキソヌクレアーゼ活性を
測定し、両者の活性比率を天然型ＫＯＤポリメラーゼを
比較することにより、３’−５’エキソヌクレアーゼ活
性の低下した酵素をスクリーニングすることができる。The modified polymerase gene obtained as described above is transformed into, for example, Escherichia coli and then applied to an agar medium containing a drug such as ampicillin to form colonies. Colonies are fed to a nutrient medium such as LB medium or 2xY
After inoculating into T medium and culturing at 37 ° C. for 12 to 20 hours,
The microbial cells are crushed and the crude enzyme solution is extracted. Any known method may be used as a method for disrupting the cells, and for example, a physical disruption method such as ultrasonication or glass beer disruption, or a lytic enzyme such as lysozyme may be used. The crude enzyme is heat-treated, for example, at 80 ° C. for 30 minutes to inactivate the polymerase derived from the host, and the DNA polymerase activity is measured. Next, the 3'-5 'exonuclease activity is measured, and the activity ratio of both is compared with that of natural KOD polymerase, whereby an enzyme having a reduced 3'-5' exonuclease activity can be screened.

【００２３】上記方法により選抜された菌株から精製Ｄ
ＮＡポリメラーゼを取得する方法は、公知のいかなる手
法を用いても良く、例えば下記方法がある。栄養培地に
培養して得られた菌体を回収した後、酵素的または物理
的破砕法により破砕抽出して粗酵素液を得る。得られた
粗酵素抽出液から熱処理、例えば８０℃、３０分間処理
し、その後、硫安沈殿によりＫＯＤポリメラーゼ画分を
回収する。この粗酵素液をセファデックスＧ−２５（フ
ァルマシア・バイオテク）ゲル濾過等の方法により脱塩
を行うことができる。この操作の後、Ｑセファロース、
ヘパリンセファロースなどのカラムクロマトグラフィー
により分離、精製し、精製酵素標品を得ることができ
る。この精製酵素標品はＳＤＳ−ＰＡＧＥによってほぼ
単一のバンドを示す程度に純化される。Purified D from the strain selected by the above method
As a method for obtaining NA polymerase, any known method may be used, and examples thereof include the following method. After the cells obtained by culturing in a nutrient medium are collected, they are crushed and extracted by an enzymatic or physical crushing method to obtain a crude enzyme solution. The crude enzyme extract thus obtained is heat-treated, for example, at 80 ° C. for 30 minutes, and then the KOD polymerase fraction is collected by ammonium sulfate precipitation. This crude enzyme solution can be desalted by a method such as Sephadex G-25 (Pharmacia Biotech) gel filtration. After this operation, Q Sepharose,
A purified enzyme preparation can be obtained by separating and purifying by column chromatography using heparin sepharose or the like. The purified enzyme preparation was purified by SDS-PAGE to the extent that it showed a substantially single band.

【００２４】本発明の改変された耐熱性ＤＮＡポリメラ
ーゼを使用して、ＤＮＡを鋳型とし、プライマー、ｄＮ
ＴＰを反応させて、プライマーを伸長して、ＤＮＡプラ
イマー伸長物を合成することができる。プライマーは２
種のオリゴヌクレオチドであって、１方は他方のＤＮＡ
伸長生成物に相補的であるプライマーであることが好ま
しい。また、加熱および冷却を繰り返す。本発明のＤＮ
Ａポリメラーゼは、その活性を維持するために、マグネ
シウムイオンおよびアンモニウムイオンおよび／または
カリウムイオンを共存させることが好ましい。また、Ｐ
ＣＲ反応液には、緩衝液およびこれらのイオンを含むと
もに、ＢＳＡ、非イオン界面活性剤、例えばTriton X-1
00および緩衝液が存在していてもよい。Using the modified thermostable DNA polymerase of the present invention, DNA is used as a template, primer, dN
TP can be reacted to extend the primer and synthesize a DNA primer extension. 2 primer
Oligonucleotides of one species, one of which is the DNA of the other
It is preferred that the primer is complementary to the extension product. Also, heating and cooling are repeated. DN of the present invention
In order to maintain its activity, A polymerase preferably coexists with magnesium ions and ammonium ions and / or potassium ions. Also, P
The CR reaction solution contains a buffer solution and these ions, BSA, a nonionic surfactant such as Triton X-1.
00 and buffer may be present.

【００２５】次に、実施例を用いて本発明を詳細に説明
する。参考例１ 超好熱始原菌ＫＯＤ由来のＤＮＡポリメラーゼ遺伝子の
クローニング 鹿児島県子宝島にて単離した超好熱始原菌ＫＯＤ１株を
９５℃にて培養後、菌体を回収した。得られた菌体から
常法に従い、超好熱始原菌ＫＯＤ株の染色体ＤＮＡを調
製した。パイロコッカス・フリオサス(Pyrococcus furi
osus) 由来のＤＮＡポリメラーゼ(Pfuポリメラーゼ) の
保存領域アミノ酸配列に基づき、２種のプライマー(5'-
GGATTAGTATAGTGCCAATGGSSGGCGA-3' および5'-GAGGGCAGA
AGTTTATTCCGAGCTT-3')を合成した。この２種のプライマ
ーを使用し、調製したＤＮＡを鋳型として、ＰＣＲ反応
を行った。Next, the present invention will be described in detail with reference to examples. Reference Example 1 of DNA polymerase gene derived from hyperthermophilic archaeon KOD
Cloning The hyperthermophilic archaeon KOD1 strain isolated in Kobojima, Kagoshima Prefecture was cultured at 95 ° C., and then the cells were collected. Chromosomal DNA of the hyperthermophilic archaeon KOD strain was prepared from the obtained bacterial cells by a conventional method. Pyrococcus furi
osus) -derived DNA polymerase (Pfu polymerase) based on the amino acid sequence of the conserved region, two primers (5'-
GGATTAGTATAGTGCCAATGGSSGGCGA-3 'and 5'-GAGGGCAGA
AGTTTATTCCGAGCTT-3 ') was synthesized. A PCR reaction was carried out using these two types of primers and the prepared DNA as a template.

【００２６】ＰＣＲ増幅ＤＮＡ断片の塩基配列を決定
し、アミノ酸配列を決定した後、この増幅ＤＮＡ断片を
プローブとして、ＫＯＤ１株染色体ＤＮＡ制限酵素処理
産物に対してサザンハイブリダイゼーションを行い、Ｄ
ＮＡポリメラーゼをコードする断片のサイズを求めた
（約４〜７Ｋｂｐ）。さらに、この大きさのＤＮＡ断片
をアガロースゲルから回収し、プラスミドｐＢＳ（スト
ラタジーン社製）に挿入し、これらの混合物より大腸菌
(E.coli JM109)を形質転換して、ライブラリーを作製し
た。サザンハイブリダイゼーションに使用したプローブ
を用いて、コロニーハイブリダイゼーションを行い、上
記ライブラリーから、ＫＯＤ１株由来のＤＮＡポリメラ
ーゼ遺伝子を含有すると考えられるクローン株(E.coli
JM109/pSBKOD1)を取得した。After the nucleotide sequence of the PCR amplified DNA fragment was determined and the amino acid sequence was determined, Southern hybridization was carried out on the KOD1 strain chromosomal DNA restriction enzyme treated product using this amplified DNA fragment as a probe, and D
The size of the fragment encoding NA polymerase was determined (approximately 4-7 Kbp). Further, a DNA fragment of this size was recovered from an agarose gel, inserted into a plasmid pBS (manufactured by Stratagene), and E. coli was extracted from these mixtures.
(E. coli JM109) was transformed to prepare a library. Colony hybridization was performed using the probe used for Southern hybridization, and a clone strain (E. coli) that was considered to contain a DNA polymerase gene derived from the KOD1 strain was obtained from the above library.
JM109 / pSBKOD1) was obtained.

【００２７】参考例１で取得したクローン株、(E.coli
JM109/pSBKOD1)よりプラスミド、ｐＳＢＫＯＤ１を回収
し、常法に従い、塩基配列を決定した。さらに求められ
た塩基配列からアミノ酸配列を推定した。ＫＯＤ１株由
来のＤＮＡポリメラーゼ遺伝子は５０１０塩基からな
り、１６７０個のアミノ酸がコードされていた（配列番
号１）。The clone strain obtained in Reference Example 1, (E. coli
The plasmid pSBKOD1 was recovered from JM109 / pSBKOD1) and the nucleotide sequence was determined according to a conventional method. Further, the amino acid sequence was deduced from the determined nucleotide sequence. The DNA polymerase gene derived from the KOD1 strain consisted of 5010 bases and encoded 1670 amino acids (SEQ ID NO: 1).

【００２８】完全なポリメラーゼ遺伝子を作成するた
め、２箇所の介在配列（１３７４〜２４５３ｂｐ：２７
０８〜４３１６ｂｐ）をＰＣＲ融合法により取り除い
た。ＰＣＲ融合法では、クローン株より回収したプラス
ミドを鋳型に、３組のプライマーを組み合わせて、各々
ＰＣＲを行い、介在配列を除いた３断片を増幅した。こ
の際、ＰＣＲに用いるプライマーは、他の断片と結合す
る側に結合相手と同様な配列がくるように設計した。ま
た、両端には別々な制限酵素サイト（Ｎ末端側：Ｅｃｏ
ＲＶ、Ｃ末端側：ＢａｍＨＩ）が創出されるように設計
した。次いで、ＰＣＲ増幅断片中、構造***に位置す
る断片と、Ｎ末端側に位置する断片を混合し、ＰＣＲを
各々の断片をプライマーとして行った。また、同様に構
造上、中央に位置する断片と、Ｃ末端側に位置する断片
を混合し、ＰＣＲを各々の断片をプライマーとして行っ
た。このようにして得られた２種の断片を用いて再度Ｐ
ＣＲを行い、介在配列が取り除かれ、Ｎ末端にＥｃｏＲ
Ｖ、Ｃ末端にＢａｍＨＩサイトを有するＫＯＤ１株由来
のＤＮＡポリメラーゼをコードする完全な形の遺伝子断
片を取得した。更に、同遺伝子をＴ７プロモーターで誘
導可能な発現ベクター、ｐＥＴ−８ｃのＮｃｏＩ／Ｂａ
ｍＨＩサイト、先に創出した制限酵素サイトを利用し、
サブクローニングして、組換え発現ベクター（ｐＥＴ−
ｐｏｌ）を得た。なお、Ｅ．ｃｏｌｉ ＢＬ２１（ＤＥ
３）／ｐＥＴ−ｐｏｌは、生命工学工業研究所へ寄託さ
れている（ＦＥＲＭ ＢＰ−５５１３）。Two intervening sequences (1374-2453 bp: 27) were prepared in order to create a complete polymerase gene.
08-4316bp) was removed by PCR fusion method. In the PCR fusion method, using the plasmid recovered from the clone strain as a template, 3 sets of primers were combined, and each PCR was performed to amplify 3 fragments excluding intervening sequences. At this time, the primers used for PCR were designed so that the sequence similar to that of the binding partner would be present on the side binding to other fragments. Separate restriction enzyme sites (N-terminal side: Eco
RV, C-terminal side: BamHI) was designed to be created. Next, among the PCR-amplified fragments, the fragment located in the center of the structure and the fragment located on the N-terminal side were mixed, and PCR was performed using each fragment as a primer. Similarly, structurally, the fragment located at the center and the fragment located at the C-terminal side were mixed, and PCR was performed using each fragment as a primer. Using the two types of fragments thus obtained, P
Perform CR to remove intervening sequences and add EcoR to the N-terminus
A complete gene fragment encoding the DNA polymerase derived from the KOD1 strain having BamHI sites at the V and C ends was obtained. Furthermore, an expression vector capable of inducing the same gene with a T7 promoter, NcoI / Ba of pET-8c
Using the mHI site and the restriction enzyme site created earlier,
Subcloning and recombinant expression vector (pET-
pol) was obtained. In addition, E. coli BL21 (DE
3) / pET-pol has been deposited with the Institute of Biotechnology (FERM BP-5513).

【００２９】実施例１ ＫＯＤポリメラーゼ遺伝子のサブクローニング 耐熱性ＤＮＡポリメラーゼを改変するために、プラスミ
ドｐＥＴ−ｐｏｌからＫＯＤポリメラーゼ遺伝子を切り
出し、ｐＢｌｕｅｓｃｒｉｐｔにサブクローニングし
た。すなわちｐＥＴ−ｐｏｌを制限酵素、ＸｂａＩとＢ
ａｍＨＩ（東洋紡製）で切断し、約２．３ｋｂのＫＯＤ
ポリメラーゼ遺伝子を切り出した。次にこのＤＮＡ断片
をライゲーションキット（東洋紡製 Ligation high) を
用いて、ＸｂａＩとＢａｍＨＩで切断したプラスミドｐ
Ｂｌｕｅｓｃｒｉｐｔ ＳＫ（−）と連結した。次に、
市販のコンピテントセル（東洋紡製 competent high JM
109)を用いて形質転換を行った。１００μｇ／ｍｌのア
ンピシリンを含んだＬＢ寒天培地（１％バクトトリプト
ン、０．５％イーストエキストラクト、０．５％塩化ナ
トリウム、１．５％寒天、ギブコ社製）で３５℃で１６
時間培養し、得られたコロニーからプラスミドを調製し
た。さらに、部分塩基配列を確認してＫＯＤポリメラー
ゼ遺伝子を含むプラスミドｐＫＯＤ１を得た。 Example 1 Subcloning of KOD Polymerase Gene In order to modify the thermostable DNA polymerase, the KOD polymerase gene was excised from the plasmid pET-pol and subcloned into pBluescript. That is, pET-pol is a restriction enzyme, XbaI and B
Cut with amHI (manufactured by Toyobo), KOD of about 2.3 kb
The polymerase gene was excised. Next, this DNA fragment was digested with XbaI and BamHI using a ligation kit (Ligation high manufactured by Toyobo) to obtain a plasmid p.
Ligated with Bluescript SK (-). next,
Commercially available competent cell (Toyobo's competent high JM
109) was used for transformation. LB agar medium containing 100 μg / ml of ampicillin (1% bactotryptone, 0.5% yeast extract, 0.5% sodium chloride, 1.5% agar, Gibco) at 35 ° C. 16
After culturing for a period of time, a plasmid was prepared from the obtained colony. Furthermore, the partial base sequence was confirmed to obtain a plasmid pKOD1 containing the KOD polymerase gene.

【００３０】実施例２ 改変型遺伝子（ＤＡ）の作製及び改変型耐熱性ＤＮＡポ
リメラーゼの精製 実施例１で得られたプラスミドｐＫＯＤ１を用いて、配
列表２に記載のＫＯＤポリメラーゼの１４１番目のアス
パラギン酸をアラニンに置換した改変型耐熱性ＤＮＡポ
リメラーゼ遺伝子を持つプラスミドを作製した（ｐＫＯ
ＤＤＡ）。作製はカメレオン site-directed mutagenes
isキット（ストラタジーン社製）を用いた。方法は取扱
説明書に準じて行った。選択プライマーとしては配列番
号４に記載のプライマーを使用した。変異プライマーは
配列番号６に記載のプライマーを用いた。なお、変異体
の確認は塩基配列の解読で行った。得られたプラスミド
で大腸菌ＪＭ１０９を形質転換し、ＪＭ１０９（ｐＫＯ
ＤＤＡ）を得た。 Example 2 Preparation of modified gene (DA) and modified thermostable DNA
Purification of Limerase Using the plasmid pKOD1 obtained in Example 1, a plasmid having a modified thermostable DNA polymerase gene in which the 141st aspartic acid of KOD polymerase shown in Sequence Listing 2 was replaced with alanine was prepared (pKO
DDA). Made by Chameleon site-directed mutagenes
The is kit (manufactured by Stratagene) was used. The method was performed according to the instruction manual. The primer described in SEQ ID NO: 4 was used as a selection primer. As the mutant primer, the primer shown in SEQ ID NO: 6 was used. The mutants were confirmed by decoding the nucleotide sequence. Escherichia coli JM109 was transformed with the obtained plasmid to obtain JM109 (pKO
DDA) was obtained.

【００３１】滅菌処理した１００μｇ／ｍｌのアンピシ
リンを含んだＴＢ培地（MolecularCloning 、ｐ．Ａ．
２に記載）６Ｌを１０Ｌジャーファーメンターに分注し
た。この培地に予め１００μｇ／ｍｌのアンピシリンを
含んだ５０ｍｌのＬＢ培地（１％バクトトリプトン、
０．５％イーストエキストラクト、０．５％塩化ナトリ
ウム、ギブコ社製）で３０℃、１６時間培養した大腸菌
ＪＭ１０９（ｐＫＯＤＤＡ）（５００ｍｌ坂口フラスコ
使用）を接種し、３５℃で１２時間通気攪拌培養した。
培養液より菌体を遠心分離により回収し、４００ｍｌの
破砕緩衝液（10mMTris-HCl(pH8.0), 80mM KCl, 5mM 2-
メルカプトエタノール、1mMEDTA) に懸濁後、超音波処
理によって菌体を破砕し、細胞破砕液を得た。次に、細
胞破砕液を８５℃にて３０分処理した後、遠心分離にて
不溶性画分を除去した。さらにポリエチレンイミンを用
いた除核酸処理、硫安分画、ヘパリンセファロースクロ
マトグラフィーを行い、最後に保存緩衝液（50mM Tris-
HCl (pH8.0), 50mM 塩化カリウム、1mM ジチオスレイト
ール、0.1% Tween20, 0.1%ノニデットP40, 50%グリセリ
ン）に置換し、改変型耐熱性ＤＮＡポリメラ−ゼ（Ｄ
Ａ）を得た。上記精製工程のＤＮＡポリメラーゼ活性は
以下の操作で行った。また、酵素活性が高い場合には、
保存緩衝液でサンプルを希釈して測定を行った。TB medium containing sterilized 100 μg / ml ampicillin (Molecular Cloning, p.A.
(Described in 2) 6 L was dispensed into a 10 L jar fermenter. 50 ml of LB medium (1% bactotryptone, containing 100 μg / ml ampicillin in advance in this medium)
Escherichia coli JM109 (pKODDA) (500 ml Sakaguchi flask used) was inoculated with 0.5% yeast extract, 0.5% sodium chloride (manufactured by Gibco) at 30 ° C for 16 hours and inoculated with aeration stirring at 35 ° C for 12 hours. did.
The bacterial cells were recovered from the culture solution by centrifugation, and 400 ml of disruption buffer solution (10 mM Tris-HCl (pH8.0), 80 mM KCl, 5 mM 2-
After suspending in mercaptoethanol and 1 mM EDTA), the cells were disrupted by ultrasonication to obtain a cell disruption solution. Next, the cell lysate was treated at 85 ° C. for 30 minutes, and the insoluble fraction was removed by centrifugation. Nucleic acid removal treatment with polyethyleneimine, ammonium sulfate fractionation, and heparin sepharose chromatography were performed, and finally the storage buffer (50 mM Tris-
HCl (pH8.0), 50 mM potassium chloride, 1 mM dithiothreitol, 0.1% Tween20, 0.1% nonidet P40, 50% glycerin), and modified heat-resistant DNA polymerase (D
A) was obtained. The DNA polymerase activity in the above purification step was performed by the following procedure. If the enzyme activity is high,
The measurement was performed by diluting the sample with a storage buffer.

