JP4794887B2 - 蛍光蛋白質 - Google Patents
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Description
(a)配列番号2に記載のアミノ酸配列;又は、
(b)配列番号2に記載のアミノ酸配列において1から数個のアミノ酸の欠失、置換及び/又は付加を有するアミノ酸配列を有し、蛍光を有し、かつ単量体で存在するアミノ酸配列:
(a)配列番号2に記載のアミノ酸配列をコードするDNA;又は、
(b)配列番号2に記載のアミノ酸配列において1から数個のアミノ酸の欠失、置換及び/又は付加を有するアミノ酸配列を有し、単量体で存在する蛍光蛋白質をコードするDNA:
(a)配列番号1に記載の塩基配列;又は、
(b)配列番号1に記載の塩基配列において1から数個の塩基の欠失、置換及び/又は付加を有する塩基配列を有し、単量体で存在する蛍光蛋白質をコードする塩基配列:
本発明のさらに別の側面によれば、本発明のDNA又は組み換えベクターを有する形質転換体が提供される。
本発明のさらに別の側面によれば、本発明の蛍光蛋白質の蛍光スペクトルとFRET(蛍光共鳴エネルギー移動)を引き起こすのに十分な重なりをもつ吸収スペクトルを示す色素蛋白質と、本発明の蛍光蛋白質とをリンカー配列を介して連結することにより得られるFRET測定用融合蛋白質が提供される。
(a)配列番号4に記載のアミノ酸配列;又は、
(b)配列番号4に記載のアミノ酸配列において1から数個のアミノ酸の欠失、置換及び/又は付加を有するアミノ酸配列を有し、配列番号4に記載のアミノ酸配列と同等の吸光特性を有するアミノ酸配列:
(1)本発明の蛍光蛋白質
イシサンゴに属するミドリイシ(Acropora sp.)より単離された青緑蛍光蛋白質MiCyは2量体を形成する。細胞をラベルする際には蛍光蛋白質が何量体を形成しようと、蛍光蛋白質自身は細胞質中に漂っているだけなので問題は起こらないが、分子をラベルする際には問題が生じてくる場合が容易に考えられる。例えば、ラベルしたい分子が多量体を形成する場合、ターゲット分子と蛍光蛋白質分子が互いに多量体を形成し合い、巨大なポリマーを形成してしまう可能性がある。また、どちらかの多量体形成が阻害された時には、その多量体形成できない分子が本来の性質を失うことになりかねない。蛍光蛋白質を複数用いた分子内FRET(蛍光エネルギー共鳴移動)のプローブにおいても同じく、多量体形成蛍光蛋白質同士を一本のペプチド鎖として発現させた場合に、互いが多量体形成をしあうためにFRETの観測は困難をきわめる。それらを解決すべく、2量体を形成するMiCyに変異導入し、単量体として機能するmMiCyの作製を行った。
(a)配列番号2に記載のアミノ酸配列;又は、
(b)配列番号2に記載のアミノ酸配列において1から数個のアミノ酸の欠失、置換及び/又は付加を有するアミノ酸配列を有し、蛍光を有し、かつ単量体で存在するアミノ酸配列:
本発明の蛍光蛋白質をコードするDNAの具体例としては、以下の何れかのDNAが挙げられる。
(a)配列番号2に記載のアミノ酸配列をコードするDNA;又は、
(b)配列番号2に記載のアミノ酸配列において1から数個のアミノ酸の欠失、置換及び/又は付加を有するアミノ酸配列を有し、単量体で存在する蛍光蛋白質をコードするDNA:
(a)配列番号1に記載の塩基配列;又は、
(b)配列番号1に記載の塩基配列において1から数個の塩基の欠失、置換及び/又は付加を有する塩基配列を有し、単量体で存在する蛍光蛋白質をコードする塩基配列:
本発明のDNAは適当なベクター中に挿入して使用することができる。本発明で用いるベクターの種類は特に限定されず、例えば、自立的に複製するベクター(例えばプラスミド等)でもよいし、あるいは、宿主細胞に導入された際に宿主細胞のゲノムに組み込まれ、組み込まれた染色体と共に複製されるものであってもよい。
糸状菌細胞で作動可能なプロモータの例としては、ADH3プロモータまたはtpiAプロモータなどがある。
本発明の組み換えベクターは更に、該ベクターが宿主細胞内で複製することを可能にするDNA配列を具備してもよく、その一例としてはSV40複製起点(宿主細胞が哺乳類細胞のとき)が挙げられる。
本発明のDNA、プロモータ、および所望によりターミネータおよび/または分泌シグナル配列をそれぞれ連結し、これらを適切なベクターに挿入する方法は当業者に周知である。
本発明のDNA又は組み換えベクターを適当な宿主に導入することによって形質転換体を作製することができる。
本発明のDNAまたは組み換えベクターを導入される宿主細胞は、本発明のDNA構築物を発現できれば任意の細胞でよく、細菌、酵母、真菌および高等真核細胞等が挙げられる。
哺乳類細胞の例としては、HEK293細胞、HeLa細胞、COS細胞、BHK細胞、CHL細胞またはCHO細胞等が挙げられる。哺乳類細胞を形質転換し、該細胞に導入されたDNA配列を発現させる方法も公知であり、例えば、エレクトロポレーション法、リン酸カルシウム法、リポフェクション法等を用いることができる。
昆虫細胞としては、Spodoptera frugiperdaの卵巣細胞であるSf9、Sf21〔バキュロウイルス・エクスプレッション・ベクターズ、ア・ラボラトリー・マニュアル、ダブリュー・エイチ・フリーマン・アンド・カンパニー(W. H. Freeman and Company)、ニューヨーク(New York)、(1992)〕、Trichoplusia niの卵巣細胞であるHiFive(インビトロジェン社製)等を用いることができる。
組換えウイルスを調製するための、昆虫細胞への組換え遺伝子導入ベクターと上記バキュロウイルスの共導入方法としては、例えば、リン酸カルシウム法又はリポフェクション法等を挙げることができる。
例えば、本発明の蛋白質が、細胞内に溶解状態で発現した場合には、培養終了後、細胞を遠心分離により回収し水系緩衝液に懸濁後、超音波破砕機等により細胞を破砕し、無細胞抽出液を得る。該無細胞抽出液を遠心分離することにより得られた上清から、通常の蛋白質の単離精製法、即ち、溶媒抽出法、硫安等による塩析法、脱塩法、有機溶媒による沈殿法、ジエチルアミノエチル(DEAE)セファロース等のレジンを用いた陰イオン交換クロマトグラフィー法、S-Sepharose FF(ファルマシア社製)等のレジンを用いた陽イオン交換クロマトグラフィー法、ブチルセファロース、フェニルセファロース等のレジンを用いた疎水性クロマトグラフィー法、分子篩を用いたゲルろ過法、アフィニティークロマトグラフィ一法、クロマトフォーカシング法、等電点電気泳動等の電気泳動法等の手法を単独あるいは組み合わせて用い、精製標品を得ることができる。
本発明は蛍光蛋白質を他の蛋白質と融合させることにより、融合蛍光蛋白質を構築することができる。
本発明の融合蛍光蛋白質の取得方法については特に制限はなく、化学合成により合成した蛋白質でもよいし、遺伝子組み換え技術による作製した組み換え蛋白質でもよい。
組み換え蛋白質を作製する場合には、先ず当該蛋白質をコードするDNAを入手することが必要である。本明細書の配列表の配列番号2に記載したアミノ酸配列及び配列番号1に記載した塩基配列の情報を利用することにより適当なプライマーを設計し、本発明の蛍光蛋白質の遺伝子を含むDNA断片を鋳型にしてPCRを行うことにより、本発明の蛍光蛋白質をコードするDNAを構築するのに必要なDNA断片を作製することができる。また同様に、融合すべき蛋白質をコードするDNA断片も入手する。
また、例えば、神経細胞の軸索、樹状突起などは発生途中の個体の中で著しく複雑な走向の変化を示すので、こういった部位を蛍光ラベルすることにより動的解析が可能になる。
顕微鏡の種類は目的に応じて適宜選択できる。経時変化を追跡するなど頻回の観察を必要とする場合には、通常の落射型蛍光顕微鏡が好ましい。細胞内の詳細な局在を追及したい場合など、解像度を重視する場合は、共焦点レーザー顕微鏡の方が好ましい。顕微鏡システムとしては、細胞の生理状態を保ち、コンタミネーションを防止する観点から、倒立型顕微鏡が好ましい。正立顕微鏡を使用する場合、高倍率レンズを用いる際には水浸レンズを用いることができる。
(a)配列番号4に記載のアミノ酸配列;又は、
(b)配列番号4に記載のアミノ酸配列において1から数個のアミノ酸の欠失、置換及び/又は付加を有するアミノ酸配列を有し、配列番号4に記載のアミノ酸配列と同等の吸光特性を有するアミノ酸配列:
本発明によれば、本明細書に記載した蛍光蛋白質、融合蛍光蛋白質、DNA、組み換えベクター又は形質転換体から選択される少なくとも1種以上を含むことを特徴とする、蛍光試薬キットが提供される。本発明のキットは、それ自体既知の通常用いられる材料及び手法で調製することができる。
蛍光蛋白質又はDNAなどの試薬は、適当な溶媒に溶解することにより保存に適した形態に調製することができる。溶媒としては、水、エタノール、各種緩衝液などを用いることができる。
以下の実施例により本発明を具体的に説明するが、本発明は実施例によって限定されるものではない。
イシサンゴに属するミドリイシ(Acropora sp.)より単離された青緑蛍光蛋白質MiCyは2量体を形成する。このような2量体を形成する蛍光蛋白質をFRETにおいてそのまま利用すると観測が困難になる等の問題が生じることから、2量体を形成するMiCyに変異導入し、単量体として機能するmMiCyの作製を行った。
MiCyのアミノ酸配列(国際公開WO2004/111235号公報)から多量体形成界面を予測し、多量体形成界面のアミノ酸を置換し、なおかつ蛍光特性を保持するようMiCyの単量体化を行った。点変異導入はMiCyを挿入した大腸菌発現ベクター(pRSET B)に点変異導入プライマーをもちいてPCRをかけることにより行った。PCRに用いたプライマーは5’側のリン酸化を行った。
反応液の組成:
100μM primer 2μl
10× T4 polynucleotide kinase buffer 5μl
100μM ATP 0.5μl
滅菌水 41.5μl
T4 polynucleotide kinase (10 U/μl) 1μl
gaagggaaac agacgtccga attagtg (配列番号5)
gtgatcatca agnnnaaggg aaaacccctt (配列番号6)
atactgtcaa cannntttca atatggaaac (配列番号7)
catctttcat ggcacaaact ttcccgctg (配列番号8)
gggataagtc ctccgaaaaa atgagtg (配列番号9)
cagagatgcc agttacactc cacttac (配列番号10)
(nはa,t,g又はcを示す)
反応液の組成:
5'リン酸化プライマー 4μl
template(MiCy−pRSET B) 100ng
10× polymerase buffer 2.5μl
10× DNA ligase buffer 2.5μl
2.5mM dNTPs 1μl
polymerase(pfu)2.5U/μl 1μl
Taq DNA ligase 40U/μl 0.5μl
滅菌水で計50μlとする。
サーマルサイクラーはGeneAmp PCR system 9700を使用した。
1)65℃ 5 min
2)95℃ 2 min
3)95℃ 20 sec
4)52℃ 20 sec
5)65℃ 8 min
6)75℃ 7 min
7)4℃ hold
3)〜5)を25サイクル
PCR後のサンプルにDpn1を1μl加えて37℃に1時間インキュベートしてテンプレートプラスミドを切断した。
Dpn1処理後のサンプルを大腸菌JM109(DE3)に形質転換して変異導入後のMiCyを発現させた。
変異導入後のMiCy変異体の塩基配列を解析し、アミノ酸配列を決定した。結果として43番目のメチオニン(M)をトレオニン(T)に、51番目のセリン(S)をプロリン(P)に、66番目のアラニン(A)をバリン(V)に、129番目のバリン(V)をトレオニン(T)に、149番目のフェニルアラニン(F)をセリン(S)に、179番目のフェニルアラニン(F)をロイシン(L)に置換されたmMiCyを得た。mMiCyの塩基配列を配列番号1に示し、アミノ酸配列を配列番号2に示す。
pH4、5 : 酢酸バッファー
pH6 : MESバッファー
pH7、8 : HEPESバッファー
pH9、10 : グリシンバッファー
pH11 : リン酸バッファー
mMiCyをpRSETBのBamH1―EcoR1サイトにサブクローニングし大腸菌JM109(DE3)に形質転換してリコンビナント蛋白質を発現させた。発現させたリコンビナント蛋白質はNi-NTAで精製した。精製したリコンビナント蛋白質mMiCyの分子量を超遠心分析により求めた。mMiCy蛋白質溶液を150mM KCl、50mM HEPES-KOH pH7.4で調製した。機器は超遠心機XL-1(ベックマン・コールター)を使用した。25000rpmで22時間遠心し、mMiCyの吸収極大(470nm)の吸収値を測定した。その結果、精製用タグ等の配列を含んだその分子量は34.8kDaと算出され、これはアミノ酸配列から予測される29.8kDaとほぼ一致し、mMiCyは多量体を形成しない単量体であることが確認された。2量体を形成するMiCyは超遠心分析から59.7kDaと算出された(図5及び6)。
現行のFRET測定系では蛍光物質または発光物質をドナーに、蛍光物質にアクセプターを使用するのが通常であるが、アクセプター側は吸収特性があれば蛍光を発しなくてもFRETの測定は可能である。逆にアクセプターが蛍光を発しなければ、それだけ蛍光取得の波長域に空きができるために、複数のFRET測定が可能となる。また、蛍光寿命を測定するライフタイムイメージングではFRETのドナーの蛍光寿命を測定すればFRETが起こっているか否かを判別できるため、同じく、蛍光を発しない色素蛋白質をアクセプターに用いたFRET系は同時に複数のFRETを測定可能とする。そこでドナーに蛍光蛋白質(mMiCy)を用い、アクセプターに色素蛋白質(KP)を用いたFRET測定系の構築を行なった。
以下、8種(3-3〜5-4)のリンカー(アミノ酸配列)を設計し、KPとmMiCyの間に挿入し発現させた。
3-3: SGGDEVDGTG (配列番号11)
3-4: SGGDEVDGTGG (配列番号12)
3-5: SGGDEVDGTGGS (配列番号13)
4-3: GSGGDEVDGTG (配列番号14)
4-4: GSGGDEVDGTGG (配列番号15)
4-5: GSGGDEVDGTGGS (配列番号16)
5-3: GGSGGDEVDGTG (配列番号17)
5-4: GGSGGDEVDGTGG (配列番号18)
設計したリンカーのN末端側にmMiCyを、C末端側にKP(国際公開WO03/104461号公報)をつなぎ、[mMiCy-リンカー-KP]の形でpRSETBのBamH1―EcoR1サイトにサブクローニングし大腸菌JM109(DE3)に形質転換してリコンビナント蛋白質を発現させた。発現させたリコンビナント融合蛋白質はNi-NTAで精製した。精製したリコンビナント融合蛋白質を用いてCaspase3の活性測定を行った。
Caspase3の活性はFRETの変化としてmMiCyの蛍光値の増加から得た。活性測定にはリコンビナントActive−Caspase3(MBL:BV-1083-9)を用いた。20mM HEPES-KOH pH7.4、100mM NaCl、0.1%CHAPS、10%sucrose溶液中に各リコンビナント融合蛋白質を0.25mg/mlになるようにして、リコンビナントActive−Caspase3を1unit加えて30度で反応させた。反応液を30分ごとにサンプリングし50倍希釈して440nmで励起したときの蛍光の増加を測定した。mMiCyの蛍光ピーク(496nm)をプロットした(図8及び9)。また、蛍光増加の比が高かったリンカー3-4とリンカー4-5を用いたリコンビナント融合蛋白質でCaspase3によるペプチド鎖の切断を行い、12.5%アクリルアミドゲルで電気泳動した。60kDa(融合蛋白質)が約30kDaの2本のバンド(KPとmMiCy(両者のアミノ酸数はほとんど変わらないが、mMiCyの方が3.8kDaの精製用タグ配列等のついた分大きい))として検出され、Caspase3によるペプチド鎖の切断を確認した(図10)。色素蛋白質KPと蛍光蛋白質mMiCyを用いたFRET測定系で1波長励起1波長測光によるCaspase3の活性測定ができた。
<110> RIKEN
<120> Fluorescent protein
<130> A41845A
<160> 34
<210> 1
<211> 696
<212> DNA
<213> Acropora sp.
