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1﹒将兔子癌坏死因于多予以编码之具有硷 排列之DNA,即由下述式[A']或( B']SerAlaSerArgAla LeuXAspLysProLeuAl aHisValValAlaAsnPr oGlnValGluGlyGlnLe uGlnTrpLeuSerGlnAr gAlaAsnAlaLeuLeuAl aAsnGlyMetLysLeuTh rAspAsnGlnLeuValVa lPrOAlaAspGlyLeuTy rLeuIleTyrSerGlnVa lLeuPheSerGlyGlnGl yCysArgSerTyrValLe uLeuTurHisThrValSe rArgPheAlaValSerTy rProAsnLysValASnLe ULeuSerAlaIleLysSe rProCysHisArgGluTh rProGluGluAlaGluPr oMetAlaTrpTyrGluPr oIleTyrLeuGlyCryVa lPheGlnLeuGluLysGl yAspArgLeUSerThrGl uValAsnGlnProGluTy rLeuAspLeuAlaGluSe rGlyGlnValTyrPheGl y1IleIleAlaLeu[A'] (式中X为Ser或Gly)SerTh rGluSerMetIleArgAs pValGluLeuAlaGluGl yProLeuProLysLysAl aGlyGlyProGlnGlySe rLysArgCysLeuCysLe uSerLeuPheSerPheLe uLeuValAlaGlyAlaTh rThrLeuPheCysLeuLe uHisPheArgValIieGl yProGlnGluGluGluGl nSerPrOASnASnLeUHi SLeuValAsnProValAl aGinMetValThrLeuAr gSerAlaSerArgAlaLe uXAspLysProLeuAlaH iSValValAlaASnPrOG lnValGluGlyGlnLeuC lnTrpLenSerGlnArgA laAsnAlaLeuLeuAlaA snCryMetLysLeuThrA spAsnGlnLeuValValP roAlaAspGlyLeuTyrL euIieTyrSerGlnValL enPheSerGlyGlnGlyC ysArgSerTyrValLeuL euThrHisThrValSerA rgPheAlaValSerTyrP roAsnLysValAsnLeuL euSerAlaIieLysSerP roCysHisArgGluThrP roCluGluAlaGluProM rtAlaTrpTyrGluProI ieTyeLeuGlyGlyValP heGlnLeuGluLysGlyA spArgLeUSerTheGluV aIAsnGlnProGluTyrL euAspLeuAlaGluSerG lyGinValTyrPheGlyI ieIieAlaLeu[B"](式中 X系表示Ser或Gly)所示对应于胺 基酸排列所具有之硷排列,或于制造含有 DNA之际,以进行如下(1)至(8) 步骤为为其特征之该DNA之制造方法( 1)兔子投与以如propioniba cteriumacnes之网内系赋活 剂,(2)从该兔子之组织取出巨噬细胞 ,(3)将巨噬细胞在环己醯亚胺之存在 下与内毒素,phorbol酯诱导剂一 起培养,(4)从该巨噬细胞以olig o(dT)纤维素附柱层层析分离含兔癌 坏死因子mRNA,(5)从该mRNA 以反转录合成单股cDNA后,转变为 双股cDNA,(6)将该DNA以Po ly(dG)─poly(de)hom opolymer伸长法插入于pBR3 22胞质体,(7)将该重组胞质体导入 如大肠菌(E﹒coli)宿主进行质体 转换而作成C─DNA库,(8)从该库 依杂交法将兔癌坏死因子[A']或[ B`]编码之cDNAb予以同源化。 