CN1370178A - 多核苷酸及其编码的与乙酰肝素酶同源较远的多肽 - Google Patents
多核苷酸及其编码的与乙酰肝素酶同源较远的多肽 Download PDFInfo
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Abstract
本发明涉及新型多核苷酸,它编码与乙酰肝素酶同源较远的多肽;包括该多核苷酸的核酸构建体;其基因修饰的表达细胞;其编码的重组蛋白,该蛋白具有乙酰肝素酶或其它糖苷水解酶活性;识别该重组蛋白的抗体;来自多核苷酸的寡核苷酸和寡核苷酸类似物及包含它的核酶。
Description
发明领域与发明背景
本发明涉及新型多核苷酸,它编码与乙酰肝素酶同源较远的多肽;包括该多核苷酸的核酸构建体;其基因修饰的表达细胞;其编码的重组蛋白,该蛋白具有乙酰肝素酶或其它糖苷水解酶活性;识别该重组蛋白的抗体;来自多核苷酸的寡核苷酸和寡核苷酸类似物及包含它的核酶。
不能认为该申请中所引用的参考文献均为本发明的现有技术。
氨基聚糖(GAGs)
氨基聚糖是由重复二糖单位构成的多聚体,二糖单位由糖醛酸和氨基己糖构成。除透明质酸外,GAGs的生物合成始于核心蛋白。蛋白聚糖可包含几个相同或不同家族的GAG侧链。GAGs作为同聚物被合成,然后可进行N-去乙酰化和N-硫酸化修饰,接着进行葡萄糖醛酸到艾杜糖醛酸的C5差向异构化和O-硫酸化。不同组织来源的GAGs的化学组成差异很大。
动物体内GAGs的自然代谢通过水解进行。通常,GAGs通过两步降解。首先,蛋白聚糖被胞内体内化,在胞内体中GAG链开始解聚。这一步主要是水解产生寡糖。当与溶酶体融合后进行进一步降解,此时发生去硫酸化和外切解聚成单糖(42)。
目前唯一清楚的降解哺乳动物GAG的内切酶为透明质酸酶。透明质酸酶是1-4内切氨基葡萄糖苷酶家族,它解聚透明质酸和硫酸软骨素。编码精子相关的PH-20(Hyal3)及溶酶体透明质酸酶Hyal1和Hyal2的cDNAs已被克隆并公开。这些酶具有40%的同源性,并具有不同的组织特异性、细胞定位和适宜PH。
外切水解酶的特性较为清楚,其中有β-葡萄糖苷酸酶,α-L-艾杜糖苷酸酶,和β-N乙酰氨基葡萄糖苷酶。除多糖链糖苷键的水解外,GAG解聚还包括去硫酸化,它是由N-乙酰氨基半乳糖-4-硫酸酯酶,艾杜糖醛酸-2-硫酸酯酶和肝素磺酰胺酶等几种溶酶体硫酸酯酶催化的。任何一种溶酶体GAG降解酶的缺乏均会导致溶酶体贮积性疾病-粘多糖病。
糖苷水解酶:
糖苷水解酶是普遍存在的一组水解两个或多个糖分子间O-糖苷键或者糖部分与非糖部分间O-糖苷键的酶。糖苷键的酶性水解主要通过应用一个或两个导致异构体构象整体转化或保留的机制而发生。在两种机制中,催化均包含两个残基:一个质子供体和一个核物质。根据氨基酸的相似性将糖苷水解酶分成58个家族。1,2,5,10,17,30,35,39和42家族的糖苷水解酶作用于大量不同的底物,但是,它们均通过温和的酸性催化机制水解糖苷键,保留异构体构象。该机制包含质子供体和核物质两个谷氨酸残基,而天冬酰胺总是位于质子供体之前。对该组糖苷水解酶进行一系列已知3D结构分析的结构表明,尽管它们序列一致性很低,但其催化功能区均应用相似的分别位于β4和β7链C末端由质子供体和核物质构成的(α/β)8折叠体。已经证实在溶酶体贮积性疾病中存在溶酶体糖苷水解酶功能性保守氨基酸的突变。
包括β-葡萄糖苷酸酶、β-甘露糖苷酶、β-葡糖脑苷脂酶、β-半乳糖苷酶和α-L-艾杜糖苷酸酶在内的溶酶体糖苷水解酶均为外切糖苷水解酶,属于GH-A家族,有类似的催化位点。但是,许多不同有机体来源的外切糖苷酶,如细菌和真菌的木聚糖酶和纤维素酶享有该催化功能区(1)。
硫酸乙酰肝素蛋白聚糖(HSPGs)
HSPGs是与大范围的脊椎动物和非脊椎动物组织的细胞表面和细胞外基质(ECM)相关的普遍大分子(3-7)。基本的HSPG结构由蛋白核心构成,几个线性的硫酸乙酰肝素链共价连接到核心蛋白。经典的多糖链由重复的己醛糖和D-氨基葡糖二糖单位构成,它们被不同程度的N-和O-连接的硫酸部分和N-连接的乙酰基所替代(3-7)。ECM分子参与细胞黏附、生长和分化的研究揭示了HSPGs在形态发生、血管生成、转移、轴突生长和组织修复中的核心作用(3-7)。硫酸乙酰肝素(HS)具有独特的与众多蛋白质结合的能力,保证广泛的效应分子黏着到细胞表面(6-8)。HSPGs也是血管的主要成分(5)。在大血管中它们大部分积聚在内膜和内中层,而在毛细血管内它们主要位于内皮下基底膜,支持内皮细胞的增殖和移动,并稳定毛细血管壁的结构。HSPGs与胶原、层黏连蛋白和纤黏连蛋白等ECM大分子之间,及与质膜上不同黏附位点相互作用的能力表明了该蛋白聚糖在自身组装和ECM成分不溶性及细胞黏附和运动等方面的关键作用。因此HS的裂解会导致内皮下ECM的解组装,并因此在正常和恶性的外渗中扮演决定性角色(9-11)。在炎症、损伤修复、糖尿病和肿瘤转移中发现HS降解代谢,这提示降解HS的酶在病理进程中起着重要作用。
乙酰肝素酶
乙酰肝素酶是参与某些氨基聚糖分解代谢的糖基化酶。它是内切葡糖苷酸酶,在特异的链间位点分裂硫酸乙酰肝素(12-15)。T、B淋巴细胞、血小板、粒细胞、巨噬细胞和肥大细胞与内皮下细胞外基质(ECM)之间的相互作用与乙酰肝素酶对硫酸乙酰肝素的降解活性相关(16)。发现结缔组织活化的肽III(CTAP),一种c趋化因子,具有乙酰肝素酶样活性。胎盘乙酰肝素酶作为黏附分子或作为降解性酶取决于微环境中的pH(17)。
乙酰肝素酶是对各种活化信号(如,凝血酶,钙离子载体,免疫复合体,抗原和丝裂原)产生应答从细胞内器室(例如,溶酶体,特异性颗粒)中释放出来的,这表明它参与炎症和细胞免疫应答的调节(16)。
还证明在没有额外刺激的情况下,4℃孵育60分钟,乙酰肝素酶可从人中性粒细胞中容易地释放出来(18)。
明胶酶,另一个ECM降解酶,与乙酰肝素酶一起在人中性粒细胞的三级颗粒中发现的,是在对佛波醇12-肉豆蔻酸13-醋酸盐(PMA)应答中从中性粒细胞释放出的(19-20)。
与此相对,各种肿瘤细胞似乎以组成性方式表达和分泌乙酰肝素酶,这与其转移潜能相关(21)。
乙酰肝素酶对硫酸乙酰肝素的降解导致肝素-结合生长因子、酶和质膜蛋白的释放,它们曾被基底膜、细胞外基质和细胞表面的硫酸乙酰肝素所隐蔽(22-23)。
在一些细胞类型中发现乙酰肝素酶活性,它们包括培养的皮肤成纤维细胞,人中性粒细胞,活化的大鼠T淋巴细胞,正常和赘生的鼠B淋巴细胞,人单核细胞和人脐静脉内皮细胞,SK肝癌细胞,人胎盘和人血小板。
有人报道从SK肝癌细胞和人胎盘(美国专利No.54362641)和人血小板(62)纯化天然乙酰肝素酶的方法。
乙酰肝素酶基因的克隆和表达
用胰蛋白酶消化从人肝癌细胞分离出的纯化的乙酰肝素酶。用高压液相色谱(HPLC)分离肽并进行微量测序。应用其中一肽的序列筛选与相应的后翻译的DNA序列的同源数据库。该步骤鉴定出一包含1020碱基对(bp)插入片段的克隆,该片段包括963bp的开放阅读框,其后有27bp的3’未翻译区和polyA尾。将该新基因命名为hpa.未得到的hpa5’端的克隆通过Marathon RACE由胎盘cDNA混合物进行。连接的hpacDNA(也记作phpa)片段包含一开放阅读框,它编码一543个氨基酸的多肽,其分子量为61192道尔顿(2)。在美国专利申请号08/92217009/109386和09/258892中有对克隆过程的详细描述,后者是专利1998,8,31备案的PCT/US98/17954,的部分延续,在此引入作为参考。
编码乙酰肝素酶的基因组位点大约40kb.它定位于人的4号染色体,由12个外显子构成,被11个内含子分割。
hpa基因产物体外催化硫酸乙酰肝素(HS)降解的活性,通过在High five和Sf21昆虫细胞以及人293胚胎肾细胞表达系统中表达hpa的整个开放阅读框进行了检测。感染或转染细胞的提取物用于分析乙酰肝素酶催化活性。为了达到该目的,细胞裂解物与硫酸酯标记的ECM来源的HSPG(峰1)共同孵育,随后对反应混合物进行凝胶过滤分析(Sepharose 6B)。尽管底物自身由高分子量的物质构成,但HSPG底物与用包含hpa的载体所转染或感染的细胞提取物孵育会导致高分子量的底物完全转化为低分子量的标记的硫酸乙酰肝素降解片段(如,美国专利No.09/071618,在此引入作为参考)。
其它实验证实,用pF hpa病毒感染的细胞所表达的乙酰肝素酶能降解HS与其它存在于自然产生的完整ECM(参照美国专利No.09/071618,在此引入作为参考)中的大分子成分所形成的复合体(例如,纤黏连蛋白,层黏连蛋白,胶原蛋白),其作用方式与报道的高度转移的肿瘤细胞或免疫系统的活化细胞相似(7,8)。
hpa基因在人乳腺癌和肝细胞癌中的优先表达
应用半定量RT-PCR来评估不同转移程度的人乳腺癌细胞系中hpa基因的表达。发现hpa基因表达显著增高与非转移性MCF-7乳腺癌,中度转移的MDA231和高度转移的MDA435乳腺癌细胞系的转移能力相关。重要的是,hpa基因表达的差异情况与乙酰肝素酶的活性情况相关。
通过用石蜡包埋的人乳腺组织切片进行原位杂交证实hpa基因在人乳腺癌中表达。乙酰肝素酶反义核糖探针对侵袭性管道癌组织切片进行杂交的结果显示出对癌细胞的特异性强染色。在出现囔变性纤维瘤的癌旁区域也有hpa基因的表达。来自复位性乳房成形术的正常乳腺组织没有表达hpa基因。在人肝细胞癌标本的组织切片中也看到hpa基因的高表达,而在正常肝组织中没有表达。此外,用hpa RNA探针证实在卵巢腺癌,宫颈鳞状细胞癌和结肠腺癌的组织标本中也存在强染,而在相应的非恶性对照组织中hpa mRNA染色很浅(2)。
用抗乙酰肝素酶的单克隆抗体,通过用石蜡包埋的切片进行免疫组化也证实乙酰肝素酶优先表达于人肿瘤而非相应的正常组织。在结肠癌的癌细胞中发现胞浆内阳性染色,并在同一标本的管状绒毛腺瘤的发育不良的上皮细胞内发现阳性染色,而在远离肿瘤的看似正常的结肠上皮中没有染色或染色极少。尤为重要的是,与周围的正常肝组织相比转移到肝脏的结肠腺癌细胞强染。
乙酰肝素酶蛋白的潜在形式和活化形式
在杆状病毒表达系统中产生的重组酶的分子大小为65kDa。该多肽包含有6个潜在的N-糖基化位点。通过肽N-糖苷酶处理去糖基化以后,该蛋白出现在57kDa的条带处。其分子量与所推导的去除预测的3kDa信号肽序列后hpa全长cDNA编码的543个氨基酸多肽的分子量(61192道尔顿〕相符。同时用O-糖苷酶和神经氨酸酶处理后,未发现该N-去糖基化蛋白分子大小的减少。去糖基化对酶活性无可检测到的影响。
与杆状病毒酶不同,乙酰肝素酶多肽全长在哺乳动物细胞(例如,293肾细胞,CHO)的表达是,在细胞裂解物中产生一50 kDa的主要蛋白,并产生一65 kDa的次要蛋白。在一些转染的CHO克隆中发现其65 kDa形式的蛋白优先释放到培养基中。应用半定量凝胶过滤方法比较两种形式的酶活性,发现50 kDa的酶大约100倍于65 kDa形式的酶。当重组的65 kDa杆状病毒酶与从人血小板、SK-hep-1细胞或胎盘纯化出的50 kDa的乙酰肝素酶进行特异性活性比较时也发现类似的差异。血小板乙酰肝素酶氨基端测序表明在glu157-lys158之间存在裂解。正如乙酰肝素酶的疏水性图所显示的,该位点处于一个亲水峰,易于被暴露,因而易接近蛋白酶。
乙酰肝素酶参与肿瘤细胞侵袭和转移
