CN1145636A - 血管内皮生长因子2 - Google Patents
血管内皮生长因子2 Download PDFInfo
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- CN1145636A CN1145636A CN94195053A CN94195053A CN1145636A CN 1145636 A CN1145636 A CN 1145636A CN 94195053 A CN94195053 A CN 94195053A CN 94195053 A CN94195053 A CN 94195053A CN 1145636 A CN1145636 A CN 1145636A
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- polypeptide
- vegf2
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Abstract
本发明公开了人VEGF2多肽和编码这种VEGF2多肽的DNA(RNA),还提供了通过重组技术生产这种多肽的方法,以及抗这种多肽的抗体和拮抗剂。这种多肽可以和合适的药物学载体或稀释剂结合以提供针对各种疾病的诊断、治疗和/或预防效果。本发明还提供了为治疗目的使用所述抗体和拮抗剂抑制VEGF2的作用的方法。
Description
本发明涉及新鉴别的多核苷酸、由该多核苷酸编码的多肽、这些多核苷酸和多肽的用途,以及这些多核苷酸和多肽的生产。更具体地,本发明的多肽为人血管内皮生长因子2。本发明还涉及抑制这种多肽的作用。
新血管的形成,或称血管生成,对于胚胎发育、随后的生长以及组织修复是非常重要的。血管生成是人实体瘤生长的一个重要部分,异常血管生成伴随有其它疾病如风湿性关节炎、银屑病、以及糖尿病性视网膜疾病(Folkman,J.and Klagsbrun,M.,Science 235:442-447,(1987))。
血管生成涉及几种因子,酸性和碱性成纤维细胞生长因子分子是内皮细胞和其它细胞类型的促***原。促血管生成素和血管生成素可以诱导血管生成,但它们的功能尚不清楚(Folkman,J.,1993,Cancer Medicine pp.153-170,Le and FebigerPress)。一种对血管内皮细胞具有高选择性的促***原是血管内皮生长因子或称VEGF(Ferrara,N.,et al.,Endocr.Rev.13:19-32,(1992))。血管内皮生长因子是一种分泌的血管生成促***原,其靶细胞特异性似乎限制在血管内皮细胞。已鉴定了鼠VEGF基因并分析了其在胚胎发生中的表达模式。在相邻于有孔内皮的内皮细胞中观察到了VEGF的持续表达,例如在脉络丛和肾小球中,该资料与VEGF作为内皮细胞生长和分化的多功能调控因子的作用是相符合的,见Breier,G.et al.Development,114:521-532(1992)。
VEGF促进血管生成,VEGF与人血小板衍生的生长因子PDGFα和PDGFβ的序列具有同源性(Leung,D.W.,et al.,Science,1306-1309,(1989)),同源程度分别为约21%和24%,在这三者之间有8个半胱氨酸是保守的,尽管它们是相似的,但在VEGF和PDGF之间也有特异的差异。PDGF是***的主要生长因子,而VEGF对内皮细胞高度特异。VEGF已知是血管渗透因子(VPM)以及卵泡星状衍生的生长因子,其是结合肝素的二聚体多肽。
由于不同剪接,VEGF具有121、165、189和206个氨基酸四种不同形式,VEGF121和VEGF165是可溶的并能促进血管生成,而VEGF189和VEGF206在细胞表面结合于含肝素的粘蛋白。VEGF的时间和空间表达与血管的生理增殖有关(Gajdusek,C.M.,and Carbon,S.J.,Cell Physiol.,139:570-579,(1989));McNeil,P.L.,Muthukrishnan,L.,Warder,E.,D’Amore,P.A.,J.Cell.Biol.,109:811-822,(1989))。其高亲和结合位点仅位于组织的内皮细胞中(Jakeman,L.B.,et al.,Clin.Invest.89:244-253,(1989))。该因子可从垂体细胞和若干种肿瘤细胞系中分离,并被包含在一些人神经胶质瘤中(Plate,K.H.Nature 359:845-848,(1992))。令人感兴趣的是,VEGF121或VEGF165的表达使中国仓鼠卵巢细胞能在裸鼠中形成肿瘤(Ferrara,N.,et al.,J.Clin.Invest.91:160-170,(1993))。最后,用抗VEGF单克隆抗体抑制VEGF的功能已证实在免疫缺陷小鼠中抑制肿瘤生长(Kim,K.J.,Nature 362:841-844,(1993))。
已发现血管渗透因子,也称VEGF,甚至在损伤停止后负责血浆蛋白的持续微血管超渗透性,这是正常伤口愈合的特征。这一点暗示VPF(或VEGF)在伤口愈合中是重要的因子,见Brown,L.F.et al.,J.Exp.Med.,176:1375-9(1992)。
USP5,073,492,1991年12月17日授予Chen等人,公开了在合适的环境中协同促进内皮细胞生长的方法,该方法包括向该环境中加入VEGF、效应子和源于血清的因子。另外,如欧洲专利申请92302750.2,1992年9月30日公开,披露了通过聚合酶链反应技术制备血管内皮细胞生长因子C亚基DNA,该DNA编码一种异二聚体或同二聚体的蛋白质。所述的蛋白质是哺乳动物血管内皮细胞促***原,因此,可以用于促进血管发育和修复。
根据本发明的一个方面,提供了新的成熟多VEGF2,以及该多肽的片段、类似物和衍生物,本发明的VEGF2是人源的。
根据本发明的另一个方面,提供了编码这些多肽的多核苷酸(DNA或RNA)。
根据本发明的又一方面,提供了一种通过重组技术生产这些多肽的方法。
根据本发明的再一方面,提供了为治疗目的而使用这种多肽、或使用编码这种多肽的多核苷酸的方法,这些多肽或多核苷酸用作伤口愈合剂以促进内皮化并用于诊断肿瘤、癌症治疗以及确定和分离未知的VEGF2受体。
根据本发明的另一方面,提供了抗VEGF2的抗体和生产这种抗体的方法。
根据本发明的又一方面,提供了VEGF2的拮抗剂/抑制剂,其可用于抑制这种多肽的作用,例如防止肿瘤血管生成。
本发明的这些及其它方面根据本文的教导对本领域熟练技术人员将是显而易见的。
下述附图旨在说明本发明的实施方案,并非限制本发明的范围。
图1示出编码VEGF2′的多核苷酸序列全长VEGF2多肽的的相应的推导的氨基酸序列包括350个氨基酸残基,其中开始的约24个氨基酸代表前导序列。氨基酸由标准三字母缩写表示。
