CN1154143A - 合成前导肽序列 - Google Patents
合成前导肽序列 Download PDFInfo
- Publication number
- CN1154143A CN1154143A CN95194183A CN95194183A CN1154143A CN 1154143 A CN1154143 A CN 1154143A CN 95194183 A CN95194183 A CN 95194183A CN 95194183 A CN95194183 A CN 95194183A CN 1154143 A CN1154143 A CN 1154143A
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- Prior art keywords
- seq
- sequence
- ser
- leu
- expression cassette
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- 108010076504 Protein Sorting Signals Proteins 0.000 title claims abstract description 71
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 80
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 78
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 76
- 229920001184 polypeptide Polymers 0.000 claims abstract description 36
- 150000001413 amino acids Chemical class 0.000 claims description 124
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Abstract
本发明涉及用于在酵母中分泌多肽的合成前导肽序列。
Description
本发明涉及用于在酵母中分泌多肽的合成前导肽序列。
酵母有机体能产生多种蛋白质,这些蛋白质在胞内合成,但具有胞外功能。这种胞外蛋白质被称为分泌性蛋白质。这种分泌性蛋白最初以前体或前蛋白体形式在胞内表达,所述前体带有一个前序列,以确保有效地引导表达产物通过内质网(ER)膜。所述前序列(一般被称为信号肽)通常在转运过程中从目的产物上断裂下来。该蛋白质一旦进入分泌途径,就被转运到高尔基体,再从高尔基体循不同途径进入诸如细胞液泡或细胞膜之类的细胞隔室中,或是排出细胞外,分泌到外部培养基里(Pfeffer,S.R.和Rothman)J.E.Ann.Rev.Biochem.56(1987),829-852)。
业已提出了几种在酵母中表达并分泌与之异源的蛋白质的方法。欧洲专利申请公开NO.88632中披露了一种表达、加工和分泌与酵母异源的蛋白质的方法,该方法包括:用一种具有编码目的蛋白质和信号肽的DNA的表达载体转化酵母有机体;制备所述转化有机体的培养物;生长该培养物并从培养基中回收所述蛋白质。所述信号肽可以是目的蛋白质本身的信号肽、一种异源信号肽或天然信号肽与异源信号肽的杂合体。
采用与酵母异源的信号肽所遇到的问题可能是,异源信号肽不能确保有效转运和/或在该信号肽之后断裂。
酿酒酵母(Saccharomyces cerevisiae)MFα1(α-因子)是以有165个氨基酸的前原形式合成的,它包括19个氨基酸的信号肽或前肽,接下来是一个有64个氨基酸的“前导序列”或前肽,它包括3个N-连接的糖基化位点,后接(LysArg((Asp/Glu)Ala)2-3α-因子)4(Kurjan,J.和Herskowitz,I.Cell30(1982)933-943)。这种前原MFα1的信号前导序列部分已被广泛用于在酿酒酵母中合成并分泌异源蛋白质。
与酵母同源的信号/前导肽的应用披露于下列专利文件中:美国专利说明书NO.4,546,082,欧洲专利申请公开号:116201、123294、123544、163529和123289,以及丹麦专利申请NO.3614/83。
EP123289披露了利用酿酒酵母α-因子前体分泌外源蛋白的方法,而WO84/01153披露了用酿酒酵母转化酶信号肽分泌外源蛋白质的方法,DK3614/83披露了用酿酒酵母PH05信号肽分泌外源蛋白质的方法。
美国专利说明书NO.4,546,082、EP16201、EP123294、EP123544和EP163529披露了将来自酿酒酵母的α-因子信号-前导序列(MFα1或MFα2)用于在酵母中表达的异源蛋白质的分泌过程的方法。该方法通过把编码酿酒酵母MFα1信号/前导序列的DNA序列融合在编码目的蛋白质的基因的5′末端,证实了该目的蛋白质的分泌和加工。
EP206783披露了一种利用一种α-因子前导序列由酿酒酵母分泌多肽的系统,所述前导序列已被截短,去掉了上述天然前导序列上的4个α-因子单位,留下前导肽本身,通过α-因子加工位点LysArgGluAlaGluAla同一种异源多肽融合。这种结构被证明能导致较小肽(少于50个氨基酸)的有效加工。为了分泌和加工较大的多肽,已经过天然α-因子的前导序列截短,在前导肽和多肽之间留下一或两个α-因子单位。
多种分泌性蛋白质都要经由一个蛋白酶解加工系统进行加工,该系统可以裂解位于两个连续的碱性氨基酸的羧基末端的肽键。这种酶解活性发生在酿酒酵母中,由KEX2基因编码(Julius,D.A.等,Cell37(1984b)1075)。为了分泌活性酿酒酵母交配因子α1(MFα1或α-因子),需要由KEX2蛋白酶对所述产物进行加工,但KEX2不参与活性酿酒酵母交配因子α的分泌过程。
在培养用一种在上述参考文献中构建的载体转化的酵母有机体时,有时会实现希望分泌的多肽的分泌和正确加工。不过,在多数情况下分泌的水平极低或无分泌,或者上述蛋白酶解加工是不正确的或不完全的。因此,本发明的目的是提供能确保多肽更有效地表达和/或加工的前导肽。
令人吃惊的是,业已发现一种能在酵母中以高产率分泌多肽的新型前导肽。
因此,本发明涉及一种包括以下序列的DNA表达盒:
5′-P-SP-LS-PS-*基因*-(T)i-3′
其中,
P是一个启动子序列,
SP是一个编码一种信号肽的DNA序列,
LS是一个编码一种具有通式I的前导肽的DNA序列:
GlnProIle(Asp/Glu)(Asp/Glu)X1(Glu/Asp)X2AsnZ(Thr/Ser)X3(I)
其中,
X1是一个肽键或一个可编码氨基酸;
X2是一个肽键、一个可编码氨基酸或一个有多达4个可编码氨基酸的序列,这些氨基酸可以相同或不同,
Z是除Pro以外的可编码氨基酸;和
X3是一个有4~30个可编码氨基酸的序列,这些氨基酸可以相同或不同;
PS是编码一个加工位点的DNA序列;
*基因*是编码一种多肽的DNA序列;
T是一个终止序列;而
i是0或1。
在本文中,“前导肽”这一表述方式是指一种肽,其功能是把表达的多肽从内质网引导至高尔基体,并进一步引导至分泌小泡,以便分泌到培养基里(即:表达的多肽被输出到细胞壁外或至少穿过细胞膜而进入细胞的壁膜间隙)。与前导肽连用的“合成”一词是指这种前导肽不是天然存在的。
“信号肽”一词是指一种主要呈疏水性的前序列,以在酵母中表达的一种胞外蛋白质的前体的N-末端序列形式出现。该信号肽的功能是让表达的蛋白质分泌到内质网里。信号肽通常就是在这一过程中断裂下来的。该信号肽可以与产生上述蛋白质的酵母有机体异源或同源。
“多肽”指一种异源多肽,即不是由宿主酵母有机体天然产生的多肽;也可以指一种同源多肽,即由宿主酵母天然产生的多肽及其任何前体形式。在一个优选实施方案中,本发明的表达盒编码一种异源多肽。
“可编码氨基酸”是指可以由核苷酸的三联体(“密码子”)编码的氨基酸。
在本说明书的氨基酸序列中,两个氨基酸的三字母密码由一条斜线分开,并在括号中给出,例如(Asp/Glu),它用于表示所述序列中的相应位置上为这两个氨基酸中的一个或另一个。
另一方面,本发明涉及一种在酵母中生产一种多肽的方法,该方法包括在合适的培养基中培养一种酵母细胞,该细胞能够表达一种多肽并用一种包括本发明的一个前导肽序列的上述酵母表达载体转化过,以实现所述多肽的表达与分泌,此后,从培养基中回收所述多肽。
下面结合附图对本发明做进一步说明。其中:
图1是质粒pAK492的示意图;
图2是编码信号肽/前导序列/MI3胰岛素前体的DNA序列的一部分;
图3表示质粒pAK546的结构;
图4表示前导序列SEQ ID No.4的氨基酸序列及其编码DNA序列;
图5表示酿酒酵母表达质粒pAK546编码YAP3信号肽,前导序列SEQ ID No.4和MI3胰岛素前体的DNA序列和所编码的氨基酸序列;
图6表示前导序列SEQ ID No.6的氨基酸序列及其编码DNA序列;
图7表示前导序列SEQ ID No.8的氨基酸序列及其编码DNA序列;
图8表示前导序列SEQ ID No.17的氨基酸序列及其编码DNA序列;
图9表示前导序列SEQ ID No.16的氨基酸序列及其编码DNA序列;
图10表示前导序列SEQ ID No.19的氨基酸序列及其编码DNA序列;
图11表示前导序列SEQ ID No.20的氨基酸序列及其编码DNA序列;
图12表示前导序列SEQ ID No.21的氨基酸序列及其编码DNA序列;
图13表示pAK527的DNA片段,它在SEQ ID No.4和6的构建中被用作直接模板;
图14表示pAK531的DNA片段,它在SEQ ID No.8的构建中被用作直接模板;
图15表示pAK555的DNA片段,它在SEQ ID No.16和17的构建中被用作直接模板;
图16表示pAK559的DNA片段,它在SEQ ID No.19和20的构建中被用作直接模板;
图17表示pAK562的DNA片段,它在SEQ ID No.21的构建中被用作直接模板;
图18表示前导序列SEQ ID No.27的氨基酸序列及其编码DNA序列SEQ ID No.66;
图19表示一种N-末端延伸的MI3胰岛素前体的氨基酸序列SEQ ID No.70,及其编码DNA序列SEQ ID No.71;
图20表示前导序列SEQ ID No.67的氨基酸序列及其编码DNA序列SEQ ID No.69;
图21表示pAK614的DNA片段SEQ ID No.72,它在SEQ IDNo.27的构建中被用作直接模板;以及
图22表示pAK625的DNA片段SEQ ID No.73,它在SEQ IDNo.67的构建中被用作直接模板。
当通式I中的X1表示氨基酸时,它最好是Ser、Thr或Ala。当通式I中的X2表示氨基酸时,它最好是Ser、Thr或Ala。当通式I中的X2表示有两个氨基酸的序列时,它最好是SerIle。当通式I中的X2表示有3个氨基酸的序列时,它最好是SerAlaIle。当通式I中的X2表示有4个氨基酸的序列时,它最好是SerPheAlaThr。在一种优选实施方案中,X3是一种通式II的氨基酸序列:
X4-X5-X6 (II)其中,X4是有1~21个可编码氨基酸的一种序列,这些氨基酸可以相同或不同;X5是Pro或是氨基酸序列ValAsnLeu或LeuAlaAs-nValAlaMetAla中的一个;而X6是有1~8个可编码氨基酸的序列,这些氨基酸可以相同或不同。
在通式II中,X4最好是一种氨基酸序列,它包括基序Leu-ValAsnLeu、SerValAsnLeu、MetAlaAsp、ThrGluSer、ArgPheAlaThr或ValAlaMetAla中的一个或多个;或者X4是包括序列AsnSerThr或AsnThrThr的一种氨基酸序列;或者X4是包括下述序列的一种氨基酸序列:
(Ser/Leu)ValAsnLeu,
(Ser/Leu)ValAsnLeuMetAlaAsp,
(Ser/Leu)ValAsnLeuMetAlaAspAsp,
(Ser/Leu)ValAsnLeuMetAlaAspAspThrGluSer,
(Ser/Leu)ValAsnLeuMetAlaAspAspThrGluSerIle或
(Ser/Leu)ValAsnLeuMetAlaAspAspThrGluSerArgPheAlaThr;
或者X4是包括以下序列的一种氨基酸序列:
Asn(Thr/Ser)ThrLeu,
Asn(Thr/Ser)ThrLeuAsnLeu或
Asn(Thr/Ser)ThrLeuValAsnLeu;
或这些序列的任意组合。
在通式II中,X5最好是Pro或是一种氨基酸序列,该氨基酸序列包括序列ValAsnLeu、LeuAlaAsnValAlaMetAla、LeuAspVal-ValAsnLeuProGly或LeuAspValValAsnLeuIleSerMet。
在通式II中,当X6表示一个氨基酸时,它最好是Ala、Gly、Leu、Thr、Val或Ser。当通式II中的X6表示有2个氨基酸的序列时,它最好是GlyAla或SerAla。当通式II中的X6表示有3个氨基酸的序列时,它最好是AlaValAla。当通式II中的X6表示有8个氨基酸的序列时,它最好是GlyAlaAspSerLysThrValGlu。
由DNA序列LS编码的优选前导肽的例子有:SEQ ID No.1 GlnProIleAspGluAspAsnAspThrSerValAsnLeuProAla;SEQ ID No.2 GlnProIleAspAspGluAsnThrThrSerValAsnLeuProAla;SEQ ID No.3 GlnProIleAspAspGluSerAsnThrThrSerValAsnLeuProAla;SEQ ID No.4 GlnProIleAspAspGluAsnThrThrSerValAsnLeuProVal;SEQ ID No.5 GlnProIleAspAspThrGluAsnThrThrSerValAsnLeuProAla;SEQ ID No.6 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuPro-
Ala;SEQ ID No.7 GlnProIleAspAspGluAsnThrThrSerValAsnLeuMetAla;SEQ ID No.8 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuPro-
GlyAla;SEQ ID No.9 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMet-
Ala;SEQ ID No.10 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnValPro-
Thr;SEQ ID No.11 GlnProIleAspAspThrGluSerAsnThrThrLeuValAsnValPro-
Thr;SEQ ID No.12 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuPro-
Thr;SEQ ID No.13 GlnProIleAspAspThrGluSerAsnThrThrLeuValAsnValPro-
GlyAla;SEQ ID No.14 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMet-
AlaProAlaValAla;SEQ ID No.15 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMet-
AspLeuAlaValGlyLeuProGlyAla;SEQ ID No.16 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMet-
AlaAspAspThrGluSerIleAsnThrThrLeuValAsnLeuProGly-
Ala;SEQ ID No.17 GlnProIleAspAspThrGluSerIleAsnThrThrLeuValAsnLeu-
ProGlyAla;SEQ ID No.18 GlnProIleAspAspThrGluSerAsnThrThrLeuValAsnLeuPro-