【００３２】 （試薬） Ａ： ４０ｍＭ Ｔｒｉｓ−ＨＣｌ（ｐＨ７．５） １６ｍＭ 塩化マグネシウム １５ｍＭ ジチオスレイトール １００μｇ／ｍｌ ＢＳＡ Ｂ： ２μｇ／μｌ 活性化仔牛胸腺ＤＮＡ Ｃ： １．５ｍＭ ｄＮＴＰ（２５０cpm/pmol〔 ³Ｈ〕ｄＴＴＰ） Ｄ： ２０％ トリクロロ酢酸（２ｍＭピロリン酸ナトリウム） Ｅ： １μｇ／μｌ キャリアーＤＮＡ(Reagent) A: 40 mM Tris-HCl (pH 7.5) 16 mM magnesium chloride 15 mM dithiothreitol 100 μg / ml BSA B: 2 μg / μl activated calf thymus DNA C: 1.5 mM dNTP (250 cpm / pmol [ ^3] H] dTTP) D: 20% trichloroacetic acid (2 mM sodium pyrophosphate) E: 1 μg / μl carrier DNA

【００３３】（方法）Ａ液２５μｌ、Ｂ液およびＣ液各
５μｌおよび滅菌水１０μｌをエッペンドルフチューブ
に加えて攪拌混合した後、上記酵素液５μｌを加えて７
５℃で１０分間反応する。その後、氷冷し、Ｅ液５０μ
ｌ、Ｄ液１００μｌを加えて、攪拌後、さらに１０分間
氷冷する。この液をガラスフィルター（ワットマンＧＦ
／Ｃフィルター）で濾過し、Ｄ液及びエタノールで充分
洗浄し、フィルターの放射活性を液体シンチレーション
カウンター（パッカード社製）で計測し、鋳型ＤＮＡへ
のヌクレオチドの取り込みを測定した。酵素活性の１単
位はこの条件下で３０分あたり１０ｎモルのヌクレオチ
ドを酸不溶性画分に取り込む酵素量とした。(Method) Solution A (25 μl), solution B and solution C (5 μl each) and sterilized water (10 μl) were added to an Eppendorf tube and mixed by stirring, and then the above enzyme solution (5 μl) was added to the mixture.
React for 10 minutes at 5 ° C. Then, cool with ice, and E solution 50μ
1, and 100 μl of D solution were added, and the mixture was stirred and ice-cooled for 10 minutes. This liquid was added to a glass filter (Whatman GF
/ C filter), thoroughly washed with solution D and ethanol, the radioactivity of the filter was measured with a liquid scintillation counter (manufactured by Packard), and nucleotide incorporation into the template DNA was measured. One unit of enzyme activity was defined as the amount of enzyme that incorporated 10 nmole of nucleotide into the acid-insoluble fraction per 30 minutes under these conditions.

【００３４】実施例３ 変異体（ＥＡ）遺伝子の作製及び改変型耐熱性ＤＮＡポ
リメラーゼの精製 実施例２と同様の方法にて、配列表２に記載のＫＯＤポ
リメラーゼの１４３番目のグルタミン酸をアラニンに置
換した改変型耐熱性ＤＮＡポリメラーゼ遺伝子を持つプ
ラスミドを作製した（ｐＫＯＤＥＡ）。選択プライマー
としては配列番号５に記載のプライマーを使用した。変
異プライマーは配列番号８に記載のプライマーを用い
た。更に実施例２同様の精製方法にて改変型耐熱性ＤＮ
Ａポリメラ−ゼ（ＥＡ）を得た。 Example 3 Construction of mutant (EA) gene and modified thermostable DNA
Purification of Limerase In the same manner as in Example 2, a plasmid having a modified thermostable DNA polymerase gene in which glutamic acid at position 143 of KOD polymerase shown in Sequence Listing 2 was replaced with alanine was prepared (pKODEA). The primer described in SEQ ID NO: 5 was used as a selection primer. As the mutant primer, the primer shown in SEQ ID NO: 8 was used. Furthermore, a modified heat-resistant DN was prepared by the same purification method as in Example 2.
A polymerase (EA) was obtained.

【００３５】実施例４ 変異体（ＤＥＡ）遺伝子の作製及び改変型耐熱性ＤＮＡ
ポリメラーゼの精製 実施例２と同様の方法にて配列表２に記載のＫＯＤポリ
メラーゼの１４１番目のアスパラギン酸及び１４３番目
のグルタミン酸をアラニンに置換した改変型耐熱性ＤＮ
Ａポリメラーゼ遺伝子を持つプラスミドを作製した（ｐ
ＫＯＤＤＥＡ）。選択プライマーとしては配列番号４に
記載のプライマーを使用した。変異プライマーは配列番
号６に記載のプライマーを用いた。更に実施例２と同様
の精製方法にて改変型耐熱性ＤＮＡポリメラ−ゼ（ＤＥ
Ａ）を得た。 Example 4 Construction of mutant (DEA) gene and modified thermostable DNA
Purification of Polymerase In the same manner as in Example 2, a modified thermostable DN in which the 141st aspartic acid and the 143rd glutamic acid of KOD polymerase shown in Sequence Listing 2 were replaced with alanine.
A plasmid having the A polymerase gene was prepared (p
KODDEA). The primer described in SEQ ID NO: 4 was used as a selection primer. As the mutant primer, the primer shown in SEQ ID NO: 6 was used. Further, a modified thermostable DNA polymerase (DE) was prepared by the same purification method as in Example 2.
A) was obtained.

【００３６】実施例５ 変異体（ＮＤ）遺伝子の作製及び改変型耐熱性ＤＮＡポ
リメラーゼの精製 実施例２と同様の方法にて、配列表２に記載のＫＯＤポ
リメラーゼの２１０番目のアスパラギンをアスパラギン
酸に置換した改変型耐熱性ＤＮＡポリメラーゼ遺伝子を
持つプラスミドを作製した（ｐＫＯＤＮＤ）。選択プラ
イマーとしては配列番号４に記載のプライマーを使用し
た。変異プライマーは配列番号９に記載のプライマーを
用いた。更に実施例２同様の精製方法にて改変型耐熱性
ＤＮＡポリメラ−ゼ（ＮＤ）を得た。 Example 5 Preparation of mutant (ND) gene and modified thermostable DNA
Purification of Limerase In the same manner as in Example 2, a plasmid having a modified thermostable DNA polymerase gene in which the 210th asparagine of KOD polymerase shown in Sequence Listing 2 was replaced with aspartic acid was prepared (pKODND). The primer described in SEQ ID NO: 4 was used as a selection primer. As the mutant primer, the primer described in SEQ ID NO: 9 was used. Further, a modified thermostable DNA polymerase (ND) was obtained by the same purification method as in Example 2.

【００３７】実施例６ 変異体（ＹＦ）遺伝子の作製及び改変型耐熱性ＤＮＡポ
リメラーゼの精製 実施例２と同様の方法にて配列表２に記載のＫＯＤポリ
メラーゼの３１１番目のチロシンをフェニルアラニンに
置換した改変型耐熱性ＤＮＡポリメラーゼ遺伝子を持つ
プラスミドを作製した（ｐＫＯＤＹＦ）。選択プライマ
ーとしては配列番号４に記載のプライマーを使用した。
変異プライマーは配列番号１０に記載のプライマーを用
いた。更に実施例２同様の精製方法にて改変型耐熱性Ｄ
ＮＡポリメラ−ゼ（ＹＦ）を得た。 Example 6 Preparation of mutant (YF) gene and modified thermostable DNA
Purification of Limerase In the same manner as in Example 2, a plasmid having a modified thermostable DNA polymerase gene in which the 311th tyrosine of KOD polymerase shown in Sequence Listing 2 was replaced with phenylalanine was prepared (pKODYF). The primer described in SEQ ID NO: 4 was used as a selection primer.
As the mutant primer, the primer described in SEQ ID NO: 10 was used. Further, by the same purification method as in Example 2, the modified heat resistance D
NA polymerase (YF) was obtained.

【００３８】実施例７ 改変型耐熱性ＤＮＡポリメラーゼのエキソヌクレアーゼ
活性の確認 上記実施例２〜６で得られた改変型耐熱性ＤＮＡポリメ
ラーゼのエキソヌクレアーゼ活性を以下の方法にて測定
した。対照として、天然型のＫＯＤポリメラーゼ（東洋
紡製）を用いた。５０μｌの反応液（120mM Tris-HCl(p
H8.8 at 25℃),10mM KCl, 6mM 硫酸アンモニウム，1mM
MgCl₂, 0.1% Triton X-100, 0.001% BSA, 5 μg トリ
チウムラベルされた大腸菌ＤＮＡ）を１．５ｍｌのエッ
ペンチューブにに分注し、ＤＮＡポリメラーゼをそれぞ
れ２５単位、５０単位、１００単位加えた。なお、天然
型のＫＯＤポリメラーゼは０．２５単位、０．５単位、
１単位用いた。７５℃で１０分間反応させた後、氷冷に
よって反応を停止し、次にキャリアーとして、０．１％
のＢＳＡを５０μｌ加え、さらに１０％のトリクロロ酢
酸、２％ピロリン酸ナトリウム溶液を１００μｌ加え混
合した。氷上で１５分放置した後、１２，０００回転で
１０分間遠心し沈殿を分離した。上清１００μｌの放射
活性を液体シンチレーションカウンター（パッカード社
製）で計測し、酸可溶性画分に遊離したヌクレオチド量
を測定した。図１に各ポリメラーゼのポリメラーゼ活性
とＤＮＡの分解率を示した。この結果では改変酵素ＤＥ
Ａ、ＤＡ、ＥＡの３種類のポリメラーゼはエキソヌクレ
アーゼ活性が検出できなかった。また、改変酵素ＮＤは
天然型酵素の約０．１％、改変酵素ＹＦは約０．０１％
のエキソヌクレアーゼ活性を示した。 Example 7 Modified Thermostable DNA Polymerase Exonuclease
Confirmation of activity The exonuclease activity of the modified thermostable DNA polymerases obtained in Examples 2 to 6 was measured by the following method. As a control, a natural KOD polymerase (manufactured by Toyobo) was used. 50 μl of reaction solution (120 mM Tris-HCl (p
H8.8 at 25 ℃), 10mM KCl, 6mM ammonium sulfate, 1mM
MgCl ₂ , 0.1% Triton X-100, 0.001% BSA, 5 μg tritium-labeled E. coli DNA) was dispensed into a 1.5 ml Eppendorf tube, and 25 units, 50 units, and 100 units of DNA polymerase were added, respectively. . The natural KOD polymerase is 0.25 unit, 0.5 unit,
1 unit was used. After reacting at 75 ° C for 10 minutes, the reaction was stopped by cooling with ice, and then 0.1% was used as a carrier.
50 μl of BSA in Example 2 was added, and 100 μl of 10% trichloroacetic acid and 2% sodium pyrophosphate solution was further added and mixed. After leaving it on ice for 15 minutes, it was centrifuged at 12,000 rpm for 10 minutes to separate the precipitate. The radioactivity of 100 μl of the supernatant was measured with a liquid scintillation counter (manufactured by Packard) to measure the amount of nucleotides released in the acid-soluble fraction. FIG. 1 shows the polymerase activity of each polymerase and the DNA degradation rate. This result shows that the modified enzyme DE
The exonuclease activity of three kinds of polymerases A, DA and EA could not be detected. In addition, the modifying enzyme ND is about 0.1% of the natural enzyme, and the modifying enzyme YF is about 0.01%.
Showed exonuclease activity of.

【００３９】実施例８ 熱安定性の確認 実施例２〜６で得られた改変型ＤＮＡポリメラーゼの熱
安定性を以下の方法にて測定した。精製した改変型ＤＮ
Ａポリメラーゼ５単位を１００μｌの緩衝液（20mMTris
-HCl pH8.8 at 25℃、10mM塩化カリウム，10mM 硫酸
アンモニウム，2mM 硫酸マグネシウム, 0.1% Triton X-
100, 0.1mg/ml BSA, 5mM 2- メルカプトエタノール）に
混合し、９５℃でプレインキュベートとした。この混合
液から経時的に試料を採取し、実施例２記載の方法にて
ポリメラーゼ活性を測定した。比較として、Ｔａｑポリ
メラーゼ（東洋紡製）およびび天然型ＫＯＤポリメラー
ゼ（東洋紡製）も同様の操作を行った。図２に示したよ
うに、いずれの改変型ポリメラーゼも天然型ＫＯＤポリ
メラーゼと同様に、９５℃、６時間の処理で６０％以上
の残存活性を示した。それに対してＴａｑポリメラーゼ
は１５％以下の残存活性であった。 Example 8 Confirmation of thermostability The thermostability of the modified DNA polymerases obtained in Examples 2 to 6 was measured by the following method. Purified modified DN
5 units of A polymerase is added to 100 μl of buffer solution (20 mM Tris
-HCl pH8.8 at 25 ℃, 10mM potassium chloride, 10mM ammonium sulfate, 2mM magnesium sulfate, 0.1% Triton X-
100, 0.1 mg / ml BSA, 5 mM 2-mercaptoethanol) was mixed and pre-incubated at 95 ° C. Samples were taken from this mixed solution over time, and the polymerase activity was measured by the method described in Example 2. For comparison, Taq polymerase (manufactured by Toyobo) and natural KOD polymerase (manufactured by Toyobo) were subjected to the same operation. As shown in FIG. 2, all the modified polymerases showed residual activity of 60% or more after treatment at 95 ° C. for 6 hours, like the natural KOD polymerase. In contrast, Taq polymerase had a residual activity of 15% or less.

【００４０】実施例９ ＤＮＡ合成速度測定 実施例２〜６で得られた改変型ＤＮＡポリメラーゼのＤ
ＮＡ合成速度を以下の方法で測定した。精製した改変型
ＤＮＡポリメラーゼ１単位を１０μｌの反応液(20mM Tr
is-HCl(pH7.5), 8mM塩化マグネシウム, 7.5mM ジチオス
レイトール, 100 μg/ml BSA, 0.1mM dNTP, 0.2 μCi
[α-³²P]dCTP)で０．２μｇの配列番号１５のプライマ
ーをアニーリングさせたＭ１３ｍｐ１８１本鎖ＤＮＡと
７５℃で２０秒、４０秒、６０秒間反応させた。反応停
止は等量の反応停止液（50mM 水酸化ナトリウム，10mM
EDTA, 5% フィコール, 0.05% ブロモフェノールブル
ー) を加えることにより行った。比較としてＴａｑポリ
メラーゼ（東洋紡製）および天然型のＫＯＤポリメラー
ゼ（東洋紡製）も同様の操作を行った。 Example 9 Measurement of DNA synthesis rate D of the modified DNA polymerases obtained in Examples 2 to 6
The NA synthesis rate was measured by the following method. 1 unit of purified modified DNA polymerase (10 μl) was added to the reaction mixture (20 mM Tr
is-HCl (pH 7.5), 8 mM magnesium chloride, 7.5 mM dithiothreitol, 100 μg / ml BSA, 0.1 mM dNTP, 0.2 μCi
0.2 μg of the primer of SEQ ID NO: 15 was annealed with [α- ³² P] dCTP) and reacted with M13mp181 single-stranded DNA at 75 ° C. for 20 seconds, 40 seconds, and 60 seconds. To stop the reaction, use the same amount of the stop solution (50mM sodium hydroxide, 10mM
This was done by adding EDTA, 5% Ficoll, 0.05% bromophenol blue). For comparison, Taq polymerase (manufactured by Toyobo) and natural KOD polymerase (manufactured by Toyobo) were subjected to the same operation.

【００４１】上記反応にて合成されたＤＮＡをアルカリ
アガロースゲル電気泳動にて分画した後、ゲルを乾燥さ
せオートラジオグラフィーを行った。ＤＮＡサイズマー
カーとしてはラベルしたλ/HindIIIを用いた。このマー
カーのバンドを指標として合成されたＤＮＡのサイズを
測定することによって、ＤＮＡ合成速度を求めた。その
結果、いずれの改変型ポリメラーゼも天然型のＫＯＤポ
リメラーゼと同様に、約１２０塩基／秒の合成速度を有
していた。それに対してＴａｑポリメラーゼは約６０塩
基／秒の合成速度であった。The DNA synthesized in the above reaction was fractionated by alkaline agarose gel electrophoresis, the gel was dried and autoradiography was performed. Labeled λ / HindIII was used as a DNA size marker. The rate of DNA synthesis was determined by measuring the size of the synthesized DNA using the band of this marker as an index. As a result, all modified polymerases had a synthesis rate of about 120 bases / second, similar to the natural KOD polymerase. In contrast, Taq polymerase had a synthesis rate of about 60 bases / sec.