图2示出生长因子PDGFα、PDGFβ、VEGF和VEGF2在氨基酸水平的同源性。
图3以表格形式示出PDGFα、PDGFβ、VEGF和VEGF2的同源性百分数。
图4示出在乳腺癌细胞系中存在VEGF2的mRNA。
图5为在成人组织中VEGF2的Northern印迹分析的结果。
图6示出在体外转录/翻译后进行VEGF2的SDS-PAGE凝胶电泳的结果。在35S-甲硫氨酸存在下,在一偶联的反应中将全长和部分的VEGF2 cDNA转录和翻译,用4-20%梯度SDS PAGE分析翻译产物并在X光胶片上曝光。
根据本发明的一个方面,提供了分离出的核酸(多核苷酸),其编码具有图1的推导的氨基酸序列的成熟多肽,或者编码由ATCC保藏号___的克隆的cDNA编码的成熟多肽。
编码本发明的多肽的多核苷酸可以得自早期人胚胎(8-9周)破骨细胞瘤、成人心脏或几种乳腺癌细胞系。本发明的多核苷酸由衍生自9周早期人胚胎的cDNA文库中发现,其与VEGF/PDGF家族在结构上相关,含有一编码约350个氨基酸残基的蛋白质的开放读框,其中开始的24个氨基酸残基可能是前导序列,因此成熟蛋白包括326个氨基酸,该蛋白与血管内皮生长因子具有最高的同源性(30%同一性),其次是PDGFα(23%)和PDGFβ(22%)(见图3)。特别重要的是在该家族的所有四个成员中所有的8个半胱氨酸都是保守的(见图2的框区)。另外,PDGF/VEGF家族的特征,PXCVXXXRCXGCCN,在VEGF2中是保守的(见图2)。VEGF2、VEGF和两种PDGF之间的同源性是蛋白质序列水平的,未测出核苷酸序列的同源性,因此,很难通过简单的程序如低严格性杂交来分离VEGF2。
本发明的多核苷酸可以是RNA形式,或DNA形式,DNA包括cDNA、基因组DNA和合成DNA,DNA可以是双链或单链,并且如果是单链,则可以是编码链或非编码(反义)链。编码成熟多肽的编码序列可以与图1所示的编码序列相同,或与保藏克隆的编码序列相同,或者也可以是不同的编码序列,该编码序列由于遗传密码的丰余性或简并性,与图1的DNA或保藏的cDNA编码相同的成熟多肽。
编码图1的成熟多肽或编码由保藏的cDNA编码的成熟多肽的多核苷酸可以包括:成熟多肽的编码序列;成熟多肽的编码序列和另外的编码序列如前导序列或分泌序列或蛋白原序列;成熟多肽的编码序列(和任选的另外的编码序列)和非编码序列如内含子或成熟多肽编码序列的5’和/或3’非编码序列。
因此,术语“编码多肽的多核苷酸”包含如下的多核苷酸,即仅包括多肽编码序列的多核苷酸,以及包括另外的编码和/或非编码序列的多核苷酸。
本发明还涉及上述多核苷酸的变异体,其编码具有图1的推导的氨基酸序列的多肽或编码由保藏的克隆的cDNA编码的多肽的片段、类似物和衍生物。这些多核苷酸的变异体可以是天然发生的该多核苷酸的等位基因变异体,或是该多核苷酸的非天然发生的变异体。
因此,本发明包括编码图1所示的相同成熟多肽或由保藏克隆的cDNA编码的相同成熟多肽的多核苷酸,以及这些多核苷酸的变异体,所述变异体编码图1的多肽或由保藏克隆的cDNA编码的多肽的片段、衍生物或类似物。这些核苷酸变异体包括缺失变异体、置换变异体和增加或***变异体。
如上所述,多核苷酸可以具有为图1所示的编码序列的天然发生的等位基因变异体的编码序列,或保藏克隆的编码序列的天然发生变异体的编码序列。如本领域所公知的,等位基因变异体是多核苷酸的另一种形式,其可以置换、缺失或增加一个或多个核苷酸,但基本上不改变编码多肽的功能。
本发明还包括多核苷酸,其中成熟多肽的编码序列可以在同一读框内与有助于多肽从宿主细胞表达和分泌的多核苷酸融合,例如前导序列,其是用于控制多肽从细胞转运的分泌序列。具有前导序列的多肽是一种前蛋白(preprotein),前导序列可以由宿主细胞切下以形成多肽的成熟形式。多核苷酸还可编码抑制蛋白原(proprotein),其是成熟蛋白加上另外的5’氨基酸残基。具有序列原(prosequence)的成熟蛋白为蛋白原并是蛋白质的非活性形式,一旦序列原被切掉,则保留一活性成熟蛋白。
因此,例如,本发明的多核苷酸可以编码一成熟蛋白,或编码一具有序列原的蛋白或一具有序列原和前序列(前导序列)的蛋白。
本发明的多核苷酸还可以具有在读框内与标记序列融合的编码序列,其可用于纯化本发明的多肽。标记序列可以是,例如,由pQE-9载体提供的六组氨酸标记物用于在细菌宿主中纯化与该标记物融合的成熟多肽,或者,例如,当使用哺乳动物宿主如COS-7细胞时,标记序列可以是血凝素标记物(HA)。HA标记物相应于由流感病毒血凝素蛋白衍生的表位(Wilson,I.,et al.,Cell,37:767(1984))。
本发明还涉及可与上述序列杂交的多核苷酸,条件是序列间具有至少50%、优选70%的同一性。本发明特别涉及在严格条件下与上述多核苷酸杂交的多核苷酸。如本文所用的,术语“严格条件”是指杂交仅发生在序列间具有至少95%、优选97%同一性的情况下。在一优选的实施方案中,与上述多核苷酸杂交的多核苷酸编码基本上保持了由图1的cDNA或保藏的cDNA编码的成熟多肽的相同的生物学功能或活性的多肽。
本文所指的保藏物将根据国际承认的用于专利程序的微生物保藏的布达佩斯条约的规定期限保藏。这些保藏仅为本领域技术人员提供方便,而不是对根据35U.S.C 112索取保藏物的许可。保藏物中所含的多核苷酸的序列以及该序列编码的多肽的氨基酸序列引入本文作参考,并且在与本文的序列描述有抵触时,其是参照。制造、使用或销售保藏物需经许可,而本文不授予这种许可。
本发明还涉及具有图1的推导的氨基酸序列或具有由保藏的cDNA编码的氨基酸序列的VEGF2多肽,以及该多肽的片段、类似物和衍生物。
当指图1的多肽或由保藏的cDNA编码的多肽时,术语“片段”、“衍生物”和“类似物”是指基本上保持这些多肽的相同的生物学功能或活性的多肽。因此,类似物包括蛋白原,其可通过裂解蛋白原部分而激活以生产有活性的成熟多肽。
本发明的多肽可以是重组多肽、天然多肽或合成多肽,优选重组多肽。
图1的多肽或由保藏的cDNA编码的多肽的片段、衍生物或类似物可以是(i)其中的一或多个氨基酸残基被保守或非保守的氨基酸残基取代(优选由保守的氨基酸残基取代),并且这种取代的氨基酸残基可以由遗传密码编码,也可不是由遗传密码编码,或者(ii)其中的一或多个氨基酸残基包括一取代基,或者(iii)其中的成熟多肽与另一种化合物融合,所述化合物例如为增加多肽的半寿期的化合物(例如聚乙二醇),或者(iv)其中有附加的氨基酸与成熟多肽融合,例如前导序列或分泌序列或用于纯化成熟多肽获得序列或者蛋白原序列。这种片段、衍生物和类似物被视为属于本领域熟练技术人员根据本文的教导而理解的范围。
本发明的多肽和多核苷酸优选地以分离的形式提供,并优选纯化至均一性。