GlyAla;SEQ ID No.19 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMet-
AlaAspAspThrGluSerArgPheAlaThrAsnThrThrLeuValAsn-
LeuProLeu;SEQ ID No.20 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMet-
AlaAspAspThrGluSerIleAsnThrThrLeuValAsnLeuAlaAsn-
ValAlaMetAla;SEQ ID No.21 GlnProIleAspAspThrGluSerAlaIleAsnThrThrLeuValAsn-
LeuProGlyAla;SEQ ID No.22 GlnProIleAspAspThrGluSerPheAlaThrAsnThrThrLeuVal-
AsnLeuProGlyAla;SEQ ID No.23 GlnProIleAspAspThrGluSerIleAsnThrThrLeuValAsnLeu-
MetAlaAspAspThrGluSerArgPheAlaThrAsnThrThrLeuVal-
AsnLeuProLeu;SEQ ID No.24 GlnProIleAspAspThrGluSerIleAsnThrThrLeuValAsnLeu-
MetAlaAspAspThrGluSerArgPheAlaThrAsnThrThrLeuAsp-
ValValAsnLeuProGlyAla;SEQ ID No.25 GlnProIleAspAspThrGluSerAlaAlaIleAsnThrThrLeuVal-
AsnLeuProGlyAla;SEQ ID No.26 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMet-
AlaAspAspThrGluSerArgPheAlaThrAsnThrThrLeuValAsn-
LeuAlaAsnValAlaMetAla;SEQ ID No.27 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMet-
AlaAspAspThrGluSerArgPheAlaThrAsnThrThrLeuAspVal-
ValAsnLeuIleSerMetAla;SEQ ID No.28 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMet-
AlaAsnThrThrGluSerArgPheAlaThrAsnThrThrLeuAspVal-
ValAsnLeuIleSerMetAla;和SEQ ID No.67 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMet-
AlaAspAspThrGluSerArgPheAlaThrAsnThrThrLeuAlaLeu-
AspValValAsnLeuIleSerMetAlaLysArg.
由DNA序列LS编码的特别优选的前导肽是:SEQ ID No.15 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMet-
AspLeuAlaValGlyLeuProGlyAla;SEQ ID No.16 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMet-
AlaAspAspThrGluSerIleAsnThrThrLeuValAsnLeuProGly-
Ala;SEQ ID No.17 GlnProIleAspAspThrGluSerIleAsnThrThrLeuValAsnLeu-
ProGlyAla;SEQ ID No.18 GlnProIleAspAspThrGluSerAsnThrThrLeuValAsnLeuPro-
GlyAla;SEQ ID No.19 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMet-
AlaAspAspThrGluSerArgPheAlaThrAsnThrThrLeuValAsn-
LeuProLeu;SEQ ID No.20 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMet-
AlaAspAspThrGluSerIleAsnThrThrLeuValAsnLeuAlaAsn-
ValAlaMetAla;SEQ ID No.21 GlnProIleAspAspThrGluSerAlaIleAsnThrThrLeuValAsn-
LeuProGlyAla;SEQ ID No.22 GlnProIleAspAspThrGluSerPheAlaThrAsnThrThrLeuVal-
AsnLeuProGlyAla;SEQ ID No.23 GlnProIleAspAspThrGluSerIleAsnThrThrLeuValAsnLeu-
MetAlaAspAspThrGluSerArgPheAlaThrAsnThrThrLeuVal-
AsnLeuProLeu;SEQ ID No.24 GlnProIleAspAspThrGluSerIleAsnThrThrLeuValAsnLeu-
MetAlaAspAspThrGluSerArgPheAlaThrAsnThrThrLeuAsp-
ValValAsnLeuProGlyAla;SEQ ID No.25 GlnProIleAspAspThrGluSerAlaAlaIleAsnThrThrLeuVal-
AsnLeuProGlyAla;SEQ ID No.26 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMet-
AlaAspAspThrGluSerArgPheAlaThrAsnThrThrLeuValAsn-
LeuAlaAsnValAlaMetAla;和SEQ ID No.28 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMet-
AlaAsnThrThrGluSerArgPheAlaThrAsnThrThrLeuAspVal-
ValAsnLeuIleSerMetAla.SEQ ID No.67 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMet-
AlaAspAspThrGluSerArgPheAlaThrAsnThrThrLeuAlaLeu-
AspValValAsnLeuIleSerMetAlaLysArg.
所述信号序列(SP)能够编码任何能确保有效引导表达的多肽进入细胞的分泌途径的信号肽。所述信号肽可以是一种天然存在的信号肽或其功能部分,或者是一种合成肽。已发现的合适信号肽为α-因子信号肽、小鼠唾液淀粉酶信号肽、修饰的羧肽酶信号肽、酵母BAR1信号肽或Humicola Lanuginosa脂肪酶信号肽或其衍生物。小鼠唾液淀粉酶信号肽序列由Hagenbuchle,O.等披露(Nature289(1981),643-646)。羧肽酶信号序列由Valls,L.A.等披露(Cell48(1987),887-897)。 BAR1信号肽披露于WO87/02670中。酵母天冬氨酸蛋白酶3信号肽披露于丹麦专利申请No.0828/93中。
由DNA序列PS编码的酵母加工位点可以是Lys和Arg的任意成对组合,如LysArg、ArgLys、ArgArg或LysLys,它使得酿酒酵母的KEX2蛋白酶或其它酵母菌种的相同蛋白酶能够加工所述多肽(Julius,D.A.等,Cell37(1984),1075)。如果KEX2加工不方便,例如,由于在目的产物内部出现2个连续的碱性氨基酸而导致该多肽产物断裂的话,可以选择另一种蛋白酶的加工位点,它包括该多肽产物中所没有的氨基酸组合,例如FXa的加工位点IleGluGlyArg(参见Sambrook,J.,Fritssh,E.F.和Maniatis,T.,Molecular Cloning:ALaboratory Manual,Cold Spring Harbor Laboratory Press,New York,1989)。
由本发明方法生产的蛋白质可以是任何蛋白,该蛋白可有利地在酵母中生产。这种蛋白质的例子有异源蛋白质,如抑蛋白酶肽、组织因子途径抑制剂或其它蛋白酶抑制剂、胰岛素或胰岛素前体、人或小牛生长激素、白介素、胰高血糖素、GLP-1、IGF-I、IGF-II、组织纤溶酶原激活物、转化生长因子α或β、血小板衍生的生长因子、酶或其功能性类似物。在本文中,“功能性类似物”一词是指与某种天然蛋白质具有类似功能的一种蛋白质(是指与天然蛋白质性质有关,而不是生物活性水平)。该蛋白质可在结构上与天然蛋白相似,并可以通过以下方式由天然蛋白衍生而来:在天然蛋白的C-末端或N-末端或同时在两末端增加一个或多个氨基酸;在天然氨基酸序列一个或多个不同位点上取代一个或多个氨基酸;在天然蛋白的一端或两端或该氨基酸序列中的一个或几个位点上缺失一个或多个氨基酸;或在天然氨基酸序列中的一个或多个位点上插入一个或多个氨基酸。对上述几种蛋白质来说,这类修饰作用是公知的。而且,还可用本发明方法生产其它蛋白质的前体或中间产物。这种前体的一个例子是MI3胰岛素前体,它包括氨基酸序列B(1-29)AlaAlaLysA(1-21),其中A(1-21)是人胰岛素的A链,而B(1-29)是人胰岛素的B链,B链上缺少了Thr(B30)。
编码前导序列的优选DNA结构示于图4-12中,或者是其适当的修饰形式。对上述DNA序列进行适当修饰的例子有核苷酸取代,它不会导致另一种氨基酸序列的蛋白质出现,但会影响插入了该DNA结构的酵母有机体的密码子使用;或核苷酸取代,它会导致不同氨基酸序列的出现,因此,可能出现不同的蛋白质结构。可行修饰的其它例子有:将一个或多个密码子插入上述序列中,在该序列的任一端增加一个或多个密码子,以及在该序列的任一端或在该序列中缺失一个或多个密码子。
所述重组表达载体带有这样的表达盒:
5′-P-SP-LS-PS-*基因*-(T)i-3′
其中,P、SP、LS、*基因*、T和i的含义与上文相同,它可以是能在酵母有机体内复制的任何载体。该启动子可以是在酵母中具有转录活性的任何DNA序列,并可由编码与酵母同源或异源的蛋白质的基因衍生而来。该启动子最好由编码一种与酵母同源的蛋白质的基因衍生而来。合适启动子的例子有酿酒酵母MFα1、TPI、ADH或PGK启动子。
上述序列最好还应是与一个合适的终止子,如TPI终止子可操作连接(参见Alber,T.和Kawasaki,G.,J.Mol.Appl.Genet.1(1982)419-434)。
本发明的重组表达载体还包括一个使得该载体能在酵母中复制的DNA序列。这种序列的例子有酵母质粒2μ复制基因REP1-3和复制起点。这种载体还可包括一个选择标记,例如,由Russell,P.R.所披露的粟酒裂殖酵母(Schizosaccharomyces)TPI基因(Gene40(1985)125-130)。
用于连接序列5′-P-SP-LS-PS-*基因*-(T)i-3′的方法,以及将其插入带有酵母复制所需的信息的合适的酵母载体中的方法是本领域技术人员所熟知的(例如Sambrook,J.,Fritsch,E.F.和Maniatis,T.,在所引书中)。应当知道,所述载体既可以通过首先制备带有5′-P-SP-LS-PS-*基因*-(T)i-3′完整序列的DNA结构,接着再将该片段插入合适的表达载体构建而成,又可以通过依次将DNA片断插入带有个体因子的遗传信息(如启动子序列、信号肽、前导序列GlnProIle(Asp/Glu)(Asp/Glu)X1(Glu/Asp)X2AsnZ(Thr/Ser)X3、加工位点、多肽,以及终止序列(如果有的话))的合适载体,接着进行连接。
本发明方法所用的酵母有机体可以是任何合适的酵母有机体,在培养时这种酵母能产生大量的目的多肽。举例来说,合适的酵母有机体可以是酿酒酵母、克鲁弗酵母(Saccharomyces Kluyveri)、粟酒裂殖酵母或葡萄汁酵母(Saccharomyces uvarum)酵母菌的菌株。举例来说,所述酵母细胞的转化可以这样实现:先制成原生质体,接着再用已知方法转化。用于培养所述细胞的培养基可以是适于生长酵母有机体的任何常规培养基。可用常规方法从培养基中回收分泌的多肽,这些多肽大部分以正确加工形式存在于培养基中。所述回收方法包括以下步骤:通过离心或过滤从培养基中分离酵母细胞;用盐,如硫酸铵沉淀上清液或滤液中的蛋白质成分;随后,用各种层析方法,如离子交换层析、亲和层析等方法进行纯化。
在下面的实施例中将对本发明做进一步说明,这些实施例并不构成对所要求保护的发明范围的限定。
实施例
质粒和DNA材料
所有表达质粒都是C-POT型的。这种质粒披露于欧洲专利申请No.171142中,其特征是带有用于质粒选择和稳定的非洲粟酒裂殖酵母丙糖磷酸异构酶基因(POT)。带有POT-基因的质粒可以从一种保藏的大肠杆菌(E.Coli)菌株(ATCC39685)中获取。这种质粒还带有酿酒酵母丙糖磷酸异构酶启动子和终止子(PTPI和TTPI)。除了包括编码信号/前导序列/产物的区在内的由EcoRI-XbaI限制位点所限定的区域之外,这种质粒与pMT242(Egel-Mi-tani,M.等,Gene73(1988),113-120)相同(见图1)。
在实施例中所述的构建前导序列的PCR反应中,质粒pAK527、pAK531、pAK555、pAK559、pAK562、pAK614和pAK625被用作DNA模板。用作直接模板的合成DNA片段如图13-17所示。除了所示DNA区域之外,这些质粒与图1所示的pAK492相同。
合成DNA片段是在一台自动DNA合成仪(Applied Biosystemsmodel380A)合成的,采用亚磷酰胺化合物和市售试剂(Beaucage,S.L.和Caruthers,M.H.,Tetrahedron Letters22(1981)1859-1869)。
其它所有方法和材料在本领域都是公知的(例如,Sambrook,J.,Fritsch,E.F.和Maniatis,T.,Molecular Cloning:A Laboratory Man-ual,Cold Spring Harbor Laboratory Press,New York,1989)。实施例1
合成用于在酿酒酵母(菌株yAK546)中表达MI3胰岛素前体的前导序列SEQ ID No.4。
前导序列SEQ ID No.4的氨基酸序列如下:
GlnProIleAspAspGluAsnThrThrSerValAsnLeuProVal
合成了以下寡核苷酸:
#94 5′-TAAATCTATAACTACAAAAAACACATA-3′ SEQ ID No.29
#333 5′-GACTCTCTTAACTGGCAAGTTGACA-3′ SEQ ID No.30
#312 5′-AAGTACAAAGCTTCAACCAAGTGAGAACCACACAAGTGTT
GGTTAACGAATCTCTT-3′ SEQ ID No.31
#1845 5′-CATACACAATATAAACGACGG-3′ SEQ ID No.32
采用Gene Amp PCR试剂盒(Perkin Elmer,761 Main Avewalk,CT06859,USA),按照生产商提供的使用说明进行以下聚合酶链式反应(PCR)。在反应期间,用100μl矿物油(Sigma Chemical CO,St.Louis MO,USA覆盖在PCR混合物上:
聚合酶链式反应No.1
5μl寡核苷酸#94(50pmol)
5μl寡核苷酸#333(50pmol)
10μl 10×PCR缓中液
16μl dNTP混合液
0.5μl Taq酶
0.5μl pAK527质粒(图13)作为模板(0.2μgDNA)
63μl水
总共进行12个循环,一个循环包括:94℃下1分钟;37℃下2分钟;72℃下3分钟。然后将PCR混合物加注到2%琼脂糖凝胶上,并采用标准技术电泳(Sambrook,J.,Fritsch,E.F.和Maniatis,T.,见所引书目)。把所得到的DNA片段从琼脂糖凝胶上切下来,并用Gene Clean试剂盒(Bio 101 inc.,PO BOX 2284,La Jolla,CA92038,USA)按照生产商的使用说明分离DNA片断。
