CN1346888A - 生产转基因非人类哺乳动物的方法 - Google Patents
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Abstract
本发明涉及作为胆盐刺激的脂酶(BSSL;EC3.1.1.1)变异体的新多肽。还涉及编码所述多肽的DNA分子,及含所述DNA分子的亚产物。本发明还涉及生产所述BSSL变异体以及生产能表达BSSL变异体的转基因非人类哺乳动物的方法。此外,本发明涉及所述的转基因动物以及含有来自所述转基因动物的乳的婴儿配方。本发明还涉及含所述多肽的药物组合物,以及所述多肽和DNA分子对于药物制造的用途。
Description
本申请是申请日为1994年2月25日,申请号为94191848.3,发明名称为“胆盐刺激的脂酶变异体、编码其之DNA分子和转基因非人类哺乳动物”的发明专利申请的分案申请。
技术领域
本发明涉及作为胆盐刺激的脂酶(BSSL;EC 3.1.1.1.)变异体的新的多肽。也涉及编码所述多肽的DNA分子,涉及含所述DNA分子的亚产品。此外本发明涉及生产所述BSSL变异体和生产能表达BSSL变异体的转基因非人类哺乳动物的方法。而且本发明涉及所述的转基因动物及含此转基因动物乳的婴儿膳食配方。本发明还涉及含所述多肽的药物组合物,所述多肽及制造药物的DNA分子的用途。
背景技术膳食脂类的水解
膳食脂类是一种重要的能量来源。富含能量的三脂酰甘油占这些脂类的95%以上。一些脂类,如某些脂肪酸和脂溶性维生素,是必需的膳食成分。在胃肠吸收之前,三脂酰甘油和微量成分,如酯化的脂溶性维生素、胆固醇及二脂酰磷脂酰甘油,需要酯键水解以得到疏水性弱、可吸收的产物。通过称为脂酶的特异性酶类催化这些反应。
在人类中,相关的必需脂酶有,胃脂酶、共脂酶依赖的胰脂酶(水解三-和二脂酰甘油)、胰磷脂酶A2(水解二脂酰磷脂酰甘油)和羧基酯水解酶(CEH)(水解固醇酯和脂溶性维生素酯及三、二和单脂酰甘油)。对哺乳的新生儿,胆盐刺激的脂酶(BSSL)在水解上述提到的几种脂类过程中发挥着重要的作用。脂类消化产物和胆盐一起形成混合的吸收微团或单层吸收囊泡(Hernell et al.,1990)。胆盐刺激的脂酶
胆盐刺激的脂酶(BSSL)在有限几个种类,如人类、大猩猩、猫和狗中是乳中成分(Hernell et al.,1989,Hamosh et al.,1986)。在上部小肠内容物中与胆汁混合后,BSSL被初级胆盐(Hernell,1975)特异地激活。约占总乳蛋白1%的BSSL,和乳一起经过胃时不降解,在十二指肠内容物中胆盐保护其不被如胰蛋白酶和糜蛋白酶的胰蛋白酶类失活。
加热处理人乳(巴斯德消毒法62.5℃,30分钟),使BSSL完全失活(Bjrksten et al.,1980),使早产儿的脂肪吸收系数降低约1/3(Williamson et al.,1978,Atkinson et al.,1981),因此由于BSSL和同样脂肪成分的婴儿膳食配方比,新鲜人乳中三脂酰甘油较好利用(Hernell et al.,1991,Chapell et al.,1986)。
BSSL是一种非特异性脂酶(EC 3.1.1.1),它不但大量水解三脂酰甘油,而且水解二和单脂酰甘油,胆固醇酯类及脂溶性维生素酯类(Blckberg & Hernell,1983)。因此,活化后,BSSL本身有能力水解人乳中的大部分脂类,纵使人乳三脂酰甘油的最有效利用需要胃脂酶(EC 3.1.1.3),共脂酶依赖的胰脂酶(EC 3.1.1.3)和BSSL(Bernbck et al.,1990)的协同作用。
最近研究表明,乳酶对于新生儿利用长链多不饱和脂肪酸是特别重要的(Hernell et al.,1993)。这些脂肪酸是eicosanoids的重要前体并有助于神经系统的发育。新生儿,尤其早产儿,从前体合成这些脂肪酸的能力有限。因此,认为出生后在一段还未确定的时期内仍然需要它们。
最近从几个实验室研究中已描述了乳脂酶和胰羧基酯水解酶(CEH)(E.C.3.1.1.1)两者cDNA结构的特征(Baba et al.,1991;Hui et al.,1991;Nilsson et al.,1990;Reue etal.,1991),结论为,乳酶和胰酶是同一基因的产物。BSSL或CEH基因的cDNA序列及推导出的氨基酸序列(SEQ ID NO:1)也公开在WO91/15234(Oklahoma Medical Research Foundation)和WO 91/18923(Aktiebolaget Astra)中。
BSSL是一种单链糖蛋白。推导出的蛋白质(SEQ ID NO:3)含722个氨基酸残基,而且高度糖基化(Abouakil et al.,1989)。这种蛋白质N-端部分的一半同乙酰胆碱脂酶及其它一些脂酶有显著同源性(Nilsson et al.,1990)。
一个暂定的丝氨基残基活性位点定位在第194位丝氨酸;这个丝氨酸两侧的序列同丝氨酸水解酶的共有活性位点序列是一致的。一个暂定的N-糖基化位点、在活性位点丝氨酸N-端的仅7个残基处(Nilsson et al.,1990)。
BSSL在其C-端含16个脯氨酸丰富的重复序列,每个重复序列由11个氨基酸残基组成。重复序列数量的不同似乎主要解释了源于不同种之相应酶之间分子大小和氨基酸构成上的差异(Han et al.,1987;Fontaine et al.,1991;Kyger et al.,1989)。这些重复序列带有占此蛋白15-20%的糖类的大部分(Baba et al.,1991;Abouakil et al.,1989)。
在BSSL和典型的脂酶之间独一无二的结构差别在多肽链的C-端,即由11个氨基酸残基组成的16个富含脯氨酸的重复序列。牛和大鼠相应的胰酶分别只有3和4个重复序列(Han et al.,1987;Kyger et al.,1989)。因此,可以假设即,C-端,或至少部分C-端对于脂酶活性抗乳化的长链三脂酰甘油的活性是必不可少的。脂类吸收异常
脂类吸收异常及由此引起的营养不良,一般原因是管腔内共脂酶依赖的胰脂酶和/或胆盐的水平降低。这种脂酶缺乏的典型例子是囊性纤维性变患者,其中80%的人由于普遍的遗传性紊乱导致终生缺陷,再就是慢性胰腺炎,通常由慢性酒精中毒引起。
目前,治疗胰脂酶缺乏的患者,是口服很大剂量的猪胰酶粗制品。然而,共脂酶依赖的胰脂酶通常由于胃内pH低而失活。使用大剂量的酶不能完全克服此影响。因此大剂量口服胰酶对多数患者是不够的,而且该制品不纯且口味差。
已制定配方的某种药片,能通过胃的酸性区,只在空肠的相对碱性环境中释放出酶。但是,许多胰病患者空肠呈不正常的酸性,在那些情况下药片不能释放酶。
而且,由于目前市场上的制品为非人类来源,有产生免疫反应的危险性,对患者产生有害作用或降低疗效。
此制品进一步的弊端是,和共脂酶依赖的脂酶比,其它脂解活性的内容物尚未陈述。实际上,它们多半合很低水平的BSSL/CEH活性。这可能是许多患囊性纤维性变的患者尽管用补充疗法,但仍患有脂溶性维生素和必需脂肪酸缺乏的一个原因。
因此,强烈需要具有人类脂酶特性和结构并具有广泛底物特异性的产品。此产品能给患一或几种胰脂解酶缺乏的患者口服。使用本发明的产品本身或同含其它脂酶的制品结合满足了这种需要。
本发明构思简述
按照本发明,重组BSSL变异体有稳定的催化活性,但比全长BSSL含较少糖基化位点,由此其生产可能降低糖类的异质性程度。这种复杂性的降低使此重组蛋白易于纯化及描述特征,将导致更有效地生产具有BSSL活性的多肽。
另一方面,糖基化程度的降低减少了对宿主的要求,并允许几种宿主细胞有较高水平的生产。但在另一方面,BSSL变异体糖基化位点数的降低,允许较低等真核生物进行高效生产并限制可引起免疫反应的异常糖基化的潜在危险。这种大小的降低,糖基化复杂性较低也表明与具有很复杂及含大量糖类部分的蛋白质相比较,宿主范围比较广阔。
治疗用BSSL变异体的大小较小,但是活性相等,这意谓着补充所需的物质重量减少了。进一步,重组BSSL变异体缺乏大多数或全部O-糖基化重复单位,可能的优点是降低接受个体中产生免疫反应的危险性。这是因为依赖于产生它的细胞,O-连接的糖可能是很有异源性的。
科学文献指出,天然BSSL粘着在小肠粘液中并通过其吸收。筛选的降低了吸收率的BSSL变异体在较长时期内对膳食脂类底物是有活性的,进而产生高效的腔内消化。这种变异体的例子是具有降低的糖基化的分子。
如上所述,已证实BSSL对新生儿神经系统发育上发挥重要作用的长链多不饱和脂肪酸(Hernell et al.,1993)和维生素的利用是特别重要的。
按照本发明,在此方面更有效的BSSL变异体可经已知方法筛选。截短的酶可能有不同的构象,所述构象影响对不同的脂类底物的特异性。本发明公开
一方面,本发明涉及编码短于722个氨基酸的BSSL变异体的核酸分子,所说的BSSL变异体含SEQ ID NO:3中所示536-722残基部分氨基酸序列。
术语“部分氨基酸序列”理解为含一个氨基酸及几个氨基酸的序列或几个结合的所述序列。
术语“BSSL变异体”理解为多肽具有BSSL活性,并且含序列表SEQ ID NO:3所示的人BSSL之部分氨基酸序列。
术语“具有BSSL活性的多肽”理解为至少含有下列特性的多肽
(a)适于口服;
(b)能被特异性胆盐活化;
(c)在小肠内容物中起非特异性脂酶的作用,即能水解脂类,
相对地与其化学结构和物理状态(乳化的、微团的、可溶
的)无关;
并且可选择地有一或更多下述特性:
(d)水解有不同链长度及不同不饱和程度之脂肪酸的三脂酰甘
油的能力;
(e)也有水解二脂酰甘油、单脂酰甘油胆甾烯基酯、溶血磷脂
酰甘油、视黄基和其它脂溶性维生素酯的能力;
(f)不但有水解三脂酰甘油的sn-1(3)酯键的能力,而且
有水解sn-2酯键的能力;
(g)不但具有和初级胆盐相互作用的能力,而且有和次级胆盐
相互作用的能力;
(h)最佳活性依赖胆盐;
(i)在胃内容物中稳定,对其催化效率的影响不会达到任何实
质程度;
(j)若胆盐存在,稳定地抗胰酶,如胰蛋白酶失活;
(k)结合肝素和肝素衍生物,如硫酸肝素的能力;
(l)和脂-水界面结合的能力;
(m)对低压冻干法足够稳定;
(n)当和食物成分,如人乳或乳膳食成分混合时稳定。
另一些方面,本发明涉及如上所述的核酸分子,其中所说BSSL变异体在其C-端位置有一个苯丙氨酸残基,或在其C-端部分含一个谷氨酰胺-蛋氨酸-脯氨酸(Gln-Met-Pro)序列,另一方面在其C-端部分可替换含有SEQ ID NO:3中第712-722残基所示的氨基酸序列。
在本发明中,术语“C-端位置”命名为最后的C-端残基位置,而术语“C-端部分”可理解为组成BSSL变异体C-端的约50个氨基酸残基。
如上所述本发明进一步涉及核酸分子,其中所说的BSSL变异体含不到16个的重复单位。本发明中术语“重复单位”命名为33个核苷酸的重复单位之一,所述每一核苷酸均在序列表SEQ ID NO:1中有说明。
另一些方面,本发明涉及上述编码一种多肽的核酸分子,所述多肽的氨基酸序列与序列表中的SEQ ID NO:5、6或9所示的氨基酸序列有至少90%的同源,并涉及编码一种多肽的核酸分子,所述多肽的氨基酸序列与序列表SEQ ID NO:7所示的氨基酸序列至少有90%的同源,但编码187位置上有一个天冬酰胺的多肽的那些核酸分子除外。
本发明也涉及序列表中SEQ ID NO:5、6、7、或9所示的多肽以及如上所说的核酸序列编码的多肽。
本发明还涉及一种含上述核酸分子的杂种基因,一种含所述杂种基因的可复制的表达载体以及含有所述杂种基因的细胞。所述细胞可以是原核细胞、单细胞真核有机体或源于多细胞有机体如哺乳动物的细胞。
在本发明中,“杂种基因”意思是核酸序列,它一方面包含编码如上定义的BSSL变异体的核酸序列,另一方面含有能调控所述杂种基因产物表达的基因之核酸序列。术语“基因”意思是在所需组织能调控和定位杂种基因表达的一个完整的基因和其亚序列。通常,所说的亚序列至少有一或多个启动子区、一个转录起始位点、3′和5′非编码区和结构序列。
优选地,杂种基因通过在体外将编码BSSL变异体的核酸序列插入到用现有技术能调控表达的基因中而形成的。另外,编码BSSL变异体的核酸序列在体内可通过同源重组插入。
在本发明中,术语“可复制的”意思是指载体能在给定类型的它被导入的宿主细胞中复制。紧接核酸的序列上游可提供一个编码信号肽的序列,信号肽的存在保证了由含载体的宿主细胞表达的BSSL变异体的分泌。信号序列可以是天然和核酸序列有关的,或是其它来源的。
载体可以是适于重组DNA方法的任何载体,载体的选择通常依赖于它被导入的宿主细胞。因此,载体可以是自主复制载体,即作为染色体外单位存在的载体,其复制不依赖染色体复制;此类载体的例子有质粒、噬菌体、粘粒、微小染色体或病毒。另外,载体可以是这样的载体,当其引入宿主细胞时,整合进宿主细胞基因组中,与其整合的染色体一起复制。适宜的载体例子是细菌表达载体和酵母表达载体。本发明的载体可携带上述定义的本发明的任何核酸序列。
另一方面,本发明涉及重组多肽的生产方法,所说方法包括(i)在能于特异性宿主细胞或有机体中复制的杂种基因中插入上述核酸分子;(ii)将所得的重组杂种基因导入宿主细胞或有机体;(iii)在培养基内或上使所得的细胞生长,或鉴定并繁殖有机体,以表达多肽;和(iv)回收多肽。
用于生长细胞的培养基可以是适于此目的的任何常规培养基。合适的载体可以是上述任何载体,而合适的宿主细胞可以是上述列出的任何类型细胞。用于构建载体和使其引入宿主细胞的方法可以是重组DNA领域中用于此目的的任何已知方法。可以分泌细胞表达的人重组BSSL变异体,即通过细胞膜输出,这依赖于细胞的类型和载体的构成。
如果BSSL变异体由重组宿主细胞内产生,即,细胞不分泌出BSSL变异体,则可通过标准方法,包括通过机械方法,如声处理或匀浆化,或通过酶学或化学方法使细胞裂解,接着进行纯化而回收。
为了分泌,编码BSSL变异体的DNA序列之前应有一编码信号肽的序列,其存在保证了BSSL变异体从细胞中分泌,以致将至少相当比例的表达的BSSL变异体分泌到培养基中并回收。
本发明也涉及到一种表达系统,包括在含有所说的杂种基因的宿主细胞或有机体中可以表达的杂种基因,当杂种基因表达时产生重组多肽,通过将上述核酸序列插入能调节所说杂种基因表达的基因中产生所述的杂种基因。
生产本发明的重组BSSL变异体可能的方法是使用能将BSSL变异体分泌到其乳中的转基因非人类哺乳动物。转基因非人类哺乳动物的使用,其优点是可以以合理的成本获得高产率的重组BSSL变异体,尤其若非人类哺乳动物是母牛时,在乳中生产作为例如婴儿膳食配方的正常组分的重组BSSL变异体以便当使用重组BSSL变异体作为以乳为基本的产品中的营养添加剂时不必过度纯化。
而且在较高等有机体如非人类哺乳动物中生产通常会对哺乳动物蛋白进行正确的加工,如有关上述的翻译后加工过程和适当折叠,也可以得到大量基本上纯的BSSL变异体。
因此,上述表达系统可以是哺乳动物表达系统,其包括编码BSSL变异体的DNA序列,此序列插入到编码非人类哺乳动物乳蛋白的基因中,以便形成在成年哺乳动物雌性乳腺内可表达的杂种基因,所述哺乳动物含有所说杂种基因。
通常认为,作为表达组织的乳腺和编码乳蛋白的基因特别适合于在转基因非人类哺乳动物中生产异源蛋白质,由于乳蛋白在乳腺中通常以高表达水平生产。而且易于大量收集和使用乳。在本发明中,在重组BSSL变异体的生产中使用乳蛋白基因的进一步的益处是,利用表达调节及生产位置(乳腺)在类似其自然生产条件的条件下进行生产。
当在转基因哺乳动物中使用时,上述杂种基因优选地含有编码信号肽的序列,以便能将杂种基因产物正确地分泌到乳腺内。信号肽典型地通常是在所述研究的乳蛋白基因中发现的或是与编码BSSL变异体的DNA序列相关的。但是,能调节杂种基因产物使其分泌到乳腺内的其它信号序列,也是相关的。当然,杂种基因的各种元件应以如此方式融合,以便使基因产物进行正确表达及加工。因此,应将编码所需信号肽的DNA序列与编码BSSL变异体的DNA序列的N-末端部分精确融合。在杂种基因中,编码BSSL变异体的DNA序列,通常含有其终止密码子,但不是其自身的信息裂解和聚腺苷酸位点。通常保留编码BSSL变异体的DNA序列的下游,乳蛋白基因的mRNA加工序列。
许多因素被认为对某个特别杂种基因的实际表达水平有影响。启动子及上述其它调节序列的能力、哺乳动物基因组中表达系统的整合位点、编码BSSL变异体的DNA序列在乳蛋白编码基因中的整合位点、赋予转录后调节元件及其它类似因子对获得的表达水平是相当重要的。根据对影响杂种基因表达水平的各种因子的认识,本领域技术人员知道怎样设计用于本发明目的的表达体系。
所用的乳蛋白基因可源于与表达体系所插入的乳蛋白基因之种相同,或源于别的种。就此而论已表明,乳腺靶基因表达的调节元件在功能上跨跃了种的界限,这也许是由于可能的共同祖先(Hennighausenet al.1990)。
在构建本发明表达体系中所用的编码乳蛋白适宜的基因或其有效的亚序列通常发现于不同哺乳类源的乳清蛋白之中,如乳清酸蛋白(WAP)基因,优选的是鼠源的;和β-乳球蛋白基因,优选的是羊源的。而且可能发现不同来源的酪蛋白基因也适于BSSL变异体的转基因生产,如牛αS1-酪蛋白和兔β-酪蛋白。目前优选的基因是鼠WAP基因,因发现其可在不同转基因动物乳中使许多外源人类蛋白质的高水平表达(Hennighausen et al.1990)。
另一个和本发明表达系统相关的优选序列是能调节高水平表达的所谓表达稳定序列。强有力证据表明,此稳定序列发现于乳蛋白基因附近和上游。
本发明也包括生产能表达BSSL变异体的转基因非人类哺乳动物的方法,包括(a)将上述表达系统进入受精卵或非人类哺乳动物胚胎细胞以便使表达系统掺入哺乳动物种系;和(b)使所得的受精卵或胚胎发育成为成年雌性非人类哺乳动物。
可使用任何合适的技术,如在“Manipulating the MouseEmbryo”,A Laboratory Manual,Cold Spring HarborLaboratory Press 1986中描述的将表达系统掺入哺乳动物种系。例如,几百个分子的表达系统可直接注射进受精卵,如受精的一个细胞卵或其卵原核,或所用哺乳动物的胚胎,此后将微注射的卵转入假孕代育母亲的输卵管,使其发育。
生产能表达BSSL变异体的转基因非人类哺乳动物的方法也包括其中所说的哺乳动物基本上不能表达来自哺乳动物本身的BSSL的方法。所述方法包括(a)损害哺乳动物的BSSL表达能力以便基本上不表达哺乳动物BSSL,并按上述将一个表达系统以使在哺乳动物中表达BSSL变异体的方式插入哺乳动物种系,和/或(b)用如上限定的表达系统取代哺乳动物BSSL基因或其部分。
通过在导致BSSL表达的DNA序列中引入突变可以方便地破坏哺乳动物的BSSL表达能力。所述突变可以包括使DNA序列脱离框架、引入终止密码、缺乏DNA序列的一或多个核苷酸。
可用上述的表达系统或通过熟知的同源重组原理用编码BSSL变异体的DNA序列取代哺乳动物BSSL基因或其部分。
在更重要的方面,本发明涉及在其基因组中有如上DNA序列的转基因非人类哺乳动物。所说DNA序列可优选地存在于哺乳动物的胚系及哺乳动物的乳蛋白基因中。优选地可从包括小鼠、大鼠、兔、羊、猪和牛筛选转基因非人类哺乳动物。
本发明也包括如上转基因非人类哺乳动物的后代及从所述转基因非人类哺乳动物获得的乳。
本发明进一步涉及含如上乳的婴儿配方及上述含BSSL变异体的婴儿配方。婴儿配方可使用常规方法制备,而且可含任何必需的添加剂矿物质、维生素等。
进一步,本发明涉及含如上BSSL变异体的药物组合物及所述BSSL变异体在治疗中的应用。
更进一步,本发明涉及用上述BSSL变异体制造用于治疗与外分泌胰腺功能不全:胆囊纤维化、慢性胰腺炎、脂肪吸收障碍、脂溶性维生素吸收不良、由于病理原因造成的脂肪吸收不良相关的病理状态之药物。本发明也涉及用于药物制造的BSSL变异体对于改善膳食脂类利用的用途,尤其在早产儿中。