术语“分离的”是指从其原始环境(例如,若其是天然存在的则是天然环境)中脱离的物质。例如,存在于活体动物中的天然发生的多核苷酸或多肽不是分离的,但从一些或所有共存物质中分离的相同的多核苷酸或DNA或多肽则是分离的。这种多核苷酸可以是载体的一部分和/或这种多核苷酸或多肽可以是某种组合物的一部分,并且如果这种载体或组合物不是其天然环境的一部分,则其还是分离的。
本发明还涉及包括本发明的多核苷酸的载体,用本发明的载体遗传工程化的宿主细胞,以及用重组技术生产本发明的多肽。
宿主细胞用本发明的载体遗传工程化(转导或转化或转染),所述的载体可以是克隆载体或表达载体。载体可以是质粒、病毒颗粒、噬菌体等。遗传工程化的宿主细胞可以在改良的传统营养培养基中培养,改良培养基是为了激活启动子,选择转化子或扩增VEGF2基因。培养条件如温度、pH等是先前用于选择用来表达的宿主细胞的条件,其对本领域普通技术人员是显而易见的。
本发明的多核苷酸可通过重组技术用于生产多肽,因此,例如,该多核苷酸可以包括在任一种表达载体中特别是载体或质粒中以表达多肽。这些载体包括染色体的、非染色体的和合成的DNA序列,例如SV40衍生物;细菌质粒;噬菌体DNA;杆状病毒;酵母质粒;衍生于质粒和噬菌体DNA的结合体的载体;病毒DNA如痘苗病毒、腺病毒、禽痘病毒和拟狂犬病毒。然而,也可使用任何其它载体,只要其在宿主中可复制和生存。
如上所述,可通过多种程序将合适的DNA序列***载体,通常,通过本领域公知的程序将DNA序列***合适的限制性内切酶位点。这些和其它程序属于本领域熟练技术人员的范畴。
表达载体中的DNA序列与合适的表达调控序列(启动子)连接以指导mRNA合成,作为这些启动子的代表性例子,可提及的有:LTR或SV40启动子,E.colilac或trp启动子,λ噬菌体PL启动子和其它公知用于控制基因在原核细胞或真核细胞或者病毒中表达的启动子。表达载体还含有核糖体结合位点用于翻译起始和转录终止子。载体还可包括用于扩增表达的合适的序列。
另外,表达载体优选含有一个用于为选择转化的宿主细胞提供表型标记的基因,如对于真核细胞培养为二氢叶酸还原酶或新霉素抗性,而在E.coli中例如为四环素或氨苄青霉素抗性。
可采用含有如上所述合适DNA序列以及合适启动子或调控序列的载体转化合适的宿主以使宿主表达蛋白质。作为合适的宿主的例子,可以提及的有:细菌细胞,如E.coli、链霉菌、鼠伤寒沙门氏杆菌;真菌细胞,如酵母;昆虫细胞如果蝇和Sf9;动物细胞如CHO、COS或Bowes黑素瘤;植物细胞等。选择合适的宿主细胞被视为属于本领域熟练技术人员根据本文的教导而理解的范围。
更具体地,本发明还包括重组构建体,所述构建体包含一或多种上述序列。该构建体包括载体,如质粒或病毒载体,在该载体中以正向或反向***本发明的序列。在这一实施方案的一个优选方面,该构建体还包括调节序列,包括例如,与所述序列连接的启动子。本领域熟练技术人员已知大量的合适的载体和启动子,它们是可商购的。以下举出载体的例子,细菌的载体:pQE70,pQE-9(Qiagen,Inc.),pBs,phagescript,PsiX174,pBluescript SK,pBsKS,pNH8a,pNH16a,pNH18a,pNH46a(Stratagene);pTrc99A,pKK223-3,pKK233-3,pDR540,pRIT5(Pharmacia)。真核载体:pWLneo,pSV2cat,pOG44,pXT1,pSG(Stratagene),pSVK3,pBPV,pMSG,pSVL(Pharmacia)。然而,也可使用其它质粒或载体,只要其可在宿主中复制和生存即可。
启动子区可以选自使用CAT(氯霉素转移酶)的载体或其它带有选择性标记的载体的任何所需基因,两个合适的载体是pKK232-8和pCM7。特别提到的细菌启动子包括lacI,lacZ,T3,T7,gpt,lambda PR,PL和trp;真核启动子包括CMV立即早期,HSV胸腺嘧啶激酶,早期和晚期SV40,反转录病毒的LTR,和鼠金属硫蛋白-I。选择合适的载体和启动子属于本领域普通技术人员的水平范畴。
在另一实施方案中,本发明涉及含有上述构建体的宿主细胞,所述的宿主细胞可以是高等真核细胞,如哺乳细胞,或低等真核细胞,如酵母细胞,或者宿主细胞可以是原核细胞,如细菌细胞。可通过磷酸钙转染、DEAE-葡聚糖介导的转染,或电穿孔(Davis,L.,Dibner,M.,Battey,I.,Basic Methods in MolecularBiology,(1986))将构建体导入宿主细胞。
可以用传统的方式使用宿主细胞中的构建体以生产由重组序列编码的基因产物。或者,也可通过常规的肽合成仪合成生产本发明的多肽。
在合适的启动子的控制下,可以在哺乳细胞、酵母、细菌或其它细胞中表达成熟蛋白质,也可采用无细胞翻译***使用由本发明的DNA构建体衍生的RNA生产这种蛋白质。原核和真核宿主使用的合适的克隆和表达载体见Sambrook,et al,Molecular Clonning:A Laboratory Manual,Second Edition,Cold Spring Harbor,N.Y.,(1989),该书引入本文作参考。
高等真核生物对编码本发明的多肽的DNA的转录可通过在载体中***一增强子序列而得以增加,增强子是约10-300bp的顺式作用的DNA因子,其作用于启动子以增加其转录。其例子包括位于复制起点晚期侧(100-270bp处)的SV40增强子、巨细胞病毒早期启动子增强子、位于复制起点晚期侧的多瘤病毒增强子,以及腺病毒增强子。
通常,重组表达载体包括复制起点和允许宿主细胞的转化的选择标记,如E.coli的氨苄青霉素抗性基因和酿酒酵母的TRP1基因,以及衍生于高表达基因的启动子以指导下游结构序列的转录。这种启动子可以衍生于编码糖酵解酶如3-磷酸甘油酸激酶(PGK)、α-因子、酸性磷酸酶、或热休克蛋白的操纵子。在合适的阶段,杂合结构序列装配上翻译起始和终止序列,以及优选地,装配一能指导翻译的蛋白质进入周质空间或胞外培养基的前导序列。任选地,杂合序列可以编码一包括N-末端鉴别肽的融合蛋白质,以赋予所需的特征,如表达的重组产物的稳定性和纯化简便性。
用于细菌的表达载体的构建是通过将编码所需蛋白质的结构DNA序列与合适的翻译起始和终止信号一起***带有功能启动子的可操纵阅读相而完成。载体包括一或多种表型选择标记及一个复制起点以确证载体保持在宿主中,并且,如果需要,可以在宿主中扩增。合适的用于转化的原核宿主包括E.coli、枯草芽孢杆菌、鼠伤寒沙门氏杆菌以及假单胞菌属、链霉菌属和葡萄球菌属的各菌种,当然,也可采用其它菌。
作为代表性而非限制性的例子,有用的细菌表达载体可以包括衍生于包含熟知的克隆载体pBR322(ATCC 37017)的遗传因子的商购质粒的选择标记和细菌复制起点,这些商购质粒包括,例如,pKK223-3(Pharmacia Fine Chemicals,Uppsala,Sweden)和GEM1(Promega Biotec,Madi son,WI,USA)。这些pBR322“骨架”部分与合适的启动子及待表达的结构序列结合。