聚合酶链式反应No.2
5μl寡核苷酸#312(50pmol)
5μl寡核苷酸#94(50pmol)
10μl 10×PCR缓冲液
16μl dNTP混合液
0.5μl Taq酶
10μl来自PCR No.1的纯化DNA片段
53.5μl水
总共进行12个循环,一个循环包括:94℃下1分钟;37℃下2分钟;72℃下3分钟。使用Gene Clean试剂盒(Bio 101 inc.,PO BOX2284,La Jolla,CA92038,USA)按照生产商的使用说明分离并提纯聚合酶链式反应No.2的DNA片段。
将纯化的PCR DNA片段溶于10μl水和限制酶缓冲液中,并按照标准技术(Sambrook,J.,Fritsch,E.F.和Maniatis,T.见所引书目),在15μl的总体积中用限制酶Asp718和HindIII进行酶切。在琼脂糖凝胶上对167bp的Asp718/HindIIIDNA片段进行电泳处理,并用上所述Gene Clean试剂盒进行提纯。酿酒酵母表达质粒pAK492(如图1所示)是上述质粒pMT742的衍生物,其中,编码信号/前导序列/胰岛素前体的片段已被图2所示的EcoRI-XbaI片段所取代。该片段是在一台Applied Biosystem DNA合成仪上按照生产商的使用说明合成的。用限制酶Asp718和XbaI对质粒pAK492进行酶切,并分离10986pb的载体片段。用限制酶HindIII和XbaI对质粒pAK492进行酶切,并分离编码MI3胰岛素前体的一部分的140bp的DNA片段。在标准条件下(Sambrook,J.,Fritsch,E.F.和Maniatis,T.,见所引书目),用T4 DNA连接酶将上述三个DNA片段连接在一起。然后,把连接混合物转化入感受态大肠杆菌菌株(R-,M+),并通过氨苄青霉素抗性鉴定转化体。用标准DNA小量制备技术(Sambrook,J.,Fritsch,E.F.和Maniatis,T.,见下文)从所得到的大肠杆菌菌落中分离质粒,并用合适的限制酶,即EcoRI、XbaI、NcoI和HindIII进行检测。采用引物#94对选择的质粒pAK546进行DNA序列分析(Sequenase,U.S.BiochemicalCorp.),结果表明该质粒带有编码前导序列SEQ ID No.4的DNA序列。编码前导序列SEQ ID,No.4的DNA序列如图4所示。将质粒pAK546转化入欧洲专利申请公开号No.214826中所披露的酿酒酵母菌株MT663中,所得到的菌株被命名为yAK546。该表达质粒的蛋白质编码区的DNA序列如图5所示。实施例2
合成用于在酿酒酵母(菌株yAK531)中表达MI3胰岛素前体的前导序列SEQ ID No.6。
前导序列SEQ ID No.6的氨基酸序列如下:
GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuProAla
合成了以下寡核苷酸:
#331 5′-GAATCTCTTAGCTGGCAAGTTGACAGAAGTAGTGTTAG
TTTCAGAGTCGTCAATT-3′ SEQ ID No.33
按照实施例1方法进行聚合酶链式反应,所不同的是,用寡核苷酸#331代替寡核苷酸#333。
按照实施例1方法在琼脂糖凝胶上对168bp的Asp718/HindIIIDNA片段进行电泳和纯化。按照实施例1的方法把Asp718/HindIIIDNA片段亚克隆到酿酒酵母表达载体中。用实施例1的方法对选择的质粒pAK531进行DNA序列分析,证实其带有编码前导序列SEQ ID No.6的DNA序列。关于编码前导序列SEQ ID No.6的DNA序列,见图6。将质粒pAK531转化入披露于欧洲专利申请86306721.1中的酿酒酵母菌株MT663中,所得到的菌株被命名为yAK531。编码信号肽和胰岛素前体MI3的DNA序列与图5中所示序列相同。实施例3
合成用于在酿酒酵母(菌株yAK547)中表达MI3胰岛素前体的前导序列SEQ ID No.8。
前导序列SEQ ID No.8的氨基酸序列如下:
GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuProGlyAla
合成了以下寡核苷酸:
#345 5′-AACGAATCTCTTAGCACCTGGCAAGTTGACAGAAGT-3′SEQ ID No.34
按照实施例1中所述的方法进行聚合酶链式反应,所不同的是,用寡核苷酸#345取代了寡核苷酸#333,并以质粒pAK531(图14)为模板。
按照实施例1所述方法,在琼脂糖凝胶上对171bp的Asp718/HindIIIDNA片段进行电泳并纯化。按照实施例1所述的方法把Asp718/HindIIIDNA片段亚克隆到酿酒酵母表达质粒中。按照实施例1的方法对选择的质粒pAK547进行DNA序列分析,证实其含有编码前导序列SEQ ID No.8的DNA序列。编码前导序列SEQ IDNo.8的DNA序列如图7所示。将质粒pAK547转化入欧洲专利申请No.86306721.1中所披露的酿酒酵母菌株MT663中,所得到的菌株被命名为yAK547。编码信号肽和胰岛素前体MI3的DNA序列与图5中所示序列相同。实施例4
合成用于在酿酒酵母(菌株yAK561)中表达MI3胰岛素前体的前导序列SEQ ID No.17。
前导序列SEQ ID No.17的氨基酸序列如下:
GlnProIleAspAspThrGluSerIleAsnThrThrLeuValAsnLeuProGlyAla
合成了以下寡核苷酸:
#376 5′-AACGAATCTCTTAGCACCTGGCAAGTTGACCAAAGTAG
TGTTGATAGATTCAGTGTCGTC-3′ SEQ ID No.35
按照实施例1中所述方法进行聚合酶链式反应,所不同的是,用寡核苷酸#376取代了寡核苷酸#333,并以质粒pAK555(图15)为模板。
按照实施例1所述方法在琼脂糖凝胶上对180bp的Asp718/HindIIIDNA片段进行电泳处理并提纯。按照实施例1所述方法把Asp718/HindIIIDNA片段亚克隆到酿酒酵母表达质粒中。按照实施例1,对选择的质粒pAK561进行DNA序列分析,证实其带有编码前导序列SEQ ID No.17的DNA序列。编码前导序列SEQ ID No.17的DNA序列如图8所示。将质粒pAK561转化入在欧洲专利申请No.86306721.1中所披露的酿酒酵母菌株MT663中,所得到的菌株被命名为yAK561。编码信号肽和胰岛素前体MI3的DNA序列与图5中所示序列相同。实施例5
合成用于在酿酒酵母(菌株yAK559)中表达MI3胰岛素前体的前导序列SEQ ID No.16。
前导序列SEQ ID No.16的氨基酸序列如下:GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMetAlaAspAspThr-GluSerIleAsnThrThrLeuValAsnLeuProGlyAla
合成了以下寡核苷酸:
#375 5′-AACGAATCTCTTAGCACCTGGCAAGTTAACCAAAGTAGT
GTTGATAGATTCAGTGTCGTCAGCCATCAAGTTGAC-3′ SEQ ID No.36
用实施例1的方法进行聚合酶链式反应,所不同的是,用寡核苷酸#375取代了寡核苷酸#333,并以质粒pAK555(图15)为模板。
用实施例1的方法在琼脂糖凝胶上对222bp的Asp718/HindIIIDNA片段进行电泳和提纯。用实施例1的方法将Asp718/HindIIIDNA片段亚克隆到酿酒酵母表达质粒中。用实施例1的方法对选择的质粒pAK559进行DNA序列分析,证实其带有编码前导序列SEQ ID No.16的DNA序列。编码前导序列SEQ ID No.16的DNA序列如图9所示。将质粒pAK559转化入在欧洲专利申请No.86306721.1中所披露的酿酒酵母菌株MT663中,所得到的菌株被命名为yAK559。编码信号肽和胰岛素前体MI3的DNA序列与图5中所示序列相同。实施例6
合成用于在酿酒酵母(菌株yAK580)中表达MI3胰岛素前体的前导序列SEQ ID No.19。
前导序列SEQ ID No.19的氨基酸序列如下:GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMetAlaAspAspThr-GluSerArgPheAlaThrAsnThrThrLeuValAsnLeuProLeu
合成了以下的寡核苷酸:
#384 5′-AACGAATCTCTTCAATGGCAAGTTAACCAAAGTAGTGT
TAGTAGCGAATCTAGATTCAGTGTCGTCAGCCAT-3′ SEQ ID No.37
用实施例1的方法进行聚合酶链式反应,所不同的是,用寡核苷酸#384取代了寡核苷酸#333,并以质粒pAK559(图16)为模板。
用实施例1的方法,在琼脂糖凝胶上对228bp的Asp718/HindIIIDNA片段进行电泳和提纯。用实施例1的方法把Asp718/HindIIIDNA片段亚克隆到酿酒酵母表达质粒中。用实施例1的方法对选择的质粒pAK580进行DNA序列分析,证实其带有编码前导序列SEQ ID No.19的DNA序列。编码前导序列SEQ ID No.19的DNA序列如图10所示。将质粒pAK580转化入在欧洲专利申请No.86306721.1中所披露的酿酒酵母菌株MT663中,所得到的菌株被命名为yAK580。编码信号肽和胰岛素前体MI3的DNA序列与图5中所示序列相同。实施例7
合成用于在酿酒酵母(菌株yAK583)中表达MI3胰岛素前体的前导序列SEQ ID No.20。
前导序列SEQ ID No.20的氨基酸序列如下:GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMetAlaAspAspThr-GluSerIleAsnThrThrLeuValAsnLeuAlaAsnValAlaMetAla
合成了以下的寡核苷酸:#390 5′-AACGAATCTCTTAGCCATGGCAACGTTAGCCAAGTTAA
CCAAAGT-3′ SEQ ID No.38
用实施例1的方法进行聚合酶链式反应,所不同的是,用寡核苷酸#390取代了寡核苷酸#333,并以质粒pAK559(图16)为模板。
用实施例1的方法在琼脂糖凝胶上对231bp的Asp718/HindIIIDNA片段进行电泳并提纯。用实施例1的方法把Asp718/HindIIIDNA片段亚克隆到酿酒酵母表达质粒中。用实施例1的方法对选择的质粒pAK583进行DNA序列分析,证实其带有编码前导序列SEQ ID No.20的DNA序列。编码前导序列SEQ ID No.20的DNA序列如图11所示。将质粒pAK583转化入在欧洲专利申请No.86306721.1中所披露的酿酒酵母菌株MT663中,所得到的菌株被命名为yAK583。编码信号肽和胰岛素前体MI3的DNA序列与图5中所示的序列相同。实施例8
合成用于在酿酒酵母(菌株yAK586)中表达MI3胰岛素前体的前导序列SEQ ID No.21。
前导序列SEQ ID No.21的氨基酸序列如下:
GlnProIleAspAspThrGluSerAlaIleAsnThrThrLeuValAsnLeuProGlyAla
合成了以下寡核苷酸:
#401 5′-AACGAATCTCTTAGCACCTGGCAAGTTGACCAAAGTAG
TGTTGATAGCAGATTCAGTGTCG-3′ SEQ ID No.39
用实施例1的方法进行聚合酶链式反应,所不同的是,用寡核苷酸#401取代了寡核苷酸#333,并以质粒pAK562(图17)为模板。
用实施例1的方法在琼脂糖凝胶上对183bp的Asp718/HindIIIDNA片段进行电泳并提纯。用实施例1的方法把Asp718/HindIIIDNA片段亚克隆到酿酒酵母表达质粒中。用实施例1的方法对选择的质粒pAK586进行DNA序列分析,证实其带有编码前导序列SEQ ID No.21的DNA序列,见图12。将质粒pAK586转化入在欧洲专利申请No.86306721.1中所披露的酿酒酵母菌株MT663中,所得到的菌株被命名为yAK586。编码信号肽和胰岛素前体MI3的DNA序列与图5中所示的序列相同。实施例9
用本发明所选择的前导序列表达MI3胰岛素前体。
在YPD培养基(Sherman,F.等,Methods in Yeast Genetics,ColdSpring Harbor Laboratory Press,1981)上生长带有上述质粒的酵母菌株。每一菌株有6个5ml的培养物在30℃下摇动培养72小时,使最终OD600大约为15。离心之后取出上清液进行HPLC分析,通过Snel,L.等所披露的方法(Chromatographia24(1987)329-332)测定分泌的胰岛素前体的浓度。
在表1中,用本发明选择的前导序列所获得的胰岛素前体MI3的表达水平,是以用pMT742的转化体所获得的表达水平的百分比形式给出的,后者是采用酿酒酵母的MFα(1)前导序列。
表1
实施例10
前导序列 | 表达水平% |
MT748α-前导序列 | 100 |
SEQ ID No.15 | 87 |
SEQ ID No.16 | 215 |
SEQ ID No.17 | 157 |
SEQ ID No.19 | 166 |
SEQ ID No.20 | 86 |
SEQ ID No.21 | 145 |
SEQ ID No.22 | 137 |
SEQ ID No.23 | 121 |
合成用于在酿酒酵母(菌株yAK677)中表达伸展的MI3胰岛素前体的前导序列SEQ ID No.27。
前导序列SEQ ID No.27具有以下氨基酸序列:
GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMetAlaAspAspThr-
GluSerArgPheAlaThrAsnThrThrLeuAspValValAsnLeuIleSerMetAla
合成了下列寡核苷酸:#440 5′-GGTTAACGAACTTTGGAGCTTCAGCTTCAGCTTCTTCTCTCTTAGCCAT
GGAGATCAAGTTAACAACATCCAAAGTAGTGTT-3′ SEQ ID No.64和#441 5′-CAAGTACAAAGCTTCAACCAAGTGGGAACCGCACAAGTGTTGGTTAACG
AACTT-3′ SEQ ID No.65
用实施例1的方法进行聚合酶链式反应,所不同的是,用寡核苷酸#440代替寡核苷酸#333,并以质粒pAK614为模板。在另一个聚合酶链式反应中,用寡核苷酸#441取代寡核苷酸#312。
用实施例1的方法分离纯化的PCR DNA片段,并用限制酶Asp718和HindIII消化。用实施例1的方法在琼脂糖凝胶上对268bp的Asp718/HindIIIDNA片段进行电泳并纯化。用实施例1的方法把Asp718/HindIIIDNA片段亚克隆到酿酒酵母表达质粒中。所不同的是,140bp的HindIII/XbaI DNA片段是pAK602衍生而来,它编码AspB28人胰岛素。用实施例1的方法对选择的质粒pAK616进行DNA序列分析,证实其带有编码前导序列SEQ IDNo.27的DNA序列。编码前导序列SEQ ID No.27的DNA序列SEQ ID No.66如图18所示。从pAK616中分离268bp的Asp718/HindIIIDNA片段,并将其同来自pAK601的10986bp的Asp718/XbaI DNA片段和来自pAK464(编码伸展形式的AspB28人胰岛素)的140bp的HindIII/XbaI DNA片段连接,并命名为pAK625。从pAK625中分离180bp的Asp718/NcoI DNA片段,并将其同来自pJB146(编码伸展形式的胰岛素前体)的221bp的NcoI/XbaI DNA片段和来自pAK601的10824bp的Asp718/XbaI DNA片段连接,所得到的质粒被命名为pAK677。将质粒pAK677转化入在欧洲专利申请86306721.1中所披露的酿酒酵母菌株MT663中,所得到的菌株被命名为yAK677 。除了编码前导序列的DNA序列之外,编码信号肽的DNA序列如图5所示。编码伸展的MI3胰岛素前体的DNA序列如图19所示。实施例11