实施例1.重组BSSL在真核及原核细胞中的表达1.1实验方法1.1.1重组质粒
被克隆进pUC19中的含2.3kb人BSSL cDNA(Nilsson et al.1990)的质粒pS146经HindIII和SalI酶解,将BSSL cDNA导入牛乳头瘤病毒(BPV)表达载体,pS147(图1)。此载体含有位于鼠金属硫因1(mMT-1)控制增强子及启动元件之下(Paylakis & Hamer,1983)的人BSSL cDNA。mRNA加工信号通过含部分外显子II、内含子II、外显子III及兔β-球蛋白基因下游元件的基因组片段提供。将该转录单位克隆进含整个BPV基因组的载体中。对BPV和BSSL转录单位来说,转录是单方向的。为了使此载体在大肠杆菌中繁殖,该载体还包含pML2d,它是pBR322的衍生物(Sarver et al.1982)。
表达载体pS147和在Harrey肉瘤病毒5′-长未端重复序列及SV40多腺苷酸化信号(Lusd & Botchan,1984)驱动下的编码新霉素抗性基因的载体共转染。
为了在大肠杆菌中表达BSSL,从质粒pT7-7(Ausubel et al,1992)中将BSSL cDNA作为NdeI-BamHI片段亚克隆到pGEMEX-1(Promega,Madison,WI,USA)(Studier & Moffat,1986)中。用这种克隆方法,T7基因10编码序列被前面加起始密码子的编码成熟蛋白质的BSSL基因置换。最终表达载体pGEMEX/BSSL通过使用特异的BSSL内引物,经DNA测序证实。1.1.1.2诱变
指定起始密码ATG中的A为核苷酸1号。对于氨基酸序号,信号肽中的第一个蛋氨酸号为-23,将成熟蛋白质中第一个氨基酸残基,丙氨酸,指定为1号。
为构建缺失变异体A(SEQ ID NO:4),合成了两个PCR引物,PCR-1和PCR-2(表1)。为了克隆进不同的质粒,产生了HindIII、SalI和BamHI位点。没有改变氨基酸序列,在BSSL序列中产生了BclI位点。这样做为了便于加入合成的DNA,以获得其它变异体。引物PCR-2含两个合成的终止密码子。所得的PCR片段经BamHI和HindIII酶解并克隆进pUC18以便序列分析。该质粒命名为pS157。正确的PCR片段通过在独一无二的Asp700位点(BSSL cDNA中1405位置)和β-球蛋白基因片段前部的SalI位点与BSSL序列融合而插入到BPV表达载体,结果产生pS257。
通过使用3、4、7和8号寡核苷酸(表1)获得了B-变异体构建体(SEQ ID NO:5)。退火的寡核苷酸正好编码C-未端氨基酸序列相当于全长蛋白质中的赖氨酸712至苯丙氨酸722。此片段和谷氨酰胺535融合。一个翻译终止子直接插入到最后一个苯丙氨酸后。此片段在5′端含一个BclI位点并在3′端含一个SalI位点,使其能够导入pS157。所得的质粒用Asp700和SalI酶解,将313bp片段导入上述的表达载体。产生的质粒命名为pS258。表1.
用于构建BSSL变异体的合成的寡核苷酸在限制性位点的核苷酸下面划了线。翻译终止信号由黑体字标出。变异体N中可变密码在PCR-3中用黑体字和星号标出。
寡核苷酸 | 序列(5′-3′) |
PCR-1 | CGGGATCCGAAGCCCTTCGCCACCCCCACG |
PCR-2 | CGAAGCTTGTCGACTTACTACTGATCAGTCACTGTGGGCAGCGCCAG |
PCR-3 | GGGAATTCTGGCCATTGCTTGGGTGAAGAGGAATATCGCGGCCTTCGGGGGGGACCCCAACCAGATCACGCTCTTCGGGGAGTCT |
PCR-4 | CGGGATCCCACATAGTGCAGCATGGGGTACTCCAGGCC |
1 | GATCAGGGGGCCCCCCCCGTGCCGCCCACGGGTGACTCCGGG |
2 | GCCCCCCCCGTGCCGCCCACGGGTGACTCCAAGGAAGCTCAGA |
3 | TGCCTGCAGTCATTAGGTTTTAGTAAGTCGACA |
4 | AGCTTGTCGACTTACTAAAACCTAATGACTG |
5 | CAGGCATCTGAGCTTCCTTGGAGTCACCCGTGGGCGGCACGGGGGGGGCCCCGGA |
6 | GTCACCCGTGGGCGGCACGGGGGGGGCCCCCT |
7 | GATCAGAAGGAAGCTCAGA |
8 | CAGGCATCTGAGCTTCCTTC T |
为了构建编码C-变异体(SEQ ID NO:6)的基因,使用1至6号寡核苷酸(表1)。退火的DNA片段含两个重复序列,编码11个氨基酸与共有序列(Nilsson et al.1990)一致,在谷氨酰胺535和赖氨酸712-苯丙氨酸722序列之间插入。此片段也含一个5′端的BclI位点及一个3′端SalI位点,以允许与上述相同的克隆策略。所得的质粒命名为pS259。
为构建变异体N(非-N-糖基化的变异体,SEQ ID NO:7),合成了两个PCR引物(PCR-3和PCR-4,表1)。产生EcoRI和BamHI位点,以使360bp的PCR产物克隆进pUC19中进行序列分析。将潜在N-连接的糖基化位点,在天冬酰胺187,变成谷氨酰胺。分离作为BalI-HindIII片段的修饰的序列然后与含mMT-1启动子和5′端BSSL cDNA的SacI和BalI片段一起克隆进SacI和HindIII消化过的pUC19,一个约1.2kb的SacI-DraIII片段由此质粒分离到,然后分别插入到在表达载体之内的mMT-1元件和BSSL cDNA序列中。所得的质粒命名为pS299。1.1.3.哺乳动物细胞培养和转染
根据磷酸钙沉淀法(Graham & Van der Eb,1973)将载体共转染进鼠细胞系C127(ATCC CRL 1616)。
在补充10%胎牛血清的Ham′s F12-Dulbecco′s ModifiedEagle′s培养基(DMEM)(1∶1)中培养C127细胞。用1.5mg×ml-1的G418筛选新霉素抗性细胞克隆,10-15天后,抗性细胞克隆从主平皿中分离,传代,用于分析。1.1.4细菌菌株和培养条件
为了表达实验将载体pGEMEX/BSSL转化到大肠杆菌菌株JM109(DE3)和BL21(DE3)pLysS中。表达实验按Studier等人(1986)的描述进行。收集细菌后,通过离心(4℃,5,000xg 10分钟,)沉淀细胞。为制备周质和胞质部分,将沉淀重悬于每克沉淀4ml20mM Tris-Cl/20%蔗糖,pH 8.0,200μl 0.1MEDTA及40μl的溶菌酶(15mg/每ml水)中。重悬液置冰上40分钟。每克沉淀物加160μl 0.5M MgCl2,此后在12000xg,离心悬液20分钟,产生的上清液含周质蛋白质,沉淀物代表细胞质部分。另外,为制备可溶性蛋白质,将细胞悬于40mM Tris-Cl,0.1mMEDTA,0.5mM(苯基甲基磺酰氟化物pH8.2),冻融和声处理几次使其溶解。离心细胞溶解液(25℃,30,000xg 30分钟)。1.1.5.核酸分析
从分离的哺乳动物细胞系或大肠杆菌细胞(Ausubel et al,1992)制备RNA和DNA。在琼脂糖凝胶中使RNA或DNA分离,转印到GeneScreen Plus(New England Nuclear)上,按供应商的说明杂交。1.1.6.天然酶的制备
按前述的方法(Blckberg & Hernell,1981)从人乳中纯化胆盐刺激的脂酶。纯化的制品经SDS-PAGE判断同源性,当用长链三脂酰甘油为底物检测时,比活性为每分钟,每mg释放100μmol脂肪酸。1.1.7酶分析
使用阿拉伯树胶乳化的三油酸甘油酯为底物,按所述的方法(Blckberg & Hernell,1981)进行酶检测。用10mM胆酸钠为激活的胆盐,进行培养。当检测胆盐依赖性时,加入胆盐(胆酸钠或脱氧胆酸钠,Sigma Chem.Co)达到表3中所给的浓度。1.1.8 Western印迹
为了在印迹实验中获得有意义的反应,通过Blue Sepharose(Pharmaci LKB Biotechnology)层析浓缩条件培养基。将各个培养基和Blue Sepharose(每ml凝胶约10ml培养基)混合。用含0.1MKCl的0.5M Tris-Cl缓冲液,pH7.4洗涤凝胶(每ml胶10ml)。酶活性用同一缓冲液的1.5M KCl洗脱。用此方法浓缩了25-30倍,纯化了3-5倍。按Laemmli(1970)方法,必要地在10%聚丙烯酰胺凝胶上进行SDS-PAGE。转移至硝酸纤维素膜上,与抗纯化的BSSL的多克隆兔抗血清一起孵育后,用和碱性磷酸酶结合的山羊抗兔IgG及来自Bio-Rad的发育试剂盒进行测定结果。1.1.9用N-糖苷酶F处理
加1μl 1M的β-巯基乙醇和0.5μl 10%(w/v)的SDS到含BSSL活性为每分钟释放2.5μmol脂肪酸的10μl变异体B中。煮沸5分钟后,加入10μl 0.1M磷酸钠缓冲液pH8.0,6μl0.1M EDTA,4μl 7.5%(w/v)Nonidet P40和5μl(IU)N-糖苷酶F(Boechringer Mannheim)。作为对照,处理了同等量的变异体B,除无糖苷酶加入外。在37℃温育过夜后样品经SDS-PAGE电泳,并使用多克隆兔BSSL抗血清进行印迹。1.2.结果1.2.1构建BSSL变异体
在表2和图1中概括了关于全长BSSL,BSSL变异体的修饰。用于产生这些变异体的策略在1.1.部分描述了。对变异体A(SEQ IDNO:4)而言,终止密码引入到谷氨酰胺535位置后,去除了全长蛋白质的最后187个氨基酸。对变异体B(SEQ ID NO:5)而言,编码正巧11个氨基酸的区域和原始翻译终止子与谷氨酰胺-535融合,因此,这种变异体缺乏所有的重复序列。对变异体C(SEQ IDNO:6)而言,含两个重复序列的片段,插入到谷氨酰胺-535和赖氨酸-712至苯丙氨酸722序列之间。所述重复序列含有与共有序列(Nilsson et al,1990)相同的序列。
为了分析唯一不明确的N-连接的碳水化合物结构(其位置接近活性位点丝氨酸-194)的重要性构建变异体。通过将天冬酰胺-187变成谷氨酰胺而改变潜在的N-糖基化位点获得变异体N(SEQ IDNO:7)。表2和人类BSSL相关的BSSL变异体的氨基酸序列
1.2.2哺乳动物细胞系重组DNA的特征描述
变异体 | 缺失的残基 | 变化的残基 |
A(SEQ ID NO:4) | 536-722 | |
B(SEQ ID NO:5) | 536-711 | |
C(SEQ ID NO:6) | 536-568,591-711 | |
N(SEQ ID NO:7) | Asn 187→Gln |
重组DNA样品从用编码不同BSSL变异体的表达载体转染的细胞系中制备。制备的DNA用BamHI消化,在琼脂糖凝胶上分开,并转移至杂交膜上。使用的探针是32P标记的BSSL cDNA。杂交结果证实了重组基因的存在,而且载体拷贝数在不同的细胞系大约相等(图2)。杂交片段的位置反映了各种BSSL序列的长度并与期望大小一致。位置也类似于起源于细菌的用于转染实验的DNA,表明在细胞系中没有主要的载体DNA的重排(图2)。代表变异体A的DNA一样品中上边的杂交信号可能归因于部分消化。1.2.3在哺乳动物细胞中全长和突变BSSL mRNA的表达
为分析不同重组BSSL基因的表达,从分离的细胞系中制备了mRNA。Northern印迹实验和用32P-标记的BSSL cDNA杂交实验表明,在含有BSSL载体(图3)的所有细胞系中都可检测到重组mRNA。在对照样品中没有杂交,所述对照样品得自含除BSSL cDNA外的相同载体的细胞系(图3)。
杂交mRNA的不同长度与cDNAs的修饰一致。在不同样品中重组BSSLmRNA变异体的稳定状态水平除了变异体A(图3)外,几乎是相同的。变异体A mRNA积累降低的原因尚未知晓,但观察到两个细胞系群及分离的克隆。在不同样品中等量RNA的存在,通过用鼠β-actin探针(图3,较低组)杂交证实1.2.4生产哺乳动物全长和BSSL变异体
收集了源于单个克隆的用全长BSSL及其不同突变形式转染的C127-细胞培养基并分析BSSL的活性(图4)。对于全长分子和变异体N、B和C,最高表达克隆的活性范围是每分钟每毫升培养基释放0.7-2.3μmol脂肪酸。和天然人乳中BSSL的比活性比较,此表达水平为每毫升培养基7-23μg。对变异体A而言,所有分析的克隆其活性低于每分钟每毫升培养基释放0.05μmol脂肪酸。在Blue-Sepharose柱上浓缩和冷冻干燥显示最高活性的克隆,表明的确表达了有活性的酶,尽管表达水平较低不能排除。通过相当低比活性解释以变异体A部分获得的低活性可能性。
源于不同转染实验克隆的Western印迹显示于图5A。BSSL变异体明显的Mr正如所望。但是应注意到,对全长BSSL及变异体B和C而言,出现了双条带。因为所有这三个都有单一完整的N-糖基化位点,而没出现双条带的变异体N缺少N-糖基化位点,所以可能解释为,双条带产生于N-糖基化的差别。因此变异体B应该用N-糖苷酶F消化。如图5B所示,上面的带只保留痕量,而下面的带强度增加,表明只有部分表达的变异体N-糖基化了。
BSSL的特性之一是其可通过初级胆盐,如胆酸盐(Hernell,1975)将异性活化。所有不同重组形式的BSSL证明了胆酸盐活化的相同浓度依赖性(图6)。在使用的实验体系中约10mM获得了最大活性。当将胆酸盐换成脱氧胆酸盐(次级胆盐),无此活化产生。因此,重组全长及不同的变异体证明了胆盐活化的同一特异性。1.2.5.大肠杆菌中全长BSSL的表达及生化特性
用含位于T7启动子控制下的人BSSL cDNA的表达载体pGEMEX/BSSL转化两个大肠杆菌菌株JM109(DE3)和BL21(DE3)pLysS(Studjer et al.,1986)。鉴别来自于两个菌株的转化体,将其培养并用IPTG诱导约90分钟(Studier et al.,1986)。通过使用BSSL cDNA为32P标记的探针,经Northern印迹分析总mRNA表明,两个菌株中均有效地诱导了表达而且转录受到密切调节(图7A)。明显的重组BSSL mRNA的大小约2.4kb,和期望长度一致。蛋白质样品的SDS-PAGE分离及用抗BSSL抗体的免疫测定表明在大肠杆菌中有效地产生了全长BSSL(图7B)。BL21(DE3)pLysS菌株比JM109(DE)(图7B)菌株有更多的蛋白质被分泌到周质。
IPTG诱导的大肠杆菌培养基含相当于0.5-4μg蛋白质/ml培养基的活性可溶的BSSL。Western印迹证明20-60%的反应物在不溶的沉淀物中。未诱导的细菌不包含任何明显的BSSL活性。
来自培养细菌的脂酶活性表明和天然乳中BSSL有同样的胆盐依赖性。2.重组全长和突变的胆盐刺激脂酶的纯化及特征描述2.1.实验方法2.1.1.酶和酶变异体
如上所述构建和表达了重组全长BSSL和BSSL变异体B、C和N。和天然的酶比较,变异体B(SEQ ID NO:5)缺少所有16个独一无二的,O-糖基化的,富含脯氨酸、C-末端重复序列(aa 536-711),但含有大部分的融合于谷氨酰胺-535的C-未端片段(aa712-722)。变异体C(SEQ ID NO:6)合同样的C-未端片段和在谷氨酰胺-535与赖氨酸-712之间的两个11个残基的重复单位。在变异体N(非-N-糖基化变异体,SEQ ID NO:7),负责唯一连接糖的天冬酰胺-187换成了谷氨酰胺残基。天然BSSL从人乳中按所述方法纯化(Blckberg & Hernell,1981)。2.1.2.酶分析
用阿拉伯乳化的三油酸甘油酯为底物,如所述(Blckberg &Hernell,1981)测定了脂酶活性,用胆酸钠(10mM)为活化胆盐。此测定的不同变化在附图说明中给出。2.1.3.制备免疫吸附剂
按制造者的描述用CMBr,使纯化的乳BSSL(5mg)结合到Sepharose上。抗纯化的乳BSSL兔产生的多克隆抗血清40ml通过此柱。用0.1M甘氨酸-HCl pH2.5洗脱特异抗体。用固态Tris使pH立即调至8左右。脱盐和冻干后,将6mg亲和纯化抗体与上述Sepharose结合。2.1.4.纯化方法
含5-25μg重组表达BSSL或BSSL变异体的条件培养基,以每ml配好凝胶10ml培养基与Blue Sepharose混合(Pharmacia,Swedan)。彻底混合30分钟后,用0.05M Tris-Cl pH7.0,0.05MKCl流洗凝胶;脂酶活性用0.05M Tris-Cl,pH7.0,1.5MKCl洗脱。收集活性峰,对5mM佛多那钠,pH7.4,0.05M NaCl透析。透析液用于肝素-Sepharose柱。此柱用在5mM佛多那钠缓冲液,pH7.4中的梯度0.05-1.0M NaCl洗脱。收集合脂酶活性的组分,用于免疫吸附柱,用0.05M Tris-Cl pH7.5,0.15M NaCl流洗后,0.1M甘氨酸-HCl、pH2.5洗脱结合的脂酶。用固态Tris使组分的pH立即调到8左右。2.1.5.电泳
基本根据Laemmli(1970)方法,进行了十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)蛋白质用考马斯亮蓝染色。2.1.6.N-未端序列分析
在Applied Biosystems公司的477A脉冲液相测序仪及带有来于制造商的规范循环程序和化学品的连网苯硫乙内酰脲120A分析仪上进行氨基酸序列分析。从测得的标准蛋白质序列(β-乳球蛋白)计算,最初及重复产量分别为47%和97%。2.2.结果2.2.1.重组BSSL和BSSL变异体的纯化
主要用在Blue Sepharose柱上层析条件培养基作为浓缩步骤。接着在肝素-Sepharose柱上层析主要是去除培养基中存在的大部分白蛋白得到初步纯化。此步骤也说明,重组BSSL分子都具有肝素结合能力。免疫吸附后,所有BSSL变异体,象经SDS-PAGE(图8)制定的那样,纯度超过90%。全长酶及变异体B和C作为成对物迁移。不同变异体的表观Mr列于表3。N-未端序列分析对所有8个循环的变异体测出了一个单一顺序:Ala-Lys-Leu-Gly-Ala-Val-Tyr-Thr-。2.2.2.脂酶活性
表3显示了不同制品的表观分子量。制品的比活性范围是每分钟每毫克蛋白质释放游离脂肪酸75-120μmol。因此没有观察到全长BSSL和BSSL变异体间活性的显著差别。
所有制品说明绝对需要对抗长链三脂酰甘油的初级胆汁盐(胆酸钠)活性(图9A)。脱氧胆酸钠不能提供任何变异体活性(资料未列出)。但是,一旦结合不同的胆盐,脱氧胆酸盐就有双重作用(图9B和C)。首先它降低活化必需的胆酸盐浓度,其次在较高胆盐浓度下抑制酶的活性。表3
重组全长BSSL和BSSL变异体的表观Mr
2.2.3.重组BSSL和BSSL变异体的稳定性
酶 | Mr(KDa)通过SDS-PAGE测定 |
全 长 | 105,107 |
变异体B | 63,65 |
变异体C | 60,62 |
变异体N | 95 |
重组BSSL和BSSL变异体与天然乳BSSL显示了同样的pH稳定性(图10)。pH在2.5-3左右所有情况下都有失活发生。pH在3以上,所有变异体是完全稳定的,提供的蛋白质浓度足够高。这通过加入小牛血清白蛋白或卵清蛋白达到(资料未列出)在所有测试的PH,稀释的样品都是不太稳定的,但阈值是一样的(资料未列出)。图11显示和天然乳酶比较,重组酶的热稳定性。在37-40℃此活性开始降低。变异体(B、C、N)稳定性比全长重组酶和乳酶略低。但是,如果蛋白质浓度通过加入小牛血清白蛋自得到提高,所有变异体在40℃也是稳定的(图11)。