停止在毛细血管床的循环血流中的肿瘤细胞经常附着在或邻近相邻内皮细胞间的细胞间连接处。紧随转移性细胞附着之后就是连接的破坏,内皮细胞边界的回缩,通过内皮细胞内的裂口向暴露在其下的基底膜(BM)移动(24)。一旦位于内皮细胞和基底膜之间,侵袭的细胞必然降解内皮下糖蛋白和BM的蛋白聚糖,以游出管腔。人们认为有几种细胞酶(例如,胶原酶IV,纤溶酶原激活剂,组织蛋白酶B,弹性蛋白酶,等)参与BM的降解(25)。这些酶中乙酰肝素酶在特异的链内位点裂解HS(16,11)。人们发现降解HS的乙酰肝素酶的表达与小鼠淋巴瘤(26)、纤维肉瘤和黑色素瘤(21)细胞的转移潜能相关。而且,检测到在荷有转移性肿瘤的动物和黑色素瘤患者(21)的血清中,以及癌症患者的肿瘤活检(12)中乙酰肝素酶的水平增高。
检测了各种非抗凝剂种类的肝素对乙酰肝素酶的抑制效果,以观察其对阻止成血细胞外渗的潜在应用。用乙酰肝素酶抑制剂处理实验动物能显著降低(>90%)B16黑色素瘤、Lewis肺癌和乳房的腺癌细胞引发的肺转移的发生率(12,13,28)。与抗凝血酶III有高和低亲和力的肝素因子有相当高的抗转移活性,这提示是肝素的乙酰肝素酶抑制活性而非抗凝活性在多糖的抗转移特性中发挥作用(12)。
乙酰肝素酶在肿瘤转移中的直接作用被两个实验系统所证实。鼠T淋巴瘤细胞系Eb没有可检测到的乙酰肝素酶活性。将hpa基因导入Eb细胞观察其是否能造成这些细胞的转移行为。为了达到该目的,用人的全长hpa cDNA转染Eb细胞。稳定转染的细胞显示出乙酰肝素酶mRNA的高表达和酶活性。将hpa转染的Eb细胞和空转染的mock细胞皮下注射到DAB/2小鼠,检测小鼠的存活期和肝转移。所有(n=20)注射了mock细胞的小鼠在实验的第一个4周全部存活,而接种了hpacDNA转染的Eb细胞的小鼠死亡率达50%。接种了hpa转染细胞的小鼠的肝脏有大量的Eb淋巴瘤细胞浸润,肝脏表面的肉眼观察及组织切片的显微镜检测均能证实。与此相对,接种了空转染的Eb对照细胞的小鼠肝脏肉眼观察不到转移灶。很少或没有淋巴瘤细胞浸润到肝组织。在不同的肿瘤转移模型中,将乙酰肝素酶基因瞬时转染到低转移的B16-F1小鼠黑色素瘤细胞,任何静脉内接种,肺转移增加4-5倍。
最后,与对照相比,外面黏附了乙酰肝素酶的B16-F1小鼠黑色素瘤细胞增加C57BL小鼠的肺转移(参照,美国专利No.09/260037,提名为INTRODUCING A BIOLOGICAL MATERIAL INTO A PATIENT,是美国专利No.09/140888的部分延续,在此引入作为参考)。
乙酰肝素酶可能参与肿瘤的血管生成
成纤维细胞生长因子是一个结构上与特征为与肝素有高亲和力的多肽相关的家族(29)。它们是血管内皮细胞的强丝裂原,是新血管生成的最有力诱导剂(29-30)。碱性成纤维细胞生长因子(bFGF)是从体外制备的内皮下ECM(31)和角膜的基底膜(32)中提取出的,提示ECM可作为bFGF的储存库。免疫组化染色显示bFGF定位于多种组织和血管的基底膜(23)。尽管bFGF在正常组织普遍存在,但内皮细胞在这些组织中的增殖通常非常低,提示bFGF通过某种机制隐藏于其作用部位。bFGF与ECM间相互作用的研究表明bFGF结合ECM中的HSPG,通过HS降解酶以活性形式释放(32,33,34)。证实,血小板、肥大细胞、中性粒细胞和淋巴瘤相比表达的乙酰肝素酶活性参与活性bFGF从ECM和基底膜释放(35),这提示也许乙酰肝素酶活性不仅作用于细胞的迁移和侵袭,而且可诱导间接的新血管反应。这些结果提示ECM HSPG为bFGF和可能的其它肝素-结合生长促进因子提供天然的储存库(36,37〕。取代在基底膜和ECM中的储存的bFGF会提供一在正常和病理性部位诱导新血管生成的新机制。
最近的研究表明肝素和HS参与bFGF结合到高亲和力的细胞表面受体,并参与bFGF细胞信号传导(38,39)。此外,HS始于最佳效应的大小与由乙酰肝素酶释放的HS片段相似(40)。用血管内皮细胞生长因子(VEGF)也得到类似的结果(41),这提示双受体机制的运作使HS参与细胞与肝素-结合生长的相互作用。因此可以设想,ECM中内皮细胞生长因子的限制阻止它们对血管内皮的系统作用,于是保持内皮细胞的更新和血管生长率很低。另一方面,bFGF作为与HS片段的复合体从ECM储存库释放,会诱导局部内皮细胞增殖和损伤愈合、炎症和肿瘤发生等过程中的新血管生成(36,37)。
乙酰肝素酶参与的其它生理过程及其潜在的治疗性应用
除参与肿瘤细胞转移、炎症和自身免疫以外,哺乳动物的乙酰肝素酶可用于调节肝素-结合生长因子的生物利用度;细胞对肝素-结合生长因子(如,bFGF,VEGF)和细胞因子(IL-8)的细胞应答(44,41);细胞与质膜脂蛋白的相互作用(49);细胞对某种病毒、细菌和原虫感染的敏感性(45-47);淀粉样斑块的分解(48)。
病毒感染:已经证实硫酸乙酰肝素在细胞表面的存在是单纯疱疹病毒(45)和登革热(46)病毒结合到细胞表面和进行随后的细胞感染的基本要求。因此,用乙酰肝素酶移去细胞表面的硫酸乙酰肝素可阻止病毒感染。事实上,用细菌的乙酰肝素酶(降解硫酸乙酰肝素)或肝素酶(降解乙酰肝素)处理细胞会降低两个相关动物疱疹病毒对细胞的结合,并能至少部分给予细胞对病毒感染的对抗(45)。有一些研究表明细胞表面的硫酸乙酰肝素还参与HIV感染(47)。
神经变性的疾病:在Genstmann-Straussler综合征,Creutzfeldt-Jak疾病和Scrape的朊病毒蛋白淀粉样斑块中鉴定出硫酸乙酰肝素蛋白聚糖。硫酸乙酰肝素酶可分解这些淀粉样斑块,这些淀粉样斑块在阿尔茨海默病的病变过程中也起着一定作用。
再狭窄和动脉粥样硬化:在动脉粥样硬化和再狭窄的病变过程中基本事件是内皮损伤而造成的动脉平滑肌细胞(SMCs)增殖和富含胆固醇的脂蛋白堆积(50)。除了在SMC增殖中作为肝素-结合生长因子的低亲和力受体外,HS还参与脂蛋白结合、保留和摄取(51)。已证实HSPG和脂蛋白脂酶参与新的代谢途径,该途径可导致富含胆固醇的脂蛋白在细胞和间质大量堆积(49)。预期后一条路径通过促进富含apoB和apoE的脂蛋白(如,LDL,VLDL,乳糜微粒)堆积而高度致动脉硬化,尽管通过细胞胆固醇含量可产生反馈抑制。因此,期望通过乙酰肝素酶移去SMC HS抑制SMC增殖和脂类堆积,由此来停止再狭窄和动脉粥样硬化的进程。
肺的疾病:从文献获得的数据提示GAG降解酶在缩短感染和炎症率的相关应用中降低窦道黏度和呼吸道分泌的潜在作用,如乙酰肝素酶,结缔组织活化肽,肝素酶,透明质酸酶,硫酸酯酶和软骨素酶等,但并不限定于此。CF患者的痰中至少含3%的GAGs,它在其量和黏性特征上起作用。重组的乙酰肝素酶可降低CF患者痰的黏性程度(参照美国专利No.09/046475)。
总而言之,乙酰肝素酶证实在损伤愈合、血管生成、再狭窄、动脉粥样硬化、炎症、神经变性疾病和病毒感染等情况下有用。哺乳动物的乙酰肝素酶可用于中和质膜肝素,作为潜在的鱼精蛋白替代物。抗乙酰肝素酶的抗体可用于免疫检测,和微转移、活检标本中自身免疫损伤和肾衰,质膜样品和体液的诊断。
因此,广泛共识是需要其它具有糖苷水解酶活性的分子,并且它将大大有利,因为这些分子可对某些底物有更高的特异性或比已知的乙酰肝素酶有不同的底物特异性。
发明概述
根据本发明的一个方面,它提供分离的核酸,包括能与SEQ ID NO1、4、6或其部分杂交的多核苷酸,杂交条件是:68℃6×SSC,1%SDS,5×Denharts,10%硫酸葡聚糖,100ug/ml鲑精DNA,32p标记的探针,用3×SSC,0.1%SDS在68℃洗涤。
根据本发明的另一个方面,它提供分离的核酸,包括能与SEQ IDNO 1、4、6或其部分杂交的多核苷酸,杂交条件是:68℃6×SSC,1%SDS,5×Denharts,10%硫酸葡聚糖,100ug/ml鲑精DNA,32p标记的探针,用1×SSC,0.1%SDS在68℃洗涤。
根据本发明的另一个方面,它提供分离的核酸,包括能与SEQ IDNO 1、4、6或其部分杂交的多核苷酸,杂交条件是:68℃6×SSC,1%SDS,5×Denharts,10%硫酸葡聚糖,100ug/ml鲑精DNA,32p标记的探针,用0.1×SSC,0.1%SDS在68℃洗涤。
根据本发明的另一个方面,它提供分离的核酸,包括多核苷酸,当用DNA序列分析软件包的Bestfit程序分析时它至少与SEQ ID NO 1、4、6或其部分有60%的一致性,该软件包由Wisconsin大学的GeneticComputer Group(GCG)开发(间隙产生补偿-50,间隙扩展补偿-3)。
根据本发明的另一个方面,它提供分离的核酸,包括编码多肽的多核苷酸,当用DNA序列分析软件包的Bestfit程序分析时所述多肽至少与SEQ ID NO 3、5、7或其部分有60%的一致性,该软件包由Wisconsin大学的Genetic Computer Group(GCG)开发(间隙产生补偿-50,间隙扩展补偿-3)。
下述本发明优选实施方案的进一步特征是,多核苷酸如SEQ ID NO1、4、6或其部分所示。
根据本发明的另一个方面,它提供重组蛋白,包括由所述多核苷酸编码的多肽。
根据本发明的另一个方面,它提供重组蛋白,包括多肽,当用DNA序列分析软件包的Bestfit程序分析时它至少与SEQ ID NO 3、5、7或其部分有60%的一致性,该软件包由Wisconsin大学的GeneticComputer Group(GCG)开发(间隙产生补偿-50,间隙扩展补偿-3)。
下述本发明优选实施方案的进一步特征是,多肽如SEQ ID NO 3、5、7或其部分所示。
根据本发明的另一个方面,它提供核酸构建体,包括所述分离的核酸。
根据本发明的另一个方面,它提供核酸构建体,包括编码所述重组蛋白的多核苷酸。
根据本发明的另一个方面,它提供宿主细胞,包括所述多核苷酸或构建体,和/或表达所述的重组蛋白。
根据本发明的另一个方面,它提供反义寡核苷酸或核酸构建体,构建体包括多核苷酸或多核苷酸类似物,在生理条件下,它至少10个碱基能与下列序列体内杂交:(i)多核苷酸链的一部分,当用DNA序列分析软件包的Bestfit程序分析时,该多核苷酸编码的多肽至少与SEQ IDNO 3、5、7或其部分有60%的一致性,该软件包由Wisconsin大学的Genetic Computer Group(GCG)开发(间隙产生补偿-50,间隙扩展补偿-3);(ii)多核苷酸链的一部分,当用DNA序列分析软件包的Bestfit程序分析时,它至少与SEQ ID NO 1、4、6或其部分有60%的一致性,该软件包由Wisconsin大学的Genetic Computer Group(GCG)开发(间隙产生补偿-50,间隙扩展补偿-3)。
根据本发明的另一个方面,它提供包括所述反义寡核苷酸的核酶和核酶序列。
本发明提供多核苷酸和多肽,它们属于GH-A家族的asp-glu糖苷水解酶类,基于与乙酰肝素酶同源,可能是GAG降解酶。
附图简述
这里仅通过实施例参照附图对本发明进行描述,其中,
图1显示了核苷酸序列(SEQ ID NO 1-2)及推导的hnhp1的氨基酸序列(SEQ ID NO 2-3)。
图2是比较推导出的hnhp1的氨基酸序列(SEQ ID NO 2-3)和乙酰肝素酶的氨基酸序列(SEQ ID NO9)。应用GCG软件包的Gap程序进行比较(间隙产生补偿-50,间隙扩展补偿-3)。
图3阐释了hnhp1转录的多变性。HnhpI的扩增是从胎盘和睾丸marathon ready cDNA文库,用基因特异的引物pn9-312u(SEQ ID NO:14)和hnI1-230 (SEQ ID NO:11)进行的。
图4是一动物斑点图。用EcoRI酶切来自不同种类的基因组DNA10μg,用0.7%琼脂糖-TBE凝胶分离。电泳后,用HCl处理胶,然后用NaOH处理,用0.4N NaOH将DNA片段向下转移到尼龙膜(Hybond N+,Amersham)上。将膜与包含hnhpI cDNA(pn9)的1.7KbDNA探针杂交。泳道顺序为:H-人;M-小鼠;Rt-大鼠;P-猪;Cw-牛; Hr-马;S-羊;Rb-兔; D-狗; Ch-鸡; F-鱼。分子大小标志物(λBste II)在左边。