合成用于在酿酒酵母(yAK680)中表达伸展的MI3胰岛素前体的前导序列SEQ ID No.67。
前导序列SEQ ID No.67的氨基酸序列如下:
GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeuMetAlaAspAspThr
GluSerArgPheAlaThrAsnThrThrLeuAlaLeuAspValValAsnLeuIleSerMet
Ala
合成了下列寡核苷酸:
#577 5′-TCTCTTAGCCATGGAGATCAAGTTAACAACATCCAAAG
CCAAAGTAGTGTT-3′ SEQ ID No.68
用实施例1的方法进行PCR,所不同的是,用寡核苷酸#577代替寡核苷酸#333,并以质粒pAK625为模板,而不进行第二个PCR。按照实施例1的方法用限制酶Asp718和NcoI消化PCR片段。
用实施例1的方法在琼脂糖凝胶上对190bp的Asp718/NcoIDNA片段进行电泳和提纯。不同的是,10824bp的Asp718/XbaI载体DNA片段是从pAK601上分离的。用实施例1的方法把上述190bp的Asp718/NcoI DNA片段亚克隆到酿酒酵母表达质粒中,不同的是,221bp的NcoI/XbaI DNA片段(编码伸展形式的MI3胰岛素前体)是从pAK677上分离的,并用它代替HindIII/XbaI DNA片段。用实施例1的方法对选择的质粒进行DNA序列分析,证实其带有编码前导序列SEQ ID No.67的DNA序列,并将其命名为pAK680。编码前导序列SEQ ID No.67的DNA序列SEQ ID No.69如图20所示。将质粒pAK680转化入在欧洲专利申请86306721.1中所披露的酿酒酵母菌株MT663中,所得到的菌株被命名为yAK680。除了编码前导序列的DNA序列之外,编码信号肽的DNA序列如图5所示,而伸展的胰岛素前体MI3DNA序列如图19所示。实施例12
用本发明的前导序列SEQ ID No.27和SEQ ID No.67表达N-末端伸展的MI3胰岛素前体。
在YPD培养基(Sherman,F.等,Methods in Yeast Genetics,ColdSpring Harbor Laboratory Press,1981)中生长带有上述质粒的酵母菌株。对每一菌株,用6个5ml的培养物在30℃下摇动培养72小时,使最终OD600大约为15。离心以后取上清液作HPLC分析,通过由Snel,L.等所披露的方法(Chromatographia24(1987)329-332)测定所分泌的胰岛素前体的浓度。
在表2中,通过使用本发明的前导序列SEQ ID No.27和SEQID No.67所获得的一些N-末端伸展的MI3胰岛素前体的表达水平,是以用pMT742的转化体所获得的表达水平的百分比形式给出的,后者使用的是酿酒酵母的MFα(1)前导序列。
表2
菌株 | 信号肽 | 前导序列 | 伸展 | 相对于MT748 |
MT748 | α | α | ||
yAK675 | YAP3 | SEQ IDNo.27 | EEAEAEAPK | 251% |
yAK677 | YAP3 | SEQ IDNo.27 | EEAEAEAEPK | 224% |
yAK681 | YAP3 | SEQ IDNo.67 | EEAEAEAPK | 248% |
yAK680 | YAP3 | SEQ IDNo.67 | EEAEAEAEPK | 362% |
序列表(1)一般信息:
(I)申请人:
(A)名称:Novo Nordisk A/S
(B)街道:Novo alle
(C)城市:DK-2880 Bagsvaerd
(E)国家:丹麦
(G)电话:+45 44448888
(H)传真:+45 44490555
(I)电传:37173
(II)发明名称:合成前导肽序列
(III)序列数:73
(IV)有关地址:
(A)地址:Novo Nordisk A/S
Corporate Patents
(B)街道:Novo Allé
(C)城市:DK-2880 Bagsvaerd
(E)国家:丹麦
(V)计算机可读形式:
(A)介质类型:软盘
(B)计算机:IBM PC兼容机
(C)操作系统:PC-DOS/MS-DOS
(D)软件:Patent In Release#1.0,Version#1.25
(VI)本申请的资料:
(A)申请号:
(B)申请日:
(C)分类号:
(VII)优选权申请资料:
(A)申请号:DK0705/94和US08/282,852
(B)申请日:16-06-1994和29-07-1994
(VIII)律师/代理人资料:
(A)姓名:Jrgensen,Dan等
(C)资料/案卷号:4085-WO,DJ
(IX)通讯信息:
(A)电话:+45 4444 8888
(B)传真:+45 44493256(2)有关SEQ ID No:1的信息
(i)序列特征:
(A)长度:15个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ii)分子类型:肽
(xi)序列表达:SEQ ID No:1:
Gln Pro Ile Asp Glu Asp Asn Asp Thr Ser Val Asn Leu Pro Ala
1 5 10 15(2)有关SEQ ID No:2的信息
(i)序列特征:
(A)长度:15个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ii)分子类型:肽
(xi)序列表达:SEQ ID No:2
Gln Pro Ile Asp Asp Glu Asn Thr Thr Ser Val Asn Leu Pro Ala
1 5 10 15(2)有关SEQ ID No:3的信息
(i)序列特征:
(A)长度:16个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ii)分子类型:肽
(xi)序列表达:SEQ ID No:3
Gln Pro Ile Asp Asp Glu Ser Asn Thr Thr Ser Val Asn Leu Pro Ala
1 5 10 15(2)有关SEQ ID No:4的信息
(i)序列特征:
(A)长度:15个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ii)分子类型:肽
(xi)序列表达:SEQ ID No:4
Gln Pro Ile Asp Asp Glu Asn Thr Thr Ser Val Asn Leu Pro Val
1 5 10 15(2)有关SEQ ID No:5的信息
(i)序列特征:
(A)长度:16个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ii)分子类型:肽
(xi)序列表达:SEQ ID No:5
Gln Pro Ile Asp Asp Thr Glu Asn Thr Thr Ser Val Asn Leu Pro Ala
1 5 10 15(2)有关SEQ ID No:6的信息
(i)序列特征:
(A)长度:17个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ii)分子类型:肽
(xi)序列表达:SEQ ID No:6
Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Ser Val Asn Leu Pro
1 5 10 15
Ala(2)有关SEQ ID No:7的信息
(i)序列特征:
(A)长度:15个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ii)分子类型:肽
(xi)序列表达:SEQ ID No:7
Gln Pro Ile Asp Asp Glu Asn Thr Thr Ser Val Asn Leu Met Ala
1 5 10 15(2)有关SEQ ID No:8的信息
(i)序列特征:
(A)长度:18个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ii)分子类型:肽
(xi)序列表达:SEQ ID No:8
Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Ser Val Asn Leu Pro
1 5 10 15
Gly Ala(2)有关SEQ ID No:9的信息
(i)序列特征:
(A)长度:17个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ii)分子类型:肽
(xi)序列表达:SEQ ID No:9
Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Ser Val Asn Leu Met
1 5 10 15
Ala(2)有关SEQ ID No:10的信息
(i)序列特征:
(A)长度:17个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ii)分子类型:肽
(xi)序列表达:SEQ ID No:10
Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Ser Val Asn Val Pro
1 5 10 15
Thr(2)有关SEQ ID No:11的信息
(i)序列特征:
(A)长度:17个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ii)分子类型:肽
(xi)序列表达:SEQ ID No:11
Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Leu Val Asn Val Pro
1 5 10 15
Thr(2)有关SEQ ID No:12的信息
(i)序列特征:
(A)长度:17个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ii)分子类型:肽
(xi)序列表达:SEQ ID No:12
Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Ser Val Asn Leu Pro
1 5 10 15
Thr(2)有关SEQ ID No:13的信息
(i)序列特征:
(A)长度:18个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ii)分子类型:肽
(xi)序列表达:SEQ ID No:13
Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Leu Val Asn Val Pro
1 5 10 15
Gly Ala(2)有关SEQ ID No:14的信息
(i)序列特征:
(A)长度:21个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ii)分子类型:肽
(xi)序列表达:SEQ ID No:14
Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Ser Val Asn Leu Met
1 5 10 15
Ala Pro Ala Val Ala
20(2)有关SEQ ID No:15的信息
(i)序列特征:
(A)长度:25个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ii)分子类型:肽
(xi)序列表达:SEQ ID No:15
Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Ser Val Asn Leu Met
1 5 10 15
Asp Leu Ala Val Gly Leu Pro Gly Ala
20 25(2)有关SEQ ID No:16的信息
(i)序列特征:
(A)长度:33个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ii)分子类型:肽
(xi)序列表达:SEQ ID No:16
Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Ser Val Asn Leu Met
1 5 10 15
Ala Asp Asp Thr Glu Ser Ile Asn Thr Thr Leu Val Asn Leu Pro Gly
20 25 30
Ala(2)有关SEQ ID No:17的信息
(i)序列特征:
(A)长度:19个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ii)分子类型:肽
(xi)序列表达:SEQ ID No:17
Gln Pro Ile Asp Asp Thr Glu Ser Ile Asn Thr Thr Leu Val Asn Leu
1 5 10 15
Pro Gly Ala(2)有关SEQ ID No:18的信息
(i)序列特征:
(A)长度:18个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ii)分子类型:肽
(xi)序列表达:SEQ ID No:18
Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Leu Val Asn Leu pro
1 5 10 15
Gly Ala(2)有关SEQ ID No:19的信息
(i)序列特征:
(A)长度:35个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ii)分子类型:肽
(xi)序列表达:SEQ ID No:19
Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Ser Val Asn Leu Met
1 5 10 15
Ala Asp Asp Thr Glu Ser Arg Phe Ala Thr Asn Thr Thr Leu Val Asn
20 25 30Leu Pro Leu
35(2)有关SEQ ID No:20的信息
(i)序列特征:
(A)长度:36个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ii)分子类型:肽
(xi)序列表达:SEQ ID No:20
Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Ser Val Asn Leu Met
1 5 10 15
Ala Asp Asp Thr Glu Ser Ile Asn Thr Thr Leu Val Asn Leu Ala Asn
20 25 30
Val Ala Met Ala
35(2)有关SEQ ID No:21的信息
(i)序列特征:
(A)长度:20个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ii)分子类型:肽
(xi)序列表达:SEQ ID No:21
Gln Pro Ile Asp Asp Thr Glu Ser Ala Ile Asn Thr Thr Leu Val Asn
1 5 10 15
Leu Pro Gly Ala
20(2)有关SEQ ID No:22的信息
(i)序列特征:
(A)长度:21个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ii)分子类型:肽
(xi)序列表达:SEQ ID No:22
Gln Pro Ile Asp Asp Thr Glu Ser Phe Ala Thr Asn Thr Thr Leu Val
1 5 10 15
Asn Leu Pro Gly Ala
20(2)有关SEQ ID No:23的信息
(i)序列特征:
(A)长度:36个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ii)分子类型:肽
(xi)序列表达:SEQ ID No:23