天然乳BSSL和所有重组变异体对胰蛋白酶都敏感。一种依赖时间的失活获得了(图12)。但是,如果胆盐,即胆酸盐存在于缓冲液中,脂酶变异体就得到了保护,脂酶活性保留下来(图12)。
因此,鉴于许多体外特征,即胆盐活化、肝素结合、pH和温度稳定性及对蛋白酶失活的胆盐保护将不同的BSSL变异体与天然乳BSSL比较,没有发现显著差异。3.在转基因动物中表达3.1.表达载体的构建
为了构建在转基因动物乳中生产重组人BSSL变异体的表达载体,使用了下列策略(图13)。
应用Lidberg等人(1992)叙述的方法,获得了含人BSSL基因不同部分的质粒(pS309、pS310、pS311)。质粒pS309含一个SphI片段,此片段包含5′-非转录区至第四内含子的部分序列。质粒pS310含一个SacI片段,此片段含有一从第一内含子的部分序列至第六内含子的部分序列的BSSL变异体基因序列。最后质粒pS311含一个BamHI片段,此片段含BSSL基因的第五内含子的主要部分及缺失第十一外显子的内含子/外显子结构的其余部分。缺失的序列是231bp,它能产生一个编码BSSL变异体的序列,此变异体实际上含77个氨基酸或比全长BSSL少七个重复单位。产生BSSL变异体(变异体T)的核苷酸序列列于序列表SEQ ID NO:8中。变异体T的氨基酸序列列于序列表SEQ ID NO:9中。
由于在入BSSL基因第十一外显子有高度的重复序列,在这种序列克隆进质粒或在细菌中繁殖时,可设想有相当高的重排频度。根据这种假设,含有截短第十一外显子的所需BSSL变异体基因得到了鉴定、分离、进行了序列分析。另一个质粒pS283,含部分在HindIII和SacI位点克隆进pUC19的部分人BSSL cDNA,用于基因组序列的融合。质粒pS283也用于获得合适的限制酶酶切位点,KpnI位于BSSL 5′非翻译前导序列中。
用NcoI和SacI消化质粒pS283,通过电泳分离到一约2.7kb的片段。用NcoI和BspEI消化质粒pS309,分离到一包含BSSL基因5′-部分约2.3kb的片段。用BspEI和SacI消化质粒pS310,分离到一含BSSL基因中间段部分约2.7kb的片段。这三个片段连接在一起,转化感受态大肠杆菌菌株TG2,经氨基苄青霉素筛选分离转化体。
从许多转化体制备了质粒,一个含所要构建物的质粒,叫pS312(图14),用于进一步研究。
为了获得pS311的修饰形式,位于终止密码下游的BamHI位点换成SalI位点,便于进一步克隆,使用了下列方法:通过BamHI部分消化,使质粒pS311线性化。分离到线性化的片段,将BamHI换成SalI位点的合成DNA接头(5′-GATCGTCGAC-3′)插入,破坏了BamHI位点。由于有两个潜在的用于整合合成接头的位置,所以通过限制酶切割分析所得的质粒。在11外显子下游理想位置插入接头的质粒得到分离命名为pS313。
为了获得含有人BSSL变异体基因组序列的最终表达载体构建体,使用现有的表达载体pS314,设计它来调节泌乳期乳腺细胞的阶段和组织特异性表达。质粒pS314含一个克隆为NotI片段的鼠乳清酸性蛋白(WAP)基因(Campbell et al.1984)的基因组片段。
基因组片段含约4.5kb的上游调节序列(URS),所有四个鼠WAP外显子及所有内含子序列、约3kb的最后外显子的下游序列。独一无二的KpnI位点位于天然WAP翻译起始密码上游第一个外显子24bp内。另一个独一无二的限制酶位点位于第三外显子内SalI位点。
人BSSL变异体基因组序列用下列方法插入到KpnI和SalI位点间,第一,用KpnI和SalI消化pS314,电泳分离代表裂解质粒的片段,第二,用KpnI和BamHI消化pS312一个约4.7kb,代表BSSL基因5′部分的片段得到分离。第三,用BamHI和SalI消化pS313,分离到人BSSL基因的3′一部分。这三个片段连接起来,转化感受态大肠杆菌,经氨基苄青霉素选择后分离转化体。
质粒从几个转化体中得到制备,通过限制酶图谱和序列分析仔细分析。确定了代表所需的表达载体的一个质粒,命名为pS317(图15)。
为了移去原核质粒序列,用NotI消化pS317。此后,含人BSSL变异体基因组片段和侧翼的鼠WAP序列的重组载体因子经琼脂糖电泳分离。分离的片段在注进鼠胚胎之前进一步经电洗脱纯化。
在转基因鼠乳中表达人BSSL变异体的重组基因显示于图16。3.2.转基因动物的产生
按照3.1.部分从质粒pS317分离一个NotI片段。此DNA片段含连接到编码人BSSL变异体基因组序列的鼠WAP启动子。这种分离片段以3ng/μl的浓度,将其注射进从供体小鼠获取的350C57B1/6J×CBA/2J-f2胚胎的原核中,供体小鼠预先用5IU怀孕母马牛清用于排卵过速的促性腺激素处理。C57B1/6J×CBA/2J-f1动物从Bomholtgard Breeding和Research Center LTD,Ry,Denmark获得。从输卵管采集胚胎后,用M2培养基(Hogan etal.1986)中的透明质酸酶处理,从丘细胞分离胚胎。清洗后,将胚胎转至M16培养基(Hogan et al,1986),保存在5%CO2孵箱中。使用液压微操作器和带有Nomarski镜片的Nikon倒置显微镜,在轻石蜡油下的M2微粒中进行注射。注射后,267个看上去健康的胚胎埋植进12个假孕的,经腹膜内注射0.3个ml,2.5%AvertinC57B1/6J×CBA/2J-f1受体中。从动物生后三周获得的尾尸检标本DNA,经PCR分析,识别整合转基因的小鼠。用Southern Blot分析证实了阳性结果。
为收集乳,雌性泌乳期动物用2IU催产素经腹膜内注射,十分钟后用0.40ml,2.5%的Avertin腹膜内麻醉。集乳器通过一个硅化过的管附在乳头上,通过轻轻按摩哺乳动物腺体,将乳收集到一个1.5ml的Eppendorf管中。乳量随泌乳天数改变,每个小鼠及每次收集在0.1和0.5ml之间。3.3.转基因小鼠中BSSL变异体的表达
通过剪尾样本制备的DNA分析鉴别转基因鼠。组织样品与蛋白酶K温育,经酚/氯仿抽提。如存在代表表达载体片段的异源引入的DNA扩增特异的片段的带引物的聚合酶链反应中使用分离的DNA。用DNA杂交实验分析动物以证实PCR数据并检测可能的重排,整合载体元件的结构并得到有关整合载体元件拷贝数的资料。
在一套实验中,用两种方法分析了31只小鼠,结果表明,一只鼠带有pS317的异源DNA载体元件。PCR分析和杂交实验的结果一致(图17)。
总共65个测试动物,有10个是pS317转基因的。
将鉴定带有载体DNA元件(建立者动物)的小鼠配对,F1动物用同样的方法进行转基因分析。
通过琼脂糖福尔马林凝胶电泳分离在泌乳期从pS317转基因雌性动物不同组织中分离的RNA,然后转至滤纸上,以32P-标记的BSSLcDNA为探针杂交。获得的结果证明,在泌乳期表达在乳腺表达受限制(图18)。
从麻醉的用催产素处理诱导泌乳的建立者动物收集乳样品,分析重组BSSL变异体的存在。此是通过SDS-PAGE电泳,转移至硝酸纤维素膜,及与产生的抗天然人BSSL的多克隆抗体孵育实现。
获得的结果证实了转基因小鼠乳中重组人BSSL变异体的表达。图19证实了转基因小鼠乳中重组BSSL变异体的存在。SDS-PAGE分离及源于不同pS317转基因小鼠的乳样品免疫印迹分析显示,和全长源于转基因pS314的小鼠乳中重组BSSL比较,带有还原性表观分子量的重组BSSL产量高。质粒pS314类似pS317,不同的是,pS314含全长人BSSL cDNA,不含基因组变异体。出现在所有鼠乳样品中的双带代表鼠BSSL,并因此显出抗血清的交叉反应。出现在图19的第9泳道明显的双带(包含纯化的鼠BSSL)的观察进一步支持这个结论。
稳定的转基因动物系产生了。
用相似的方法,也可制备能表达人BSSL变异体的其它转基因动物,如兔、奶牛或绵羊。保藏
根据布达佩斯条约在DSM(Deutsche Sammlung vonMikroorganismen und Zellkulturen)保藏了下列质粒
质粒 | 保藏号 | 保藏日 |
pS309pS310pS311pS317 | DSM 7101DSM 7102DSM 7103DSM 7104 | 1992.6.12 |
pS147pS257pS299 | DSM 7495DSM 7496DSM 7497 | 1993.2.26 |
pS258pS259 | DSM 7501DSM 7502 | 1993.3.3 |
附图概述图1A.用于表达不同BSSL变异体的BPV为基本载体的图谱。B.分析不同BSSL变异体的图例说明。FL表示全长BSSL。活性位点用圆圈表示,潜在的N-连接的碳水化合物位点用三角形表示。含重复序列的区域用条线区表示,保守的C-未端用涂满黑色区表示。图2
源于表达BSSL变异体细胞系的DNA的Southern印迹分析。分析了从表达全长BSSL(FL)、变异体A(A)、变异体B(B)、变异体C(C)和变异体N(N)制备的DNA。用BamHI消化5μg各自制备的细胞DNA(左)及lng纯化的细菌载体DNA(右)。DNA样品在琼脂糖凝胶上分离,转移至GeneScreen Plus膜上,与32P-标记的人BSSL cDNA杂交。图3
从表达BSSL变异体的细胞系分离的RNA的Northern印迹分析。分析了从生产全长BSSL(FL)、变异体A(A)、变异体B(B)、变异体C(C)、变异体N(N)的细胞系制备的10μg总RNA。用来自C127细胞系的RNA作负对照(-)(上边组)所述细胞系含有与图1中载体相同的BPV-载体,除了编码一个与BSSL不相干的蛋白质外,用32P-标记的BSSL cDNA与滤膜杂交。用鼠β-肌动蛋白cDNA为探针,与滤膜再次杂交。β-肌动蛋白mRNA信号(下边组)用作上样到每一泳道mRNA量的内标对照。图4
在用全长和突变形式的人BSSL转染的C127细胞中BSSL的表达活性。C127细胞用不同的BSSL构建体转染:全长BSSL(FL)、变异体N(N)、变异体C(C)、变异体B(B)、变异体A(A)。起始生长期后,筛选个别克隆,使之成长至汇合。筛选的克隆(n)数表示在图中。在条件培养基上确定脂酶活性。其数值用每ml条件培养基每分钟释放的游离脂肪酸的μmol表示。图5A.全长和突变的重组BSSL的Western印迹分析。脂酶活性的量表示为,每分钟释放脂肪酸的μmol,用于凝胶是,全长0.2(泳道1)、变异体N0.16(泳道2)、变异体C0.6(泳道3)、变异体B0.8(泳道4)及天然BSSL 0.1(泳道5)。所用的抗血清是在抗从人乳纯化的BSSL的兔中产生的。分子大小标记(Prestained SDS-PAGEStandard,Low Range,BioRad)标在左边。B.N-糖苷酶F处理的变异体B的Western印迹。用实验方法所述的N-糖苷酶F消化变异体B。泳道1是未处理的,泳道2是处理的变异体B。图6
全长及突变BSSL的胆盐依赖性。在改变浓度的胆酸钠(实线)或脱氧胆酸钠(断线)存在的情况下,在来自全长重组BSSL(*)、变异体A(□)、变异体B(▲)变异体C(■)、变异体N(●)及纯化的人乳BSSL(○)的条件培养基上确定脂酶活性。对A变异体而言,按实验方法所述,在Blue Sepharose上浓缩条件培养基。除了最大活性只有其它酶的1/10变异体A外,选择各来源的酶量以获得同样的最大活性水平。对照实验表明生长培养基不影响活性水平或天然BSSL的胆盐依赖性(数据未列出)。图7A.使用pGEMEX通过不同的大肠杆菌菌株产生的BSSL Northern印迹。细菌用实验方法中所述的IPTG进行诱导。
实验条件见图2所示。泳道1,菌株BL21(DE3)pLysS,未诱导的;泳道2,菌株BL21(DE3)pLysS,诱导的;泳道3,菌株JM109(DE3),未诱导的;泳道4,菌株JM109(DE3),诱导的。B.使用抗纯化的乳BSSL的抗体8-18%SDS-PAGE Western印迹表明,使用pGEMEX在不同大肠杆菌菌株中重组BSSL的表达。用IPTG诱导细菌,按实验方法所述从裂解液制备细胞质和周质蛋白质。上样至泳道2-5(周质制品)和7-10(胞质制品)的细菌蛋白质量代表了使菌株和生产水平成比例的相同培养量。泳道1,Pharmacia分子大小标记;泳道2和8,菌株JM109(DE3),诱导的;泳道3和7,菌株JM109(DE3),未诱导的;泳道4和10,菌株BL21(DE3)pLysS,诱导的;泳道5和9,菌株BL21(DE3)pLysS,未诱导的;泳道6,25ng纯化的天然BSSL。图8
纯化的重组BSSL及BSSL变异体的SDS-PAGE。全长重组BSSL(FL)和BSSL变异体N、B及C按所述纯化。除了B用1.5μg外,每种用3μg纯化的天然乳BSSL(NAT)用5μg。分子大小标记的位置在左边。图9
通过胆酸钠,脱氧胆酸钠对重组BSSL及BSSL活化的作用。在无(左边组)或存在5mM(中间组)或10mM(右边组)脱氧胆酸盐的条件下,用不同浓度的胆酸钠分析了纯化的重组全长BSSL(●)、重组BSSL变异体B(○)、C(■)和N(▲)、及纯化的天然乳BSSL(□)制品的脂酶活性。图10
重组BSSL和BSSL变异体在不同pH的稳定性。在37℃,pH2-8的不同缓冲液中温育天然BSSL、重组全长BSSL及BSSL变异体。所有缓冲液均含1mg/ml牛血清白蛋白,30分钟后,撤去分装品,分析脂酶活性,符号的解释见图9的说明。图11
重组BSSL和BSSL变异体的热稳定性。在50mM Tris-Cl缓冲液pH7.5,指定的温度下温育纯化的重组全长BSSL,BSSL变异体及天然乳BSSL。一组牛血清白蛋白(BSA)样品加至1mg/ml。30分钟后撤出样品并分析脂酶活性。活性表示为在0分钟每份样品的活性百分比,符号的解释见图9的说明。图12
胆盐对通过胰蛋白酶使重组BSSL及BSSL变异体失活的影响。在25℃,有(实线)无(断线)10mM胆酸钠存在条件下,将纯化重组全长BSSL、BSSL变异体及天然乳BSSL(15μl含14μg)加到含10μg胰蛋白酶(TPCK-胰蛋白酶、Boehringer-Mannheim)60μl1.0M Tris-Cl pH7.4的溶液中。在指定时间,撤除测定样品,分析脂酶活性。数值表示为无胰蛋白酶条件下对照温育中获得值的百分比。符号解释见图9的说明。图13
生产质粒pS317的方法,更详细资料见3.1部分图14
质粒pS312的图示结构。图15
质粒pS317的图示结构。图16
表示3.1部分所述WAP第一外显子内人BSSL变异体基因组结构物理上引入的物理图谱。图17A.用于转基因动物鉴定的PCR引物的定位图示。5′-引物位于在融合上游位置-148bp开始的WAP序列内,所述融合在WAP和BSSL变异体之间。3′-引物位于编码融合点下游400bp的第一BSSL变异体内含子中。B.使用的PCR引物序列C.琼脂糖凝胶显示潜在建立者动物典型的PCR分析。M:分子量标记。泳道1:产生于质粒pS317的对照PCR产物。泳道2-13;从潜在建立者动物制备的DNA进行PCR反应。图18
从pS317转基因雌性小鼠分离的不同组织制备的RNA Northern印迹分析。在泌乳第四天分离组织。通过琼脂糖-甲醛分离分析源于每一组织的10μg总RNA,转移其至滤膜上,与32P-标记的人BSSLcDNA进行杂交。泳道含Mg:乳腺;Li:肝;Ki:肾;Sp:脾;He:心;Lu:肺;Sg:唾液腺;Br:脑。RNA的核苷酸大小在右边标出。图19
从pS317转基因小鼠、全长cDNA载体pS314转基因小鼠及对照动物乳的Western印迹。样品经SDS-PAGE分离,转移至Immobilon滤膜上,用抗天然人BSSL产生的抗血清进行兔疫印迹分析。泳道1:分子量标记;泳道2、3和4:2μl来自3个pS317建立者FO#91F1雌仔的乳(F1 30、31和33)。泳道5:2μl来自建立者#90pS317的乳。泳道6、7和8:2μl源于非BSSL转基因动物乳;泳道9:纯化的鼠BSSL;泳道10:纯化的人天然BSSL。
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序列表(1)一般资料:
(i)申请人:
(A)名字:AB ASTRA
(B)街道:Kvarnbergagatan 16
(C)市:Sodertalje
(E)国家:Sweden
(F)邮编(ZIP):S-151 85
(G)电话:+46-8-553 260 00
(H)电传:+46-8-553 288 20
(I)电挂:19237 astra s
(ii)发明题目:新多肽
(iii)序列数:9
(iv)计算机可读形式:
(A)介质类型:软盘
(B)计算机:IBM PC可容机
(C)操作系统:PC-DOS/MS-DOS
(D)软件:PatentIn Release #1.0,Version #1.25(EPO)
(vi)在先申请资料:
(A)申请号:SE 9300686-4
(B)申请日:1993.3.1
(vi)在先申请资料:
(A)申请号:SE 9300722-7
(B)申请日:1993.3.4(2)SEQ ID NO:1的资料:
(i)序列特征:
(A)长度:2428bp
(B)类型:核酸
(C)链型:双链
(D)拓扑结构:线性
(ii)分子类型:cDNA到mRNA
(iii)假设:无
(iii)反义:无
(vi)来源:
(A)有机体:Homo sapiens
(F)组织类型:乳腺
(ix)特征:
(A)名称/关键词:CDS
(B)位置:82..2319
(D)其它资料:/产物=“胆盐刺激的脂酶”
(ix)特征:
(A)名称/关键词:外显子
(B)位置:985..1173
(ix)特征:
(A)名称/关键词:外显子
(B)位置:1174..1377
(ix)特征:
(A)名称/关键词:外显子
(B)位置:1378..1575
(ix)特征:
(A)名称/关键词:外显子
(B)位置:1576..2415
(ix)特征:
(A)名称/关键词:mat_肽
(B)位置:151..2316
(ix)特征:
(A)名称/关键词:polyA_信号
(B)位置:2397..2402
(ix)特征:
(A)名称/关键词:重复_区域
(B)位置:1756..2283
(ix)特征:
(A)名称/关键词:5′UTR
(B)位置:1..81
(ix)特征:
(A)名称/关键词:重复_单位
(B)位置:1756..1788
(ix)特征:
(A)名称/关键词:重复_单位
(B)位置:1789..1821
(ix)特征:
(A)名称/关键词:重复_单位
(B)位置:1822..1854
(ix)特征:
(A)名称/关键词:重复_单位
(B)位置:1855..1887
(ix)特征:
(A)名称/关键词:重复_单位
(B)位置:1888..1920
(ix)特征:
(A)名称/关键词:重复_单位
(B)位置:1921..1953
(ix)特征:
(A)名称/关键词:重复_单位
(B)位置:1954..1986
(ix)特征:
(A)名称/关键词:重复_单位
(B)位置:1987..2019
(ix)特征:
(A)名称/关键词:重复_单位
(B)位置:2020..2052
(ix)特征:
(A)名称/关键词:重复_单位
(B)位置:2053..2085
(ix)特征:
(A)名称/关键词:重复_单位
(B)位置:2086..2118
(ix)特征:
(A)名称/关键词:重复_单位
(B)位置:2119..2151
(ix)特征:
(A)名称/关键词:重复_单位
(B)位置:2152..2184
(ix)特征:
(A)名称/关键词:重复_单位
(B)位置:2185..2217
(ix)特征:
(A)名称/关键词:重复_单位