<220>
<221> CDS
<222> (1)..(696)
<223>
<400> 1
atg tct tat tca aag caa ggc atc gca caa gaa atg cgg acg aaa tac 48
Met Ser Tyr Ser Lys Gln Gly Ile Ala Gln Glu Met Arg Thr Lys Tyr
1 5 10 15
cgt atg gaa ggc agt gtc aat ggc cat gag ttc acg atc gaa ggt gta 96
Arg Met Glu Gly Ser Val Asn Gly His Glu Phe Thr Ile Glu Gly Val
20 25 30
gga act gga aac cct tac gaa ggg aaa cag acg tcc gaa tta gtg atc 144
Gly Thr Gly Asn Pro Tyr Glu Gly Lys Gln Thr Ser Glu Leu Val Ile
35 40 45
atc aag cct aag gga aaa ccc ctt cca ttc tcc ttt gac ata ctg tca 192
Ile Lys Pro Lys Gly Lys Pro Leu Pro Phe Ser Phe Asp Ile Leu Ser
50 55 60
aca gtc ttt caa tat gga aac aga tgc ttc aca aag tac cct gca gac 240
Thr Val Phe Gln Tyr Gly Asn Arg Cys Phe Thr Lys Tyr Pro Ala Asp
65 70 75 80
atg cct gac tat ttc aag caa gca ttc cca gat gga atg tca tat gaa 288
Met Pro Asp Tyr Phe Lys Gln Ala Phe Pro Asp Gly Met Ser Tyr Glu
85 90 95
agg tca ttt cta ttt gag gat gga gga gtt gct aca gcc agc tgg agc 336
Arg Ser Phe Leu Phe Glu Asp Gly Gly Val Ala Thr Ala Ser Trp Ser
100 105 110
att cgt ctc gaa gga aat tgc ttc atc cac aat tcc atc tat cat ggc 384
Ile Arg Leu Glu Gly Asn Cys Phe Ile His Asn Ser Ile Tyr His Gly
115 120 125
aca aac ttt ccc gct gat gga ccc gta atg aag aag cag aca att ggc 432
Thr Asn Phe Pro Ala Asp Gly Pro Val Met Lys Lys Gln Thr Ile Gly
130 135 140
tgg gat aag tcc tcc gaa aaa atg agt gtg gct aaa gag gtg cta aga 480
Trp Asp Lys Ser Ser Glu Lys Met Ser Val Ala Lys Glu Val Leu Arg
145 150 155 160
ggt gat gtg act cag ttt ctt ctg ctc gaa gga ggt ggt tac cag aga 528
Gly Asp Val Thr Gln Phe Leu Leu Leu Glu Gly Gly Gly Tyr Gln Arg
165 170 175
tgc cag tta cac tcc act tac aaa aca gag aag cca gtc gca atg ccc 576
Cys Gln Leu His Ser Thr Tyr Lys Thr Glu Lys Pro Val Ala Met Pro
180 185 190
ccg agt cat gtc gta gaa cat caa att gtg agg acc gac ctt ggc caa 624
Pro Ser His Val Val Glu His Gln Ile Val Arg Thr Asp Leu Gly Gln
195 200 205
act gca aaa ggc ttc aag gtc aag ctg gaa gaa cat gct gag gct cat 672
Thr Ala Lys Gly Phe Lys Val Lys Leu Glu Glu His Ala Glu Ala His
210 215 220
gtt aac cct ttg aag gtt aaa taa 696
Val Asn Pro Leu Lys Val Lys
225 230
<210> 2
<211> 231
<212> PRT
<213> Acropora sp.
<400> 2
Met Ser Tyr Ser Lys Gln Gly Ile Ala Gln Glu Met Arg Thr Lys Tyr
1 5 10 15
Arg Met Glu Gly Ser Val Asn Gly His Glu Phe Thr Ile Glu Gly Val
20 25 30
Gly Thr Gly Asn Pro Tyr Glu Gly Lys Gln Thr Ser Glu Leu Val Ile
35 40 45
Ile Lys Pro Lys Gly Lys Pro Leu Pro Phe Ser Phe Asp Ile Leu Ser
50 55 60
Thr Val Phe Gln Tyr Gly Asn Arg Cys Phe Thr Lys Tyr Pro Ala Asp
65 70 75 80
Met Pro Asp Tyr Phe Lys Gln Ala Phe Pro Asp Gly Met Ser Tyr Glu
85 90 95
Arg Ser Phe Leu Phe Glu Asp Gly Gly Val Ala Thr Ala Ser Trp Ser
100 105 110
Ile Arg Leu Glu Gly Asn Cys Phe Ile His Asn Ser Ile Tyr His Gly
115 120 125
Thr Asn Phe Pro Ala Asp Gly Pro Val Met Lys Lys Gln Thr Ile Gly
130 135 140
Trp Asp Lys Ser Ser Glu Lys Met Ser Val Ala Lys Glu Val Leu Arg
145 150 155 160
Gly Asp Val Thr Gln Phe Leu Leu Leu Glu Gly Gly Gly Tyr Gln Arg
165 170 175
Cys Gln Leu His Ser Thr Tyr Lys Thr Glu Lys Pro Val Ala Met Pro
180 185 190
Pro Ser His Val Val Glu His Gln Ile Val Arg Thr Asp Leu Gly Gln
195 200 205
Thr Ala Lys Gly Phe Lys Val Lys Leu Glu Glu His Ala Glu Ala His
210 215 220
Val Asn Pro Leu Lys Val Lys
225 230
<210> 3
<211> 690
<212> DNA
<213> Cnidopus japonicus
<400> 3
atg gct tca atg att aaa gac gtt atg cgc ttc aag atg aac atg gaa 48
Met Ala Ser Met Ile Lys Asp Val Met Arg Phe Lys Met Asn Met Glu
1 5 10 15
ggg acg gtt aac ggc cac cac ttc aag tgt gaa gct gat gga gag ggc 96
Gly Thr Val Asn Gly His His Phe Lys Cys Glu Ala Asp Gly Glu Gly
20 25 30
aag cca tat gaa ggg act caa ctc gga aga ata agg gtc acc gaa ggc 144
Lys Pro Tyr Glu Gly Thr Gln Leu Gly Arg Ile Arg Val Thr Glu Gly
35 40 45
ggg cca ttg ccg ttt tct ttc gac atc ttg tcg act tgc act caa tat 192
Gly Pro Leu Pro Phe Ser Phe Asp Ile Leu Ser Thr Cys Thr Gln Tyr
50 55 60
gga agc aag acc atg atc aag cat ttg tcc ggg att cca gac tac ttt 240
Gly Ser Lys Thr Met Ile Lys His Leu Ser Gly Ile Pro Asp Tyr Phe
65 70 75 80
aag cag tct ttt cca gaa gga ttc acc tgg gaa agg aac aca atc tat 288
Lys Gln Ser Phe Pro Glu Gly Phe Thr Trp Glu Arg Asn Thr Ile Tyr
85 90 95
gag gat ggt ggt cac ctc aca gct cac caa gac act agt ctt aag gga 336
Glu Asp Gly Gly His Leu Thr Ala His Gln Asp Thr Ser Leu Lys Gly
100 105 110
gat tgc ctg atc tat aaa gtg aaa gtc ctt gga ggt aat ttt cct gcc 384
Asp Cys Leu Ile Tyr Lys Val Lys Val Leu Gly Gly Asn Phe Pro Ala
115 120 125
aat ggc cct gtg atg cag aag aag agc aaa gga tgg gaa ccc tgt acc 432
Asn Gly Pro Val Met Gln Lys Lys Ser Lys Gly Trp Glu Pro Cys Thr
130 135 140
gaa atg ctt tat cca cgt gat gga gtg ctt tgt ggc caa aca ttg atg 480
Glu Met Leu Tyr Pro Arg Asp Gly Val Leu Cys Gly Gln Thr Leu Met
145 150 155 160
gca ctt aaa tgc gcc aat ggt aaa aat ctg act tgc cag cta aga tct 528
Ala Leu Lys Cys Ala Asn Gly Lys Asn Leu Thr Cys Gln Leu Arg Ser
165 170 175
act tac agg tcc aga aaa caa gcc agt gca ttg cag aca cca ggc ttc 576
Thr Tyr Arg Ser Arg Lys Gln Ala Ser Ala Leu Gln Thr Pro Gly Phe
180 185 190
cat ttc gga gac cat cgt att gag ata ctc aag gaa gca gag ggg ggc 624
His Phe Gly Asp His Arg Ile Glu Ile Leu Lys Glu Ala Glu Gly Gly
195 200 205
aat tac ttt gag cag cac gaa aca tca gtc gcc agg tac tgt gat gtt 672
Asn Tyr Phe Glu Gln His Glu Thr Ser Val Ala Arg Tyr Cys Asp Val
210 215 220
gcc ccg tca aag cac tga 690
Ala Pro Ser Lys His
225
<210> 4
<211> 229
<212> PRT
<213> Cnidopus japonicus
<400> 4
Met Ala Ser Met Ile Lys Asp Val Met Arg Phe Lys Met Asn Met Glu
1 5 10 15
Gly Thr Val Asn Gly His His Phe Lys Cys Glu Ala Asp Gly Glu Gly
20 25 30
Lys Pro Tyr Glu Gly Thr Gln Leu Gly Arg Ile Arg Val Thr Glu Gly
35 40 45
Gly Pro Leu Pro Phe Ser Phe Asp Ile Leu Ser Thr Cys Thr Gln Tyr
50 55 60
Gly Ser Lys Thr Met Ile Lys His Leu Ser Gly Ile Pro Asp Tyr Phe
65 70 75 80
Lys Gln Ser Phe Pro Glu Gly Phe Thr Trp Glu Arg Asn Thr Ile Tyr
85 90 95
Glu Asp Gly Gly His Leu Thr Ala His Gln Asp Thr Ser Leu Lys Gly
100 105 110
Asp Cys Leu Ile Tyr Lys Val Lys Val Leu Gly Gly Asn Phe Pro Ala
115 120 125
Asn Gly Pro Val Met Gln Lys Lys Ser Lys Gly Trp Glu Pro Cys Thr
130 135 140
Glu Met Leu Tyr Pro Arg Asp Gly Val Leu Cys Gly Gln Thr Leu Met
145 150 155 160
Ala Leu Lys Cys Ala Asn Gly Lys Asn Leu Thr Cys Gln Leu Arg Ser
165 170 175
Thr Tyr Arg Ser Arg Lys Gln Ala Ser Ala Leu Gln Thr Pro Gly Phe
180 185 190
His Phe Gly Asp His Arg Ile Glu Ile Leu Lys Glu Ala Glu Gly Gly
195 200 205
Asn Tyr Phe Glu Gln His Glu Thr Ser Val Ala Arg Tyr Cys Asp Val
210 215 220
Ala Pro Ser Lys His
225
<210> 5
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<400> 5
gaagggaaac agacgtccga attagtg 27
<210> 6
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<220>
<222> (13), (14) and (15)
<223> any nucleotide
<400> 6
gtgatcatca agnnnaaggg aaaacccctt 30
<210> 7
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<220>
<222> (13), (14) and (15)
<223> any nucleotide
<400> 7
atactgtcaa cannntttca atatggaaac 30
<210> 8
<211> 29
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<400> 8
catctttcat ggcacaaact ttcccgctg 29
<210> 9
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<400> 9
gggataagtc ctccgaaaaa atgagtg 27
<210> 10
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Synthetic DNA
<400> 10
cagagatgcc agttacactc cacttac 27
<210> 11
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Linker peptide
<400> 11
Ser Gly Gly Asp Glu Val Asp Gly Thr Gly
1 5 10
<210> 12
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Linker peptide
<400> 12
Ser Gly Gly Asp Glu Val Asp Gly Thr Gly Gly
1 5 10
<210> 13
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Linker peptide
<400> 13
Ser Gly Gly Asp Glu Val Asp Gly Thr Gly Gly Ser
1 5 10
<210> 14
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Linker peptide
<400> 14
Gly Ser Gly Gly Asp Glu Val Asp Gly Thr Gly
1 5 10
<210> 15
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Linker peptide
<400> 15
Gly Ser Gly Gly Asp Glu Val Asp Gly Thr Gly Gly
1 5 10
<210> 16
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Linker peptide
<400> 16
Gly Ser Gly Gly Asp Glu Val Asp Gly Thr Gly Gly Ser
1 5 10
<210> 17
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Linker peptide
<400> 17
Gly Gly Ser Gly Gly Asp Glu Val Asp Gly Thr Gly
1 5 10
<210> 18
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Linker peptide
<400> 18
Gly Gly Ser Gly Gly Asp Glu Val Asp Gly Thr Gly Gly
1 5 10
<210> 19
<211> 1413
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Recombinant fused DNA
<220>
<221> CDS
<222> (1)..(1413)
<223>
<400> 19
atg tct tat tca aag caa ggc atc gca caa gaa atg cgg acg aaa tac 48
Met Ser Tyr Ser Lys Gln Gly Ile Ala Gln Glu Met Arg Thr Lys Tyr
1 5 10 15
cgt atg gaa ggc agt gtc aat ggc cat gag ttc acg atc gaa ggt gta 96
Arg Met Glu Gly Ser Val Asn Gly His Glu Phe Thr Ile Glu Gly Val
20 25 30
gga act gga aac cct tac gaa ggg aaa cag acg tcc gaa tta gtg atc 144
Gly Thr Gly Asn Pro Tyr Glu Gly Lys Gln Thr Ser Glu Leu Val Ile
35 40 45
atc aag cct aag gga aaa ccc ctt cca ttc tcc ttt gac ata ctg tca 192
Ile Lys Pro Lys Gly Lys Pro Leu Pro Phe Ser Phe Asp Ile Leu Ser