【００４２】実施例１０ 改変型ＤＮＡポリメラーゼを用いたＰＣＲ反応（プラス
ミド） 天然型ＫＯＤポリメラーゼおよび改変型耐熱性ＤＮＡポ
リメラーゼを用いてＰＣＲ反応を行った。５０μｌの反
応液（120mM Tris-HCl(pH8.0 at 25℃), 10mMKCl, 6mM
硫酸アンモニウム, 1mM MgCl₂, 0.2mM dNTP, 0.1% Trit
on X-100, 0.001% BSA, 1ng の制限酵素ＳｃａＩで直鎖
状にしたプラスミドpBR322, 10ピコモルの配列表１３，
１４記載のプライマー）に各酵素を２．５単位加えてＰ
ＣＲ反応を行った。サーマルサイクラーはパーキンエル
マー社製のモデルＰＪ２０００を用いた。また、反応条
件は９４℃、３０秒 → ６８℃、２分３０秒を２５サ
イクル行った。また、Ｔａｑポリメラーゼ（東洋紡製）
も同様にしてＰＣＲ反応を行った。ただし、反応液組成
は（10mM Tris-HCl (pH8.8 at 25℃), 50mM KCl, 1.5mM
MgCl₂, 0.2mM dNTP, 0.1% Triton X-100, 1ng の制限
酵素ＳｃａＩで直鎖状にしたプラスミドpBR322, 10ピコ
モルの配列表１２及び１３記載のプライマー）で行っ
た。反応終了後、５μｌの反応液についてアガロースゲ
ル電気泳動を行い約４．３Ｋｂのターゲットの増幅を確
認した。図３にアガロースゲル電気泳動の結果を示し
た。この結果、改変型ＤＮＡポリメラーゼを用いてＰＣ
Ｒを行った場合、天然型ＫＯＤポリメラーゼを用いるよ
りも良好な増幅であった。さらに、Ｔａｑポリメラーゼ
よりも良好な増幅であった。 Example 10 PCR Reaction Using Modified DNA Polymerase (Plus
Mid) A PCR reaction was performed using a natural KOD polymerase and a modified thermostable DNA polymerase. 50 μl of reaction solution (120 mM Tris-HCl (pH 8.0 at 25 ° C), 10 mM KCl, 6 mM
Ammonium sulfate, 1 mM MgCl ₂ , 0.2 mM dNTP, 0.1% Trit
on X-100, 0.001% BSA, plasmid pBR322 linearized with 1 ng of the restriction enzyme ScaI, 10 picomoles of Sequence Listing 13,
14) and 2.5 units of each enzyme to P
A CR reaction was performed. As the thermal cycler, Model PJ2000 manufactured by Perkin Elmer was used. The reaction conditions were 94 ° C., 30 seconds → 68 ° C., 25 minutes, 25 cycles. Also, Taq polymerase (manufactured by Toyobo)
PCR reaction was performed in the same manner. However, the composition of the reaction solution is (10mM Tris-HCl (pH8.8 at 25 ℃), 50mM KCl, 1.5mM
The plasmid pBR322 was linearized with MgCl ₂ , 0.2 mM dNTP, 0.1% Triton X-100, and 1 ng of the restriction enzyme ScaI, and 10 pmol of the primer described in Sequence Listing 12 and 13). After completion of the reaction, 5 μl of the reaction solution was subjected to agarose gel electrophoresis to confirm amplification of a target of about 4.3 Kb. The results of agarose gel electrophoresis are shown in FIG. As a result, PC was modified using the modified DNA polymerase.
With R, the amplification was better than with native KOD polymerase. Furthermore, the amplification was better than that of Taq polymerase.

【００４３】実施例１１ 改変型ＤＮＡポリメラーゼを用いたＰＣＲ反応（ヒトゲ
ノム） 改変型耐熱性ＤＮＡポリメラーゼを用いてＰＣＲ反応を
行った。５０μｌの反応液（120mM Tris-HCl(pH8.0 at
25℃), 10mM KCl, 6mM硫酸アンモニウム, 1mMMgCl₂, 0.
2mM dNTP, 0.1% Triton X-100, 0.001% BSA, 100ng の
ヒト胎盤由来のゲノムＤＮＡ（クローンテック社製）、
10ピコモルの配列表１１，１２記載のプライマー）に各
酵素を２．５単位加えてＰＣＲ反応を行った。サーマル
サイクラーはパーキンエルマー社製のモデルＰＪ２００
０を用いた。また、反応条件は９４℃、３０秒 → ６
８℃、３分を２５サイクル行った。比較として、Ｔａｑ
ポリメラーゼ（東洋紡製）も同様にしてＰＣＲ反応を行
った。ただし、反応液組成は（10mM Tris-HCl(pH8.8 at
25 ℃), 50mM KCl, 1.5mMMgCl₂, 0.2mM dNTP, 0.1% Tr
iton X-100, 100ngのヒト胎盤由来のゲノムＤＮＡ（ク
ローンテック社製、10ピコモルの配列表１２及び１３記
載のプライマー）で行った。反応終了後、５μｌの反応
液についてアガロースゲル電気泳動を行い、約４ｋｂの
ターゲットの増幅を確認した。図４にアガロースゲル電
気泳動の結果を示した。この結果、改変型ＤＮＡポリメ
ラーゼを用いてＰＣＲを行った場合、Ｔａｑポリメラー
ゼよりも良好な増幅であった。 Example 11 PCR reaction using modified DNA polymerase (human
Nome) PCR reaction was performed using a modified thermostable DNA polymerase. 50 μl of reaction solution (120 mM Tris-HCl (pH 8.0 at
25 ° C), 10 mM KCl, 6 mM ammonium sulfate, 1 mM MgCl ₂ , 0.
2 mM dNTP, 0.1% Triton X-100, 0.001% BSA, 100 ng of human placenta-derived genomic DNA (Clontech),
2.5 units of each enzyme was added to 10 picomoles of the primers shown in Sequence Listings 11 and 12 and PCR was performed. Thermal cycler model PJ200 made by Perkin Elmer
0 was used. The reaction conditions are 94 ° C, 30 seconds → 6
25 cycles of 8 ° C. and 3 minutes were performed. For comparison, Taq
A polymerase (manufactured by Toyobo) was also used for the PCR reaction in the same manner. However, the composition of the reaction solution is (10 mM Tris-HCl (pH 8.8 at
25 ℃), 50mM KCl, 1.5mM MgCl ₂ , 0.2mM dNTP, 0.1% Tr
Iton X-100, 100 ng of human placenta-derived genomic DNA (manufactured by Clontech, 10 picomoles of primers shown in Sequence Listing 12 and 13) were used. After completion of the reaction, 5 μl of the reaction solution was subjected to agarose gel electrophoresis to confirm amplification of a target of about 4 kb. The results of agarose gel electrophoresis are shown in FIG. As a result, when PCR was performed using the modified DNA polymerase, the amplification was better than that of Taq polymerase.

【００４４】[0044]

【発明の効果】本発明の改変された耐熱性ＤＮＡポリメ
ラーゼは、改変前の酵素に比べて、３’−５’エキソヌ
クレアーゼ活性が低下しているこにより、改変前の酵素
に比べて、増幅効率に優れたＰＣＲを行うことができ
る。INDUSTRIAL APPLICABILITY The modified thermostable DNA polymerase of the present invention has a decreased 3′-5 ′ exonuclease activity as compared with the enzyme before modification, and thus has a higher amplification than the enzyme before modification. PCR with excellent efficiency can be performed.

【００４５】[0045]

【配列表】[Sequence list]