(B)位置:2218..2250
(ix)特征:
(A)名称/关键词:重复_单位
(B)位置:2251..2283
(xi)序列描述:SEQ ID NO:1:ACCTTCTGTA TCAGTTAAGT GTCAAGATGG AAGGAACAGC AGTCTCAAGA TAATGCAAAG 60AGTTTATTCA TCCAGAGGCT G ATG CTC ACC ATG GGG CGC CTG CAA CTG GTT 111
Met Leu Thr Met Gly Arg Leu Gln Leu Val
-23 -20 -15GTG TTG GGC CTC ACC TGC TGC TGG GCA GTG GCG AGT GCC GCG AAG CTG 159Val Leu Gly Leu Thr Cys Cys Trp Ala Val Ala Ser Ala Ala Lys Leu
-10 -5 1GGC GCC GTG TAC ACA GAA GGT GGG TTC GTG GAA GGC GTC AAT AAG AAG 207Gly Ala Val Tyr Thr Glu Gly Gly Phe Val Glu Gly Val Asn Lys Lys
5 10 15CTC GGC CTC CTG GGT GAC TCT GTG GAC ATC TTC AAG GGC ATC CCC TTC 255Leu Gly Leu Leu Gly Asp Ser Val Asp Ile Phe Lys Gly Ile Pro Phe20 25 30 35GCA GCT CCC ACC AAG GCC CTG GAA AAT CCT CAG CCA CAT CCT GGC TGG 303Ala Ala Pro Thr Lys Ala Leu Glu Asn Pro Gln Pro His Pro Gly Trp
40 45 50CAA GGG ACC CTG AAG GCC AAG AAC TTC AAG AAG AGA TGC CTG CAG GCC 351Gln Gly Thr Leu Lys Ala Lys Asn Phe Lys Lys Arg Cys Leu Gln Ala
55 60 65ACC ATC ACC CAG GAC AGC ACC TAC GGG GAT GAA GAC TGC CTG TAC CTC 399Thr Ile Thr Gln Asp Ser Thr Tyr Gly Asp Glu Asp Cys Leu Tyr Leu
70 75 80AAC ATT TGG GTG CCC CAG GGC AGG AAG CAA GTC TCC CGG GAC CTG CCC 447Asn Ile Trp Val Pro Gln Gly Arg Lys Gln Val Ser Arg Asp Leu Pro
85 90 95GTT ATG ATC TGG ATC TAT GGA GGC GCC TTC CTC ATG GGG TCC GGC CAT 495Val Met Ile Trp Ile Tyr Gly Gly Ala Phe Leu Met Gly Ser Gly His100 105 110 115GGG GCC AAC TTC CTC AAC AAC TAC CTG TAT GAC GGC GAG GAG ATC GCC 543Gly Ala Asn Phe Leu Asn Asn Tyr Leu Tyr Asp Gly Glu Glu Ile Ala
120 125 130ACA CGC GGA AAC GTC ATC GTG GTG ACC TTC AAC TAC CGT GTC GGC CCC 591Thr Arg Gly Asn Val Ile Val Val Thr Phe Asn Tyr Arg Val Gly Pro
135 140 145CTT GGG TTC CTC AGC ACT GGG GAC GCC AAT CTG CCA GGT AAC TAT GGC 639Leu Gly Phe Leu Ser Thr Gly Asp Ala Asn Leu Pro Gly Asn Tyr Gly
150 155 160CTT CGG GAT CAG CAC ATG GCC ATT GCT TGG GTG AAG AGG AAT ATC GCG 687Leu Arg Asp Gln His Met Ala Ile Ala Trp Val Lys Arg Asn Ile Ala
165 170 175GCC TTC GGG GGG GAC CCC AAC AAC ATC ACG CTC TTC GGG GAG TCT GCT 735Ala Phe Gly Gly Asp Pro Asn Asn Ile Thr Leu Phe Gly Glu Ser Ala180 185 190 195GGA GGT GCC AGC GTC TCT CTG CAG ACC CTC TCC CCC TAC AAC AAG GGC 783Gly Gly Ala Ser Val Ser Leu Gln Thr Leu Ser Pro Tyr Asn Lys Gly
200 205 210CTC ATC CGG CGA GCC ATC AGC CAG AGC GGC GTG GCC CTG AGT CCC TGG 831Leu Ile Arg Arg Ala Ile Ser Gln Ser Gly Val Ala Leu Ser Pro Trp
215 220 225GTC ATC CAG AAA AAC CCA CTC TTC TGG GCC AAA AAG GTG GCT GAG AAG 879Val Ile Gln Lys Asn Pro Leu Phe Trp Ala Lys Lys Val Ala Glu Lys
230 235 240GTG GGT TGC CCT GTG GGT GAT GCC GCC AGG ATG GCC CAG TGT CTG AAG 927Val Gly Cys Pro Val Gly Asp Ala Ala Arg Met Ala Gln Cys Leu Lys
245 250 255GTT ACT GAT CCC CGA GCC CTG ACG CTG GCC TAT AAG GTG CCG CTG GCA 975Val Thr Asp Pro Arg Ala Leu Thr Leu Ala Tyr Lys Val Pro Leu Ala260 265 270 275GGC CTG GAG TAC CCC ATG CTG CAC TAT GTG GGC TTC GTC CCT GTC ATT 1023Gly Leu Glu Tyr Pro Met Leu His Tyr Val Gly Phe Val Pro Val Ile
280 285 290GAT GGA GAC TTC ATC CCC GCT GAC CCG ATC AAC CTG TAC GCC AAC GCC 1071Asp Gly Asp Phe Ile Pro Ala Asp Pro Ile Asn Leu Tyr Ala Asn Ala
295 300 305GCC GAC ATC GAC TAT ATA GCA GGC ACC AAC AAC ATG GAC GGC CAC ATC 1119Ala Asp Ile Asp Tyr Ile Ala Gly Thr Asn Asn Met Asp Gly His Ile
310 315 320TTC GCC AGC ATC GAC ATG CCT GCC ATC AAC AAG GGC AAC AAG AAA GTC 1167Phe Ala Ser Ile Asp Met Pro Ala Ile Asn Lys Gly Asn Lys Lys Val
325 330 335ACG GAG GAG GAC TTC TAC AAG CTG GTC AGT GAG TTC ACA ATC ACC AAG 1215Thr Glu Glu Asp Phe Tyr Lys Leu Val Ser Glu Phe Thr Ile Thr Lys340 345 350 355GGG CTC AGA GGC GCC AAG ACG ACC TTT GAT GTC TAC ACC GAG TCC TGG 1263Gly Leu Arg Gly Ala Lys Thr Thr Phe Asp Val Tyr Thr Glu Ser Trp
360 365 370GCC CAG GAC CCA TCC CAG GAG AAT AAG AAG AAG ACT GTG GTG GAC TTT 1311Ala Gln Asp Pro Ser Gln Glu Ash Lys Lys Lys Thr Val Val Asp Phe
375 380 385GAG ACC GAT GTC CTC TTC CTG GTG CCC ACC GAG ATT GCC CTA GCC CAG 1359Glu Thr Asp Val Leu Phe Leu Val Pro Thr Glu Ile Ala Leu Ala Gln
390 395 400CAC AGA GCC AAT GCC AAG AGT GCC AAG ACC TAC GCC TAC CTG TTT TCC 1407His Arg Ala Asn Ala Lys Ser Ala Lys Thr Tyr Ala Tyr Leu Phe Ser
405 410 415CAT CCC TCT CGG ATG CCC GTC TAC CCC AAA TGG GTG GGG GCC GAC CAT 1455His Pro Ser Arg Met Pro Val Tyr Pro Lys Trp Val Gly Ala Asp His420 425 430 435GCA GAT GAC ATT CAG TAC GTT TTC GGG AAG CCC TTC GCC ACC CCC ACG 1503Ala Asp Asp Ile Gln Tyr Val Phe Gly Lys Pro Phe Ala Thr Pro Thr
440 445 450GGC TAC CGG CCC CAA GAC AGG ACA GTC TCT AAG GCC ATG ATC GCC TAC 1551Gly Tyr Arg Pro Gln Asp Arg Thr Val Ser Lys Ala Met Ile Ala Tyr
455 460 465TGG ACC AAC TTT GCC AAA ACA GGG GAC CCC AAC ATG GGC GAC TCG GCT 1599Trp Thr Asn Phe Ala Lys Thr Gly Asp Pro Asn Met Gly Asp Ser Ala
470 475 480GTG CCC ACA CAC TGG GAA CCC TAC ACT ACG GAA AAC AGC GGC TAC CTG 1647Val Pro Thr His Trp Glu Pro Tyr Thr Thr Glu Asn Ser Gly Tyr Leu
485 490 495GAG ATC ACC AAG AAG ATG GGC AGC AGC TCC ATG AAG CGG AGC CTG AGA 1695Glu Ile Thr Lys Lys Met Gly Ser Ser Ser Met Lys Arg Ser Leu Arg500 505 510 515ACC AAC TTC CTG CGC TAC TGG ACC CTC ACC TAT CTG GCG CTG CCC ACA 1743Thr Asn Phe Leu Arg Tyr Trp Thr Leu Thr Tyr Leu Ala Leu Pro Thr
520 525 530GTG ACC GAC CAG GAG GCC ACC CCT GTG CCC CCC ACA GGG GAC TCC GAG 1791Val Thr Asp Gln Glu Ala Thr Pro Val Pro Pro Thr Gly Asp Ser Glu
535 540 545GCC ACT CCC GTG CCC CCC ACG GGT GAC TCC GAG ACC GCC CCC GTG CCG 1839Ala Thr Pro Val Pro Pro Thr Gly Asp Ser Glu Thr Ala Pro Val Pro
550 555 560CCC ACG GGT GAC TCC GGG GCC CCC CCC GTG CCG CCC ACG GGT GAC TCC 1887Pro Thr Gly Asp Ser Gly Ala Pro Pro Val Pro Pro Thr Gly Asp Ser
565 570 575GGG GCC CCC CCC GTG CCG CCC ACG GGT GAC TCC GGG GCC CCC CCC GTG 1935Gly Ala Pro Pro Val Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val580 585 590 595CCG CCC ACG GGT GAC TCC GGG GCC CCC CCC GTG CCG CCC ACG GGT GAC 1983Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val Pro Pro Thr Gly Asp
600 605 610TCC GGG GCC CCC CCC GTG CCG CCC ACG GGT GAC TCC GGG GCC CCC CCC 2031Ser Gly Ala Pro Pro Val Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro
615 620 625GTG CCG CCC ACG GGT GAC TCC GGC GCC CCC CCC GTG CCG CCC ACG GGT 2079Val Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val Pro Pro Thr Gly
630 635 640GAC GCC GGG CCC CCC CCC GTG CCG CCC ACG GGT GAC TCC GGC GCC CCC 2127Asp Ala Gly Pro Pro Pro Val Pro Pro Thr Gly Asp Ser Gly Ala Pro
645 650 655CCC GTG CCG CCC ACG GGT GAC TCC GGG GCC CCC CCC GTG ACC CCC ACG 2175Pro Val Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val Thr Pro Thr660 665 670 675GGT GAC TCC GAG ACC GCC CCC GTG CCG CCC ACG GGT GAC TCC GGG GCC 2223Gly Asp Ser Glu Thr Ala Pro Val Pro Pro Thr Gly Asp Ser Gly Ala
680 685 690CCC CCT GTG CCC CCC ACG GGT GAC TCT GAG GCT GCC CCT GTG CCC CCC 2271Pro Pro Val Pro Pro Thr Gly Asp Ser Glu Ala Ala Pro Val Pro Pro
695 700 705ACA GAT GAC TCC AAG GAA GCT CAG ATG CCT GCA GTC ATT AGG TTT TAGCGTCCCA 2326Thr Asp Asp Ser Lys Glu Ala Gln Met Pro Ala Val Ile Arg Phe
710 715 720TGAGCCTTGG TATCAAGAGG CCACAAGAGT GGGACCCCAG GGGCTCCCCT CCCATCTTGA 2366GCTCTTCCTG AATAAAGCCT CATACCCCTA AA 2428(2)SEQ ID NO:2的资料:
(i)序列特征:
(A)长度:745个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ii)分子类型:蛋白质
(xi)序列描述:SEQ ID NO:2:Met Leu Thr Met Gly Arg Leu Gln Leu Val Val Leu Gly Leu Thr Cys-23 -20 -15 -10Cys Trp Ala Val Ala Ser Ala Ala Lys Leu Gly Ala Val Tyr Thr Glu
-5 1 5Gly Gly Phe Val Glu Gly Val Asn Lys Lys Leu Gly Leu Leu Gly Asp10 15 20 25Ser Val Asp Ile Phe Lys Gly Ile Pro Phe Ala Ala Pro Thr Lys Ala
30 35 40Leu Glu Asn Pro Gln Pro His Pro Gly Trp Gln Gly Thr Leu Lys Ala
45 50 55Lys Asn Phe Lys Lys Arg Cys Leu Gln Ala Thr Ile Thr Gln Asp Ser
60 65 70Thr Tyr Gly Asp Glu Asp Cys Leu Tyr Leu Asn Ile Trp Val Pro Gln
75 80 85Gly Arg Lys Gln Val Ser Arg Asp Leu Pro Val Met Ile Trp Ile Tyr90 95 100 105Gly Gly Ala Phe Leu Met Gly Ser Gly His Gly Ala Asn Phe Leu Asn
110 115 120Asn Tyr Leu Tyr Asp Gly Glu Glu Ile Ala Thr Arg Gly Asn Val Ile
125 130 135Val Val Thr Phe Asn Tyr Arg Val Gly Pro Leu Gly Phe Leu Ser Thr
140 145 150Gly Asp Ala Asn Leu Pro Gly Asn Tyr Gly Leu Arg Asp Gln His Met
155 160 165Ala Ile Ala Trp Val Lys Arg Asn Ile Ala Ala Phe Gly Gly Asp Pro170 175 l80 185Asn Asn Ile Thr Leu Phe Gly Glu Ser Ala Gly Gly Ala Ser Val Ser
190 195 200Leu Gln Thr Leu Ser Pro Tyr Asn Lys Gly Leu Ile Arg Arg Ala Ile
205 210 215Ser Gln Ser Gly Val Ala Leu Ser Pro Trp Val Ile Gln Lys Asn Pro
220 225 230Leu Phe Trp Ala Lys Lys Val Ala Glu Lys Val Gly Cys Pro Val Gly