Gln Pro Ile Asp Asp Thr Glu Ser Ile Asn Thr Thr Leu Val Asn Leu
1 5 10 15
Met Ala Asp Asp Thr Glu Ser Arg Phe Ala Thr Asn Thr Thr Leu Val
20 25 30
Asn Leu Pro Leu
35(2)有关SEQ ID No:24的信息
(i)序列特征:
(A)长度:39个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ii)分子类型:肽
(xi)序列表达:SEQ ID No:24
Gln Pro Ile Asp Asp Thr Glu Ser Ile Asn Thr Thr Leu Val Asn Leu
1 5 10 15
Met Ala Asp Asp Thr Glu Ser Arg Phe Ala Thr Asn Thr Thr Leu Asp
20 25 30
Val Val Asn Leu Pro Gly Ala
35(2)有关SEQ ID No:25的信息
(i)序列特征:
(A)长度:21个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ii)分子类型:肽
(xi)序列表达:SEQ ID No:25
Gln Pro Ile Asp Asp Thr Glu Ser Ala Ala Ile Asn Thr Thr Leu Val
1 5 10 15
Asn Leu Pro Gly Ala
20(2)有关SEQ ID No:26的信息
(i)序列特征:
(A)长度:39个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ii)分子类型:肽
(xi)序列表达:SEQ ID No:26
Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Ser Val Asn Leu Met
1 5 10 15
Ala Asp Asp Thr Glu Ser Arg Phe Ala Thr Asn Thr Thr Leu Val Asn
20 25 30
Leu Ala Asn Val Ala Met Ala
35(2)有关SEQ ID No:27的信息
(i)序列特征:
(A)长度:39个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ii)分子类型:肽
(xi)序列表达:SEQ ID No:27
Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Ser Val Asn Leu Met
1 5 10 15
Ala Asp Asp Thr Glu Ser Arg Phe Ala Thr Asn Thr Thr Leu Asp Val
20 25 30
Val Asn Leu Ile Ser Met Ala
35(2)有关SEQ ID No:28的信息
(i)序列特征:
(A)长度:39个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ii)分子类型:肽
(xi)序列表达:SEQ ID No:28
Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Ser Val Asn Leu Met
1 5 10 15
Ala Asn Thr Thr Glu Ser Arg Phe Ala Thr Asn Thr Thr Leu Asp Val
20 25 30
Val Asn Leu Ile Ser Met Ala
35(2)有关SEQ ID No:29的信息
(i)序列特征:
(A)长度:27bp
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:cDNA
(xi)序列表达:SEQ ID No:29TAAATCTATA ACTACAAAAA ACACATA 27(2)有关SEQ ID No:30的信息
(i)序列特征:
(A)长度:25bp
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:cDNA
(xi)序列表达:SEQ ID No:30GACTCTCTTA ACTGGCAAGT TGACA 25(2)有关SEQ ID No:31的信息
(i)序列特征:
(A)长度:56bp
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:cDNA
(xi)序列表达:SEQ ID No:31AAGTACAAAG CTTCAACCAA GTGAGAACCA CACAAGTGTT GGTTAACGAA TCTCTT 56(2)有关SEQ ID No:32的信息
(i)序列特征:
(A)长度:21bp
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:cDNA
(xi)序列表达:SEQ ID No:32CATACACAAT ATAAACGACG G 21(2)有关SEQ ID No:33的信息
(i)序列特征:
(A)长度:55bp
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:cDNA
(xi)序列表达:SEQ ID No:33GAATCTCTTA GCTGGCAAGT TGACAGAAGT AGTGTTAGTT TCAGAGTCGT CAATT 55(2)有关SEQ ID No:34的信息
(i)序列特征:
(A)长度:36bp
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:cDNA
(xi)序列表达:SEQ ID No:34AACGAATCTC TTAGCACCTG GCAAGTTGAC AGAAGT 36(2)有关SEQ ID No:35的信息
(i)序列特征:
(A)长度:60bp
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:cDNA
(xi)序列表达:SEQ ID No:35AACGAATCTC TTAGCACCTG GCAAGTTGAC CAAAGTAGTG TTGATAGATT CAGTGTCGTC 60(2)有关SEQ ID No:36的信息
(i)序列特征:
(A)长度:75bp
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:cDNA
(xi)序列表达:SEQ ID No:36AACGAATCTC TTAGCACCTG GCAAGTTAAC CAAAGTAGTG TTGATAGATT CAGTGTCGTC 60AGCCATCAAG TTGAC 75(2)有关SEQ ID No:37的信息
(i)序列特征:
(A)长度:72bp
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:cDNA
(xi)序列表达:SEQ ID No:37AACGAATCTC TTCAATGGCA AGTTAACCAA AGTAGTGTTA GTAGCGAATC TAGATTCAGT 60GTCGTCAGCC AT 72(2)有关SEQ ID No:38的信息
(i)序列特征:
(A)长度:45bp
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:cDNA
(xi)序列表达:SEQ ID No:38AACGAATCTC TTAGCCATGG CAACGTTAGC CAAGTTAACC AAAGT 45(2)有关SEQ ID No:39的信息
(i)序列特征:
(A)长度:61bp
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:cDNA
(xi)序列表达:SEQ ID No:39AACGAATCTC TTAGCACCTG GCAAGTTGAC CAAAGTAGTG TTGATAGCAG ATTCAGTGTC 60G 61(2)有关SEQ ID No:40的信息
(i)序列特征:
(A)长度:372bp
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:cDNA
(ix)特征:
(A)名称/关键词:CDS
(B)位置:82..351
(xi)序列表达:SEQ ID No:40GAATTCATTC AAGAATAGTT CAAACAAGAA GATTACAAAC TATCAATTTC ATACACAATA 60TAAACGACGG GTACCAAAAT A ATG AAA CTG AAA ACT GTA AGA TCT GCG GTC 111
Met Lys Leu Lys Thr Val Arg Ser Ala Val
1 5 10CTT TCG TCA CTC TTT GCA TCT CAG GTC CTT GGC CAA CCA ATA GAC GAA 159Leu Ser Ser Leu Phe Ala Ser Gln Val Leu Gly Gln Pro Ile Asp Glu
15 20 25GAC AAC GAC ACT TCT TCC ATG GCT AAG AGA TTC GTT AAC CAA CAC TTG 207Asp Asn Asp Thr Ser Ser Met Ala Lys Arg Phe Val Ash Gln His Leu
30 35 40TGC GGT TCC CAC TTG GTT GAA GCT TTG TAC TTG GTT TGC GGT GAA AGA 255Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg
45 50 55GGT TTC TTC TAC ACT CCT AAG GCT GCT AAG GGT ATT GTC GAG CAA TGC 303Gly Phe Phe Tyr Thr Pro Lys Ala Ala Lys Gly Ile Val Glu Gln Cys
60 65 70TGT ACC TCC ATC TGC TCC TTG TAC CAA TTG GAA AAC TAC TGC AAC TAGACGCAGC358Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn75 80 85 90CCGCAGGCTC TAGA 372(2)有关SEQ ID No:41的信息
(i)序列特征:
(A)长度:89个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ii)分子类型:肽
(xi)序列表达:SEQ ID No:41Met Lys Leu Lys Thr Val Arg Ser Ala Val Leu Ser Ser Leu Phe Ala1 5 10 15Ser Gln Val Leu Gly Gln Pro Ile Asp Glu Asp Asn Asp Thr Ser Ser
20 25 30Met Ala Lys Arg Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val
35 40 45Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro
50 55 60Lys Ala Ala Lys Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser65 70 75 80Leu Tyr Gln Leu Glu Asn Tyr Cys Asn
85(2)有关SEQ ID No:42的信息
(i)序列特征:
(A)长度:45bp
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:cDNA
(ix)特征:
(A)名称/关键词:CDS
(B)位置:1..45
(xi)序列表述:SEQ ID No:42CAA CCA ATT GAC GAC GAA AAC ACT ACT TCT GTC AAC TTG CCA GTT 45Gln Pro Ile Asp Asp Glu Asn Thr Thr Ser Val Asn Leu Pro Val1 5 10 15(2)有关SEQ ID No:43的信息
(i)序列特征:
(A)长度:15个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ii)分子类型:肽
(xi)序列表述:SEQ ID No:43Gln Pro Ile Asp Asp Glu Asn Thr Thr Ser Val Asn Leu Pro Val1 5 10 15(2)有关SEQ ID No:44的信息
(i)序列特征:
(A)长度:297bp
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:cDNA
(ix)特征:
(A)名称/关键词:CDS
(B)位置:1..276
(xi)序列表述:SEQ ID No:44ATG AAA CTG AAA ACT GTA AGA TCT GCG GTC CTT TCG TCA CTC TTT GCA 48Met Lys Leu Lys Thr Val Arg Ser Ala Val Leu Ser Ser Leu Phe Ala1 5 10 15TCT CAG GTC CTT GGC CAA CCA ATT GAC GAC GAA AAC ACT ACT TCT GTC 96Ser Gln Val Leu Gly Gln Pro Ile Asp Asp Glu Asn Thr Thr Ser Val
20 25 30AAC TTG CCA GTT AAG AGA TTC GTT AAC CAA CAC TTG TGT GGT TCT CAC 144Asn Leu Pro Val Lys Arg Phe Val Asn Gln His Leu Cys Gly Ser His
35 40 45TTG GTT GAA GCT TTG TAC TTG GTT TGC GGT GAA AGA GGT TTC TTC TAC 192Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr
50 55 60ACT CCT AAG GCT GCT AAG GGT ATT GTC GAA CAA TGC TGT ACC TCC ATC 240Thr Pro Lys Ala Ala Lys Gly Ile Val Glu Gln Cys Cys Thr Ser Ile65 70 75 80TGC TCC TTG TAC CAA TTG GAA AAC TAC TGC AAC TAGACGCAGC CCGCAGGCTC 293Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn
85 90TAGA 297(2)有关SEQ ID No:45的信息
(i)序列特征:
(A)长度:91个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ii)分子类型:肽
(xi)序列表达:SEQ ID No:45Met Lys Leu Lys Thr Val Arg Ser Ala Val Leu Ser Ser Leu Phe Ala1 5 10 15Ser Gln Val Leu Gly Gln Pro Ile Asp Asp Glu Asn Thr Thr Ser Val
20 25 30Asn Leu Pro Val Lys Arg Phe Val Asn Gln His Leu Cys Gly Ser His
35 40 45Leu Val Glu Ala Leu Tyr Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr
50 55 60Thr Pro Lys Ala Ala Lys Gly Ile Val Glu Gln Cys Cys Thr Ser Ile65 70 75 80Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys Asn
85 90(2)有关SEQ ID No:46的信息
(i)序列特征:
(A)长度:51bp
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:cDNA
(ix)特征:
(A)名称/关键词:CDS
(B)位置:1..51
(xi)序列表达:SEQ ID No:46CAA CCA ATT GAC GAC ACT GAA TCT AAC ACT ACT TCT GTC AAC TTG CCA 48Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Ser Val Asn Leu Pro1 5 10 15GCT 51Ala(2)有关SEQ ID No:47的信息
(i)序列特征:
(A)长度:17个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ii)分子类型:肽
(xi)序列表达:SEQ ID No:47Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Ser Val Asn Leu Pro1 5 10 15Ala(2)有关SEQ ID No:48的信息
(i)序列特征:
(A)长度:54bp
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:cDNA
(ix)特征:
(A)名称/关键词:CDS
(B)位置:1..54
(xi)序列表达:SEQ ID No:48CAA CCA ATT GAC GAC ACT GAA TCT AAC ACT ACT TCT GTC AAC TTG CCA 48Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Ser Val Asn Leu Pro1 5 10 15GGT GCT 54Gly Ala(2)有关SEQ ID No:49的信息
(i)序列特征:
(A)长度:57bp
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:cDNA
(ix)特征:
(A)名称/关键词:CDS
(B)位置:1..57
(xi)序列表达:SEQ ID No:49CAA CCA ATT GAC GAC ACT GAA TCT ATC AAC ACT ACT TTG GTC AAC TTG 48Gln Pro Ile Asp Asp Thr Glu Ser Ile Asn Thr Thr Leu Val Asn Leu1 5 10 15CCA GGT GCTPro Gly Ala 57(2)有关SEQ ID No:50的信息
(i)序列特征:
(A)长度:99bp
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:cDNA
(ix)特征:
(A)名称/关键词:CDS
(B)位置1..f99
(xi)序列表达:SEQ ID No:50CAA CCA ATT GAC GAC ACT GAA TCT AAC ACT ACT TCT GTC AAC TTG ATG 48Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Ser Val Asn Leu Met1 5 10 15GCT GAC GAC ACT GAA TCT ATC AAC ACT ACT TTG GTT AAC TTG CCA GGT 96Ala Asp Asp Thr Glu Ser Ile Asn Thr Thr Leu Val Asn Leu Pro Gly
20 25 30GCT 99Ala(2)有关SEQ ID No:51的信息
(i)序列特征:
(A)长度:105bp
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:cDNA
(ix)特征:
(A)名称/关键词:CDS
(B)位置:1..105
(xi)序列表达:SEQ ID No:51CAA CCA ATT GAC GAC ACT GAA TCT AAC ACT ACT TCT GTC AAC TTG ATG 48Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Ser Val Asn Leu Met1 5 10 15GCT GAC GAC ACT GAA TCT AGA TTC GCT ACT AAC ACT ACT TTG GTT AAC 96Ala Asp Asp Thr Glu Ser Arg Phe Ala Thr Asn Thr Thr Leu Val Asn
20 25 30TTG CCA TTG 105Leu Pro Leu
35(2)有关SEQ ID No:52的信息
(i)序列特征:
(A)长度:108bp
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:cDNA
(ix)特征:
(A)名称/关键词:CDS
(B)位置:1..108
(xi)序列表达:SEQ ID No:52CAA CCA ATT GAC GAC ACT GAA TCT AAC ACT ACT TCT GTC AAC TTG ATG 48Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Ser Val Asn Leu Met1 5 10 15GCT GAC GAC ACT GAA TCT ATC AAC ACT ACT TTG GTT AAC TTG GCT AAC 96Ala Asp Asp Thr Glu Ser Ile Asn Thr Thr Leu Val Asn Leu Ala Asn
20 25 30GTT GCC ATG GCT 108Val Ala Met Ala
35(2)有关SEQ ID No:53的信息
(i)序列特征:
(A)长度:60bp
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:cDNA
(ix)特征:
(A)名称/关键词:CDS
(B)位置:1..60
(xi)序列表达:SEQ ID No:53CAA CCA ATT GAC GAC ACT GAA TCT GCT ATC AAC ACT ACT TTG GTC AAC 48Gln Pro Ile Asp Asp Thr Glu Ser Ala Ile Asn Thr Thr Leu Val Asn1 5 10 15TTG CCA GGT GCT 60Leu Pro Gly Ala
20(2)有关SEQ ID No:54的信息
(i)序列特征:
(A)长度:276bp
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:cDNA
(ix)特征:
(A)名称/关键词:CDS
(B)位置:113..274
(xi)序列表达:SEQ ID No:54TTAAATCTAT AACTACAAAA AACACATACA GGAATTCATT CAAGAATAGT TCAAACAAGA 60AGATTACAAA CTATCAATTT CATACACAAT ATAAACGACG GGTACCAAAA TA ATG 115
Met
1AAA CTG AAA ACT GTA AGA TCT GCG GTC CTT TCG TCA CTC TTT GCA TCT 163Lys Leu Lys Thr Val Arg Ser Ala Val Leu Ser Ser Leu Phe Ala Ser
5 10 15CAG GTC CTT GGC CAA CCA ATT GAC GAC GAA AAC ACT ACT TCT GTT AAC 211Gln Val Leu Gly Gln Pro Ile Asp Asp Glu Asn Thr Thr Ser Val Asn
20 25 30TTG CCA GCT AAG AGA TTC GTT AAC CAA CAC TTG TGC GGT TCC CAC TTG 259Leu Pro Ala Lys Arg Phe Val Asn Gln His Leu Cys Gly Ser His Leu
35 40 45GTT GAA GCT TTG TAC TT 276Val Glu Ala Leu Tyr50(2)有关SEQ ID No:55的信息
(i)序列特征:
(A)长度:54个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ii)分子类型:肽
(xi)序列表达:SEQ ID No:55Met Lys Leu Lys Thr Val Arg Ser Ala Val Leu Ser Ser Leu Phe Ala1 5 10 15Ser Gln Val Leu Gly Gln Pro Ile Asp Asp Glu Asn Thr Thr Ser Val
20 25 30Asn Leu Pro Ala Lys Arg Phe Val Asn Gln His Leu Cys Gly Ser His
35 40 45Leu Val Glu Ala Leu Tyr
50(2)有关SEQ ID No:56的信息
(i)序列特征:
(A)长度:282bp
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:cDNA
(ix)特征:
(A)名称/关键词:CDS
(B)位置:113..280
(xi)序列表达:SEQ ID No:56TTAAATCTAT AACTACAAAA AACACATACA GGAATTCATT CAAGAATAGT TCAAACAAGA 60AGATTACAAA CTATCAATTT CATACACAAT ATAAACGACG GGTACCAAAA TA ATG 115
Met
1AAA CTG AAA ACT GTA AGA TCT GCG GTC CTT TCG TCA CTC TTT GCA TCT 163Lys Leu Lys Thr Val Arg Ser Ala Val Leu Ser Ser Leu Phe Ala Ser
5 10 15CAG GTC CTT GGC CAA CCA ATT GAC GAC ACT GAA TCT AAC ACT ACT TCT 211Gln Val Leu Gly Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Ser
20 25 30GTC AAC TTG CCA GCT AAG AGA TTC GTT AAC CAA CAC TTG TGC GGT TCC 259Val Asn Leu Pro Ala Lys Arg Phe Val Asn Gln His Leu Cys Gly Ser
35 40 45CAC TTG GTT GAA GCT TTG TAC TT 282His Leu Val Glu Ala Leu Tyr50 55(2)有关SEQ ID No:57的信息
(i)序列特征:
(A)长度:56个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ii)分子类型:肽
(xi)序列表达:SEQ ID No:57Met Lys Leu Lys Thr Val Arg Ser Ala Val Leu Ser Ser Leu Phe Ala1 5 10 15Ser Gln Val Leu Gly Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr
20 25 30Ser Val Asn Leu Pro Ala Lys Arg Phe Val Asn Gln His Leu Cys Gly
35 40 45Ser His Leu Val Glu Ala Leu Tyr
50 55(2)有关SEQ ID No:58的信息
(i)序列特征:
(A)长度:282bp
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:cDNA
(ix)特征:
(A)名称/关键词:CDS
(B)位置:113..280
(xi)序列表达:SEQ ID No:58TTAAATCTAT AACTACAAAA AACACATACA GGAATTCATT CAAGAATAGT TCAAACAAGA 60AGATTACAAA CTATCAATTT CATACACAAT ATAAACGACG GGTACCAAAA TA ATG 115
Met
1AAA CTG AAA ACT GTA AGA TCT GCG GTC CTT TCG TCA CTC TTT GCA TCT 163Lys Leu Lys Thr Val Arg Ser Ala Val Leu Ser Ser Leu Phe Ala Ser
5 10 15CAG GTC CTT GGC CAA CCA ATT GAC GAC ACT GAA TCT AAC ACT ACT TCT 211Gln Val Leu Gly Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Ser
20 25 30GTC AAC TTG ATG GCT AAG AGA TTC GTT AAC CAA CAC TTG TGC GGT TCC 259Val Asn Leu Met Ala Lys Arg Phe Val Asn Gln His Leu Cys Gly Ser
35 40 45CAC TTG GTT GAA GCT TTG TAC TT 282His Leu Val Glu Ala Leu Tyr50 55(2)有关SEQ ID No:59的信息
(i)序列特征:
(A)长度:56个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ii)分子类型:肽
(xi)序列表达:SEQ ID No:59Met Lys Leu Lys Thr Val Arg Ser Ala Val Leu Ser Ser Leu Phe Ala1 5 10 15Ser Gln Val Leu Gly Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr
20 25 30Ser Val Asn Leu Met Ala Lys Arg Phe Val Asn Gln His Leu Cys Gly
35 40 45Ser His Leu Val Glu Ala Leu Tyr
50 55(2)有关SEQ ID No:60的信息
(i)序列特征:
(A)长度:330bp
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:cDNA
(ix)特征:
(A)名称/关键词:CDS
(B)位置:113..328
(xi)序列表达:SEQ ID No:60TTAAATCTAT AACTACAAAA AACACATACA GGAATTCATT CAAGAATAGT TCAAACAAGA 60AGATTACAAA CTATCAATTT CATACACAAT ATAAACGACG GGTACCAAAA TA ATG 115
Met
1AAA CTG AAA ACT GTA AGA TCT GCG GTC CTT TCG TCA CTC TTT GCA TCT 163Lys Leu Lys Thr Val Arg Ser Ala Val Leu Ser Ser Leu Phe Ala Ser
5 10 15CAG GTC CTT GGC CAA CCA ATT GAC GAC ACT GAA TCT AAC ACT ACT TCT 211Gln Val Leu Gly Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Ser