图5阐述了hpa和hnhpI之间的交叉杂交。Hpa是通过PCR从marathon ready胎盘cDNA文库中扩增的。HnhpI是从睾丸marathonready cDNA文库扩增的。PCR产物双份跑琼脂糖胶,并转移到尼龙膜上。一张膜用32p标记的hpa cDNA探针,另一张用hnhpI,克隆pn9。
图6是乙酰肝素酶和hnhpI的疏水性比较图。曲线是根据Kyte和Dulittle方法计算出的,每窗口显示17个氨基酸。
图7是用Westem blot分析在人胚胎肾293细胞中表达的重组hnhpI。A是对照乙酰肝素酶-FLAG前体;B-D为用对照pSI载体(B)、pSI-pn6(C)和pSI-pn9(D)转染的293细胞。通过SDS-PAGE分离细胞裂解物,转移到Immobilon-P尼龙膜(Millipore)上。膜与抗FLAG Flag抗体1∶1000(Kodak anti Flag M2 cat:IB131025)孵育。
优选实施方案
本发明涉及新型多核苷酸,它编码与乙酰肝素酶同源较远的多肽;包括该多核苷酸的核酸构建体;其基因修饰的表达细胞;其编码的具有乙酰肝素酶或其它糖苷水解酶活性的重组蛋白;识别重组蛋白的抗体;来自该多核苷酸的寡核苷酸和寡核苷酸类似物以及包含它们的核酶。
参照附图和相应的描述会更好的理解本发明的原则和操作。
在详细解释本发明的至少一个实施方案之前,应该知道本发明的应用并不限定于如下描述或附图中阐述的构建和安排。本发明可有其它的实施方案或以不同的方式执行或实施。同样,应该知道这里应用措辞和术语的目的是为了便于描述,而非限定于此。
为了使本发明可实施,用人乙酰肝素酶的整个氨基酸序列(SEQ IDNO:9)在人EST库中进行了同源序列的筛选。筛选出一同源较远的片段,登录号为A1222323,IMAGE克隆号为1843155,来自睾丸B细胞和胎儿肺制备的Soares NFL T GBC SI Homo Sapiens cDNA文库。该克隆包含一560bp(SEQ ID NO:23)的插入片段,该片段的3’端与编码人乙酰肝素酶的hpa基因同源。用来自新鉴定出的克隆的引物分离出几个包括几个开放阅读框的cDNAs,这反映了在开放阅读框中的可变剪接,其中最长的是pn6,显示于图1中(SEQ ID NO:1,2,3),它有2060个核苷酸,包含一个1776个核苷酸的开放阅读框,编码592个氨基酸的多肽,其分子量为66.5kDa。将新克隆的基因命名为hnhpI。SEQ ID NO:4,6和SEQ ID NO:5,7中分别列出了较短形式的pn9和pn5及其推导出的氨基酸序列,在后述的实施例部分进行进一步描述。hnhpI和乙酰肝素酶之间氨基酸序列的比较见图3。两个蛋白质之间同源52.8%或55.3%这取决于所应用的软件。即便在很温和的洗涤条件下,hpa和hnhpI之间也未见到交叉杂交(图5)。动物图谱分析证明hnhpI和其它相关基因或许组成一新的基因家族,存在于包括哺乳动物和禽类在内的其它有机体的基因组中。用G3放射性杂交确定hnhpI的染色体定位,它定位于人10号染色体,紧邻标志物SHGC-57721。该结果也提示该基因可能存在第二个拷贝或相关基因。hnhpI在淋巴结、脾脏、结肠和卵巢低表达;在前列腺和小肠稍高表达;在睾丸中显著高表达。用同样的方法在骨髓、肝脏、胸腺、扁桃体或白细胞中未检测到表达。用乙酰肝素酶及hnhpI的氨基酸序列从小鼠的EST库中查到一小鼠EST克隆(克隆1378452,登录号AI019269,来自小鼠胸腺,SEQID NO:8)。但是,该克隆包括两个读码框的移码突变,这阻碍了其开放阅读框。
乙酰肝素酶和hnhpI氨基酸序列间的整体同源提示这两个蛋白质享有类似的功能。两个蛋白质之间的同源性集中在几个区域。它们也许代表蛋白质的功能区。变异性提示在底物识别、细胞定位和活性参数上的潜在差异。
尽管在乙酰肝素酶和其它糖苷水解酶之间缺乏整体的同源性,但在乙酰肝素酶的224和225位置发现氨基酸对asp-glu(NE,SEQ ID NO:13),这是GH-A家族的糖苷水解酶质子供体的特征。在其它家族成员中,该NE对位于β链末端。如2所示,在预测的hnhpI氨基酸序列中围绕NE对的区域是保守的。这提示hnhpI产物是一糖苷水解酶。这一定义包括任何多糖降解酶,外切或内切糖苷酶,根据其与乙酰肝素酶的相似性它好象编码一GAG降解酶。
此外,乙酰肝素酶和hnhpI疏水性图谱的重叠表明蛋白内的交叉。糖苷水解酶的氨基酸序列特征是位于亲水性峰内,在直线蛋白内位于同一位置。在疏水性图谱中的显著差异是在乙酰肝素酶的157,158位氨基酸附近,这构成了酶的加工位点。在乙酰肝素酶中,该位点位于亲水性峰的头上,而hnhpI的相同区域是相当不亲水的。在乙酰肝素酶110氨基酸附近出现的峰也在hnhpI的130氨基酸附近出现。在该区域切开乙酰肝素酶产生酶活性。hnhpI的相同区域也许是可能的处理位点。
67kDa的乙酰肝素酶的N末端有一潜在的信号肽。这两种蛋白质在N末端的同源性低,并且在hnhpI多肽内未找到信号肽。
根据本发明的一个方面,它提供分离的核酸,包括能与SEQ ID NO1、4、6或其部分杂交的多核苷酸,杂交条件是:68℃ 6×SSC,1%SDS,5×Denharts,10%硫酸葡聚糖,100ug/ml鲑精DNA,32p标记的探针,用3×SSC,1×SSC,或0.1×SSC和0.1%SDS在68℃洗涤。
这里及以后的权利要求部分中应用的“部分”是指氨基酸或核酸的顺向伸展。这样的部分包括,例如,至少90个核苷酸(等同于至少30个氨基酸),至少120个核苷酸(等同于至少40个氨基酸),至少150个核苷酸(等同于至少50个氨基酸),至少180个核苷酸(等同于至少60个氨基酸),至少210个核苷酸(等同于至少70个氨基酸),至少300个核苷酸(等同于至少100个氨基酸),至少600个核苷酸(等同于至少200个氨基酸),至少900个核苷酸(等同于至少300个氨基酸),至少1200个核苷酸(等同于至少400个氨基酸),至少1500个核苷酸(等同于至少500个氨基酸),或更多。
根据本发明的另一个方面,它提供分离的核酸,包括多核苷酸,当用DNA序列分析软件包的Bestfit程序分析时它至少与SEQ ID NO 1、4、6或其部分有60%的一致性,该软件包由Wisconsin大学的GeneticComputer Group(GCG)开发(间隙产生补偿-50,间隙扩展补偿-3),优选至少65%的一致性,更优选至少70%,还优选至少75%,还优选至少80%,更优选至少85%,更优选至少90%,最优选至少95%-100%。
根据本发明的另一个方面,它提供分离的核酸,包括编码多肽的多核苷酸,当用DNA序列分析软件包的Bestfit程序分析时该多肽至少与SEQ ID NO 3、5、7或其部分有60%的一致性,该软件包由Wisconsin大学的Genetic Computer Group(GCG)开发(间隙产生补偿-50,间隙扩展补偿-3),优选至少65%的一致性,更优选至少70%,还优选至少75%,还优选至少80%,更优选至少85%,更优选至少90%,最优选至少95%-100%。
这里及以后的权利要求部分中应用的“同源”是指一致+相似。
根据本发明的另一个方面,它提供重组蛋白,包括由所述多核苷酸编码的多肽。
根据本发明,核苷酸可以是互补的多核苷酸序列,基因组多核苷酸序列或混合的多核苷酸序列。
这里所用的“互补的多核苷酸序列”包括最初用逆转录酶或任何其它的RNA依赖性DNA聚合酶,从信使RNA逆转录而来的序列。这些序列可用DNA依赖性DNA聚合酶体内或体外扩增。
这里所用的“基因组多核苷酸序列”包括最初源自染色体并反映染色体的邻近部分的序列。
这里所用的“混合多核苷酸序列”包括至少部分互补的序列和至少部分基因组序列。混合序列可包括一些编码多肽所需要的外显子序列,以及一些位于它们之间的内含子序列。内含子序列可以是任何来源,包括其它基因,经典的会包括保守的剪接信号序列。这些内含子还可包括调节表达的顺式元件。
于是,本发明的这一方面包含(1)SEQ ID NO:1,4,6所示的多核苷酸;(2)其片段或部分;(3)与其杂交的序列;(4)其同源序列;(5)其相应的基因组序列和混合序列;(6)用不同密码子编码相似多肽的序列;(7)通过突变而产生的变异序列,如通过一个或多个核苷酸的缺失、插入或替代而产生突变,可自然方式或人为诱导,可以随机或打靶方式。
根据本发明的另一个方面,它提供重组蛋白质,当用DNA序列分析软件包的Bestfit程序分析时,其包括的多肽至少与SEQ ID NO 3、5、7或其部分有60%的同源性,该软件包由Wisconsin大学的GeneticComputer Group(GCG)开发(间隙产生补偿-50,间隙扩展补偿-3),优选至少65%,更优选至少70%,还优选至少75%,还优选至少80%,更优选至少85%,更优选至少90%,最优选至少95%-100%。
根据本发明的另一个方面,它提供核酸构建体,包括这里所描述的分离的核酸。
根据本发明的优选实施方案,该核酸构建体还包括正义或反义方向调节分离的核酸表达的启动子。已知这样的启动子有转录需要的顺式作用元件,它们结合DNA依赖性RNA聚合酶,转录其下游的序列。其下游序列或处于其可能的两个方向中的一种,以导致通过核酶机制可翻译的正义RNA转录,或通常不包含可翻译序列的反义RNA转录;或与内源性序列或mRNA或染色体DNA形成双螺旋或三螺旋阻抑基因表达,以下进行详述。
尽管所述分离的核酸是本发明的基本元素,它可调整并能用于不同背景。第二重要的是用于与本发明连接的启动子的选择,它将包括任何适宜的启动子。但是,该领域的技术人员会赞同有必要确保转录起始位点位于开放阅读框的上游。在本发明的一优选实施方案中,所选启动子包括在特殊目标宿主细胞中活化的元件。可从转录调节因子中选择这些元件,在应激或饥饿的情况下,转录调节子激活基因的转录对于这些细胞的存活是必要的,如热休克蛋白,但并不限定于此。
本发明的构建体优选还包括适当的可选择的标志物。在本发明的更优选实施方案中,构建体还包括复制的起点。在本发明的另一最优选实施方案中,构建体是一穿梭载体,它既可在大肠杆菌(其中,构建体包括一适当的可选择的标志物和复制的起点)中增殖,又与细胞内的增殖相容,或整合到所选有机体的基因组中。依照本发明的这一方面,构建体可以是,例如质粒、菌粒、噬菌粒、黏粒、噬菌体、病毒或人工染色体。
另外,依照本发明的这一方面,核酸构建体还包括阳性和阴性选择标志,由此可用于选择同源重组事件,包括在knock-in和knock-out过程中用于的同源重组,但并不限定于此。该领域的技术人员可容易地设计包括阳性和阴性选择基因的knock-in或knock-out构建体,以用该构建体有效筛选经受同源重组事件的转染的胚胎干细胞。可将这些细胞导入发育的胚胎以产生嵌合体,检测其后代是否携有knock-in或knock-out构建体。按照本发明,knock-in和/或knock-out构建体还可用于研究新基因的功能。这些构建体还可用于体细胞和/或生殖细胞的基因治疗,以破坏缺陷者的活性,获得功能性等位基因或替代有机体内沉默等位基因活性的缺失,然后按要求来下调或上调其活性。与构建体相关的详细内容以及knock-in和knock-out构建体的应用可在下列文献中找到:Fukushige,S.Ikeda,J.E.:Trapping of mammalian promoters byCre-Iox site-specific recombination.DNA Res 3(1996)73-80;Bedell,M.A.,Jenkins,N.A.,Copeland,N.G.:Mouse models of human disease.Part I:Techniques and resources for genetic analysis in mice.Genes andDevelopment 11(1997)1-11;Bermingham J.J.,Scherer,S.S.,O’Connel,S.,Arroyo,E.,Kalla,K.A.,Powell,F.L,Rosenfeld M.G.:Tst-1/Oct-6/SCIPregulates a unique step in peripheral myelination and is required for normalrespiration.Genes Dev 10(1996)1751-62。引用这些文献作为参考。
