CN1326467A - 表达以及分泌制管张素和endostatin的免疫融合物 - Google Patents
表达以及分泌制管张素和endostatin的免疫融合物 Download PDFInfo
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Abstract
所公开的内容为编码一种免疫球蛋白Fc-血管生成抑制剂融合蛋白的核苷酸序列,例如DNA序列或RNA序列。所述血管生成抑制剂可以是制管张素、endostatin、具有制管张素活性的纤溶酶原片段或具有endostatin活性的胶原蛋白ⅩⅧ片段。可以将所述核苷酸序列插入到合适的表达载体中并在哺乳动物细胞中表达。此外所公开的内容还有通过表达这种核苷酸序列生产一系列免疫球蛋白Fc-血管生成抑制剂融合蛋白。所公开的内容还有使用这种核苷酸序列和融合蛋白治疗血管生成性疾病的方法。
Description
发明领域
本发明一般涉及制备和使用含有血管生成抑制剂的融合蛋白的方法和组合物。更具体地讲,本发明涉及制备和使用含有免疫球蛋白Fc区和血管生成抑制剂的称为免疫融合物(immunofusin)的融合蛋白的方法和组合物。
发明背景
人们发现两种有效血管生成抑制剂制管张素(O’Reilly等(1994)Cell 79:315)和endostatin(O’Reilly等(1997)Cell 88:277)由原发性肿瘤天然产生。这两种蛋白是内皮细胞增生的特异性抑制剂并通过阻断血管生成(阻断供给肿瘤营养的新血管的形成)而抑制肿瘤生长。研究表明,这些血管生成抑制剂甚至在非常高剂量下都是无毒的,它们可以抑制转移瘤的生长并可以使原发性肿瘤退化到显微休眠状态。已鉴定出这两种抑制剂是非常大的完整分子的蛋白酶解片段。已经发现制管张素是纤溶酶原的一个片段,而endostatin是胶原蛋白XVIII的一个片段。
在癌症领域人们已非常关注这两种蛋白,因为已经证实它们抑制小鼠中的许多不同类型肿瘤的生长,而没有明显的副作用或抗药性。传统的化学治疗通常导致主要由癌细胞遗传不稳定性引起的获得性抗药性。使用血管生成抑制剂的治疗不是把癌细胞作为目标而是针对支持肿瘤生长的正常内皮细胞。因为内皮细胞在遗传上是稳定的,所以血管生成抑制剂治疗可减少抗药性。这些研究表明,采用endostatin的延长的抗血管生成治疗的小鼠不发生抗药性。因此,对小鼠周期性重复的endostatin治疗延长肿瘤休眠期以及停止治疗后肿瘤不复发(Boehm等(1997)Nature 390:404)。
尽管用小鼠获得有希望的结果,但是仍然不能用大肠杆菌、杆状病毒、酵母和哺乳动物表达系统大批量生产临床级可溶的、有活性的制管张素和endostatin。用大肠杆菌表达获得不确定组合物的不溶性蛋白聚集物,不能将这种蛋白注射到人体内。其它生产方法诸如杆状病毒和哺乳动物表达系统得到非常低水平的重组蛋白(O’Reilly等(1997)Cell 88:277)。
迄今可以将该低得率表达系统解释为制管张素和endostatin这两种是非常大的蛋白内部片段。在缺乏从前体分子中切割的残基时,该截短蛋白可能没有进行合适的折叠。例如,制管张素有26个形成大量二硫键的半胱氨酸残基。在分泌途径中,制管张素自身的表达不能提供最适环境以正确地形成这些大量的二硫键。此外,以大肠杆菌生产的重组endostatin蛋白在透析期间沉淀,可能是由于endostatin的疏水性引起(O’Reilly等(1997)Cell 88:277)。
以其现存形式使用制管张素和endostatin主要障碍是不得不将相对大量的蛋白每天注射,持续数星期至数月以达到所需的临床效果。例如,目前在小鼠模型中,需要剂量20mg/kg/天的endostatin以证实最佳药效(Boehm等(1997)Nature 390:404)。已知急需在临床上试验endostatin和制管张素,重要是的得到可以产生大量临床级物质的生产方法。
在哺乳动物细胞中,已经用来生产高水平表达融合蛋白的一种表达系统是编码信号序列、免疫球蛋白Fc区和靶蛋白的DNA构成物。这种构成物的融合产物统称为“免疫融合物”。已经将几种靶蛋白成功地表达为免疫融合物,它们包括:IL2、CD26、Tat、Rev、OSF-2、βIG-H3、IgE受体、PSMA和gp120。这些表达构成物公开于美国专利第5,541,087号和美国专利第5,726,044号,其公开内容均通过引用结合到本文中。
在哺乳动物细胞中,表达重组融合蛋白的主要目的一直是试图对所述杂种分子赋予新的或有用的特性,例如合适的折叠、增加溶解度、体内引导细胞因子或毒素、Fc受体结合、补体活化、A蛋白结合、延长循环半寿期和增加穿过血-脑屏障的能力。在哺乳动物细胞中所产生的重组融合蛋白的实例包括细胞因子免疫缀合物(Gillies等(1992)Proc.Natl Acad Sci USA 89:1428;Gillies等(1993)Bioconjugate Chemistry 4:230)、免疫粘附素(Capon等(1989)Nature337:525)、免疫毒素(Chaudhary等(1989)Nature 339:394)和神经生长因子缀合物(Friden等(1993)Science 259:373)。上述每个出版物都通引用结合到本文中。
本发明的一个目的是提供有利于在各种哺乳动物宿主细胞中有效地生产和分泌血管生成抑制剂的新的DNA。本发明的另一个目的是提供治疗哺乳动物的方法,该治疗方法采用编码血管生成抑制剂蛋白的核酸或氨基酸序列明确的血管生成抑制剂蛋白,包括非天然的、生物合成或其它人工蛋白诸如合理设计产生的蛋白。
发明概述
本发明的特征是用于制备和使用含有血管生成抑制剂蛋白的融合蛋白的方法和组合物。所述融合蛋白可以促进高水平表达有生物活性的血管生成抑制剂蛋白。然后在给予哺乳动物(例如人)之前,可以将血管生成抑制剂蛋白从所述融合蛋白中切下并将其与药学上可接受的载体组合。或者,可以将编码含有血管生成抑制剂的融合蛋白的核酸序列或氨基酸序列明确的含有血管生成抑制剂的融合蛋白与药学上可接受的载体组合,然后给予所述哺乳动物。
在一方面,本发明提供编码本发明融合蛋白的核酸分子,例如DNA或RNA分子。所述核酸分子编码信号序列、免疫球蛋白Fc区和至少一种靶蛋白,本文也称为血管生成抑制剂蛋白,所述蛋白选自制管张素、endostatin、具有制管张素活性的纤溶酶原片段、具有endostatin活性的胶原蛋白XVIII片段和它们的组合物。在一个优选的实施方案中,所述核酸分子从5’到3’方向依次编码信号序列、免疫球蛋白Fc区和靶蛋白序列。在另一个优选的实施方案中,所述核酸分子从5’到3’方向顺序地编码信号序列、靶序列和免疫球蛋白Fc区。
在另一个优选的实施方案中,所述免疫球蛋白Fc区包括免疫球蛋白铰链区,而最好是包括至少一个免疫球蛋白重链恒定区,例如免疫球蛋白重链恒定区第2(CH2)结构域、免疫球蛋白重链恒定区第3(CH3)结构域并且根据用来产生Fc区的免疫球蛋白的类型,任选包括免疫球蛋白重链恒定区第4(CH4)结构域。在一个更优选的实施方案中,所述免疫球蛋白Fc区包括绞链区、CH2结构域和CH3结构域。在某些情况下,所述免疫球蛋白Fc区最好是缺乏至少CH1结构域。尽管所述免疫球蛋白Fc区可以基于任何免疫球蛋白种类,例如IgA、IgD、IgE、IgG和IgM,但是优选为基于IgG的免疫球蛋白Fc区。
在另一个实施方案中,可以有效地结合将本发明的核酸掺入到复制型表达载体中,然后可以将其转染到哺乳动物宿主细胞中。在另一个优选的实施方案中,本发明提供包含本发明的这种核酸序列的宿主细胞。
在另一方面,本发明提供包含或者直接通过多肽键或者用多肽接头的方式,与靶蛋白连接的免疫球蛋白Fc区的融合蛋白,所述靶蛋白选自制管张素、endostatin、具有制管张素活性的纤溶酶原片段、具有endostatin活性的胶原蛋白XVIII片段和它们的组合物。可以将所述靶蛋白通过其C-末端融合到免疫球蛋白Fc区的N-末端。然而,在一个更优选的实施方案中,将所述靶蛋白通过其N-末端融合到免疫球蛋白Fc区的C-末端,
在另一个实施方案中,所述融合蛋白可以包含选自制管张素、endostatin、具有制管张素活性的纤溶酶原片段和具有endostatin活性的胶原蛋白XVIII片段的第二靶蛋白。在这种类型的构成物中,第一和第二靶蛋白可以是相同或不相同的蛋白。例如,在一个优选的实施方案中,所述融合蛋白包含第一靶蛋白制管张素、免疫球蛋白Fc区和第二靶蛋白endostatin。可以将第一和第二靶蛋白或者直接地或者用多肽接头的方连接在一起。另一方面,可以将这两种靶蛋白或者直接地或者通过多肽接头连接到所述免疫球蛋白Fc区。在后一种情况下,将第一靶蛋白连接到免疫球蛋白Fc区的N-末端,而将第二靶蛋白连接到免疫球蛋白Fc区的C-末端。
在另一个实施方案中,两种融合蛋白可以或者通过共价键(例如二硫键或肽键)或者通过非共价键结合产生多聚体蛋白。在一个优选的实施方案中,将两种融合蛋白通过半胱氨酸残基(最好是位于排列在这两条链的免疫球蛋白Fc区内的免疫球蛋白铰链区内)的一个或多个二硫键共价结合。
在一个优选的实施方案中,所述靶蛋白包含分子量约40kD的纤溶酶原片段,并任选地包含SEQ ID NO:3所示的氨基酸序列。在另一个优选的实施方案中,所述靶蛋白包含具有SEQ ID NO:1所示氨基酸序列的胶原蛋白XVIII片段。此外,所述靶蛋白可以是全长制管张素或endostatin或它们的生物活性片段。在制备某些融合蛋白中所述靶蛋白的来源将取决于所述靶蛋白预期的用途。例如,如果是将所述靶蛋白给予人,则所述靶蛋白最好是人源靶蛋白。
在另一方面,本发明提供生产包含免疫球蛋白Fc区和靶蛋白的融合蛋白的方法,所述靶蛋白选自制管张素、endostatin、具有制管张素活性的纤溶酶原片段和具有endostatin活性的胶原蛋白XVIII片段。所述方法包括步骤(a)提供含有编码这种融合蛋白的DNA分子(或者有或者没有信号序列)的哺乳动物细胞和(b)培养所述哺乳动物细胞以生产所述融合蛋白。然后可以将所生产的融合蛋白收集、重折叠,必要时,采用众所周知的常规纯化技术纯化,然后在本领域中使用。假设所述融合蛋白包含在免疫球蛋白Fc区和所述靶蛋白之间排列的蛋白水解酶切割位点,则可以采用常规蛋白水解酶将所述靶蛋白从所述融合蛋白中切除,并且必要时,在使用之前纯化。
在另一方面,本发明提供治疗需要血管生成抑制剂基本治疗的哺乳动物(例如人)的方法。例如,设想可以将本发明的血管生成抑制剂给予肿瘤患者。用血管生成抑制剂治疗可以使肿瘤生长减慢或停止,并在某些情况下,可以引起肿瘤消退。治疗可以包括给予所述哺乳动物足量的血管生成抑制剂以使肿瘤生长减慢或停止。可以以融合蛋白的形式或核酸、最好是与表达载体有效结合核酸与药学上可接受的载体组合,提供所述血管生成抑制剂。
从以下详细描述、附图和权利要求书中,本发明的前面所述和其它目的、特点和优点将更加显而易见。
附图简述
图1A-1F是按照本发明构建的典型血管生成抑制剂融合蛋白的示意图(参见实施例10-15)。所述图分别描述了,图1A,制管张素的Fc-Kringle 1;图1B,制管张素的Fc-内部Kringle 1;图1C,制管张素的Fc-Endostatin-GlySer接头-内部Kringle 1;图1D,制管张素的Fc-Endostatin-GlySer接头-Kringle 1;图1 E,Fc-Endostatin-GlySer接头-制管张素;图1F,制管张素-Fc-Endostatin。垂直线代表连接排列在所述Fc分子绞链区内的连接半胱氨酸残基(C)任选的二硫键。
发明详述
本发明提供融合蛋白,本文是指免疫融合物,所述融合蛋白可用于大批量生产临床级血管生成抑制剂。在使用之前,可以将所述血管生成抑制剂从免疫融合物蛋白构成物中切下。然而,设想可以将所述免疫融合物或编码所述免疫融合物的核酸直接给予需要用血管生成抑制剂治疗的哺乳动物。
因此,本发明提供包含免疫球蛋白Fc区和至少一种靶蛋白的融合蛋白,靶蛋白在本文是指血管生成抑制剂。血管生成抑制剂最好是选自制管张素、endostatin、具有制管张素活性的纤溶酶原片段、具有endostatin活性的胶原蛋白XVIII片段。然而,设想可以将具有血管生成抑制剂活性的其它多肽(目前已知的或以后发现的)表达成为本文所述类型的融合蛋白。
在如图1A-1F的附图中,说明了体现本发明蛋白构成物的6个典型的实施方案。因为优选为二聚体构成物,所以用通过半胱氨酸在相邻的亚基之间的一对二硫键交联的二聚体进行所有说明。在所述附图中,图示二硫键通过免疫球蛋白铰链区将两个免疫球蛋白Fc部分连接在一起,因此具有这些分子的天然形式的特征。当优选包括Fc铰链区的构成物并且该构成物已经显示有希望作为治疗剂的同时,本发明设想可以根据需要选择在其它位置上进行交联。此外,在某些情况下,用于实施本发明的二聚体和多聚体可以由非共价结合而产生,例如,疏水性相互作用。
因为同源二聚体构成物是本发明重要的实施方案,所以图1说明了这样的构成物。应当认为异源二聚体结构也是有用的,但正如本领域技术人员已知的,通常难以将它们纯化。然而,可以构建用于在包括人类在内的各种哺乳动物物种中对抑制血管生成有活性的构成物,包括同源二聚体和异源二聚体的混合物。例如,所述异源二聚体结构的一条链可以包含endostatin,而另一条链则可以包含制管张素。
图1A说明按照实施例10中所述方法生产的二聚体构成物。所述二聚体的每个单体包含包括铰链区、CH2结构域和CH3结构域的免疫球蛋白Fc区1。直接与Fc区I的C端连接的是制管张素2的第一Kringle区,内环和外环两种。图1B显示包含如图1A中的相同Fc区的本发明的第二个实施方案(参见实施例11),这时仅具有与Fc区1的C末端连接的制管张素3的Kringle 1的内环。从图1C到1E描述了本发明所述蛋白构成物的各种实施方案,所述蛋白构成物包括作为靶蛋白的串联排列和接头连接的多种血管生成抑制剂。图1C中,所述靶蛋白包含全长endostatin 4、多肽接头5和制管张素3的Kringle1的内环。图1D图示蛋白包含如图1A相同的Fc区以及包含全长endostatin 4、多肽接头5和全部Kringle 1区的制管张素(内环和外环两种)2的靶蛋白。因为最大C端蛋白结构域包含全长拷贝制管张素7中,所以图1E与图1D的构成物不同。
尽管图1A-1E表示Fc-X型构成物,其中X为所述靶蛋白,但是设想X-Fc型构成物也可用于实施本发明。此外,设想本发明有用的蛋白也可用公式X-Fc-X表示,其中X代表相同或不相同的靶蛋白。图1F描述了这样的一种构成物,所述构成物包含N-至C-端方向的全长人制管张素7、包括铰链区的人免疫球蛋白Fc区6和全长人endostatin结构域4。
本文所用的术语“血管生成抑制剂”是指减少或抑制哺乳动物中的新血管形成的任何多肽链。关于癌症治疗,所述血管生成抑制剂减少或抑制肿瘤内或表面、最好是在实体瘤的里面或表面的新血管形成。设想采用众所周知的多种常用方法可以鉴定有用的血管生成抑制剂。这样的方法包括,例如牛毛细血管内皮细胞增生测定、鸡尿囊绒膜(CAM)测定或小鼠角膜测定。然而,优选为CAM测定(参见,例如O’Reilly等(1994)Cell 79:315-328和O’Reilly等(1997)Cell 88:277-285,其公开内容均通过引用结合到本文中)。概括地讲,从受精三天卵白中取出具有完整卵黄的胚胎,然后置于培养皿中。于37℃、3%CO2温育三天后,将各个胚胎尿囊绒膜置于含有推定的血管生成抑制剂的甲基纤维素板中。在温育约48小时之后,于显微镜下观察所述尿囊绒膜以提供抑制区的证据。
用于实施本发明优选的血管生成抑制剂包括,例如制管张素(O’Reilly等(1994)Cell 79:315-328和美国专利第5,733,876、5,837,682和5,885,795号)和endostatin(O’Reilly等(1997)Cell 88:277-285和美国专利第5,854,205号)。如前所述,制管张素和endostatin是内皮细胞增生的特异性抑制剂并能够通过阻断血管生成而抑制肿瘤生长,即阻断供给肿瘤营养的新血管的形成。
已经鉴定制管张素为纤溶酶原的蛋白酶解片段(O’Reilly等(1994)Cell 79:315-328和美国专利第5,733,876、5,837,682和5,885,795号,这些公开内容都通过引用结合到本文中)。具体地讲,制管张素是含有至少三个纤溶酶原Kringle区的纤溶酶原的38 kDa内部片段。已经鉴定endostatin为胶原蛋白XVIII的蛋白酶解片段(O’Reilly等(1997)Cell 88:277-285,其公开内容通过引用结合到本文中)。具体地讲,endostatin是胶原蛋白XVIII的20 kDa C-末端片段。本文所用的术语“制管张素”和“endostatin”不仅是指全长蛋白,而且还分别指其变异体和生物活性片段,以及纤溶酶原和胶原蛋白XVIII的生物活性片段。术语生物活性片段,就制管张素而论是指纤溶酶原或制管张素的任何蛋白片段,根据CAM测定所述片段至少具有全长制管张素活性的30%,更优选至少70%和最优选至少90%。术语生物活性片段,就endostatin而论是指胶原蛋白XVIII或endostatin的任何蛋白片段,根据CAM测定所述片段至少具有全长endostatin活性的30%,更优选至少70%和最优选至少90%。
术语变异体包括特定变异体和等位变异体,以及其它天然产生或非天然产生的变异体,例如由常规基因工程方法产生的变异体,它们或者与本文公开的endostatin天然产生的序列或者与本文公开的制管张素天然产生序列至少70%相似或60%相同,更优选至少75%相似或65%相同和最优选的80%相似和70%相同。
为了确定候选多肽是否与参比多肽具有必不可少的相似性或同一性的百分率,首先将候选氨基酸序列和参比氨基酸序列对比,所述序列对比采用Smith和Waterman(1981),J.Mol.Biol.147:195-197中所描述的的动态编程算法(dynamic programming algorithm)和Henikoff和Henikoff(l992)“蛋白质组件的氨基酸取代矩阵”,Proc.NatlAcad Sci.USA 89:10915-10919的图2中所描述的BLOSUM62取代矩阵。对于本发明,缺口插入补偿参数(gap insertion penalty)的合适值为-12,而缺口延伸补偿参数(gap extension penalty)的合适值为-4。采用Smith-Waterman算法和BLOSUM62矩阵诸如GCG程序组(Oxford Molecular Group,Oxford,英国)进行序列对比的计算机程序是市场上可得到的并被本领域技术人员广泛使用。
一旦在候选序列和参比序列之间进行了序列对比,就可以计算出相似性的百分数。将每条序列的各个氨基酸按照它们与其它氨基酸的相似性依次比较。如果相对于两个对比的氨基酸的BLOSUM62矩阵值为零或负数,则相似性的配对数为0;否则相似性的配对数为1.0。相似性的原始数是序列对比的氨基酸相似性的配对数的总和。然后用较小候选序列或参比序列的氨基酸数除原始数,将原始数标准化。所述标准化原始数为相似性的百分率。或者,为了计算同一性的百分率,将每条序列的对比氨基酸依次进行比较。如果所述氨基酸为不相同,同一性配对数为0;否则同一性配对数为1.0。原始同一性的配对数为序列对比的相同氨基酸的总和。然后用较小候选序列或参比序列中的氨基酸数除原始数,将原始数标准化。所述标准化原始数为同一性的百分率。为了计算相似性和同一性的百分率,将插入和缺失忽略不计。因此,尽管在原始序列对比中使用缺口补偿参数,但它们不用于本计算。