50 55 60
aca gtc ttt caa tat gga aac aga tgc ttc aca aag tac cct gca gac 240
Thr Val Phe Gln Tyr Gly Asn Arg Cys Phe Thr Lys Tyr Pro Ala Asp
65 70 75 80
atg cct gac tat ttc aag caa gca ttc cca gat gga atg tca tat gaa 288
Met Pro Asp Tyr Phe Lys Gln Ala Phe Pro Asp Gly Met Ser Tyr Glu
85 90 95
agg tca ttt cta ttt gag gat gga gga gtt gct aca gcc agc tgg agc 336
Arg Ser Phe Leu Phe Glu Asp Gly Gly Val Ala Thr Ala Ser Trp Ser
100 105 110
att cgt ctc gaa gga aat tgc ttc atc cac aat tcc atc tat cat ggc 384
Ile Arg Leu Glu Gly Asn Cys Phe Ile His Asn Ser Ile Tyr His Gly
115 120 125
aca aac ttt ccc gct gat gga ccc gta atg aag aag cag aca att ggc 432
Thr Asn Phe Pro Ala Asp Gly Pro Val Met Lys Lys Gln Thr Ile Gly
130 135 140
tgg gat aag tcc tcc gaa aaa atg agt gtg gct aaa gag gtg cta aga 480
Trp Asp Lys Ser Ser Glu Lys Met Ser Val Ala Lys Glu Val Leu Arg
145 150 155 160
ggt gat gtg act cag ttt ctt ctg ctc gaa gga ggt ggt tac cag aga 528
Gly Asp Val Thr Gln Phe Leu Leu Leu Glu Gly Gly Gly Tyr Gln Arg
165 170 175
tgc cag tta cac tcc act tac aaa aca gag aag cca gtc gca atg ccc 576
Cys Gln Leu His Ser Thr Tyr Lys Thr Glu Lys Pro Val Ala Met Pro
180 185 190
ccg agt cat gtc gta gaa cat caa att gtg agg acc gac ctt ggc caa 624
Pro Ser His Val Val Glu His Gln Ile Val Arg Thr Asp Leu Gly Gln
195 200 205
act gca aaa ggc ttc aag gtc aag ctg gaa gaa cat gct gag gct cat 672
Thr Ala Lys Gly Phe Lys Val Lys Leu Glu Glu His Ala Glu Ala His
210 215 220
gtt aac cct ttg aag gtt aaa agc ggt ggc gac gag gtg gac ggt acc 720
Val Asn Pro Leu Lys Val Lys Ser Gly Gly Asp Glu Val Asp Gly Thr
225 230 235 240
ggt atg gct tca atg att aaa gac gtt atg cgc ttc aag atg aac atg 768
Gly Met Ala Ser Met Ile Lys Asp Val Met Arg Phe Lys Met Asn Met
245 250 255
gaa ggg acg gtt aac ggc cac cac ttc aag tgt gaa gct gat gga gag 816
Glu Gly Thr Val Asn Gly His His Phe Lys Cys Glu Ala Asp Gly Glu
260 265 270
ggc aag cca tat gaa ggg act caa ctc gga aga ata agg gtc acc gaa 864
Gly Lys Pro Tyr Glu Gly Thr Gln Leu Gly Arg Ile Arg Val Thr Glu
275 280 285
ggc ggg cca ttg ccg ttt tct ttc gac atc ttg tcg act tgc act caa 912
Gly Gly Pro Leu Pro Phe Ser Phe Asp Ile Leu Ser Thr Cys Thr Gln
290 295 300
tat gga agc aag acc atg atc aag cat ttg tcc ggg att cca gac tac 960
Tyr Gly Ser Lys Thr Met Ile Lys His Leu Ser Gly Ile Pro Asp Tyr
305 310 315 320
ttt aag cag tct ttt cca gaa gga ttc acc tgg gaa agg aac aca atc 1008
Phe Lys Gln Ser Phe Pro Glu Gly Phe Thr Trp Glu Arg Asn Thr Ile
325 330 335
tat gag gat ggt ggt cac ctc aca gct cac caa gac act agt ctt aag 1056
Tyr Glu Asp Gly Gly His Leu Thr Ala His Gln Asp Thr Ser Leu Lys
340 345 350
gga gat tgc ctg atc tat aaa gtg aaa gtc ctt gga ggt aat ttt cct 1104
Gly Asp Cys Leu Ile Tyr Lys Val Lys Val Leu Gly Gly Asn Phe Pro
355 360 365
gcc aat ggc cct gtg atg cag aag aag agc aaa gga tgg gaa ccc tgt 1152
Ala Asn Gly Pro Val Met Gln Lys Lys Ser Lys Gly Trp Glu Pro Cys
370 375 380
acc gaa atg ctt tat cca cgt gat gga gtg ctt tgt ggc caa aca ttg 1200
Thr Glu Met Leu Tyr Pro Arg Asp Gly Val Leu Cys Gly Gln Thr Leu
385 390 395 400
atg gca ctt aaa tgc gcc aat ggt aaa aat ctg act tgc cag cta aga 1248
Met Ala Leu Lys Cys Ala Asn Gly Lys Asn Leu Thr Cys Gln Leu Arg
405 410 415
tct act tac agg tcc aga aaa caa gcc agt gca ttg cag aca cca ggc 1296
Ser Thr Tyr Arg Ser Arg Lys Gln Ala Ser Ala Leu Gln Thr Pro Gly
420 425 430
ttc cat ttc gga gac cat cgt att gag ata ctc aag gaa gca gag ggg 1344
Phe His Phe Gly Asp His Arg Ile Glu Ile Leu Lys Glu Ala Glu Gly
435 440 445
ggc aat tac ttt gag cag cac gaa aca tca gtc gcc agg tac tgt gat 1392
Gly Asn Tyr Phe Glu Gln His Glu Thr Ser Val Ala Arg Tyr Cys Asp
450 455 460
gtt gcc ccg tca aag cac tga 1413
Val Ala Pro Ser Lys His
465 470
<210> 20
<211> 470
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Recombinant fused protein
<400> 20
Met Ser Tyr Ser Lys Gln Gly Ile Ala Gln Glu Met Arg Thr Lys Tyr
1 5 10 15
Arg Met Glu Gly Ser Val Asn Gly His Glu Phe Thr Ile Glu Gly Val
20 25 30
Gly Thr Gly Asn Pro Tyr Glu Gly Lys Gln Thr Ser Glu Leu Val Ile
35 40 45
Ile Lys Pro Lys Gly Lys Pro Leu Pro Phe Ser Phe Asp Ile Leu Ser
50 55 60
Thr Val Phe Gln Tyr Gly Asn Arg Cys Phe Thr Lys Tyr Pro Ala Asp
65 70 75 80
Met Pro Asp Tyr Phe Lys Gln Ala Phe Pro Asp Gly Met Ser Tyr Glu
85 90 95
Arg Ser Phe Leu Phe Glu Asp Gly Gly Val Ala Thr Ala Ser Trp Ser
100 105 110
Ile Arg Leu Glu Gly Asn Cys Phe Ile His Asn Ser Ile Tyr His Gly
115 120 125
Thr Asn Phe Pro Ala Asp Gly Pro Val Met Lys Lys Gln Thr Ile Gly
130 135 140
Trp Asp Lys Ser Ser Glu Lys Met Ser Val Ala Lys Glu Val Leu Arg
145 150 155 160
Gly Asp Val Thr Gln Phe Leu Leu Leu Glu Gly Gly Gly Tyr Gln Arg
165 170 175
Cys Gln Leu His Ser Thr Tyr Lys Thr Glu Lys Pro Val Ala Met Pro
180 185 190
Pro Ser His Val Val Glu His Gln Ile Val Arg Thr Asp Leu Gly Gln
195 200 205
Thr Ala Lys Gly Phe Lys Val Lys Leu Glu Glu His Ala Glu Ala His
210 215 220
Val Asn Pro Leu Lys Val Lys Ser Gly Gly Asp Glu Val Asp Gly Thr
225 230 235 240
Gly Met Ala Ser Met Ile Lys Asp Val Met Arg Phe Lys Met Asn Met
245 250 255
Glu Gly Thr Val Asn Gly His His Phe Lys Cys Glu Ala Asp Gly Glu
260 265 270
Gly Lys Pro Tyr Glu Gly Thr Gln Leu Gly Arg Ile Arg Val Thr Glu
275 280 285
Gly Gly Pro Leu Pro Phe Ser Phe Asp Ile Leu Ser Thr Cys Thr Gln
290 295 300
Tyr Gly Ser Lys Thr Met Ile Lys His Leu Ser Gly Ile Pro Asp Tyr
305 310 315 320
Phe Lys Gln Ser Phe Pro Glu Gly Phe Thr Trp Glu Arg Asn Thr Ile
325 330 335
Tyr Glu Asp Gly Gly His Leu Thr Ala His Gln Asp Thr Ser Leu Lys
340 345 350
Gly Asp Cys Leu Ile Tyr Lys Val Lys Val Leu Gly Gly Asn Phe Pro
355 360 365
Ala Asn Gly Pro Val Met Gln Lys Lys Ser Lys Gly Trp Glu Pro Cys
370 375 380
Thr Glu Met Leu Tyr Pro Arg Asp Gly Val Leu Cys Gly Gln Thr Leu
385 390 395 400
Met Ala Leu Lys Cys Ala Asn Gly Lys Asn Leu Thr Cys Gln Leu Arg
405 410 415
Ser Thr Tyr Arg Ser Arg Lys Gln Ala Ser Ala Leu Gln Thr Pro Gly
420 425 430
Phe His Phe Gly Asp His Arg Ile Glu Ile Leu Lys Glu Ala Glu Gly
435 440 445
Gly Asn Tyr Phe Glu Gln His Glu Thr Ser Val Ala Arg Tyr Cys Asp
450 455 460
Val Ala Pro Ser Lys His
465 470
<210> 21
<211> 1416
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Recombinant fused DNA
<220>
<221> CDS
<222> (1)..(1416)
<223>
<400> 21
atg tct tat tca aag caa ggc atc gca caa gaa atg cgg acg aaa tac 48
Met Ser Tyr Ser Lys Gln Gly Ile Ala Gln Glu Met Arg Thr Lys Tyr
1 5 10 15
cgt atg gaa ggc agt gtc aat ggc cat gag ttc acg atc gaa ggt gta 96
Arg Met Glu Gly Ser Val Asn Gly His Glu Phe Thr Ile Glu Gly Val
20 25 30
gga act gga aac cct tac gaa ggg aaa cag acg tcc gaa tta gtg atc 144
Gly Thr Gly Asn Pro Tyr Glu Gly Lys Gln Thr Ser Glu Leu Val Ile
35 40 45
atc aag cct aag gga aaa ccc ctt cca ttc tcc ttt gac ata ctg tca 192
Ile Lys Pro Lys Gly Lys Pro Leu Pro Phe Ser Phe Asp Ile Leu Ser
50 55 60
aca gtc ttt caa tat gga aac aga tgc ttc aca aag tac cct gca gac 240
Thr Val Phe Gln Tyr Gly Asn Arg Cys Phe Thr Lys Tyr Pro Ala Asp
65 70 75 80
atg cct gac tat ttc aag caa gca ttc cca gat gga atg tca tat gaa 288
Met Pro Asp Tyr Phe Lys Gln Ala Phe Pro Asp Gly Met Ser Tyr Glu
85 90 95
agg tca ttt cta ttt gag gat gga gga gtt gct aca gcc agc tgg agc 336
Arg Ser Phe Leu Phe Glu Asp Gly Gly Val Ala Thr Ala Ser Trp Ser
100 105 110
att cgt ctc gaa gga aat tgc ttc atc cac aat tcc atc tat cat ggc 384
Ile Arg Leu Glu Gly Asn Cys Phe Ile His Asn Ser Ile Tyr His Gly
115 120 125
aca aac ttt ccc gct gat gga ccc gta atg aag aag cag aca att ggc 432
Thr Asn Phe Pro Ala Asp Gly Pro Val Met Lys Lys Gln Thr Ile Gly
130 135 140
tgg gat aag tcc tcc gaa aaa atg agt gtg gct aaa gag gtg cta aga 480
Trp Asp Lys Ser Ser Glu Lys Met Ser Val Ala Lys Glu Val Leu Arg
145 150 155 160
ggt gat gtg act cag ttt ctt ctg ctc gaa gga ggt ggt tac cag aga 528
Gly Asp Val Thr Gln Phe Leu Leu Leu Glu Gly Gly Gly Tyr Gln Arg
165 170 175
tgc cag tta cac tcc act tac aaa aca gag aag cca gtc gca atg ccc 576
Cys Gln Leu His Ser Thr Tyr Lys Thr Glu Lys Pro Val Ala Met Pro
180 185 190
ccg agt cat gtc gta gaa cat caa att gtg agg acc gac ctt ggc caa 624
Pro Ser His Val Val Glu His Gln Ile Val Arg Thr Asp Leu Gly Gln
195 200 205
act gca aaa ggc ttc aag gtc aag ctg gaa gaa cat gct gag gct cat 672
Thr Ala Lys Gly Phe Lys Val Lys Leu Glu Glu His Ala Glu Ala His
210 215 220
gtt aac cct ttg aag gtt aaa agc ggt ggc gac gag gtg gac ggt acc 720
Val Asn Pro Leu Lys Val Lys Ser Gly Gly Asp Glu Val Asp Gly Thr
225 230 235 240
ggt ggc atg gct tca atg att aaa gac gtt atg cgc ttc aag atg aac 768
Gly Gly Met Ala Ser Met Ile Lys Asp Val Met Arg Phe Lys Met Asn
245 250 255
atg gaa ggg acg gtt aac ggc cac cac ttc aag tgt gaa gct gat gga 816
Met Glu Gly Thr Val Asn Gly His His Phe Lys Cys Glu Ala Asp Gly
260 265 270
gag ggc aag cca tat gaa ggg act caa ctc gga aga ata agg gtc acc 864
Glu Gly Lys Pro Tyr Glu Gly Thr Gln Leu Gly Arg Ile Arg Val Thr
275 280 285
gaa ggc ggg cca ttg ccg ttt tct ttc gac atc ttg tcg act tgc act 912
Glu Gly Gly Pro Leu Pro Phe Ser Phe Asp Ile Leu Ser Thr Cys Thr
290 295 300
caa tat gga agc aag acc atg atc aag cat ttg tcc ggg att cca gac 960
Gln Tyr Gly Ser Lys Thr Met Ile Lys His Leu Ser Gly Ile Pro Asp