转化合适的宿主菌株并将宿主菌株培养至合适的细胞密度后,用合适的方法(如温度改变或化学诱导)诱导选定的启动子,并继续培养细胞一段时间。
典型地,细胞由离心收集,用物理或化学方法破碎,保留得到的粗提物以进一步纯化。
用于表达蛋白质的微生物细胞可用任何常规方法破碎,如冻-融循环、超声处理、机械破碎,或使用细胞裂解试剂。
也可使用各种哺乳细胞培养***表达重组蛋白,哺乳细胞表达***包括由Gluzman,Cell,23:175(1981)描述的猴肾成纤维细胞COS-7细胞系,以及其它能表达相容载体的细胞系,如C127,3T3,CHO,Hela和BHK细胞系。哺乳类表达载体包括复制起点,合适的启动子和增强子,以及任何必需的核糖体结合位点,多腺苷酸化位点,剪接供体和受***点,转录终止序列,和5’旁侧非转录序列。可使用源自SV40病毒基因组的DNA序列,例如SV40起点、早期启动子、增强子、剪接体和多腺苷酸化位点以提供所需的非转录遗传因子。
可通过硫酸铵或乙醇沉淀、酸抽提、阳离子或阴离子交换层析、磷酸纤维素层析、疏水作用层析、亲和层析、羟基磷灰石层析和凝集素层析等方法从重组细胞培养物中回收并纯化VEGF2。优选的是在纯化过程中存在低浓度(约0.1-5mM)的钙离子(Price,et al.,J.Biol.Chem.,244:917(1969))。如果需要,可使用蛋白质重折叠步骤以完成成熟蛋白的构型,最后,可采用高效液相色谱(HPLC)进行最后的纯化步骤。
本发明的多肽可以是天然纯化的产物,或是化学合成过程的产物,或者通过重组技术从原核或真核宿主(例如,通过培养细菌、酵母、高等植物、昆虫和哺乳类细胞)而生产。根据在重组生产过程中采用的宿主,本发明的多肽可以用哺乳类或其它真核类的碳水化合物糖基化或是非糖基化的。
VEGF2可用作伤口愈合剂,特别是当需要对损伤组织进行再血管化时或当新的毛细血管生成是非常重要的情况下。因此,其可用于治疗深度创伤如真皮溃疡,包括压伤、静脉溃疡和糖尿病溃疡。另外,其可用于治疗深度烧伤和损伤,即在需要生成血管以在损伤位点的烧伤处进行植皮和瓣膜的情况。在这种情况下,其可直接用于患处。类似地,当烧伤、其它损伤后需要重建或者甚至是用于化妆目的时,VEGF2可用于整形外科。
VEGF2还可用于诱导损伤的骨组织、牙周组织或韧带组织的生长,其可用于牙周疾病,在这种情况下,VEGF2是包含在甲基纤维素凝胶中施用到病牙的牙根中,这种治疗随着胶原纤维的向内生长能形成新骨和牙骨质。其还可用于再生由于疾病和损伤而损坏的牙支持组织,如牙槽骨、牙骨质和牙周韧带。
由于血管生成对于保持伤口清洁和不被感染是很重要的,所以VEGF2可用于外科手术和伤口修复后使用,其特别适用于治疗可能高发感染的腹部伤口。
在血管移植手术中VEGF2可用于促进内皮化,当血管移植物是移植材料或合成材料时,VEGF2可以施用到移植物的表面或连接处以促进血管内皮细胞的生长。这种方法的一种衍化是VEGF2可以通过刺激移植组织的生长而用于修复心肌梗塞及其它需要冠状动脉搭桥手术的情况。与此相关的是局部缺血后用VEGF2修复心血管***。
VEGF2的鉴定可以用于产生一些血管内皮生长因子的抑制剂。由于血管生成和新血管化是实体肿瘤生长的非常重要的步骤,所以抑制血管内皮生长因子的血管生成活性对于防止实体肿瘤的进一步生长、延迟或甚至抑制肿瘤是很有用的。虽然VEGF2在正常组织包括乳腺中的表达水平是非常低的,但是发现其在由恶性肿瘤衍生的至少两个乳腺癌细胞系中以中等水平表达,因此,VEGF2可能与肿瘤血管生成和生长有关。
VEGF2可用于体外培养血管内皮细胞,其可以10pg/ml-10ng/ml的浓度加入到条件培养基中。
本发明的多肽还可通过在体内表达这种多肽而使用,这种方法通常称为“基因疗法”。
因此,例如,可用编码来自体内的多肽的多核苷酸(DNA或RNA)对细胞如骨髓细胞进行工程化,随后将工程化的细胞再提供给需用多肽治疗的患者,这些方法是本领域公知的。例如,可用含编码本发明的多肽的RNA的反转录病毒颗粒利用本领域熟知的程序对细胞进行工程化。
类似地,例如也可通过本领域公知的程序在体内对细胞进行工程化以在体内表达多肽。如本领域所公知的,可将生产含编码本发明的多肽的RNA的反转录病毒颗粒的生产者细胞给予患者以在体内进行细胞的工程化并在体内表达多肽。通过这种方法给予本发明的多肽的这些和其它方法对于本领域熟练技术人员根据本发明的教导是显而易见的。例如,用于工程化细胞的表达载体可以不是反转录病毒,而是例如腺病毒,其在与合适的输送载体结合后可用于在体内工程化细胞。
本发明的多肽可以与合适的药物载体结合应用,这种组合物包括药物有效量的蛋白质以及药物学可接受的载体或赋形剂。这种载体包括但不限于盐水、缓冲的盐水、葡萄糖、水、甘油、乙醇及其结合物。配制品应适合给药方式。
本发明还提供了一种药物包装盒或试剂盒,其包括一或多个填充有一或多种本发明的药物组合物成分的容器。与这种容器在一起的还有由控制药品或生物产品的生产、使用或销售的政府机构出具的通知,该通知反应了该机构许可其为人体用而生产、使用或销售。另外,本发明的多肽可以与其它药物化合物结合应用。
所述的药物组合物可以以传统的方式给药,如口服和注射给药,优选的是局部给药。给予个体的VEGF2的量和剂量模式取决于数种因素,如给药方式、需治疗的病情的性质、需治疗个体的体重和开药医生的判断。通常,例如,其是以药物有效剂量至少约10μg/kg体重而给予,在大多数情况下,给药量不超过8mg/kg体重/天,考虑到给药途径、症状等,优选的剂量是约每天为10μg/kg体重-1mg/kg体重。
本发明的序列还可用于染色体鉴定,该序列特异地定向于个别的人染色体的特定位置并可与之杂交。另外,目前需要鉴定染色体上的特定位点,而现在只有很少几种基于实际序列资料(重复多态性)的染色体标记试剂可以商购用于标记染色***置。本发明的将DNA定位于染色体是将这些序列与相关疾病的基因联系起来的非常重要的第一步。
简而言之,可通过从cDNA制备PCR引物(优选15-25bp)而将序列定位在染色体上。使用cDNA的计算机分析快速选择长度不超过基因组DNA的一个外显子并因而不会使扩增复杂化的引物,随后使用这些引物对含有个别的人染色体的体细胞杂合子进行PCR筛选。只有那些含有相应于引物的人基因的杂合子能得到扩增产物。
体细胞杂合子的PCR作图是将特定DNA定位于特定染色体的一个快速程序。采用本发明相同的寡核苷酸引物,可以类似的方式将来自特定染色体的一组片段或大基因组克隆的集合体进行亚定位。其它的可类似用于染色体作图的作图策略包括原位杂交、用标记的流选的染色体进行的预筛选,以及通过杂交预选以构建染色体-特异cDNA文库。