235 240 245Asp Ala Ala Arg Met Ala Gln Cys Leu Lys Val Thr Asp Pro Arg Ala250 255 260 265Leu Thr Leu Ala Tyr Lys Val Pro Leu Ala Gly Leu Glu Tyr Pro Met
270 275 280Leu His Tyr Val Gly Phe Val Pro Val Ile Asp Gly Asp Phe Ile Pro
285 290 295Ala Asp Pro Ile Asn Leu Tyr Ala Asn Ala Ala Asp Ile Asp Tyr Ile
300 305 310Ala Gly Thr Asn Asn Met Asp Gly His Ile Phe Ala Ser Ile Asp Met
315 320 325Pro Ala Ile Asn Lys Gly Asn Lys Lys Val Thr Glu Glu Asp Phe Tyr330 335 340 345Lys Leu Val Ser Glu Phe Thr Ile Thr Lys Gly Leu Arg Gly Ala Lys
350 355 360Thr Thr Phe Asp Val Tyr Thr Glu Ser Trp Ala Gln Asp Pro Ser Gln
365 370 375Glu Asn Lys Lys Lys Thr Val Val Asp Phe Glu Thr Asp Val Leu Phe
380 385 390Leu Val Pro Thr Glu Ile Ala Leu Ala Gln His Arg Ala Asn Ala Lys
395 400 405Ser Ala Lys Thr Tyr Ala Tyr Lau Phe Ser His Pro Ser Arg Met Pro410 415 420 425Val Tyr Pro Lys Trp Val Gly Ala Asp His Ala Asp Asp Ile Gln Tyr
430 435 440Val Phe Gly Lys Pro Phe Ala Thr Pro Thr Gly Tyr Arg Pro Gln Asp
445 450 455Arg Thr Val Ser Lys Ala Met Ile Ala Tyr Trp Thr Asn Phe Ala Lys
460 465 470Thr Gly Asp Pro Asn Met Gly Asp Ser Ala Val Pro Thr His Trp Glu
475 480 485Pro Tyr Thr Thr Glu Asn Ser Gly Tyr Leu Glu Ile Thr Lys Lys Met490 495 500 505Gly Ser Ser Ser Met Lys Arg Ser Leu Arg Thr Asn Phe Leu Arg Tyr
510 515 520Trp Thr Leu Thr Tyr Leu Ala Leu Pro Thr Val Thr Asp Gln Glu Ala
525 530 535Thr Pro Val Pro Pro Thr Gly Asp Ser Glu Ala Thr Pro Val Pro Pro
540 545 550Thr Gly Asp Ser Glu Thr Ala Pro Val Pro Pro Thr Gly Asp Ser Gly
555 560 565Ala Pro Pro Val Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val Pro570 575 580 585Pro Thr Gly Asp Ser Gly Ala Pro Pro Val Pro Pro Thr Gly Asp Ser
590 595 600Gly Ala Pro Pro Val Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val
605 610 615Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val Pro Pro Thr Gly Asp
620 625 630Ser Gly Ala Pro Pro Val Pro Pro Thr Gly Asp Ala Gly Pro Pro Pro
635 640 645Val Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val Pro Pro Thr Gly650 655 660 665Asp Ser Gly Ala Pro Pro Val Thr Pro Thr Gly Asp Ser Glu Thr Ala
670 675 680Pro Val Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val Pro Pro Thr
685 690 695Gly Asp Ser Glu Ala Ala Pro Val Pro Pro Thr Asp Asp Ser Lys Glu
700 705 710Ala Gln Met Pro Ala Val Ile Arg Phe
715 720(2)SEQ ID NO:3的资料:
(i)序列特征:
(A)长度:722个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ii)分子类型:蛋白质
(iii)假设:无
(vi)来源
(A)有机体:Homo sapiens
(F)组织类型:乳腺
(xi)序列描述:SEQ ID NO:3:Ala Lys Leu Gly Ala Val Tyr Thr Glu Gly Gly Phe Val Glu Gly Val1 5 10 15Asn Lys Lys Leu Gly Leu Leu Gly Asp Ser Val Asp Ile Phe Lys Gly
20 25 30Ile Pro Phe Ala Ala Pro Thr Lys Ala Leu Glu Asn Pro Gln Pro His
35 40 45Pro Gly Trp Gln Gly Thr Leu Lys Ala Lys Asn Phe Lys Lys Arg Cys
50 55 60Leu Gln Ala Thr Ile Thr Gln Asp Ser Thr Tyr Gly Asp Glu Asp Cys65 70 75 80Leu Tyr Leu Asn Ile Trp Val Pro Gln Gly Arg Lys Gln Val Ser Arg
85 90 95Asp Leu Pro Val Met Ile Trp Ile Tyr Gly Gly Ala Phe Leu Met Gly
100 105 110Ser Gly His Gly Ala Asn Phe Leu Asn Asn Tyr Leu Tyr Asp Gly Glu
115 120 125Glu Ile Ala Thr Arg Gly Asn Val Ile Val Val Thr Phe Asn Tyr Arg
130 135 140Val Gly Pro Leu Gly Phe Leu Ser Thr Gly Asp Ala Asn Leu Pro Gly145 150 155 160Asn Tyr Gly Leu Arg Asp Gln His Met Ala Ile Ala Trp Val Lys Arg
165 170 175Asn Ile Ala Ala Phe Gly Gly Asp Pro Asn Asn Ile Thr Leu Phe Gly
180 185 190Glu Ser Ala Gly Gly Ala Ser Val Ser Leu Gln Thr Leu Ser Pro Tyr
195 200 205Asn Lys Gly Leu Ile Arg Arg Ala Ile Ser Gln Ser Gly Val Ala Leu
210 215 220Ser Pro Trp Val Ile Gln Lys Asn Pro Leu Phe Trp Ala Lys Lys Val225 230 235 240Ala Glu Lys Val Gly Cys Pro Val Gly Asp Ala Ala Arg Met Ala Gln
245 250 255Cys Leu Lys Val Thr Asp Pro Arg Ala Leu Thr Leu Ala Tyr Lys Val
260 265 270Pro Leu Ala Gly Leu Glu Tyr Pro Met Leu His Tyr Val Gly Phe Val
275 280 285Pro Val Ile Asp Gly Asp Phe Ile Pro Ala Asp Pro Ile Asn Leu Tyr
290 295 300Ala Asn Ala Ala Asp Ile Asp Tyr Ile Ala Gly Thr Asn Asn Met Asp305 310 315 320Gly His Ile Phe Ala Ser Ile Asp Met Pro Ala Ile Asn Lys Gly Asn
325 330 335Lys Lys Val Thr Glu Glu Asp Phe Tyr Lys Leu Val Ser Glu Phe Thr
340 345 350Ile Thr Lys Gly Leu Arg Gly Ala Lys Thr Thr Phe Asp Val Tyr Thr
355 360 365Glu Ser Trp Ala Gln Asp Pro Ser Gln Glu Asn Lys Lys Lys Thr Val
370 375 380Val Asp Phe Glu Thr Asp Val Leu Phe Leu Val Pro Thr Glu Ile Ala385 390 395 400Leu Ala Gln His Arg Ala Asn Ala Lys Ser Ala Lys Thr Tyr Ala Tyr
405 410 415Leu Phe Ser His Pro Ser Arg Met Pro Val Tyr Pro Lys Trp Val Gly
420 425 430Ala Asp His Ala Asp Asp Ile Gln Tyr Val Phe Gly Lys Pro Phe Ala
435 440 445Thr Pro Thr Gly Tyr Arg Pro Gln Asp Arg Thr Val Ser Lys Ala Met
450 455 460Ile Ala Tyr Trp Thr Asn Phe Ala Lys Thr Gly Asp Pro Asn Met Gly465 470 475 480Asp Ser Ala Val Pro Thr His Trp Glu Pro Tyr Thr Thr Glu Asn Ser
485 490 495Gly Tyr Leu Glu Ile Thr Lys Lys Met Gly Ser Ser Ser Met Lys Arg
500 505 510Ser Leu Arg Thr Asn Phe Leu Arg Tyr Trp Thr Leu Thr Tyr Leu Ala
515 520 525Leu Pro Thr Val Thr Asp Gln Glu Ala Thr Pro Val Pro Pro Thr Gly
530 535 540Asp Ser Glu Ala Thr Pro Val Pro Pro Thr Gly Asp Ser Glu Thr Ala545 550 555 560Pro Val Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val Pro Pro Thr
565 570 575Gly Asp Ser Gly Ala Pro Pro Val Pro Pro Thr Gly Asp Ser Gly Ala
580 585 590Pro Pro Val Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val Pro Pro
595 600 605Thr Gly Asp Ser Gly Ala Pro Pro Val Pro Pro Thr Gly Asp Ser Gly
610 615 620Ala Pro Pro Val Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val Pro625 630 635 640Pro Thr Gly Asp Ala Gly Pro Pro Pro Val Pro Pro Thr Gly Asp Ser
645 650 655Gly Ala Pro Pro Val Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val
660 665 670Thr Pro Thr Gly Asp Ser Glu Thr Ala Pro Val Pro Pro Thr Gly Asp
675 680 685Ser Gly Ala Pro Pro Val Pro Pro Thr Gly Asp Ser Glu Ala Ala Pro
690 695 700Val Pro Pro Thr Asp Asp Ser Lys Glu Ala Gln Met Pro Ala Val Ile705 710 715 720Arg Phe(2)SEQ ID NO:4的资料:
(i)序列特征:
(A)长度:535个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ii)分子类型:蛋白质
(iii)假设:无
(vi)来源
(A)有机体:Homo sapiens
(F)组织类型:乳腺
(ix)特征:
(A)名称/关键词:肽
(B)位置:1..535
(D)其它资料:/标记=变异体_A
(xi)序列描述:SEQ ID NO:4:Ala Lys Leu Gly Ala Val Tyr Thr Glu Gly Gly Phe Val Glu Gly Val1 5 10 15Asn Lys Lys Leu Gly Leu Leu Gly Asp Ser Val Asp Ile Phe Lys Gly
20 25 30Ile Pro Phe Ala Ala Pro Thr Lys Ala Leu Glu Asn Pro Gln Pro His
35 40 45Pro Gly Trp Gln Gly Thr Leu Lys Ala Lys Asn Phe Lys Lys Arg Cys
50 55 60Leu Gln Ala Thr Ile Thr Gln Asp Ser Thr Tyr Gly Asp Glu Asp Cys65 70 75 80Leu Tyr Leu Asn Ile Trp Val Pro Gln Gly Arg Lys Gln Val Ser Arg
85 90 95Asp Leu Pro Val Met Ile Trp Ile Tyr Gly Gly Ala Phe Leu Met Gly
100 105 110Ser Gly His Gly Ala Asn Phe Leu Asn Asn Tyr Leu Tyr Asp Gly Glu
115 120 125Glu Ile Ala Thr Arg Gly Asn Val Ile Val Val Thr Phe Asn Tyr Arg
130 135 140Val Gly Pro Leu Gly Phe Leu Ser Thr Gly Asp Ala Asn Leu Pro Gly145 150 155 160Asn Tyr Gly Leu Arg Asp Gln His Met Ala Ile Ala Trp Val Lys Arg
165 170 175Asn Ile Ala Ala Phe Gly Gly Asp Pro Asn Asn Ile Thr Leu Phe Gly
l80 185 190Glu Ser Ala Gly Gly Ala Ser Val Ser Leu Gln Thr Leu Ser Pro Tyr
195 200 205Asn Lys Gly Leu Ile Arg Arg Ala Ile Ser Gln Ser Gly Val Ala Leu
210 215 220Ser Pro Trp Val Ile Gln Lys Asn Pro Leu Phe Trp Ala Lys Lys Val225 230 235 240Ala Glu Lys Val Gly Cys Pro Val Gly Asp Ala Ala Arg Met Ala Gln
245 250 255Cys Leu Lys Val Thr Asp Pro Arg Ala Leu Thr Leu Ala Tyr Lys Val
260 265 270Pro Leu Ala Gly Leu Glu Tyr Pro Met Leu His Tyr Val Gly Phe Val
275 280 285Pro Val Ile Asp Gly Asp Phe Ile Pro Ala Asp Pro Ile Asn Leu Tyr
290 295 300Ala Asn Ala Ala Asp Ile Asp Tyr Ile Ala Gly Thr Asn Asn Met Asp305 310 315 320Gly His Ile Phe Ala Ser Ile Asp Met Pro Ala Ile Asn Lys Gly Asn
325 330 335Lys Lys Val Thr Glu Glu Asp Phe Tyr Lys Leu Val Ser Glu Phe Thr
340 345 350Ile Thr Lys Gly Leu Arg Gly Ala Lys Thr Thr Phe Asp Val Tyr Thr
355 360 365Glu Ser Trp Ala Gln Asp Pro Ser Gln Glu Asn Lys Lys Lys Thr Val
370 375 380Val Asp Phe Glu Thr Asp Val Leu Phe Leu Val Pro Thr Glu Ile Ala385 390 395 400Leu Ala Gln His Arg Ala Asn Ala Lys Ser Ala Lys Thr Tyr Ala Tyr
405 410 415Leu Phe Ser His Pro Ser Arg Met Pro Val Tyr Pro Lys Trp Val Gly
420 425 430Ala Asp His Ala Asp Asp Ile Gln Tyr Val Phe Gly Lys Pro Phe Ala
435 440 445Thr Pro Thr Gly Tyr Arg Pro Gln Asp Arg Thr Val Ser Lys Ala Met
450 455 460Ile Ala Tyr Trp Thr Asn Phe Ala Lys Thr Gly Asp Pro Asn Met Gly465 470 475 480Asp Ser Ala Val Pro Thr His Trp Glu Pro Tyr Thr Thr Glu Asn Ser
485 490 495Gly Tyr Leu Glu Ile Thr Lys Lys Met Gly Ser Ser Ser Met Lys Arg