305 310 315 320
tac ttt aag cag tct ttt cca gaa gga ttc acc tgg gaa agg aac aca 1008
Tyr Phe Lys Gln Ser Phe Pro Glu Gly Phe Thr Trp Glu Arg Asn Thr
325 330 335
atc tat gag gat ggt ggt cac ctc aca gct cac caa gac act agt ctt 1056
Ile Tyr Glu Asp Gly Gly His Leu Thr Ala His Gln Asp Thr Ser Leu
340 345 350
aag gga gat tgc ctg atc tat aaa gtg aaa gtc ctt gga ggt aat ttt 1104
Lys Gly Asp Cys Leu Ile Tyr Lys Val Lys Val Leu Gly Gly Asn Phe
355 360 365
cct gcc aat ggc cct gtg atg cag aag aag agc aaa gga tgg gaa ccc 1152
Pro Ala Asn Gly Pro Val Met Gln Lys Lys Ser Lys Gly Trp Glu Pro
370 375 380
tgt acc gaa atg ctt tat cca cgt gat gga gtg ctt tgt ggc caa aca 1200
Cys Thr Glu Met Leu Tyr Pro Arg Asp Gly Val Leu Cys Gly Gln Thr
385 390 395 400
ttg atg gca ctt aaa tgc gcc aat ggt aaa aat ctg act tgc cag cta 1248
Leu Met Ala Leu Lys Cys Ala Asn Gly Lys Asn Leu Thr Cys Gln Leu
405 410 415
aga tct act tac agg tcc aga aaa caa gcc agt gca ttg cag aca cca 1296
Arg Ser Thr Tyr Arg Ser Arg Lys Gln Ala Ser Ala Leu Gln Thr Pro
420 425 430
ggc ttc cat ttc gga gac cat cgt att gag ata ctc aag gaa gca gag 1344
Gly Phe His Phe Gly Asp His Arg Ile Glu Ile Leu Lys Glu Ala Glu
435 440 445
ggg ggc aat tac ttt gag cag cac gaa aca tca gtc gcc agg tac tgt 1392
Gly Gly Asn Tyr Phe Glu Gln His Glu Thr Ser Val Ala Arg Tyr Cys
450 455 460
gat gtt gcc ccg tca aag cac tga 1416
Asp Val Ala Pro Ser Lys His
465 470
<210> 22
<211> 471
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Recombinant fused protein
<400> 22
Met Ser Tyr Ser Lys Gln Gly Ile Ala Gln Glu Met Arg Thr Lys Tyr
1 5 10 15
Arg Met Glu Gly Ser Val Asn Gly His Glu Phe Thr Ile Glu Gly Val
20 25 30
Gly Thr Gly Asn Pro Tyr Glu Gly Lys Gln Thr Ser Glu Leu Val Ile
35 40 45
Ile Lys Pro Lys Gly Lys Pro Leu Pro Phe Ser Phe Asp Ile Leu Ser
50 55 60
Thr Val Phe Gln Tyr Gly Asn Arg Cys Phe Thr Lys Tyr Pro Ala Asp
65 70 75 80
Met Pro Asp Tyr Phe Lys Gln Ala Phe Pro Asp Gly Met Ser Tyr Glu
85 90 95
Arg Ser Phe Leu Phe Glu Asp Gly Gly Val Ala Thr Ala Ser Trp Ser
100 105 110
Ile Arg Leu Glu Gly Asn Cys Phe Ile His Asn Ser Ile Tyr His Gly
115 120 125
Thr Asn Phe Pro Ala Asp Gly Pro Val Met Lys Lys Gln Thr Ile Gly
130 135 140
Trp Asp Lys Ser Ser Glu Lys Met Ser Val Ala Lys Glu Val Leu Arg
145 150 155 160
Gly Asp Val Thr Gln Phe Leu Leu Leu Glu Gly Gly Gly Tyr Gln Arg
165 170 175
Cys Gln Leu His Ser Thr Tyr Lys Thr Glu Lys Pro Val Ala Met Pro
180 185 190
Pro Ser His Val Val Glu His Gln Ile Val Arg Thr Asp Leu Gly Gln
195 200 205
Thr Ala Lys Gly Phe Lys Val Lys Leu Glu Glu His Ala Glu Ala His
210 215 220
Val Asn Pro Leu Lys Val Lys Ser Gly Gly Asp Glu Val Asp Gly Thr
225 230 235 240
Gly Gly Met Ala Ser Met Ile Lys Asp Val Met Arg Phe Lys Met Asn
245 250 255
Met Glu Gly Thr Val Asn Gly His His Phe Lys Cys Glu Ala Asp Gly
260 265 270
Glu Gly Lys Pro Tyr Glu Gly Thr Gln Leu Gly Arg Ile Arg Val Thr
275 280 285
Glu Gly Gly Pro Leu Pro Phe Ser Phe Asp Ile Leu Ser Thr Cys Thr
290 295 300
Gln Tyr Gly Ser Lys Thr Met Ile Lys His Leu Ser Gly Ile Pro Asp
305 310 315 320
Tyr Phe Lys Gln Ser Phe Pro Glu Gly Phe Thr Trp Glu Arg Asn Thr
325 330 335
Ile Tyr Glu Asp Gly Gly His Leu Thr Ala His Gln Asp Thr Ser Leu
340 345 350
Lys Gly Asp Cys Leu Ile Tyr Lys Val Lys Val Leu Gly Gly Asn Phe
355 360 365
Pro Ala Asn Gly Pro Val Met Gln Lys Lys Ser Lys Gly Trp Glu Pro
370 375 380
Cys Thr Glu Met Leu Tyr Pro Arg Asp Gly Val Leu Cys Gly Gln Thr
385 390 395 400
Leu Met Ala Leu Lys Cys Ala Asn Gly Lys Asn Leu Thr Cys Gln Leu
405 410 415
Arg Ser Thr Tyr Arg Ser Arg Lys Gln Ala Ser Ala Leu Gln Thr Pro
420 425 430
Gly Phe His Phe Gly Asp His Arg Ile Glu Ile Leu Lys Glu Ala Glu
435 440 445
Gly Gly Asn Tyr Phe Glu Gln His Glu Thr Ser Val Ala Arg Tyr Cys
450 455 460
Asp Val Ala Pro Ser Lys His
465 470
<210> 23
<211> 1419
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Recombinant fused DNA
<220>
<221> CDS
<222> (1)..(1419)
<223>
<400> 23
atg tct tat tca aag caa ggc atc gca caa gaa atg cgg acg aaa tac 48
Met Ser Tyr Ser Lys Gln Gly Ile Ala Gln Glu Met Arg Thr Lys Tyr
1 5 10 15
cgt atg gaa ggc agt gtc aat ggc cat gag ttc acg atc gaa ggt gta 96
Arg Met Glu Gly Ser Val Asn Gly His Glu Phe Thr Ile Glu Gly Val
20 25 30
gga act gga aac cct tac gaa ggg aaa cag acg tcc gaa tta gtg atc 144
Gly Thr Gly Asn Pro Tyr Glu Gly Lys Gln Thr Ser Glu Leu Val Ile
35 40 45
atc aag cct aag gga aaa ccc ctt cca ttc tcc ttt gac ata ctg tca 192
Ile Lys Pro Lys Gly Lys Pro Leu Pro Phe Ser Phe Asp Ile Leu Ser
50 55 60
aca gtc ttt caa tat gga aac aga tgc ttc aca aag tac cct gca gac 240
Thr Val Phe Gln Tyr Gly Asn Arg Cys Phe Thr Lys Tyr Pro Ala Asp
65 70 75 80
atg cct gac tat ttc aag caa gca ttc cca gat gga atg tca tat gaa 288
Met Pro Asp Tyr Phe Lys Gln Ala Phe Pro Asp Gly Met Ser Tyr Glu
85 90 95
agg tca ttt cta ttt gag gat gga gga gtt gct aca gcc agc tgg agc 336
Arg Ser Phe Leu Phe Glu Asp Gly Gly Val Ala Thr Ala Ser Trp Ser
100 105 110
att cgt ctc gaa gga aat tgc ttc atc cac aat tcc atc tat cat ggc 384
Ile Arg Leu Glu Gly Asn Cys Phe Ile His Asn Ser Ile Tyr His Gly
115 120 125
aca aac ttt ccc gct gat gga ccc gta atg aag aag cag aca att ggc 432
Thr Asn Phe Pro Ala Asp Gly Pro Val Met Lys Lys Gln Thr Ile Gly
130 135 140
tgg gat aag tcc tcc gaa aaa atg agt gtg gct aaa gag gtg cta aga 480
Trp Asp Lys Ser Ser Glu Lys Met Ser Val Ala Lys Glu Val Leu Arg
145 150 155 160
ggt gat gtg act cag ttt ctt ctg ctc gaa gga ggt ggt tac cag aga 528
Gly Asp Val Thr Gln Phe Leu Leu Leu Glu Gly Gly Gly Tyr Gln Arg
165 170 175
tgc cag tta cac tcc act tac aaa aca gag aag cca gtc gca atg ccc 576
Cys Gln Leu His Ser Thr Tyr Lys Thr Glu Lys Pro Val Ala Met Pro
180 185 190
ccg agt cat gtc gta gaa cat caa att gtg agg acc gac ctt ggc caa 624
Pro Ser His Val Val Glu His Gln Ile Val Arg Thr Asp Leu Gly Gln
195 200 205
act gca aaa ggc ttc aag gtc aag ctg gaa gaa cat gct gag gct cat 672
Thr Ala Lys Gly Phe Lys Val Lys Leu Glu Glu His Ala Glu Ala His
210 215 220
gtt aac cct ttg aag gtt aaa agc ggt ggc gac gag gtg gac ggt acc 720
Val Asn Pro Leu Lys Val Lys Ser Gly Gly Asp Glu Val Asp Gly Thr
225 230 235 240
ggt ggc agc atg gct tca atg att aaa gac gtt atg cgc ttc aag atg 768
Gly Gly Ser Met Ala Ser Met Ile Lys Asp Val Met Arg Phe Lys Met
245 250 255
aac atg gaa ggg acg gtt aac ggc cac cac ttc aag tgt gaa gct gat 816
Asn Met Glu Gly Thr Val Asn Gly His His Phe Lys Cys Glu Ala Asp
260 265 270
gga gag ggc aag cca tat gaa ggg act caa ctc gga aga ata agg gtc 864
Gly Glu Gly Lys Pro Tyr Glu Gly Thr Gln Leu Gly Arg Ile Arg Val
275 280 285
acc gaa ggc ggg cca ttg ccg ttt tct ttc gac atc ttg tcg act tgc 912
Thr Glu Gly Gly Pro Leu Pro Phe Ser Phe Asp Ile Leu Ser Thr Cys
290 295 300
act caa tat gga agc aag acc atg atc aag cat ttg tcc ggg att cca 960
Thr Gln Tyr Gly Ser Lys Thr Met Ile Lys His Leu Ser Gly Ile Pro
305 310 315 320
gac tac ttt aag cag tct ttt cca gaa gga ttc acc tgg gaa agg aac 1008
Asp Tyr Phe Lys Gln Ser Phe Pro Glu Gly Phe Thr Trp Glu Arg Asn
325 330 335
aca atc tat gag gat ggt ggt cac ctc aca gct cac caa gac act agt 1056
Thr Ile Tyr Glu Asp Gly Gly His Leu Thr Ala His Gln Asp Thr Ser
340 345 350
ctt aag gga gat tgc ctg atc tat aaa gtg aaa gtc ctt gga ggt aat 1104
Leu Lys Gly Asp Cys Leu Ile Tyr Lys Val Lys Val Leu Gly Gly Asn
355 360 365
ttt cct gcc aat ggc cct gtg atg cag aag aag agc aaa gga tgg gaa 1152
Phe Pro Ala Asn Gly Pro Val Met Gln Lys Lys Ser Lys Gly Trp Glu
370 375 380
ccc tgt acc gaa atg ctt tat cca cgt gat gga gtg ctt tgt ggc caa 1200
Pro Cys Thr Glu Met Leu Tyr Pro Arg Asp Gly Val Leu Cys Gly Gln
385 390 395 400
aca ttg atg gca ctt aaa tgc gcc aat ggt aaa aat ctg act tgc cag 1248
Thr Leu Met Ala Leu Lys Cys Ala Asn Gly Lys Asn Leu Thr Cys Gln
405 410 415
cta aga tct act tac agg tcc aga aaa caa gcc agt gca ttg cag aca 1296
Leu Arg Ser Thr Tyr Arg Ser Arg Lys Gln Ala Ser Ala Leu Gln Thr
420 425 430
cca ggc ttc cat ttc gga gac cat cgt att gag ata ctc aag gaa gca 1344
Pro Gly Phe His Phe Gly Asp His Arg Ile Glu Ile Leu Lys Glu Ala
435 440 445
gag ggg ggc aat tac ttt gag cag cac gaa aca tca gtc gcc agg tac 1392
Glu Gly Gly Asn Tyr Phe Glu Gln His Glu Thr Ser Val Ala Arg Tyr
450 455 460
tgt gat gtt gcc ccg tca aag cac tga 1419
Cys Asp Val Ala Pro Ser Lys His
465 470
<210> 24
<211> 472
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Recombinant fused protein
<400> 24
Met Ser Tyr Ser Lys Gln Gly Ile Ala Gln Glu Met Arg Thr Lys Tyr
1 5 10 15
Arg Met Glu Gly Ser Val Asn Gly His Glu Phe Thr Ile Glu Gly Val
20 25 30
Gly Thr Gly Asn Pro Tyr Glu Gly Lys Gln Thr Ser Glu Leu Val Ile
35 40 45
Ile Lys Pro Lys Gly Lys Pro Leu Pro Phe Ser Phe Asp Ile Leu Ser
50 55 60
Thr Val Phe Gln Tyr Gly Asn Arg Cys Phe Thr Lys Tyr Pro Ala Asp
65 70 75 80
Met Pro Asp Tyr Phe Lys Gln Ala Phe Pro Asp Gly Met Ser Tyr Glu
85 90 95
Arg Ser Phe Leu Phe Glu Asp Gly Gly Val Ala Thr Ala Ser Trp Ser
100 105 110
Ile Arg Leu Glu Gly Asn Cys Phe Ile His Asn Ser Ile Tyr His Gly
115 120 125
Thr Asn Phe Pro Ala Asp Gly Pro Val Met Lys Lys Gln Thr Ile Gly
130 135 140
Trp Asp Lys Ser Ser Glu Lys Met Ser Val Ala Lys Glu Val Leu Arg
145 150 155 160
Gly Asp Val Thr Gln Phe Leu Leu Leu Glu Gly Gly Gly Tyr Gln Arg