按照本发明的另一方面,它提供宿主细胞或动物,它们包括所述核酸构建体或其一部分。该领域的技术人员熟知用核酸构建体转化宿主细胞的方法,原核和真核和有机体,以及筛选转化体(例如,转化的细胞或转基因动物)的方法。此外,一旦被转染,这些细胞和有机体可指导重组蛋白的足量生产,然后用已知的方法对蛋白进行纯化,包括各种色谱和凝胶电泳的方法,但并不限定于此。纯化的重组蛋白可象以后详述的那样用于诱导抗体。转化细胞和有机体的方法在文献参考43中有详细论述,52中详细论述了纯化重组蛋白的方法,在此引入作为参考。
按照本发明的另一方面,它提供寡核苷酸,它至少有17,至少18,至少19,至少20,至少22,至少25,至少30或至少40个碱基与所述分离的核酸特异性杂交。
较短核苷酸(200bp以下,如17-40bp)的杂交受严紧、中度或轻度杂交的影响,其中严紧杂交受到杂交溶液的影响:6×SSC和1%SDS或3M TMACI,0.01M磷酸钠(pH6.8)1mMEDTA(pH7.6),0.5%SDS,100ug/ml变性鲑精DNA,0.1%脱脂奶粉,杂交温度低于Tm 1-1.5℃,最后的洗涤溶液为3M TMACI,0.01M磷酸钠(pH6.8)1mMEDTA(pH7.6),0.5%SDS,洗涤温度低于Tm 1-1.5℃;中度杂交受到杂交溶液的影响:6×SSC和1%SDS或3M TMACI,0.01M磷酸钠(pH6.8)1mMEDTA(pH7.6),0.5%SDS,100ug/ml变性鲑精DNA,0.1%脱脂奶粉,杂交温度低于Tm 2-2.5℃,最后的洗涤溶液为3MTMACI,0.01M磷酸钠(pH6.8)1mMEDTA(pH7.6),0.5%SDS,洗涤温度低于Tm 1-1.5℃,最后的洗涤溶液为6×SSC,最后在22℃洗涤;轻度杂交受到杂交溶液的影响:6×SSC和1%SDS或3M TMACI,0.01M磷酸钠(pH6.8)1mMEDTA(pH7.6),0.5%SDS,100ug/ml变性鲑精DNA,0.1%脱脂奶粉,杂交温度37℃,最后的洗涤溶液为6×SSC,最后在22℃洗涤。
按照本发明的另一方面,它提供寡核苷酸对,每一寡核苷酸至少各有17,至少18,至少19,至少20,至少22,至少25,至少30或至少40个碱基以相反方向与所述分离的核酸特异性杂交,这样在核苷酸扩增反应,如聚合酶链式反应中,可以指导其部分以指数扩增。该领域的技术人员熟知聚合酶链式反应和其它核苷酸扩增反应,这里无需再描述。优选该寡核苷酸对的解链温度(Tm)相容,例如解链温度相差不到7℃,优选小于5℃,更优选小于4℃,最优选小于3℃,理想的是在3-0℃之间。按照本发明的另一方面,它提供用所述引物对而获得的寡核苷酸扩增产物。可用凝胶电泳或任何其它基于大小的分离技术来分离该核苷酸扩增产物。另外,可通过亲和分离方法来分离该核苷酸扩增产物,或用标准亲和或用序列亲和。此外,一旦分离,该产物还可通过显著性酶切、连接等进行基因操作,以作为与活性的上和/或下调相关的许多应用中的任何一种。
按照本发明的另一方面,它提供反义寡核苷酸,包含在生理条件下至少有10,优选10-15,更优选50-20,最优选至少17,至少18,至少19,至少20,至少22,至少25,至少30或至少40个碱基体内与下列核苷酸杂交的多核苷酸或多核苷酸类似物,(1)多核苷酸链的一部分,当用DNA序列分析软件包的Bestfit程序分析时,该多核苷酸编码的多肽至少与SEQ ID NO 3、5、7或其部分有60%的同源性,优选至少65%,更优选至少70%,还优选至少75%,还优选至少80%,更优选至少85%,更优选至少90%,最优选至少95%-100%。该软件包由Wisconsin大学的Genetic Computer Group(GCG)开发(间隙产生补偿-50,间隙扩展补偿-3);或〔2〕多核苷酸链的一部分,当用DNA序列分析软件包的Bestfit程序分析时,它至少与SEQ ID NO 1、4、6或其部分有60%的一致性,优选至少65%,更优选至少70%,还优选至少75%,还优选至少80%,更优选至少85%,更优选至少90%,最优选至少95%-100%。该软件包由Wisconsin大学的Genetic ComputerGroup(GCG)开发(间隙产生补偿-12,间隙扩展补偿-4)。
如以后进一步阐述的,该反义寡核苷酸可用于下调基因的表达。可应用固相寡核苷酸合成很容易地合成该反义寡核苷酸。
化学合成的寡核苷酸和其类似物的能力是它含有预先确定好的序列,提供下调基因表达的手段。可以考虑三类基因表达调控的策略。
在转录水平,通过链替代或通过三股螺旋的形成而结合到基因组DNA的反义或正义寡核苷酸或其类似物可抑制转录。在转录本水平,结合了目标mRNA分子的反义寡核苷酸或其类似物通过细胞内RnaseH造成杂交体的酶解。在这种情况下,通过与目标mRNA杂交,反义寡核苷酸或其类似物提供的双杂交体被Rnase H酶识别并破坏。杂交体的形成会干扰正常的剪接。结果,在这两种情况下,等待翻译的目标mRNA完整转录本的数目减少或消失。在翻译水平,结合了目标mRNA分子的反义寡核苷酸或其类似物,通过立体阻碍,抑制必要的转录因子(核糖体)结合到目标mRNA,使该mRNA无能翻译,这是该领域知道的一个现象-杂交阻抑。
因此,反义序列,如上所述可阻抑任何内源性和/或外源性基因的表达,这依赖于它们的特殊序列,这引起致力于将反义路径开发成一新的药理学工具的科学家和药理学家的高度注意。
例如,已知有几种反义寡核苷酸阻抑造血细胞增殖、生长,进入细胞循环的S期,生存期减少,抑制受体介导的应答。
为了使反义寡核苷酸或其类似物体内更有效的抑制基因表达,反义寡核苷酸或其类似物必须满足以下要求:(1)结合靶序列的特异性足够;(2)溶于水;(3)对胞内和胞外的核酸酶稳定;(4)能穿过细胞膜;(5)当用于治疗有机体时,毒性低。
未经过修饰的寡核苷酸通常不适于用作反义序列,因为它们体内的半衰期短,其间,它们很快被核酸酶降解。而且,难于制备到毫克级以上。此外,这样的寡核苷酸穿透细胞膜的能力很弱。
很明显,为了达到上述要求,需要将寡核苷酸设计成适宜的方式。因此,开始了大量修饰寡核苷酸的研究。
例如,由双链DNA(dsDNA)通过三股螺旋形成来进行识别所引发的问题,通过一聪明的“夹心背”化学连接而解决,由是一条链上一个多嘌呤的序列被识别,通过“夹心背”另一条链上的同嘌呤被识别。通过人工碱基也可获得很好的螺旋形成,由此可提高与离子强度和pH相关的结合条件。
此外,为了提高半衰期和膜的穿透性,对多核苷酸骨架进行大量的改变,然而收效甚微。
可在碱基、糖或磷酸部位对寡核苷酸进行修饰。这些修饰包括,例如应用甲基膦酸盐,单硫磷酸盐,二硫磷酸盐,磷酰胺盐,磷酸酯,桥化的磷酸硫盐,桥化的磷酰胺盐,桥化的甲叉膦酸盐,去磷酸核苷酸间类似物,它有硅氧烷桥、碳酸桥、羧甲基酯桥、乙酰胺桥、氨基甲酸酯桥、硫醚桥、硫氧桥、磺基桥,各种“可塑”DNA,α-异构体桥和甲硼烷衍生物。
国际专利申请WO 89/12060公开了许多合成寡核苷酸类似物的结构单元,及按一定顺序连接这些结构单元而形成的寡核苷酸类似物。结构单元可以是“刚硬的”(如含有环状结构)或“可弯曲的”(如缺乏环状结构)。无论哪种情况,结构单元包含一羟基和一巯基,通过它们结构单元连接形成寡核苷酸类似物。寡核苷酸类似物中的连接部位选自硫化物(-S)、氧硫化物(-SO-)和砜(-SO2-)。
国际专利申请WO 89/20702描述了一包括肽骨架的无环寡核苷酸,在骨架上选择的任何化学核碱基或其类似物都是线性的,并具有在天然DNA或RNA内的编码特征。这些新化合物叫做肽核苷酸(PNAs),在细胞内不仅比其天然状态的对应物稳定得多,而且与天然DNA和RNA的结合50-100倍高于彼此相邻的天然核苷酸。可通过Merrifield固相肽合成,从包含胸腺嘧啶、胞嘧啶、腺嘌呤和鸟嘌呤的四个保护性单体来合成PNA寡聚体。为了提高水溶性,抑制凝集,将赖氨酸的氨基放置在C末端,并被固定。
反义技术要求信使RNA与寡核苷酸配对以形成抑制翻译的双股螺旋。反义寡核苷酸介导基因治疗的概念早在1978就引入肿瘤治疗。该方法是基于某些基因对于肿瘤细胞的生长及分裂是很关键的。合成遗传物质DNA的片段可达到此目的。这些分子与肿瘤细胞RNA中的靶基因分子结合,由此抑制基因的翻译,导致这些细胞的功能障碍性生长。也设想了其它的作用机制。在肿瘤和其它疾病,包括病毒和其它感染性疾病,的治疗中应用了这些策略,并取得一些成功。通常合成的寡核苷酸长13-30个核苷酸。寡核苷酸分子在血液中的寿命相当短。因此,必须对它们进行化学修饰以阻止它们在血液中被普遍存在的核酸酶破坏。磷酸硫盐(phosphorthioates)进入临床试验的反义寡核苷酸中广泛应用的修饰。新一代的反义分子由与合成的DNA的中心部分杂交的反义寡核苷酸构成,而在DNA每一末端的4个碱基用2’O-甲基核糖修饰以模仿RNA。在临床前的动物实验中证实,与第一代未经修饰的磷酸硫盐相比,这样的化合物在机体组织内的代谢更稳定,安全性提高。其它核苷酸类似物也在反义技术中试用。
RNA寡核苷酸也可用于反义抑制,因为它们与目标物形成稳定的RNA-RNA双螺旋,显示足够的抑制。但是,由于它们稳定性低,RNA寡核苷酸通常在应用了为该目的而设计的载体的细胞内表达。当目标mRNA为编码大量且长效的蛋白质时,此方法受到欢迎。
最近的科学出版物在肝炎、肿瘤、冠状动脉狭窄和其它疾病的动物模型中证实了反义化合物的效力。最近FDA通过了第一个反义药物。该药物-Fomivirsen由Isis开发,用于AIDS患者的巨细胞病毒的局部治疗,该患者不能耐受针对CMV视网膜炎的其它治疗或有禁忌症,或对先前针对CMV视网膜炎的治疗反应不够(Pharmacotherapy NewsNetwork)。
现在几种反义化合物在美国正进行临床试用。包括局部施用的抗病毒药,全身性肿瘤治疗药物。反义治疗药物有许多传统药物不及的优点,有望治疗许多危及生命的疾病。传统的药物在疾病引发蛋白形成后介入。然而,反义治疗药物阻断mRNA转录/翻译,在蛋白形成前介入,并且因为反义治疗药物仅瞄准一个特异的mRNA,所以它们比目前的蛋白抑制疗法更有效而副作用更小。
在转录水平阻碍基因表达的第二个选择是应用能与双链DNA杂交的合成寡核苷酸。形成三股螺旋。这样的寡核苷酸可抑制转录因子结合到基因的启动子,从而抑制转录。另外,它们可抑制双股螺旋展开,因此,基因转录在三股螺旋结构内。
因此,依照本发明,它提供一种包括所述反义寡核苷酸的药物组分和可入药的载体。可入药的载体的载体可以是如负载了反义寡核苷酸的脂质体。局部施用的制剂可包括洗剂、软膏、胶体、乳液、栓剂、滴剂、液体、雾剂和粉剂。作为传统的药物载体,水溶液、粉剂或油脂、增稠剂等是必须的或需要的。口服施用的组分包括粉剂或颗粒剂,溶于水或非水性介质的的悬液或溶液,香囊,胶囊或片剂。也许需要增稠剂、稀释剂、香味剂、助分散剂、乳化剂或黏合剂。非肠道施用的制剂包括可包含缓冲液、稀释剂和其它适宜添加剂的灭菌水溶液,但并不限定于此。