配列番号：1 配列の長さ：5342 配列の型：核酸（ＤＮＡ） 鎖の数：２本鎖 トポロジー：直鎖状 配列の種類：genomic ＤＮＡ 起源：超好熱始原菌 株名：ＫＯＤ１ 配列の特徴 １５６−５１６５ Ｐ ＣＤＳ １３７４−２４５３ 介在配列 ２７０８−４３１６ 介在配列 配列 GCTTGAGGGC CTGCGGTTAT GGGACGTTGC AGTTTGCGCC TACTCAAAGA TGCCGGTTTT 60 ATAACGGAGA AAAATGGGGA GCTATTACGA TCTCTCCTTG ATGTGGGGTT TACAATAAAG 120 CCTGGATTGT TCTACAAGAT TATGGGGGAT GAAAG ATG ATC CTC GAC ACT GAC 173 Met Ile Leu Asp Thr Asp 1 5 TAC ATA ACC GAG GAT GGA AAG CCT GTC ATA AGA ATT TTC AAG AAG GAA 221 Tyr Ile Thr Glu Asp Gly Lys Pro Val Ile Arg Ile Phe Lys Lys Glu 10 15 20 AAC GGC GAG TTT AAG ATT GAG TAC GAC CGG ACT TTT GAA CCC TAC TTC 269 Asn Gly Glu Phe Lys Ile Glu Tyr Asp Arg Thr Phe Glu Pro Tyr Phe 25 30 35 TAC GCC CTC CTG AAG GAC GAT TCT GCC ATT GAG GAA GTC AAG AAG ATA 317 Tyr Ala Leu Leu Lys Asp Asp Ser Ala Ile Glu Glu Val Lys Lys Ile 40 45 50 ACC GCC GAG AGG CAC GGG ACG GTT GTA ACG GTT AAG CGG GTT GAA AAG 365 Thr Ala Glu Arg His Gly Thr Val Val Thr Val Lys Arg Val Glu Lys 55 60 65 70 GTT CAG AAG AAG TTC CTC GGG AGA CCA GTT GAG GTC TGG AAA CTC TAC 413 Val Gln Lys Lys Phe Leu Gly Arg Pro Val Glu Val Trp Lys Leu Tyr 75 80 85 TTT ACT CAT CCG CAG GAC GTC CCA GCG ATA AGG GAC AAG ATA CGA GAG 461 Phe Thr His Pro Gln Asp Val Pro Ala Ile Arg Asp Lys Ile Arg Glu 90 95 100 CAT GGA GCA GTT ATT GAC ATC TAC GAG TAC GAC ATA CCC TTC GCC AAG 509 His Gly Ala Val Ile Asp Ile Tyr Glu Tyr Asp Ile Pro Phe Ala Lys 105 110 115 CGC TAC CTC ATA GAC AAG GGA TTA GTG CCA ATG GAA GGC GAC GAG GAG 557 Arg Tyr Leu Ile Asp Lys Gly Leu Val Pro Met Glu Gly Asp Glu Glu 120 125 130 CTG AAA ATG CTC GCC TTC GAC ATT GAA ACT CTC TAC CAT GAG GGC GAG 605 Leu Lys Met Leu Ala Phe Asp Ile Glu Thr Leu Tyr His Glu Gly Glu 135 140 145 150 GAG TTC GCC GAG GGG CCA ATC CTT ATG ATA AGC TAC GCC GAC GAG GAA 653 Glu Phe Ala Glu Gly Pro Ile Leu Met Ile Ser Tyr Ala Asp Glu Glu 155 160 165 GGG GCC AGG GTG ATA ACT TGG AAG AAC GTG GAT CTC CCC TAC GTT GAC 701 Gly Ala Arg Val Ile Thr Trp Lys Asn Val Asp Leu Pro Tyr Val Asp 170 175 180 GTC GTC TCG ACG GAG AGG GAG ATG ATA AAG CGC TTC CTC CGT GTT GTG 749 Val Val Ser Thr Glu Arg Glu Met Ile Lys Arg Phe Leu Arg Val Val 185 190 195 AAG GAG AAA GAC CCG GAC GTT CTC ATA ACC TAC AAC GGC GAC AAC TTC 797 Lys Glu Lys Asp Pro Asp Val Leu Ile Thr Tyr Asn Gly Asp Asn Phe 200 205 210 GAC TTC GCC TAT CTG AAA AAG CGC TGT GAA AAG CTC GGA ATA AAC TTC 845 Asp Phe Ala Tyr Leu Lys Lys Arg Cys Glu Lys Leu Gly Ile Asn Phe 215 220 225 230 GCC CTC GGA AGG GAT GGA AGC GAG CCG AAG ATT CAG AGG ATG GGC GAC 893 Ala Leu Gly Arg Asp Gly Ser Glu Pro Lys Ile Gln Arg Met Gly Asp 235 240 245 AGG TTT GCC GTC GAA GTG AAG GGA CGG ATA CAC TTC GAT CTC TAT CCT 941 Arg Phe Ala Val Glu Val Lys Gly Arg Ile His Phe Asp Leu Tyr Pro 250 255 260 GTG ATA AGA CGG ACG ATA AAC CTG CCC ACA TAC ACG CTT GAG GCC GTT 989 Val Ile Arg Arg Thr Ile Asn Leu Pro Thr Tyr Thr Leu Glu Ala Val 265 270 275 TAT GAA GCC GTC TTC GGT CAG CCG AAG GAG AAG GTT TAC GCT GAG GAA 1037 Tyr Glu Ala Val Phe Gly Gln Pro Lys Glu Lys Val Tyr Ala Glu Glu 280 285 290 ATA ACA CCA GCC TGG GAA ACC GGC GAG AAC CTT GAG AGA GTC GCC CGC 1085 Ile Thr Pro Ala Trp Glu Thr Gly Glu Asn Leu Glu Arg Val Ala Arg 295 300 305 310 TAC TCG ATG GAA GAT GCG AAG GTC ACA TAC GAG CTT GGG AAG GAG TTC 1133 Tyr Ser Met Glu Asp Ala Lys Val Thr Tyr Glu Leu Gly Lys Glu Phe 315 320 325 CTT CCG ATG GAG GCC CAG CTT TCT CGC TTA ATC GGC CAG TCC CTC TGG 1181 Leu Pro Met Glu Ala Gln Leu Ser Arg Leu Ile Gly Gln Ser Leu Trp 330 335 340 GAC GTC TCC CGC TCC AGC ACT GGC AAC CTC GTT GAG TGG TTC CTC CTC 1229 Asp Val Ser Arg Ser Ser Thr Gly Asn Leu Val Glu Trp Phe Leu Leu 345 350 355 AGG AAG GCC TAT GAG AGG AAT GAG CTG GCC CCG AAC AAG CCC GAT GAA 1277 Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala Pro Asn Lys Pro Asp Glu 360 365 370 AAG GAG CTG GCC AGA AGA CGG CAG AGC TAT GAA GGA GGC TAT GTA AAA 1325 Lys Glu Leu Ala Arg Arg Arg Gln Ser Tyr Glu Gly Gly Tyr Val Lys 375 380 385 390 GAG CCC GAG AGA GGG TTG TGG GAG AAC ATA GTG TAC CTA GAT TTT AGA 1373 Glu Pro Glu Arg Gly Leu Trp Glu Asn Ile Val Tyr Leu Asp Phe Arg 395 400 405 TGC CAT CCA GCC GAT ACG AAG GTT GTC GTC AAG GGG AAG GGG ATT ATA 1421 Cys His Pro Ala Asp Thr Lys Val Val Val Lys Gly Lys Gly Ile Ile 410 415 420 AAC ATC AGC GAG GTT CAG GAA GGT GAC TAT GTC CTT GGG ATT GAC GGC 1469 Asn Ile Ser Glu Val Gln Glu Gly Asp Tyr Val Leu Gly Ile Asp Gly 425 430 435 TGG CAG AGA GTT AGA AAA GTA TGG GAA TAC GAC TAC AAA GGG GAG CTT 1517 Trp Gln Arg Val Arg Lys Val Trp Glu Tyr Asp Tyr Lys Gly Glu Leu 440 445 450 GTA AAC ATA AAC GGG TTA AAG TGT ACG CCC AAT CAT AAG CTT CCC GTT 1565 Val Asn Ile Asn Gly Leu Lys Cys Thr Pro Asn His Lys Leu Pro Val 455 460 465 470 GTT ACA AAG AAC GAA CGA CAA ACG AGA ATA AGA GAC AGT CTT GCT AAG 1613 Val Thr Lys Asn Glu Arg Gln Thr Arg Ile Arg Asp Ser Leu Ala Lys 475 480 485 TCT TTC CTT ACT AAA AAA GTT AAG GGC AAG ATA ATA ACC ACT CCC CTT 1661 Ser Phe Leu Thr Lys Lys Val Lys Gly Lys Ile Ile Thr Thr Pro Leu 490 495 500 TTC TAT GAA ATA GGC AGA GCG ACA AGT GAG AAT ATT CCA GAA GAA GAG 1709 Phe Tyr Glu Ile Gly Arg Ala Thr Ser Glu Asn Ile Pro Glu Glu Glu 505 510 515 GTT CTC AAG GGA GAG CTC GCT GGC ATA CTA TTG GCT GAA GGA ACG CTC 1757 Val Leu Lys Gly Glu Leu Ala Gly Ile Leu Leu Ala Glu Gly Thr Leu 520 525 530 TTG AGG AAA GAC GTT GAA TAC TTT GAT TCA TCC CGC AAA AAA CGG AGG 1805 Leu Arg Lys Asp Val Glu Tyr Phe Asp Ser Ser Arg Lys Lys Arg Arg 535 540 545 550 ATT TCA CAC CAG TAT CGT GTT GAG ATA ACC ATT GGG AAA GAC GAG GAG 1853 Ile Ser His Gln Tyr Arg Val Glu Ile Thr Ile Gly Lys Asp Glu Glu 555 560 565 GAG TTT AGG GAT CGT ATC ACA TAC ATT TTT GAG CGT TTG TTT GGG ATT 1901 Glu Phe Arg Asp Arg Ile Thr Tyr Ile Phe Glu Arg Leu Phe Gly Ile 570 575 580 ACT CCA AGC ATC TCG GAG AAG AAA GGA ACT AAC GCA GTA ACA CTC AAA 1949 Thr Pro Ser Ile Ser Glu Lys Lys Gly Thr Asn Ala Val Thr Leu Lys 585 590 595 GTT GCG AAG AAG AAT GTT TAT CTT AAA GTC AAG GAA ATT ATG GAC AAC 1997 Val Ala Lys Lys Asn Val Tyr Leu Lys Val Lys Glu Ile Met Asp Asn 600 605 610 ATA GAG TCC CTA CAT GCC CCC TCG GTT CTC AGG GGA TTC TTC GAA GGC 2045 Ile Glu Ser Leu His Ala Pro Ser Val Leu Arg Gly Phe Phe Glu Gly 615 620 625 630 GAC GGT TCA GTA AAC AGG GTT AGG AGG AGT ATT GTT GCA ACC CAG GGT 2093 Asp Gly Ser Val Asn Arg Val Arg Arg Ser Ile Val Ala Thr Gln Gly 635 640 645 ACA AAG AAC GAG TGG AAG ATT AAA CTG GTG TCA AAA CTG CTC TCC CAG 2141 Thr Lys Asn Glu Trp Lys Ile Lys Leu Val Ser Lys Leu Leu Ser Gln 650 655 660 CTT GGT ATC CCT CAT CAA ACG TAC ACG TAT CAG TAT CAG GAA AAT GGG 2189 Leu Gly Ile Pro His Gln Thr Tyr Thr Tyr Gln Tyr Gln Glu Asn Gly 665 670 675 AAA GAT CGG AGC AGG TAT ATA CTG GAG ATA ACT GGA AAG GAC GGA TTG 2237 Lys Asp Arg Ser Arg Tyr Ile Leu Glu Ile Thr Gly Lys Asp Gly Leu 680 685 690 ATA CTG TTC CAA ACA CTC ATT GGA TTC ATC AGT GAA AGA AAG AAC GCT 2285 Ile Leu Phe Gln Thr Leu Ile Gly Phe Ile Ser Glu Arg Lys Asn Ala 695 700 705 710 CTG CTT AAT AAG GCA ATA TCT CAG AGG GAA ATG AAC AAC TTG GAA AAC 2333 Leu Leu Asn Lys Ala Ile Ser Gln Arg Glu Met Asn Asn Leu Glu Asn 715 720 725 AAT GGA TTT TAC AGG CTC AGT GAA TTC AAT GTC AGC ACG GAA TAC TAT 2381 Asn Gly Phe Tyr Arg Leu Ser Glu Phe Asn Val Ser Thr Glu Tyr Tyr 730 735 740 GAG GGC AAG GTC TAT GAC TTA ACT CTT GAA GGA ACT CCC TAC TAC TTT 2429 Glu Gly Lys Val Tyr Asp Leu Thr Leu Glu Gly Thr Pro Tyr Tyr Phe 745 750 755 GCC AAT GGC ATA TTG ACC CAT AAC TCC CTG TAC CCC TCA ATC ATC ATC 2477 Ala Asn Gly Ile Leu Thr His Asn Ser Leu Tyr Pro Ser Ile Ile Ile 760 765 770 ACC CAC AAC GTC TCG CCG GAT ACG CTC AAC AGA GAA GGA TGC AAG GAA 2525 Thr His Asn Val Ser Pro Asp Thr Leu Asn Arg Glu Gly Cys Lys Glu 775 780 785 790 TAT GAC GTT GCC CCA CAG GTC GGC CAC CGC TTC TGC AAG GAC TTC CCA 2573 Tyr Asp Val Ala Pro Gln Val Gly His Arg Phe Cys Lys Asp Phe Pro 795 800 805 GGA TTT ATC CCG AGC CTG CTT GGA GAC CTC CTA GAG GAG AGG CAG AAG 2621 Gly Phe Ile Pro Ser Leu Leu Gly Asp Leu Leu Glu Glu Arg Gln Lys 810 815 820 ATA AAG AAG AAG ATG AAG GCC ACG ATT GAC CCG ATC GAG AGG AAG CTC 2669 Ile Lys Lys Lys Met Lys Ala Thr Ile Asp Pro Ile Glu Arg Lys Leu 825 830 835 CTC GAT TAC AGG CAG AGG GCC ATC AAG ATC CTG GCA AAC AGC ATC CTA 2717 Leu Asp Tyr Arg Gln Arg Ala Ile Lys Ile Leu Ala Asn Ser Ile Leu 840 845 850 CCC GAG GAA TGG CTT CCA GTC CTC GAG GAA GGG GAG GTT CAC TTC GTC 2765 Pro Glu Glu Trp Leu Pro Val Leu Glu Glu Gly Glu Val His Phe Val 855 860 865 870 AGG ATT GGA GAG CTC ATA GAC CGG ATG ATG GAG GAA AAT GCT GGG AAA 2813 Arg Ile Gly Glu Leu Ile Asp Arg Met Met Glu Glu Asn Ala Gly Lys 875 880 885 GTA AAG AGA GAG GGC GAG ACG GAA GTG CTT GAG GTC AGT GGG CTT GAA 2861 Val Lys Arg Glu Gly Glu Thr Glu Val Leu Glu Val Ser Gly Leu Glu 890 895 900 GTC CCG TCC TTT AAC AGG AGA ACT AAC AAG GCC GAG CTC AAG AGA GTA 2909 Val Pro Ser Phe Asn Arg Arg Thr Asn Lys Ala Glu Leu Lys Arg Val 905 910 915 AAG GCC CTG ATT AGG CAC GAT TAT TCT GGC AAG GTC TAC ACC ATC AGA 2957 Lys Ala Leu Ile Arg His Asp Tyr Ser Gly Lys Val Tyr Thr Ile Arg 920 925 930 CTG AAG TCG GGG AGG AGA ATA AAG ATA ACC TCT GGC CAC AGC CTC TTC 3005 Leu Lys Ser Gly Arg Arg Ile Lys Ile Thr Ser Gly His Ser Leu Phe 935 940 945 950 TCT GTG AGA AAC GGG GAG CTC GTT GAA GTT ACG GGC GAT GAA CTA AAG 3053 Ser Val Arg Asn Gly Glu Leu Val Glu Val Thr Gly Asp Glu Leu Lys 955 960 965 CCA GGT GAC CTC GTT GCA GTC CCG CGG AGA TTG GAG CTT CCT GAG AGA 3101 Pro Gly Asp Leu Val Ala Val Pro Arg Arg Leu Glu Leu Pro Glu Arg 970 975 980 AAC CAC GTG CTG AAC CTC GTT GAA CTG CTC CTT GGA ACG CCA GAA GAA 3149 Asn His Val Leu Asn Leu Val Glu Leu Leu Leu Gly Thr Pro Glu Glu 985 990 995 GAA ACT TTG GAC ATC GTC ATG ACG ATC CCA GTC AAG GGT AAG AAG AAC 3197 Glu Thr Leu Asp Ile Val Met Thr Ile Pro Val Lys Gly Lys Lys Asn 1000 1005 1010 TTC TTT AAA GGG ATG CTC AGG ACT TTG CGC TGG ATT TTC GGA GAG GAA 3245 Phe Phe Lys Gly Met Leu Arg Thr Leu Arg Trp Ile Phe Gly Glu Glu 1015 1020 1025 1030 AAG AGG CCC AGA ACC GCG AGA CGC TAT CTC AGG CAC CTT GAG GAT CTG 3293 Lys Arg Pro Arg Thr Ala Arg Arg Tyr Leu Arg His Leu Glu Asp Leu 1035 1040 1045 GGC TAT GTC CGG CTT AAG AAG ATC GGC TAC GAA GTC CTC GAC TGG GAC 3341 Gly Tyr Val Arg Leu Lys Lys Ile Gly Tyr Glu Val Leu Asp Trp Asp 1050 1055 1060 TCA CTT AAG AAC TAC AGA AGG CTC TAC GAG GCG CTT GTC GAG AAC GTC 3389 Ser Leu Lys Asn Tyr Arg Arg Leu Tyr Glu Ala Leu Val Glu Asn Val 1065 1070 1075 AGA TAC AAC GGC AAC AAG AGG GAG TAC CTC GTT GAA TTC AAT TCC ATC 3437 Arg Tyr Asn Gly Asn Lys Arg Glu Tyr Leu Val Glu Phe Asn Ser Ile 1080 1085 1090 CGG GAT GCA GTT GGC ATA ATG CCC CTA AAA GAG CTG AAG GAG TGG AAG 3485 Arg Asp Ala Val Gly Ile Met Pro Leu Lys Glu Leu Lys Glu Trp Lys 1095 1100 1105 1110 ATC GGC ACG CTG AAC GGC TTC AGA ATG AGA AAG CTC ATT GAA GTG GAC 3533 Ile Gly Thr Leu Asn Gly Phe Arg Met Arg Lys Leu Ile Glu Val Asp 1115 1120 1125 GAG TCG TTA GCA AAG CTC CTC GGC TAC TAC GTG AGC GAG GGC TAT GCA 3581 Glu Ser Leu Ala Lys Leu Leu Gly Tyr Tyr Val Ser Glu Gly Tyr Ala 1130 1135 1140 AGA AAG CAG AGG AAT CCC AAA AAC GGC TGG AGC TAC AGC GTG AAG CTC 3629 Arg Lys Gln Arg Asn Pro Lys Asn Gly Trp Ser Tyr Ser Val Lys Leu 1145 1150 1155 TAC AAC GAA GAC CCT GAA GTG CTG GAC GAT ATG GAG AGA CTC GCC AGC 3677 Tyr Asn Glu Asp Pro Glu Val Leu Asp Asp Met Glu Arg Leu Ala Ser 1160 1165 1170 AGG TTT TTC GGG AAG GTG AGG CGG GGC AGG AAC TAC GTT GAG ATA CCG 3725 Arg Phe Phe Gly Lys Val Arg Arg Gly Arg Asn Tyr Val Glu Ile Pro 1175 1180 1185 1190 AAG AAG ATC GGC TAC CTG CTC TTT GAG AAC ATG TGC GGT GTC CTA GCG 3773 Lys Lys Ile Gly Tyr Leu Leu Phe Glu Asn Met Cys Gly Val Leu Ala 1195 1200 1205 GAG AAC AAG AGG ATT CCC GAG TTC GTC TTC ACG TCC CCG AAA GGG GTT 3821 Glu Asn Lys Arg Ile Pro Glu Phe Val Phe Thr Ser Pro Lys Gly Val 1210 1215 1220 CGG CTG GCC TTC CTT GAG GGG TAC TCA TCG GCG ATG GCG ACG TCC ACC 3869 Arg Leu Ala Phe Leu Glu Gly Tyr Ser Ser Ala Met Ala Thr Ser Thr 1225 1230 1235 GAA CAA GAG ACT CAG GCT CTC AAC GAA AAG CGA GCT TTA GCG AAC CAG 3917 Glu Gln Glu Thr Gln Ala Leu Asn Glu Lys Arg Ala Leu Ala Asn Gln 1240 1245 1250 CTC GTC CTC CTC TTG AAC TCG GTG GGG GTC TCT GCT GTA AAA CTT GGG 3965 Leu Val Leu Leu Leu Asn Ser Val Gly Val Ser Ala Val Lys Leu Gly 1255 1260 1265 1270 CAC GAC AGC GGC GTT TAC AGG GTC TAT ATA AAC GAG GAG CTC CCG TTC 4013 His Asp Ser Gly Val Tyr Arg Val Tyr Ile Asn Glu Glu Leu Pro Phe 1275 1280 1285 GTA AAG CTG GAC AAG AAA AAG AAC GCC TAC TAC TCA CAC GTG ATC CCC 4061 Val Lys Leu Asp Lys Lys Lys Asn Ala Tyr Tyr Ser His Val Ile Pro 1290 1295 1300 AAG GAA GTC CTG AGC GAG GTC TTT GGG AAG GTT TTC CAG AAA AAC GTC 4109 Lys Glu Val Leu Ser Glu Val Phe Gly Lys Val Phe Gln Lys Asn Val 1305 1310 1315 AGT CCT CAG ACC TTC AGG AAG ATG GTC GAG GAC GGA AGA CTC GAT CCC 4157 Ser Pro Gln Thr Phe Arg Lys Met Val Glu Asp Gly Arg Leu Asp Pro 1320 1325 1330 GAA AAG GCC CAG AGG CTC TCC TGG CTC ATT GAG GGG GAC GTA GTG CTC 4205 Glu Lys Ala Gln Arg Leu Ser Trp Leu Ile Glu Gly Asp Val Val Leu 1335 1340 1345 1350 GAC CGC GTT GAG TCC GTT GAT GTG GAA GAC TAC GAT GGT TAT GTC TAT 4253 Asp Arg Val Glu Ser Val Asp Val Glu Asp Tyr Asp Gly Tyr Val Tyr 1355 1360 1365 GAC CTG AGC GTC GAG GAC AAC GAG AAC TTC CTC GTT GGC TTT GGG TTG 4301 Asp Leu Ser Val Glu Asp Asn Glu Asn Phe Leu Val Gly Phe Gly Leu 1370 1375 1380 GTC TAT GCT CAC AAC AGC TAC TAC GGT TAC TAC GGC TAT GCA AGG GCG 4349 Val Tyr Ala His Asn Ser Tyr Tyr Gly Tyr Tyr Gly Tyr Ala Arg Ala 1385 1390 1395 CGC TGG TAC TGC AAG GAG TGT GCA GAG AGC GTA ACG GCC TGG GGA AGG 4397 Arg Trp Tyr Cys Lys Glu Cys Ala Glu Ser Val Thr Ala Trp Gly Arg 1400 1405 1410 GAG TAC ATA ACG ATG ACC ATC AAG GAG ATA GAG GAA AAG TAC GGC TTT 4445 Glu Tyr Ile Thr Met Thr Ile Lys Glu Ile Glu Glu Lys Tyr Gly Phe 1415 1420 1425 1430 AAG GTA ATC TAC AGC GAC ACC GAC GGA TTT TTT GCC ACA ATA CCT GGA 4493 Lys Val Ile Tyr Ser Asp Thr Asp Gly Phe Phe Ala Thr Ile Pro Gly 1435 1440 1445 GCC GAT GCT GAA ACC GTC AAA AAG AAG GCT ATG GAG TTC CTC AAC TAT 4541 Ala Asp Ala Glu Thr Val Lys Lys Lys Ala Met Glu Phe Leu Asn Tyr 1450 1455 1460 ATC AAC GCC AAA CTT CCG GGC GCG CTT GAG CTC GAG TAC GAG GGC TTC 4589 Ile Asn Ala Lys Leu Pro Gly Ala Leu Glu Leu Glu Tyr Glu Gly Phe 1465 1470 1475 TAC AAA CGC GGC TTC TTC GTC ACG AAG AAG AAG TAT GCG GTG ATA GAC 4637 Tyr Lys Arg Gly Phe Phe Val Thr Lys Lys Lys Tyr Ala Val Ile Asp 1480 1485 1490 GAG GAA GGC AAG ATA ACA ACG CGC GGA CTT GAG ATT GTG AGG CGT GAC 4685 Glu Glu Gly Lys Ile Thr Thr Arg Gly Leu Glu Ile Val Arg Arg Asp 1495 1500 1505 1510 TGG AGC GAG ATA GCG AAA GAG ACG CAG GCG AGG GTT CTT GAA GCT TTG 4733 Trp Ser Glu Ile Ala Lys Glu Thr Gln Ala Arg Val Leu Glu Ala Leu 1515 1520 1525 CTA AAG GAC GGT GAC GTC GAG AAG GCC GTG AGG ATA GTC AAA GAA GTT 4781 Leu Lys Asp Gly Asp Val Glu Lys Ala Val Arg Ile Val Lys Glu Val 1530 1535 1540 ACC GAA AAG CTG AGC AAG TAC GAG GTT CCG CCG GAG AAG CTG GTG ATC 4829 Thr Glu Lys Leu Ser Lys Tyr Glu Val Pro Pro Glu Lys Leu Val Ile 1545 1550 1555 CAC GAG CAG ATA ACG AGG GAT TTA AAG GAC TAC AAG GCA ACC GGT CCC 4877 His Glu Gln Ile Thr Arg Asp Leu Lys Asp Tyr Lys Ala Thr Gly Pro 1560 1565 1570 CAC GTT GCC GTT GCC AAG AGG TTG GCC GCG AGA GGA GTC AAA ATA CGC 4925 His Val Ala Val Ala Lys Arg Leu Ala Ala Arg Gly Val Lys Ile Arg 1575 1580 1585 1590 CCT GGA ACG GTG ATA AGC TAC ATC GTG CTC AAG GGC TCT GGG AGG ATA 4973 Pro Gly Thr Val Ile Ser Tyr Ile Val Leu Lys Gly Ser Gly Arg Ile 1595 1600 1605 GGC GAC AGG GCG ATA CCG TTC GAC GAG TTC GAC CCG ACG AAG CAC AAG 5021 Gly Asp Arg Ala Ile Pro Phe Asp Glu Phe Asp Pro Thr Lys His Lys 1610 1615 1620 TAC GAC GCC GAG TAC TAC ATT GAG AAC CAG GTT CTC CCA GCC GTT GAG 5069 Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn Gln Val Leu Pro Ala Val Glu 1625 1630 1635 AGA ATT CTG AGA GCC TTC GGT TAC CGC AAG GAA GAC CTG CGC TAC CAG 5117 Arg Ile Leu Arg Ala Phe Gly Tyr Arg Lys Glu Asp Leu Arg Tyr Gln 1640 1645 1650 AAG ACG AGA CAG GTT GGT TTG AGT GCT TGG CTG AAG CCG AAG GGA ACT 5165 Lys Thr Arg Gln Val Gly Leu Ser Ala Trp Leu Lys Pro Lys Gly Thr 1655 1660 1665 1670 TGACCTTTCC ATTTGTTTTC CAGCGGATAA CCCTTTAACT TCCCTTTCAA AAACTCCCTT 5225 TAGGGAAAGA CCATGAAGAT AGAAATCCGG CGGCGCCCGG TTAAATACGC TAGGATAGAA 5285 GTGAAGCCAG ACGGCAGGGT AGTCGTCACT GCCCCGAGGG TTCAACGTTG AGAAGTT 5342 SEQ ID NO: 1 Array length: 5342 Sequence type: nucleic acid (DNA) Number of chains: double-stranded Topology: linear Sequence type: genomic DNA Origin: Hyperthermophilic archaeon Share name: KOD1 Sequence features 156-5165 P CDS 1374-2453 intervening sequence 2708-4316 Intervening sequence Array GCTTGAGGGC CTGCGGTTAT GGGACGTTGC AGTTTGCGCC TACTCAAAGA TGCCGGTTTT 60 ATAACGGAGA AAAATGGGGA GCTATTACGA TCTCTCCTTG ATGTGGGGTT TACAATAAAG 120 CCTGGATTGT TCTACAAGAT TATGGGGGAT GAAAG ATG ATC CTC GAC ACT GAC 173                                        