165 170 175
Cys Gln Leu His Ser Thr Tyr Lys Thr Glu Lys Pro Val Ala Met Pro
180 185 190
Pro Ser His Val Val Glu His Gln Ile Val Arg Thr Asp Leu Gly Gln
195 200 205
Thr Ala Lys Gly Phe Lys Val Lys Leu Glu Glu His Ala Glu Ala His
210 215 220
Val Asn Pro Leu Lys Val Lys Ser Gly Gly Asp Glu Val Asp Gly Thr
225 230 235 240
Gly Gly Ser Met Ala Ser Met Ile Lys Asp Val Met Arg Phe Lys Met
245 250 255
Asn Met Glu Gly Thr Val Asn Gly His His Phe Lys Cys Glu Ala Asp
260 265 270
Gly Glu Gly Lys Pro Tyr Glu Gly Thr Gln Leu Gly Arg Ile Arg Val
275 280 285
Thr Glu Gly Gly Pro Leu Pro Phe Ser Phe Asp Ile Leu Ser Thr Cys
290 295 300
Thr Gln Tyr Gly Ser Lys Thr Met Ile Lys His Leu Ser Gly Ile Pro
305 310 315 320
Asp Tyr Phe Lys Gln Ser Phe Pro Glu Gly Phe Thr Trp Glu Arg Asn
325 330 335
Thr Ile Tyr Glu Asp Gly Gly His Leu Thr Ala His Gln Asp Thr Ser
340 345 350
Leu Lys Gly Asp Cys Leu Ile Tyr Lys Val Lys Val Leu Gly Gly Asn
355 360 365
Phe Pro Ala Asn Gly Pro Val Met Gln Lys Lys Ser Lys Gly Trp Glu
370 375 380
Pro Cys Thr Glu Met Leu Tyr Pro Arg Asp Gly Val Leu Cys Gly Gln
385 390 395 400
Thr Leu Met Ala Leu Lys Cys Ala Asn Gly Lys Asn Leu Thr Cys Gln
405 410 415
Leu Arg Ser Thr Tyr Arg Ser Arg Lys Gln Ala Ser Ala Leu Gln Thr
420 425 430
Pro Gly Phe His Phe Gly Asp His Arg Ile Glu Ile Leu Lys Glu Ala
435 440 445
Glu Gly Gly Asn Tyr Phe Glu Gln His Glu Thr Ser Val Ala Arg Tyr
450 455 460
Cys Asp Val Ala Pro Ser Lys His
465 470
<210> 25
<211> 1416
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Recombinant fused DNA
<220>
<221> CDS
<222> (1)..(1416)
<223>
<400> 25
atg tct tat tca aag caa ggc atc gca caa gaa atg cgg acg aaa tac 48
Met Ser Tyr Ser Lys Gln Gly Ile Ala Gln Glu Met Arg Thr Lys Tyr
1 5 10 15
cgt atg gaa ggc agt gtc aat ggc cat gag ttc acg atc gaa ggt gta 96
Arg Met Glu Gly Ser Val Asn Gly His Glu Phe Thr Ile Glu Gly Val
20 25 30
gga act gga aac cct tac gaa ggg aaa cag acg tcc gaa tta gtg atc 144
Gly Thr Gly Asn Pro Tyr Glu Gly Lys Gln Thr Ser Glu Leu Val Ile
35 40 45
atc aag cct aag gga aaa ccc ctt cca ttc tcc ttt gac ata ctg tca 192
Ile Lys Pro Lys Gly Lys Pro Leu Pro Phe Ser Phe Asp Ile Leu Ser
50 55 60
aca gtc ttt caa tat gga aac aga tgc ttc aca aag tac cct gca gac 240
Thr Val Phe Gln Tyr Gly Asn Arg Cys Phe Thr Lys Tyr Pro Ala Asp
65 70 75 80
atg cct gac tat ttc aag caa gca ttc cca gat gga atg tca tat gaa 288
Met Pro Asp Tyr Phe Lys Gln Ala Phe Pro Asp Gly Met Ser Tyr Glu
85 90 95
agg tca ttt cta ttt gag gat gga gga gtt gct aca gcc agc tgg agc 336
Arg Ser Phe Leu Phe Glu Asp Gly Gly Val Ala Thr Ala Ser Trp Ser
100 105 110
att cgt ctc gaa gga aat tgc ttc atc cac aat tcc atc tat cat ggc 384
Ile Arg Leu Glu Gly Asn Cys Phe Ile His Asn Ser Ile Tyr His Gly
115 120 125
aca aac ttt ccc gct gat gga ccc gta atg aag aag cag aca att ggc 432
Thr Asn Phe Pro Ala Asp Gly Pro Val Met Lys Lys Gln Thr Ile Gly
130 135 140
tgg gat aag tcc tcc gaa aaa atg agt gtg gct aaa gag gtg cta aga 480
Trp Asp Lys Ser Ser Glu Lys Met Ser Val Ala Lys Glu Val Leu Arg
145 150 155 160
ggt gat gtg act cag ttt ctt ctg ctc gaa gga ggt ggt tac cag aga 528
Gly Asp Val Thr Gln Phe Leu Leu Leu Glu Gly Gly Gly Tyr Gln Arg
165 170 175
tgc cag tta cac tcc act tac aaa aca gag aag cca gtc gca atg ccc 576
Cys Gln Leu His Ser Thr Tyr Lys Thr Glu Lys Pro Val Ala Met Pro
180 185 190
ccg agt cat gtc gta gaa cat caa att gtg agg acc gac ctt ggc caa 624
Pro Ser His Val Val Glu His Gln Ile Val Arg Thr Asp Leu Gly Gln
195 200 205
act gca aaa ggc ttc aag gtc aag ctg gaa gaa cat gct gag gct cat 672
Thr Ala Lys Gly Phe Lys Val Lys Leu Glu Glu His Ala Glu Ala His
210 215 220
gtt aac cct ttg aag gtt aaa ggc agc ggt ggc gac gag gtg gac ggt 720
Val Asn Pro Leu Lys Val Lys Gly Ser Gly Gly Asp Glu Val Asp Gly
225 230 235 240
acc ggt atg gct tca atg att aaa gac gtt atg cgc ttc aag atg aac 768
Thr Gly Met Ala Ser Met Ile Lys Asp Val Met Arg Phe Lys Met Asn
245 250 255
atg gaa ggg acg gtt aac ggc cac cac ttc aag tgt gaa gct gat gga 816
Met Glu Gly Thr Val Asn Gly His His Phe Lys Cys Glu Ala Asp Gly
260 265 270
gag ggc aag cca tat gaa ggg act caa ctc gga aga ata agg gtc acc 864
Glu Gly Lys Pro Tyr Glu Gly Thr Gln Leu Gly Arg Ile Arg Val Thr
275 280 285
gaa ggc ggg cca ttg ccg ttt tct ttc gac atc ttg tcg act tgc act 912
Glu Gly Gly Pro Leu Pro Phe Ser Phe Asp Ile Leu Ser Thr Cys Thr
290 295 300
caa tat gga agc aag acc atg atc aag cat ttg tcc ggg att cca gac 960
Gln Tyr Gly Ser Lys Thr Met Ile Lys His Leu Ser Gly Ile Pro Asp
305 310 315 320
tac ttt aag cag tct ttt cca gaa gga ttc acc tgg gaa agg aac aca 1008
Tyr Phe Lys Gln Ser Phe Pro Glu Gly Phe Thr Trp Glu Arg Asn Thr
325 330 335
atc tat gag gat ggt ggt cac ctc aca gct cac caa gac act agt ctt 1056
Ile Tyr Glu Asp Gly Gly His Leu Thr Ala His Gln Asp Thr Ser Leu
340 345 350
aag gga gat tgc ctg atc tat aaa gtg aaa gtc ctt gga ggt aat ttt 1104
Lys Gly Asp Cys Leu Ile Tyr Lys Val Lys Val Leu Gly Gly Asn Phe
355 360 365
cct gcc aat ggc cct gtg atg cag aag aag agc aaa gga tgg gaa ccc 1152
Pro Ala Asn Gly Pro Val Met Gln Lys Lys Ser Lys Gly Trp Glu Pro
370 375 380
tgt acc gaa atg ctt tat cca cgt gat gga gtg ctt tgt ggc caa aca 1200
Cys Thr Glu Met Leu Tyr Pro Arg Asp Gly Val Leu Cys Gly Gln Thr
385 390 395 400
ttg atg gca ctt aaa tgc gcc aat ggt aaa aat ctg act tgc cag cta 1248
Leu Met Ala Leu Lys Cys Ala Asn Gly Lys Asn Leu Thr Cys Gln Leu
405 410 415
aga tct act tac agg tcc aga aaa caa gcc agt gca ttg cag aca cca 1296
Arg Ser Thr Tyr Arg Ser Arg Lys Gln Ala Ser Ala Leu Gln Thr Pro
420 425 430
ggc ttc cat ttc gga gac cat cgt att gag ata ctc aag gaa gca gag 1344
Gly Phe His Phe Gly Asp His Arg Ile Glu Ile Leu Lys Glu Ala Glu
435 440 445
ggg ggc aat tac ttt gag cag cac gaa aca tca gtc gcc agg tac tgt 1392
Gly Gly Asn Tyr Phe Glu Gln His Glu Thr Ser Val Ala Arg Tyr Cys
450 455 460
gat gtt gcc ccg tca aag cac tga 1416
Asp Val Ala Pro Ser Lys His
465 470
<210> 26
<211> 471
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Recombinant fused protein
<400> 26
Met Ser Tyr Ser Lys Gln Gly Ile Ala Gln Glu Met Arg Thr Lys Tyr
1 5 10 15
Arg Met Glu Gly Ser Val Asn Gly His Glu Phe Thr Ile Glu Gly Val
20 25 30
Gly Thr Gly Asn Pro Tyr Glu Gly Lys Gln Thr Ser Glu Leu Val Ile
35 40 45
Ile Lys Pro Lys Gly Lys Pro Leu Pro Phe Ser Phe Asp Ile Leu Ser
50 55 60
Thr Val Phe Gln Tyr Gly Asn Arg Cys Phe Thr Lys Tyr Pro Ala Asp
65 70 75 80
Met Pro Asp Tyr Phe Lys Gln Ala Phe Pro Asp Gly Met Ser Tyr Glu
85 90 95
Arg Ser Phe Leu Phe Glu Asp Gly Gly Val Ala Thr Ala Ser Trp Ser
100 105 110
Ile Arg Leu Glu Gly Asn Cys Phe Ile His Asn Ser Ile Tyr His Gly
115 120 125
Thr Asn Phe Pro Ala Asp Gly Pro Val Met Lys Lys Gln Thr Ile Gly
130 135 140
Trp Asp Lys Ser Ser Glu Lys Met Ser Val Ala Lys Glu Val Leu Arg
145 150 155 160
Gly Asp Val Thr Gln Phe Leu Leu Leu Glu Gly Gly Gly Tyr Gln Arg
165 170 175
Cys Gln Leu His Ser Thr Tyr Lys Thr Glu Lys Pro Val Ala Met Pro
180 185 190
Pro Ser His Val Val Glu His Gln Ile Val Arg Thr Asp Leu Gly Gln
195 200 205
Thr Ala Lys Gly Phe Lys Val Lys Leu Glu Glu His Ala Glu Ala His
210 215 220
Val Asn Pro Leu Lys Val Lys Gly Ser Gly Gly Asp Glu Val Asp Gly
225 230 235 240
Thr Gly Met Ala Ser Met Ile Lys Asp Val Met Arg Phe Lys Met Asn
245 250 255
Met Glu Gly Thr Val Asn Gly His His Phe Lys Cys Glu Ala Asp Gly
260 265 270
Glu Gly Lys Pro Tyr Glu Gly Thr Gln Leu Gly Arg Ile Arg Val Thr
275 280 285
Glu Gly Gly Pro Leu Pro Phe Ser Phe Asp Ile Leu Ser Thr Cys Thr
290 295 300
Gln Tyr Gly Ser Lys Thr Met Ile Lys His Leu Ser Gly Ile Pro Asp
305 310 315 320
Tyr Phe Lys Gln Ser Phe Pro Glu Gly Phe Thr Trp Glu Arg Asn Thr
325 330 335
Ile Tyr Glu Asp Gly Gly His Leu Thr Ala His Gln Asp Thr Ser Leu
340 345 350
Lys Gly Asp Cys Leu Ile Tyr Lys Val Lys Val Leu Gly Gly Asn Phe
355 360 365
Pro Ala Asn Gly Pro Val Met Gln Lys Lys Ser Lys Gly Trp Glu Pro
370 375 380
Cys Thr Glu Met Leu Tyr Pro Arg Asp Gly Val Leu Cys Gly Gln Thr
385 390 395 400
Leu Met Ala Leu Lys Cys Ala Asn Gly Lys Asn Leu Thr Cys Gln Leu
405 410 415
Arg Ser Thr Tyr Arg Ser Arg Lys Gln Ala Ser Ala Leu Gln Thr Pro
420 425 430
Gly Phe His Phe Gly Asp His Arg Ile Glu Ile Leu Lys Glu Ala Glu
435 440 445
Gly Gly Asn Tyr Phe Glu Gln His Glu Thr Ser Val Ala Arg Tyr Cys
450 455 460
Asp Val Ala Pro Ser Lys His
465 470
<210> 27
<211> 1419
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Recombinant fused DNA
<220>
<221> CDS
<222> (1)..(1419)
<223>
<400> 27
atg tct tat tca aag caa ggc atc gca caa gaa atg cgg acg aaa tac 48
Met Ser Tyr Ser Lys Gln Gly Ile Ala Gln Glu Met Arg Thr Lys Tyr
1 5 10 15
cgt atg gaa ggc agt gtc aat ggc cat gag ttc acg atc gaa ggt gta 96
Arg Met Glu Gly Ser Val Asn Gly His Glu Phe Thr Ile Glu Gly Val
20 25 30
gga act gga aac cct tac gaa ggg aaa cag acg tcc gaa tta gtg atc 144
Gly Thr Gly Asn Pro Tyr Glu Gly Lys Gln Thr Ser Glu Leu Val Ile
35 40 45
atc aag cct aag gga aaa ccc ctt cca ttc tcc ttt gac ata ctg tca 192
Ile Lys Pro Lys Gly Lys Pro Leu Pro Phe Ser Phe Asp Ile Leu Ser
50 55 60
aca gtc ttt caa tat gga aac aga tgc ttc aca aag tac cct gca gac 240
Thr Val Phe Gln Tyr Gly Asn Arg Cys Phe Thr Lys Tyr Pro Ala Asp
65 70 75 80