依照本发明的另一方面,它提供包含所述反义寡核苷酸的核酶和核酶序列。通过固相寡核苷酸合成可容易地合成这样的核酶。
核酶在基因表达的序列特异性抑制方面的应用日渐增加,核酶通过裂解编码目标蛋白的mRNA而抑制基因表达。设计裂解任何特异性目标RNA的核酶的可能性使它们成为在基础研究和治疗性应用两方面的有价值的工具。在治疗性方面,核酶被用于感染性疾病的目标病毒RNAs肿瘤中的决定性癌基因和遗传性疾病中的特异性体细胞性突变。最引入注意的是,几个用于HIV患者的核酶基因治疗方案已用于I期临床。最近,核酶用于转基因动物的研究,基因打靶的效力和路径说明。几种核酶正用于不同阶段的临床试验。ANGIOZYME是在人临床试验中被研究的第一个合成的核酶。ANGIOZYME特异性抑制VEGF-r(血管内皮生长因子受体),该受体是血管生成路径中的一个关键成分。Ribozyme Pharmaceutials公司和其它公司在动物模型中证实了抗血管生成治疗性药物的重要性。HEPTAZYME是设计的选择性破坏肝炎病毒C(HCV)的核酶,发现在细胞培养方法中,它能有效降低肝炎C病毒RNA(Ribozyme Pharmaceutials公司-WEB首页)。
依照本发明的另一个实施方案,它提供抗体,包括特异性识别并结合多肽的免疫球蛋白,当用DNA序列分析软件包的Bestfit程序分析时该多肽至少与SEQ ID NO 3、5、7或其部分有60%的同源性(一致+相似),优选至少65%,更优选至少70%,还优选至少75%,还优选至少80%,更优选至少85%,更优选至少90%,最优选至少95%-100%。该软件包由Wisconsin大学的Genetic Computer Group(GCG)开发(间隙产生补偿-50,间隙扩展补偿-3)。依照本发明的一个优选实施方案,抗体特异性识别并结合SEQ ID NO 3、5、7所示的多肽或其部分。
本发明可利用血清免疫球蛋白、多克隆抗体或其片段(即抗体的免疫反应性衍生物)或单克隆抗体或其片段。单克隆抗体或单克隆抗体的纯化片段至少有一个抗原结合区域,包括Fv,F(ab1)2,Fab片段(Harlow,Lane,1988 Antibody,Cold Spring Harbor),单链抗体(美国专利4946778),嵌和的或人源抗体,补体决定区域(CDR),可用传统的方法制备。可用该领域的技术人员熟知的方法来纯化这些血清免疫球蛋白抗体和片段,包括硫酸铵沉淀或硫酸钠沉淀,然后对生理盐水透析;离子交换色谱,亲和或免疫亲和色谱及凝胶过滤,区带低于等(参见Goding,Monoclonal Antibodies:Principles and Practice,第二版,pp104-126,1986,Prlando,Fla.,Academic出版)。正常生理条件下,可在血浆和其它体液中以及某些细胞的膜中发现抗体,它由B型淋巴细胞或其功能性等同体产生。IgG类抗体由4个靠二硫键连接在一起的多糖链构成。完整IgG分子的4条链是由两条相同的重链,称作H链,和两条相同的轻链,称作L链构成的。其余类型的抗体包括IgD,IgA,IgE,IgM和相关蛋白。
单抗的产生和筛选方法在该领域内是熟知的,如在综述中总结的,如Tramontano and Schloeder,Methods in Enzymology 178,551-568,1989。本发明的重组蛋白可用于体外产生抗体。更优选本发明的重组蛋白用于体内诱生抗体。总的来说,就是用本发明的重组蛋白免疫适宜的动物宿主。有利的是,所用动物是近交系小鼠。通常用包括溶于生理性溶剂的本发明的重组蛋白溶液和任何适宜佐剂的混合物免疫动物,佐剂能增强机体对免疫原的免疫应答。例如,通常是用本发明重组蛋白的溶液与完全氟氏佐剂的混合物进行初次免疫,所述混合物制备成油包水乳剂。经典的是进行肌内、皮内、皮下、腹腔内、足掌内或任何适当的途径施用。免疫原的免疫计划表可按要求进行调整,但习惯上要用温和的佐剂,如不完全氟氏佐剂进行几次追加或二次免疫。在免疫过程中可应用任何常规方法检测抗体的滴度和结合重组蛋白的特异性,这些方法有,例如放免法或酶联免疫吸附法,即ELISA法。正如业内所知,当达到适宜的抗体滴度时,从免疫动物获取抗体产生性淋巴细胞,进行培养、筛选和克隆。经典的是从免疫动物的脾脏获取大量的淋巴细胞,但也可从循环血流、淋巴结或其它淋巴器官收获它们。然后让淋巴细胞与任何适宜的骨髓瘤细胞系融合,产生杂交瘤。另外,可用业内所知的方法在培养中刺激淋巴细胞生长,并使其固定,包括按照已经建立的方法让淋巴细胞接触病毒、化学物质或核苷酸,如癌基因。融合后,在适宜的培养条件下培养杂交瘤,例如在多孔板中,筛选培养上清以鉴定出包含识别所选半抗原的抗体的培养物。在适当的培养条件下,通过无限稀释和扩展克隆分泌识别本发明的重组蛋白的抗体的杂交瘤。纯化抗体,用免疫球蛋白类型和结合亲和力表示其特性。
不想对本发明的其它目的、优点和新特征进行限制,通过下面的实施例,该领域的技术人员会很清楚。此外,如上面所描述的以及在权利要求部分所要求的本发明的不同实施方案和方面会在以下实施例中找到实验支持。
实施例
参照以下的实施例,与上面的描述一起,以非限制性方式阐明本发明。
概括地讲,这里所应用的术语和下面所述的在重组DNA技术中的实验室步骤在该领域内是熟知并通常应用的。应用了标准的技术进行克隆、DNA和RNA提取、扩增和纯化。包括DNA连接酶、DNA聚合酶、限制性内切酶等的酶反应通常按照厂家的说明进行。这些技术和许多其它技术通常按Sambrook等,Molecular Cloning-A LaboratoryManual,Cold Spring Harbor Laboratory,Cold Spring Harbor,N.Y.(1989)进行,在此引入它作为参考。本申请文件中还提供了其它一般的参考。相信其中的步骤是该领域内是熟知的,在此提供是为了读者的便利。其中包含的所有信息在此引入作为参考。
材料和实验方法
下面的实施例中参照了以下方法和实验细节。
引物列表:hnI1116 5′-GGAGAGCAAGTCTGTGTTGATTC-3′ (SEQ ID NO:10)hnI1230 5′-CACTGGTAGCCATGAGTGTGAG-3′ (SEQ ID NO:11 )hnIu350 5′-TTGGTCATCCCTCCAGTCACCA-3′ (SEQ ID NO:12)pn9-312u 5′-CTTGCCTGTAGACAGAGCTGCAG-3′ (SEQ ID NO:14)hpu-685 5′-GAGCAGCCAGGTGAGCCCAAGA-3′ (SEQ ID NO:16)hp1967 5′-TCAGATGCAAGCAGCAACTTTGGC-3′ (SEQ ID NO:17)mnlu118 5′-CACCCTGATGTCATGCTGGAG-3′ (SEQ ID NO:18)mn11563 5′-CATCTAGGAGAGCAATGACGTTC-3′ (SEQ ID NO:19)Ap1 5′-CCATCCTAATACGACTCACTATAGGGC-3′ (SEQ ID NO:20)Ap2 5′-ACTCACTATAGGGCTCGAGCGGC-3′ (SEQ ID NO:21)
Southern分析
用血液和细胞培养物DNA maxi kit(Qiagene)从动物或人血液中提取基因组DNA。用EcoR I酶切DNA,用凝胶电泳分离,转移到尼龙膜Hybond N+(Amersham)上。PCR产物也进行同样的处理。杂交在68℃6×SSC,1%SDS,5×Denharts,10%硫酸葡聚糖,100ug/ml鲑精DNA,32p标记的探针的条件下进行。Pn9,1.7kb的片段,除162位核苷酸缺失外包含整个开放阅读框,用它作探针。杂交后,用3×SSC,0.1%SDS,在68℃洗膜,曝光到X线胶片3天。然后用0.1×SSC,0.1%SDS,68℃洗膜,再曝光4天。
RT-PCR
按照厂家说明,用TRI-Reagent(Molecular research center Inc.)制备RNA。取1.25ug用SuperScriptII逆转录酶(Gibco BRL)和Oligo(dT)15引物(SEQ ID NO:22)(Promega)进行逆转录反应。用Taq聚合酶(Promega)或EXpand high fidelity(Boehringer Mannheim)扩增所得第一条cDNA链。
cDNA序列分析
序列确定是用载体特异的和基因特异的引物,用自动DNA测序仪(Applied Biosystems,373A型)进行。至少通过两个独立的引物来读核苷酸。计算、序列分析和排列对比应用DNA序列分析软件包进行,它是由Wisconsin大学的Genetic Computer Group(GCG)开发的。两个序列的排列对比应用Bestfit(间隙产生-12,间隙扩展-4)或用Gap程序(间隙产生-50,间隙扩展-3)进行。
组织分布
通过半定量PCR来检测hnhp I转录本的组织分布。cDNA模板来自Clotech。用基因特异的引物hnIu350(SEQ ID NO:12)和hnII116(SEQID NO:10)进行PCR。PCR程序是:94℃,3分钟,然后是40个循环的94℃,45秒,64℃,1分钟,72℃,1分钟。用于其它分析的样品进行25、30、35和40个循环。
染色体定位
用放射性杂交模板Stanford G3来进行hnhp I的染色体定位。该模板用Weizmann Institute的人类基因组中心提供。用基因特异的引物hnIu350(SEQ ID NO:12)和hnII116(SEQ ID NO:10)扩增出hnhp I基因的一225bp的基因组片段。PCR程序是:94℃,3分钟,然后是39个循环的94℃,45秒,64℃,1分钟,72℃,1分钟。通过Stanford人类基因组中心的RH服务器进行结果分析。
实施例1
为新的乙酰肝素酶基因克隆一EST
用人乙酰肝素酶(SEQ ID NO:9)的整个氨基酸序列筛选人EST数据库,寻找同源序列。NCBI-基本BLAST查询-tblastn程序下,用BLAST2.0服务器进行筛选。
筛选出一同源较远的片段,登录号为AI222323,IMAGE克隆号为1843155,来自从睾丸B细胞和胚胎肺制备的Soares_NFL_T_GBC_SIHomo Sapiens cDNA文库。该序列的搜索值如下:Score=38.3bits(87),EXpect=0.15,Identities=16/36(44%),Positive=22/36(60%)。登录号为AI222323的序列包含1843155(与SEQ ID NO:23的165-543互补)克隆株3’的378个核苷酸。
该克隆购自IMAGE集团。它包含一560bp(SEQ ID NO:23)的插入片段。对整个氨基酸序列进行了确定并与hpa cDNA编码的人乙酰肝素酶进行了对比。1843155克隆与hpa cDNA之间的同源性仅限于cDNA克隆的3’端。1843155克隆的99-275核苷酸(SEQ ID NO:23)与hpa的1532-1708(SEQ ID NO:24)之间有59%的同源性。该区域的推导氨基酸序列与人乙酰肝素酶的氨基酸88-542(SEQ ID NO:9)60%同源(一致+相似)。下游区域(核苷酸276-560,(SEQ ID NO:23)代表了3’未翻译区和polyA尾。上游序列,核苷酸1-98(SEQ ID NO:23)与乙酰肝素酶无关。发现该无关序列与来自相同文库的不同cDNA克隆一致。因此推测从IMAGE集团获得的人EST 1843155克隆是一嵌和体,它包含连接到一个载体上的两个无关部分cDNA.