20 25 30GTC AAC TTG ATG GCT GAC GAC ACT GAA TCT ATC AAC ACT ACT TTG GTT 259Val Asn Leu Met Ala Asp Asp Thr Glu Ser Ile Asn Thr Thr Leu Val
35 40 45AAC TTG CCA GGT GCT AAG AGA TTC GTT AAC CAA CAC TTG TGC GGT TCC 307Asn Leu Pro Gly Ala Lys Arg Phe Val Asn Gln His Leu Cys Gly Ser50 55 60 65CAC TTG GTT GAA GCT TTG TAC TT 330His Leu Val Glu Ala Leu Tyr
70(2)有关SEQ ID No:61的信息
(i)序列特征:
(A)长度:72个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ii)分子类型:肽
(xi)序列表达:SEQ ID No:61Met Lys Leu Lys Thr Val Arg Ser Ala Val Leu Ser Ser Leu Phe Ala1 5 10 15Ser Gln Val Leu Gly Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr
20 25 30Ser Val Asn Leu Met Ala Asp Asp Thr Glu Ser Ile Asn Thr Thr Leu
35 40 45Val Asn Leu Pro Gly Ala Lys Arg Phe Val Asn Gln His Leu Cys Gly
50 55 60Ser His Leu Val Glu Ala Leu Tyr65 70(2)有关SEQ ID No:62的信息
(i)序列特征:
(A)长度:288bp
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:cDNA
(ix)特征:
(A)名称/关键词:CDS
(B)位置:113..286
(xi)序列表达:SEQ ID No:62TTAAATCTAT AACTACAAAA AACACATACA GGAATTCATT CAAGAATAGT TCAAACAAGA 60AGATTACAAA CTATCAATTT CATACACAAT ATAAACGACG GGTACCAAAA TA ATG 115
Met
1AAA CTG AAA ACT GTA AGA TCT GCG GTC CTT TCG TCA CTC TTT GCA TCT 163Lys Leu Lys Thr Val Arg Ser Ala Val Leu Ser Ser Leu Phe Ala Ser
5 10 15CAG GTC CTT GGC CAA CCA ATT GAC GAC ACT GAA TCT ATC AAC ACT ACT 211Gln Val Leu Gly Gln Pro Ile Asp Asp Thr Glu Ser Ile Asn Thr Thr
20 25 30TTG GTC AAC TTG CCA GGT GCT AAG AGA TTC GTT AAC CAA CAC TTG TGC 259Leu Val Asn Leu Pro Gly Ala Lys Arg Phe Val Asn Gln His Leu Cys
35 40 45GGT TCC CAC TTG GTT GAA GCT TTG TAC TT 288Gly Ser His Leu Val Glu Ala Leu Tyr50 55(2)有关SEQ ID No:63的信息
(i)序列特征:
(A)长度:58个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ii)分子类型:肽
(xi)序列表达:SEQ ID No:63Met Lys Leu Lys Thr Val Arg Ser Ala Val Leu Ser Ser Leu Phe Ala1 5 10 15Ser Gln Val Leu Gly Gln Pro Ile Asp Asp Thr Glu Ser Ile Asn Thr
20 25 30Thr Leu Val Asn Leu Pro Gly Ala Lys Arg Phe Vel Asn Gln His Leu
35 40 45Cys Gly Ser His Leu Val Glu Ala Leu Tyr
50 55(2)有关SEQ ID No:64的信息
(i)序列特征:
(A)长度:82bp
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:cDNA
(xi)序列表达:SEQ ID No:64GGTTAACGAA CTTTGGAGCT TCAGCTTCAG CTTCTTCTCT CTTAGCCATG GAGATCAAGT 60TAACAACATC CAAAGTAGTG TT 82(2)有关SEQ ID No:65的信息
(i)序列特征:
(A)长度:54bp
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:cDNA
(xi)序列表达:SEQ ID No:65CAAGTACAAA GCTTCAACCA AGTGGGAACC GCACAAGTGT TGGTTAACGA ACTT 54(2)有关SEQ ID No:66的信息
(i)序列特征:
(A)长度:117bp
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:cDNA
(xi)序列表达:SEQ ID No:66CAACCAATTG ACGACACTGA ATCTAACACT ACTTCTGTCA ACTTGATGGC TGACGACACT 60GAATCTAGAT TCGCTACTAA CACTACTTTG GATGTTGTTA ACTTGATCTC CATGGCT 117(2)有关SEQ ID No:67的信息
(i)序列特征:
(A)长度:41个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ii)分子类型:肽
(xi)序列表达:SEQ ID No:67Gln Pro Ile Asp Asp Thr Glu Ser Asn Thr Thr Ser Val Asn Leu Met1 5 10 15Ala Asp Asp Thr Glu Ser Arg Phe Ala Thr Asn Thr Thr Leu Ala Leu
20 25 30Asp Val Val Asn Leu Ile Ser Met Ala
35 40(2)有关SEQ ID No:68的信息
(i)序列特征:
(A)长度:51bp
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:cDNA
(xi)序列表达:SEQ ID No:68TCTCTTAGCC ATGGAGATCA AGTTAACAAC ATCCAAAGCC AAAGTAGTGT T 51(2)有关SEQ ID No:69的信息
(i)序列特征:
(A)长度:123bp
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:cDNA
(xi)序列表达:SEQ ID No:69CAACCAATTG ACGACACTGA ATCTAACACT ACTTCTGTCA ACTTGATGGC TGACGACACT 60GAATCTAGAT TCGCTACTAA CACTACTTTG GCTTTGGATG TTGTTAACTT GATCTCCATG 120GCT 123(2)有关SEQ ID No:70的信息
(i)序列特征:
(A)长度:65个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ii)分子类型:肽
(xi)序列表达:SEQ ID No:70Lys Arg Glu Glu Ala Glu Ala Glu Ala Glu Pro Lys Phe Val Asn Gln1 5 10 15His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys Gly
20 25 30Glu Arg Gly Phe Phe Tyr Thr Pro Lys Ala Ala Lys Gly Ile Val Glu
35 40 45Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn Tyr Cys
50 55 60Asn65(2)有关SEQ ID No:71的信息
(i)序列特征:
(A)长度:219bp
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:cDNA
(v)片段类型:内部片段
(xi)序列表达:SEQ ID No:71AAGAGAGAAG AAGCTGAAGC TGAAGCTGAA CCAAAGTTCG TTAACCAACA CTTGTGTGGT 60TCTCACTTGG TTGAAGCTTT GTACTTGGTT TGCGGTGAAA GAGGTTTCTT CTACACTCCT 120AAGGCTGCTA AGGGTATTGT CGAACAATGC TGTACCTCCA TCTGCTCCTT GTACCAATTG 180GAAAACTACT GCAACTAGAC GCAGCCCGCA GGCTCTAGA 219(2)有关SEQ ID No:72的信息
(i)序列特征:
(A)长度:348bp
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:cDNA
(xi)序列表达:SEQ ID No:72TTAAATCTAT AACTACAAAA AACACATACA GGAATTCCAT TCAAGAATAG TTCAAACAAG 60AAGATTACAA ACTATCAATT TCATACACAA TATAAACGAC GGTACCAAAA TAATGAAACT 120GAAAACTGTA AGATCTGCGG TCCTTTCGTC ACTCTTTGCA TCTCAGGTCC TTGGCCAACC 180AATTGACGAC ACTGAATCTA ACACTACTTC TGTCAACTTG ATGGCTGACG ACACTGAATC 240TAGATTCGCT ACTAACACTA CTTTGGTTAA CTTGGCTAAC GTTGCCAACC AACACTTGTG 300TGGTTCTCAC TTGGTTGAAG CTTTGTACTT ATGGCTAAGA GATTCGTT 348(2)有关SEQ ID No:73的信息
(i)序列特征:
(A)长度:379bp
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(ii)分子类型:cDNA
(xi)序列表达:SEQ ID No:73TTAAATCTAT AACTACAAAA AACACATACA GGAATTCCAT TCAAGAATAG TTCAAACAAG 60AAGATTACAA ACTATCAATT TCATACACAA TATAAACGAC GGTACCAAAA TAATGAAACT 120GAAAACTGTA AGATCTGCGG TCCTTTCGTC ACTCTTTGCA TCTCAGGTCC TTGGCCAACC 180AATTGACGAC ACTGAATCTA ACACTACTTC TGTCAACTTG ATGGCTGACG ACACTGAATC 240TAGATTCGCT ACTAACACTA CTTTGGATGT TGTTAACTTG ATCTCCATGG CTAAGAGAGA 300AGAAGCTGAA GCTGAAGCTG AACCAAAGTT CGTTAACCAA CACTTGTGTG GTTCTCACTT 360GGTTGAAGCT TTGTACTTG 379
Claims (27)
1.一种DNA表达盒,包括如下序列:
5′-P-SP-LS-PS-*基因*-(T)i-3′
其中,
P是一个启动子序列,
SP是一个编码一种信号肽的DNA序列,
LS是一个编码一种通式I前导肽的DNA序列,
GlnProIle(Asp/Glu)(Asp/Glu)X1(Glu/Asp)X2AsnZ(Thr/Ser)X3 (I)
其中,
X1是一个肽键或一个可编码氨基酸;
X2是一个肽键、一个可编码氨基酸或一个有多达4个可编码氨基酸的序列,这些氨基酸可以相同或不同;
Z是一个除Pro之外的可编码氨基酸;和
X3是一个有4~30个可编码氨基酸的序列,这些氨基酸可以相同或不同;
PS是编码一个加工位点的DNA序列;
*基因*是编码一种多肽的DNA序列;
T是一个终止序列;和
i是0或1。
2.一种如权利要求1的表达盒,其中,通式I中的X1是Ser、Thr或Ala。
3.一种如权利要求1的表达盒,其中,通式I中的X2是Ser、Thr或Ala。
4.一种如权利要求1的表达盒,其中,通式I中的X2是SerIle。
5.一种如权利要求1的表达盒,其中,通式I中的X2是Ser-AlaIle。
6.一种如权利要求1的表达盒,其中,通式I中的X2是Ser-PheAlaThr。
7.一种如权利要求1的表达盒,其中,通式I中的X3是通式II所示的一个氨基酸序列:
X4-X5-X6 (II)
其中X4是一个有1~21个可编码氨基酸的序列;X5是Pro或是包括氨基酸序列ValAsnLeu、LeuAlaAs-nValAlaMetAla、LeuAspValValAsnLeuProGly或LeuAspVal-ValAsnLeuIleSerMet的一个氨基酸序列;以及X6是一个有1~8个可编码氨基酸的序列。
8.一种如权利要求7的表达盒,其中,通式II中的X4是包括下列基序中的一个或多个的氨基酸序列:LeuValAsnLeu、Ser-ValAsnLeu、MetAlaAsp、ThrGluSer、ArgPheAlaThr和ValAlaMetA-la。
9.一种如权利要求7的表达盒,其中,通式II中的X4是一个包括序列AsnSerThr或AsnThrThr的氨基酸序列。
10.一种如权利要求7的表达盒,其中,通式II中的X4是一个包括以下序列的氨基酸序列:
(Ser/Leu)ValAsnLeu,
(Ser/Leu)ValAsnLeuMetAlaAsp,
(Ser/Leu)ValAsnLeuMetAlaAspAsp,
(Ser/Leu)ValAsnLeuMetAlaAspAspThrGluSer,
(Ser/Leu)ValAsnLeuMetAlaAspAspThrGluSerIle,或
(Ser/Leu)ValAsnLeuMetAlaAspAspThrGluSerArgPheAlaThr。
11.一种如权利要求7的表达盒,其中,通式II中的X4是一个包括以下序列的氨基酸序列:
Asn(Thr/Ser)ThrLeu,
Asn(Thr/Ser)ThrLeuAsnLeu,或
Asn(Thr/Ser)ThrLeuValAsnLeu。
12.一种如权利要求7的表达盒,其中,通式II中的X5是Pro。
13.一种如权利要求7的表达盒,其中,通式II中的X5是氨基酸序列ValAsnLeu。
14.一种如权利要求7的表达盒,其中,通式II中的X5是氨基酸序列LeuAlaAsnValAlaMetAla。
15.一种如权利要求7的表达盒,其中,通式II中的X5是氨基酸序列LeuAspValValAsnLeuProGly。
16.一种如权利要求7的表达盒,其中,通式II中的X5是氨基酸序列LeuAspValValAsnLeuIleSerMet。
17.一种如权利要求7的表达盒,其中,通式II中的X6是Ala、Gly、Leu、Thr、Val或Ser。
18.一种如权利要求7的表达盒,其中,通式II中的X6是GlyAla或SerAla。
19.一种如权利要求7的表达盒,其中,通式II中的X6是AlaValAla。
20.一种如权利要求7的表达盒,其中,通式II中的X6是GlyAlaAspSerLysThrValGlu。