cDNA克隆与中期染色体涂片的荧光原位杂交(FISH)可以用来提供精确的一步法染色体定位。这一技术可使用短至500或600个碱基cDNA;然而,大于2000bp的克隆更易于与独特的染色***置结合以具有足够的信号强度便于检测。FISH需要使用衍生EST的克隆,越长越好。例如,2000bp是好的,更好为4000bp,而超过4000则可能不是获得好结果所必须的,因其时间不合理。此技术的综述见Vermaet al.,Human Chromosomes:a Manual of Basic Techniques,Pergamon Press,New York(1988)。
一旦某一序列已被精确定位于染色体某一位置,则可比较该序列在染色体上的物理位置与遗传图谱资料的关系,这种资料例如可在V.McKusick,MendelianInheritance in Man中发现(可在线从Johns Hopkins University Welch MedicalLibrary获得)。随后可通过连锁分析(物理相邻基因的共遗传性)鉴别基因与已定位于相同染色体区的疾病之间的关系。
接着,必须确定感染个体和未感染个体之间的cDNA或基因组序列的差异,如果在一些或所有感染个体中观察到某一突变而未在任何正常个体中观察到这种突变,则该突变可能是该疾病的致病原。
应用目前的物理作图和遗传作图技术的分辨率,一种精确定位于与疾病有关的染色体区的cDNA可以是50-500个潜在的致病基因中的一个(这假定作图分辨率为1兆碱基,并且每20kb为一个基因)。
感染个体和未感染个体的比较通常是首先寻找染色体的结构改变,如可从染色体涂片上观察到的或者用基于该cDNA序列的PCR检测到的缺失或易位。最后,需要对来自一些个体的基因进行全测序以证实存在突变并从多态性中区分突变。
本发明还涉及用反义技术体内抑制VEGF2,反义技术通过三螺旋形成或反义DNA或RNA可用于控制基因表达,这两种方法均基于多核苷酸与DNA或RNA的结合。例如,用编码本发明的多肽的多核苷酸序列的5’编码区设计长度为10-40bp的反义RNA寡核苷酸。设计一DNA寡核苷酸以互补于转录涉及的基因的某区域(三螺旋-见Lee et al.,Nucl.Acids Res.,6:3073(1979);Cooney et al,Science,241:456(1988);and Dervan et al,Science,251:1360(1991)),从而防止VEGF2的转录和生产。反义RNA寡核苷酸与mRNA体内杂交并阻断mRNA分子翻译成VEGF2(反义-Okano,J.Neurochem.,56:560(1991);Oligodeoxynucleotides asAntisense Inhibitors of Gene Expression,CRC Press,Boca Raton,FL(1988))。
或者,上述寡核苷酸可以通过本领域公知的程序输送到细胞,从而反义RNA或DNA可以体内表达以通过上述方式抑制VEGF2的生产。
因此,VEGF2的反义构建体可以抑制VEGF2的血管生成活性并防止实体瘤的进一步生长甚至抑制之,这是因为血管生成和新血管化是实体瘤生长的重要步骤,这些反义构建体还可以用于治疗风湿性关节炎、银屑病和糖尿病性视网膜疾病,这些病的特征都是异常血管生成。
多肽、其片段或其它衍生物、或其类似物、或表达其的细胞可用作免疫原生产抗体,这些抗体可以是例如多克隆抗体或单克隆抗体。本发明还包括嵌合抗体、单链抗体、人源化抗体,以及Fab片段,或者Fab表达文库的产物。可使用现有技术中公知的各种程序生产这些抗体和片段。
抗对应于本发明的序列的多肽的抗体可以通过将多肽直接注射给动物或将多肽给予动物、优选非人而获得,如此获得的抗体随后与多肽本身结合。以这种方式,仅编码多肽的一个片段的序列也可用于生产与完整天然多肽结合的抗体,这种抗体随后可用于从表达该多肽的组织中分离该多肽。为制备单克隆抗体,可使用任何提供由连续细胞系培养物生产的抗体的技术,例如杂交瘤技术(Kohler and Milstein,1975,Nature,256:495-497),三瘤技术,人B细胞杂交瘤技术(kozbor et al.,1983,Immunology Today 4:72),以及生产人单克隆多肽的EBV-杂交瘤技术(Cole,etal.,1985,in Monoclonal Antibodies and Cancer Therapy,Alan R.Liss,Inc.,pp.77-96)。
用于生产单链抗体的技术(USP4.946,778)可以用来生产本发明的免疫原性多肽产物的单链抗体。另外,转基因小鼠也可用来表达本发明的免疫原性多肽产物的人源化单链抗体。
可以鉴别并应用中和抗体来掩蔽血管内皮生长因子,这一点已在抗VEGF的小鼠模型***中得以证实。VEGF2还可通过基因内的一些显性失活突变体进行失活。已知PDGFα和β形成异二聚体或同二聚体,VEGF形成同二聚体,VEGF2之间的类似的相互作用也可以预期。因此,这些抗体可用于阻断VEGF2的血管生成活性,并延迟实体瘤的生长,这些抗体还可以用于治疗由于VEGF2的存在而导致的增加的血管通透性引起的炎症。
这些抗体还可以进一步用于免疫分析以检测个体中是否存在肿瘤,酶免疫分析可用采自个体的血样进行,VEGF2水平升高可认为是癌症的诊断标志。
本发明还涉及本发明的多肽的拮抗剂/抑制剂,该拮抗剂/抑制剂抑制或消除该多肽的功能。
因此,例如,拮抗剂与本发明的多肽结合并抑制或消除其功能,拮抗剂例如可以是与多肽结合的抗体,或在某些情况下是寡核苷酸。抑制剂的一个例子是与多肽结合并占据多肽的催化位点的小分子从而使催化位点不能与底物接触,进而防止正常的生物学活性,这些小分子的例子包括但不限于小肽或类肽分子。
还可以生产VEGF2截短的产物,其能与野生型VEGF2相互作用形成不能激活内皮细胞生长的二聚体,因此使内源性VEGF2失活。或者,VEGF2的突变形式本身形成二聚体并占据靶细胞表面的合适的酪氨酸激酶的配位体结合区,但却不能激活细胞生长。
或者,可以采用与受体(本发明的多肽正常与该受体结合)结合的本发明的多肽的拮抗剂,该拮抗剂可以是紧密相关的蛋白质以便它们识别并结合于天然蛋白质的受***点,但它们是天然蛋白质的无活性形式并由此因为受***点被占据而防止VEGF2的作用。以这种方式,VEGF2的作用被防止,拮抗剂/抑制剂通过占据由VEGF2识别的肿瘤的受***点或失活VEGF2本身而可用作抗肿瘤药物。拮抗剂/抑制剂还可用于防止由VEGF2的增加血管通透性引起的炎症,拮抗剂/抑制剂还可用于治疗实体肿瘤生长、糖尿病性视网膜疾病、银屑病和风湿性关节炎。
拮抗剂/抑制剂可与例如上述的药物学可接受载体形成组合物而应用。
以下将参照实施例进一步描述本发明,但是应理解的是,本发明不受这些实施例的限制。除非另有说明,所有的份或量均为重量份或重量。
为促进对下述实施例的理解,以下将描述常用的方法和/或术语。