将本文所公开的靶蛋白表达作为具有免疫球蛋白Fc区的融合蛋白。已知每个免疫球蛋白重链恒定区由4或5个结构域组成。所述结构域依次命名如下:CH1-铰合部-CH2-CH3(-CH4)。所述重链结构域的DNA序列在免疫球蛋白种类之间存在残余同源,例如IgG的CH2结构域与IgA和IgD的CH2结构域以及与IgM和IgE的CH2结构域同源。
本文所用的术语“免疫球蛋白Fc区”不用说是指免疫球蛋白链恒定区的羧基端部分,最好是免疫球蛋白重链恒定区或它们的一部分。例如,免疫球蛋白Fc区可以包含1)CH1结构域、CH2结构域和CH3结构域,2)CH1结构域和CH2结构域,3)CH1结构域和CH3结构域,4)CH2结构域和CH3结构域或5)两个或多个结构域和免疫球蛋白铰链区的组合。在一个优选的实施方案中,在所述DNA构成物中所用的Fc区包括至少免疫球蛋白铰链区CH2结构域和CH3结构域,而最好是缺乏至少CH1结构域。
目前衍生重链恒定区的优选的免疫球蛋白种类为IgG(Igγ)(γ亚类1、2、3或4)。可以使用其它种类的免疫球蛋白IgA(Igα)、IgD(Igδ)、IgE(Igε)和IgM(Igμ)。在美国专利第5,541,087和5,726,044号中详细地讨论了选择合适免疫球蛋白重链恒定区。从某些免疫球蛋白种类和亚类中选择特定免疫球蛋白重链恒定区序列以获得特定的结果被认为是在本领域中的技术水平内。编码免疫球蛋白Fc区的DNA构成物部分最好是包括至少一部分铰合部结构域和最好至少一部分Fcγ的CH3结构域或在任何IgA、IgD、IgE和IgM中的同源结构域。
根据应用情况,人们可以使用人类以外的物种例如小鼠或大鼠的恒定区基因。用作免疫融合物DNA构成物中的融合配偶体的Fc区一般可以来自任何物种的哺乳动物。当人们不希望在宿主细胞或动物中激发抗Fc区的免疫应答时,所述Fc区可以得自与宿主细胞或动物的相同物种。例如,当宿主动物或细胞是人时,可以使用人Fc;同样,当宿主动物或细胞是小鼠时,可以使用鼠Fc。此外,这些恒定区构成物的置换或缺失也将是有作用的,其中置换或缺失所述恒定区结构域的一个或多个氨基酸残基。一个实例是引入在CH2区上段中的氨基酸置换以产生与Fc受体的亲和力降低的Fc变异体(Cole等(1997)J.Immunol.159:3613)。其中本领域中的一般技术人员可以采用众所周知的分子生物学技术制备这样的构成物。
使用人Fcγ1作为Fc区序列有几个优点。例如,如果血管生成抑制剂Fc融合蛋白被用作生物药物,则Fcγ1结构域可以对所述融合蛋白赋予效应子功能活性。所述效应子功能活性包括生物活性诸如补体活化、涉及抗体的细胞的细胞毒性、胎盘转运和增加血清半寿期。Fc结构域还提供通过抗-Fc ELISA来检测和通过结合到金黄色葡萄球菌(Staphylococcus aureus)A蛋白(“A蛋白”)来纯化。然而,在某些应用情况下,最好是从Fc区消除特异性的效应子功能,诸如Fc受体结合或补体活化。
就血管生成抑制剂免疫融合物而论,免疫球蛋白Fc融合配偶体的一种功能是有利于血管生成抑制剂蛋白的合适折叠以产生活性血管生成抑制剂蛋白并影响所述活性部分至少在细胞外培养基中的溶解度。由于该Fc融合配偶体是亲水性的,所以血管生成抑制剂免疫融合物溶解性好,与例如大肠杆菌中产生的重组endostatm不同(O’Reilly(1997)Cell 88:277)。在本文所公开的所有实施例中,都获得高产率的免疫融合物。血管生抑制剂免疫融合物通常以浓度约30-100μg/ml分泌到培养基中,并可通过A蛋白层析容易地纯化成同质性。另外,可以将血管生成抑制剂免疫融合物切割,然后采用常规纯化方法进一步纯化,采用例如肝素琼脂糖凝胶法、赖氨酸琼脂糖凝胶法或亲和纯化法。
除高水平表达外,本发明融合蛋白还显示较长的血清半寿期,推测可能是由于它们的分子大小较大。例如,在小鼠中,人Fc-人制管张素的血清半寿期为33小时,与之相比人制管张素为4-6小时(O’Reilly等(1996)Nature Medicine 2:689)。相信分子量40kD的制管张素和分子量20kD的endostatin都太小以至通过肾过滤有效地清除。相比之下,Fc-制管张素的二聚体形式和二聚体Fc-endostatin分别是145kD和100kD,因为有两个连接于或者两个制管张素分子或者两个endostatin分子的两个免疫球蛋白Fc区。这样的二价体结构与制管张素受体和endostatin受体的结合亲和力较高。如果血管生成抑制活性是受体介导的,则Fc融合蛋白比单价体制管张素或单价体endostatin自身潜在性更有效地抑制肿瘤。此外,如果制管张素和/或endostatin属于二聚体蛋白配体类,则Fc对制管张素或endostatin产生的物理性限制使二聚作用成为一种分子内过程,因此,动态平衡有利于二聚体并增强其与受体的结合。也可以通过标准重组DNA技术将半胱氨酸残基导入到合适位置的单体上以通过共价二硫键形成而稳定该二聚体。
本文所用的术语“多价体”是指掺入两个或多个生物学活性片段的重组分子。形成该多价体分子的蛋白片段可以通过多肽肽接头连接,所述接头连接组分部分而不会引起移码并使每个分子独立起作用。
本文所用的术语“二价体”是指在本发明融合构成物中含有两个靶蛋白的多价体重组分子,例如Fc-X分子,其中X独立地选自制管张素、endostatin或它们的变异体。由于有两个X部分与免疫球蛋白Fc区(通常其自身是包括至少一部分铰链区和CH3结构域,以及任选的CH2结构域的重链片段的二聚体)融合,因此所述分子为二价体(参见,例如图1A)。如果本发明的融合构成物的形式为Fc-X-X,则所产生的Fc二聚体分子为四价体。可以通过肽接头将形成Fc-X-X分子的这两种蛋白连接。二价体分子可以增加所述分子和其受体之间的表观结合亲和力。例如,Fc-endostatin的一个endostatin部分可以以一定亲和力结合到细胞上的受体上,同一Fc-endostatin的第二个endostatin部分可以以非常高的亲和力(表观亲和力)结合到相同细胞上的第二个受体上。这是因为第一个endostatin部分结合后,第二个endostatin部分与所述受体的物理邻近效应所致。在抗体结合到抗原的情况下,表观亲和力增加至少104。
本文所用的术语“多聚体”和“多聚体的”是指或者例如通过共价性相互作用(例如通过二硫键)共价地或者例如通过疏水性相互作用非共价地使两条或多条多肽链的稳定结合。术语多聚体包括同源多聚体(其中所述多肽是相同的)以及异源多聚体(其中所述多肽是不相同的)。
本文所用的术语“二聚体的”是指特异性多聚体分子,其中两条蛋白多肽链通过共价或非共价性相互作用的稳定连接。应当理解,免疫球蛋白Fc区Fc片段自身通常为包括至少一部分铰链区和CH3结构域和任选的CH2结构域的重链片段的二聚体。已知许多蛋白配体是以二聚体结合到它们的受体上。如果蛋白配体X天然地二聚体化,则在Fc-X分子中的X部分将在更大程度上二聚体化,因为二聚体形成过程是浓度依赖性的。通过相联的免疫球蛋白Fc区连接的两个X部分的物理邻近效应将使二聚体形成成为分子内过程,使动态平衡有利于二聚体形成并增强其结合到受体上。
不用说,本发明采用常规重组DNA方法来产生用于实施本发明的Fc融合蛋白。所述Fc融合构成物最好是在DNA水平上产生,并且将所产生的DNA整合到表达载体中,然后表达产生所述免疫融合物。本文所用的术语“载体”不用说是指包含核苷酸序列的任何核酸,所述核苷酸序列可以掺入到宿主细胞中并重组及整合到所述宿主细胞基因组中或作为附加体自主复制。这样的载体包括线型核酸、质粒、噬菌粒、粘粒、RNA载体、病毒载体等。病毒载体非限制性实例包括反转录病毒、腺病毒和腺伴随病毒。本文所用的术语靶蛋白的“基因表达”或“表达”,不用说是指转录DNA序列、翻译mRNA转录物和分泌Fc融合蛋白产物。
有用的表达载体为pdCs(Lo等(1988)Protein Engineering11:495,其公开内容通过引用结合到本文中),其中Fc-X基因的转录采用人巨细胞病毒的增强子/启动子和猿猴病毒40多腺苷酸化信号。所用的人巨细胞病毒的增强子序列和启动子序列得自Boshart等,1985,Cell 41:521中提供的核苷酸-601至+7序列,该文献公开内容通过引用结合到本文中。所述载体也含有突变二氢叶酸还原酶基因作为选择标记(Simonsen和Levinson(1983)Proc.Nat Acad Sci USA80:2495,其公开内容通过引用结合到本文中)。
可以用本发明的DNA序列将合适的宿主细胞转化或转染,并且用来表达和分泌靶蛋白。目前用于本发明的优选宿主细胞包括无限增殖杂交瘤细胞、NS/O骨髓瘤细胞、293细胞、中国仓鼠卵巢细胞、海拉(Hela)细胞和COS细胞。
本发明的融合蛋白最好是用常规重组DNA方法产生。所述融合蛋白最好是由编码信号序列、免疫球蛋白Fc区和靶蛋白(本文也称为血管生成抑制剂)的DNA分子在宿主细胞中表达产生。优选的构成物可以从5’至3’方向编码信号序列、免疫球蛋白Fc区和靶蛋白。或者,所述构成物可以从5’至3’方向编码信号序列、靶蛋白和免疫球蛋白Fc区。
本文所用的术语“信号序列”不用说是指肽区段,所述肽区段在所述宿主细胞中引导分泌血管生成抑制剂免疫融合物蛋白并在翻译后再切除。本发明的信号序列是一种多核苷酸,它编码引发转运蛋白质穿过内质网膜的氨基酸序列。用于本发明的信号序列包括抗体轻链信号序列例如抗体14.18 (Gillies等,1989,Jour.of ImmunolMeth.,125:191-202)、抗体重链信号序列例如MOPC141抗体重链信号序列(Sakano等,1980,Nature 286:5774)和本领域中已知的任何其它信号序列(参见例如,Watson,1984,Nucleic Acids Research12:5145)。这些参考文献都通过引用结合到本文中。
本领域中,信号序列的特征已经很清楚,并且已知通常含有16-30个氨基酸残基,以及可以含有更多或更少的氨基酸残基。通常信号肽由三个区组成:碱性N-端区、中央疏水区和更强极性的C-端区。中央疏水区含有4-12个疏水性残基,在转运新生多肽期间,所述疏水性残基锚定信号肽穿过膜脂层双分子层。引发后,通常称为信号肽酶的细胞酶在内质网腔内酶切信号肽。潜在的信号肽切割位点一般在“(-3,-1)规则(rule)”后,因此,典型信号肽在位置-1至-3具有少量中性氨基酸残基,并在该区域内缺乏脯氨酸残基。信号肽酶在-1和+1氨基酸之间酶切这样的一种信号肽。因此,可以将编码信号序列的DNA部分在分泌期间从所述免疫融合蛋白的氨基端切除。这导致分泌由Fc区和所述靶蛋白组成的免疫融合蛋白。von Heijne(1986)Nucleic Acids Res.,14:4683对信号肽序列进行了详细讨论,其公开内容通过引用结合到本文中。
本领域技术人员显而易见的是,对用于本发明的特定信号序列的适应性可能需要进行某些常规实验。这种实验将包括确定信号序列引导分泌免疫融合物的能力,还要确定所用的序列的最佳构型、基因组或cDNA以达到有效分泌免疫融合物。另外,根据von Heijne所述规则(上述参考文献),本领域技术人员能够产生合成信号肽,并用常规实验测试这样的合成信号序列的效能。信号序列也称为“信号肽”、“前导序列”或“前导肽”。
所述信号序列和免疫球蛋白Fc区的融合物本文有时称为分泌盒。用于实施本发明的典型分泌盒是从5’至3’方向编码免疫球蛋轻链基因的信号序列和人免疫球蛋白γ1基因的Fcγ1区的多核苷酸。免疫球蛋白Fcγ1基因的Fcγ1区最好是包括至少一部分铰合结构域和至少一部分CH3结构域,或者至少部分铰合结构域、CH2结构域和CH3结构域。编码所述分泌盒的DNA可以是其基因组构型或其cDNA构型。
在另一个实施方案中,所述DNA序列编码所述分泌盒和血管生成抑制剂蛋白之间插入的蛋白酶切割位点。切割位点用于所编码融合蛋白的蛋白酶剪切,因此从血管生成抑制剂蛋白中分离Fc结构域。本文所用的“蛋白酶切割位点”不用说是指通过蛋白酶或其它蛋白剪切剂优先剪切的氨基酸序列。有用的蛋白酶切割位点包括蛋白酶诸如胰蛋白酶、纤溶酶或肠激酶K识别的氨基酸序列。现在已知许多切割位点/剪切剂对。参见例如美国专利第5,726,044号,其公开内容通过引用结合到本文中。其中所述靶蛋白序列是制管张素、endostatin或其活性变异体的前体分子,通过用内源蛋白酶诸如弹性蛋白酶或纤溶酶或尿激酶剪切产生所需蛋白产物。
本发明也包括含重组制管张素和endostatin或其片段的不同组合的融合蛋白,所述融合蛋白可以大量制备。尽管在抑制肿瘤生长方面已证实有效,但制管张素和endostatin怎样阻断血管生成的机制没有完全弄清楚。制管张素具有几种Kringle结构,而endostatin具有不同结构的基序,其中每种都可以单独引起或促使所述蛋白结合到内皮细胞,然后发挥抗血管生成作用。因此,本发明包括制管张素的生物活性片段(诸如Kringle 1、Kringle 2、Kringle 3及其组合物)和endostatin的生物活性片段的靶蛋白,所述生物活性片段与天然产生的全长制管张素和endostatin生理作用相似。
本发明的另一个实施方案提供血管生成抑制剂的双功能杂种构成物。本文所用的双功能杂种分子或构成物是指通过组合两种蛋白亚基产生的蛋白,其中所述两种亚基可以得自不同的蛋白。每种蛋白亚基具有其自身独立的功能,所以在所述杂种分子中,该两种亚基的功能可以是相加或协同的功能。这样功能的杂种蛋白使得可以在动物模型中测试制管张素和endostatin的协同作用。优选的双功能杂种蛋白包含或者直接串联或者通过多肽接头串联至少两种不同的血管生成抑制剂。例如,在一个优选的实施方案中,所述靶序列编码按读框与至少一部分endostatin连接的至少一部分制管张素,而制管张素结构域和endostatin结构域均具有抗血管生成活性或血管生成抑制作用。这两个单位均可以通过多肽接头连接。
本文所用的术语“多肽接头”不用说是指可以将两个蛋白质或一个蛋白质和一个Fc区连接在一起的肽序列。所述多肽接头最好是包含多种氨基酸诸如甘氨酸和/或丝氨酸。最好是,所述多肽接头包括一系列长度约为10-15个残基的甘氨酸和丝氨酸的肽。参见例如美国专利第5,258,698号,其公开内容通过引用结合到本文中。然而,设想可以通过常规实验法确定所述最佳接头的长度和氨基酸组分。
发现如果将制管张素的不同部分表达作为Fc融合分子,则获得高水平表达,推测可能是因为Fc部分作为一种载体,帮助所述多肽在C-端正确地折叠。此外,Fc区可以糖基化和在生理pH时高度带电,因此Fc区可以帮助溶解疏水性蛋白。
本发明也提供生产非人类物种的制管张素和endostatin的Fc融合蛋白的方法。非人类血管生成抑制剂融合蛋白对血管生成抑制剂临床前期的研究是有作用的,因为一种蛋白质药物效能和毒性研究在对试验之前必须在动物模型系统中进行。人蛋白可能在小鼠模型中不产生作用,因为所述蛋白可能诱发免疫应答,和/或具有不同的药物动力学特征,而扭曲试验结果。因此,等效鼠蛋白是在小鼠模型中试验的人蛋白最好的替代物。
标准Lewis肺癌小鼠模型(O’Reilly等(1997)Cell 88:277)用来比较可溶性人Fc-人制管张素、人Fc-人Endostatin、鼠Fc-鼠制管张素、鼠Fc-鼠Endostatin与大肠杆菌表达系统产生的所述不可溶的蛋白。在Lewis肺癌模型中在抑制肿瘤生长方面,可溶的Fc融合蛋白比用大肠杆菌产生的相应蛋白更有效。此外,将实验小鼠近亲交配,其肿瘤是诱发而不是自发产生。因此,在小鼠模型中的效能可能与抗人肿瘤的效能无关。采用狗的临床前期研究将提供关于这些血管生成抑制剂对自发性肿瘤的效能更准确的信息,因为有大量天然发生的自发性狗肿瘤。生产本发明的鼠(mu)Fc-鼠制管张素、鼠Fc-鼠endostatm和狗(ca)Fc-狗制管张素、狗Fc-狗endostatin的方法将有利于血管生成抑制剂在鼠和狗两个系统中的临床前研究。
本发明提供通过给予本发明的DNA、RNA或蛋白治疗血管生成性疾病(condition mediated by angiogenesis)的方法。血管生成性疾病包括,例如实体瘤;血源性肿瘤(blood born tumors)、转移瘤、良性瘤包括血管瘤、听神经瘤、神经纤维瘤、粒性结膜炎和脓性肉芽肿;类风湿性关节炎;牛皮癣;眼部血管生成性疾病(糖尿病性视网膜病、早产儿视网膜病、黄斑变性、角膜移植排斥、新生血管性青光眼);晶状体后纤维组织形成、发红(rubeosis)、遗传性出血性毛细血管扩张综合征;心肌血管生成;斑性新血管形成;毛细管扩张;血友病患者关节血管纤维瘤和创伤性肉芽;以及内皮细胞过度或异常刺激、肠粘连、动脉硬化、硬皮病性疤痕和肥大性疤痕即疤痕疙瘩。
本文公开的DNA构成物可用于基因治疗,其中将endostatin基因或制管张素基因通过各种方式之一传递到细胞中,例如与启动子连接的天然DNA或病毒载体内的DNA。进入细胞后,则表达制管张素基因和/或endostatin基因或基因片段,而所述蛋白在体内产生以发挥其正常生物功能。本发明的DNA构成物可高水平表达所述融合蛋白。本发明的融合蛋白也可用于治疗血管生成性疾病,而且比天然血管生成抑制剂和其它重组血管生成抑制剂具有更好的临床效果,因为本发明的血管生成抑制剂免疫融合物比所述其它重组血管生成抑制剂或单独的天然血管生成抑制剂具有更长的血清半寿期。由于所述二价体和二聚体结构,本发明的二价体形式和二聚体形式应具有更强的结合亲和力。由于通过所融合的Fc区或柔性多肽接头连接的两种不同血管生成抑制剂可能的协同作用,本发明的双功能杂种分子可具有更好的临床效果。
可以将本发明的组合物通过任何合适的方式给予动物,所述给药为直接(例如,局部注射、植入或局部给予组织部位)或全身性(例如胃肠外或口服)给予。如果所述组合物以胃肠外提供,诸如静脉内、皮下、眼、腹膜内、肌内、颊、直肠、阴道、眼框内、脑内、颅内、椎管内、心室内、鞘内、脑池内、囊内、鼻内或通过气雾剂给予,所述组合物最好是包含部分水或生理上可匹配的液体悬浮液或溶液。因此,所述载体(carrier)或媒介物(vehicle)为生理上可接受的以便除了传递所需组合物给患者之外,没有另外负面影响患者的电解质和/或容积平衡。用于所述制剂的液体介质因此可以包括正常生理盐水(例如,9.85%NaCl水溶液,0.15M,pH7-7.4)。
每次给予所述免疫融合物的剂量范围优选为50ng/m2-lg/m2,更优选为5μg/m2-200 mg/m2,而最优选为0.1mg/m2-50 mg/m2。每次给予编码所述免疫融合物的核酸的剂量范围优选为1μg/m2-100mg/m2,更优选为20μg/m2-10mg/m2,而最优选为400μg/m2-4mg/m2。然而设想在本领域技术水平内,通过常规实验法可以很好确定给予的最佳方式和剂量。
通过以下非限制的实施例进一步说明本发明。
实施例实施例1.人Fc-人Endostatin的表达