2﹒如申请专利范围第1项之制法,其系以下 述式[I']或[II'](5')─TC AGCTTCTCGCGCCCTGX' GACAAGCCTCTACCCCAC GTAGTAGCAAACCCGCAA GTGGAGGGCCAGCTCCAG TGGCTGAGCCAGCGTGCG AACGCCCTGCTGGCCAAC GGCATGAAGCTCACGGAC AACCAGCTGGTGGTGCCG GCCGACGGGCTGTACCTC ATCTACTCCCAGGTTCTC TTCACCGGTCAAGGCTGC CGCTCCTACGTGCTCCTC ACTCACACTGTCAGCCGC TTCGCCGTCTCCTACCCG AACAAGGTCAACCTCCTC TCTGCCATCAAGAGCCCC TGCCACCGGGAGACCCCC GAGGAGGCTGAGCCCATG GCCTGGTACGAGCCCATC TACCTGGGcGGCGTCTTC CAGTTGGAGAAGCGTGAC cGGcTcAGCAccGAGGTc AACCAGCCTGAGTACCTG GACCTTGCCGAGTCCGGG CAGGTCTACTTTGGGATC ATTGCCCTG─(3')1I(式 中x'系表示AGT或GGT),(5') ─AGCACTGAGAGTATGAT CCGGGACGTCGAGCTGGC GGAGGGGCCGCTCCCCAA GAAGGCAGGGGGGCCCCA GGGCTCCAAGCGCTGCCT CTGCCTcAGcCTCTTCTC TTTCCTGCTCGTOGCTGG AGcCACCACGCTCTTCTG CCTGCTGCACTTCAMGTG ATCGGCCCTcAGGACGAA GAGcAGTccCCAAACAAC CTCCATCTAGTCAACCCT GTCGCcCAGATGCTCAcC CTCAGATCAGCTTCTCGC GCCCTGx'GACAAGCCTC TACCCCAcGTAGTAGCAA ACCCGCAAOTGGAGGGCC AGCTCcAGTGGCTGAGCC AGCGTGCCAAcGCCCTCC TGGCCAACGGCATGAACC TCACGGAcAAccACCTGG TGCTGCCGCCcGAcGGGC TGTAcCTCATCTACTCCC AGGTTcTCTTCAGCGCTc AAGGCTGccGCTCCTAcG TGcTCCTcAcTCACAcTC TcAGccGCTTcGccGTCT CCTACCCGAAcAAGGTCA ACCTCCTCTCTGCCATCA AGAGCCCCTGCCACCGGG AGACCCCCCAGGAGGCTG AGCCCATGCCCTGGTACG AGCCCATCTACCTGGGCG GCGTCTTCCAGTTGGAGA AGGGTGACCGGCTCAGCA CCGAGGTCAACCAGCCTG AGTACCTGGACCTTGCCC AGTCCGGGCAGGTCTACT TTMGATCATTCCCCTG─( 3')[I@(式中X'为表示AGT或G GT)所示之以制造具有硷排列或含有之 DNA为其特征者。 3﹒免癌坏死因子多,即由下述式[A'] SerAlaSerArgAlaLeu xASPLysProLenAlaHi sValValAlaAsnProGl nValGluGlyGlnLeuGl nTrpLeuSerGlnArgAl aAsnAlaLeuLeuAlaAs nGlyMetLysLenThrAs pAsnGlnLeuValValPr oAlaAspCryLeuTyrLe uIleTyrSerGlnValLe uPheSerGlyGlnGlyCy sArgSerTyrValLeuLe uTurHisThrValSerAr gPhrAlaValSerTyrPr oAsnLysValAsnLeuLe nSerAlaIleLysSerPr oCysHisArgGluThrPr oGluCluAlaGluProMe tAlaTrpTyrGluProIl eTyrLeuGlyGlyValPh eGlnLeuGluLysGlyAs pArgLeuSerThrgluVa lAsnGlnPreGluTyrLe uAspLeuAlaGluSerGl yGlnValTyrPheGlyIi eIleAlaLeu[A'](式中X 系表不Ser或Gly)所示之以制造胺 基酸排列或其胺基酸排列之N─末端具有 结合以Met之胺基酸排列或含有多生 产用形质转换体,由以下(1)至(3) 步骤为其特征之形质转换体的制造方法(1)式[A']所 示之兔癌坏死因子多 编码之硷排列5'─末端具有起始生殖 因子型(codon)'3,─末端有终 止生殖因子型(codon)DNA以t ac促进质,具有促进质硷排列DNA断 片结合于其下侧(2)将该DNA插入于 如胞质体pBR322媒体(3)将该重 组胞质体之媒体导入以如E﹒coli宿 主以制造兔癌坏死因子多生产用之形质 转换体。 