Met Ile Leu Asp Thr Asp                                          1 5 TAC ATA ACC GAG GAT GGA AAG CCT GTC ATA AGA ATT TTC AAG AAG GAA 221 Tyr Ile Thr Glu Asp Gly Lys Pro Val Ile Arg Ile Phe Lys Lys Glu              10 15 20 AAC GGC GAG TTT AAG ATT GAG TAC GAC CGG ACT TTT GAA CCC TAC TTC 269 Asn Gly Glu Phe Lys Ile Glu Tyr Asp Arg Thr Phe Glu Pro Tyr Phe          25 30 35 TAC GCC CTC CTG AAG GAC GAT TCT GCC ATT GAG GAA GTC AAG AAG ATA 317 Tyr Ala Leu Leu Lys Asp Asp Ser Ala Ile Glu Glu Val Lys Lys Ile      40 45 50 ACC GCC GAG AGG CAC GGG ACG GTT GTA ACG GTT AAG CGG GTT GAA AAG 365 Thr Ala Glu Arg His Gly Thr Val Val Thr Val Lys Arg Val Glu Lys  55 60 65 70 GTT CAG AAG AAG TTC CTC GGG AGA CCA GTT GAG GTC TGG AAA CTC TAC 413 Val Gln Lys Lys Phe Leu Gly Arg Pro Val Glu Val Trp Lys Leu Tyr                  75 80 85 TTT ACT CAT CCG CAG GAC GTC CCA GCG ATA AGG GAC AAG ATA CGA GAG 461 Phe Thr His Pro Gln Asp Val Pro Ala Ile Arg Asp Lys Ile Arg Glu              90 95 100 CAT GGA GCA GTT ATT GAC ATC TAC GAG TAC GAC ATA CCC TTC GCC AAG 509 His Gly Ala Val Ile Asp Ile Tyr Glu Tyr Asp Ile Pro Phe Ala Lys         105 110 115 CGC TAC CTC ATA GAC AAG GGA TTA GTG CCA ATG GAA GGC GAC GAG GAG 557 Arg Tyr Leu Ile Asp Lys Gly Leu Val Pro Met Glu Gly Asp Glu Glu     120 125 130 CTG AAA ATG CTC GCC TTC GAC ATT GAA ACT CTC TAC CAT GAG GGC GAG 605 Leu Lys Met Leu Ala Phe Asp Ile Glu Thr Leu Tyr His Glu Gly Glu 135 140 145 150 GAG TTC GCC GAG GGG CCA ATC CTT ATG ATA AGC TAC GCC GAC GAG GAA 653 Glu Phe Ala Glu Gly Pro Ile Leu Met Ile Ser Tyr Ala Asp Glu Glu                 155 160 165 GGG GCC AGG GTG ATA ACT TGG AAG AAC GTG GAT CTC CCC TAC GTT GAC 701 Gly Ala Arg Val Ile Thr Trp Lys Asn Val Asp Leu Pro Tyr Val Asp             170 175 180 GTC GTC TCG ACG GAG AGG GAG ATG ATA AAG CGC TTC CTC CGT GTT GTG 749 Val Val Ser Thr Glu Arg Glu Met Ile Lys Arg Phe Leu Arg Val Val         185 190 195 AAG GAG AAA GAC CCG GAC GTT CTC ATA ACC TAC AAC GGC GAC AAC TTC 797 Lys Glu Lys Asp Pro Asp Val Leu Ile Thr Tyr Asn Gly Asp Asn Phe     200 205 210 GAC TTC GCC TAT CTG AAA AAG CGC TGT GAA AAG CTC GGA ATA AAC TTC 845 Asp Phe Ala Tyr Leu Lys Lys Arg Cys Glu Lys Leu Gly Ile Asn Phe 215 220 225 230 GCC CTC GGA AGG GAT GGA AGC GAG CCG AAG ATT CAG AGG ATG GGC GAC 893 Ala Leu Gly Arg Asp Gly Ser Glu Pro Lys Ile Gln Arg Met Gly Asp                 235 240 245 AGG TTT GCC GTC GAA GTG AAG GGA CGG ATA CAC TTC GAT CTC TAT CCT 941 Arg Phe Ala Val Glu Val Lys Gly Arg Ile His Phe Asp Leu Tyr Pro             250 255 260 GTG ATA AGA CGG ACG ATA AAC CTG CCC ACA TAC ACG CTT GAG GCC GTT 989 Val Ile Arg Arg Thr Ile Asn Leu Pro Thr Tyr Thr Leu Glu Ala Val         265 270 275 TAT GAA GCC GTC TTC GGT CAG CCG AAG GAG AAG GTT TAC GCT GAG GAA 1037 Tyr Glu Ala Val Phe Gly Gln Pro Lys Glu Lys Val Tyr Ala Glu Glu     280 285 290 ATA ACA CCA GCC TGG GAA ACC GGC GAG AAC CTT GAG AGA GTC GCC CGC 1085 Ile Thr Pro Ala Trp Glu Thr Gly Glu Asn Leu Glu Arg Val Ala Arg 295 300 305 310 TAC TCG ATG GAA GAT GCG AAG GTC ACA TAC GAG CTT GGG AAG GAG TTC 1133 Tyr Ser Met Glu Asp Ala Lys Val Thr Tyr Glu Leu Gly Lys Glu Phe                 315 320 325 CTT CCG ATG GAG GCC CAG CTT TCT CGC TTA ATC GGC CAG TCC CTC TGG 1181 Leu Pro Met Glu Ala Gln Leu Ser Arg Leu Ile Gly Gln Ser Leu Trp             330 335 340 GAC GTC TCC CGC TCC AGC ACT GGC AAC CTC GTT GAG TGG TTC CTC CTC 1229 Asp Val Ser Arg Ser Ser Thr Gly Asn Leu Val Glu Trp Phe Leu Leu         345 350 355 AGG AAG GCC TAT GAG AGG AAT GAG CTG GCC CCG AAC AAG CCC GAT GAA 1277 Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala Pro Asn Lys Pro Asp Glu     360 365 370 AAG GAG CTG GCC AGA AGA CGG CAG AGC TAT GAA GGA GGC TAT GTA AAA 1325 Lys Glu Leu Ala Arg Arg Arg Gln Ser Tyr Glu Gly Gly Tyr Val Lys 375 380 385 390 GAG CCC GAG AGA GGG TTG TGG GAG AAC ATA GTG TAC CTA GAT TTT AGA 1373 Glu Pro Glu Arg Gly Leu Trp Glu Asn Ile Val Tyr Leu Asp Phe Arg                 395 400 405 TGC CAT CCA GCC GAT ACG AAG GTT GTC GTC AAG GGG AAG GGG ATT ATA 1421 Cys His Pro Ala Asp Thr Lys Val Val Val Lys Gly Lys Gly Ile Ile             410 415 420 AAC ATC AGC GAG GTT CAG GAA GGT GAC TAT GTC CTT GGG ATT GAC GGC 1469 Asn Ile Ser Glu Val Gln Glu Gly Asp Tyr Val Leu Gly Ile Asp Gly         425 430 435 TGG CAG AGA GTT AGA AAA GTA TGG GAA TAC GAC TAC AAA GGG GAG CTT 1517 Trp Gln Arg Val Arg Lys Val Trp Glu Tyr Asp Tyr Lys Gly Glu Leu     440 445 450 GTA AAC ATA AAC GGG TTA AAG TGT ACG CCC AAT CAT AAG CTT CCC GTT 1565 Val Asn Ile Asn Gly Leu Lys Cys Thr Pro Asn His Lys Leu Pro Val 455 460 465 470 GTT ACA AAG AAC GAA CGA CAA ACG AGA ATA AGA GAC AGT CTT GCT AAG 1613 Val Thr Lys Asn Glu Arg Gln Thr Arg Ile Arg Asp Ser Leu Ala Lys                 475 480 485 TCT TTC CTT ACT AAA AAA GTT AAG GGC AAG ATA ATA ACC ACT CCC CTT 1661 Ser Phe Leu Thr Lys Lys Val Lys Gly Lys Ile Ile Thr Thr Pro Leu             490 495 500 TTC TAT GAA ATA GGC AGA GCG ACA AGT GAG AAT ATT CCA GAA GAA GAG 1709 Phe Tyr Glu Ile Gly Arg Ala Thr Ser Glu Asn Ile Pro Glu Glu Glu         505 510 515 GTT CTC AAG GGA GAG CTC GCT GGC ATA CTA TTG GCT GAA GGA ACG CTC 1757 Val Leu Lys Gly Glu Leu Ala Gly Ile Leu Leu Ala Glu Gly Thr Leu     520 525 530 TTG AGG AAA GAC GTT GAA TAC TTT GAT TCA TCC CGC AAA AAA CGG AGG 1805 Leu Arg Lys Asp Val Glu Tyr Phe Asp Ser Ser Arg Lys Lys Arg Arg 535 540 545 550 ATT TCA CAC CAG TAT CGT GTT GAG ATA ACC ATT GGG AAA GAC GAG GAG 1853 Ile Ser His Gln Tyr Arg Val Glu Ile Thr Ile Gly Lys Asp Glu Glu                 555 560 565 GAG TTT AGG GAT CGT ATC ACA TAC ATT TTT GAG CGT TTG TTT GGG ATT 1901 Glu Phe Arg Asp Arg Ile Thr Tyr Ile Phe Glu Arg Leu Phe Gly Ile             570 575 580 ACT CCA AGC ATC TCG GAG AAG AAA GGA ACT AAC GCA GTA ACA CTC AAA 1949 Thr Pro Ser Ile Ser Glu Lys Lys Gly Thr Asn Ala Val Thr Leu Lys         585 590 595 GTT GCG AAG AAG AAT GTT TAT CTT AAA GTC AAG GAA ATT ATG GAC AAC 1997 Val Ala Lys Lys Asn Val Tyr Leu Lys Val Lys Glu Ile Met Asp Asn     600 605 610 ATA GAG TCC CTA CAT GCC CCC TCG GTT CTC AGG GGA TTC TTC GAA GGC 2045 Ile Glu Ser Leu His Ala Pro Ser Val Leu Arg Gly Phe Phe Glu Gly 615 620 625 630 GAC GGT TCA GTA AAC AGG GTT AGG AGG AGT ATT GTT GCA ACC CAG GGT 2093 Asp Gly Ser Val Asn Arg Val Arg Arg Ser Ile Val Ala Thr Gln Gly                 635 640 645 ACA AAG AAC GAG TGG AAG ATT AAA CTG GTG TCA AAA CTG CTC TCC CAG 2141 Thr Lys Asn Glu Trp Lys Ile Lys Leu Val Ser Lys Leu Leu Ser Gln             650 655 660 CTT GGT ATC CCT CAT CAA ACG TAC ACG TAT CAG TAT CAG GAA AAT GGG 2189 Leu Gly Ile Pro His Gln Thr Tyr Thr Tyr Gln Tyr Gln Glu Asn Gly         665 670 675 AAA GAT CGG AGC AGG TAT ATA CTG GAG ATA ACT GGA AAG GAC GGA TTG 2237 Lys Asp Arg Ser Arg Tyr Ile Leu Glu Ile Thr Gly Lys Asp Gly Leu     680 685 690 ATA CTG TTC CAA ACA CTC ATT GGA TTC ATC AGT GAA AGA AAG AAC GCT 2285 Ile Leu Phe Gln Thr Leu Ile Gly Phe Ile Ser Glu Arg Lys Asn Ala 695 700 705 710 CTG CTT AAT AAG GCA ATA TCT CAG AGG GAA ATG AAC AAC TTG GAA AAC 2333 Leu Leu Asn Lys Ala Ile Ser Gln Arg Glu Met Asn Asn Leu Glu Asn                 715 720 725 AAT GGA TTT TAC AGG CTC AGT GAA TTC AAT GTC AGC ACG GAA TAC TAT 2381 Asn Gly Phe Tyr Arg Leu Ser Glu Phe Asn Val Ser Thr Glu Tyr Tyr             730 735 740 GAG GGC AAG GTC TAT GAC TTA ACT CTT GAA GGA ACT CCC TAC TAC TTT 2429 Glu Gly Lys Val Tyr Asp Leu Thr Leu Glu Gly Thr Pro Tyr Tyr Phe         745 750 755 GCC AAT GGC ATA TTG ACC CAT AAC TCC CTG TAC CCC TCA ATC ATC ATC 2477 Ala Asn Gly Ile Leu Thr His Asn Ser Leu Tyr Pro Ser Ile Ile Ile     760 765 770 ACC CAC AAC GTC TCG CCG GAT ACG CTC AAC AGA GAA GGA TGC AAG GAA 2525 Thr His Asn Val Ser Pro Asp Thr Leu Asn Arg Glu Gly Cys Lys Glu 775 780 785 790 TAT GAC GTT GCC CCA CAG GTC GGC CAC CGC TTC TGC AAG GAC TTC CCA 2573 Tyr Asp Val Ala Pro Gln Val Gly His Arg Phe Cys Lys Asp Phe Pro                 795 800 805 GGA TTT ATC CCG AGC CTG CTT GGA GAC CTC CTA GAG GAG AGG CAG AAG 2621 Gly Phe Ile Pro Ser Leu Leu Gly Asp Leu Leu Glu Glu Arg Gln Lys             810 815 820 ATA AAG AAG AAG ATG AAG GCC ACG ATT GAC CCG ATC GAG AGG AAG CTC 2669 Ile Lys Lys Lys Met Lys Ala Thr Ile Asp Pro Ile Glu Arg Lys Leu         825 830 835 CTC GAT TAC AGG CAG AGG GCC ATC AAG ATC CTG GCA AAC AGC ATC CTA 2717 Leu Asp Tyr Arg Gln Arg Ala Ile Lys Ile Leu Ala Asn Ser Ile Leu     840 845 850 CCC GAG GAA TGG CTT CCA GTC CTC GAG GAA GGG GAG GTT CAC TTC GTC 2765 Pro Glu Glu Trp Leu Pro Val Leu Glu Glu Gly Glu Val His Phe Val 855 860 865 870 AGG ATT GGA GAG CTC ATA GAC CGG ATG ATG GAG GAA AAT GCT GGG AAA 2813 Arg Ile Gly Glu Leu Ile Asp Arg Met Met Glu Glu Asn Ala Gly Lys                 875 880 885 GTA AAG AGA GAG GGC GAG ACG GAA GTG CTT GAG GTC AGT GGG CTT GAA 2861 Val Lys Arg Glu Gly Glu Thr Glu Val Leu Glu Val Ser Gly Leu Glu             890 895 900 GTC CCG TCC TTT AAC AGG AGA ACT AAC AAG GCC GAG CTC AAG AGA GTA 2909 Val Pro Ser Phe Asn Arg Arg Thr Asn Lys Ala Glu Leu Lys Arg Val         905 910 915 AAG GCC CTG ATT AGG CAC GAT TAT TCT GGC AAG GTC TAC ACC ATC AGA 2957 Lys Ala Leu Ile Arg His Asp Tyr Ser Gly Lys Val Tyr Thr Ile Arg     920 925 930 CTG AAG TCG GGG AGG AGA ATA AAG ATA ACC TCT GGC CAC AGC CTC TTC 3005 Leu Lys Ser Gly Arg Arg Ile Lys Ile Thr Ser Gly His Ser Leu Phe 935 940 945 950 TCT GTG AGA AAC GGG GAG CTC GTT GAA GTT ACG GGC GAT GAA CTA AAG 3053 Ser Val Arg Asn Gly Glu Leu Val Glu Val Thr Gly Asp Glu Leu Lys                 955 960 965 CCA GGT GAC CTC GTT GCA GTC CCG CGG AGA TTG GAG CTT CCT GAG AGA 3101 Pro Gly Asp Leu Val Ala Val Pro Arg Arg Leu Glu Leu Pro Glu Arg             970 975 980 AAC CAC GTG CTG AAC CTC GTT GAA CTG CTC CTT GGA ACG CCA GAA GAA 3149 Asn His Val Leu Asn Leu Val Glu Leu Leu Leu Gly Thr Pro Glu Glu         985 990 995 GAA ACT TTG GAC ATC GTC ATG ACG ATC CCA GTC AAG GGT AAG AAG AAC 3197 Glu Thr Leu Asp Ile Val Met Thr Ile Pro Val Lys Gly Lys Lys Asn     1000 1005 1010 TTC TTT AAA GGG ATG CTC AGG ACT TTG CGC TGG ATT TTC GGA GAG GAA 3245 Phe Phe Lys Gly Met Leu Arg Thr Leu Arg Trp Ile Phe Gly Glu Glu 1015 1020 1025 1030 AAG AGG CCC AGA ACC GCG AGA CGC TAT CTC AGG CAC CTT GAG GAT CTG 3293 Lys Arg Pro Arg Thr Ala Arg Arg Tyr Leu Arg His Leu Glu Asp Leu                 1035 1040 1045 GGC TAT GTC CGG CTT AAG AAG ATC GGC TAC GAA GTC CTC GAC TGG GAC 3341 Gly Tyr Val Arg Leu Lys Lys Ile Gly Tyr Glu Val Leu Asp Trp Asp             1050 1055 1060 TCA CTT AAG AAC TAC AGA AGG CTC TAC GAG GCG CTT GTC GAG AAC GTC 3389 Ser Leu Lys Asn Tyr Arg Arg Leu Tyr Glu Ala Leu Val Glu Asn Val         1065 1070 1075 AGA TAC AAC GGC AAC AAG AGG GAG TAC CTC GTT GAA TTC AAT TCC ATC 3437 Arg Tyr Asn Gly Asn Lys Arg Glu Tyr Leu Val Glu Phe Asn Ser Ile     1080 1085 1090 CGG GAT GCA GTT GGC ATA ATG CCC CTA AAA GAG CTG AAG GAG TGG AAG 3485 Arg Asp Ala Val Gly Ile Met Pro Leu Lys Glu Leu Lys Glu Trp Lys 1095 1100 1105 1110 ATC GGC ACG CTG AAC GGC TTC AGA ATG AGA AAG CTC ATT GAA GTG GAC 3533 Ile Gly Thr Leu Asn Gly Phe Arg Met Arg Lys Leu Ile Glu Val Asp                 1115 1120 1125 GAG TCG TTA GCA AAG CTC CTC GGC TAC TAC GTG AGC GAG GGC TAT GCA 3581 Glu Ser Leu Ala Lys Leu Leu Gly Tyr Tyr Val Ser Glu Gly Tyr Ala             1130 1135 1140 AGA AAG CAG AGG AAT CCC AAA AAC GGC TGG AGC TAC AGC GTG AAG CTC 3629 Arg Lys Gln Arg Asn Pro Lys Asn Gly Trp Ser Tyr Ser Val Lys Leu         1145 1150 1155 TAC AAC GAA GAC CCT GAA GTG CTG GAC GAT ATG GAG AGA CTC GCC AGC 3677 Tyr Asn Glu Asp Pro Glu Val Leu Asp Asp Met Glu Arg Leu Ala Ser     1160 1165 1170 AGG TTT TTC GGG AAG GTG AGG CGG GGC AGG AAC TAC GTT GAG ATA CCG 3725 Arg Phe Phe Gly Lys Val Arg Arg Gly Arg Asn Tyr Val Glu Ile Pro 1175 1180 1185 1190 AAG AAG ATC GGC TAC CTG CTC TTT GAG AAC ATG TGC GGT GTC CTA GCG 3773 Lys Lys Ile Gly Tyr Leu Leu Phe Glu Asn Met Cys Gly Val Leu Ala                 1195 1200 1205 GAG AAC AAG AGG ATT CCC GAG TTC GTC TTC ACG TCC CCG AAA GGG GTT 3821 Glu Asn Lys Arg Ile Pro Glu Phe Val Phe Thr Ser Pro Lys Gly Val             1210 1215 1220 CGG CTG GCC TTC CTT GAG GGG TAC TCA TCG GCG ATG GCG ACG TCC ACC 3869 Arg Leu Ala Phe Leu Glu Gly Tyr Ser Ser Ala Met Ala Thr Ser Thr         1225 1230 1235 GAA CAA GAG ACT CAG GCT CTC AAC GAA AAG CGA GCT TTA GCG AAC CAG 3917 Glu Gln Glu Thr Gln Ala Leu Asn Glu Lys Arg Ala Leu Ala Asn Gln     1240 1245 1250 CTC GTC CTC CTC TTG AAC TCG GTG GGG GTC TCT GCT GTA AAA CTT GGG 3965 Leu Val Leu Leu Leu Asn Ser Val Gly Val Ser Ala Val Lys Leu Gly 1255 1260 1265 1270 CAC GAC AGC GGC GTT TAC AGG GTC TAT ATA AAC GAG GAG CTC CCG TTC 4013 His Asp Ser Gly Val Tyr Arg Val Tyr Ile Asn Glu Glu Leu Pro Phe                 1275 1280 1285 GTA AAG CTG GAC AAG AAA AAG AAC GCC TAC TAC TCA CAC GTG ATC CCC 4061 Val Lys Leu Asp Lys Lys Lys Asn Ala Tyr Tyr Ser His Val Ile Pro             1290 1295 1300 AAG GAA GTC CTG AGC GAG GTC TTT GGG AAG GTT TTC CAG AAA AAC GTC 4109 Lys Glu Val Leu Ser Glu Val Phe Gly Lys Val Phe Gln Lys Asn Val         1305 1310 1315 AGT CCT CAG ACC TTC AGG AAG ATG GTC GAG GAC GGA AGA CTC GAT CCC 4157 Ser Pro Gln Thr Phe Arg Lys Met Val Glu Asp Gly Arg Leu Asp Pro     1320 1325 1330 GAA AAG GCC CAG AGG CTC TCC TGG CTC ATT GAG GGG GAC GTA GTG CTC 4205 Glu Lys Ala Gln Arg Leu Ser Trp Leu Ile Glu Gly Asp Val Val Leu 1335 1340 1345 1350 GAC CGC GTT GAG TCC GTT GAT GTG GAA GAC TAC GAT GGT TAT GTC TAT 4253 Asp Arg Val Glu Ser Val Asp Val Glu Asp Tyr Asp Gly Tyr Val Tyr                 1355 1360 1365 GAC CTG AGC GTC GAG GAC AAC GAG AAC TTC CTC GTT GGC TTT GGG TTG 4301 Asp Leu Ser Val Glu Asp Asn Glu Asn Phe Leu Val Gly Phe Gly Leu             1370 1375 1380 GTC TAT GCT CAC AAC AGC TAC TAC GGT TAC TAC GGC TAT GCA AGG GCG 4349 Val Tyr Ala His Asn Ser Tyr Tyr Gly Tyr Tyr Gly Tyr Ala Arg Ala         1385 1390 1395 CGC TGG TAC TGC AAG GAG TGT GCA GAG AGC GTA ACG GCC TGG GGA AGG 4397 Arg Trp Tyr Cys Lys Glu Cys Ala Glu Ser Val Thr Ala Trp Gly Arg     1400 1405 1410 GAG TAC ATA ACG ATG ACC ATC AAG GAG ATA GAG GAA AAG TAC GGC TTT 4445 Glu Tyr Ile Thr Met Thr Ile Lys Glu Ile Glu Glu Lys Tyr Gly Phe 1415 1420 1425 1430 AAG GTA ATC TAC AGC GAC ACC GAC GGA TTT TTT GCC ACA ATA CCT GGA 4493 Lys Val Ile Tyr Ser Asp Thr Asp Gly Phe Phe Ala Thr Ile Pro Gly                 1435 1440 1445 GCC GAT GCT GAA ACC GTC AAA AAG AAG GCT ATG GAG TTC CTC AAC TAT 4541 Ala Asp Ala Glu Thr Val Lys Lys Lys Ala Met Glu Phe Leu Asn Tyr             1450 1455 1460 ATC AAC GCC AAA CTT CCG GGC GCG CTT GAG CTC GAG TAC GAG GGC TTC 4589 Ile Asn Ala Lys Leu Pro Gly Ala Leu Glu Leu Glu Tyr Glu Gly Phe         1465 1470 1475 TAC AAA CGC GGC TTC TTC GTC ACG AAG AAG AAG TAT GCG GTG ATA GAC 4637 Tyr Lys Arg Gly Phe Phe Val Thr Lys Lys Lys Tyr Ala Val Ile Asp     1480 1485 1490 GAG GAA GGC AAG ATA ACA ACG CGC GGA CTT GAG ATT GTG AGG CGT GAC 4685 Glu Glu Gly Lys Ile Thr Thr Arg Gly Leu Glu Ile Val Arg Arg Asp 1495 1500 1505 1510 TGG AGC GAG ATA GCG AAA GAG ACG CAG GCG AGG GTT CTT GAA GCT TTG 4733 Trp Ser Glu Ile Ala Lys Glu Thr Gln Ala Arg Val Leu Glu Ala Leu                 1515 1520 1525 CTA AAG GAC GGT GAC GTC GAG AAG GCC GTG AGG ATA GTC AAA GAA GTT 4781 Leu Lys Asp Gly Asp Val Glu Lys Ala Val Arg Ile Val Lys Glu Val             1530 1535 1540 ACC GAA AAG CTG AGC AAG TAC GAG GTT CCG CCG GAG AAG CTG GTG ATC 4829 Thr Glu Lys Leu Ser Lys Tyr Glu Val Pro Pro Glu Lys Leu Val Ile         1545 1550 1555 CAC GAG CAG ATA ACG AGG GAT TTA AAG GAC TAC AAG GCA ACC GGT CCC 4877 His Glu Gln Ile Thr Arg Asp Leu Lys Asp Tyr Lys Ala Thr Gly Pro     1560 1565 1570 CAC GTT GCC GTT GCC AAG AGG TTG GCC GCG AGA GGA GTC AAA ATA CGC 4925 His Val Ala Val Ala Lys Arg Leu Ala Ala Arg Gly Val Lys Ile Arg 1575 1580 1585 1590 CCT GGA ACG GTG ATA AGC TAC ATC GTG CTC AAG GGC TCT GGG AGG ATA 4973 Pro Gly Thr Val Ile Ser Tyr Ile Val Leu Lys Gly Ser Gly Arg Ile                 1595 1600 1605 GGC GAC AGG GCG ATA CCG TTC GAC GAG TTC GAC CCG ACG AAG CAC AAG 5021 Gly Asp Arg Ala Ile Pro Phe Asp Glu Phe Asp Pro Thr Lys His Lys             1610 1615 1620 TAC GAC GCC GAG TAC TAC ATT GAG AAC CAG GTT CTC CCA GCC GTT GAG 5069 Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn Gln Val Leu Pro Ala Val Glu         1625 1630 1635 AGA ATT CTG AGA GCC TTC GGT TAC CGC AAG GAA GAC CTG CGC TAC CAG 5117 Arg Ile Leu Arg Ala Phe Gly Tyr Arg Lys Glu Asp Leu Arg Tyr Gln     1640 1645 1650 AAG ACG AGA CAG GTT GGT TTG AGT GCT TGG CTG AAG CCG AAG GGA ACT 5165 Lys Thr Arg Gln Val Gly Leu Ser Ala Trp Leu Lys Pro Lys Gly Thr 1655 1660 1665 1670 TGACCTTTCC ATTTGTTTTC CAGCGGATAA CCCTTTAACT TCCCTTTCAA AAACTCCCTT 5225 TAGGGAAAGA CCATGAAGAT AGAAATCCGG CGGCGCCCGG TTAAATACGC TAGGATAGAA 5285 GTGAAGCCAG ACGGCAGGGT AGTCGTCACT GCCCCGAGGG TTCAACGTTG AGAAGTT 5342