“质粒”的命名是将小写字母p置于前面和/或后跟大写字母和/或数字。本文的起始质粒或者是商购得到的,或者是公众可以不受限制地得到的,或者可以根据公布的程序由可得到的质粒构建的。另外,与所述的这些等效的质粒是本领域公知的,并对本领域普通技术人员是显而易见的。
DNA的“消化”是指用仅作用于DNA的特定序列的限制性酶对DNA的催化裂解。本文所用的各种限制性酶是可商购的,其反应条件、辅因子和其它要求对于本领域普通技术人员是公知的。为分析目的,典型地,1μg质粒或DNA片段使用约2单位的酶,反应体积为20μl缓冲液;为分离DNA片段用于构建质粒的目的,典型地,在较大体积中用20-250单位酶消化5-50μgDNA。对特定限制性酶的合适的缓冲液和底物由厂商提供。通常使用37℃1小时的温育时间,但可根据供应商的指示而变化。消化后,将反应物直接在聚丙烯酰胺凝胶上电泳以分离所需片段。
裂解片段的大小分离是根据Goeddel,D.et al.,Nucleic Acids Res.,8:4057(1980)所述在8%聚丙烯酰胺凝胶上进行。
“寡核苷酸”是指可化学合成的单链聚脱氧核苷酸或两个互补的聚脱氧核苷酸链。这种合成的寡核苷酸没有5’磷酸,并因此如果不在激酶的存在下用ATP加上磷酸则不能与另一寡核苷酸连接。合成的寡核苷酸将与没有经过脱磷酸化的片段连接。
“连接”是指在两个双链核酸片段之间形成磷酸二酯键的过程(Maniatis,T.,et al.,Id.,p.146)。除非另有说明,连接可以采用公知的缓冲液,在每0.5μg约等摩尔的待连接DNA片段使用10单位T4 DNA连接酶(“连接酶”)的条件下进行。
除非另有说明,使用Graham,F.and Van der Eb.A.,Virology,52:456-457(1973)的方法进行转化。实施例1 VEGF2在人组织和乳腺癌细胞系中的表达模式
进行Northern印迹分析以检测VEGF2在人组织和在人组织的乳腺癌细胞系中的表达水平。用RNAzolTMB***(Biotecx Laboratories,Inc.)分离总细胞RNA样品,从每个乳腺组织和特异细胞系中分离出的约10μg总RNA在1%琼脂糖凝胶上分离并转印到尼龙滤膜上(Molecular Cloning,Sambrook Fritsch,andManiatis,Cold Spring Harbor Press,1989)。根据Stratagene Prime-It试剂盒用50ng DNA片段进行标记反应,用5’Prime-3 Prime,Inc.的Select-G-50柱纯化标记的DNA。然后,滤膜与1,000,000cpm/ml放射标记的全长VEGF2基因在0.5M NaPO4和7%SDS中于65℃杂交过夜,用0.5×SSC,0.1%SDS在室温和60℃各洗两次后,用增感屏将滤膜暴露在-70℃下过夜。在2个乳腺癌细胞系中观察到1.6Kd的信号,泳道4代表一株不依赖于***而生长的具有非常强的致瘤性的细胞系,见图4。同样,得自10个人成年组织的10μg总RNA在琼脂糖凝胶上分离并转印到尼龙滤膜上,随后滤膜与放射标记的VEGF2探针在7%SDS,0.5M NaPO4,pH7.2;1%BSA中于65℃杂交过夜,接着在0.2×SSC中于65℃洗,用增感屏将滤膜在-70℃下暴露于胶片24天,见图5。实施例2通过体外转录和翻译表达VEGF2
体外转录和翻译VEGF2 cDNA以确定由全长和部分VEGF2 cDNA编码的可翻译多肽的大小。使用3套引物,1)M13-反向和正向引物;2)M13-反向引物和VEGF引物F4;3)M13-反向引物和VEGF引物F5,用PCR扩增***在pBluescriptSK载体中的VEGF2的全长和部分cDNA***片段。这些引物的序列如下。M13-2反向引物:5’-ATGCTTCCGGCTCGTATG-3’这一序列位于pBluescript载体中的VEGF2 cDNA***片段的5’端的上游并与cDNA呈反义方向,在这一引物与VEGF2 cDNA之间有一T3启动子序列。M13-2正向引物:5’-GGGTTTTCCCAGTCACGAC-3’这一序列位于pBluescript载体中的VEGF2 cDNA***片段的3’端的下游并与cDNA呈反义方向。VEGF引物F4:5’-CCACATGGTTCAGGAAAGACA-3’这一序列以反义方向位于VEGF2 cDNA内的1259-1239bp处,其距离终止密码子约169bp,并在该cDNA最后一个核苷酸的266bp之前。
用所有三对引物的PCR反应产生在cDNA***片段之前带有T3启动子序列的扩增产物。第一和第三对引物产生的PCR产物编码VEGF2的全长多肽,第二对引物产生的PCR产物缺少了VEGF2多肽的C-末端的36个氨基酸的编码序列。
使用约0.5μg第一对引物的PCR产物、1μg第二对引物的PCR产物、1μg第三对引物的PCR产物进行体外转录/翻译,该体外转录/翻译反应的反应体积为25μl,使用TNTTM Coupled Reticulocyte Lysate Systems(Promega,CAT#L4950)。具体地,反应体系含有12.5μl TNT兔网织红细胞裂解物,2μl TNT反应缓冲液,1μl T3聚合酶,1μl 1mM氨基酸混合物(无甲硫氨酸),4μl 35S-甲硫氨酸(>1000Ci/mmol,10mCi/ml),1μl 40U/μl的RNasin核糖核酸酶抑制剂,0.5或1μg PCR产物,加入无核酸酶的水至体积为25μl,在30℃温育反应物2小时,在4-20%梯度SDS-PAGE凝胶上分析5μl反应产物,在25%异丙醇和10%乙酸中固定后,干燥凝胶并在70℃将凝胶在X-光胶片上曝光过夜。
如图6所示,含有全长VEGF2 cDNA和在3’-非翻译区(3’-UTR)缺少了266bp的cDNA的PCR产物产生相同长度的翻译产物,其分子量计算为38-40dk(泳道1 & 3),缺少了全部3’UTR和编码C-末端36个氨基酸的序列的cDNA翻译成计算分子量为36-38kd的多肽(泳道2)。
序列表(1)一般信息:(i)申请人:HU,ET AL。(ii)发明名称:血管内皮生长因子2(iii)序列数:2(iv)联系地址:
(A)联系人:CARELLA,BYRNE,BAIN,GILFILLAN,CECCHI,STEWART &OLSTEIN
(B)街道:6 BECKER FARM ROAD
(C)城市:ROSELAND
(D)州:新泽西州
(E)国家:美国
(F)邮编:07068(v)计算机可读形式:
(A)媒介类型:3.5寸软盘
(B)计算机:IBM PS/2
(C)操作***:MS-DOS
(D)软件:WORD PERFECT 5.1(vi)本申请资料
(A)申请号:08/207,550
(B)申请日:1994年3月8日
(C)分类:(v)在先申请资料
(A)申请号:
(B)申请日:(vi)律师/代理人信息:
(A)姓名:FERRARO,GREGORY D.