4﹒兔癌坏死忱多子,即由下述式[A'S erAlaSerArgAlaLeuX AspLysProLeuAlaHis ValValAlaAsnProGln ValGluGlyGlnLeuGln TrpLeuSerGlnArgAla AsnAlaLeuLeuAlaAsn GlyMetLysLeuThrAsp AsnGlnLeuValValPro AlaAspGlyLeuTyrLeu IleTyrSerGlnValLeu PheSerGlyGlnGlyCys ArgSerTyrValLeuLeu TurHisThrValSerArg PheAlaValSerTyrPro AsnLysValASnLeULeu SerAlaIleLysSerPro CysHisArgGluThrPro GluGluAlaGluProMet AlaTrpTyrGluProIle TyrLeuGlyGlyValPhe GlnLeuGluLysGlyAsp ArgLeUSerThrGluVal AsnGlnProGluTyrLeu AspLeuAlaGluSerGly GlnValTyrPheGlyIle IleAlaLeu[A'](式中X为 Ser或Gly)所示胺基酸排列或其胺 基酸排列之N─末端结合有Met之胺基 酸排列或含有制造多之际,进行以下1 至5步骤为其特征之该多之制造方法(1)由申请专 利范围第1项所获得之式 [A']所不乏兔癌坏死因子多予以编 码之硷排列5─末端具有起始生殖因子型 (codon),3,─末端有终止生殖 因子型(codon)DNA以tac促 进质(promoter)具有促进质硷 排列DNA断片结合于其下侧(2)将该 DNA插入于如胞质体pBR322媒体(3)将该组织胞质体导 入以如E﹒co li宿主,获得形质转换体。(4):培养该形质转换体。 (5)于该培养菌体中采取产生之式[A']所 示之胺基酸排列或于该N─末端结合有M et胺基酸排列或含有多。 5﹒一种DNA,具有或含有与如下胺基酸排 列对应之硷排列之将兔子癌坏死因子多 予以编码之具有硷排列之DNA,即由下 述式[A']或[B']SerAlaSe rArgAlaLeuXAspLysP roLeuAlaHisValValA laAsnProClnValGluG lyGlnLeuGlnTrpLeuS erGlnArgAlaAsnAlaL euLeuAlaAsnGlyMetL ysLeuThrAspAsnGlnL euValValProAlaAspG lyLeuTyrLeuIleTyrS erGlnValLeuPheSerG ryGlnGlyCysArgSerT yrValLeuLeuTurHisT hrValSerArgPheAlaV alSerTyrProAsnLysV alASnLeULeuSerAlaI leLysSerProCysHisA rgGluThrProGluGluA laGluProMetAlaTrpT yrGluProIleTyrLeuG lyGlyValPheGlnLeuG luLysGlyAspArgLeuS erThrGluValAsnGlnP roGluTyrLeuAspLeuA laGluSerGlyGlnValT yrPheGlyIleIleAlaL eu[A'](式中X为Ser或Gly )SerThrGluSerMetIl eArgAspValGluLenAl aGluGlyProLeuProLy sLysAlaGlyGlyProGl nGlySerLysArgCysLe uCysLeuSerLeuPheSe rPheLeuLenValAlaGl yAlaThrThrLeuPheCy sLeULeUHisPheArgVa lIleGlyProGlnGluGl uGluGlnSerProAsnAs nLeuHisLeuValAsnPr oValAlaClnMetValTh rLenArgSerAlaSerAr gAlaLenXAspLysProL euAlaHisValValAlaA snProGlnValGluGlyG lnLeuGlnTrpLeuSerG lnArgAlaAsnAlaLeuL euAlaAsnGlyMetLysL euThrAspAsnGlnLeuV alValProAlaAspGlyL euTyrLeuIleTyrSerG lnValLeuPheSerGlyG lnGlyCysArgSerTyrV alLeuLeuThrHisThrV alSerArgPheAlaValS erTyrProAsnLysValA snLeuLeuSerAlaIleL ysSerProCysHisArgG luThrProGluGluAlaG luProMetAlaTrpTyrG luProIleTyeLeuGlyG lYValPheGlnLeuGluL ysGlyAspArgLeuSerT