【００４６】配列番号：2 配列の長さ：774 配列の型：アミノ酸 トポロジー：直鎖状 配列の種類：蛋白質 配列 Met Ile Leu Asp Thr Asp Tyr Ile Thr Glu Asp Gly Lys Pro Val Ile 1 5 10 15 Arg Ile Phe Lys Lys Glu Asn Gly Glu Phe Lys Ile Glu Tyr Asp Arg 20 25 30 Thr Phe Glu Pro Tyr Phe Tyr Ala Leu Leu Lys Asp Asp Ser Ala Ile 35 40 45 Glu Glu Val Lys Lys Ile Thr Ala Glu Arg His Gly Thr Val Val Thr 50 55 60 Val Lys Arg Val Glu Lys Val Gln Lys Lys Phe Leu Gly Arg Pro Val 65 70 75 80 Glu Val Trp Lys Leu Tyr Phe Thr His Pro Gln Asp Val Pro Ala Ile 85 90 95 Arg Asp Lys Ile Arg Glu His Gly Ala Val Ile Asp Ile Tyr Glu Tyr 100 105 110 Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Val Pro 115 120 125 Met Glu Gly Asp Glu Glu Leu Lys Met Leu Ala Phe Asp Ile Glu Thr 130 135 140 Leu Tyr His Glu Gly Glu Glu Phe Ala Glu Gly Pro Ile Leu Met Ile 145 150 155 160 Ser Tyr Ala Asp Glu Glu Gly Ala Arg Val Ile Thr Trp Lys Asn Val 165 170 175 Asp Leu Pro Tyr Val Asp Val Val Ser Thr Glu Arg Glu Met Ile Lys 180 185 190 Arg Phe Leu Arg Val Val Lys Glu Lys Asp Pro Asp Val Leu Ile Thr 195 200 205 Tyr Asn Gly Asp Asn Phe Asp Phe Ala Tyr Leu Lys Lys Arg Cys Glu 210 215 220 Lys Leu Gly Ile Asn Phe Ala Leu Gly Arg Asp Gly Ser Glu Pro Lys 225 230 235 240 Ile Gln Arg Met Gly Asp Arg Phe Ala Val Glu Val Lys Gly Arg Ile 245 250 255 His Phe Asp Leu Tyr Pro Val Ile Arg Arg Thr Ile Asn Leu Pro Thr 260 265 270 Tyr Thr Leu Glu Ala Val Tyr Glu Ala Val Phe Gly Gln Pro Lys Glu 275 280 285 Lys Val Tyr Ala Glu Glu Ile Thr Pro Ala Trp Glu Thr Gly Glu Asn 290 295 300 Leu Glu Arg Val Ala Arg Tyr Ser Met Glu Asp Ala Lys Val Thr Tyr 305 310 315 320 Glu Leu Gly Lys Glu Phe Leu Pro Met Glu Ala Gln Leu Ser Arg Leu 325 330 335 Ile Gly Gln Ser Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu 340 345 350 Val Glu Trp Phe Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala 355 360 365 Pro Asn Lys Pro Asp Glu Lys Glu Leu Ala Arg Arg Arg Gln Ser Tyr 370 375 380 Glu Gly Gly Tyr Val Lys Glu Pro Glu Arg Gly Leu Trp Glu Asn Ile 385 390 395 400 Val Tyr Leu Asp Phe Arg Ser Leu Tyr Pro Ser Ile Ile Ile Thr His 405 410 415 Asn Val Ser Pro Asp Thr Leu Asn Arg Glu Gly Cys Lys Glu Tyr Asp 420 425 430 Val Ala Pro Gln Val Gly His Arg Phe Cys Lys Asp Phe Pro Gly Phe 435 440 445 Ile Pro Ser Leu Leu Gly Asp Leu Leu Glu Glu Arg Gln Lys Ile Lys 450 455 460 Lys Lys Met Lys Ala Thr Ile Asp Pro Ile Glu Arg Lys Leu Leu Asp 465 470 475 480 Tyr Arg Gln Arg Ala Ile Lys Ile Leu Ala Asn Ser Tyr Tyr Gly Tyr 485 490 495 Tyr Gly Tyr Ala Arg Ala Arg Trp Tyr Cys Lys Glu Cys Ala Glu Ser 500 505 510 Val Thr Ala Trp Gly Arg Glu Tyr Ile Thr Met Thr Ile Lys Glu Ile 515 520 525 Glu Glu Lys Tyr Gly Phe Lys Val Ile Tyr Ser Asp Thr Asp Gly Phe 530 535 540 Phe Ala Thr Ile Pro Gly Ala Asp Ala Glu Thr Val Lys Lys Lys Ala 545 550 555 560 Met Glu Phe Leu Asn Tyr Ile Asn Ala Lys Leu Pro Gly Ala Leu Glu 565 570 575 Leu Glu Tyr Glu Gly Phe Tyr Lys Arg Gly Phe Phe Val Thr Lys Lys 580 585 590 Lys Tyr Ala Val Ile Asp Glu Glu Gly Lys Ile Thr Thr Arg Gly Leu 595 600 605 Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln Ala 610 615 620 Arg Val Leu Glu Ala Leu Leu Lys Asp Gly Asp Val Glu Lys Ala Val 625 630 635 640 Arg Ile Val Lys Glu Val Thr Glu Lys Leu Ser Lys Tyr Glu Val Pro 645 650 655 Pro Glu Lys Leu Val Ile His Glu Gln Ile Thr Arg Asp Leu Lys Asp 660 665 670 Tyr Lys Ala Thr Gly Pro His Val Ala Val Ala Lys Arg Leu Ala Ala 675 680 685 Arg Gly Val Lys Ile Arg Pro Gly Thr Val Ile Ser Tyr Ile Val Leu 690 695 700 Lys Gly Ser Gly Arg Ile Gly Asp Arg Ala Ile Pro Phe Asp Glu Phe 705 710 715 720 Asp Pro Thr Lys His Lys Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn Gln 725 730 735 Val Leu Pro Ala Val Glu Arg Ile Leu Arg Ala Phe Gly Tyr Arg Lys 740 745 750 Glu Asp Leu Arg Tyr Gln Lys Thr Arg Gln Val Gly Leu Ser Ala Trp 755 760 765 Leu Lys Pro Lys Gly Thr 770SEQ ID NO: 2 Array length: 774 Sequence type: Amino acid Topology: linear Sequence Type: Protein Array Met Ile Leu Asp Thr Asp Tyr Ile Thr Glu Asp Gly Lys Pro Val Ile   1 5 10 15 Arg Ile Phe Lys Lys Glu Asn Gly Glu Phe Lys Ile Glu Tyr Asp Arg              20 25 30 Thr Phe Glu Pro Tyr Phe Tyr Ala Leu Leu Lys Asp Asp Ser Ala Ile          35 40 45 Glu Glu Val Lys Lys Ile Thr Ala Glu Arg His Gly Thr Val Val Thr      50 55 60 Val Lys Arg Val Glu Lys Val Gln Lys Lys Phe Leu Gly Arg Pro Val  65 70 75 80 Glu Val Trp Lys Leu Tyr Phe Thr His Pro Gln Asp Val Pro Ala Ile                  85 90 95 Arg Asp Lys Ile Arg Glu His Gly Ala Val Ile Asp Ile Tyr Glu Tyr             100 105 110 Asp Ile Pro Phe Ala Lys Arg Tyr Leu Ile Asp Lys Gly Leu Val Pro         115 120 125 Met Glu Gly Asp Glu Glu Leu Lys Met Leu Ala Phe Asp Ile Glu Thr     130 135 140 Leu Tyr His Glu Gly Glu Glu Phe Ala Glu Gly Pro Ile Leu Met Ile 145 150 155 160 Ser Tyr Ala Asp Glu Glu Gly Ala Arg Val Ile Thr Trp Lys Asn Val                 165 170 175 Asp Leu Pro Tyr Val Asp Val Val Ser Thr Glu Arg Glu Met Ile Lys             180 185 190 Arg Phe Leu Arg Val Val Lys Glu Lys Asp Pro Asp Val Leu Ile Thr         195 200 205 Tyr Asn Gly Asp Asn Phe Asp Phe Ala Tyr Leu Lys Lys Arg Cys Glu     210 215 220 Lys Leu Gly Ile Asn Phe Ala Leu Gly Arg Asp Gly Ser Glu Pro Lys 225 230 235 240 Ile Gln Arg Met Gly Asp Arg Phe Ala Val Glu Val Lys Gly Arg Ile                 245 250 255 His Phe Asp Leu Tyr Pro Val Ile Arg Arg Thr Ile Asn Leu Pro Thr             260 265 270 Tyr Thr Leu Glu Ala Val Tyr Glu Ala Val Phe Gly Gln Pro Lys Glu         275 280 285 Lys Val Tyr Ala Glu Glu Ile Thr Pro Ala Trp Glu Thr Gly Glu Asn     290 295 300 Leu Glu Arg Val Ala Arg Tyr Ser Met Glu Asp Ala Lys Val Thr Tyr 305 310 315 320 Glu Leu Gly Lys Glu Phe Leu Pro Met Glu Ala Gln Leu Ser Arg Leu                 325 330 335 Ile Gly Gln Ser Leu Trp Asp Val Ser Arg Ser Ser Thr Gly Asn Leu             340 345 350 Val Glu Trp Phe Leu Leu Arg Lys Ala Tyr Glu Arg Asn Glu Leu Ala         355 360 365 Pro Asn Lys Pro Asp Glu Lys Glu Leu Ala Arg Arg Arg Gln Ser Tyr     370 375 380 Glu Gly Gly Tyr Val Lys Glu Pro Glu Arg Gly Leu Trp Glu Asn Ile 385 390 395 400 Val Tyr Leu Asp Phe Arg Ser Leu Tyr Pro Ser Ile Ile Ile Thr His                 405 410 415 Asn Val Ser Pro Asp Thr Leu Asn Arg Glu Gly Cys Lys Glu Tyr Asp             420 425 430 Val Ala Pro Gln Val Gly His Arg Phe Cys Lys Asp Phe Pro Gly Phe         435 440 445 Ile Pro Ser Leu Leu Gly Asp Leu Leu Glu Glu Arg Gln Lys Ile Lys     450 455 460 Lys Lys Met Lys Ala Thr Ile Asp Pro Ile Glu Arg Lys Leu Leu Asp 465 470 475 480 Tyr Arg Gln Arg Ala Ile Lys Ile Leu Ala Asn Ser Tyr Tyr Gly Tyr                 485 490 495 Tyr Gly Tyr Ala Arg Ala Arg Trp Tyr Cys Lys Glu Cys Ala Glu Ser             500 505 510 Val Thr Ala Trp Gly Arg Glu Tyr Ile Thr Met Thr Ile Lys Glu Ile         515 520 525 Glu Glu Lys Tyr Gly Phe Lys Val Ile Tyr Ser Asp Thr Asp Gly Phe     530 535 540 Phe Ala Thr Ile Pro Gly Ala Asp Ala Glu Thr Val Lys Lys Lys Ala 545 550 555 560 Met Glu Phe Leu Asn Tyr Ile Asn Ala Lys Leu Pro Gly Ala Leu Glu                 565 570 575 Leu Glu Tyr Glu Gly Phe Tyr Lys Arg Gly Phe Phe Val Thr Lys Lys             580 585 590 Lys Tyr Ala Val Ile Asp Glu Glu Gly Lys Ile Thr Thr Arg Gly Leu         595 600 605 Glu Ile Val Arg Arg Asp Trp Ser Glu Ile Ala Lys Glu Thr Gln Ala     610 615 620 Arg Val Leu Glu Ala Leu Leu Lys Asp Gly Asp Val Glu Lys Ala Val 625 630 635 640 Arg Ile Val Lys Glu Val Thr Glu Lys Leu Ser Lys Tyr Glu Val Pro                 645 650 655 Pro Glu Lys Leu Val Ile His Glu Gln Ile Thr Arg Asp Leu Lys Asp             660 665 670 Tyr Lys Ala Thr Gly Pro His Val Ala Val Ala Lys Arg Leu Ala Ala         675 680 685 Arg Gly Val Lys Ile Arg Pro Gly Thr Val Ile Ser Tyr Ile Val Leu     690 695 700 Lys Gly Ser Gly Arg Ile Gly Asp Arg Ala Ile Pro Phe Asp Glu Phe 705 710 715 720 Asp Pro Thr Lys His Lys Tyr Asp Ala Glu Tyr Tyr Ile Glu Asn Gln                 725 730 735 Val Leu Pro Ala Val Glu Arg Ile Leu Arg Ala Phe Gly Tyr Arg Lys             740 745 750 Glu Asp Leu Arg Tyr Gln Lys Thr Arg Gln Val Gly Leu Ser Ala Trp         755 760 765 Leu Lys Pro Lys Gly Thr     770