实施例2
为新的乙酰肝素酶基因克隆cDNA
为了分离完整的cDNA,根据1843155克隆的序列设计了3种引物。参照厂家的说明,用Marathon RACE(cDNA末端的快速扩增)(Clontech,Palo Alto,California)从胎盘cDNA扩增cDNA。第一个循环用基因特异的引物hnI1116(SEQ ID NO:10)和通用引物ApI(SEQ IDNO:20)进行。第二个循环用基因特异的引物hnI1230(SEQ ID NO:11)和通用引物Ap2(SEQ ID NO:21)进行。扩增后,在接近1.7kb处获得双融合条带。将该cDNA扩增产物亚克隆到pGEM T-easy(Promega,Madison,WI),并测定3个独立克隆pn5,pn6,pn9的核苷酸序列。最长cDNA pn6的共有序列如所示(SEQ ID NO:1,2,3)。它长2060个核苷酸,包含1776个核苷酸的开放阅读框,该阅读框编码592个氨基酸的多肽,推测其分子量为66.5kDa。新克隆的基因命名为hnhpI。两个较短的cDNA-pn5,pn9及它们的氨基酸序列分别列于SEQ ID NO 4和6及SEQ ID NO 5和7。pn5,pn9与pn6等同,但由于可变剪接它们中每一个均在阅读框中有一缺失。pn9包含162个核苷酸的缺失,SEQ ID NO:1中的473-634,对应的氨基酸是SEQ ID NO:3的150-230。结果,pn9编码一538个氨基酸的多肽(SEQ ID NO:5),分子量60.4kDa。pn5包含336个核苷酸的缺失,SEQ ID NO:1中的473-808,对应的氨基酸是SEQ ID NO:3的150-261。因此,它编码一480个氨基酸的多肽(SEQ ID NO:7),分子量53.9kDa。SEQ ID NO:3中第11个氨基酸是甲硫氨酸。通常认为,第一个甲硫氨酸在哺乳动物中作为翻译起始位点。但是,第二个ATG周围的核苷酸较好符合作为翻译起始位点的Kozak共有序列。因此,翻译可能在第二个甲硫氨酸开始,产生一581个氨基酸的蛋白质,分子量65.4kDa。不同长度转录本的存在被hnlhpcDNA的扩增PCR所证实,PCR中应用了两个基因特异的引物:pn9-312u(SEQ ID NO:14),它的位置接近于5’末端,和引物hnI1230(SEQID NO:11),它覆盖了开放阅读框3’末端的终止密码子。扩增从由胎盘和睾丸制备的Marathon ready cDNA进行。PCR产物见图3。从胎盘获得4条带:1.45kb和1.6kb的两条主带,与pn9和pn6的两条次要条带相似;一条1.35kb与pn5相似的条带和1.8kb的一条。后者的序列还未确定。睾丸cDNA的扩增显示出不同方式。看到1.35,1.65,1.85 2.05kb四条带和一1.5kb的次要条带。各种形式似乎代表了可变剪接的产物。因为缺失的特征是保持了开放阅读框,所以各种cDNA的翻译产物可构成一蛋白质家族。hnhpo和乙酰肝素酶氨基酸序列之间的比较见图3。应用GCG程序的gap程序排列整个氨基酸序列,两个蛋白质主带的同源性是45.5%等同,7.3%相似,总的同源性是52.8%(间隙产生-50,间隙扩展-3)。BestFit程序确定两个序列间最同源的区域。应用该程序,两个氨基酸序列间的同源性始于hnIhpI的63位(SEQ ID NO:3)和乙酰肝素酶的41位(SEQ ID NO:9),47.5%等同,7.8%相似,即同源性55.3%。通过BestFit程序计算,hnhpI和hpa核苷酸序列间的同源性为57%。同源区域位于hnhpI的648-1812位核苷酸(SEQ ID NO:1)和hpa的564-1708(SEQ ID NO:24)位核苷酸之间。应用Gap程序,整个序列间的同源性为51%(间隙产生-50,间隙扩展-3)。
实施例3
动物图谱
用hnhpI cDNA作探针来检测在人DNA和各种动物DNA中的同源序列。Southem分析的放射自显影示于图4。在人DNA中检测到几条带。在所有的动物中均检测到几条强带,而在鸡中检测到弱的条带。这与人和被检动物之间的种系发生的关系相关。强带表明hnhpI在哺乳动物及遗传学上较远的生命体中是保守的。多重条带表明在所有动物中hnhpI位点占据大的基因组区域。在严紧洗涤后,几条特异条带消失。这些代表同源序列,并提示存在一个基因家族,根据这里报道的它们与人的同源性可分离之。
实施例4
通过交叉杂交与乙酰肝素酶比较
为了检查hpa和hnhpI在较低严紧度下进行交叉杂交的能力,通过PCR扩增人hpa和hnhpI的整个编码区域。应用引物hpu-685(SEQ IDNO:16)和hp1967(SEQ ID NO:17),通过RT-PCR从血小板mRNA扩增人hpa;应用引物hn11230(SEQ ID NO:11)和pn9-312u(SEQ IDNO:14),从睾丸扩增hnhpI。对产物进行定量,取100pg和1ng样品跑琼脂糖胶,然后进行Southern杂交。用32p标记的hpa cDNA和hnhpIcDNA作探针。即便曝光5天也未见到交叉杂交(图5)。因为hpa是目前知道的与hnhpI最相似的序列,该实验证实在图4的放射自显影中检测到的条带是hnhpI基因或未知的与其同源的序列,也许构成一个基因家族。这还表明用hnhpI作探针筛选相关的文库或用hnhpI来源的PCR引物扩增相关的cDNA或DNA序列可分离出这些序列。
实施例5
染色体定位
应用G3放射性杂交模板来确定hnhpI的染色体定位。从83人/小鼠放射性杂交中扩增出hnhpI。用RH服务器分析结果,hnhpI基因位于10号染色体,紧邻标记SHGC-57721。该结果也提示该基因可能存在第二个拷贝。
实施例6
hnhpI的表达谱
应用校准的人cDNA模板(Clontech,Palo Alto,Ca)确定hnhpI转录本的组织分布。结果如表1所示。表达水平普遍低。在40个循环以后才能清楚的看到PCR产物。
表1
组织 hnI(40循环)
骨髓
肝
淋巴结 +
白细胞
脾脏 +
胸腺
扁桃体
结肠 +
卵巢 +
前列腺 ++
小肠 ++
睾丸 +++
实施例7
小鼠同源物的克隆
用乙酰肝素酶及hnhpI的氨基酸序列筛选小鼠EST数据库查出一小鼠EST克隆,它与乙酰肝素酶有较远的同源性,而与hnhpI有相当高的同源性。EST克隆1378452登录号AI019269,来自小鼠胸腺,351个核苷酸长,列于SEQ ID NO:8。用GCG软件包的BestFit程序检验,它与人(SEQ ID NO:24)和小鼠(SEQ ID NO:15)hpa核苷酸序列的161个核苷酸(191-351,SEQ ID NO:8)有61-63%的一致性,与hnhpI核苷酸序列(SEQ ID NO:15)有93%的一致性。该克隆的核苷酸序列不包含开放阅读框。与hnhpI核苷酸序列相比,在EST数据库中找到的序列中鉴定出2个移码。随后,通过该克隆的核苷酸序列分析,以及从BL6小鼠黑色素瘤细胞分离该片段并确定其核苷酸序列证实了该移码。该小鼠基因转录水平极低。从小鼠cDNA模板(Clontech,Palo Alto,Ca)进行40个循环的PCR也无扩增产物也提示其表达水平低。该cDNA模板包括小鼠心脏、脑、脾脏、肺、肝脏、骨骼肌、肾脏、睾丸和7,11,15和17天胚胎的cDNA。扩增应用基因特异的引物nmlu118(SEQ ID NO:18)和nm11563(SEQ ID NO:19)。
实施例8
hnhpI在哺乳动物细胞的表达
为了在人细胞中过表达hnhpI,构建哺乳动物表达载体。为了能检测H nhpI翻译产物,设计的hnhpI表达载体编码C末端有tag的hnI蛋白。编码8个氨基酸FLAG(Kodak)的DNA序列与hnhpI开放阅读框的3’末端融合。
FLAG序列与hnhpI编码序列的融合通过PCR扩增进行,应用引物hnI-c-flag:5’-A-3’(SEQ ID NO:25)和引物pn9-312u(SEQ ID NO:14)进行扩增。PCR的程序是:94℃3分,随后5个循环的94℃45秒,72℃2分,然后32个循环的94℃45秒,64℃45秒,72℃2分。
扩增产物亚克隆到pGEM-T-easy,验证序列。所得质粒命名为pGEM-pn6F和pGEM-pn9F。
在pSI哺乳动物表达载体(Promega)中产生两个构建体:第一个包含整个hnhpI序列(pn6),第二个包含可变剪接形式(pn9)。pSI-pn6表达载体的构建是通过下列片段的三次连接:EcoRI-BamHI片段,它包含hnI-pn6的5’端,切自gGem-T-easy-pn9;BamHI-NotI片段,包含3’FLAG tagged hbnhpI,切自pGEM-pn6F,和用EcoRI-NotI酶切的pSI。pSI-pn9表达载体的构建与此相似,通过下列片段的三次连接:EcoRI-SspI片段,它包含hnhpI-pn6的5’端,切自gGem-T-easy-pn9;SspI-NotI片段,包含3’FLAG tagged hbnhpI,切自pGem-pn6F,和用EcoRI-NotI酶切的pSI。
用Fugene转染试剂(Boehringer Mannheim)将所得质粒转染到人胚胎肾293细胞。48小时后收集转染细胞,通过western blot分析蛋白质。通过SDS-PAGE分离2.5×105细胞的裂解物,转移到尼龙膜上,并与1∶1000稀释的抗FLAG抗体(Kodak anti FLAG M2 cat:IB13025,终浓度10ug/ml)孵育。在用pSI-pn6F和pSI-pn9F转染的细胞中,分别检测到大约65kDa和60kDa的蛋白。这些蛋白的大小与通过计算相应开放阅读框翻译产物的分子量而预测的相似。这证明整个hnhpI cDNA和pn9剪接形式均可成功地在人293细胞转录和翻译。但是,与乙酰肝素酶不同,HnhpI蛋白产物在这些细胞中不进行主要加工。
尽管是通过具体实施例对本发明进行了描述,但很明显,该领域的技术人员会很清楚其许多替代、调整和变化形式。因此,本发明旨在包括在所附权利要求书实质和广阔范围内的所有替代、调整和变化形式。这里引用的所有出版物在此引入作为参考。
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apolipoprotein E-enriched remnant lipoproteins by cultured cells. J.
Biol.Chem.,268,10160-10167.52. Current protocols in protein science(1996-1999).Coligan,J.E.,
Dunn,B.M.,Ploegh,H.L.,Speicher,D.W.and Wingfield,P.T.Eds.
John Wiley & Sons Inc.
序列表(1)-般资料:
(i) 申请人: I.佩克等(Iris Pecker et al.)
(ii) 发明名称: 多核苷酸和其编码的多肽
(iii)序列数: 24
(iv) 通讯地址:
(A)联系人: Sol Sheinbein c/o Anthony Castorina
(B)街道: 2001 Jefferson Davis Highway,Suite 207
(C)城市: Arlington
(D)州: Virginia
(E)国家 United States of America
(F)邮编: 22202
(v)计算机可读形式:
(A)介质类型: 1.44M,3.5”英寸软盘
(B)计算机: Twinhead*Slimnote-890TX
(C)操作系统: MS DOS version 6.2,
Windows version 3.11
(D)软件: Word for Windows version 2.0
转变成ASCI文件
(vi)本申请数据:
(A)申请号:
(B)申请日:
(C)分类号:
(vii)在先申请数据:
(A)申请号: 60/140,801
(B)申请日:1999年6月25日
(viii)代理人信息:
(A)姓名: Sheinbein,Sol
(B)注册号: 25,457
(C)文档号: 20105
(ix)通讯信息:
(A)电话: 972-3-6127676
(B)传真: 972-3-6127575
(C)电传:(2)SEQ ID NO:1信息
(i)序列特征:
(A)长度:2060
(B)类型:核酸
(C)链型:双链
(D)拓扑结构:线性
(xi)序列描述: SEQ ID NO:1:CGCTTAATTC TAGAAGAGGG ATTGAATGAG GGTGCTTTGT GCCTTCCCTG 50AAGCCATGCC CTCCAGCAAC TCCCGCCCCC CCGCGTGCCT AGCCCCGGGG 100GCTCTCTACT TGGCTCTGTT GCTCCATCTC TCCCTTTCCT CCCAGGCTGG 150AGACAGGAGA CCCTTGCCTG TAGACAGAGC TGCAGGTTTG AAGGAAAAGA 200CCCTGATTCT ACTTGATGTG AGCACCAAGA ACCCAGTCAG GACAGTCAAT 250GAGAACTTCC TCTCTCTGCA GCTGGATCCG TCCATCATTC ATGATGGCTG 300GCTCGATTTC CTAAGCTCCA AGCGCTTGGT GACCCTGGCC CGGGGACTTT 350CGCCCGCCTT TCTGCGCTTC GGGGGCAAAA GGACCGACTT CCTGCAGTTC 400CAGAACCTGA GGAACCCGGC GAAAAGCCGC GGGGGCCCGG GCCCGGATTA 450CTATCTCAAA AACTATGAGG ATGACATTGT TCGAAGTGAT GTTGCCTTAG 500ATAAACAGAA AGGCTGCAAG ATTGCCCAGC ACCCTGATGT TATGCTGGAG 550CTCCAAAGGG AGAAGGCAGC TCAGATGCAT CTGGTTCTTC TAAAGGAGCA 600ATTCTCCAAT ACTTACAGTA ATCTCATATT AACAGCCAGG TCTCTAGACA 650AACTTTATAA CTTTGCTGAT TGCTCTGGAC TCCACCTGAT ATTTGCTCTA 700AATGCACTGC GTCGTAATCC CAATAACTCC TGGAACAGTT CTAGTGCCCT 750GAGTCTGTTG AAGTACAGCG CCAGCAAAAA GTACAACATT TCTTGGGAAC 800TGGGTAATGA GCCAAATAAC TATCGGACCA TGCATGGCCG GGCAGTAAAT 850GGCAGCCAGT TGGGAAAGGA TTACATCCAG CTGAAGAGCC TGTTGCAGCC 900CATCCGGATT TATTCCAGAG CCAGCTTATA TGGCCCTAAT ATTGGGCGGC 950CGAGGAAGAA TGTCATCGCC CTCCTAGATG GATTCATGAA GGTGGCAGGA 1000AGTACAGTAG ATGCAGTTAC CTGGCAACAT TGCTACATTG ATGGCCGGGT 1050GGTCAAGGTG ATGGACTTCC TGAAAACTCG CCTGTTAGAC ACACTCTCTG 1100ACCAGATTAG GAAAATTCAG AAAGTGGTTA ATACATACAC TCCAGGAAAG 1150AAGATTTGGC TTGAAGGTGT GGTGACCACC TCAGCTGGAG GCACAAACAA 1200TCTATCCGAT TCCTATGCTG CAGGATTCTT ATGGTTGAAC ACTTTAGGAA 1250TGCTGGCCAA TCAGGGCATT GATGTCGTGA TACGGCACTC ATTTTTTGAC 1300CATGGATACA ATCACCTCGT GGACCAGAAT TTTAACCCAT TACCAGACTA 1350CTGGCTCTCT CTCCTCTACA AGCGCCTGAT CGGCCCCAAA GTCTTGGCTG 1400TGCATGTGGC TGGGCTCCAG CGGAAGCCAC GGCCTGGCCG AGTGATCCGG 1450GACAAACTAA GGATTTATGC TCACTGCACA AACCACCACA ACCACAACTA 1500CGTTCGTGGG TCCATTACAC TTTTTATCAT CAACTTGCAT CGATCAAGAA 1550AGAAAATCAA GCTGGCTGGG ACTCTCAGAG ACAAGCTGGT TCACCAGTAC 1600CTGCTGCAGC CCTATGGGCA GGAGGGCCTA AAGTCCAAGT CAGTGCAACT 1650GAATGGCCAG CCCTTAGTGA TGGTGGACGA CGGGACCCTC CCAGAATTGA 1700AGCCCCGCCC CCTTCGGGCC GGCCGGACAT TGGTCATCCC TCCAGTCACC 1750ATGGGCTTTT TTGTGGTCAA GAATGTCAAT GCTTTGGCCT GCCGCTACCG 1800ATAAGCTATC CTCACACTCA TGGCTACCAG TGGGCCTGCT GGGCTGCTTC 1850CACTCCTCCA CTCCAGTAGT ATCCTCTGTT TTCAGACATC CTAGCAACCA 1900GCCCCTGCTG CCCCATCCTG CTGGAATCAA CACAGACTTG CTCTCCAAAG 1950AGACTAAATG TCATAGCGTG ATCTTAGCCT AGGTAGGCCA CATCCATCCC 2000AAAGGAAAAT GTAGACATCA CCTGTACCTA TATAAGGATA AAGGCATGTG 2050TATAGAGCAA 2060(2)SEQ ID NO:2信息