按照Lo等的学说((1998)Protein Engineering 11:495),将人endostatin表达作为人Fc-人endostatin(huFc-huEndo)融合蛋白。Fc是指人免疫球蛋白γ的Fc片段(SEQ ID NO:1所示为其DNA序列;SEQ IDNO:2所示为其氨基酸序列)。(聚合酶链式反应PCR)用来连接endostatin cDNA (SEQ ID NO:3;其氨基酸序列公开于SEQ ID NO:4中),用于表达Fc-Endo融合蛋白。正向引物或者是5’-CC CCG GGTAAA CAC AGC CAC CGC GAC TTC C(SEQ ID NO:5;所编码的氨基酸公开于SEQ ID NO:6中)或者是5’-C AAG CTT CAC AGC CACCGC GAC TTC C(SEQ ID NO:7;所编码的氨基酸公开于SEQ IDNO:8中),其中XmaI位点或HindIII位点后是编码endostatin N-端的序列。具有XmaI位点的引物使endostatin cDNA连接到IgGFc区CH3结构域末端的XmaI位点。具有HindIII位点的引物使endostatin cDNA连接到pdCs-Fc(D4K)载体的HindIII位点,所述载体在所述融合蛋白连接点含有肠激酶识别位点Asp4-Lys(LaVallie等(1993)J.Biol.Chem.268:23311-23317)。所述反向引物是5’-C CTC GAG CTA CTT GGAGGC AGT CAT G(SEQ ID NO:9),将所述引物设计置于翻译终止密码子(STOP codon)(反密码子,CTA)紧接在endostatin C-端之后,而这之后是XhoI位点。将所述PCR产物克隆并测序,并将XmaI-XhoI片段连接到XmaI和XhoI消化产生的pdCs-Fc载体上。同样,将编码endostatin的HindIII-XhoI片段连接到经适当消化的pdCs-huFc(D4K)载体中。通过电穿孔NS/O细胞,随后在含有100nM氨甲蝶呤的生长培养基中选择,获得稳定表达Fc-endo或Fc(D4K)-endostatin的克隆。通过抗-人Fc ELISA (实施例3)测定并通过SDS-PAGE证实蛋白表达水平,其显示约52kD蛋白产物。通过有限稀释将最佳生产克隆亚克隆。实施例2.细胞培养和转染
关于瞬时转染,通过质粒DNA与磷酸钙共沉淀(Sambrook等(1989)Molecular Cloning-ALaboratory Manual,Cold Spring Harbor,NY)或按照供应商的方法通过采用脂转染胺+(LipofectAMINE Plus)(LifeTechnologies,Gaithersburg,MD)的脂转染,将所述质粒引入人肾293细胞中。
为了获得稳定转染的克隆,通过电穿孔将质粒DNA引入小鼠骨髓瘤NS/O细胞中。将NS/O细胞生长于用10%胎牛血清补充的Dulbecco改进的Eagle培养基中。将约5×106细胞用PBS洗涤一次,并再次悬浮于0.5mlPBS中。然后将10μg线状化质粒DNA与所述细胞一起在冰上的基因脉冲发生器容器(Gene Pulser Cuvette)(0.4cm电极缺口(electrode gap)BioRad,Hercules,CA)中温育10分钟。采用设定0.25V和500μF的基因脉冲发生器(Gene Pulser)(BioRad,Hercules,CA)进行电穿孔。将它们重新悬浮于生长培养基中之后,使细胞在冰上恢复10分钟,然后平板接种到两个96孔板上。通过生长在100nM氨甲蝶呤(MTX)中选择稳定转染的克隆,MTX在转染后2天加入。给所述细胞每3天更换培养基1次,连续3次以上,然后在2-3周出现MTX-抗性克隆。通过抗-Fc ELISA测定克隆上清液,以鉴定出高生产克隆。分离高生产克隆,然后在含100nM MTX生长培养基中培养。
实施例3.ELISA方法
三种不同的ELISA用来测定MTX-抗性克隆上清液和其它试验样品中的蛋白产物浓度。抗-人Fc(huFc)ELISA用来测量含人Fc蛋白的量。抗-鼠Fc(muFc)抗体和抗-狗Fc(caFc)抗体用于ELISA以分别测量含鼠-Fc蛋白和含狗-Fc蛋白的量。以下详细描述用于抗-人FcELISA的方法。A.包被板
用96孔板(Nunc-Immuno plate MaxiSorpTM,Nalge NuncInternational,Rochester,NY),以5μg/ml AffiniPure山羊抗-人IgG(H+L)(Jackson ImmunoResearch Laboratories,West Grove,PA)的PBS按100μl/孔包被ELISA板。覆盖包被板,并于4℃温育过夜。然后将板用含0.05%吐温20的PBS洗涤4次,然后用含1%BSA/1%山羊血清的PBS封闭(200μl/孔)。与封闭缓冲液一起于37℃温育2小时后,所述板用含0.05%吐温的PBS洗涤4次,然后将所述板在纸毛巾上吸干。B.与试验祥品和第二抗体一起温育
用样品缓冲液将试验样品稀释到合适浓度,样品缓冲液为含1%BSA/1%山羊血清/0.05%吐温的PBS。用其浓度已知的嵌合抗体(含人Fc)制备标准曲线。为了制备标准曲线,用所述样品缓冲液进行系列稀释以得到范围为125ng/ml-3.9ng/ml的标准曲线。将所述稀释样品和标准样品加到所述板(100μl/孔)中,然后将所述板于37℃温育2小时。在温育后,将所述板用含0.05%吐温的PBS洗涤8次。然后向每个孔加入100μl第二抗体、结合辣根过氧化物酶(HRP)的抗-人IgG(Jackson ImmunoResearch Laboratories,Inc West Grove,PA),用样品缓冲液稀释约120,000倍。必须对每批结合HRP的抗-人IgG测定第二抗体的准确稀释度。于37℃温育2小时后。将所述板用含0.05%吐温的PBS洗涤8次。C.显色
将30mg(1片)邻苯二胺二氢氯化物(OPD)溶解到15ml含新鲜加入的0.03%H2O2的0.025M柠檬酸/0.05M Na2HPO4缓冲液,pH5中,制备底物溶液。将所述底物溶液以100μl孔加到所述板中。于室温在黑暗中使所述板显色反应30分钟。所述显色反应的时间可以根据所述包被板的批间差异、第二抗体等而改变。加入4N H2SO4(100μl/孔)终止所述反应。用均设定为490nm和650nm的板读出器读出所述板,并编程以使490nm OD减去背景650nm OD。
用于抗-muFc ELISA的方法相似,只是将ELISA板用PBS中0.5μg/ml AffiniPure山羊抗-鼠IgG(H+L)(Jackson ImmunoResearch,WestGrove,PA)以100μl孔进行包被;而第二抗体为结合辣根过氧化物酶的山羊抗-muIgG(Fcγ)(Jackson ImmunoResearch,West Grove,PA),以1:5000稀释倍数使用。对于抗-caFc ELISA,ELISA板同样用PBS中的5μg/ml AffiniPure兔抗-狗IgG(Fc片段特异性)(JacksonImmunoResearch,West Grove,PA)以100μl孔进行包被;而第二抗体为结合辣根过氧化物酶的AffinPure兔抗-狗IgG(Fc片段特异性)(Jackson ImmunoResearch,West Grove,PA),以1:5000稀释倍数使用。实施例4.人Fc-人制管张素的表达
基本上按实施1所述将人制管张素(DNA序列见SEQ ID NO:10;氨基酸序列见SEQ ID NO:11)表达为人Fc-人制管张素(huFc-huAngio)融合蛋白。PCR用来连接制管张素cDNA(SEQ ID NO:3),以用pdCs-huFc或pdCs-huFc(D4K)载体表达。相应的正向引物为5’-CC CCGGGT AAG AAA GTG TAT CTC TCA GAG(SEQ ID NO:12;所编码的氨基酸见SEQ ID NO:13)和5’-C CCC AAG CTT AAA GTG TATCTC TCA GAG (SEQ ID NO:14;所编码的氨基酸见SEQ ID NO:15),其中XmaI位点或HindIII位点之后为编码制管张素N-端的序列。反向引物是5’-CCC CTC GAG CTA CGC TTC TGT TCC TGA GCA(SEQ ID NO:16),设计所述引物使翻译终止密码子(STOP codon)(反密码子,CTA)紧接在制管张素C-端之后,而再后是XhoI位点。将所述PCR产物克隆并测序,然后将所产生的编码制管张素的XmaI-XhoI片段和HindIII-XhoI片段分别连接到pdCs-huFc和pdCs-huFc(D4K)载体上。按实施例2和3所述,选择并测定稳定表达huFc-huAngio和huFc(D4K)-huAngio的NS/0克隆。实施例5.鼠Fc-鼠-Endostatin的表达
基本上按实施1所述将鼠endostatm(DNA序列见SEQ IDNO:17;氨基酸序列见SEQ ID NO:18)和鼠Fc(DNA序列见SEQ IDNO:19;所编码的氨基酸见SEQ ID NO:20)表达为鼠Fc-鼠endostatin(muFc-muEndo)融合蛋白。PCR用来连接endostatin cDNA(SEQ IDNO:4),以用pdCs-muFc(D4K)载体表达。正向引物为5’-C CCC AAGCTT CAT ACT CAT CAG GAC TTT C(SEQ ID NO:21;所编码的氨基酸见SEQ ID NO:22),其中HindIII位点之后为编码endostatin N-端的序列。反向引物为5’-CCC CTC GAG CTA TTT GGA GAA AGAGGT C(SEQ ID NO:23),设计所述引物使翻译终止密码子(STOPcodon)(反密码子,CTA)紧接在endostatin C-端之后,而这之后是XhoI位点。将所述PCR产物克隆并测序,然后将所产生的编码endostatin的HindIII-XhoI片段连接到pdCs-muFc(D4K)载体上。如实施例2和3中所述,选择并测定(抗-muFc ELISA)稳定表达muFc(D4K)-muEndo的NS/O克隆。实施例6.鼠Fc-鼠制管张素的表达
基本上按实施例所述将鼠制管张素(DNA序列见SEQ ID NO:24;氨基酸序列见SEQ ID NO:25)表达为鼠Fc-鼠制管张素(muFc-muAngio)融合蛋白。PCR用来连接所述制管张素cDNA(SEQ ID NO:6),以便以pdCs-Fc(D4K)载体表达。正向引物为5’-C CCC AAG CTT GTG TATCTG TCA GAA TGT AAG CCC TCC TGT CTC TGA GCA(SEQ IDNO:26;所编码的氨基酸见SEQ ID NO:27),其中HindIII位点之后为编码制管张素N-端的序列。反向引物是5’-CCC CTC GAG CTACCC TCC TGT CTC TGA GCA(SEQ ID NO:28),设计所述引物使翻译终止密码子(STOP codon)(反密码子,CTA)紧接在制管张素C-端之后,而这之后是XhoI位点(CTCGAG)。将所述PCR产物克隆并测序,然后将编码制管张素的HindIII-XhoI片段连接到pdCs-muFc(D4K)载体上。如实施例2和3所述,选择并测定(抗-muFc ELISA)稳定表达muFc(D4K)-muAngio的NS/O克隆。实施例7.狗Fc(caFc)的表达
将从狗血中分离的狗外周血单核细胞(PBMC)用来制备mRNA。用逆转录酶和寡聚物(dT)合成第一条cDNA链后,采用正向引物5’-CC TTA AGC GAA AAT GGA AGA GTT CCT CGC(SEQ ID NO:29;所编码的氨基酸见SEQ ID NO:30),其中紧靠编码狗Fc铰链区序列的上游导入AfIII位点;以及反向引物5’-C CTC GAG TCA TTT ACCCGG GGA ATG GGA GAG GGA TTT CTG(SEQ ID NO:31),其中在狗Fc翻译终止密码子(STOP codon)(反密码子,TCA)之后导入XhoI位点,进行PCR以扩增狗Fc(Kazuhiko等,(1992)JP 1992040894-A1)。所述反向引物还导入沉默突变以产生XmaI限制酶切位点,该位点有利于通过接头-连接物构建pdCs-caFc(D4K)载体,然后连接到编码狗endostatm或制管张素的DNA构成物上。用同样的方法构建pdCs-huFc,Lo等(Lo等,Protem Engineering(1998)11:495)对其进行了详细地论述,如下构建pdCs-huFc的表达载体。将编码狗Fc的AflII-XhoI片段连接到编码所述轻链信号肽的XbaI-AflII片段以及XbaI-XhoI消化的pdCs载体。所产生的pdCs-caFc表达载体然后用来转染293细胞。转染后约3天,通过A蛋白层析纯化所述上清液。采用结合过氧化物酶的兔抗-狗IgG(Fc片段特异性)(Jackson ImmunoResearch,West Grove,PA),在SDS-PAGE后再通过蛋白质印迹分析,确定狗Fc (DNA序列见SEQ ID NO:32;氨基酸序列见SEQ ID NO:33)的表达。实施例8.狗Fc-狗Endostatin的表达
基本按实施例5所述将狗endostatin的编码序列(DNA序列见SEQID NO:34;氨基酸序列见SEQ ID NO:35)连接到HindIII-XhoI片段上以表达Fc融合蛋白。在3’端,引入终止密码子(STOP codon),例如通过PCR紧接在编码C-末端赖氨酸残基的密码子之后引入终止密码子,而这之后是NotI限制酶切位点。然而在5’端,有一个DraIII限制酶切位点便于重组。化学合成一个HindIII粘端和一个DraIII粘端组成的寡核苷酸双链体,然后用来连接编码其余的狗endostatin cDNA的DraIII-XhoI限制性片段。所用的双链体如下所示:HindIII5'-AGCTT CAC ACC CAC CAG GAC TT C CAG CCG GTG CTG CAC CTG(SEQID NO:36)A GTG TGG GTG GTC CTG AAG GTC GGC CAC GAC GTG-5’(SEQID NO:38)
DraIII
在所述双链体中第一个CAC编码狗endostatin N-端组氨酸残基。因此编码狗全长endostatin的HindIII-XhoI片段可连接到HindIII-XhoI消化的pdCs-caFc载体(参见实施例7)用于表达。如实施例2和3中所述,选择并通过抗-caFc ELISA测定稳定表达caFc-caEndo的NS/O克隆。在SDS-PAGE上分析并通过蛋白质印迹分析确定所述蛋白产物。实施例9.狗Fc-狗制管张素的表达
基本上按以上实施例所述,连接编码狗全长制管张素的cDNA(DNA序列见SEQ ID NO:39;氨基酸序列见SEQ ID NO:40)以表达caFc融合蛋白。简单地说,在3’端引入终止密码子(STOP codon),例如通过PCR紧接在编码C-端赖氨酸残基的密码子之后引入终止密码子,而这之后是NotI限制酶切位点而不是XhoI位点。因为在狗制管张素的cDNA中有一个内部XhoI限制酶切位点。在5’端,紧接在制管张素N-端的上游按读框引入HindIII位点。然后将编码狗全长制管张素的HindIII-NotI片段连接到HindIII-NotI消化的pdCs-caFc载体(其中通过接头连接将NotI位点引入XhoI位点处)用于表达。如实施例2和3中所述,选择并通过抗-caFc ELISA测定稳定表达caFc-caAngio的NS/O克隆。在SDS-PAGE上分析并通过蛋白质印迹分析确定所述蛋白产物。实施例10.鼠制管张素的鼠Fc-K1的表达
制管张素包含纤溶酶原的5个Kringle结构域中的前4个。为了测定是否任何一个或几个Kringle结构域决定所观察到的制管张素的抗-血管生成活性,则可产生单个Kringle结构域或其组合物用于测试。为了证实Fc作为融合蛋白配偶体的作用,鼠制管张素的第一个Kringle结构域(K1)的表达用以下方式实现。第一个Kringle结构域终止于鼠制管张素的Glu-87处(SEQ ID NO:25)。在该位置的cDNA中有一个合适的NsiI限制酶切位点,以便NsiI消化后,将第4个碱基3’-突出端通过T4聚合酶去除以产生一个平端。将翻译终止密码子通过连接到回文接头TGA CTC GAG TCA(SEQ ID NO:41)引入紧接在编码Glu-87的GAA下游,其中所述终止密码子TGA之后是XhoI位点。然后将编码该截短制管张素的HindIII-XhoI片段即只是第一个Kringle,连接到pdCs-muFc(D4K)载体中用于表达。按抗-muFc ELISA和SDS-PAGE分析,在瞬时表达和稳定表达中均获得高水平表达。实施例11.鼠制管张素的鼠Fc-innerK1的表达
Kringle结构域由多个环组成,包括外环和内环。在鼠制管张素的第一个Kringle中,所述内环明确为Cys 55和Cys 79,它们在所述环的碱基处一起形成一个二硫键。内环Cys-67与外环的一个Cys残基形成另一个二硫键以产生所述Kringle结构。为了测试内环是否具有抗-血管生成活性,将它如下表达为muFc-innerK1(Kringle 1)。用编码第一个Kringle的DNA片段作模板,具有序列5’GGG CCT TGGAGC TAC ACT ACA(SEQ ID NO:42;所编码的氨基酸见SEQ IDNO:43)的诱变引物用来通过PCR使TGC(Cys-67)诱变为AGC(Ser)。如果将Kringle 1的内环表达但不表达其外环,则这保证Cys-67不形成二硫键。具有序列5’GCGGATCCAAGCTT AGT ACA CAT CCCAAT GAG GG (SEQ ID NO:44;所编码的氨基酸见SEQ ID NO:45)的上游引物用来紧接5’Ser-43(AGT)的密码子按读框引入HindIII位点。将一个BamHI位点也引入紧接在HindIII位点的上游。所述BamHI位点用于连接以下实施例12所示柔性Gly-Ser接头末端处的BamHI位点。因此,通过鼠制管张素Glu-87将编码Ser-43的HindIII-XhoI DNA片段连接于pdCs-muFc(D4K)载体用于表达。用抗-muFc ELISA和SDS-PAGE分析,在瞬时表达和稳定表达中均获得高水平表达muFc-innerK1。实施例12.鼠制管张素的鼠Fc-鼠Endo-GlySer接头-innerK1的表达
杂种分子鼠Fc-鼠Endo-innerK1包含通过含甘氨酸和丝氨酸残基的一个多肽接头连接到鼠制管张素第一个Kringle内环的鼠Fc-鼠Endo。如下装配所述DNA构成物。
在鼠endostatin cDNA的3’末端有一个BspHI位点。为了在鼠endostain的C-端引入一个甘氨酸和丝氨酸残的柔性接头,将编码endostatin的540-bp HindIII-BspHI片段连接到SEQ ID NO:46和SEQID NO:48中公开的寡核苷酸形成的重叠寡核苷酸双链体。SEQ IDNO:46编码的氨基酸接头见SEQ ID NO:47。
将编码鼠endostatin和Gly-Ser接头HindIII-BamHI的片段亚克隆到标准克隆载体中。然后将BamHI位点用来引入编码实施例11中的innerK1的BamHI-XhoI片段。将所产生的编码muEndo-GlySer接头-innerK1的HindIII-XhoI片段连接到pdCs-muFc(D4K)载体用于表达。以抗-muFc ELISA和SDS-PAGE分析,在瞬时表达和稳定表达中均获得高水平表达muFc-muEndo-GlySer接头-innerK1。实施例13.鼠制管张素的鼠Fc-鼠Endo-GlySer接头-K1的表达
杂种分子鼠Fc-鼠Endo-K1包含通过含甘氨酸和丝酸残基的多肽接头连接到鼠制管张素的第一个Krmgle的鼠Fc-鼠Endo。将所述DNA构成物如下装配。
将编码鼠Endo-GlySer接头的HindIII-BamHI片段的BamHI末端(实施例12)通过以下连接物连接到编码鼠制管张素Kringle 1的HindIII-XhoI片段(实施例10)。BamHI5’GA TCC TCA GGC C (SEQ ID NO:49)
G AGT CCG GTCGA (SEQ ID NO:50)
HindIII