21.一种如权利要求1的表达盒,其中,由DNA序列LS编码的前导肽选自下组:SEQ ID No.1 GlnProIleAspGluAspAsnAspThrSerValAsnLeuProAla;SEQ ID No.2 GlnProIleAspAspGluAsnThrThrSerValAsnLeuProAla;SEQ ID No.3 GlnProIleAspAspGluSerAsnThrThrSerValAsnLeuPro-
Ala;SEQ ID No.4 GlnProIleAspAspGluAsnThrThrSerValAsnLeuProVal;SEQ ID No.5 GlnProIleAspAspThrGluAsnThrThrSerValAsnLeuPro-
Ala;SEQ ID No.6 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeu-
ProAla;SEQ ID No.7 GlnProIleAspAspGluAsnThrThrSerValAsnLeuMetAla;SEQ ID No.8 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeu
ProGlyAla;SEQ ID No.9 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeu-
MetAla;SEQ ID No.10 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnVal-
ProThr;SEQ ID No.11 GlnProIleAspAspThrGluSerAsnThrThrLeuValAsnVal-
ProThr;SEQ ID No.12 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeu-
ProThr;SEQ ID No.13 GlnProIleAspAspThrGluSerAsnThrThrLeuValAsnVal-
ProGlyAla;SEQ ID No.14 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeu-
MetAlaProAlaValAla;SEQ ID No.15 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeu-
MetAspLeuAlaValGlyLeuProGlyAla;SEQ ID No.16 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeu-
MetAlaAspAspThrGluSerIleAsnThrThrLeuValAsnLeu-
ProGlyAla;SEQ ID No.17 GlnProIleAspAspThrGluSerIleAsnThrThrLeuValAsn-
LeuProGlyAla;SEQ ID No.18 GlnProIleAspAspThrGluSerAsnThrThrLeuValAsnLeu-
ProGlyAla;SEQ ID No.19 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeu-
MetAlaAspAspThrGluSerArgPheAlaThrAsnThrThrLeu
ValAsnLeuProLeu;SEQ ID No.20 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeu-
MetAlaAspAspThrGluSerIleAsnThrThrLeuValAsnLeu-
AlaAsnValAlaMetAla;SEQ ID No.21 GlnProIleAspAspThrGluSerAlaIleAsnThrThrLeuVal-
AsnLeuProGlyAla;SEQ ID No.22 GlnProIleAspAspThrGluSerPheAlaThrAsnThrThr-
LeuValAsnLeuProGlyAla;SEQ ID No.23 GlnProIleAspAspThrGluSerIleAsnThrThrLeuValAsn-
LeuMetAlaAspAspThrGluSerArgPheAlaThrAsnThrThr-
LeuValAsnLeuProLeu;SEQ ID No.24 GlnProIleAspAspThrGluSerIleAsnThrThrLeuVal-
AsnLeuMetAlaAspAspThrGluSerArgPheAlaThrAsnThr-
ThrLeuAspValValAsnLeuProGlyAla;SEQ ID No.25 GlnProIleAspAspThrGluSerAlaAlaIleAsnThrThrLeu-
ValAsnLeuProGlyAla;SEQ ID No.26 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeu-
MetAlaAspAspThrGluSerArgPheAlaThrAsnThrThrLeu-
ValAsnLeuAlaAsnValAlaMetAla;SEQ ID No.27 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeu-
MetAlaAspAspThrGluSerArgPheAlaThrAsnThrThrLeu-
AspVal-ValAsnLeuIleSerMetAla;SEQ ID No.28 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeu-
MetAlaAsnThrThrGluSerArgPheAlaThrAsnThrThrLeu-
AspValValAsnLeuIleSerMetAla;和SEQ ID No.67 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeu-
MetAlaAspAspThrGluSerArgPheAlaThrAsnThrThrLeu-
AlaLeuAspValValAsnLeuIleSerMetAlaLysArg,
由DNA序列LS编码的特别优选的前导肽如下:SEQ ID No.15 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeu
MetAspLeuAlaValGlyLeuProGlyAla;SEQ ID No.16 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeu-
MetAlaAspAspThrGluSerIleAsnThrThrLeuValAsnLeu-
ProGlyAla;SEQ ID No.17 GlnProIleAspAspThrGluSerIleAsnThrThrLeuValAsn-
LeuProGlyAla;SEQ ID No.18 GlnProIleAspAspThrGluSerAsnThrThrLeuValAsnLeu
ProGlyAla;SEQ ID No.19 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeu-
MetAlaAspAspThrGluSerArgPheAlaThrAsnThrThrLeu-
ValAsnLeuProLeu;SEQ ID No.20 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeu-
MetAlaAspAspThrGluSerIleAsnThrThrLeuValAsnLeu-
AlaAsnValAlaMetAla;SEQ ID No.21 GlnProIleAspAspThrGluSerAlaIleAsnThrThrLeuVal-
AsnLeuProGlyAla;SEQ ID No.22 GlnProIleAspAspThrGluSerPheAlaThrAsnThrThrLeu-
ValAsnLeuProGlyAla;SEQ ID No.23 GlnProIleAspAspThrGluSerIleAsnThrThrLeuValAsn-
LeuMetAlaAspAspThrGluSerArgPheAlaThrAsnThrThr-
LeuValAsnLeuProLeu;SEQ ID No.24 GlnProIleAspAspThrGluSerIleAsnThrThrLeuValAsn-
LeuMetAlaAspAspThrGluSerArgPheAlaThrAsnThrThr-
LeuAspValValAsnLeuProGlyAla;SEQ ID No.25 GlnProIleAspAspThrGluSerAlaAlaIleAsnThrThrLeu-
ValAsnLeuProGlyAla;SEQ ID No.26 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeu-
MetAlaAspAspThrGluSerArgPheAlaThrAsnThrThrLeu-
ValAsnLeuAlaAsnValAlaMetAla;和SEQ ID No.28 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeu-
MetAlaAsnThrThrGluSerArgPheAlaThrAsnThrThrLeu-
AspValValAsnLeuIleSerMetAla;SEQ ID No.67 GlnProIleAspAspThrGluSerAsnThrThrSerValAsnLeu-
MetAlaAspAspThrGluSerArgPheAlaThrAsnThrThrLeu-
AlaLeuAspValValAsnLeuIleSerMetAlaLysArg.
22.一种如权利要求1的表达盒,其中,SP是一个编码α-因子信号肽、小鼠唾液淀粉酶信号肽、羧肽酶信号肽、酵母天冬氨酸蛋白酶3信号肽或酵母BAR1信号肽的DNA序列。
23.一种如权利要求1的表达盒,其中,PS是一个编码LysArg、ArgLys、ArgArg、LysLys或Ile-GluGlyArg的DNA序列。
24.一种如权利要求1的表达盒,其中,所述多肽选自抑蛋白酶肽、组织因子途径抑制剂、或其它蛋白酶抑制剂、胰岛素或胰岛素前体、胰岛素样生长因子I、胰岛素样生长因子II、人或小牛生长激素、白介素、胰高血糖素、类胰高血糖素肽-1、组织纤溶酶原激活物、转化生长因子α或β、由血小板衍生的生长因子、酶、或以上物质的功能性类似物。
25.一种酵母表达载体,它包括如上述权利要求中任一项所述的一个表达盒。
26.一种酵母细胞,它能够表达一种多肽、并且是用权利要求25所述的酵母表达载体转化过的。
27.一种用于在酵母中生产一种多肽的方法,该方法包括:在一种合适的培养基中培养一种酵母细胞,这种细胞能够表达目的多肽,而且是用权利要求25所述的酵母表达载体转化过的,以获得所述多肽的表达和分泌,然后,从所述培养基中回收所述多肽。
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
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DK0705/1994 | 1994-06-16 | ||
DK70594 | 1994-06-16 | ||
DK0705/94 | 1994-06-16 | ||
US28285294A | 1994-07-29 | 1994-07-29 | |
US08/282,852 | 1994-07-29 |
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CN1154143A true CN1154143A (zh) | 1997-07-09 |
CN1115413C CN1115413C (zh) | 2003-07-23 |
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CN95194183A Expired - Lifetime CN1115413C (zh) | 1994-06-16 | 1995-06-16 | 合成前导肽序列 |
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EP (1) | EP0763117B1 (zh) |
JP (1) | JP3676369B2 (zh) |
KR (1) | KR100251054B1 (zh) |
CN (1) | CN1115413C (zh) |
AT (1) | ATE208823T1 (zh) |
AU (1) | AU693569B2 (zh) |
BR (1) | BR9508033A (zh) |
CZ (1) | CZ290069B6 (zh) |
DE (1) | DE69523915T2 (zh) |
DK (1) | DK0763117T3 (zh) |
ES (1) | ES2168367T3 (zh) |
FI (1) | FI965005A (zh) |
HU (1) | HU221118B1 (zh) |
MX (1) | MX9606492A (zh) |
NO (1) | NO965362L (zh) |
PL (1) | PL317722A1 (zh) |
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WO2019154311A1 (zh) * | 2018-02-09 | 2019-08-15 | 江苏恒瑞医药股份有限公司 | 一种密码子优化的人胰岛素类似物前体基因和信号肽基因 |
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DE69523915T2 (de) | 2002-08-22 |
BR9508033A (pt) | 1999-06-29 |
JPH10501413A (ja) | 1998-02-10 |
KR100251054B1 (ko) | 2000-04-15 |
WO1995034666A1 (en) | 1995-12-21 |
AU2733495A (en) | 1996-01-05 |
EP0763117B1 (en) | 2001-11-14 |
DK0763117T3 (da) | 2002-02-18 |
FI965005A (fi) | 1997-02-13 |
NO965362L (no) | 1997-02-13 |
FI965005A0 (fi) | 1996-12-13 |
ATE208823T1 (de) | 2001-11-15 |
PL317722A1 (en) | 1997-04-28 |
CZ290069B6 (cs) | 2002-05-15 |
HUT76378A (en) | 1997-08-28 |
HU9603477D0 (en) | 1997-02-28 |
MX9606492A (es) | 1997-03-29 |
EP0763117A1 (en) | 1997-03-19 |
US5795746A (en) | 1998-08-18 |
NO965362D0 (no) | 1996-12-13 |
UA40648C2 (uk) | 2001-08-15 |
DE69523915D1 (en) | 2001-12-20 |
JP3676369B2 (ja) | 2005-07-27 |
ES2168367T3 (es) | 2002-06-16 |
PT763117E (pt) | 2002-05-31 |
CZ364196A3 (en) | 1997-05-14 |
HU221118B1 (en) | 2002-08-28 |
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AU693569B2 (en) | 1998-07-02 |
US5639642A (en) | 1997-06-17 |
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