(i)序列特征:
(A)长度:2060
(B)类型:核酸
(C)链型:双链
(D)拓扑结构:线性
(xi)序列描述: SEQ ID NO:2:
C GCT TAA TTC TAG AAG AGG GAT TGA 25ATG AGG GTG CTT TGT GCC TTC CCT GAA GCC ATG CCC TCC AGC AAC 70Met Arg Val Leu Cys Ala Phe Pro Glu Ala Met Pro Ser Ser Asn
5 10 15TCC CGC CCC CCC GCG TGC CTA GCC CCG GGG GCT CTC TAC TTG GCT 115Ser Arg Pro Pro Ala Cys Leu Ala Pro Gly Ala Leu Tyr Leu Ala
20 25 30CTG TTG CTC CAT CTC TCC CTT TCC TCC CAG GCT GGA GAC AGG AGA 160Leu Leu Leu His Leu Ser Leu Ser Ser Gln Ala Gly Asp Arg Arg
35 40 45CCC TTG CCT GTA GAC AGA GCT GCA GGT TTG AAG GAA AAG ACC CTG 205Pro Leu Pro Val Asp Arg Ala Ala Gly Leu Lys Glu Lys Thr Leu
50 55 60ATT CTA CTT GAT GTG AGC ACC AAG AAC CCA GTC AGG ACA GTC AAT 250Ile Leu Leu Asp Val Ser Thr Lys Asn Pro Val Arg Thr Val Asn
65 70 75GAG AAC TTC CTC TCT CTG CAG CTG GAT CCG TCC ATC ATT CAT GAT 295Glu Asn Phe Leu Ser Leu Gln Leu Asp Pro Ser Ile Ile His Asp
80 85 90GGC TGG CTC GAT TTC CTA AGC TCC AAG CGC TTG GTG ACC CTG GCC 340Gly Trp Leu Asp Phe Leu Ser Ser Lys Arg Leu Val Thr Leu Ala
95 100 105CGG GGA CTT TCG CCC GCC TTT CTG CGC TTC GGG GGC AAA AGG ACC 385Arg Gly Leu Ser Pro Ala Phe Leu Arg Phe Gly Gly Lys Arg Thr
110 115 120GAC TTC CTG CAG TTC CAG AAC CTG AGG AAC CCG GCG AAA AGC CGC 430Asp Phe Leu Gln Phe Gln Asn Leu Arg Asn Pro Ala Lys Ser Arg
125 130 135GGG GGC CCG GGC CCG GAT TAC TAT CTC AAA AAC TAT GAG GAT GAC 475Gly Gly Pro Gly Pro Asp Tyr Tyr Leu Lys Asn Tyr Glu Asp Asp
140 145 150ATT GTT CGA AGT GAT GTT GCC TTA GAT AAA CAG AAA GGC TGC AAG 520Ile Val Arg Ser Asp Val Ala Leu Asp Lys Gln Lys Gly Cys Lys
155 160 165ATT GCC CAG CAC CCT GAT GTT ATG CTG GAG CTC CAA AGG GAG AAG 565Ile Ala Gln His Pro Asp Val Met Leu Glu Leu Gln Arg Glu Lys
170 175 180GCA GCT CAG ATG CAT CTG GTT CTT CTA AAG GAG CAA TTC TCC AAT 610Ala Ala Gln Met His Leu Val Leu Leu Lys Glu Gln Phe Ser Asn
185 190 195ACT TAC AGT AAT CTC ATA TTA ACA GCC AGG TCT CTA GAC AAA CTT 655Thr Tyr Ser Asn Leu Ile Leu Thr Ala Arg Ser Leu Asp Lys Leu
200 205 210TAT AAC TTT GCT GAT TGC TCT GGA CTC CAC CTG ATA TTT GCT CTA 700Tyr Asn Phe Ala Asp Cys Ser Gly Leu His Leu Ile Phe Ala Leu
215 220 225AAT GCA CTG CGT CGT AAT CCC AAT AAC TCC TGG AAC AGT TCT AGT 745Asn Ala Leu Arg Arg Asn Pro Asn Asn Ser Trp Asn Ser Ser Ser
230 235 240GCC CTG AGT CTG TTG AAG TAC AGC GCC AGC AAA AAG TAC AAC ATT 790Ala Leu Ser Leu Leu Lys Tyr Ser Ala Ser Lys Lys Tyr Asn Ile
245 250 255TCT TGG GAA CTG GGT AAT GAG CCA AAT AAC TAT CGG ACC ATG CAT 835Ser Trp Glu Leu Gly Asn Glu Pro Asn Asn Tyr Arg Thr Met His
260 265 270GGC CGG GCA GTA AAT GGC AGC CAG TTG GGA AAG GAT TAC ATC CAG 880Gly Arg Ala Val Asn Gly Ser Gln Leu Gly Lys Asp Tyr Ile Gln
275 280 285CTG AAG AGC CTG TTG CAG CCC ATC CGG ATT TAT TCC AGA GCC AGC 925Leu Lys Ser Leu Leu Gln Pro Ile Arg Ile Tyr Ser Arg Ala Ser
290 295 300TTA TAT GGC CCT AAT ATT GGG CGG CCG AGG AAG AAT GTC ATC GCC 970Leu Tyr Gly Pro Asn Ile Gly Arg Pro Arg Lys Asn Val Ile Ala
305 310 315CTC CTA GAT GGA TTC ATG AAG GTG GCA GGA AGT ACA GTA GAT GCA 1015Leu Leu Asp Gly Phe Met Lys Val Ala Gly Ser Thr Val Asp Ala
320 325 330GTT ACC TGG CAA CAT TGC TAC ATT GAT GGC CGG GTG GTC AAG GTG 1060Val Thr Trp Gln His Cys Tyr Ile Asp Gly Arg Val Val Lys Val
335 340 345ATG GAC TTC CTG AAA ACT CGC CTG TTA GAC ACA CTC TCT GAC CAG 1105Met Asp Phe Leu Lys Thr Arg Leu Leu Asp Thr Leu Ser Asp Gln
350 355 360ATT AGG AAA ATT CAG AAA GTG GTT AAT ACA TAC ACT CCA GGA AAG 1150Ile Arg Lys Ile Gln Lys Val Val Asn Thr Tyr Thr Pro Gly Lys
365 370 375AAG ATT TGG CTT GAA GGT GTG GTG ACC ACC TCA GCT GGA GGC ACA 1195Lys Ile Trp Leu Glu Gly Val Val Thr Thr Ser Ala Gly Gly Thr
380 385 390AAC AAT CTA TCC GAT TCC TAT GCT GCA GGA TTC TTA TGG TTG AAC 1240Asn Asn Leu Ser Asp Ser Tyr Ala Ala Gly Phe Leu Trp Leu Asn
395 400 405ACT TTA GGA ATG CTG GCC AAT CAG GGC ATT GAT GTC GTG ATA CGG 1285Thr Leu Gly Met Leu Ala Asn Gln Gly Ile Asp Val Val Ile Arg
410 415 420CAC TCA TTT TTT GAC CAT GGA TAC AAT CAC CTC GTG GAC CAG AAT 1330His Ser Phe Phe Asp His Gly Tyr Asn His Leu Val Asp Gln Asn
425 430 435TTT AAC CCA TTA CCA GAC TAC TGG CTC TCT CTC CTC TAC AAG CGC 1375Phe Asn Pro Leu Pro Asp Tyr Trp Leu Ser Leu Leu Tyr Lys Arg
440 445 450CTG ATC GGC CCC AAA GTC TTG GCT GTG CAT GTG GCT GGG CTC CAG 1420Leu Ile Gly Pro Lys Val Leu Ala Val His Val Ala Gly Leu Gln
455 460 465CGG AAG CCA CGG CCT GGC CGA GTG ATC CGG GAC AAA CTA AGG ATT 1465Arg Lys Pro Arg Pro Gly Arg Val Ile Arg Asp Lys Leu Arg Ile
470 475 480TAT GCT CAC TGC ACA AAC CAC CAC AAC CAC AAC TAC GTT CGT GGG 1510Tyr Ala His Cys Thr Asn His His Asn His Asn Tyr Val Arg Gly
485 490 495TCC ATT ACA CTT TTT ATC ATC AAC TTG CAT CGA TCA AGA AAG AAA 1555Ser Ile Thr Leu Phe Ile Ile Asn Leu His Arg Ser Arg Lys Lys
500 505 510ATC AAG CTG GCT GGG ACT CTC AGA GAC AAG CTG GTT CAC CAG TAC 1600Ile Lys Leu Ala Gly Thr Leu Arg Asp Lys Leu Val His Gln Tyr
515 520 525CTG CTG CAG CCC TAT GGG CAG GAG GGC CTA AAG TCC AAG TCA GTG 1645Leu Leu Gln Pro Tyr Gly Gln Glu Gly Leu Lys Ser Lys Ser Val
530 535 54DCAA CTG AAT GGC CAG CCC TTA GTG ATG GTG GAC GAC GGG ACC CTC 1690Gln Leu Asn Gly Gln Pro Leu Val Met Val Asp Asp Gly Thr Leu
545 550 555CCA GAA TTG AAG CCC CGC CCC CTT CGG GCC GGC CGG ACA TTG GTC 1735Pro Glu Leu Lys Pro Arg Pro Leu Arg Ala Gly Arg Thr Leu Val
560 565 570ATC CCT CCA GTC ACC ATG GGC TTT TTT GTG GTC AAG AAT GTC AAT 1780Ile Pro Pro Val Thr Met Gly Phe Phe Val Val Lys Asn Val Asn
575 580 585GCT TTG GCC TGC CGC TAC CGA TAA GCT ATC CTC ACA CTC ATG GCT 1825Ala Leu Ala Cys Arg Tyr Arg
590ACC AGT GGG CCT GCT GGG CTG CTT CCA CTC CTC CAC TCC AGT AGT 1870ATC CTC TGT TTT CAG ACA TCC TAG CAA CCA GCC CCT GCT GCC CCA 1915TCC TGC TGG AAT CAA CAC AGA CTT GCT CTC CAA AGA GAC TAA ATG 1960TCA TAG CGT GAT CTT AGC CTA GGT AGG CCA CAT CCA TCC CAA AGG 2005AAA ATG TAG ACA TCA CCT GTA CCT ATA TAA GGA TAA AGG CAT GTG 2050TAT AGA GCA A 2060(2)SEQ ID NO:3信息
(i)序列特征:
(A)长度:592
(B)类型:氨基酸(C)链型:单链
(D)拓扑结构:线性
(xi)序列描述: SEQ ID NO:3:Met Arg Val Leu Cys Ala Phe Pro Glu Ala Met Pro Ser Ser Asn
5 10 15Ser Arg Pro Pro Ala Cys Leu Ala Pro Gly Ala Leu Tyr Leu Ala
20 25 30Leu Leu Leu His Leu Ser Leu Ser Ser Gln Ala Gly Asp Arg Arg
35 40 45Pro Leu Pro Val Asp Arg Ala Ala Gly Leu Lys Glu Lys Thr Leu
50 55 60Ile Leu Leu Asp Val Ser Thr Lys Asn Pro Val Arg Thr Val Asn
65 70 75Glu Asn Phe Leu Ser Leu Gln Leu Asp Pro Ser Ile Ile His Asp
80 85 90Gly Trp Leu Asp Phe Leu Ser Ser Lys Arg Leu Val Thr Leu Ala
95 100 105Arg Gly Leu Ser Pro Ala Phe Leu Arg Phe Gly Gly Lys Arg Thr
110 115 120Asp Phe Leu Gln Phe Gln Asn Leu Arg Asn Pro Ala Lys Ser Arg
125 130 135Gly Gly Pro Gly Pro Asp Tyr Tyr Leu Lys Asn Tyr Glu Asp Asp
140 145 150Ile Val Arg Ser Asp Val Ala Leu Asp Lys Gln Lys Gly Cys Lys
155 160 165Ile Ala Gln His Pro Asp Val Met Leu Glu Leu Gln Arg Glu Lys
170 175 180Ala Ala Gln Met His Leu Val Leu Leu Lys Glu Gln Phe Ser Asn
185 190 195Thr Tyr Ser Asn Leu Ile Leu Thr Ala Arg Ser Leu Asp Lys Leu
200 205 210Tyr Asn Phe Ala Asp Cys Ser Gly Leu His Leu Ile Phe Ala Leu
215 220 225Asn Ala Leu Arg Arg Asn Pro Asn Asn Ser Trp Asn Ser Ser Ser