所述连接物具有一个HindIII的粘端,连接时不需要再产生HindIII位点。因此,将所产生的编码muEndo-GlySer接头-Kringle 1的HindIII-XhoI片段连接到pdCs-muFc(D4K)载体用于表达。用抗-muFcELISA和SDS-PAGE分析,在瞬时表达和稳定表达中均获得高水平表达muFc-muEndo-GlySer接头-K1。实施例14.鼠Fc-鼠Endo-GlySer接头-鼠制管张素的表达
杂种分子鼠Fc-鼠Endo-GlySer接头-鼠制管张素包含通过含甘氨酸和丝酸残基的多肽接头连接到鼠制管张素的鼠Fc-鼠Endo。将所述DNA构成物基本上如下装配。将编码鼠Endo-GlySer接头的HindIII-BamHI片段BamHI末端(实施例12)通过实施例13中所述连接物连接到编码鼠制管张素的HindIII-XhoI片段。将所产生的编码鼠Endo-GlySer接头-鼠制管张素的HindIII-XhoI片段连接到pdCs-muFc(D4K)载体用于表达。用抗-muFc ELISA和SDS-PAGE分析,在瞬时表达和稳定表达中均获得高水平表达鼠Fc-鼠Endo-GlySer接头-鼠制管张素。实施例15.人制管张素-人Fc-人Endo的表达
杂种分子人制管张素-人Fc-人Endo包含通过肽键连接到人Fc=人Endo的人制管张素。将所述DNA构成物如下装配。首先通过PCR产生编码人制管张素但无终止密码子的HindIII-XhoI片段,以使制管张素的最后氨基酸残基的密码子紧接着是XhoI位点的CTCGAG。通过以下AflII-HindIII连接物,将5’末端的HindIII连接到轻链信号肽的XbaI-AfIII片段(Lo等,Protein Engineering(1998)11:495):AfIII5’TTAAGCGGCC (SEQ ID NO:51)
CG CGG GTCGA (SEQ ID NO:52)
HindIII’
在连接时,上述连接物的HindIII粘端不再产生HindIII位点。在3’末端,通过以下XhoI’-AfIII连接物,将XhoI位点连接到编码人Fc-人-Endo的AfIII-XhoI片段的AfIII位点:XhoI’5’TCGACTCCGGC (SEQ ID NO:53)
GAGGCCGAATT (SEQ ID NO:54)
AfIII
在连接时,上述连接物的XhoI粘端不再产生XhoI位点。所产生的编码信号肽-人制管张素-人Fc-人endostatin的XbaI-XhoI片段克隆到pdCs载体上用于表达。用抗-muFc ELISA和SDS-PAGE分析,在瞬时表达和稳定表达中均获得高水平表达。实施例16.药物代谢动力学
在一组药物代谢动力学研究中,将移植100-200mm3 Lewis肺肿瘤的C57/BL6小鼠,每只鼠注射720μg人Fc-人制管张素到其尾静脉中。根据O’Reilly所描述的实际治疗方案(O’Reilly(1996)NatureMedicine 2:689),选择在该研究中使用的肿瘤大小和人Fc-人制管张素的剂量。在注射后1/2、1、2、4、8、24和48小时从眼眶后采取血样。在抗-人Fc ELISA后再用蛋白质印迹分析分析所述血样。发现人Fc-人制管张素的循环半寿期在小鼠中约为32小时,而蛋白质印迹分析显示高于90%的人-Fc-人制管张素以完整的分子保持循环。
还以200μg/小鼠的剂量在无肿瘤的Swiss小鼠中重复所述药物代谢动力学研究。在这种情况下,发现人Fc-人制管张素的循环半寿期约为33小时。
等同实施方案
本发明可以体现为其它具体的但不偏离其精神或基本特征的形式。因此认为前述实施方案是在所有方面说明而不是限制本文所描述的发明。因此本发明的范围由附属的权利要求书限定而不是由以上所述限定,因此本发明包括在权利要求书等同目的和范围内的所有修改。
序列表<110>Lo,Kin-Ming
Li,Yue
Gillies,StePhen D<120>表达以及分泌制管张素和ENDOSTATIN的免疫融合物<130>LEX-006PC<140><141><150>US 60/097,883<151>1998-08-25<160>S4<170>PatentIn Ver.2.0<210>1<211>696<212>DNA<213>人<220><221>CDS<222>(1)..(696)<223>人免疫球蛋白γ的Fc片段<400> 1gag ccc aaa tct tct gac aaa act cac aca tgc cca ccg tgc cca gca 48Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala1 5 10 15cct gaa ctc ctg ggg gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc 96Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
20 25 30aag gac acc ctc atg arc tcc cgg acc cct gag gtc aca tgc gtg gtg 144Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45gtg gac gtg agc cac gaa gac cct gag gtc aag ttc aac tgg tac gtg 192Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
50 55 60gac ggc gtg gag gtg cat aat gcc aag aca aag ccg cgg gag gag cag 240Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln65 70 75 80tac aac agc acg tac cgt gtg gtc agc gtc ctc acc gtc ctg cac cag 288Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
85 90 95gac tgg ctg aat ggc aag gag tac aag tgc aag gtc tcc aac aaa gcc 336Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
100 105 110ctc cca gcc ccc arc gag aaa acc ate tcc aaa gcc aaa ggg cag ccc 384Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
115 120 125cga gaa cca cag gtg tac acc etg ccc cca tca cgg gag gag atg acc 432Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
130 135 140aag aac cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tat ccc agc 480Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser145 150 155 160gac atc gcc gtg gag tgg gag agc aat ggg cag ccg gag aac aac tac 528Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
165 170 175aag acc acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tat 576Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
180 185 190agc aag ctc acc gtg gac aag agc agg tgg cag cag ggg aac gtc ttc 624Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
195 200 205tca tgc tcc gtg atg cat gag gct ctg cac aac cac tac acg cag aag 672Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
210 215 220agc ctc tcc ctg tcc ccg ggt aaa 696Ser Leu Ser Leu Ser Pro Gly Lys225 230<210>2<211>232<212>PRT<213>人<400>2Glu Pro Lys Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala1 5 10 15Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
20 25 30Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val
50 55 60Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln65 70 75 80Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
85 90 95Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
100 105 110Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
115 120 125Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
130 135 140Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser145 150 155 160Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
165 170 175Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
180 185 190Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
195 200 205Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
210 215 220Ser Leu Ser Leu Ser Pro Gly Lys225 230<210>3<211>549<212>DNA<213>人<220><221>CDS<222>(1)..(549)<223>endostatin<400>3cac agc cac cgc gac ttc cag ccg gtg ctc cac ctg gtt gcg ctc aac 48His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn1 5 10 15agc ccc ctg tca ggc ggc atg cgg ggc atc cgc ggg gcc gac ttc cag 96Ser Pro Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp Phe Gln
20 25 30tgc ttc cag cag gcg cgg gcc gtg ggg ctg gcg ggc acc ttc cgc gcc 144Cys Phe Gln Gln Ala Arg Ala Val Gly Leu Ala Gly Thr Phe Arg Ala
35 40 45ttc ctg tcc tcg cgc ctg cag gac ctg tac agc atc gtg cgc cgt gcc 192Phe Leu Ser Ser Arg Leu Gln Asp Leu Tyr Ser Ile Val Arg Arg Ala
50 55 60gac cgc gca gcc gtg ccc arc gtc aac crc aag gac gag ctg ctg ttt 240Asp Arg Ala Ala Val Pro Ile Val Asn Leu Lys Asp Glu Leu Leu Phe65 70 75 80ccc agc tgg gag gct ctg ttc tca ggc tct gag ggt ccg ctg aag ccc 288Pro Ser Trp Glu Ala Leu Phe Ser Gly Ser Glu Gly Pro Leu Lys Pro
85 90 95ggg gca cgc ate ttc tcc ttt gac ggc aag gac gtc ctg agg cac ccc 336Gly Ala Arg Ile Phe Ser Phe Asp Gly Lys Asp Val Leu Arg His Pro
100 105 110acc tgg ccc cag aag agc gtg tgg cat ggc tcg gac ccc aac ggg cgc 384Thr Trp Pro Gln Lys Ser Val Trp His Gly Ser Asp Pro Asn Gly Arg
115 120 125agg ctg acc gag agc tac tgt gag acg tgg cgg acg gag gct ccc tcg 432Arg Leu Thr Glu Ser Tyr Cys Glu Thr Trp Arg Thr Glu Ala Pro Ser
130 135 140gcc acg ggc cag gcc tcc tcg ctg ctgggg ggcagg ctc ctg ggg cag 480Ala Thr Gly Gln Ala Ser Ser Leu Leu Gly Gly Arg Leu Leu Gly Gln145 150 155 160agt gcc gcg agc tgc cat cac gcc tac atc gtg ctc tgc att gag aac 528Ser Ala Ala Ser Cys His His Ala Tyr Ile Val Leu Cys Ile Glu Asn
165 170 175agc ttc atg act gcc tcc aag 549Ser Phe Met Thr Ala Ser Lys
180<210>4<211>183<212>PRT<213>人<400>4His Ser His Arg Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn1 5 10 15Ser Pro Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp Phe Gln
20 25 30Cys Phe Gln Gln Ala Arg Ala Val Gly Leu Ala Gly Thr Phe Arg Ala
35 40 45Phe Leu Ser Ser Arg Leu Gln Asp Leu Tyr Ser Ile Val Arg Arg Ala
50 55 60Asp Arg Ala Ala Val Pro Ile Val Asn Leu Lys Asp Glu Leu Leu Phe65 70 75 80Pro Ser Trp Glu Ala Leu Phe Ser Gly Ser Glu Gly Pro Leu Lys Pro
85 90 95Gly Ala Arg Ile Phe Ser Phe Asp Gly Lys Asp Val Leu Arg His Pro
100 105 110Thr Trp Pro Gln Lys Ser Val Trp His Gly Ser Asp Pro Asn Gly Arg
115 120 125Arg Leu Thr Glu Ser Tyr Cys Glu Thr Trp Arg Thr Glu Ala Pro Ser
130 135 140Ala Thr Gly Gln Ala Ser Ser Leu Leu Gly Gly Arg Leu Leu Gly Gln145 150 155 160Ser Ala Ala Ser Cys His His Ala Tyr Ile Val Leu Cys Ile Glu Asn
165 170 175Ser Phe Met Thr Ala Ser Lys
180<210>5<211>30<212>DNA<213>人工序列<220><223>人工序列的描述:人Fc-Endo的正向引物<220><221>CDS<222>(3)..(29)<400>5cc ccg ggt aaa cac agc cac cgc gac ttc c 30Pro Gly Lys His Ser His Arg Asp Phe
1 5<210>6<211>9<212>PRT<213>人工序列<400>6Pro Gly Lys His Ser His Arg Asp Phe1 5<210>7<211>26<212>DNA<213>人工序列<220><223>人工序列的描述:人Fc-Endo的正向引物<220><221>CDS<222>(2)..(25)<400>7c aag ctt cac agc cac cgc gac ttc c 26Lys Leu His Ser His Arg Asp Phe
1 5<210>8<211>8<212>PRT<213>人工序列<400>8Lys Leu His Ser His Arg Asp Phe1 5<210>9<211>26<212>DNA<213>人工序列<220><223>人工序列的描述:人Fc-endo的反向引物<400>9cctcgagcta cttggaggca gtcatg 26<210>10<211>1089<212>DNA<213>人<220><221>CDS<222>(1)..(1089)<223>制管张素<400> 10aaa gtg tat ctc tca gag tgc aag act ggg aat gga aag aac tac aga 48Lys Val Tyr Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg1 5 10 15ggg acg atg tcc aaa aca aaa aat ggc atc acc tgt caa aaa tgg agt 96Gly Thr Met Ser Lys Thr Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser
20 25 30tcc act tct ccc cac aga cct agattc tca cct gct aca cac ccc tca 144Ser Thr Ser Pro His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser
35 40 45gag gga ctg gag gag aac tac tgc agg aat cca gac aac gat ccg cag 192Glu Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln
50 55 60ggg ccc tgg tgc tat act act gat cca gaa aag aga tat gac tac tgc 240Gly Pro Trp Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys65 70 75 80gac att crt gag tgt gaa gag gaa tgt atg cat tgc agt gga gaa aac 288Asp Ile Leu Glu Cys Glu Glu Glu Cys Met His Cys Ser Gly Glu Asn
85 90 95tat gac ggc aaa att tcc aag acc atg tct gga ctg gaa tgc cag gcc 336Tyr Asp Gly Lys Ile Ser Lys Thr Met Ser Gly Leu Glu Cys Gln Ala
100 105 110tgg gac tct cag agc cca cac gct cat gga tac att cct tcc aaa ttt 384Trp Asp Ser Gln Ser Pro His Ala His Gly Tyr Ile Pro Ser Lys Phe
115 120 125cca aac aag aac ctg aag aag aat tac tgt cgt aac ccc gat agg gag 432Pro Asn Lys Asn Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu
130 135 140ctg cgg cct tgg tgt ttc acc acc gac ccc aac aag cgc tgg gaa ctt 480Leu Arg Pro Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu145 150 155 160tgc gac atc ccc cgc tgc aca aca cctcca cca tct tct ggt ccc acc 528Cys Asp Ile Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr
165 170 175tac cag tgt ctg aag gga aca ggt gaa aac tat cgc ggg aat gtg gct 576Tyr Gln Cys Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly Asn Val Ala
180 185 190gtt acc gtt tcc ggg cac acc tgt cag cac tgg agt gca cag acc cct 624Val Thr Val Ser Gly His Thr Cys Gln His Trp Ser Ala Gln Thr Pro
195 200 205cac aca cat aac agg aca cca gaa aac ttc ccc tgc aaa aat ttg gat 672His Thr His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp
210 215 220gaa aac tac tgc cgc aat cct gac gga aaa agg gcc cca tgg tgc cat 720Glu Asn Tyr Cys Arg Asn Pro Asp Gly Lys Arg Ala Pro Trp Cys His225 230 235 240aca acc aac agc caa gtg cgg tgg gag tac tgt aag ata ccg tcc tgt 768Thr Thr Asn Ser Gln Val Arg Trp Glu Tyr Cys Lys Ile Pro Ser Cys
245 250 255gac tcc tcc cca gta tcc acg gaa caa ttg gct ccc aca gca cca cct 816Asp Ser Ser Pro Val Ser Thr Glu Gln Leu Ala Pro Thr Ala Pro Pro
260 265 270gag cta acc cct gtg gtc cag gac tgc tac cat ggt gat gga cag agc 864Glu Leu Thr Pro Val Val Gln Asp Cys Tyr His Gly Asp Gly Gln Ser
275 280 285tac cga ggc aca tcc tcc acc acc acc aca gga aag aag tgt cag tct 912Tyr Arg Gly Thr Set Set Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser
290 295 300tgg tca tct atg aca cca cac cgg cac cag aag acc cca gaa aac tac 960Trp Ser Ser Met Thr Pro His Arg His Gln Lys Thr Pro Glu Asn Tyr305 310 315 320cca aat gct ggc ctg aca atg aac tac tgc agg aat cca gat gcc gat 1008Pro Asn Ala Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp
325 330 335aaa ggc ccc tgg tgt ttt acc aca gac ccc agc gtc agg tgg gag tac 1056Lys Gly Pro Trp Cys Phe Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr
340 345 350tgc aac ctg aaa aaa tgc tca gga aca gaa gcg 1089Cys Asn Leu Lys Lys Cys Ser Gly Thr Glu Ala
355 360<210>11<211>363<212>PRT<213>人<400>11Lys Val Tyr Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Asn Tyr Arg1 5 10 15Gly Thr Met Ser Lys Thr Lys Asn Gly Ile Thr Cys Gln Lys Trp Ser
20 25 30Ser Thr Ser Pro His Arg Pro Arg Phe Ser Pro Ala Thr His Pro Ser
35 40 45Glu Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Pro Gln
50 55 60Gly Pro Trp Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr Asp Tyr Cys65 70 75 80Asp Ile Leu Glu Cys Glu Glu Glu Cys Met His Cys Ser Gly Glu Asn
85 90 95Tyr Asp Gly Lys Ile Ser Lys Thr Met Ser Gly Leu Glu Cys Gln Ala
100 105 110Trp Asp Ser Gln Ser Pro His Ala His Gly Tyr Ile Pro Ser Lys Phe
115 120 125Pro Asn Lys Asn Leu Lys Lys Asn Tyr Cys Arg Asn Pro Asp Arg Glu
130 135 140Leu Arg Pro Trp Cys Phe Thr Thr Asp Pro Asn Lys Arg Trp Glu Leu145 150 155 160Cys Asp Ile Pro Arg Cys Thr Thr Pro Pro Pro Ser Ser Gly Pro Thr
165 170 175Tyr Gln Cys Leu Lys Gly Thr Gly Glu Asn Tyr Arg Gly Asn Val Ala
180 185 190Val Thr Val Ser Gly His Thr Cys Gln His Trp Ser Ala Gln Thr Pro
195 200 205His Thr His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp
210 215 220Glu Asn Tyr Cys Arg Asn Pro Asp Gly Lys Arg Ala Pro Trp Cys His225 230 235 240Thr Thr Asn Ser Gln Val Arg Trp Glu Tyr Cys Lys Ile Pro Ser Cys
245 250 255Asp Ser Ser Pro Val Ser Thr Glu Gln Leu Ala Pro Thr Ala Pro Pro
260 265 270Glu Leu Thr Pro Val Val Gln Asp Cys Tyr His Gly Asp Gly Gln Ser
275 280 285Tyr Arg Gly Thr Ser Ser Thr Thr Thr Thr Gly Lys Lys Cys Gln Ser
290 295 300Trp Ser Ser Met Thr Pro His Arg His Gln Lys Thr Pro Glu Asn Tyr305 310 315 320Pro Asn Ala Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala Asp
325 330 335Lys Gly Pro Trp Cys Phe Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr
340 345 350Cys Asn Leu Lys Lys Cys Ser Gly Thr Glu Ala
355 360<210>12<211>29<212>DNA<213>人工序列<220><223>人工序列的描述:人Fc-Angio的正向引物<220><221>CDS<222>(3)..(29)<400>12cc ccg ggt aag aaa gtg tat crc tca gag 29Pro Gly Lys Lys Val Tyr Leu Ser Glu
1 5<210>13<211>9<212>PRT<213>人工序列<400>13Pro Gly Lys Lys Val Tyr Leu Ser Glu1 5<210>14<211>28<212>DNA<213>人工序列<220><223>人工序列的描述:人Fc-Angio的正向引物<220><221>CDS<222>(2)..(28)<400>14c ccc aag ctt aaa gtg tat ctc tca gag 28Pro Lys Leu Lys Val Tyr Leu Ser Glu
1 5<210>15<211>9<212>PRT<213>人工序列<400>15Pro Lys Leu Lys Val Tyr Leu Ser Glu1 5<210>16<211>30<212>DNA<213>人工序列<220><223>人工序列的描述:人Fc-Angio的反向引物<400>16cccctcgagc tacgcttctg ttcctgagca 30<210>17<211>552<212>DNA<213>小家鼠<220><221>CDS<222>(1)..(552)<223>endostatin<400> 17cat act cat cag gac ttt cag cca gtg crc cac ctg gtg gca ctg aac 48His Thr His Gln Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn1 5 10 15acc ccc ctg tct gga ggc atg cgt ggt atc cgt gga gca gat ttc cag 96Thr Pro Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp Phe Gln
20 25 30tgc ttc cag caa gcc cga gcc gtg ggg ctg tcg ggc acc ttc cgg gct 144Cys Phe Gln Gln Ala Arg Ala Val Gly Leu Ser Gly Thr Phe Arg Ala
35 40 45ttc ctg tcc tct agg ctg cag gat ctc tat agc atc gtg cgc cgt gct 192Phe Leu Ser Ser Arg Leu Gln Asp Leu Tyr Ser Ile Val Arg Arg Ala
50 55 60gac cgg ggg tct gtg ccc arc gtc aac ctg aag gac gag gtg cta tct 240Asp Arg Gly Ser Val Pro Ile Val Asn Leu Lys Asp Glu Val Leu Ser65 70 75 80ccc agc tgg gac tcc ctg ttt tct ggc tcc cag ggt caa gtg caa ccc 288Pro Ser Trp Asp Ser Leu Phe Ser Gly Ser Gln Gly Gln Val Gln Pro
85 90 95ggg gcc cgc atc ttt tct ttt gac ggcaga gat gtc ctg aga cac cca 336Gly Ala Arg Ile Phe Ser Phe Asp Gly Arg Asp Val Leu Arg His Pro
100 105 110gcc tgg ccg cag aag agc gta tgg cac ggc tcg gac ccc agt ggg cgg 384Ala Trp Pro Gln Lys Ser Val Trp His Gly Ser Asp Pro Ser Gly Arg
115 120 125agg ctg atg gag agt tac tgt gag aca tgg cga act gaa act act ggg 432Arg Leu Met Glu Ser Tyr Cys Glu Thr Trp Arg Thr Glu Thr Thr Gly
130 135 140gct aca ggt cag gcc tcc tcc ctg ctg rca ggc agg ctc ctg gaa cag 480Ala Thr Gly Gln Ala Ser Ser Leu Leu Ser Gly Arg Leu Leu Glu Gln145 150 155 160aaa gct gcg agc tgc cac aac agc tac arc gtc ctg tgc att gag aat 528Lys Ala Ala Ser Cys His Asn Ser Tyr Ile Val Leu Cys Ile Glu Asn
165 170 175agc ttc atg acc tct ttc tcc aaa 552Ser Phe Met Thr Ser Phe Ser Lys
180<210>18<211>184<212>PRT<213>小家鼠<400>18His Thr His Gln Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn1 5 10 15Thr Pro Leu Ser Gly Gly Met Arg Gly Ile Arg Gly Ala Asp Phe Gln
20 25 30Cys Phe Gln Gln Ala Arg Ala Val Gly Leu Ser Gly Thr Phe Arg Ala
35 40 45Phe Leu Ser Ser Arg Leu Gln Asp Leu Tyr Ser Ile Val Arg Arg Ala
50 55 60Asp Arg Gly Ser Val Pro Ile Val Asn Leu Lys Asp Glu Val Leu Ser65 70 75 80Pro Ser Trp Asp Ser Leu Phe Ser Gly Ser Gln Gly Gln Val Gln Pro
85 90 95Gly Ala Arg Ile Phe Ser Phe Asp Gly Arg Asp Val Leu Arg His Pro
100 105 110Ala Trp Pro Gln Lys Ser Val Trp His Gly Ser Asp Pro Ser Gly Arg
115 120 125Arg Leu Met Glu Ser Tyr Cys Glu Thr Trp Arg Thr Glu Thr Thr Gly
130 135 140Ala Thr Gly Gln Ala Ser Ser Leu Leu Ser Gly Arg Leu Leu Glu Gln145 150 155 160Lys Ala Ala Ser Cys His Asn Ser Tyr Ile Val Leu Cys Ile Glu Asn
165 170 175Ser Phe Met Thr Ser Phe Ser Lys
180<210>19<211>699<212>DNA<213>小家鼠<220><221>CDS<222>(1)..(699)<223>Fc<400>19gag ccc aga ggg ccc aca arc aag ccc tgt cct cca tgc aaa tgc cca 48Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro1 5 10 15gca cct aac crc ttg ggt gga cca tcc gtc ttc atc ttc cct cca aag 96Ala Pro Asn Leu Leu Gly Gly Pro Set Val Phe Ile Phe Pro Pro Lys
20 25 30atc aag gat gta ctc atg arc tcc ctg agc ccc ata gtc aca tgt gtg 144Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val
35 40 45gtg gtg gat gtg agc gag gat gac cca gat gtc cag atc agc tgg ttt 192Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe
50 55 60gtg aac aac gtg gaa gta cac aca gct cag aca caa acc cat aga gag 240Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu65 70 75 80gat tac aac agt act ctc cgg gtg gtc agt gcc ctc ccc atc cag cac 288Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His
85 90 95cag gac tgg atg agt ggc aag gag ttc aaa tgc aag gtc aac aac aaa 336Gln ASp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys
100 105 110gac crc cca gcg ccc atc gag aga acc atc tca aaa ccc aaa ggg tca 384Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser
115 120 125gta aga gct cca cag gta tat gtc ttg cct cca cca gaa gaa gag atg 432Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met
130 135 140act aag aaa cag gtc act ctg acc tgc atg gtc aca gac ttc atg cct 480Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro145 150 155 160gaa gac att tac gtg gag tgg acc aacaac ggg aaa aca gag cta aac 528Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn
165 170 175tac aag aac act gaa cca gtc ctg gac tct gat ggt tct tacttc atg 576Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met
180 185 190tac agc aag ctg aga gtg gaa aag aag aac tgg gtg gaa aga aat agc 624Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser
195 200 205tac tcc tgt tca gtg gtc cac gag ggt ctg cac aat cac cac acg act 672Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr Thr
210 215 220aag agc ttc tcc cgg acc ccg ggt aaa 699Lys Ser Phe Ser Arg Thr Pro Gly Lys225 230<210>20<211>233<212>PRT<213>小家鼠<400>20Glu Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro1 5 10 15Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys
20 25 30Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val
35 40 45Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe
50 55 60Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu65 70 75 80Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His
85 90 95Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys
100 105 110Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys pro Lys Gly Ser
115 120 125Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met
130 135 140Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro145 150 155 160Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn
165 170 175Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met
180 185 190Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser
195 200 205Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr Thr
210 215 220Lys Ser Phe Ser Arg Thr Pro Gly Lys225 230<210>21<211>29<212>DNA<213>人工序列<220><223>人工序列的描述:小鼠Fc-Endo的正向引物<220><221>CDS<222>(2)..(28)<400>21c ccc aag ctt cat act cat cag gac ttt c 29Pro Lys Leu His Thr His Gln Asp Phe
1 5<210>22<211>9<212>PRT<213>人工序列<400>22Pro Lys Leu His Thr His Gln Asp Phe1 5<210>23<211>28<212>DNA<213>人工序列<220><223>人工序列的描述:小鼠Fc-Endo的反向引物<400>23cccctcgagc tatttggaga aagaggtc 28<210>24<211>1086<212>DNA<213>小家鼠<220><221>CDS<222>(1)..(1086)<223>制管张素<400> 24gtg tat ctg tca gaa tgt aag acc ggc atc ggc aac ggc tac aga gga 48Val Tyr Leu Ser Glu Cys Lys Thr Gly Ile Gly Asn Gly Tyr Arg Gly1 5 10 15acc atg tcc agg aca aag agt ggt gtt gcc tgt caa aag tgg ggt gcc 96Thr Met Ser Arg Thr Lys Ser Gly Val Ala Cys Gln Lys Trp Gly Ala
20 25 30acg ttc ccc cac gta ccc aac tac tct ccc agt aca cat ccc aat gag 144Thr Phe Pro His Val Pro Asn Tyr Ser Pro Ser Thr His Pro Asn Glu
35 40 45gga cta gaa gag aac tac tgt agg aac cca gac aat gat gaa caa ggg 192Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Glu Gln Gly
50 55 60cct tgg tgc tac act aca gat ccg gac aag aga tat gac tac tgc aac 240Pro Trp Cys Tyr Thr Thr Asp Pro Asp Lys Arg Tyr Asp Tyr Cys Asn65 70 75 80att cct gaa tgt gaa gag gaa tgc atg tac tgc agt gga gaa aag tat 288Ile Pro Glu Cys Glu Glu Glu Cys Met Tyr Cys Ser Gly Glu Lys Tyr
85 90 95gag ggc aaa atc tcc aag acc atg tct gga ctt gac tgc cag gcc tgg 336Glu Gly Lys Ile Ser Lys Thr Met Ser Gly Leu Asp Cys Gln Ala Trp
100 105 110gat tct cag agc cca cat gct cat gga tac atc cct gcc aaa ttt cca 384Asp Ser Gln Ser Pro His Ala His Gly Tyr Ile Pro Ala Lys Phe Pro
115 120 125agc aag aac ctg aag atg aat tat tgc cac aac cct gac ggg gag cca 432Ser Lys Asn Leu Lys Met Asn Tyr Cys His Asn Pro Asp Gly Glu Pro
130 135 140agg ccc tgg tgc ttc aca aca gac ccc acc aaa cgc tgg gaa tac tgt 480Arg Pro Trp Cys Phe Thr Thr Asp Pro Thr Lys Arg Trp Glu Tyr Cys145 150 155 160gac atc ccc cgc tgc aca aca ccc ccg ccc cca ccc agc cca acc tac 528Asp Ile Pro Arg Cys Thr Thr Pro Pro Pro Pro Pro Ser Pro Thr Tyr
165 170 175caa tgt ctg aaa gga aga ggt gaa aat tac cga ggg acc gtg tct gtc 576Gln Cys Leu Lys Gly Arg Gly Glu Asn Tyr Arg Gly Thr Val Ser Val
180 185 190acc gtg tct ggg aaa acc tgt cag cgc tgg agt gag caa acc cct cat 624Thr Val Ser Gly Lys Thr Cys Gln Arg Trp Ser Glu Gln Thr Pro His
195 200 205agg cac aac agg aca cca gaa aat ttc ccc tgc aaa aat ctg gaa gag 672Arg His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Glu Glu
210 215 220aac tac tgc cgg aac cca gat gga gaa act gct ccc tgg tgc tat acc 720Asn Tyr Cys Arg Asn Pro Asp Gly Glu Thr Ala Pro Trp Cys Tyr Thr225 230 235 240act gac agc cag ctg agg tgg gag tac tgt gag att cca tcc tgc gag 768Thr Asp Ser Gln Leu Arg Trp Glu Tyr Cys Glu Ile Pro Ser Cys Glu
245 250 255tcc tca gca tca cca gac cag tca gat tcc tca gtt cca cca gag gag 816Ser Ser Ala Ser Pro Asp Gln Ser Asp Ser Ser Val Pro Pro Glu Glu
260 265 270caa aca cct gtg Gtc Gag gaa tgc tac cag agc gat ggg cag agc tat 864Gln Thr Pro Val Val Gln Glu Cys Tyr Gln Ser Asp Gly Gln Ser Tyr
275 280 285cgg ggt aca tcg tcc act acc atc aca ggg aag aag tgc cag tcC tgg 912Arg Gly Thr Ser Ser Thr Thr Ile Thr Gly Lys Lys Cys Gln Ser Trp
290 295 300gca gct atg ttt cca cac agg cat tcg aag acc cca gag aac ttc cca 960Ala Ala Met Phe Pro His Arg His Ser Lys Thr Pro Glu Asn Phe Pro305 310 315 320gat gct ggc ttg gag atg aac tac tgc agg aac ccg gat ggt gac aag 1008Asp Ala Gly Leu Glu Met Asn Tyr Cys Arg Asn Pro Asp Gly Asp Lys
325 330 335ggc cct tgg tgc tac acc act gac ccg agc gtc agg tgg gaa tac tgc 1056Gly Pro Trp Cys Tyr Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr Cys
340 345 350aac ctg aag cgg tgc tca gag aca gga ggg 1086Asn Leu Lys Arg Cys Ser Glu Thr Gly Gly
355 360<210>25<211>362<212>PRT<213>小家鼠<400>25Val Tyr Leu Ser Glu Cys Lys Thr Gly Ile Gly Asn Gly Tyr Arg Gly1 5 10 15Thr Met Ser Arg Thr Lys Ser Gly Val Ala Cys Gln Lys Trp Gly Ala
20 25 30Thr Phe Pro His Val Pro Asn Tyr Ser Pro Ser Thr His Pro Asn Glu
35 40 45Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Glu Gln Gly
50 55 60Pro Trp Cys Tyr Thr Thr Asp Pro Asp Lys Arg Tyr Asp Tyr Cys Asn65 70 75 80Ile Pro Glu Cys Glu Glu Glu Cys Met Tyr Cys Ser Gly Glu Lys Tyr
85 90 95Glu Gly Lys Ile Ser Lys Thr Met Ser Gly Leu Asp Cys Gln Ala Trp
100 105 110Asp Ser Gln Ser Pro His Ala His Gly Tyr Ile Pro Ala Lys Phe Pro
115 120 125Ser Lys Asn Leu Lys Met Asn Tyr Cys His Asn Pro Asp Gly Glu Pro
130 135 140Arg Pro Trp Cys Phe Thr Thr Asp Pro Thr Lys Arg Trp Glu Tyr Cys145 150 155 160Asp Ile Pro Arg Cys Thr Thr Pro Pro Pro Pro Pro Ser Pro Thr Tyr
165 170 175Gln Cys Leu Lys Gly Arg Gly Glu Asn Tyr Arg Gly Thr Val Ser Val
180 185 190Thr Val Ser Gly Lys Thr Cys Gln Arg Trp Ser Glu Gln Thr Pro His
195 200 205Arg His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Glu Glu
210 215 220Asn Tyr Cys Arg Asn Pro Asp Gly Glu Thr Ala Pro Trp Cys Tyr Thr225 230 235 240Thr Asp Ser Gin Leu Arg Trp Glu Tyr Cys Glu Ile Pro Ser Cys Glu
245 250 255Ser Ser Ala Ser Pro Asp Gln Ser Asp Ser Ser Val Pro Pro Glu Glu
260 265 270Gln Thr Pro Val Val Gln Glu Cys Tyr Gln Ser Asp Gly Gln Ser Tyr
275 280 285Arg Gly Thr Ser Ser Thr Thr Ile Thr Gly Lys Lys Cys Gln Ser Trp
290 295 300Ala Ala Met Phe Pro His Arg His Ser Lys Thr Pro Glu Asn Phe Pro305 310 315 320Asp Ala Gly Leu Glu Met Asn Tyr Cys Arg Asn Pro Asp Gly Asp Lys
325 330 335Gly Pro Trp Cys Tyr Thr Thr Asp Pro Ser Val Arg Trp Glu Tyr Cys
340 345 350Asn Leu Lys Arg Cys Ser Glu Thr Gly Gly
355 360<210>26<211>31<212>DNA<213>人工序列<220><223>人工序列的描述:小鼠Fc-Angio的正向引物<220><221>CDS<222>(2)..(49)<400>26c ccc aag ctt gtg tat ctg tca gaa tgt aag 31Pro Lys Leu Val Tyr Leu Ser Glu Cys Lys
1 5 10<210>27<211>10<212>PRT<213>人工序列<400>27Pro Lys Leu Val Tyr Leu Ser Glu Cys Lys1 5 10<210>28<211>30<212>DNA<213>人工序列<220><223>人工序列的描述:小鼠Fc-Angio的反向引物<400>28cccctcgagctaccctcctg tctctgagca 30<210>29<211>29<212>DNA<213>人工序列<220><223>人工序列的描述:狗Fc的正向引物<220><221>CDS<222>(3)..(29)<400>29cc tta agc gaa aat gga aga gtt cct cgc 29Leu Ser Glu Asn Gly Arg Val Pro Arg