heGluValAsnGlnProG luTyrLenAspLeuAlaC luSerGlyGlnValTyrP heGlyIleIleAlaLeu[ B"](式中X系表示Ser或Gly) 6﹒具有或含有如下式[I']或[II ']盐其基排列之DNA,(5')─Tc AGCTTCTCGGGCCCTGX' CACAACCCTCTAGCCCAC CTACTAGCAAACCCGCAA GTGGAGGGCCAGCTCCAG TCGCTGACCCAGCCTGCG AACGCCCTGCTGGZCAAO GGCATGAAGCTCACGGAC AACCAOCTGGTGGTGCCG GCCCACGGGCTGTACCTC ATCTACTcCCAGGTTCTC TTCAGCGCTCAAGGC"TG cCGCTCCTACGTGcTccT CACTCACACTGTCAGCCG CTTCGCCGTcTCCTACCC GAACAAGGTcAACCTCCT CTCTGCCATCAAGAGCCC CTGCCACcGGGAGAcccc cCAGGAGFcTGAGCCCAT GGCCTGGTACGAGCccAT CTACCTGGGCGGCGTCTT CCAGTTGGAGAAGGGTGA ccCGCTCAGCACCGACCT cAACCAGCCTGAGTACCT GGACCTTGCCGAGTCCGG GCAGGTcTACTTTGGGAT CATTGCCCTG─(3")[I'] (式中X'系表示AGT或GGT)(5 ')─AGCACTGAGAGTATG ATCCGGGACGTCGAGCTG GCGGAGGGGcCGCTCCCC AAGAAGGCAGGGGGGCCC CAGGGCTCCAAGCGCTGC CTCTGCCTCAGCCTCTTC TCTTTCCTGCTCGTGGCT GGAGCCACCAcGcTCTTC TGCCTGCTGCACTTcAGG GTGATcGGCCCTCACCAG GAAGAGCAGTCCCCAAAc AAcCTCCATCTAGTCAAC CCTGTGGCCCAGATGGTC ACCCTCAGATCAGCTTcT CGGGCCCTGX'GACAAGC CTCTAGCCCACGTAGTAG CAAACCCGCAAGTGGAGG GCCAGCTCCACTGGCTGA GCCAGCGTGCGAACGCCC TCCTGGCCAACGGCATGA AGCTCACGGACAACCAGC TGGTGGTGCCGGCCGACG GGCTGTACCTCATCTACT CCCAGGTTCTCTTCAGCG GTCAAMCTGCCGCTCCTA CCTGCTCCTCACTCACAC TGTCACCCGCTTCGCCGT CTCCTACCCGAACAAGGT CAACCTCCTCTCTGCCAT CAAGAGCCCCTGCCACCG GGAGACCCCCCAGGAGGC TGAGCCCATGGCCTCCTA CGAGCCCATCTACCTGGG CGGCGTCTTCCACTTGGA GAAGGGTGACCGGCTCAG CACCGAGGTCAACCACCC TGAGTACCTGGACCTTGC CCAGTCCCGGCAGGTCTA CTTTCCCATCATTGCCCT G─(3')[II')(式中X'为表示 AGT或GGT)7﹒一种兔子癌坏死因 子多,具有或含有如下述式[A']之 胺基酸排列或其胺基酸排列之N─末端具 有结合以Met之胺基酸之排列SerA laSerArgAlaLeuXAsp LysProLeuAlaHisVal ValAlaksnProGinVal GluGlyGlnLeuGlnTrp LeuSerGlnArgAlaAsn AlaLeuLeuAlaAsnGly MetLysLeuThrAspAsn GlnLeuValValProAla AspCryLeuTyrLeuiLe TyrSerGlnValLeuPhe SerGlyGlnGlyCysArg SerTyrValLeuLeuTur HisThrValSerArgPhe AlaValSerTyrProAsn LysValAsnLeuLeuSer AlaIieLysSerProCys HisArgGluThrProGlu GluAlaGluProMetAla TrpTyrGluProIleTyr LeuGlyGlyValPheGln LeuGluLysGlyAspArg LeuSerThrGluValAsn GlnProGluTyrLenAsp LeuAlaGluSerGlyGln ValTyrPheGlyIleILA laLeu[A'](式中X为Ser或 GLy) |