atg cct gac tat ttc aag caa gca ttc cca gat gga atg tca tat gaa 288
Met Pro Asp Tyr Phe Lys Gln Ala Phe Pro Asp Gly Met Ser Tyr Glu
85 90 95
agg tca ttt cta ttt gag gat gga gga gtt gct aca gcc agc tgg agc 336
Arg Ser Phe Leu Phe Glu Asp Gly Gly Val Ala Thr Ala Ser Trp Ser
100 105 110
att cgt ctc gaa gga aat tgc ttc atc cac aat tcc atc tat cat ggc 384
Ile Arg Leu Glu Gly Asn Cys Phe Ile His Asn Ser Ile Tyr His Gly
115 120 125
aca aac ttt ccc gct gat gga ccc gta atg aag aag cag aca att ggc 432
Thr Asn Phe Pro Ala Asp Gly Pro Val Met Lys Lys Gln Thr Ile Gly
130 135 140
tgg gat aag tcc tcc gaa aaa atg agt gtg gct aaa gag gtg cta aga 480
Trp Asp Lys Ser Ser Glu Lys Met Ser Val Ala Lys Glu Val Leu Arg
145 150 155 160
ggt gat gtg act cag ttt ctt ctg ctc gaa gga ggt ggt tac cag aga 528
Gly Asp Val Thr Gln Phe Leu Leu Leu Glu Gly Gly Gly Tyr Gln Arg
165 170 175
tgc cag tta cac tcc act tac aaa aca gag aag cca gtc gca atg ccc 576
Cys Gln Leu His Ser Thr Tyr Lys Thr Glu Lys Pro Val Ala Met Pro
180 185 190
ccg agt cat gtc gta gaa cat caa att gtg agg acc gac ctt ggc caa 624
Pro Ser His Val Val Glu His Gln Ile Val Arg Thr Asp Leu Gly Gln
195 200 205
act gca aaa ggc ttc aag gtc aag ctg gaa gaa cat gct gag gct cat 672
Thr Ala Lys Gly Phe Lys Val Lys Leu Glu Glu His Ala Glu Ala His
210 215 220
gtt aac cct ttg aag gtt aaa ggc agc ggt ggc gac gag gtg gac ggt 720
Val Asn Pro Leu Lys Val Lys Gly Ser Gly Gly Asp Glu Val Asp Gly
225 230 235 240
acc ggt ggc atg gct tca atg att aaa gac gtt atg cgc ttc aag atg 768
Thr Gly Gly Met Ala Ser Met Ile Lys Asp Val Met Arg Phe Lys Met
245 250 255
aac atg gaa ggg acg gtt aac ggc cac cac ttc aag tgt gaa gct gat 816
Asn Met Glu Gly Thr Val Asn Gly His His Phe Lys Cys Glu Ala Asp
260 265 270
gga gag ggc aag cca tat gaa ggg act caa ctc gga aga ata agg gtc 864
Gly Glu Gly Lys Pro Tyr Glu Gly Thr Gln Leu Gly Arg Ile Arg Val
275 280 285
acc gaa ggc ggg cca ttg ccg ttt tct ttc gac atc ttg tcg act tgc 912
Thr Glu Gly Gly Pro Leu Pro Phe Ser Phe Asp Ile Leu Ser Thr Cys
290 295 300
act caa tat gga agc aag acc atg atc aag cat ttg tcc ggg att cca 960
Thr Gln Tyr Gly Ser Lys Thr Met Ile Lys His Leu Ser Gly Ile Pro
305 310 315 320
gac tac ttt aag cag tct ttt cca gaa gga ttc acc tgg gaa agg aac 1008
Asp Tyr Phe Lys Gln Ser Phe Pro Glu Gly Phe Thr Trp Glu Arg Asn
325 330 335
aca atc tat gag gat ggt ggt cac ctc aca gct cac caa gac act agt 1056
Thr Ile Tyr Glu Asp Gly Gly His Leu Thr Ala His Gln Asp Thr Ser
340 345 350
ctt aag gga gat tgc ctg atc tat aaa gtg aaa gtc ctt gga ggt aat 1104
Leu Lys Gly Asp Cys Leu Ile Tyr Lys Val Lys Val Leu Gly Gly Asn
355 360 365
ttt cct gcc aat ggc cct gtg atg cag aag aag agc aaa gga tgg gaa 1152
Phe Pro Ala Asn Gly Pro Val Met Gln Lys Lys Ser Lys Gly Trp Glu
370 375 380
ccc tgt acc gaa atg ctt tat cca cgt gat gga gtg ctt tgt ggc caa 1200
Pro Cys Thr Glu Met Leu Tyr Pro Arg Asp Gly Val Leu Cys Gly Gln
385 390 395 400
aca ttg atg gca ctt aaa tgc gcc aat ggt aaa aat ctg act tgc cag 1248
Thr Leu Met Ala Leu Lys Cys Ala Asn Gly Lys Asn Leu Thr Cys Gln
405 410 415
cta aga tct act tac agg tcc aga aaa caa gcc agt gca ttg cag aca 1296
Leu Arg Ser Thr Tyr Arg Ser Arg Lys Gln Ala Ser Ala Leu Gln Thr
420 425 430
cca ggc ttc cat ttc gga gac cat cgt att gag ata ctc aag gaa gca 1344
Pro Gly Phe His Phe Gly Asp His Arg Ile Glu Ile Leu Lys Glu Ala
435 440 445
gag ggg ggc aat tac ttt gag cag cac gaa aca tca gtc gcc agg tac 1392
Glu Gly Gly Asn Tyr Phe Glu Gln His Glu Thr Ser Val Ala Arg Tyr
450 455 460
tgt gat gtt gcc ccg tca aag cac tga 1419
Cys Asp Val Ala Pro Ser Lys His
465 470
<210> 28
<211> 472
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Recombinant fused protein
<400> 28
Met Ser Tyr Ser Lys Gln Gly Ile Ala Gln Glu Met Arg Thr Lys Tyr
1 5 10 15
Arg Met Glu Gly Ser Val Asn Gly His Glu Phe Thr Ile Glu Gly Val
20 25 30
Gly Thr Gly Asn Pro Tyr Glu Gly Lys Gln Thr Ser Glu Leu Val Ile
35 40 45
Ile Lys Pro Lys Gly Lys Pro Leu Pro Phe Ser Phe Asp Ile Leu Ser
50 55 60
Thr Val Phe Gln Tyr Gly Asn Arg Cys Phe Thr Lys Tyr Pro Ala Asp
65 70 75 80
Met Pro Asp Tyr Phe Lys Gln Ala Phe Pro Asp Gly Met Ser Tyr Glu
85 90 95
Arg Ser Phe Leu Phe Glu Asp Gly Gly Val Ala Thr Ala Ser Trp Ser
100 105 110
Ile Arg Leu Glu Gly Asn Cys Phe Ile His Asn Ser Ile Tyr His Gly
115 120 125
Thr Asn Phe Pro Ala Asp Gly Pro Val Met Lys Lys Gln Thr Ile Gly
130 135 140
Trp Asp Lys Ser Ser Glu Lys Met Ser Val Ala Lys Glu Val Leu Arg
145 150 155 160
Gly Asp Val Thr Gln Phe Leu Leu Leu Glu Gly Gly Gly Tyr Gln Arg
165 170 175
Cys Gln Leu His Ser Thr Tyr Lys Thr Glu Lys Pro Val Ala Met Pro
180 185 190
Pro Ser His Val Val Glu His Gln Ile Val Arg Thr Asp Leu Gly Gln
195 200 205
Thr Ala Lys Gly Phe Lys Val Lys Leu Glu Glu His Ala Glu Ala His
210 215 220
Val Asn Pro Leu Lys Val Lys Gly Ser Gly Gly Asp Glu Val Asp Gly
225 230 235 240
Thr Gly Gly Met Ala Ser Met Ile Lys Asp Val Met Arg Phe Lys Met
245 250 255
Asn Met Glu Gly Thr Val Asn Gly His His Phe Lys Cys Glu Ala Asp
260 265 270
Gly Glu Gly Lys Pro Tyr Glu Gly Thr Gln Leu Gly Arg Ile Arg Val
275 280 285
Thr Glu Gly Gly Pro Leu Pro Phe Ser Phe Asp Ile Leu Ser Thr Cys
290 295 300
Thr Gln Tyr Gly Ser Lys Thr Met Ile Lys His Leu Ser Gly Ile Pro
305 310 315 320
Asp Tyr Phe Lys Gln Ser Phe Pro Glu Gly Phe Thr Trp Glu Arg Asn
325 330 335
Thr Ile Tyr Glu Asp Gly Gly His Leu Thr Ala His Gln Asp Thr Ser
340 345 350
Leu Lys Gly Asp Cys Leu Ile Tyr Lys Val Lys Val Leu Gly Gly Asn
355 360 365
Phe Pro Ala Asn Gly Pro Val Met Gln Lys Lys Ser Lys Gly Trp Glu
370 375 380
Pro Cys Thr Glu Met Leu Tyr Pro Arg Asp Gly Val Leu Cys Gly Gln
385 390 395 400
Thr Leu Met Ala Leu Lys Cys Ala Asn Gly Lys Asn Leu Thr Cys Gln
405 410 415
Leu Arg Ser Thr Tyr Arg Ser Arg Lys Gln Ala Ser Ala Leu Gln Thr
420 425 430
Pro Gly Phe His Phe Gly Asp His Arg Ile Glu Ile Leu Lys Glu Ala
435 440 445
Glu Gly Gly Asn Tyr Phe Glu Gln His Glu Thr Ser Val Ala Arg Tyr
450 455 460
Cys Asp Val Ala Pro Ser Lys His
465 470
<210> 29
<211> 1422
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Recombinant fused DNA
<220>
<221> CDS
<222> (1)..(1422)
<223>
<400> 29
atg tct tat tca aag caa ggc atc gca caa gaa atg cgg acg aaa tac 48
Met Ser Tyr Ser Lys Gln Gly Ile Ala Gln Glu Met Arg Thr Lys Tyr
1 5 10 15
cgt atg gaa ggc agt gtc aat ggc cat gag ttc acg atc gaa ggt gta 96
Arg Met Glu Gly Ser Val Asn Gly His Glu Phe Thr Ile Glu Gly Val
20 25 30
gga act gga aac cct tac gaa ggg aaa cag acg tcc gaa tta gtg atc 144
Gly Thr Gly Asn Pro Tyr Glu Gly Lys Gln Thr Ser Glu Leu Val Ile
35 40 45
atc aag cct aag gga aaa ccc ctt cca ttc tcc ttt gac ata ctg tca 192
Ile Lys Pro Lys Gly Lys Pro Leu Pro Phe Ser Phe Asp Ile Leu Ser
50 55 60
aca gtc ttt caa tat gga aac aga tgc ttc aca aag tac cct gca gac 240
Thr Val Phe Gln Tyr Gly Asn Arg Cys Phe Thr Lys Tyr Pro Ala Asp
65 70 75 80
atg cct gac tat ttc aag caa gca ttc cca gat gga atg tca tat gaa 288
Met Pro Asp Tyr Phe Lys Gln Ala Phe Pro Asp Gly Met Ser Tyr Glu
85 90 95
agg tca ttt cta ttt gag gat gga gga gtt gct aca gcc agc tgg agc 336
Arg Ser Phe Leu Phe Glu Asp Gly Gly Val Ala Thr Ala Ser Trp Ser
100 105 110
att cgt ctc gaa gga aat tgc ttc atc cac aat tcc atc tat cat ggc 384
Ile Arg Leu Glu Gly Asn Cys Phe Ile His Asn Ser Ile Tyr His Gly
115 120 125
aca aac ttt ccc gct gat gga ccc gta atg aag aag cag aca att ggc 432
Thr Asn Phe Pro Ala Asp Gly Pro Val Met Lys Lys Gln Thr Ile Gly
130 135 140
tgg gat aag tcc tcc gaa aaa atg agt gtg gct aaa gag gtg cta aga 480
Trp Asp Lys Ser Ser Glu Lys Met Ser Val Ala Lys Glu Val Leu Arg
145 150 155 160
ggt gat gtg act cag ttt ctt ctg ctc gaa gga ggt ggt tac cag aga 528
Gly Asp Val Thr Gln Phe Leu Leu Leu Glu Gly Gly Gly Tyr Gln Arg
165 170 175
tgc cag tta cac tcc act tac aaa aca gag aag cca gtc gca atg ccc 576
Cys Gln Leu His Ser Thr Tyr Lys Thr Glu Lys Pro Val Ala Met Pro
180 185 190
ccg agt cat gtc gta gaa cat caa att gtg agg acc gac ctt ggc caa 624
Pro Ser His Val Val Glu His Gln Ile Val Arg Thr Asp Leu Gly Gln
195 200 205
act gca aaa ggc ttc aag gtc aag ctg gaa gaa cat gct gag gct cat 672
Thr Ala Lys Gly Phe Lys Val Lys Leu Glu Glu His Ala Glu Ala His
210 215 220
gtt aac cct ttg aag gtt aaa ggc agc ggt ggc gac gag gtg gac ggt 720
Val Asn Pro Leu Lys Val Lys Gly Ser Gly Gly Asp Glu Val Asp Gly
225 230 235 240
acc ggt ggc agc atg gct tca atg att aaa gac gtt atg cgc ttc aag 768
Thr Gly Gly Ser Met Ala Ser Met Ile Lys Asp Val Met Arg Phe Lys
245 250 255
atg aac atg gaa ggg acg gtt aac ggc cac cac ttc aag tgt gaa gct 816
Met Asn Met Glu Gly Thr Val Asn Gly His His Phe Lys Cys Glu Ala
260 265 270
gat gga gag ggc aag cca tat gaa ggg act caa ctc gga aga ata agg 864
Asp Gly Glu Gly Lys Pro Tyr Glu Gly Thr Gln Leu Gly Arg Ile Arg
275 280 285
gtc acc gaa ggc ggg cca ttg ccg ttt tct ttc gac atc ttg tcg act 912
Val Thr Glu Gly Gly Pro Leu Pro Phe Ser Phe Asp Ile Leu Ser Thr
290 295 300
tgc act caa tat gga agc aag acc atg atc aag cat ttg tcc ggg att 960
Cys Thr Gln Tyr Gly Ser Lys Thr Met Ile Lys His Leu Ser Gly Ile
305 310 315 320
cca gac tac ttt aag cag tct ttt cca gaa gga ttc acc tgg gaa agg 1008
Pro Asp Tyr Phe Lys Gln Ser Phe Pro Glu Gly Phe Thr Trp Glu Arg
325 330 335