1 5<210>30<211>9<212>PRT<213>人工序列<400>30Leu Ser Glu Asn Gly Arg Val Pro Arg1 5<210>31<211>40<212>DNA<213>人工序列<220><223>人工序列的描述:狗Fc的反向引物<400>31cctcgagtca tttacccggg gaatgggaga gggatttctg 40<210>32<211>702<212>DNA<213>家犬<220><221>CDS<222>(1)..(702)<223>Fc<400>32gaa aat gga aga gtt cct cgc cca cct gat tgt ccc aaa tgc cca gcc 48Glu Asn Gly Arg Val Pro Arg Pro Pro Asp Cys Pro Lys Cys Pro Ala1 5 10 15cct gaa atg ctg gga ggg cct tcg gtc ttc atc ttt ccc ccg aaa ccc 96Pro Glu Met Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro
20 25 30aag gac acc ctc ttg att gcc cga aca cct gag gtc aca tgt gtg gtg 144Lys Asp Thr Leu Leu Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45gtg gat ctg gga cca gaa gac cct gag gtg cag atc agc tgg ttc gtg 192Val Asp Leu Gly Pro Glu Asp Pro Glu Val Gln Ile Ser Trp Phe Val
50 55 60gac ggt aag cag atg caa aca gcc aag act cag cct cgt gag gag cag 240Asp Gly Lys Gln Met Gln Thr Ala Lys Thr Gln Pro Arg Glu Glu Gln65 70 75 80ttc aat ggc acc tac cgt gtg gtc agt gtc ctc ccc att ggg cac cag 288Phe Asn Gly Thr Tyr Arg Val Val Ser Val Leu Pro Ile Gly His Gln
85 90 95gac tgg ctc aag ggg aag cag ttc acg tgc aaa gtc aac aac aaa gcc 336Asp Trp Leu Lys Gly Lys Gln Phe Thr Cys Lys Val Asn Asn Lys Ala
100 105 110ctc cca tcc ccg atc gag agg acc atc tcc aag gcc aga ggg cag gcc 384Leu Pro Ser Pro Ile Glu Arg Thr Ile Ser Lys Ala Arg Gly Gln Ala
115 120 125cat cag ccc agt gtg tat gtc ctg ccg cca tcc cgg gag gag ttg agc 432His Gln Pro Ser Val Tyr Val Leu Pro Pro Ser Arg Glu Glu Leu Ser
130 135 140aag aac aca gtc agc ttg aca tgc ctg atc aaa gac ttc ttc cca cct 480Lys Asn Thr Val Ser Leu Thr Cys Leu Ile Lys Asp Phe Phe Pro Pro145 150 155 160gac att gat gtg gag tgg cag agc ast gga cag cag gag cct gag agc 528Asp Ile Asp Val Glu Trp Gln Ser Asn Gly Gln Gln Glu Pro Glu Ser
165 170 175aag tac cgc acg acc ccg ccc cag ctg gac gag gac ggg tcc tacttc 576Lys Tyr Arg Thr Thr Pro Pro Gln Leu Asp Glu Asp Gly Ser Tyr Phe
180 185 190ctg tac agc aag ctc tct gtg gac aag agc cgc tgg cag cgg gga gac 624Leu Tyr Ser Lys Leu Ser Val Asp Lys Ser Arg Trp Gln Arg Gly Asp
195 200 205acc ttc ata tgt gcg gtg atg cat gaa gct cta cac aac cac tac aca 672Thr Phe Ile Cys Ala Val Met Mis Glu Ala Leu His Ash His Tyr Thr
210 215 220cag aaa tcc ctc tcc cat tct ccg ggt aaa 702Gln Lys Ser Leu Ser His Ser Pro Gly Lys225 230<210>33<211>234<212>PRT<213>家犬<400>33Glu Asn Gly Arg Val Pro Arg Pro Pro Asp Cys Pro Lys Cys Pro Ala 1 5 10 15Pro Glu Met Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Pro
20 25 30Lys Asp Thr Leu Leu Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val
35 40 45Val Asp Leu Gly Pro Glu Asp Pro Glu Val Gln Ile Ser Trp Phe Val
50 55 60Asp Gly Lys Gln Met Gln Thr Ala Lys Thr Gln Pro Arg Glu Glu Gln65 70 75 80Phe Asn Gly Thr Tyr Arg Val Val Ser Val Leu Pro Ile Gly His Gln
85 90 95Asp Trp Leu Lys Gly Lys Gln Phe Thr Cys Lys Val Asn Asn Lys Ala
100 105 110Leu Pro Ser Pro Ile Glu Arg Thr Ile Ser Lys Ala Arg Gly Gln Ala
115 120 125His Gln Pro Ser Val Tyr Val Leu Pro Pro Ser Arg Glu Glu Leu Ser
130 135 140Lys Asn Thr Val Ser Leu Thr Cys Leu Ile Lys Asp Phe Phe Pro Pro145 150 155 160Asp Ile Asp Val Glu Trp Gln Ser Asn Gly Gln Gln Glu Pro Glu Ser
165 170 175Lys Tyr Arg Thr Thr Pro Pro Gln Leu Asp Glu Asp Gly Ser Tyr Phe
180 185 190Leu Tyr Ser Lys Leu Ser Val Asp Lys Ser Arg Trp Gln Arg Gly Asp
195 200 205Thr Phe Ile Cys Ala Val Met His Glu Ala Leu His Asn His Tyr Thr
210 215 220Gln Lys Ser Leu Ser His Ser Pro Gly Lys225 230<210>34<211>552<212>DNA<213>家犬<220><221>CDS<222>(1)..(552)<223>Endostatin<400>34cac acc cac cag gac ttc cag ccg gtg ctg cac ctg gtg gcc ctg aac 48His Thr His Gln Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn1 5 10 15agc ccg cag ccg ggc ggc atg cga ggc atc cgg gga gcg gac ttc cag 96Ser Pro Gln Pro Gly Gly Met Arg Gly Ile Arg Gly Ala Asp Phe Gln
20 25 30tgc ttc cag cag gcg cgc gcc gcg ggg ctg gcc ggc acc ttc cgg gcc 144Cys Phe Gln Gln Ala Arg Ala Ala Gly Leu Ala Gly Thr Phe Arg Ala
35 40 45ttc ctg tcg tcg cgg ctg cag gac ctc tac agc atc gtg cgc cgc gcc 192Phe Leu Ser Ser Arg Leu Gln Asp Leu Tyr Ser Ile Val Arg Arg Ala
50 55 60gac cgc acc ggg gtg ccc gtc gtc aac crc agg gac gag gtg crc ttc 240Asp Arg Thr Gly Val Pro Val Val Asn Leu Arg Asp Glu Val Leu Phe65 70 75 80ccc agc tgg gag gcc tta ttc tcg ggc tcc gag ggc cag ctg aag ccc 288Pro Ser Trp Glu Ala Leu Phe Ser Gly Ser Glu Gly Gln Leu Lys Pro
85 90 95ggg gcc cgc atc ttc tct ttc gac ggc aga gat gtc ctg cag cac ccc 336Gly Ala Arg Ile Phe Ser Phe Asp Gly Arg Asp Val Leu Gln His Pro
100 105 110gcc tgg ccc cgg aag agc gtg tgg cac ggc tcc gac ccc agc ggg cgc 384Ala Trp Pro Arg Lys Ser Val Trp His Gly Ser Asp Pro Ser Gly Arg
115 120 125cgc ctg acc gac agc tac tgc gag acg tgg cgg acg gag gcc ccg gcg 432Arg Leu Thr Asp Ser Tyr Cys Glu Thr Trp Arg Thr Glu Ala Pro Ala
130 135 140gcc acc ggg cag gcg tcg tcg ctg ctg gcg ggc agg ctg ctg gag cag 480Ala Thr Gly Gln Ala Ser Ser Leu Leu Ala Gly Arg Leu Leu Glu Gln145 150 155 160gag gcc gcg agc tgc cgc cac gcc ttc gtg gtg ctc tgc atc gag aac 528Glu Ala Ala Ser Cys Arg His Ala Phe Val Val Leu Cys Ile Glu Asn
165 170 175agc gtc atg acc tcc ttc tcc aag 552Ser Val Met Thr Ser Phe Ser Lys
180<210>35<211>184<212>PRT<213>家犬<400>35His Thr His Gln Asp Phe Gln Pro Val Leu His Leu Val Ala Leu Asn1 5 10 15Ser Pro Gln Pro Gly Gly Met Arg Gly Ile Arg Gly Ala Asp Phe Gln
20 25 30Cys Phe Gln Gln Ala Arg Ala Ala Gly Leu Ala Gly Thr Phe Arg Ala
35 40 45Phe Leu Ser Ser Arg Leu Gln Asp Leu Tyr Ser Ile Val Arg Arg Ala
50 55 60Asp Arg Thr Gly Val Pro Val Val Asn Leu Arg Asp Glu Val Leu Phe65 70 75 80Pro Ser Trp Glu Ala Leu Phe Ser Gly Ser Glu Gly Gln Leu Lys Pro
85 90 95Gly Ala Arg Ile Phe Ser Phe Asp Gly Arg Asp Val Leu Gln His Pro
100 105 11Ala Trp Pro Arg Lys Ser Val Trp His Gly Ser Asp Pro Ser Gly Arg
115 120 125Arg Leu Thr Asp Ser Tyr Cys Glu Thr Trp Arg Thr Glu Ala Pro Ala
130 135 140Ala Thr Gly Gln Ala Ser Ser Leu Leu Ala Gly Arg Leu Leu Glu Gln145 150 155 160Glu Ala Ala Ser Cys Arg His Ala Phe Val Val Leu Cys Ile Glu Asn
165 170 175Ser Val Met Thr Ser Phe Ser Lys
180<210>36<211>41<212>DNA<213>人工序列<220><223>人工序列的描述:HindIII/DraIII接头:上链<220><221>CDS<222>(3)..(41)<400>36ag ctt cac acc cac cag gac ttc cag ccg gtg ctg cac ctg 41Leu His Thr His Gln Asp Phe Gln Pro Val Leu His Leu
1 5 10<210>37<211>13<212>PRT<213>人工序列<400>37Leu His Thr His Gln Asp Phe Gln Pro Val Leu His Leu1 5 10<210>38<211>34<212>DNA<213>人工序列<220><223>人工序列的描述:HindIII/DraIII接头:下链<400>38gtgcagcacc ggctggaagt cctggtgggt gtga 34<210>39<211>1077<212>DNA<213>家犬<220><221>CDS<222>(1)..(1077)<223>人工序列<400>39ata tat ctt tca gag tgc aag act gga aat ggg aaa acc tac agg ggg 48Ile Tyr Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Thr Tyr Arg Gly1 5 10 15acc atg gcc aaa acg aag aat gat gtt gcc tgt caa aaa tgg agt gac 96Thr Met Ala Lys Thr Lys Asn Asp Val Ala Cys Gln Lys Trp Ser Asp
20 25 30aat tct ccg cac aaa cct aac tat acg cct gag aag cac ccc ttg gag 144Asn Ser Pro His Lys Pro Asn Tyr Thr Pro Glu Lys His Pro Leu Glu
35 40 45ggg ctg gag gag aac tat tgc agg aaccct gac aac gac gag aac ggg 192Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Glu Asn Gly
50 55 60ccc tgg tgc tac acc aca aac ccagac gtg agg ttc gac tac tgc aac 240Pro Trp Cys Tyr Thr Thr Asn Pro Asp Val Arg Phe Asp Tyr Cys Asn65 70 75 80att cca gaa tgt gaa gag gaa tgt atg cat tgc agt ggg gaa aat tat 288Ile Pro Glu Cys Glu Glu Glu Cys Met His Cys Ser Gly Glu Asn Tyr
85 90 95gag ggc aaa att tcc aag aca aag tct gga ctc gag tgc caa gcc tgg 336Glu Gly Lys Ile Ser Lys Thr Lys Ser Gly Leu Glu Cys Gln Ala Trp
100 105 110aac tct caa acc cca cat gct cat gga tat att cct tcc aaa ttt cca 384Asn Ser Gln Thr Pro His Ala His Gly Tyr Ile Pro Ser Lys Phe Pro
115 120 125agc aag aac ttg aag atg aat tac tgc cgt aac cct gat ggg gag ccc 432Ser Lys Asn Leu Lys Met Asn Tyr Cys Arg Asn Pro Asp Gly Glu Pro
130 135 140cgc cca tgg tgt ttc acc atg gat ccc aac aaa cgc tgg gaa ttc tgt 480Arg Pro Trp Cys Phe Thr Met Asp Pro Asn Lys Arg Trp Glu Phe Cys145 150 155 160gac att ccc cgc tgt aca aca cca cca ccc cct tcg ggc cca acg tac 528Asp Ile Pro Arg Cys Thr Thr Pro Pro Pro Pro Ser Gly Pro Thr Tyr
165 170 175cag tgt ctg aag ggc aga ggg gag agc tac cga ggg aag gtg tcc gtc 576Gln Cys Leu Lys Gly Arg Gly Glu Ser Tyr Arg Gly Lys Val Ser Val
180 185 190act gtc tct gga cat aca tgt cag cactgg agt gaa cag acc cct cac 624Thr Val Ser Gly His Thr Cys Gln His Trp Ser Glu Gln Thr Pro His