(B)注册号:36,134
(C)案号/文档号:325800-148(viii)通讯信息:
(A)电话:201-994-1700
(B)传真:201-994-1744(2)SEQ ID NO:1的信息(i)序列特征
(A)长度:1525碱基对
(B)类型:核酸
(C)链性:单链
(D)拓扑结构:线性(ii)分子类型:cDNA(xi)序列描述:SEQ ID NO:1CGAGGCCACG GCTTATGCAA GCAAAGATCT GGAGGAGCAG TTACGGTCTG TGTCCAGTGT 60AGATGAACTC ATGACTGTAC TCTACCCAGA ATATTGGAAA ATGTACAAGT GTCAGCTAAG 120GAAAGGAGGC TGGCAACATA ACAGAGAACA GGCCAACCTC AACTCAAGGA CAGAAGAGAC 180TATAAAATTT CGTGCAGCAC ATTATAATAC AGAGATCTTG AAAAGTATTG ATAATGAGTG 240GAGAAAGACT CAATGCATGC CACGGGAGGT GTGTATAGAT GTGGGGAAGG AGTTTGGAGT 300CGCGACAAAC ACCTTCTTTA AACCTCCATG TGTGTCCGTC TACAGATGTA GGGGTTGCTG 360CAATAGTGAG GGGCTGCAGT GCATGAACAC CAGCACGAGC TACCTCAGCA AGACGTTATT 420TGAAATTACA GTGCCTCTCT CTCAAGGCCC CAAACCAGTA ACAATCAGTT TTGcCAATCA 480CACTTCCTGC CGATGCATGT CTAAACTGGA TGTTTACAGA CAAGTTCATT CCATTATTAG 540ACGTTCCCTG CCAGCAACAC TACCACAGTG TCAGGCAGCG AACAAGACCT GCCCCACCAA 600TTACATGTGG AATAATCACA TCTGCAGATG CCTGGCTCAG GAAGATTTTA TGTTTTCCTC 660GGATGCTGGA GATGACTCAA CAGATGGATT CCATGACATC TGTGGACCAA ACAAGGAGCT 720GGATGAAGAG ACCTGTCAGT GTGTCTGCAG AGCGGGGCTT CGGCCTGCCA GCTGTGGACC 780CCACAAAGAA CTAGACAGAA ACTCATGCCA GTGTGTCTGT AAAAACAAAC TCTTCCCCAG 840CCAATGRGGG GCCAACCGAC AATTTGATGA AAACACATGC CAGTGTGTAT GTAAAAGAAC 900CTGCCCCAGA AATCAACCCC TAAATCCTGG AAAATGTGCC TGTGAATGTA CAGAAAGTTC 960ACAGAAATGC TTGTTAAAAG GAAAGAAGTT CCACCACCAA ACATGCAGCT GTTACAGACG 1020GCCATGTACG AACCGCCAGA AGGCTTGTGA GCCAGGATTT TCATATAGTG AAGAAGTGTG 1080TCGTTGTGTC CCTTCATATT GGCAAAGACC ACAAATGAGC TAAGATTGTA CTGTTTTCCA 1140GTTCATCGAT TTTCTATTAT GGAAAACTGT GTTGCCACAG TAGAACTGTC TGTGAACAGA 1200GAGACCCTTG TGGGTCCATG CTAACAAAGA CAAAAGTCTG TCTTTCCTGA ACCATGTGGA 1260TAACTTTACA GAAATGGACT GGAGCTCATC TGCAAAAGGC CTCTTGTAAA GACTGGTTTT 1320CTGCCAATGA CCAAACAGCC AAGATTTTCC TCTTGTGATT TCTTTAAAAG AATGACTATA 1380TAATTTATTT CCACTAAAAA TATTGTTTCT GCATTCATTT TTATAGCAAC AACAATTGGT 1440AAAACTCACT GTGATCAATA TTTTTATATC ATGCAAAATA TGTTTAAAAT AAAATGAAAA 1500TTGTATTATA AAAAAAAAAA AAAAA 1525(2)SEQ ID NO:2的信息(i)序列特征
(A)长度:350个氨基酸
(B)类型:氨基酸
(C)链性:
(D)拓扑结构:线性(ii)分子类型:蛋白质(xi)序列描述:SEQ ID NO:2Met Thr Val Lys Tyr Pro Glu Tyr Trp Lys Met Tyr Lys Cys Gln
-20 -15 -10Leu Arg Lys Gly Gly Trp Gln His Asn Arg Glu Gln Ala Asn Leu
-5 1 5Asn Ser Arg Thr Glu Glu Thr Ile Lys Phe Ala Ala Ala His Tyr
10 15 20Asn Thr Glu Ile Leu Lys Ser Ile Asp Asn Glu Trp Arg Lys Thr
25 30 35Gln Cys Met Pro Arg Glu Val Cys Ile Asp Val Gly Lys Glu Phe
40 45 50Gly Val Ala Thr Asn Thr Phe Phe Lys Pro Pro Cys Val Ser Val
55 60 65Tyr Arg Cys Gly Gly Cys Cys Asn Ser Glu Gly Leu Gln Cys Met
70 75 80Asn Thr Ser Thr Ser Tyr Leu Ser Lys Thr Leu Phe Glu Ile Thr
85 90 95Val Pro Leu Ser Gln Gly Pro Lys Pro Val Thr Ile Ser Phe Ala
100 105 110Asn His Thr Ser Cys Arg Cys Met Ser Lys Leu Asp Val Tyr Arg
115 120 125Gln Val His Ser Ile Ile Arg Arg Ser Leu Pro Ala Thr Leu Pro
130 135 140Gln Cys Gln Ala Ala Asn Lys Thr Cys Pro Thr Asn Tyr Met Trp
145 150 155Asn Asn His Ile Cys Arg Cys Leu Ala Gln Glu Asp Phe Met Phe
160 165 170Ser Ser Asp Ala Gly Asp Asp Ser Thr Asp Gly Phe His Asp Ile
175 180 185Cys Gly Pro Asn Lys Glu Leu Asp Glu Glu Thr Cys Gln Cys Val
190 195 200Cys krg Ala Gly Leu Arg Pro Ala Ser Cys Gly Pro His Lys Glu
205 210 215Leu Asp Arg Asn Ser Cys Gln Cys Val Cys Lys Asn Lys Leu phe