500 505 510Ser Leu Arg Thr Asn Phe Leu Arg Tyr Trp Thr Leu Thr Tyr Leu Ala
515 520 525Leu Pro Thr Val Thr Asp Gln
530 535(2)SEQ ID NO:5的资料:
(i)序列特征:
(A)长度:546个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ii)分子类型:蛋白质
(iii)假设:无
(vi)来源
(A)有机体:Homo sapiens
(F)组织类型:乳腺
(ix)特征:
(A)名称/关键词:肽
(B)位置:1..546
(D)其它资料:/标记=变异体_B
(xi)序列描述:SEQ ID NO:5:Ala Lys Leu Gly Ala Val Tyr Thr Glu Gly Gly Phe Val Glu Gly Val1 5 10 15Asn Lys Lys Leu Gly Leu Leu Gly Asp Ser Val Asp Ile Phe Lys Gly
20 25 30Ile Pro Phe Ala Ala Pro Thr Lys Ala Leu Glu Asn Pro Gln Pro His
35 40 45Pro Gly Trp Gln Gly Thr Leu Lys Ala Lys Asn Phe Lys Lys Arg Cys
50 55 60Leu Gln Ala Thr Ile Thr Gln Asp Ser Thr Tyr Gly Asp Glu Asp Cys65 70 75 80Leu Tyr Leu Asn Ile Trp Val Pro Gln Gly Arg Lys Gln Val Ser Arg
85 90 95Asp Leu Pro Val Met Ile Trp Ile Tyr Gly Gly Ala Phe Leu Met Gly
100 105 110Ser Gly His Gly Ala Asn Phe Leu Asn Asn Tyr Leu Tyr Asp Gly Glu
115 120 125Glu Ile Ala Thr Arg Gly Asn Val Ile Val Val Thr Phe Asn Tyr Arg
130 135 140Val Gly Pro Leu Gly Phe Leu Ser Thr Gly Asp Ala Asn Leu Pro Gly145 150 155 160Asn Tyr Gly Leu Arg Asp Gln His Met Ala Ile Ala Trp Val Lys Arg
165 170 175Asn Ile Ala Ala Phe Gly Gly Asp Pro Asn Asn Ile Thr Leu Phe Gly
l80 185 190Glu Ser Ala Gly Gly Ala Ser Val Ser Leu Gln Thr Leu Ser Pro Tyr
195 200 205Asn Lys Gly Leu Ile Arg Arg Ala Ile Ser Gln Ser Gly Val Ala Leu
210 215 220Ser Pro Trp Val Ile Gln Lys Asn Pro Leu Phe Trp Ala Lys Lys Val225 230 235 240Ala Glu Lys Val Gly Cys Pro Val Gly Asp Ala Ala Arg Met Ala Gln
245 250 255Cys Leu Lys Val Thr Asp Pro Arg Ala Leu Thr Leu Ala Tyr Lys Val
260 265 270Pro Leu Ala Gly Leu Glu Tyr Pro Met Leu His Tyr Val Gly Phe Val
275 280 285Pro Val Ile Asp Gly Asp Phe Ile Pro Ala Asp Pro Ile Asn Leu Tyr
290 295 300Ala Asn Ala Ala Asp Ile Asp Tyr Ile Ala Gly Thr Asn Asn Met Asp305 310 315 320Gly His Ile Phe Ala Ser Ile Asp Met Pro Ala Ile Asn Lys Gly Asn
325 330 335Lys Lys Val Thr Glu Glu Asp Phe Tyr Lys Leu Val Ser Glu Phe Thr
340 345 350Ile Thr Lys Gly Leu Arg Gly Ala Lys Thr Thr Phe Asp Val Tyr Thr
355 360 365Glu Ser Trp Ala Gln Asp Pro Ser Gln Glu Asn Lys Lys Lys Thr Val
370 375 380Val Asp Phe Glu Thr Asp Val Leu Phe Leu Val Pro Thr Glu Ile Ala385 390 395 400Leu Ala Gln His Arg Ala Asn Ala Lys Ser Ala Lys Thr Tyr Ala Tyr
405 410 415Leu Phe Ser His Pro Ser Arg Met Pro Val Tyr Pro Lys Trp Val Gly
420 425 430Ala Asp His Ala Asp Asp Ile Gln Tyr Val Phe Gly Lys Pro Phe Ala
435 440 445Thr Pro Thr Gly Tyr Arg Pro Gln Asp Arg Thr Val Ser Lys Ala Met
450 455 460Ile Ala Tyr Trp Thr Asn Phe Ala Lys Thr Gly Asp Pro Asn Met Gly465 470 475 480Asp Ser Ala Val Pro Thr His Trp Glu Pro Tyr Thr Thr Glu Asn Ser
485 490 495Gly Tyr Leu Glu Ile Thr Lys Lys Met Gly Ser Ser Ser Met Lys Arg
500 505 510Ser Leu Arg Thr Asn Phe Leu Arg Tyr Trp Thr Leu Thr Tyr Leu Ala
515 520 525Leu Pro Thr Val Thr Asp Gln Lys Glu Ala Gln Met Pro Ala Val Ile
530 535 540Arg Phe545(2)SEQ ID NO:6的资料:
(i)序列特征:
(A)长度:568个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ii)分子类型:蛋白质
(iii)假设:无
(vi)来源
(A)有机体:Homo sapiens
(F)组织类型:乳腺
(ix)特征:
(A)名称/关键词:肽
(B)位置:1..568
(D)其它资料:/标记=变异体_C
(xi)序列描述:SEQ ID NO:6:Ala Lys Leu Gly Ala Val Tyr Thr Glu Gly Gly Phe Val Glu Gly Val1 5 10 15Ash Lys Lys Leu Gly Leu Leu Gly Asp Ser Val Asp Ile Phe Lys Gly
20 25 30Ile Pro Phe Ala Ala Pro Thr Lys Ala Leu Glu Asn Pro Gln Pro His
35 40 45Pro Gly Trp Gln Gly Thr Leu Lys Ala Lys Asn Phe Lys Lys Arg Cys
50 55 60Leu Gln Ala Thr Ile Thr Gln Asp Ser Thr Tyr Gly Asp Glu Asp Cys65 70 75 80Leu Tyr Leu Asn Ile Trp Val Pro Gln Gly Arg Lys Gln Val Ser Arg
85 90 95Asp Leu Pro Val Met Ile Trp Ile Tyr Gly Gly Ala Pne Leu Met Gly
100 105 110Ser Gly His Gly Ala Asn Phe Leu Asn Asn Tyr Leu Tyr Asp Gly Glu
115 120 125Glu Ile Ala Thr Arg Gly Asn Val Ile Val Val Thr Phe Asn Tyr Arg
130 135 140Val Gly Pro Leu Gly Phe Leu Ser Thr Gly Asp Ala Asn Leu Pro Gly145 150 155 160Asn Tyr Gly Leu Arg Asp Gln His Met Ala Ile Ala Trp Val Lys Arg
165 170 175Asn Ile Ala Ala Phe Gly Gly Asp Pro Asn Asn Ile Thr Leu Phe Gly
180 185 190Glu Ser Ala Gly Gly Ala Ser Val Ser Leu Gln Thr Leu Ser Pro Tyr
195 200 205Asn Lys Gly Leu Ile Arg Arg Ala Ile Ser Gln Ser Gly Val Ala Leu
210 215 220Ser Pro Trp Val Ile Gln Lys Asn Pro Leu Phe Trp Ala Lys Lys Val225 230 235 240Ala Glu Lys Val Gly Cys Pro Val Gly Asp Ala Ala Arg Met Ala Gln
245 250 255Cys Leu Lys Val Thr Asp Pro Arg Ala Leu Thr Leu Ala Tyr Lys Val
260 265 270Pro Leu Ala Gly Leu Glu Tyr Pro Met Leu His Tyr Val Gly Phe Val
275 280 285Pro Val Ile Asp Gly Asp Phe Ile Pro Ala Asp Pro Ile Asn Leu Tyr
290 295 300Ala Asn Ala Ala Asp Ile Asp Tyr Ile Ala Gly Thr Asn Asn Met Asp305 310 315 320Gly His Ile Phe Ala Ser Ile Asp Met Pro Ala Ile Asn Lys Gly Asn
325 330 335Lys Lys Val Thr Glu Glu Asp Phe Tyr Lys Leu Val Ser Glu Phe Thr
340 345 350Ile Thr Lys Gly Leu Arg Gly Ala Lys Thr Thr Phe Asp Val Tyr Thr
355 360 365Glu Ser Trp Ala Gln Asp Pro Ser Gln Glu Asn Lys Lys Lys Thr Val
370 375 380Val Asp Phe Glu Thr Asp Val Leu Phe Leu Val Pro Thr Glu Ile Ala385 390 395 400Leu Ala Gln His Arg Ala Asn Ala Lys Ser Ala Lys Thr Tyr Ala Tyr
405 410 415Leu Phe Ser His Pro Ser Arg Met Pro Val Tyr Pro Lys Trp Val Gly
420 425 430Ala Asp His Ala Asp Asp Ile Gln Tyr Val Phe Gly Lys Pro Phe Ala
435 440 445Thr Pro Thr Gly Tyr Arg Pro Gln Asp Arg Thr Val Ser Lys Ala Met
450 455 460Ile Ala Tyr Trp Thr Asn Phe Ala Lys Thr Gly Asp Pro Asn Met Gly465 470 475 480Asp Ser Ala Val Pro Thr His Trp Glu Pro Tyr Thr Thr Glu Asn Ser
485 490 495Gly Tyr Leu Glu Ile Thr Lys Lys Met Gly Ser Ser Ser Met Lys Arg
500 505 510Ser Leu Arg Thr Asn Phe Leu Arg Tyr Trp Thr Leu Thr Tyr Leu Ala
515 520 525Leu Pro Thr Val Thr Asp Gln Gly Ala Pro Pro Val Pro Pro Thr Gly
530 535 540Asp Ser Gly Ala Pro Pro Val Pro Pro Thr Gly Asp Ser Lys Glu Ala545 550 555 560Gln Met Pro Ala Val Ile Arg Phe
565(2)SEQ ID NO:7的资料:
(i)序列特征:
(A)长度:722个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ii)分子类型:蛋白质
(iii)假设:无
(vi)来源
(A)有机体:Homo sapiens
(F)组织类型:乳腺
(ix)特征:
(A)名称/关键词:肽
(B)位置:1..722
(D)其它资料:/标记=变异体_N
(xi)序列描述:SEQ ID NO:7:Ala Lys Leu Gly Ala Val Tyr Thr Glu Gly Gly Phe Val Glu Gly Val1 5 10 15Asn Lys Lys Leu Gly Leu Leu Gly Asp Ser Val Asp Ile Phe Lys Gly
20 25 30Ile Pro Phe Ala Ala Pro Thr Lys Ala Leu Glu Asn Pro Gln Pro His
35 40 45Pro Gly Trp Gln Gly Thr Leu Lys Ala Lys Asn Phe Lys Lys Arg Cys
50 55 60Leu Gln Ala Thr Ile Thr Gln Asp Ser Thr Tyr Gly Asp Glu Asp Cys65 70 75 80Leu Tyr Leu Asn Ile Trp Val Pro Gln Gly Arg Lys Gln Val Ser Arg
85 90 95Asp Leu Pro Val Met Ile Trp Ile Tyr Gly Gly Ala Phe Leu Met Gly
100 105 110Ser Gly His Gly Ala Asn Phe Leu Asn Asn Tyr Leu Tyr Asp Gly Glu
115 120 125Glu Ile Ala Thr Arg Gly Asn Val Ile Val Val Thr Phe Asn Tyr Arg
130 135 140Val Gly Pro Leu Gly Phe Leu Ser Thr Gly Asp Ala Asn Leu Pro Gly145 150 155 160Asn Tyr Gly Leu Arg Asp Gln His Met Ala Ile Ala Trp Val Lys Arg
165 170 175Asn Ile Ala Ala Phe Gly Gly Asp Pro Asn Gln Ile Thr Leu Phe Gly
l80 185 190Glu Ser Ala Gly Gly Ala Ser Val Ser Leu Gln Thr Leu Ser Pro Tyr
195 200 205Asn Lys Gly Leu Ile Arg Arg Ala Ile Ser Gln Ser Gly Val Ala Leu
210 215 220Ser Pro Trp Val Ile Gln Lys Asn Pro Leu Phe Trp Ala Lys Lys Val225 230 235 240Ala Glu Lys Val Gly Cys Pro Val Gly Asp Ala Ala Arg Met Ala Gln
245 250 255Cys Leu Lys Val Thr Asp Pro Arg Ala Leu Thr Leu Ala Tyr Lys Val
260 265 270Pro Leu Ala Gly Leu Glu Tyr Pro Met Leu His Tyr Val Gly Phe Val
275 280 285Pro Val Ile Asp Gly Asp Phe Ile Pro Ala Asp Pro Ile Asn Leu Tyr
290 295 300Ala Asn Ala Ala Asp Ile Asp Tyr Ile Ala Gly Thr Asn Asn Met Asp305 310 315 320Gly His Ile Phe Ala Ser Ile Asp Met Pro Ala Ile Asn Lys Gly Asn
325 330 335Lys Lys Val Thr Glu Glu Asp Phe Tyr Lys Leu Val Ser Glu Phe Thr
340 345 350Ile Thr Lys Gly Leu Arg Gly Ala Lys Thr Thr Phe Asp Val Tyr Thr
355 360 365Glu Ser Trp Ala Gln Asp Pro Ser Gln Glu Asn Lys Lys Lys Thr Val
370 375 380Val Asp Phe Glu Thr Asp Val Leu Phe Leu Val Pro Thr Glu Ile Ala385 390 395 400Leu Ala Gln His Arg Ala Asn Ala Lys Ser Ala Lys Thr Tyr Ala Tyr
405 410 415Leu Phe Ser His Pro Ser Arg Met Pro Val Tyr Pro Lys Trp Val Gly
420 425 430Ala Asp His Ala Asp Asp Ile Gln Tyr Val Phe Gly Lys Pro Phe Ala
435 440 445Thr Pro Thr Gly Tyr Arg Pro Gln Asp Arg Thr Val Ser Lys Ala Met
450 455 460Ile Ala Tyr Trp Thr Asn Phe Ala Lys Thr Gly Asp Pro Asn Met Gly465 470 475 480Asp Ser Ala Val Pro Thr His Trp Glu Pro Tyr Thr Thr Glu Asn Ser
485 490 495Gly Tyr Leu Glu Ile Thr Lys Lys Met Gly Ser Ser Ser Met Lys Arg
500 505 510Ser Leu Arg Thr Asn Phe Leu Arg Tyr Trp Thr Leu Thr Tyr Leu Ala