195 200 205aag cac aac agg acc cca gaa aac ttc cct tgc aaa aat ttg gat gaa 672Lys His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp Glu
210 215 220aac tac tgt cgc aac cct gat gga gaa aca gct cca tgg tgc tac aca 720Asn Tyr Cys Arg Asn Pro Asp Gly Glu Thr Ala Pro Trp Cys Tyr Thr225 230 235 240acc aac agt gag gtg agg tgg gaa cac tgc cag att ccg tcc tgt gag 768Thr Asn Ser Glu Val Arg Trp Glu His Cys Gln Ile Pro Ser Cys Glu
245 250 255tcc tct cca ata acc aca gaa tat ttg gat gcc cca gct tca gtg cca 816Ser Ser Pro Ile Thr Thr Glu Tyr Leu Asp Ala Pro Ala Ser Val Pro
260 265 270cct gaa caa act cct gtg gtc cag gag tgc tac cac ggc aat ggg cag 864Pro Glu Gln Thr Pro Val Val Gln Glu Cys Tyr His Gly Asn Gly Gln
275 280 285agt tat cga ggc aca tca tcc act act atc aca gga aga aaa tgt cag 912Ser Tyr Arg Gly Thr Ser Ser Thr Thr Ile Thr Gly Arg Lys Cys Gln
290 295 300tct tgg tca tct atg aca cca cac cga cat gag aag acc cca gaa cac 960Ser Trp Ser Ser Met Thr Pro His Arg His Glu Lys Thr Pro Glu His305 310 315 320ttc ccg gag gct ggc etg aca atg aac tac tgc agg aat ccc gac gcc 1008Phe Pro Glu Ala Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala
325 330 335gac aaa agc cct tgg tgt tac acc acc gac ccc tct gtg cgc tgg gag 1056Asp Lys Ser Pro Trp Cys Tyr Thr Thr Asp Pro Ser Val Arg Trp Glu
340 345 350ttc tgt aac ttg aga aaa tgc 1077Phe Cys Asn Leu Arg Lys Cys
355<210>40<211>359<212>PRT<213>家犬<400>40Ile Tyr Leu Ser Glu Cys Lys Thr Gly Asn Gly Lys Thr Tyr Arg Gly1 5 10 15Thr Met Ala Lys Thr Lys Asn Asp Val Ala Cys Gln Lys Trp Ser Asp
20 25 30Asn Ser Pro His Lys Pro Asn Tyr Thr Pro Glu Lys His Pro Leu Glu
35 40 45Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn Asp Glu Asn Gly
50 55 60Pro Trp Cys Tyr Thr Thr Asn Pro Asp Val Arg Phe Asp Tyr Cys Asn65 70 75 80Ile Pro Glu Cys Glu Glu Glu Cys Met His Cys Ser Gly Glu Asn Tyr
85 90 95Glu Gly Lys Ile Ser Lys Thr Lys Ser Gly Leu Glu Cys Gln Ala Trp
100 105 110Asn Ser Gin Thr Pro His Ala His Gly Tyr Ile Pro Ser Lys Phe Pro
115 120 125Ser Lys Asn Leu Lys Met Asn Tyr Cys Arg Asn Pro Asp Gly Glu Pro
130 135 140Arg Pro Trp Cys Phe Thr Met Asp Pro Asn Lys Arg Trp Glu Phe Cys145 150 155 160Asp Ile Pro Arg Cys Thr Thr Pro Pro Pro Pro Ser Gly Pro Thr Tyr
165 170 175Gln Cys Leu Lys Gly Arg Gly Glu Ser Tyr Arg Gly Lys Val Ser Val
180 185 190Thr Val Ser Gly Mis Thr Cys Gln His Trp Ser Glu Gln Thr Pro His
195 200 205Lys His Asn Arg Thr Pro Glu Asn Phe Pro Cys Lys Asn Leu Asp Glu
210 215 220Asn Tyr Cys Arg Asn Pro Asp Gly Glu Thr Ala Pro Trp Cys Tyr Thr225 230 235 240Thr ASn Ser Glu Val Arg Trp Glu His Cys Gln Ile Pro Ser Cys Glu
245 250 255Ser Ser Pro Ile Thr Thr Glu Tyr Leu Asp Ala Pro Ala Ser Val Pro
260 265 270Pro Glu Gln Thr Pro Val Val Gln Glu Cys Tyr His Gly Asn Gly Gln
275 280 285Ser Tyr Arg Gly Thr Ser Ser Thr Thr Ile Thr Gly Arg Lys Cys Gln
290 295 300Ser Trp Ser Ser Met Thr Pro His Arg His Glu Lys Thr Pro Glu His305 310 315 320Phe Pro Glu Ala Gly Leu Thr Met Asn Tyr Cys Arg Asn Pro Asp Ala
325 330 335Asp Lys Ser Pro Trp Cys Tyr Thr Thr Asp Pro Ser Val Arg Trp Glu
340 345 350Phe Cys Asn Leu Arg Lys Cys
355<210>41<211>12<212>DNA<213>人工序列<220><223>人工序列的描述:其中终止密码子TGA后为XhoI终点的回文接头<400>41tgactcgagt ca 12<210>42<211>21<212>DNA<213>人工序列<220><223>人工序列的描述:用于鼠制管张素的诱变引物<220><221>CDS<222>(1)..(21)<400>42ggg cct tgg agc tac act aca 21Gly Pro Trp Ser Tyr Thr Thr1 5<210>43<211>7<212>PRT<213>人工序列<400>43Gly Pro Trp Ser Tyr Thr Thr1 5<210>44<211>34<212>DNA<213>人工序列<220><223>人工序列的描述:用于在鼠制管张素中导入HindIII的引物<220><221>CDS<222>(9)..(32)<400>44gcggatcc aag ctt agt aca cat ccc aat gag gg 34
Lys Leu Ser Thr His Pro Asn Glu
1 5<210>45<211>8<212>PRT<213>人工序列<400>45Lys Leu Ser Thr His Pro Asn Glu1 5<210>46<211>59<212>DNA<213>人工序列<220><223>人工序列的描述:BspHI/BamHI接头:上链<220><221>CDS<222>(2)..(58)<400>46c atg acc tct ttc tcc aaa tcg agc ggg ggc agc ggg ggc gga ggc agc 49Met Thr Ser Phe Ser Lys Ser Ser Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15ggc ggg ggc g 59Gly Gly Gly<210>47<211>19<212>PRT<213>人工序列<400>47Met Thr Ser Phe Ser Lys Ser Ser Gly Gly Ser Gly Gly Gly Gly Ser1 5 10 15Gly Gly Gly<210>48<211>59<212>DNA<213>人工序列<220><223>人工序列的描述:BspHI/BamHI接头:下链<400>48gatccgcccc cgccgctgcc tccgcccccg ctgcccccgc tcgatttgga gaaagaggt 59<210>49<211>12<212>DNA<213>人工序列<220><223>人工序列的描述:BamHI/HindIII接头:上链<220><221>CDS<222>(3)..(11)<400>49ga tcc tca ggc c 12Ser Ser Gly
1<210>50<211>12<212>DNA<213>人工序列<220><223>人工序列的描述:BamHI/HindIII接头:下链<400>50agctggcctg ag 12<210>51<211>10<212>DNA<213>人工序列<220><223>人工序列的描述:AflII/HindIII接头:上链<220><221>CDS<222>(1)..(9)<400>51tta agc ggcc 10Leu Ser Gly1<210>52<211>10<212>DNA<213>人工序列<220><223>人工序列的描述:AflII/HindIII接头:下链<400>52agctgggcgc 10<210>53<211>11<212>DNA<213>人工序列<220><223>人工序列的描述:XhoI/AflII接头:上链<220><221>CDS<222>(3)..(11)<400>53tc gac tcc ggc 11Asp Ser Gly
1<210>54<211>11<212>DNA<213>人工序列<220><223>人工序列的描述:XhoI/AflII接头:下链<400>54ttaagccgga g 11
Claims (31)
1.编码一种融合蛋白的DNA分子,它包括:
(a)信号序列;
(b)免疫球蛋白Fc区;和
(c)靶蛋白序列,该靶蛋白选自制管张素、endostatin、具有制
管张素活性的纤溶酶原片段、具有endostatin活性的胶原蛋白
XVIII片段和它们的组合物。
2.权利要求1的所述DNA,其中所述信号序列、所述免疫球蛋白Fc区和所述靶蛋白序列从5’到3’方向按顺序编码。
3.权利要求1的所述DNA,其中所述信号序列、所述靶蛋白序列和所述免疫球蛋白Fc区从5’到3’方向依次编码。
4.权利要求1的所述DNA,其中所述免疫球蛋白Fc区包含一个免疫球蛋白铰链区。
5.权利要求1的所述DNA,其中所述免疫球蛋白Fc区包含一个免疫球蛋白铰链区和一个免疫球蛋白重链恒定区结构域。
6.权利要求1的所述DNA,其中所述免疫球蛋白Fc区包含一个铰链区和一个CH3结构域。
7.权利要求1的所述DNA,其中所述免疫球蛋白Fc区缺乏至少所述CH1结构域。
8.权利要求1的所述DNA,其中所述免疫球蛋白Fc区编码至少一部分免疫球蛋白γ。
9.用于转染哺乳动物细胞的复制型表达载体,所述载体包含权利要求1的所述DNA。
10.包含权利要求1的所述DNA的哺乳动物细胞。
11.包含免疫球蛋白Fc区和靶蛋白的融合蛋白,其中所述靶蛋白选自制管张素、endostatin、具有制管张素活性的纤溶酶原片段、具有endostatin活性的胶原蛋白XVIII片段和它们的组合物。
12.权利要求11的所述融合蛋白,其中所述纤溶酶原片段的分子量约为40kD并包含SEQ ID NO:3所示氨基酸序列。
13.权利要求11的所述融合蛋白,其中所述靶蛋白包含SEQ IDNO:3所示氨基酸序列。
14.权利要求11的所述融合蛋白,其中所述胶原蛋白XVIII片段包含SEQ ID NO:1所示氨基酸序列。
15.权利要求11的所述融合蛋白,其中所述靶蛋白包含至少两种选自制管张素、endostatin、纤溶酶原片段和胶原蛋白XVIII片段的分子,其中将所述两种分子通过一个多肽接头连接。
16.权利要求11的所述融合蛋白,其中所述靶蛋白连接到所述免疫球蛋白Fc区的N-末端。
17.权利要求11的所述融合蛋白,其中所述靶蛋白连接到所述免疫球蛋白Fc区的C-末端。
18.一种多聚体蛋白,它包含至少两种权利要求11的通过二硫键连接的融合蛋白。
19.权利要求18的所述多聚体蛋白,其中至少一种所述融合蛋白的靶蛋白为制管张素,而至少一种所述融合蛋白的靶蛋白为endostatin。
20.权利要求18的所述多聚体蛋白,其中两种所述融合蛋白的靶蛋白均为制管张素。
21.权利要求18的所述多聚体蛋白,其中两种所述融合蛋白的靶蛋白均为endostatin。
22.权利要求11的所述融合蛋白,它还包含一个第二靶蛋白,所述第二靶蛋白选自制管张素、endostatin、具有制管张素活性的纤溶酶原片段和具有endostatin活性的胶原蛋白XVIII片段。
23.权利要求22的所述融合蛋白,其中所述第二靶蛋白通过多肽接头连接到所述第一靶蛋白。
24.权利要求22的所述融合蛋白,其中所述第一靶蛋白连接到所述免疫球蛋白Fc区的N-末端,而所述第二靶蛋白连接到所述免疫球蛋白Fc区的C-末端。
25.一种多聚体融合蛋白,它包含至少两种权利要求11的融合蛋白,其中所述融合蛋白通过多肽键连接。
26.产生融合蛋白的方法,所述方法包括以下步骤:
a)提供权利要求10的所述哺乳动物细胞;和
b)培养所述哺乳动物细胞以生产所述融合蛋白。
27.权利要求26的所述方法,它包括收集所述靶蛋白的附加步骤。
28.权利要求26的所述方法,它包括从所述靶蛋白切除所述免疫球蛋白Fc区的附加步骤。
29.治疗血管生成性疾病的方法,该方法包括将权利要求1的所述DNA给予需要血管生成抑制剂的哺乳动物的步骤。
30.治疗血管生成性疾病的方法,该方法包括将权利要求9的所述载体给予需要血管生成抑制剂的哺乳动物的步骤。
31.通过给予制管张素或endostatin缓解病情的治疗方法,该方法包括将有效量权利要求11的所述融合蛋白给予患所述疾病的哺乳动物的步骤。
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Application Number | Priority Date | Filing Date | Title |
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US9788398P | 1998-08-25 | 1998-08-25 | |
US60/097883 | 1998-08-25 |
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US (3) | US20030139365A1 (zh) |
EP (1) | EP1107989B1 (zh) |
JP (3) | JP2002523036A (zh) |
CN (2) | CN101386651A (zh) |
AT (1) | ATE462725T1 (zh) |
AU (1) | AU761027B2 (zh) |
BR (1) | BR9913331A (zh) |
CA (1) | CA2339331C (zh) |
CZ (1) | CZ302303B6 (zh) |
DE (1) | DE69942207D1 (zh) |
DK (1) | DK1107989T3 (zh) |
ES (1) | ES2342239T3 (zh) |
HK (1) | HK1042503A1 (zh) |
HU (1) | HUP0103329A3 (zh) |
NO (1) | NO20010918L (zh) |
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CN108473549A (zh) * | 2015-10-23 | 2018-08-31 | 阿珀吉科吉尼科斯股份公司 | 单链gitr受体激动剂蛋白 |
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- 1999-08-25 EP EP99942468A patent/EP1107989B1/en not_active Expired - Lifetime
- 1999-08-25 JP JP2000566305A patent/JP2002523036A/ja active Pending
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- 1999-08-25 PL PL346703A patent/PL202057B1/pl unknown
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- 1999-08-25 CN CNA2008102106518A patent/CN101386651A/zh active Pending
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- 2002-06-07 HK HK02104318.5A patent/HK1042503A1/zh unknown
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CN108430492B (zh) * | 2015-10-23 | 2022-05-03 | 阿珀吉科吉尼科斯股份公司 | 单链cd27受体激动剂蛋白 |
CN108368511B (zh) * | 2015-10-23 | 2022-12-06 | 阿珀吉科吉尼科斯股份公司 | 单链cd137受体激动剂蛋白 |
CN109824779A (zh) * | 2017-11-23 | 2019-05-31 | 中山大学 | 一种包含IgG的Fc结构域和EB病毒包膜糖蛋白胞外域的融合蛋白 |
CN109824779B (zh) * | 2017-11-23 | 2023-05-26 | 中山大学 | 一种包含IgG的Fc结构域和EB病毒包膜糖蛋白胞外域的融合蛋白 |
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