220 225 230Pro Ser Gln Cys Gly Ala Asp Arg Glu Phe Asp Glu Asn Thr Cys
235 240 245Gln Cys Val Cys Lys Arg Thr Cys Pro Arg Asn Gln Pro Leu Asn
250 255 260Pro Gly Lys Cys Ala Cys Glu Cys Thr Glu Ser Pro Gln Lys Cys
265 270 275Cys Leu Leu Lys Gly Lys Lys Phe His His Gln Thr Cys Ser Cys
280 285 290Tyr Arg Arg Pro Cys Thr Asn Arg Gln Lys Ala Cys Glu Pro Gly
295 300 305Phe Ser Tyr Ser Glu Glu Val Cys Arg Cys Val Pro Ser Tyr Trp
310 315 320Gln Arg Pro Gln Met Ser
325
Claims (21)
1、一种编码VEGF2的分离的多核苷酸,所述的多核苷酸选自如下一组:
编码具有图1的推导的氨基酸序列的VEGF2多肽或者所述多肽的活性片段、类似物或衍生物的多核苷酸;
编码具有由ATCC保藏号75698所含eDNA编码的氨基酸序列的VEGF2多肽或者所述多肽的活性片段、类似物或衍生物的多核苷酸;
2、权利要求1的多核苷酸,其中该多核苷酸是DNA。
3、权利要求1的多核苷酸,其中该多核苷酸是RNA。
4、权利要求1的多核苷酸,其中该多核苷酸是基因组DNA。
5、权利要求2的多核苷酸,其中所述的多核苷酸编码具有图1的推导的氨基酸序列的VEGF2。
6、权利要求2的多核苷酸,其中所述的多核苷酸编码由ATCC保藏号75698的cDNA编码的VEGF2多肽。
7、权利要求1的多核苷酸,具有图1所示的VEGF2的编码序列。
8、权利要求2的多核苷酸,具有ATCC保藏号75698所保藏的VEGF2的编码序列。
9、含有权利要求2的DNA的载体。
10、用权利要求9的载体遗传工程化的宿主细胞。
11、一种用于生产多肽的方法,包括:
由权利要求10的宿主细胞表达由所述DNA编码的多肽。
12、一种生产能表达多肽的细胞的方法,包括用权利要求9的载体对细胞进行遗传工程化。
13、可与权利要求2的DNA杂交并编码具有VEGF2活性的多肽的分离的DNA。
14、一种多肽,选自如下一组:(i)具有图1的推导的氨基酸序列的VEGF2多肽及其活性片段、类似物和衍生物;(ii)由ATCC保藏号75698的cDNA编码的VEGF2多肽及所述多肽的活性片段、类似物和衍生物。
15、权利要求14的多肽,其中该多肽是具有图1的推导的氨基酸序列的VEGF2。
16、抗权利要求14的多肽的抗体。
17、抗权利要求14的多肽的拮抗剂。
18、治疗需要VEGF2的患者的方法,包括:给予患者治疗有效量的权利要求14的多肽。
19、治疗需要抑制VEGF2的患者的方法,包括:给予患者治疗有效量的抗权利要求14的多肽的拮抗剂。
20、一种药物组合物,包括权利要求14的多肽和一药物学可接受载体。
21、权利要求18的方法,其中通过提供给患者编码所述多肽的DNA并在体内表达所述多肽而给予治疗有效量的多肽。
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-
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- 1994-05-12 EP EP94923874A patent/EP0751992B1/en not_active Revoked
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- 1994-05-19 ZA ZA943464A patent/ZA943464B/xx unknown
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- 2002-02-01 US US10/060,523 patent/US20020182683A1/en not_active Abandoned
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Cited By (4)
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CN101665536B (zh) * | 1997-04-07 | 2013-07-03 | 基因技术股份有限公司 | 抗-血管内皮生长因子的抗体 |
CN1328376C (zh) * | 1999-04-16 | 2007-07-25 | 杰南技术公司 | 血管内皮细胞生长因子变体及其应用 |
CN100399030C (zh) * | 1999-11-16 | 2008-07-02 | 杰南技术公司 | 用于vegf的elisa |
CN101400787B (zh) * | 2006-01-27 | 2013-01-23 | 株式会社Prostemics | 采用脂肪来源成体干细胞大量制造生长因子的方法 |
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DE69434538D1 (de) | 2005-12-15 |
US20020182683A1 (en) | 2002-12-05 |
CN1267550C (zh) | 2006-08-02 |
US7498417B2 (en) | 2009-03-03 |
US5935820A (en) | 1999-08-10 |
AU696764C (en) | 1995-09-25 |
JP4439490B2 (ja) | 2010-03-24 |
EP0751992B1 (en) | 2005-11-09 |
EP0751992A4 (en) | 1997-12-17 |
AU696764B2 (en) | 1998-09-17 |
DK0751992T3 (da) | 2006-03-06 |
JP2004000267A (ja) | 2004-01-08 |
CA2184584A1 (en) | 1995-09-14 |
ZA943464B (en) | 1995-11-20 |
EP0751992A1 (en) | 1997-01-08 |
ATE309360T1 (de) | 2005-11-15 |
JP2006238893A (ja) | 2006-09-14 |
JPH09510093A (ja) | 1997-10-14 |
AU7394194A (en) | 1995-09-25 |
DE69434538T2 (de) | 2006-08-10 |
WO1995024473A1 (en) | 1995-09-14 |
CA2184584C (en) | 2003-04-08 |
US20040214286A1 (en) | 2004-10-28 |
ES2249762T3 (es) | 2006-04-01 |
US20090192088A1 (en) | 2009-07-30 |
JP2006333865A (ja) | 2006-12-14 |
CA2414016A1 (en) | 1995-09-14 |
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