515 520 525Leu Pro Thr Val Thr Asp Gln Glu Ala Thr Pro Val Pro Pro Thr Gly
530 535 540Asp Ser Glu Ala Thr Pro Val Pro Pro Thr Gly Asp Ser Glu Thr Ala545 550 555 560Pro Val Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val Pro Pro Thr
565 570 575Gly Asp Ser Gly Ala Pro pro Val Pro Pro Thr Gly Asp Ser Gly Ala
580 585 590Pro Pro Val Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val Pro Pro
595 600 605Thr Gly Asp Ser Gly Ala Pro Pro Val Pro Pro Thr Gly Asp Ser Gly
610 615 620Ala Pro Pro Val Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val Pro625 630 635 640Pro Thr Gly Asp Ala Gly Pro Pro Pro Val Pro Pro Thr Gly Asp Ser
645 650 655Gly Ala Pro Pro Val Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val
660 665 670Thr Pro Thr Gly Asp Ser Glu Thr Ala Pro Val Pro Pro Thr Gly Asp
675 680 685Ser Gly Ala Pro Pro Val Pro Pro Thr Gly Asp Ser Glu Ala Ala Pro
690 695 700Val Pro Pro Thr Asp Asp Ser Lys Glu Ala Gln Met Pro Ala Val Ile705 710 715 720Arg Phe(2)SEQ ID NO:8的资料:
(i)序列特征:
(A)长度:2184bp
(B)类型:核酸
(C)链型:双链
(D)拓扑结构:线性
(ii)分子类型:DNA(基因组的)
(iii)假设:无
(iii)反义:无
(vi)来源
(A)有机体:Homo sapiens
(F)组织类型:乳腺
(ix)特征:
(A)名称/关键词:CDS
(B)位置:82..2088
(D)其它资料:/标记=变异体_T
(ix)特征:
(A)名称/关键词:mat_肽
(B)位置:151..2085
(ix)特征:
(A)名称/关键词:重复_区域
(B)位置:1756..2052
(ix)特征:
(A)名称/关键词:重复_单位
(B)位置:1756..1788
(ix)特征:
(A)名称/关键词:重复_单位
(B)位置:1789..1821
(ix)特征:
(A)名称/关键词:重复_单位
(B)位置:1822..1854
(ix)特征:
(A)名称/关键词:重复_单位
(B)位置:1855..1887
(ix)特征:
(A)名称/关键词:重复_单位
(B)位置:1888..1920
(ix)特征:
(A)名称/关键词:重复_单位
(B)位置:1921..1953
(ix)特征:
(A)名称/关键词:重复_单位
(B)位置:1954..1986
(ix)特征:
(A)名称/关键词:重复_单位
(B)位置:1987..2019
(ix)特征:
(A)名称/关键词:重复_单位
(B)位置:2020..2052
(xi)序列描述:SEQ ID NO:8:ACCTTCTGTA TCAGTTAAGT GTCAAGATGG AAGGAACAGC AGTCTCAAGA TAATGCAAAG 60AGTTTATTCA TCCAGAGGCT G ATG CTC ACC ATG GGG CGC CTG CAA CTG GTT 111
Met Leu Thr Met Gly Arg Leu Gln Leu Val
-23 -20 -15GTG TTG GGC CTC ACC TGC TGC TGG GCA GTG GCG AGT GCC GCG AAG CTG 159Val Leu Gly Leu Thr Cys Cys Trp Ala Val Ala Ser Ala Ala Lys Leu
-10 -5 1GGC GCC GTG TAC ACA GAA GGT GGG TTC GTG GAA GGC GTC AAT AAG AAG 207Gly Ala Val Tyr Thr Glu Gly Gly Phe Val Glu Gly Val Asn Lys Lys
5 10 15CTC GGC CTC CTG GGT GAC TCT GTG GAC ATC TTC AAG GGC ATC CCC TTC 255Leu Gly Leu Leu Gly Asp Ser Val Asp Ile Phe Lys Gly Ile Pro Phe20 25 30 35GCA GCT CCC ACC AAG GCC CTG GAA AAT CCT CAG CCA CAT CCT GGC TGG 303Ala Ala Pro Thr Lys Ala Leu Glu Asn Pro Gln Pro His Pro Gly Trp
40 45 50CAA GGG ACC CTG AAG GCC AAG AAC TTC AAG AAG AGA TGC CTG CAG GCC 351Gln Gly Thr Leu Lys Ala Lys Asn Phe Lys Lys Arg Cys Leu Gln Ala
55 60 65ACC ATC ACC CAG GAC AGC ACC TAC GGG GAT GAA GAC TGC CTG TAC CTC 399Thr Ile Thr Gln Asp Ser Thr Tyr Gly Asp Glu Asp Cys Leu Tyr Leu
70 75 80AAC ATT TGG GTG CCC CAG GGC AGG AAG CAA GTC TCC CGG GAC CTG CCC 447Asn Ile Trp Val Pro Gln Gly Arg Lys Gln Val Ser Arg Asp Leu Pro
85 90 95GTT ATG ATC TGG ATC TAT GGA GGC GCC TTC CTC ATG GGG TCC GGC CAT 495Val Met Ile Trp Ile Tyr Gly Gly Ala Phe Leu Met Gly Ser Gly His100 105 110 115GGG GCC AAC TTC CTC AAC AAC TAC CTG TAT GAC GGC GAG GAG ATC GCC 543Gly Ala Asn Phe Leu Asn Asn Tyr Leu Tyr Asp Gly Glu Glu Ile Ala
120 125 130ACA CGC GGA AAC GTC ATC GTG GTC ACC TTC AAC TAC CGT GTC GGC CCC 591Thr Arg Gly Asn Val Ile Val Val Thr Phe Asn Tyr Arg Val Gly Pro
135 140 145CTT GGG TTC CTC AGC ACT GGG GAC GCC AAT CTG CCA GGT AAC TAT GGC 639Leu Gly Phe Leu Ser Thr Gly Asp Ala Asn Leu Pro Gly Asn Tyr Gly
150 155 160CTT CGG GAT CAG CAC ATG GCC ATT GCT TGG GTG AAG AGG AAT ATC GCG 687Leu Arg Asp Gln His Met Ala Ile Ala Trp Val Lys Arg Asn Ile Ala
165 170 175GCC TTC GGG GGG GAC CCC AAC AAC ATC ACG CTC TTC GGG GAG TCT GCT 735Ala Phe Gly Gly Asp Pro Asn Asn Ile Thr Leu Phe Gly Glu Ser Ala180 185 190 195GGA GGT GCC AGC GTC TCT CTG CAG ACC CTC TCC CCC TAC AAC AAG GGC 783Gly Gly Ala Ser Val Ser Leu Gln Thr Leu Ser Pro Tyr Asn Lys Gly
200 205 210CTC ATC CGG CGA GCC ATC AGC CAG AGC GGC GTG GCC CTG AGT CCC TGG 831Leu Ile Arg Arg Ala Ile Ser Gln Ser Gly Val Ala Leu Ser Pro Trp
215 220 225GTC ATC CAG AAA AAC CCA CTC TTC TGG GCC AAA AAG GTG GCT GAG AAG 879Val Ile Gln Lys Asn Pro Leu Phe Trp Ala Lys Lys Val Ala Glu Lys
230 235 240GTG GGT TGC CCT GTG GGT GAT GCC GCC AGG ATG GCC CAG TGT CTG AAG 927Val Gly Cys Pro Val Gly Asp Ala Ala Arg Met Ala Gln Cys Leu Lys
245 250 255GTT ACT GAT CCC CGA GCC CTG ACG CTG GCC TAT AAG GTG CCG CTG GCA 975Val Thr Asp Pro Arg Ala Leu Thr Leu Ala Tyr Lys Val Pro Leu Ala260 265 270 275GGC CTG GAG TAC CCC ATG CTG CAC TAT GTG GGC TTC GTC CCT GTC ATT 1023Gly Leu Glu Tyr Pro Met Leu His Tyr Val Gly Phe Val Pro Val Ile
280 285 290GAT GGA GAC TTC ATC CCC GCT GAC CCG ATC AAC CTG TAC GCC AAC GCC 1071Asp Gly Asp Phe Ile Pro Ala Asp Pro Ile Asn Leu Tyr Ala Asn Ala
295 300 305GCC GAC ATC GAC TAT ATA GCA GGC ACC AAC AAC ATG GAC GGC CAC ATC 1119Ala Asp Ile Asp Tyr Ile Ala Gly Thr Asn Asn Met Asp Gly His Ile
310 315 320TTC GCC AGC ATC GAC ATG CCT GCC ATC AAC AAG GGC AAG AAG AAA GTC 1167Phe Ala Ser Ile Asp Met Pro Ala Ile Asn Lys Gly Asn Lys Lys Val
325 330 335ACG GAG GAG GAC TTC TAC AAG CTG GTC AGT GAG TTC ACA ATC ACC AAG 1015Tnr Glu Glu Asp Phe Tyr Lys Leu Val Ser Glu Phe Thr Ile Thr Lys340 345 350 355GGG CTC AGA GGC GCC AAG ACG ACC TTT GAT GTC TAC ACC GAG TCC TGG 1263Gly Leu Arg Gly Ala Lys Thr Thr Phe Asp Val Tyr Thr Glu Ser Trp
360 365 370GCC CAG GAC CCA TCC CAG GAG AAT AAG AAG AAG ACT GTG GTG GAC TTT 1311Ala Gln Asp Pro Ser Gln Glu Asn Lys Lys Lys Thr Val Val Asp Phe
375 380 385GAG ACC GAT GTC CTC TTC CTG GTG CCC ACC GAG ATT GCC CTA GCC CAG 1359Glu Thr Asp Val Leu Phe Leu Val Pro Thr Glu Ile Ala Leu Ala Gln
390 395 400CAC AGA GCC AAT GCC AAG AGT GCC AAG ACC TAC GCC TAC CTG TTT TCC 1407His Arg Ala Asn Ala Lys Ser Ala Lys Thr Tyr Ala Tyr Leu Phe Ser
405 410 415CAT CCC TCT CGG ATG CCC GTC TAC CCC AAA TGG GTG GGG GCC GAC CAT 1455His Pro Ser Arg Met Pro Val Tyr Pro Lys Trp Val Gly Ala Asp His420 425 430 435GCA GAT GAC ATT CAG TAC GTT TTC GGG AAG CCC TTC GCC ACC CCC ACG 1503Ala Asp Asp Ile Gln Tyr Val Phe Gly Lys Pro Phe Ala Thr Pro Thr
440 445 450GGC TAC CGG CCC CAA GAC AGG ACA GTC TCT AAG GCC ATG ATC GCC TAC 1551Gly Tyr Arg Pro Gln Asp Arg Thr Val Ser Lys Ala Met Ile Ala Tyr
455 460 465TGG ACC AAC TTT GCC AAA ACA GGG GAC CCC AAC ATG GGC GAC TCG GCT 1599Trp Thr Asn Phe Als Lys Thr Gly Asp Pro Asn Met Gly Asp Ser Ala
470 475 480GTG CCC ACA CAC TGG GAA CCC TAC ACT ACG GAA AAC AGC GGC TAC CTG 1647Val Pro Thr His Trp Glu Pro Tyr Thr Thr Glu Asn Ser Gly Tyr Leu
485 490 495GAG ATC ACC AAG AAG ATG GGC AGC AGC TCC ATG AAG CGG AGC CTG AGA 1695Glu Ile Thr Lys Lys Met Gly Ser Ser Ser Met Lys Arg Ser Leu Arg500 505 510 515ACC AAC TTC CTG CGC TAC TGG ACC CTC ACC TAT CTG GCG CTG CCC ACA 1743Thr Asn Phe Leu Arg Tyr Trp Thr Leu Thr Tyr Leu Ala Leu Pro Thr
520 525 530GTG ACC GAC CAG GAG GCC ACC CCT GTG CCC CCC ACA GGG GAC TCC GAG 1791Val Thr Asp Gln Glu Ala Thr Pro Val Pro Pro Thr Gly Asp Ser Glu
535 540 545GCC ACT CCC GTG CCC CCC ACG GGT GAC TCC GAG ACC GCC CCC GTG CCG 1839Ala Thr Pro Val Pro Pro Thr Gly Asp Ser Glu Thr Ala Pro Val Pro
550 555 560CCC ACG GGT GAC TCC GGG GCC CCC CCC GTG CCG CCC ACG GGT GAC TCC 1887Pro Thr Gly Asp Ser Gly Ala Pro Pro Val Pro Pro Thr Gly Asp Ser
565 570 575GGG GCC CCC CCC GTG CCG CCC ACG GGT GAC TCC GGG GCC CCC CCC GTG 1935Gly Ala Pro Pro Val Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val580 585 590 595CCG CCC ACG GGT GAC TCC GGG GCC CCC CCC GTG CCG CCC ACG GGT GAC 1983Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val Pro Pro Thr Gly Asp
600 605 610TCC GGG GCC CCC CCC GTG CCG CCC ACG GGT GAC TCC GGG GCC CCC CCT 2031Ser Gly Ala Pro Pro Val Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro
615 620 625GTG CCC CCC ACA GAT GAC TCC AAG GAA GCT CAG ATG CCT GCA GTC ATT 2079Val Pro Pro Thr Asp Asp Ser Lys Glu Ala Gln Met Pro Ala Val Ile
630 635 640AGG TTT TAGCGTCCCA TGAGCCTTGG TATCAAGAGG CCACAAGAGT GGGACCCCAG 2135Arg Phe
645GGGCTCCCCT CCCATCTTGA GCTCTTCCTG AATAAAGCCT CATACCCCT 2184(2)SEQ ID NO:9的资料:
(i)序列特征:
(A)长度:668个氨基酸
(B)类型:氨基酸
(D)拓扑结构:线性
(ii)分子类型:蛋白质
(xi )序列描述:SEQ ID NO:9:Met Leu Thr Met Gly Arg Leu Gln Leu Val Val Leu Gly Leu Thr Cys-23 -20 -15 -10Cys Trp Ala Val Ala Ser Ala Ala Lys Leu Gly Ala Val Tyr Thr Glu
-5 1 5Gly Gly Phe Val Glu Gly Val Asn Lys Lys Leu Gly Leu Leu Gly Asp10 15 20 25Ser Val Asp Ile Phe Lys Gly Ile Pro Phe Ala Ala Pro Thr Lys Ala
30 35 40Leu Glu Asn Pro Gln Pro His Pro Gly Trp Gln Gly Thr Leu Lys Ala
45 50 55Lys Asn Phe Lys Lys Arg Cys Leu Gln Ala Thr Ile Thr Gln Asp Ser