aac aca atc tat gag gat ggt ggt cac ctc aca gct cac caa gac act 1056
Asn Thr Ile Tyr Glu Asp Gly Gly His Leu Thr Ala His Gln Asp Thr
340 345 350
agt ctt aag gga gat tgc ctg atc tat aaa gtg aaa gtc ctt gga ggt 1104
Ser Leu Lys Gly Asp Cys Leu Ile Tyr Lys Val Lys Val Leu Gly Gly
355 360 365
aat ttt cct gcc aat ggc cct gtg atg cag aag aag agc aaa gga tgg 1152
Asn Phe Pro Ala Asn Gly Pro Val Met Gln Lys Lys Ser Lys Gly Trp
370 375 380
gaa ccc tgt acc gaa atg ctt tat cca cgt gat gga gtg ctt tgt ggc 1200
Glu Pro Cys Thr Glu Met Leu Tyr Pro Arg Asp Gly Val Leu Cys Gly
385 390 395 400
caa aca ttg atg gca ctt aaa tgc gcc aat ggt aaa aat ctg act tgc 1248
Gln Thr Leu Met Ala Leu Lys Cys Ala Asn Gly Lys Asn Leu Thr Cys
405 410 415
cag cta aga tct act tac agg tcc aga aaa caa gcc agt gca ttg cag 1296
Gln Leu Arg Ser Thr Tyr Arg Ser Arg Lys Gln Ala Ser Ala Leu Gln
420 425 430
aca cca ggc ttc cat ttc gga gac cat cgt att gag ata ctc aag gaa 1344
Thr Pro Gly Phe His Phe Gly Asp His Arg Ile Glu Ile Leu Lys Glu
435 440 445
gca gag ggg ggc aat tac ttt gag cag cac gaa aca tca gtc gcc agg 1392
Ala Glu Gly Gly Asn Tyr Phe Glu Gln His Glu Thr Ser Val Ala Arg
450 455 460
tac tgt gat gtt gcc ccg tca aag cac tga 1422
Tyr Cys Asp Val Ala Pro Ser Lys His
465 470
<210> 30
<211> 473
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Recombinant fused protein
<400> 30
Met Ser Tyr Ser Lys Gln Gly Ile Ala Gln Glu Met Arg Thr Lys Tyr
1 5 10 15
Arg Met Glu Gly Ser Val Asn Gly His Glu Phe Thr Ile Glu Gly Val
20 25 30
Gly Thr Gly Asn Pro Tyr Glu Gly Lys Gln Thr Ser Glu Leu Val Ile
35 40 45
Ile Lys Pro Lys Gly Lys Pro Leu Pro Phe Ser Phe Asp Ile Leu Ser
50 55 60
Thr Val Phe Gln Tyr Gly Asn Arg Cys Phe Thr Lys Tyr Pro Ala Asp
65 70 75 80
Met Pro Asp Tyr Phe Lys Gln Ala Phe Pro Asp Gly Met Ser Tyr Glu
85 90 95
Arg Ser Phe Leu Phe Glu Asp Gly Gly Val Ala Thr Ala Ser Trp Ser
100 105 110
Ile Arg Leu Glu Gly Asn Cys Phe Ile His Asn Ser Ile Tyr His Gly
115 120 125
Thr Asn Phe Pro Ala Asp Gly Pro Val Met Lys Lys Gln Thr Ile Gly
130 135 140
Trp Asp Lys Ser Ser Glu Lys Met Ser Val Ala Lys Glu Val Leu Arg
145 150 155 160
Gly Asp Val Thr Gln Phe Leu Leu Leu Glu Gly Gly Gly Tyr Gln Arg
165 170 175
Cys Gln Leu His Ser Thr Tyr Lys Thr Glu Lys Pro Val Ala Met Pro
180 185 190
Pro Ser His Val Val Glu His Gln Ile Val Arg Thr Asp Leu Gly Gln
195 200 205
Thr Ala Lys Gly Phe Lys Val Lys Leu Glu Glu His Ala Glu Ala His
210 215 220
Val Asn Pro Leu Lys Val Lys Gly Ser Gly Gly Asp Glu Val Asp Gly
225 230 235 240
Thr Gly Gly Ser Met Ala Ser Met Ile Lys Asp Val Met Arg Phe Lys
245 250 255
Met Asn Met Glu Gly Thr Val Asn Gly His His Phe Lys Cys Glu Ala
260 265 270
Asp Gly Glu Gly Lys Pro Tyr Glu Gly Thr Gln Leu Gly Arg Ile Arg
275 280 285
Val Thr Glu Gly Gly Pro Leu Pro Phe Ser Phe Asp Ile Leu Ser Thr
290 295 300
Cys Thr Gln Tyr Gly Ser Lys Thr Met Ile Lys His Leu Ser Gly Ile
305 310 315 320
Pro Asp Tyr Phe Lys Gln Ser Phe Pro Glu Gly Phe Thr Trp Glu Arg
325 330 335
Asn Thr Ile Tyr Glu Asp Gly Gly His Leu Thr Ala His Gln Asp Thr
340 345 350
Ser Leu Lys Gly Asp Cys Leu Ile Tyr Lys Val Lys Val Leu Gly Gly
355 360 365
Asn Phe Pro Ala Asn Gly Pro Val Met Gln Lys Lys Ser Lys Gly Trp
370 375 380
Glu Pro Cys Thr Glu Met Leu Tyr Pro Arg Asp Gly Val Leu Cys Gly
385 390 395 400
Gln Thr Leu Met Ala Leu Lys Cys Ala Asn Gly Lys Asn Leu Thr Cys
405 410 415
Gln Leu Arg Ser Thr Tyr Arg Ser Arg Lys Gln Ala Ser Ala Leu Gln
420 425 430
Thr Pro Gly Phe His Phe Gly Asp His Arg Ile Glu Ile Leu Lys Glu
435 440 445
Ala Glu Gly Gly Asn Tyr Phe Glu Gln His Glu Thr Ser Val Ala Arg
450 455 460
Tyr Cys Asp Val Ala Pro Ser Lys His
465 470
<210> 31
<211> 1419
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Recombinant fused DNA
<220>
<221> CDS
<222> (1)..(1419)
<223>
<400> 31
atg tct tat tca aag caa ggc atc gca caa gaa atg cgg acg aaa tac 48
Met Ser Tyr Ser Lys Gln Gly Ile Ala Gln Glu Met Arg Thr Lys Tyr
1 5 10 15
cgt atg gaa ggc agt gtc aat ggc cat gag ttc acg atc gaa ggt gta 96
Arg Met Glu Gly Ser Val Asn Gly His Glu Phe Thr Ile Glu Gly Val
20 25 30
gga act gga aac cct tac gaa ggg aaa cag acg tcc gaa tta gtg atc 144
Gly Thr Gly Asn Pro Tyr Glu Gly Lys Gln Thr Ser Glu Leu Val Ile
35 40 45
atc aag cct aag gga aaa ccc ctt cca ttc tcc ttt gac ata ctg tca 192
Ile Lys Pro Lys Gly Lys Pro Leu Pro Phe Ser Phe Asp Ile Leu Ser
50 55 60
aca gtc ttt caa tat gga aac aga tgc ttc aca aag tac cct gca gac 240
Thr Val Phe Gln Tyr Gly Asn Arg Cys Phe Thr Lys Tyr Pro Ala Asp
65 70 75 80
atg cct gac tat ttc aag caa gca ttc cca gat gga atg tca tat gaa 288
Met Pro Asp Tyr Phe Lys Gln Ala Phe Pro Asp Gly Met Ser Tyr Glu
85 90 95
agg tca ttt cta ttt gag gat gga gga gtt gct aca gcc agc tgg agc 336
Arg Ser Phe Leu Phe Glu Asp Gly Gly Val Ala Thr Ala Ser Trp Ser
100 105 110
att cgt ctc gaa gga aat tgc ttc atc cac aat tcc atc tat cat ggc 384
Ile Arg Leu Glu Gly Asn Cys Phe Ile His Asn Ser Ile Tyr His Gly
115 120 125
aca aac ttt ccc gct gat gga ccc gta atg aag aag cag aca att ggc 432
Thr Asn Phe Pro Ala Asp Gly Pro Val Met Lys Lys Gln Thr Ile Gly
130 135 140
tgg gat aag tcc tcc gaa aaa atg agt gtg gct aaa gag gtg cta aga 480
Trp Asp Lys Ser Ser Glu Lys Met Ser Val Ala Lys Glu Val Leu Arg
145 150 155 160
ggt gat gtg act cag ttt ctt ctg ctc gaa gga ggt ggt tac cag aga 528
Gly Asp Val Thr Gln Phe Leu Leu Leu Glu Gly Gly Gly Tyr Gln Arg
165 170 175
tgc cag tta cac tcc act tac aaa aca gag aag cca gtc gca atg ccc 576
Cys Gln Leu His Ser Thr Tyr Lys Thr Glu Lys Pro Val Ala Met Pro
180 185 190
ccg agt cat gtc gta gaa cat caa att gtg agg acc gac ctt ggc caa 624
Pro Ser His Val Val Glu His Gln Ile Val Arg Thr Asp Leu Gly Gln
195 200 205
act gca aaa ggc ttc aag gtc aag ctg gaa gaa cat gct gag gct cat 672
Thr Ala Lys Gly Phe Lys Val Lys Leu Glu Glu His Ala Glu Ala His
210 215 220
gtt aac cct ttg aag gtt aaa ggt ggc agc ggt ggc gac gag gtg gac 720
Val Asn Pro Leu Lys Val Lys Gly Gly Ser Gly Gly Asp Glu Val Asp
225 230 235 240
ggt acc ggt atg gct tca atg att aaa gac gtt atg cgc ttc aag atg 768
Gly Thr Gly Met Ala Ser Met Ile Lys Asp Val Met Arg Phe Lys Met
245 250 255
aac atg gaa ggg acg gtt aac ggc cac cac ttc aag tgt gaa gct gat 816
Asn Met Glu Gly Thr Val Asn Gly His His Phe Lys Cys Glu Ala Asp
260 265 270
gga gag ggc aag cca tat gaa ggg act caa ctc gga aga ata agg gtc 864
Gly Glu Gly Lys Pro Tyr Glu Gly Thr Gln Leu Gly Arg Ile Arg Val
275 280 285
acc gaa ggc ggg cca ttg ccg ttt tct ttc gac atc ttg tcg act tgc 912
Thr Glu Gly Gly Pro Leu Pro Phe Ser Phe Asp Ile Leu Ser Thr Cys
290 295 300
act caa tat gga agc aag acc atg atc aag cat ttg tcc ggg att cca 960
Thr Gln Tyr Gly Ser Lys Thr Met Ile Lys His Leu Ser Gly Ile Pro
305 310 315 320
gac tac ttt aag cag tct ttt cca gaa gga ttc acc tgg gaa agg aac 1008
Asp Tyr Phe Lys Gln Ser Phe Pro Glu Gly Phe Thr Trp Glu Arg Asn
325 330 335
aca atc tat gag gat ggt ggt cac ctc aca gct cac caa gac act agt 1056
Thr Ile Tyr Glu Asp Gly Gly His Leu Thr Ala His Gln Asp Thr Ser
340 345 350
ctt aag gga gat tgc ctg atc tat aaa gtg aaa gtc ctt gga ggt aat 1104
Leu Lys Gly Asp Cys Leu Ile Tyr Lys Val Lys Val Leu Gly Gly Asn
355 360 365
ttt cct gcc aat ggc cct gtg atg cag aag aag agc aaa gga tgg gaa 1152
Phe Pro Ala Asn Gly Pro Val Met Gln Lys Lys Ser Lys Gly Trp Glu
370 375 380
ccc tgt acc gaa atg ctt tat cca cgt gat gga gtg ctt tgt ggc caa 1200
Pro Cys Thr Glu Met Leu Tyr Pro Arg Asp Gly Val Leu Cys Gly Gln
385 390 395 400
aca ttg atg gca ctt aaa tgc gcc aat ggt aaa aat ctg act tgc cag 1248
Thr Leu Met Ala Leu Lys Cys Ala Asn Gly Lys Asn Leu Thr Cys Gln
405 410 415
cta aga tct act tac agg tcc aga aaa caa gcc agt gca ttg cag aca 1296
Leu Arg Ser Thr Tyr Arg Ser Arg Lys Gln Ala Ser Ala Leu Gln Thr
420 425 430
cca ggc ttc cat ttc gga gac cat cgt att gag ata ctc aag gaa gca 1344
Pro Gly Phe His Phe Gly Asp His Arg Ile Glu Ile Leu Lys Glu Ala
435 440 445
gag ggg ggc aat tac ttt gag cag cac gaa aca tca gtc gcc agg tac 1392
Glu Gly Gly Asn Tyr Phe Glu Gln His Glu Thr Ser Val Ala Arg Tyr
450 455 460
tgt gat gtt gcc ccg tca aag cac tga 1419
Cys Asp Val Ala Pro Ser Lys His
465 470
<210> 32
<211> 472
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Recombinant fused protein
<400> 32
Met Ser Tyr Ser Lys Gln Gly Ile Ala Gln Glu Met Arg Thr Lys Tyr
1 5 10 15
Arg Met Glu Gly Ser Val Asn Gly His Glu Phe Thr Ile Glu Gly Val
20 25 30
Gly Thr Gly Asn Pro Tyr Glu Gly Lys Gln Thr Ser Glu Leu Val Ile
35 40 45
Ile Lys Pro Lys Gly Lys Pro Leu Pro Phe Ser Phe Asp Ile Leu Ser
50 55 60
Thr Val Phe Gln Tyr Gly Asn Arg Cys Phe Thr Lys Tyr Pro Ala Asp
65 70 75 80
Met Pro Asp Tyr Phe Lys Gln Ala Phe Pro Asp Gly Met Ser Tyr Glu
85 90 95
Arg Ser Phe Leu Phe Glu Asp Gly Gly Val Ala Thr Ala Ser Trp Ser
100 105 110
Ile Arg Leu Glu Gly Asn Cys Phe Ile His Asn Ser Ile Tyr His Gly
115 120 125
Thr Asn Phe Pro Ala Asp Gly Pro Val Met Lys Lys Gln Thr Ile Gly
130 135 140
Trp Asp Lys Ser Ser Glu Lys Met Ser Val Ala Lys Glu Val Leu Arg
145 150 155 160
Gly Asp Val Thr Gln Phe Leu Leu Leu Glu Gly Gly Gly Tyr Gln Arg
165 170 175
Cys Gln Leu His Ser Thr Tyr Lys Thr Glu Lys Pro Val Ala Met Pro
180 185 190
Pro Ser His Val Val Glu His Gln Ile Val Arg Thr Asp Leu Gly Gln