【００４７】配列番号：3 配列の長さ：2325 配列の型：核酸（ＤＮＡ） 鎖の数：２本鎖 トポロジー：直鎖状 配列の種類：genomic ＤＮＡ 配列 ATGATCCTCG ACACTGACTA CATAACCGAG GATGGAAAGC CTGTCATAAG AATTTTCAAG 60 AAGGAAAACG GCGAGTTTAA GATTGAGTAC GACCGGACTT TTGAACCCTA CTTCTACGCC 120 CTCCTGAAGG ACGATTCTGC CATTGAGGAA GTCAAGAAGA TAACCGCCGA GAGGCACGGG 180 ACGGTTGTAA CGGTTAAGCG GGTTGAAAAG GTTCAGAAGA AGTTCCTCGG GAGACCAGTT 240 GAGGTCTGGA AACTCTACTT TACTCATCCG CAGGACGTCC CAGCGATAAG GGACAAGATA 300 CGAGAGCATG GAGCAGTTAT TGACATCTAC GAGTACGACA TACCCTTCGC CAAGCGCTAC 360 CTCATAGACA AGGGATTAGT GCCAATGGAA GGCGACGAGG AGCTGAAAAT GCTCGCCTTC 420 GACATTCAAA CTCTCTACCA TGAGGGCGAG GAGTTCGCCG AGGGGCCAAT CCTTATGATA 480 AGCTACGCCG ACGAGGAAGG GGCCAGGGTG ATAACTTGGA AGAACGTGGA TCTCCCCTAC 540 GTTGACGTCG TCTCGACGGA GAGGGAGATG ATAAAGCGCT TCCTCCGTGT TGTGAAGGAG 600 AAAGACCCGG ACGTTCTCAT AACCTACAAC GGCGACAACT TCGACTTCGC CTATCTGAAA 660 AAGCGCTGTG AAAAGCTCGG AATAAACTTC GCCCTCGGAA GGGATGGAAG CGAGCCGAAG 720 ATTCAGAGGA TGGGCGACAG GTTTGCCGTC GAAGTGAAGG GACGGATACA CTTCGATCTC 780 TATCCTGTGA TAAGACGGAC GATAAACCTG CCCACATACA CGCTTGAGGC CGTTTATGAA 840 GCCGTCTTCG GTCAGCCGAA GGAGAAGGTT TACGCTGAGG AAATAACACC AGCCTGGGAA 900 ACCGGCGAGA ACCTTGAGAG AGTCGCCCGC TACTCGATGG AAGATGCGAA GGTCACATAC 960 GAGCTTGGGA AGGAGTTCCT TCCGATGGAG GCCCAGCTTT CTCGCTTAAT CGGCCAGTCC 1020 CTCTGGGACG TCTCCCGCTC CAGCACTGGC AACCTCGTTG AGTGGTTCCT CCTCAGGAAG 1080 GCCTATGAGA GGAATGAGCT GGCCCCGAAC AAGCCCGATG AAAAGGAGCT GGCCAGAAGA 1140 CGGCAGAGCT ATGAAGGAGG CTATGTAAAA GAGCCCGAGA GAGGGTTGTG GGAGAACATA 1200 GTGTACCTAG ATTTTAGATC CCTGTACCCC TCAATCATCA TCACCCACAA CGTCTCGCCG 1260 GATACGCTCA ACAGAGAAGG ATGCAAGGAA TATGACGTTG CCCCACAGGT CGGCCACCGC 1320 TTCTGCAAGG ACTTCCCAGG ATTTATCCCG AGCCTGCTTG GAGACCTCCT AGAGGAGAGG 1380 CAGAAGATAA AGAAGAAGAT GAAGGCCACG ATTGACCCGA TCGAGAGGAA GCTCCTCGAT 1440 TACAGGCAGA GGGCCATCAA GATCCTGGCA AACAGCTACT ACGGTTACTA CGGCTATGCA 1500 AGGGCGCGCT GGTACTGCAA GGAGTGTGCA GAGAGCGTAA CGGCCTGGGG AAGGGAGTAC 1560 ATAACGATGA CCATCAAGGA GATAGAGGAA AAGTACGGCT TTAAGGTAAT CTACAGCGAC 1620 ACCGACGGAT TTTTTGCCAC AATACCTGGA GCCGATGCTG AAACCGTCAA AAAGAAGGCT 1680 ATGGAGTTCC TCAACTATAT CAACGCCAAA CTTCCGGGCG CGCTTGAGCT CGAGTACGAG 1740 GGCTTCTACA AACGCGGCTT CTTCGTCACG AAGAAGAAGT ATGCGGTGAT AGACGAGGAA 1800 GGCAAGATAA CAACGCGCGG ACTTGAGATT GTGAGGCGTG ACTGGAGCGA GATAGCGAAA 1860 GAGACGCAGG CGAGGGTTCT TGAAGCTTTG CTAAAGGACG GTGACGTCGA GAAGGCCGTG 1920 AGGATAGTCA AAGAAGTTAC CGAAAAGCTG AGCAAGTACG AGGTTCCGCC GGAGAAGCTG 1980 GTGATCCACG AGCAGATAAC GAGGGATTTA AAGGACTACA AGGCAACCGG TCCCCACGTT 2040 GCCGTTGCCA AGAGGTTGGC CGCGAGAGGA GTCAAAATAC GCCCTGGAAC GGTGATAAGC 2100 TACATCGTGC TCAAGGGCTC TGGGAGGATA GGCGACAGGG CGATACCGTT CGACGAGTTC 2160 GACCCGACGA AGCACAAGTA CGACGCCGAG TACTACATTG AGAACCAGGT TCTCCCAGCC 2220 GTTGAGAGAA TTCTGAGAGC CTTCGGTTAC CGCAAGGAAG ACCTGCGCTA CCAGAAGACG 2280 AGACAGGTTG GTTTGAGTGC TTGGCTGAAG CCGAAGGGAA CTTGA 2325SEQ ID NO: 3 Array length: 2325 Sequence type: nucleic acid (DNA) Number of chains: double-stranded Topology: linear Sequence type: genomic DNA Array ATGATCCTCG ACACTGACTA CATAACCGAG GATGGAAAGC CTGTCATAAG AATTTTCAAG 60 AAGGAAAACG GCGAGTTTAA GATTGAGTAC GACCGGACTT TTGAACCCTA CTTCTACGCC 120 CTCCTGAAGG ACGATTCTGC CATTGAGGAA GTCAAGAAGA TAACCGCCGA GAGGCACGGG 180 ACGGTTGTAA CGGTTAAGCG GGTTGAAAAG GTTCAGAAGA AGTTCCTCGG GAGACCAGTT 240 GAGGTCTGGA AACTCTACTT TACTCATCCG CAGGACGTCC CAGCGATAAG GGACAAGATA 300 CGAGAGCATG GAGCAGTTAT TGACATCTAC GAGTACGACA TACCCTTCGC CAAGCGCTAC 360 CTCATAGACA AGGGATTAGT GCCAATGGAA GGCGACGAGG AGCTGAAAAT GCTCGCCTTC 420 GACATTCAAA CTCTCTACCA TGAGGGCGAG GAGTTCGCCG AGGGGCCAAT CCTTATGATA 480 AGCTACGCCG ACGAGGAAGG GGCCAGGGTG ATAACTTGGA AGAACGTGGA TCTCCCCTAC 540 GTTGACGTCG TCTCGACGGA GAGGGAGATG ATAAAGCGCT TCCTCCGTGT TGTGAAGGAG 600 AAAGACCCGG ACGTTCTCAT AACCTACAAC GGCGACAACT TCGACTTCGC CTATCTGAAA 660 AAGCGCTGTG AAAAGCTCGG AATAAACTTC GCCCTCGGAA GGGATGGAAG CGAGCCGAAG 720 ATTCAGAGGA TGGGCGACAG GTTTGCCGTC GAAGTGAAGG GACGGATACA CTTCGATCTC 780 TATCCTGTGA TAAGACGGAC GATAAACCTG CCCACATACA CGCTTGAGGC CGTTTATGAA 840 GCCGTCTTCG GTCAGCCGAA GGAGAAGGTT TACGCTGAGG AAATAACACC AGCCTGGGAA 900 ACCGGCGAGA ACCTTGAGAG AGTCGCCCGC TACTCGATGG AAGATGCGAA GGTCACATAC 960 GAGCTTGGGA AGGAGTTCCT TCCGATGGAG GCCCAGCTTT CTCGCTTAAT CGGCCAGTCC 1020 CTCTGGGACG TCTCCCGCTC CAGCACTGGC AACCTCGTTG AGTGGTTCCT CCTCAGGAAG 1080 GCCTATGAGA GGAATGAGCT GGCCCCGAAC AAGCCCGATG AAAAGGAGCT GGCCAGAAGA 1140 CGGCAGAGCT ATGAAGGAGG CTATGTAAAA GAGCCCGAGA GAGGGTTGTG GGAGAACATA 1200 GTGTACCTAG ATTTTAGATC CCTGTACCCC TCAATCATCA TCACCCACAA CGTCTCGCCG 1260 GATACGCTCA ACAGAGAAGG ATGCAAGGAA TATGACGTTG CCCCACAGGT CGGCCACCGC 1320 TTCTGCAAGG ACTTCCCAGG ATTTATCCCG AGCCTGCTTG GAGACCTCCT AGAGGAGAGG 1380 CAGAAGATAA AGAAGAAGAT GAAGGCCACG ATTGACCCGA TCGAGAGGAA GCTCCTCGAT 1440 TACAGGCAGA GGGCCATCAA GATCCTGGCA AACAGCTACT ACGGTTACTA CGGCTATGCA 1500 AGGGCGCGCT GGTACTGCAA GGAGTGTGCA GAGAGCGTAA CGGCCTGGGG AAGGGAGTAC 1560 ATAACGATGA CCATCAAGGA GATAGAGGAA AAGTACGGCT TTAAGGTAAT CTACAGCGAC 1620 ACCGACGGAT TTTTTGCCAC AATACCTGGA GCCGATGCTG AAACCGTCAA AAAGAAGGCT 1680 ATGGAGTTCC TCAACTATAT CAACGCCAAA CTTCCGGGCG CGCTTGAGCT CGAGTACGAG 1740 GGCTTCTACA AACGCGGCTT CTTCGTCACG AAGAAGAAGT ATGCGGTGAT AGACGAGGAA 1800 GGCAAGATAA CAACGCGCGG ACTTGAGATT GTGAGGCGTG ACTGGAGCGA GATAGCGAAA 1860 GAGACGCAGG CGAGGGTTCT TGAAGCTTTG CTAAAGGACG GTGACGTCGA GAAGGCCGTG 1920 AGGATAGTCA AAGAAGTTAC CGAAAAGCTG AGCAAGTACG AGGTTCCGCC GGAGAAGCTG 1980 GTGATCCACG AGCAGATAAC GAGGGATTTA AAGGACTACA AGGCAACCGG TCCCCACGTT 2040 GCCGTTGCCA AGAGGTTGGC CGCGAGAGGA GTCAAAATAC GCCCTGGAAC GGTGATAAGC 2100 TACATCGTGC TCAAGGGCTC TGGGAGGATA GGCGACAGGG CGATACCGTT CGACGAGTTC 2160 GACCCGACGA AGCACAAGTA CGACGCCGAG TACTACATTG AGAACCAGGT TCTCCCAGCC 2220 GTTGAGAGAA TTCTGAGAGC CTTCGGTTAC CGCAAGGAAG ACCTGCGCTA CCAGAAGACG 2280 AGACAGGTTG GTTTGAGTGC TTGGCTGAAG CCGAAGGGAA CTTGA 2325