60 65 70Thr Tyr Gly Asp Glu Asp Cys Leu Tyr Leu Asn Ile Trp Val Pro Gln
75 80 85Gly Arg Lys Gln Val Ser Arg Asp Leu Pro Val Met Ile Trp Ile Tyr90 95 100 105Gly Gly Ala Phe Leu Met Gly Ser Gly His Gly Ala Asn Phe Leu Asn
110 115 120Asn Tyr Leu Tyr Asp Gly Glu Glu Ile Ala Thr Arg Gly Asn Val Ile
125 130 135Val Val Thr Phe Asn Tyr Arg Val Gly Pro Leu Gly Phe Leu Ser Thr
140 145 150Gly Asp Ala Asn Leu Pro Gly Asn Tyr Gly Leu Arg Asp Gln His Met
155 160 165Ala Ile Ala Trp Val Lys Arg Asn Ile Ala Ala Phe Gly Gly Asp Pro170 175 l80 185Asn Asn Ile Thr Leu Phe Gly Glu Ser Ala Gly Gly Ala Ser Val Ser
190 195 200Leu Gln Thr Leu Ser Pro Tyr Asn Lys Gly Leu Ile Arg Arg Ala Ile
205 210 215Ser Gln Ser Gly Val Ala Leu Ser Pro Trp Val Ile Gln Lys Asn Pro
220 225 230Leu Phe Trp Ala Lys Lys Val Ala Glu Lys Val Gly Cys Pro Val Gly
235 240 245Asp Ala Ala Arg Met Ala Gln Cys Leu Lys Val Thr Asp Pro Arg Ala250 255 260 265Leu Thr Leu Ala Tyr Lys Val Pro Leu Ala Gly Leu Glu Tyr Pro Met
270 275 280Leu His Tyr Val Gly Phe Val Pro Val Ile Asp Gly Asp Phe Ile Pro
285 290 295Ala Asp Pro Ile Asn Leu Tyr Ala Asn Ala Ala Asp Ile Asp Tyr Ile
300 305 310Ala Gly Thr Asn Asn Met Asp Gly His Ile Phe Ala Ser Ile Asp Met
315 320 325Pro Ala Ile Asn Lys Gly Asn Lys Lys Val Thr Glu Glu Asp Phe Tyr330 335 340 345Lys Leu Val Ser Glu Phe Thr Ile Thr Lys Gly Leu Arg Gly Ala Lys
350 355 360Thr Thr Phe Asp Val Tyr Thr Glu Ser Trp Ala Gln Asp Pro Ser Gln
365 370 375Glu Asn Lys Lys Lys Thr Val Val Asp Phe Glu Thr Asp Val Leu Phe
380 385 390Leu Val Pro Thr Glu Ile Ala Leu Ala Gln His Arg Ala Asn Ala Lys
395 400 405Ser Ala Lys Thr Tyr Ala Tyr Leu Phe Ser His Pro Ser Arg Met Pro410 415 420 425Val Tyr Pro Lys Trp Val Gly Ala Asp His Ala Asp Asp Ile Gln Tyr
430 435 440Val Phe Gly Lys Pro Phe Ala Thr Pro Thr Gly Tyr Arg Pro Gln Asp
445 450 455Arg Thr Val Ser Lys Ala Met Ile Ala Tyr Trp Thr Asn Phe Ala Lys
460 465 470Thr Gly Asp Pro Asn Met Gly Asp Ser Ala Val Pro Thr His Trp Glu
475 480 485Pro Tyr Thr Thr Glu Asn Ser Gly Tyr Leu Glu Ile Thr Lys Lys Met490 495 500 505Gly Ser Ser Ser Met Lys Arg Ser Leu Arg Thr Asn Phe Leu Arg Tyr
510 515 520Trp Thr Leu Thr Tyr Leu Ala Leu Pro Thr Val Thr Asp Gln Glu Ala
525 530 535Thr Pro Val Pro Pro Thr Gly Asp Ser Glu Ala Thr Pro Val Pro Pro
540 545 550Thr Gly Asp Ser Glu Thr Ala Pro Val Pro Pro Thr Gly Asp Ser Gly
555 560 565Ala Pro Pro Val Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val Pro570 575 580 585Pro Thr Gly Asp Ser Gly Ala Pro Pro Val Pro Pro Thr Gly Asp Ser
590 595 600Gly Ala Pro Pro Val Pro Pro Thr Gly Asp Ser G1y Ala Pro Pro Val
605 610 615Pro Pro Thr Gly Asp Ser Gly Ala Pro Pro Val Pro Pro Thr Asp Asp
620 625 630Ser Lys Glu Ala Gln Met Pro Ala Val Ile Arg Phe
635 640 645
Claims (17)
1.产生能表达BSSL变异体的转基因非人类哺乳动物的方法,该方法包括(a)将一种表达系统引入非人类哺乳动物受精卵或胚胎细胞,以便将表达系统掺入哺乳动物种系,(b)将所得的被引入的受精卵或胚胎发育成成年雌性非人类哺乳动物;其中所述表达系统包含能在宿主细胞或有机体中表达的杂种基因,以致于当杂种基因表达时生产重组多肽,所说杂种基因是通过将一种核酸序列插入能调节所述杂种基因表达的基因中而产生的;所述核酸序列是一种编码不到722个氨基酸的多肽的核酸分子,所述多肽是一种BSSL变异体,所说BSSL变异体含SEQ ID NO:3所示第536-722个残基的部分氨基酸序列。
2.根据权利要求1的方法,其中所说BSSL变异体在其C-末端有一个苯丙氨酸残基。
3.根据权利要求1或2的方法,其中所说BSSL变异体在其C-末端部分含有序列Gln-Met-Pro。
4.根据权利要求1-3的任一权利要求的方法,其中所说BSSL变异体在其C-末端部分含SEQ ID NO:3中残基第712-722所示的氨基酸序列。
5.根据权利要求1-4的任一权利要求的方法,其中所述BSSL变异体含不到16个重复单位。
6.根据权利要求1的方法,其中所述核酸序列编码的多肽的氨基酸序列与序列表SEQ ID NO:5、6或9所示的氨基酸序列至少90%同源。
7.根据权利要求6的方法,其中所述核酸序列编码的多肽含序列表SEQ ID NO:5、6或9所示的氨基酸序列。
8.根据权利要求1的方法,其中所述核酸序列编码的多肽的氨基酸序列与序列表SEQ ID NO:7所示的氨基酸序列至少90%同源,但编码在187位置为天冬酰胺的多肽的那些核酸分子除外。
9.根据权利要求8的方法,其中所述核酸序列编码的多肽含序列表SEQ ID NO:7所示的氨基酸序列。
10.生产能表达BSSL变异体并实质上不能由哺乳动物本身表达BSSL的转基因非人类哺乳动物的方法,包括(a)损坏哺乳动物的BSSL表达能力,以致于实质上没有哺乳动物BSSL表达,然后将权利要求1中描述的表达系统插入哺乳动物种系,这样在哺乳动物中表达BSSL变异体;和/或(b)用权利要求1中描述的表达系统替代哺乳动物BSSL基因或其部分基因。
11.在其基因组中含权利要求1-9任一权利要求中描述的DNA序列的转基因非人类哺乳动物。
12.根据权利要求11的转基因非人类哺乳动物,其中所述DNA序列存在于哺乳动物种系中。
13.根据权利要求11或12的转基因非人类哺乳动物,其中所述DNA序列存在于哺乳动物乳蛋白基因中。
14.根据权利要求11-13的任一权利要求的转基因非人类哺乳动物,选自小鼠、大鼠、兔、绵羊、猪和牛。
15.根据权利要求11-14的任一权利要求的转基因非人类哺乳动物的后代。
16.从根据权利要求11-15的任一权利要求的转基因非人类哺乳动物获取的乳。
17.含有根据权利要求16的乳的婴儿膳食配方。
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SE9300686A SE9300686D0 (sv) | 1993-03-01 | 1993-03-01 | Novel polypeptides |
SE93006864 | 1993-03-01 | ||
SE9300722A SE9300722D0 (sv) | 1993-03-04 | 1993-03-04 | Novel polypeptides ii |
SE93007227 | 1993-03-04 |
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CNB001377388A Expired - Lifetime CN1187450C (zh) | 1993-03-01 | 1994-02-25 | 生产转基因非人类哺乳动物的方法 |
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Country Status (28)
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US (2) | US5827683A (zh) |
EP (1) | EP0687296B1 (zh) |
JP (1) | JP3837444B2 (zh) |
KR (2) | KR100312825B1 (zh) |
CN (2) | CN1071790C (zh) |
AT (1) | ATE317429T1 (zh) |
AU (1) | AU675701B2 (zh) |
BR (1) | BR9406376A (zh) |
CA (1) | CA2156083C (zh) |
CZ (1) | CZ290927B6 (zh) |
DE (1) | DE69434622T2 (zh) |
DK (1) | DK0687296T3 (zh) |
EE (1) | EE9400458A (zh) |
ES (1) | ES2258262T3 (zh) |
FI (1) | FI954082A (zh) |
HU (1) | HU221119B1 (zh) |
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IS (1) | IS4130A (zh) |
NO (1) | NO953426D0 (zh) |
NZ (1) | NZ262529A (zh) |
PL (1) | PL184960B1 (zh) |
PT (1) | PT687296E (zh) |
RU (1) | RU2219239C2 (zh) |
SG (1) | SG52597A1 (zh) |
SK (1) | SK285420B6 (zh) |
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN100343391C (zh) * | 2004-12-31 | 2007-10-17 | 中国农业大学 | 卵巢注射法制备转基因动物 |
CN103088000A (zh) * | 2012-03-23 | 2013-05-08 | 北京济福霖生物技术有限公司 | 在哺乳动物乳腺中过表达胆盐激活脂酶的方法 |
Families Citing this family (16)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0795011A1 (en) * | 1994-12-01 | 1997-09-17 | Oklahoma Medical Research Foundation | Method and compositions for reducing cholesterol absorption |
US5696087A (en) * | 1994-12-01 | 1997-12-09 | Oklahoma Medical Research Foundation | Method and compositions for reducing cholesterol absorption |
US5681819A (en) * | 1994-12-01 | 1997-10-28 | Oklahoma Medical Research Foundation | Method and compositions for reducing cholesterol absorption |
US5821226A (en) * | 1994-12-01 | 1998-10-13 | Oklahoma Medical Research Foundation | BAL C-tail drug delivery molecules |
FR2733249B1 (fr) | 1995-04-20 | 1997-06-06 | Biocem | Lipase gastrique de chien recombinante et polypeptides derives produits par les plantes, leurs procedes d'obtention et leurs utilisations |
SE9501939D0 (sv) * | 1995-05-24 | 1995-05-24 | Astra Ab | DNA molecules for expression of polypeptides |
US6342218B1 (en) | 1997-02-14 | 2002-01-29 | Oklahoma Medical Research Foundation | Method for treatment of SLE |
SE9801424D0 (sv) | 1998-04-22 | 1998-04-22 | Astra Ab | Expression methods |
AU2001255469A1 (en) * | 2000-04-21 | 2001-11-07 | Monsanto Technology Llc | Purification of ace inhibiting polypeptides containing vpp from milk |
FR2868424B1 (fr) * | 2004-03-31 | 2008-04-11 | Univ Aix Marseille Ii | Glycopeptides derives de structures pancreatiques, anticorps et leurs applications en diagnostic et therapeutique |
EP1730270B1 (fr) | 2004-03-31 | 2016-03-30 | Université d'Aix-Marseille | Glycopeptides derives de structures pancreatiques, anticorps et leurs applications en diagnostic et therapeutique |
EP2039764A1 (en) * | 2007-09-19 | 2009-03-25 | Pevion Biotech AG | Truncated secretory aspartyl proteinase 2 |
EP2629789A1 (en) | 2010-10-21 | 2013-08-28 | Swedish Orphan Biovitrum AB (Publ) | Method to increase the absorption of unsaturated fatty acids by human infants |
ES2603328T3 (es) | 2010-10-21 | 2017-02-27 | Swedish Orphan Biovitrum Ab (Publ) | Método para incrementar la tasa de crecimiento de lactantes humanos |
FR2966734B1 (fr) | 2010-10-29 | 2014-07-18 | Max Rombi | Composition comprenant au moins une enzyme proteolytique pour son utilisation pour empecher la synthese des triglycerides |
CN103562383A (zh) * | 2011-05-18 | 2014-02-05 | 瑞典孤儿比奥维特鲁姆有限公司 | 低pH蛋白质纯化方法 |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5200183A (en) * | 1987-11-19 | 1993-04-06 | Oklahoma Medical Research Foundation | Recombinant bile salt activated lipases |
US4944944A (en) * | 1987-11-19 | 1990-07-31 | Oklahoma Medical Research Foundation | Dietary compositions and methods using bile salt-activated lipase |
SE9001985D0 (sv) * | 1990-06-01 | 1990-06-01 | Astra Ab | New chemical products |
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1994
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN100343391C (zh) * | 2004-12-31 | 2007-10-17 | 中国农业大学 | 卵巢注射法制备转基因动物 |
CN103088000A (zh) * | 2012-03-23 | 2013-05-08 | 北京济福霖生物技术有限公司 | 在哺乳动物乳腺中过表达胆盐激活脂酶的方法 |
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