195 200 205
Thr Ala Lys Gly Phe Lys Val Lys Leu Glu Glu His Ala Glu Ala His
210 215 220
Val Asn Pro Leu Lys Val Lys Gly Gly Ser Gly Gly Asp Glu Val Asp
225 230 235 240
Gly Thr Gly Met Ala Ser Met Ile Lys Asp Val Met Arg Phe Lys Met
245 250 255
Asn Met Glu Gly Thr Val Asn Gly His His Phe Lys Cys Glu Ala Asp
260 265 270
Gly Glu Gly Lys Pro Tyr Glu Gly Thr Gln Leu Gly Arg Ile Arg Val
275 280 285
Thr Glu Gly Gly Pro Leu Pro Phe Ser Phe Asp Ile Leu Ser Thr Cys
290 295 300
Thr Gln Tyr Gly Ser Lys Thr Met Ile Lys His Leu Ser Gly Ile Pro
305 310 315 320
Asp Tyr Phe Lys Gln Ser Phe Pro Glu Gly Phe Thr Trp Glu Arg Asn
325 330 335
Thr Ile Tyr Glu Asp Gly Gly His Leu Thr Ala His Gln Asp Thr Ser
340 345 350
Leu Lys Gly Asp Cys Leu Ile Tyr Lys Val Lys Val Leu Gly Gly Asn
355 360 365
Phe Pro Ala Asn Gly Pro Val Met Gln Lys Lys Ser Lys Gly Trp Glu
370 375 380
Pro Cys Thr Glu Met Leu Tyr Pro Arg Asp Gly Val Leu Cys Gly Gln
385 390 395 400
Thr Leu Met Ala Leu Lys Cys Ala Asn Gly Lys Asn Leu Thr Cys Gln
405 410 415
Leu Arg Ser Thr Tyr Arg Ser Arg Lys Gln Ala Ser Ala Leu Gln Thr
420 425 430
Pro Gly Phe His Phe Gly Asp His Arg Ile Glu Ile Leu Lys Glu Ala
435 440 445
Glu Gly Gly Asn Tyr Phe Glu Gln His Glu Thr Ser Val Ala Arg Tyr
450 455 460
Cys Asp Val Ala Pro Ser Lys His
465 470
<210> 33
<211> 1422
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Recombinant fused DNA
<220>
<221> CDS
<222> (1)..(1422)
<223>
<400> 33
atg tct tat tca aag caa ggc atc gca caa gaa atg cgg acg aaa tac 48
Met Ser Tyr Ser Lys Gln Gly Ile Ala Gln Glu Met Arg Thr Lys Tyr
1 5 10 15
cgt atg gaa ggc agt gtc aat ggc cat gag ttc acg atc gaa ggt gta 96
Arg Met Glu Gly Ser Val Asn Gly His Glu Phe Thr Ile Glu Gly Val
20 25 30
gga act gga aac cct tac gaa ggg aaa cag acg tcc gaa tta gtg atc 144
Gly Thr Gly Asn Pro Tyr Glu Gly Lys Gln Thr Ser Glu Leu Val Ile
35 40 45
atc aag cct aag gga aaa ccc ctt cca ttc tcc ttt gac ata ctg tca 192
Ile Lys Pro Lys Gly Lys Pro Leu Pro Phe Ser Phe Asp Ile Leu Ser
50 55 60
aca gtc ttt caa tat gga aac aga tgc ttc aca aag tac cct gca gac 240
Thr Val Phe Gln Tyr Gly Asn Arg Cys Phe Thr Lys Tyr Pro Ala Asp
65 70 75 80
atg cct gac tat ttc aag caa gca ttc cca gat gga atg tca tat gaa 288
Met Pro Asp Tyr Phe Lys Gln Ala Phe Pro Asp Gly Met Ser Tyr Glu
85 90 95
agg tca ttt cta ttt gag gat gga gga gtt gct aca gcc agc tgg agc 336
Arg Ser Phe Leu Phe Glu Asp Gly Gly Val Ala Thr Ala Ser Trp Ser
100 105 110
att cgt ctc gaa gga aat tgc ttc atc cac aat tcc atc tat cat ggc 384
Ile Arg Leu Glu Gly Asn Cys Phe Ile His Asn Ser Ile Tyr His Gly
115 120 125
aca aac ttt ccc gct gat gga ccc gta atg aag aag cag aca att ggc 432
Thr Asn Phe Pro Ala Asp Gly Pro Val Met Lys Lys Gln Thr Ile Gly
130 135 140
tgg gat aag tcc tcc gaa aaa atg agt gtg gct aaa gag gtg cta aga 480
Trp Asp Lys Ser Ser Glu Lys Met Ser Val Ala Lys Glu Val Leu Arg
145 150 155 160
ggt gat gtg act cag ttt ctt ctg ctc gaa gga ggt ggt tac cag aga 528
Gly Asp Val Thr Gln Phe Leu Leu Leu Glu Gly Gly Gly Tyr Gln Arg
165 170 175
tgc cag tta cac tcc act tac aaa aca gag aag cca gtc gca atg ccc 576
Cys Gln Leu His Ser Thr Tyr Lys Thr Glu Lys Pro Val Ala Met Pro
180 185 190
ccg agt cat gtc gta gaa cat caa att gtg agg acc gac ctt ggc caa 624
Pro Ser His Val Val Glu His Gln Ile Val Arg Thr Asp Leu Gly Gln
195 200 205
act gca aaa ggc ttc aag gtc aag ctg gaa gaa cat gct gag gct cat 672
Thr Ala Lys Gly Phe Lys Val Lys Leu Glu Glu His Ala Glu Ala His
210 215 220
gtt aac cct ttg aag gtt aaa ggt ggc agc ggt ggc gac gag gtg gac 720
Val Asn Pro Leu Lys Val Lys Gly Gly Ser Gly Gly Asp Glu Val Asp
225 230 235 240
ggt acc ggt ggc atg gct tca atg att aaa gac gtt atg cgc ttc aag 768
Gly Thr Gly Gly Met Ala Ser Met Ile Lys Asp Val Met Arg Phe Lys
245 250 255
atg aac atg gaa ggg acg gtt aac ggc cac cac ttc aag tgt gaa gct 816
Met Asn Met Glu Gly Thr Val Asn Gly His His Phe Lys Cys Glu Ala
260 265 270
gat gga gag ggc aag cca tat gaa ggg act caa ctc gga aga ata agg 864
Asp Gly Glu Gly Lys Pro Tyr Glu Gly Thr Gln Leu Gly Arg Ile Arg
275 280 285
gtc acc gaa ggc ggg cca ttg ccg ttt tct ttc gac atc ttg tcg act 912
Val Thr Glu Gly Gly Pro Leu Pro Phe Ser Phe Asp Ile Leu Ser Thr
290 295 300
tgc act caa tat gga agc aag acc atg atc aag cat ttg tcc ggg att 960
Cys Thr Gln Tyr Gly Ser Lys Thr Met Ile Lys His Leu Ser Gly Ile
305 310 315 320
cca gac tac ttt aag cag tct ttt cca gaa gga ttc acc tgg gaa agg 1008
Pro Asp Tyr Phe Lys Gln Ser Phe Pro Glu Gly Phe Thr Trp Glu Arg
325 330 335
aac aca atc tat gag gat ggt ggt cac ctc aca gct cac caa gac act 1056
Asn Thr Ile Tyr Glu Asp Gly Gly His Leu Thr Ala His Gln Asp Thr
340 345 350
agt ctt aag gga gat tgc ctg atc tat aaa gtg aaa gtc ctt gga ggt 1104
Ser Leu Lys Gly Asp Cys Leu Ile Tyr Lys Val Lys Val Leu Gly Gly
355 360 365
aat ttt cct gcc aat ggc cct gtg atg cag aag aag agc aaa gga tgg 1152
Asn Phe Pro Ala Asn Gly Pro Val Met Gln Lys Lys Ser Lys Gly Trp
370 375 380
gaa ccc tgt acc gaa atg ctt tat cca cgt gat gga gtg ctt tgt ggc 1200
Glu Pro Cys Thr Glu Met Leu Tyr Pro Arg Asp Gly Val Leu Cys Gly
385 390 395 400
caa aca ttg atg gca ctt aaa tgc gcc aat ggt aaa aat ctg act tgc 1248
Gln Thr Leu Met Ala Leu Lys Cys Ala Asn Gly Lys Asn Leu Thr Cys
405 410 415
cag cta aga tct act tac agg tcc aga aaa caa gcc agt gca ttg cag 1296
Gln Leu Arg Ser Thr Tyr Arg Ser Arg Lys Gln Ala Ser Ala Leu Gln
420 425 430
aca cca ggc ttc cat ttc gga gac cat cgt att gag ata ctc aag gaa 1344
Thr Pro Gly Phe His Phe Gly Asp His Arg Ile Glu Ile Leu Lys Glu
435 440 445
gca gag ggg ggc aat tac ttt gag cag cac gaa aca tca gtc gcc agg 1392
Ala Glu Gly Gly Asn Tyr Phe Glu Gln His Glu Thr Ser Val Ala Arg
450 455 460
tac tgt gat gtt gcc ccg tca aag cac tga 1422
Tyr Cys Asp Val Ala Pro Ser Lys His
465 470
<210> 34
<211> 473
<212> PRT
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: Recombinant fused protein
<400> 34
Met Ser Tyr Ser Lys Gln Gly Ile Ala Gln Glu Met Arg Thr Lys Tyr
1 5 10 15
Arg Met Glu Gly Ser Val Asn Gly His Glu Phe Thr Ile Glu Gly Val
20 25 30
Gly Thr Gly Asn Pro Tyr Glu Gly Lys Gln Thr Ser Glu Leu Val Ile
35 40 45
Ile Lys Pro Lys Gly Lys Pro Leu Pro Phe Ser Phe Asp Ile Leu Ser
50 55 60
Thr Val Phe Gln Tyr Gly Asn Arg Cys Phe Thr Lys Tyr Pro Ala Asp
65 70 75 80
Met Pro Asp Tyr Phe Lys Gln Ala Phe Pro Asp Gly Met Ser Tyr Glu
85 90 95
Arg Ser Phe Leu Phe Glu Asp Gly Gly Val Ala Thr Ala Ser Trp Ser
100 105 110
Ile Arg Leu Glu Gly Asn Cys Phe Ile His Asn Ser Ile Tyr His Gly
115 120 125
Thr Asn Phe Pro Ala Asp Gly Pro Val Met Lys Lys Gln Thr Ile Gly
130 135 140
Trp Asp Lys Ser Ser Glu Lys Met Ser Val Ala Lys Glu Val Leu Arg
145 150 155 160
Gly Asp Val Thr Gln Phe Leu Leu Leu Glu Gly Gly Gly Tyr Gln Arg
165 170 175
Cys Gln Leu His Ser Thr Tyr Lys Thr Glu Lys Pro Val Ala Met Pro
180 185 190
Pro Ser His Val Val Glu His Gln Ile Val Arg Thr Asp Leu Gly Gln
195 200 205
Thr Ala Lys Gly Phe Lys Val Lys Leu Glu Glu His Ala Glu Ala His
210 215 220
Val Asn Pro Leu Lys Val Lys Gly Gly Ser Gly Gly Asp Glu Val Asp
225 230 235 240
Gly Thr Gly Gly Met Ala Ser Met Ile Lys Asp Val Met Arg Phe Lys
245 250 255
Met Asn Met Glu Gly Thr Val Asn Gly His His Phe Lys Cys Glu Ala
260 265 270
Asp Gly Glu Gly Lys Pro Tyr Glu Gly Thr Gln Leu Gly Arg Ile Arg
275 280 285
Val Thr Glu Gly Gly Pro Leu Pro Phe Ser Phe Asp Ile Leu Ser Thr
290 295 300
Cys Thr Gln Tyr Gly Ser Lys Thr Met Ile Lys His Leu Ser Gly Ile
305 310 315 320
Pro Asp Tyr Phe Lys Gln Ser Phe Pro Glu Gly Phe Thr Trp Glu Arg
325 330 335
Asn Thr Ile Tyr Glu Asp Gly Gly His Leu Thr Ala His Gln Asp Thr
340 345 350
Ser Leu Lys Gly Asp Cys Leu Ile Tyr Lys Val Lys Val Leu Gly Gly
355 360 365
Asn Phe Pro Ala Asn Gly Pro Val Met Gln Lys Lys Ser Lys Gly Trp
370 375 380
Glu Pro Cys Thr Glu Met Leu Tyr Pro Arg Asp Gly Val Leu Cys Gly
385 390 395 400
Gln Thr Leu Met Ala Leu Lys Cys Ala Asn Gly Lys Asn Leu Thr Cys
405 410 415
Gln Leu Arg Ser Thr Tyr Arg Ser Arg Lys Gln Ala Ser Ala Leu Gln
420 425 430
Thr Pro Gly Phe His Phe Gly Asp His Arg Ile Glu Ile Leu Lys Glu
435 440 445
Ala Glu Gly Gly Asn Tyr Phe Glu Gln His Glu Thr Ser Val Ala Arg
450 455 460
Tyr Cys Asp Val Ala Pro Ser Lys His
465 470
Claims (9)
- 以下の何れかのアミノ酸配列を有する蛍光蛋白質。
(a)配列番号2に記載のアミノ酸配列;又は、
(b)配列番号2に記載のアミノ酸配列において1から数個のアミノ酸の欠失、置換及び/又は付加を有するアミノ酸配列を有し、蛍光を有し、かつ単量体で存在するアミノ酸配列: - 以下の何れかのDNA。
(a)配列番号2に記載のアミノ酸配列をコードするDNA;又は、
(b)配列番号2に記載のアミノ酸配列において1から数個のアミノ酸の欠失、置換及び/又は付加を有するアミノ酸配列を有し、単量体で存在する蛍光蛋白質をコードするDNA: - 以下の何れかの塩基配列を有するDNA。
(a)配列番号1に記載の塩基配列;又は、
(b)配列番号1に記載の塩基配列において1から数個の塩基の欠失、置換及び/又は付加を有する塩基配列を有し、単量体で存在する蛍光蛋白質をコードする塩基配列: - 請求項2又は3に記載のDNAを有する組み換えベクター。
- 請求項2又は3に記載のDNA又は請求項4に記載の組み換えベクターを有する形質転換体。
- 請求項1に記載の蛍光蛋白質と他の蛋白質とを融合させて得られる融合蛋白質。
- 請求項1に記載の蛍光蛋白質の蛍光スペクトルとFRET(蛍光共鳴エネルギー移動)を引き起こすのに十分な重なりをもつ吸収スペクトルを示す色素蛋白質と、請求項1に記載の蛍光蛋白質とをリンカー配列を介して連結することにより得られるFRET測定用融合蛋白質。
- 色素蛋白質が、以下の何れかのアミノ酸配列を有する色素蛋白質である、請求項7に記載のFRET測定用融合蛋白質。
(a)配列番号4に記載のアミノ酸配列;又は、
(b)配列番号4に記載のアミノ酸配列において1から数個のアミノ酸の欠失、置換及び/又は付加を有するアミノ酸配列を有し、配列番号4に記載のアミノ酸配列からなる蛋白質の吸光特性を有するアミノ酸配列: - 請求項1に記載の蛍光蛋白質、請求項2又は3に記載のDNA、請求項4に記載の組み換えベクター、請求項5に記載の形質転換体、請求項6に記載の融合蛋白質、又は請求項7若しくは8に記載のFRET測定用融合蛋白質の何れかを含む、蛍光試薬キット。
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