【００４８】配列番号：4 配列の長さ：24 配列の型：核酸（ＤＮＡ） 鎖の数：1本鎖 トポロジー：直鎖状 配列の種類：合成ＤＮＡ 配列 CTTTTGCTCA GATCTTCTTT CCTG 24SEQ ID NO: 4 Array length: 24 Sequence type: nucleic acid (DNA) Number of chains: single strand Topology: linear Sequence type: Synthetic DNA Array CTTTTGCTCA GATCTTCTTT CCTG 24

【００４９】配列番号：5 配列の長さ:36 配列の型：核酸（ＤＮＡ） 鎖の数：１本鎖 トポロジー：直鎖状 配列の種類：合成ＤＮＡ 配列 CAGGAAAGAA GATCTGAGCA AAAG 24SEQ ID NO: 5 Array length: 36 Sequence type: nucleic acid (DNA) Number of chains: single strand Topology: linear Sequence type: Synthetic DNA Array CAGGAAAGAA GATCTGAGCA AAAG 24

【００５０】配列番号：6 配列の長さ:36 配列の型：核酸（ＤＮＡ） 鎖の数：１本鎖 トポロジー：直鎖状 配列の種類：合成ＤＮＡ 配列 CTGAAAATGC TCGCCTTCGC GATTGCAACT CTCTAC 36SEQ ID NO: 6 Array length: 36 Sequence type: nucleic acid (DNA) Number of chains: single strand Topology: linear Sequence type: Synthetic DNA Array CTGAAAATGC TCGCCTTCGC GATTGCAACT CTCTAC 36

【００５１】配列番号：7 配列の長さ:33 配列の型：核酸（ＤＮＡ） 鎖の数：１本鎖 トポロジー：直鎖状 配列の種類：合成ＤＮＡ 配列 CTGAAAATGC TCGCCTTCGC GATTGAAACT CTCT 34SEQ ID NO: 7 Array length: 33 Sequence type: nucleic acid (DNA) Number of chains: single strand Topology: linear Sequence type: Synthetic DNA Array CTGAAAATGC TCGCCTTCGC GATTGAAACT CTCT 34

【００５２】配列番号：8 配列の長さ:30 配列の型：核酸（ＤＮＡ） 鎖の数：１本鎖 トポロジー：直鎖状 配列の種類：合成ＤＮＡ 配列 GCCCTCGTGG TAGAGAGTTG CAATGTCGAA 30SEQ ID NO: 8 Array length: 30 Sequence type: nucleic acid (DNA) Number of chains: single strand Topology: linear Sequence type: Synthetic DNA Array GCCCTCGTGG TAGAGAGTTG CAATGTCGAA 30

【００５３】配列番号：9 配列の長さ:32 配列の型：核酸（ＤＮＡ） 鎖の数：１本鎖 トポロジー：直鎖状 配列の種類：合成ＤＮＡ 配列 CGGACGTACT GATAACGTAC GACGGTGACA AC 32SEQ ID NO: 9 Array length: 32 Sequence type: nucleic acid (DNA) Number of chains: single strand Topology: linear Sequence type: Synthetic DNA Array CGGACGTACT GATAACGTAC GACGGTGACA AC 32

【００５４】配列番号：10 配列の長さ:32 配列の型：核酸（ＤＮＡ） 鎖の数：1本鎖 トポロジー：直鎖状 配列の種類：合成ＤＮＡ 配列 CGGACGTACT GATAACGTAC GACGGTGACA AC 32SEQ ID NO: 10 Array length: 32 Sequence type: nucleic acid (DNA) Number of chains: single strand Topology: linear Sequence type: Synthetic DNA Array CGGACGTACT GATAACGTAC GACGGTGACA AC 32

【００５５】配列番号：11 配列の長さ:35 配列の型：核酸（ＤＮＡ） 鎖の数：1本鎖 トポロジー：直鎖状 配列の種類：合成ＤＮＡ 配列 TGGCTAGCCA AGGAACCACC AGTTGATTAG CAGAG 35SEQ ID NO: 11 Array length: 35 Sequence type: nucleic acid (DNA) Number of chains: single strand Topology: linear Sequence type: Synthetic DNA Array TGGCTAGCCA AGGAACCACC AGTTGATTAG CAGAG 35

【００５６】配列番号：12 配列の長さ:35 配列の型：核酸（ＤＮＡ） 鎖の数：１本鎖 トポロジー：直鎖状 配列の種類：合成ＤＮＡ 配列 ATAAGAGGTC CCAAGACTTA GTACCTGAAG GGTGA 36SEQ ID NO: 12 Array length: 35 Sequence type: nucleic acid (DNA) Number of chains: single strand Topology: linear Sequence type: Synthetic DNA Array ATAAGAGGTC CCAAGACTTA GTACCTGAAG GGTGA 36

【００５７】配列番号：13 配列の長さ:35 配列の型：核酸（ＤＮＡ） 鎖の数：１本鎖 トポロジー：直鎖状 配列の種類：合成ＤＮＡ 配列 AAAAAGTACT CACCAGTCAC AGAAAAGCAT CTTAC 35SEQ ID NO: 13 Array length: 35 Sequence type: nucleic acid (DNA) Number of chains: single strand Topology: linear Sequence type: Synthetic DNA Array AAAAAGTACT CACCAGTCAC AGAAAAGCAT CTTAC 35

【００５８】配列番号：14 配列の長さ:34 配列の型：核酸（ＤＮＡ） 鎖の数：1本鎖 トポロジー：直鎖状 配列の種類：合成ＤＮＡ 配列 AAAAAGTACT CAACCAAGTC ATTCVTGAGA ATAGT 34SEQ ID NO: 14 Array length: 34 Sequence type: nucleic acid (DNA) Number of chains: single strand Topology: linear Sequence type: Synthetic DNA Array AAAAAGTACT CAACCAAGTC ATTCVTGAGA ATAGT 34

【００５９】配列番号：15 配列の長さ:24 配列の型：核酸（ＤＮＡ） 鎖の数：1本鎖 トポロジー：直鎖状 配列の種類：合成ＤＮＡ 配列 CGCCAGGGTT TTCCCAGTCA CGAC 24SEQ ID NO: 15 Array length: 24 Sequence type: nucleic acid (DNA) Number of chains: single strand Topology: linear Sequence type: Synthetic DNA Array CGCCAGGGTT TTCCCAGTCA CGAC 24

【図面の簡単な説明】[Brief description of drawings]

【図１】改変型ＤＮＡポリメラーゼのポリメラーゼ活性
とＤＮＡ分解率を示す図である。FIG. 1 is a diagram showing a polymerase activity and a DNA degradation rate of a modified DNA polymerase.

【図２】改変型ＤＮＡポリメラーゼの熱安定性を示す図
である。FIG. 2 is a diagram showing the thermostability of a modified DNA polymerase.

【図３】 改変型ＤＮＡポリメラーゼを用いたＰＣＲの
結果（プラスミド）を示す電気泳動の写真である。FIG. 3 is an electrophoresis photograph showing a result (plasmid) of PCR using a modified DNA polymerase.

【図４】 改変型ＤＮＡポリメラーゼを用いたＰＣＲの
結果（ヒトゲノム）を示す電気泳動の写真である。FIG. 4 is a photograph of electrophoresis showing the result of PCR (human genome) using a modified DNA polymerase.

───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.⁷ 識別記号 ＦＩ Ｃ１２Ｒ 1:01） (72)発明者 川村 良久 福井県敦賀市東洋町10番24号 東洋紡績 株式会社 敦賀バイオ研究所内 (72)発明者 高木 昌宏 大阪府吹田市青山台１丁目３Ｃ−58− 207 (72)発明者 今中 忠行 大阪府吹田市藤白台２丁目28番11号 (56)参考文献 特表 平10−503381（ＪＰ，Ａ) 米国特許5489523（ＵＳ，Ａ) 特許3112148（ＪＰ，Ｂ２) (58)調査した分野(Int.Cl.⁷，ＤＢ名) C12N 15/00 - 15/90 ＣＡ（ＳＴＮ) ＲＥＧＩＳＴＲＹ（ＳＴＮ)─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁷ Identification code FI C12R 1:01) (72) Inventor Yoshihisa Kawamura 10-24 Toyocho, Tsuruga City, Fukui Prefecture Toyobo Co., Ltd. Tsuruga Bio Research Institute (72 ) Inventor Masahiro Takagi 1-3C, Aoyamadai, Suita City, Osaka Prefecture 3C-58-207 (72) Tadayuki Imanaka 2-28-11, Fujishirodai, Suita City, Osaka Prefecture (56) References Japanese Patent Publication No. 10-503381 (JP) , A) US Patent 5489523 (US, A) Patent 3112148 (JP, B2) (58) Fields investigated (Int.Cl. ⁷ , DB name) C12N 15/00-15/90 CA (STN) REGISTRY (STN)

Claims (18)

(57)【特許請求の範囲】(57) [Claims] 【請求項１】下記理化学的性質を有する改変された耐熱
性ＤＮＡポリメラーゼ。作用：ＤＮＡ合成活性を有し、
改変前の酵素に比べて、５％以下である３’−５’エキ
ソヌクレアーゼ活性を有する。 ＤＮＡ合成速度：少なくとも３０塩基／秒 熱安定性：ｐＨ８．８（２５℃での測定値）にて９５
℃、６時間の処理で６０％以上の残存活性を保持するこ
とができる。 至適温度：約７５℃ 分子量：８８〜９０ＫＤａ アミノ酸配列：配列番号２に記載のアミノ酸配列の第１
４１、１４３、２１０および３１１番目のアミノ酸の少
なくとも１つを他のアミノ酸に置換したアミノ酸配列1. A modified thermostable DNA polymerase having the following physicochemical properties. Action: has DNA synthesis activity,
It has a 3′-5 ′ exonuclease activity that is 5% or less compared to the enzyme before modification. DNA synthesis rate: at least 30 bases / sec Thermal stability: 95 at pH 8.8 (measured value at 25 ° C)
The residual activity of 60% or more can be retained by the treatment at 6 ° C. for 6 hours. Optimum temperature: about 75 ° C. Molecular weight: 88-90 KDa Amino acid sequence: First of the amino acid sequences set forth in SEQ ID NO: 2
Amino acid sequence in which at least one of the 41st, 143rd, 210th and 311th amino acids has been replaced with another amino acid 【請求項２】下記理化学的性質を有する改変された耐熱
性ＤＮＡポリメラーゼ。作用：ＤＮＡ合成活性を有し、
３’−５’エキソヌクレアーゼ活性を有しない。 ＤＮＡ合成速度：少なくとも３０塩基／秒 熱安定性：ｐＨ８．８（２５℃での測定値）にて９５
℃、６時間の処理で６０％以上の残存活性を保持するこ
とができる。 至適温度：約７５℃ 分子量：８８〜９０ＫＤａ アミノ酸配列：配列番号２に記載のアミノ酸配列の第１
４１、１４３、２１０および３１１番目のアミノ酸の少
なくとも１つを他のアミノ酸に置換したアミノ酸配列2. A modified thermostable DNA polymerase having the following physicochemical properties. Action: has DNA synthesis activity,
It has no 3'-5 'exonuclease activity. DNA synthesis rate: at least 30 bases / sec Thermal stability: 95 at pH 8.8 (measured value at 25 ° C)
The residual activity of 60% or more can be retained by the treatment at 6 ° C. for 6 hours. Optimum temperature: about 75 ° C. Molecular weight: 88-90 KDa Amino acid sequence: First of the amino acid sequences set forth in SEQ ID NO: 2
Amino acid sequence in which at least one of the 41st, 143rd, 210th and 311th amino acids has been replaced with another amino acid 【請求項３】 ＤＮＡ合成速度が６０塩基／秒以上であ
る請求項１または２記載の耐熱性ＤＮＡポリメラーゼ。3. A process according to claim 1 or 2, wherein the thermostable DNA polymerase DNA synthesis rate of 60 bases / sec or higher. 【請求項４】 ３’−５’エキソヌクレアーゼ活性が約
１％以下に低下した請求項１記載の耐熱性ＤＮＡポリメ
ラーゼ。Wherein the 3'-5 'claim 1 Symbol placement of thermostable DNA polymerase exonuclease activity was reduced to less than about 1%. 【請求項５】 配列番号２の第１４１番目のアスパラギ
ン酸を他のアミノ酸に置換した請求項１〜４のいずれか
１項に記載の耐熱性ＤＮＡポリメラーゼ。5. The method according to claim 1, wherein the 141st aspartic acid of SEQ ID NO: 2 is substituted with another amino acid .
The thermostable DNA polymerase according to item 1 . 【請求項６】 配列番号２の第１４１番目のアスパラギ
ン酸をアラニンに置換した請求項５記載の耐熱性ＤＮＡ
ポリメラーゼ。6. The thermostable DNA according to claim 5 , wherein the 141st aspartic acid of SEQ ID NO: 2 is substituted with alanine.
Polymerase. 【請求項７】 配列番号２の１４３番目のグルタミン酸
を他のアミノ酸に置換した請求項１〜４のいずれか１項
に記載の耐熱性ＤＮＡポリメラーゼ。7. any one of claims 1-4 for the 143 th glutamic acid of SEQ ID NO: 2 is replaced with another amino acid
Thermostable DNA polymerases described. 【請求項８】 配列番号２の第１４３番目のグルタミン
酸をアラニンに置換した請求項７記載の耐熱性ＤＮＡポ
リメラーゼ。8. The thermostable DNA polymerase according to claim 7 , wherein the 143rd glutamic acid of SEQ ID NO: 2 is substituted with alanine. 【請求項９】 配列番号２の１４１番目のアスパラギン
酸と１４３番目のグルタミン酸を他のアミノ酸に置換し
た請求項１〜４のいずれか１項に記載の耐熱性ＤＮＡポ
リメラーゼ。9. The thermostable DNA polymerase according to any one of claims 1 to 4, wherein the 141st aspartic acid and the 143rd glutamic acid of SEQ ID NO: 2 are substituted with other amino acids. 【請求項１０】 配列番号２の１４１番目のアスパラギ
ン酸と１４３番目のグルタミン酸をアラニンに置換した
請求項９記載の耐熱性ＤＮＡポリメラーゼ。10. The thermostable DNA polymerase according to claim 9, wherein the 141st aspartic acid and the 143rd glutamic acid of SEQ ID NO: 2 are substituted with alanine. 【請求項１１】 配列番号２の２１０番目のアスパラギ
ンを他のアミノ酸に置換した請求項１〜４のいずれか１
項に記載の耐熱性ＤＮＡポリメラーゼ。11. one of the 210 in SEQ ID NO: 2 asparagine of claim 1-4 which is substituted with another amino acid 1
A thermostable DNA polymerase as described in the item . 【請求項１２】 配列番号２の２１０番目のアスパラギ
ンをアスパラギン酸に換した請求項１１記載の耐熱性Ｄ
ＮＡポリメラーゼ。12. The heat resistant D according to claim 11, wherein the 210th asparagine of SEQ ID NO: 2 is replaced with aspartic acid.
NA polymerase. 【請求項１３】 配列番号２の３１１番目のチロシンを
他のアミノ酸に置換した請求項１〜４のいずれか１項に
記載の耐熱性ＤＮＡポリメラーゼ。13. The thermostable DNA polymerase according to any one of claims 1 to 4, wherein the 311th tyrosine of SEQ ID NO: 2 is substituted with another amino acid. 【請求項１４】 配列番号２の３１１番目のチロシンを
フェニルアラニンに置換した請求項１３記載の耐熱性Ｄ
ＮＡポリメラーゼ。14. The heat-resistant D according to claim 13, wherein the 311th tyrosine of SEQ ID NO: 2 is substituted with phenylalanine.
NA polymerase. 【請求項１５】 ＤＮＡを鋳型とし、プライマー、ｄＮ
ＴＰおよび請求項１〜１４のいずれか１項に記載の耐熱
性ＤＮＡポリメラーゼを反応させて、プライマーを伸長
して、ＤＮＡプライマー伸長物を合成することを特徴と
する核酸増幅法。15. A primer and dN using DNA as a template.
A nucleic acid amplification method, which comprises reacting TP with the thermostable DNA polymerase according to any one of claims 1 to 14 to extend a primer to synthesize a DNA primer extension product. 【請求項１６】 プライマーが２種のオリゴヌクレオチ
ドであって、１方は他方のＤＮＡ伸長生成物に相補的で
あるプライマーである請求項１５記載の核酸増幅法。16. The nucleic acid amplification method according to claim 15, wherein the primers are two kinds of oligonucleotides, one of which is complementary to the DNA extension product of the other. 【請求項１７】 加熱および冷却を繰り返す請求項１５
または１６記載の核酸増幅法。17. The method according to claim 15, wherein heating and cooling are repeated.
Or the nucleic acid amplification method described in 16 . 【請求項１８】 １方のプライマーが他方のプライマー
のＤＮＡ伸長生成物に相補的である２種のプライマー、
ｄＮＴＰおよび請求項１〜１４のいずれか１項に記載の
耐熱性ＤＮＡポリメラーゼ、マグネシウムイオンおよび
アンモニウムイオンおよび／またはカリウムイオン、Ｂ
ＳＡおよび非イオン界面活性剤および緩衝液を含む核酸
増幅用試薬。18. Two primers, one primer of which is complementary to the DNA extension product of the other primer,
dNTP and the thermostable DNA polymerase according to any one of claims 1 to 14 , magnesium ion and ammonium ion and / or potassium ion, B
A nucleic acid amplification reagent containing SA, a nonionic surfactant, and a buffer.