230 235 240Ala Leu Ser Leu Leu Lys Tyr Ser Ala Ser Lys Lys Tyr Asn Ile
245 250 255Ser Trp Glu Leu Gly Asn Glu Pro Asn Asn Tyr Arg Thr Met His
260 265 270Gly Arg Ala Val Asn Gly Ser Gln Leu Gly Lys Asp Tyr Ile Gln
275 280 285Leu Lys Ser Leu Leu Gln Pro Ile Arg Ile Tyr Ser Arg Ala Ser
290 295 300Leu Tyr Gly Pro Asn Ile Gly Arg Pro Arg Lys Asn Val Ile Ala
305 310 315Leu Leu Asp Gly Phe Met Lys Val Ala Gly Ser Thr Val Asp Ala
320 325 330Val Thr Trp Gln His Cys Tyr Ile Asp Gly Arg Val Val Lys Val
335 340 345Met Asp Phe Leu Lys Thr Arg Leu Leu Asp Thr Leu Ser Asp Gln
350 355 360Ile Arg Lys Ile Gln Lys Val Val Asn Thr Tyr Thr Pro Gly Lys
365 370 375Lys Ile Trp Leu Glu Gly Val Val Thr Thr ser Ala Gly Gly Thr
380 385 390Asn Asn Leu Ser Asp Ser Tyr Ala Ala Gly Phe Leu Trp Leu Asn
395 400 405Thr Leu Gly Met Leu Ala Asn Gln Gly Ile Asp Val Val Ile Arg
410 415 420His Ser Phe Phe Asp His Gly Tyr Asn His Leu Val Asp Gln Asn
425 430 435Phe Asn Pro Leu Pro Asp Tyr Trp Leu Ser Leu Leu Tyr Lys Arg
440 445 450Leu Ile Gly Pro Lys Val Leu Ala Val His Val Ala Gly Leu Gln
455 460 465Arg Lys Pro Arg Pro Gly Arg Val Ile Arg Asp Lys Leu Arg Ile
470 475 480Tyr Ala His Cys Thr Asn His His Asn His Asn Tyr Val Arg Gly
485 490 495Ser Ile Thr Leu Phe Ile Ile Asn Leu His Arg Ser Arg Lys Lys
500 505 510Ile Lys Leu Ala Gly Thr Leu Arg Asp Lys Leu Val His Gln Tyr
515 520 525Leu Leu Gln Pro Tyr Gly Gln Glu Gly Leu Lys Ser Lys Ser Val
530 535 540Gln Leu Asn Gly Gln Pro Leu Val Met Val Asp Asp Gly Thr Leu
545 550 555Pro Glu Leu Lys Pro Arg Pro Leu Arg Ala Gly Arg Thr Leu Val
560 565 570Ile Pro Pro Val Thr Met Gly Phe Phe Val Val Lys Asn Val Asn
575 580 585Ala Leu Ala Cys Arg Tyr Arg
590(2)SEQ ID NO:4信息
(i)序列特征:
(A)长度:1898
(B)类型:核酸
(C)链型:双链
(D)拓扑结构:线性
(xi)序列描述: SEQ ID NO:4:CGCTTAATTC TAGAAGAGGG ATTGAATGAG GGTGCTTTGT GCCTTCCCTG 50AAGCCATGCC CTCCAGCAAC TCCCGCCCCC CCGCGTGCCT AGCCCCGGGG 100GCTCTCTACT TGGCTCTGTT GCTCCATCTC TCCCTTTCCT CCCAGGCTGG 150AGACAGGAGA CCCTTGCCTG TAGACAGAGC TGCAGGTTTG AAGGAAAAGA 200CCCTGATTCT ACTTGATGTG AGCACCAAGA ACCCAGTCAG GACAGTCAAT 250GAGAACTTCC TCTCTCTGCA GCTGGATCCG TCCATCATTC ATGATGGCTG 300GCTCGATTTC CTAAGCTCCA AGCGCTTGGT GACCCTGGCC CGGGGACTTT 350CGCCCGCCTT TCTGCGCTTC GGGGGCAAAA GGACCGACTT CCTGCAGTTC 400CAGAACCTGA GGAACCCGGC GAAAAGCCGC GGGGGCCCGG GCCCGGATTA 450CTATCTCAAA AACTATGAGG ATGCCAGGTC TCTAGACAAA CTTTATAACT 500TTGCTGATTG CTCTGGACTC CACCTGATAT TTGCTCTAAA TGCACTGCGT 550CGTAATCCCA ATAACTCCTG GAACAGTTCT AGTGCCCTGA GTCTGTTGAA 600GTACAGCGCC AGCAAAAAGT ACAACATTTC TTGGGAACTG GGTAATGAGC 650CAAATAACTA TCGGACCATG CATGGCCGGG CAGTAAATGG CAGCCAGTTG 700GGAAAGGATT ACATCCAGCT GAAGAGCCTG TTGCAGCCCA TCCGGATTTA 750TTCCAGAGCC AGCTTATATG GCCCTAATAT TGGGCGGCCG AGGAAGAATG 800TCATCGCCCT CCTAGATGGA TTCATGAAGG TGGCAGGAAG TACAGTAGAT 850GCAGTTACCT GGCAACATTG CTACATTGAT GGCCGGGTGG TCAAGGTGAT 900GGACTTCCTG AAAACTCGCC TGTTAGACAC ACTCTCTGAC CAGATTAGGA 950AAATTCAGAA AGTGGTTAAT ACATACACTC CAGGAAAGAA GATTTGGCTT 1000GAAGGTGTGG TGACCACCTC AGCTGGAGGC ACAAACAATC TATCCGATTC 1050CTATGCTGCA GGATTCTTAT GGTTGAACAC TTTAGGAATG CTGGCCAATC 1100AGGGCATTGA TGTCGTGATA CGGCACTCAT TTTTTGACCA TGGATACAAT 1150CACCTCGTGG ACCAGAATTT TAACCCATTA CCAGACTACT GGCTCTCTGT 1200CCTCTACAAG CGCCTGATCG GCCCCAAAGT CTTGGCTGTG CATGTGGCTG 1250GGCTCCAGCG GAAGCCACGG CCTGGCCGAG TGATCCGGGA CAAACTAAGG 1300ATTTATGCTC ACTGCACAAA CCACCACAAC CACAACTACG TTCGTGGGTC 1350CATTACACTT TTTATCATCA ACTTGCATCG ATCAAGAAAG AAAATCAAGC 1400TGGCTGGGAC TCTCAGAGAC AAGCTGGTTC ACCAGTACCT GCTGCAGCCC 1450TATGGGCAGG AGGGCCTAAA GTCCAAGTCA GTGCAACTGA ATGGCCAGCC 1500CTTAGTGATG GTGGACGACG GGACCCTCCC AGAATTGAAG CCCCGCCCCC 1550TTCGGGCCGG CCGGACATTG GTCATCCCTC CAGTCACCAT GGGCTTTTTT 1600GTGGTCAAGA ATGTCAATGC TTTGGCCTGC CGCTACCGAT AAGCTATCCT 1650CACACTCATG GCTACCAGTG GGCCTGCTGG GCTGCTTCCA CTCCTCCACT 1700CCAGTAGTAT CCTCTGTTTT CAGACATCCT AGCAACCAGC CCCTGCTGCC 1750CCATCCTGCT GGAATCAACA CAGACTTGCT CTCCAAAGAG ACTAAATGTC 1800ATAGCGTGAT CTTAGCCTAG GTAGGCCACA TCCATCCCAA AGGAAAATGT 1850AGACATCACC TGTACCTATA TAAGGATAAA GGCATGTGTA TAGAGCAA 18982)SEQ ID NO:5信息
(i)序列特征:
(A)长度:538
(B)类型:氨基酸
(C)链型:单链
(D)拓扑结构:线性
(xi)序列描述: SEQ ID NO:5:Met Arg Val Leu Cys Ala Phe Pro Glu Ala Met Pro Ser Ser Asn
5 10 15Ser Arg Pro Pro Ala Cys Leu Ala Pro Gly Ala Leu Tyr Leu Ala
20 25 30Leu Leu Leu His Leu Ser Leu Ser Ser Gln Ala Gly Asp Arg Arg
35 40 45Pro Leu Pro Val Asp Arg Ala Ala Gly Leu Lys Glu Lys Thr Leu
50 55 60Ile Leu Leu Asp Val Ser Thr Lys Asn Pro Val Arg Thr Val Asn
65 70 75Glu Asn Phe Leu Ser Leu Gln Leu Asp Pro Ser Ile Ile His Asp
80 85 90Gly Trp Leu Asp Phe Leu Ser Ser Lys Arg Leu Val Thr Leu Ala
95 100 105Arg Gly Leu Ser Pro Ala Phe Leu Arg Phe Gly Gly Lys Arg Thr
110 115 120Asp Phe Leu Gln Phe Gln Asn Leu Arg Asn Pro Ala Lys Ser Arg
125 130 135Gly Gly Pro Gly Pro Asp Tyr Tyr Leu Lys Asn Tyr Glu Asp Ala
140 145 150Arg Ser Leu Asp Lys Leu Tyr Asn Phe Ala Asp Cys Ser Gly Leu
155 160 165His Leu Ile Phe Ala Leu Asn Ala Leu Arg Arg Asn Pro Asn Asn
170 175 180Ser Trp Asn Ser Ser Ser Als Leu Ser Leu Leu Lys Tyr Ser Ala
185 190 195Ser Lys Lys Tyr Asn Ile Ser Trp Glu Leu Gly Asn Glu Pro Asn
200 205 210Asn Tyr Arg Thr Met His Gly Arg Ala Val Asn Gly Ser Gln Leu
215 220 225Gly Lys Asp Tyr Ile Gln Leu Lys Ser Leu Leu Gln Pro Ile Arg
230 235 240Ile Tyr Ser Arg Ala Ser Leu Tyr Gly Pro Asn Ile Gly Arg Pro
245 250 255Arg Lys Asn Val Ile Ala Leu Leu Asp Gly Phe Met Lys Val Ala
260 265 270Gly Ser Thr Val Asp Ala Val Thr Trp Gln His Cys Tyr Ile Asp
275 280 285Gly Arg Val Val Lys Val Met Asp Phe Leu Lys Thr Arg Leu Leu
290 295 300Asp Thr Leu Ser Ala Gln Ile Arg Lys Ile Gln Lys Val Val Asn
305 310 315Thr Tyr Thr Pro Gly Lys Lys Ile Trp Leu Glu Gly Val Val Thr
320 325 330Thr Ser Als Gly Gly Thr Asn Asn Leu Ser Asp Ser Tyr Ala Ala
335 340 345Gly Phe Leu Trp Leu Asn Thr Leu Gly Met Leu Ala Asn Gln Gly
350 355 360Ile Asp Val Val Ile Arg His Ser Phe Phe Asp His Gly Tyr Asn
365 370 375His Leu Val Asp Gln Asn Phe Asn Pro Leu Pro Asp Tyr Trp Leu
380 385 390Ser Leu Leu Tyr Lys Arg Leu Ile Gly Pro Lys Val Leu Ala Val
395 400 405His Val Ala Gly Leu Gln Arg Lys Pro Arg Pro Gly Arg Val Ile
410 415 420Arg Asp Lys Leu Arg Ile Tyr Ala His Cys Thr Asn His His Asn
425 430 435His Asn Tyr Val Arg Gly Ser Ile Thr Leu Phe Ile Ile Asn Leu
440 445 450His Arg Ser Arg Lys Lys Ile Lys Leu Ala Gly Thr Leu Arg Asp
455 460 465Lys Leu Val His Gln Tyr Leu Leu Gln Pro Tyr Gly Gln Glu Gly
470 475 480Leu Lys Ser Lys Ser Val Gln Leu Asn Gly Gln Pro Leu Val Met
485 490 495Val Asp Asp Gly Thr Leu Pro Glu Leu Lys Pro Arg Pro Leu Arg
500 505 510Ala Gly Arg Thr Leu Val Ile Pro Pro Val Thr Met Gly Phe Phe
515 520 525Val Val Lys Asn Val Asn Ala Leu Ala Cys Arg Tyr Arg
530 5352)SEQ ID NO:6信息
(i)序列特征:(A)长度:1724
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(xi)序列描述: SEQ ID NO:6:CGCTTAATTC TAGAAGAGGG ATTGAATGAG GGTGCTTTGT GCCTTCCCTG 50AAGCCATGCC CTCCAGCAAC TCCCGCCCCC CCGCGTGCCT AGCCCCGGGG 100GCTCTCTACT TGGCTCTGTT GCTCCATCTC TCCCTTTCCT CCCAGGCTGG 150AGACAGGAGA CCCTTGCCTG TAGACAGAGC TGCAGGTTTG AAGGAAAAGA 200CCCTGATTCT ACTTGATGTG AGCACCAAGA ACCCAGTCAG GACAGTCAAT 250GAGAACTTCC TCTCTCTGCA GCTGGATCCG TCCATCATTC ATGATGGCTG 300GCTCGATTTC CTAAGCTCCA AGCGCTTGGT GACCCTGGCC CGGGGACTTT 350CGCCCGCCTT TCTGCGCTTC GGGGGCAAAA GGACCGACTT CCTGCAGTTC 400CAGAACCTGA GGAACCCGGC GAAAAGCCGC GGGGGCCCGG GCCCGGATTA 450CTATCTCAAA AACTATGAGG ATGAGCCAAA TAACTATCGG ACCATGCATG 500GCGGGGCAGT AAATGGCAGC CAGTTGGGAA AGGATTACAT CCAGCTGAAG 550AGCCTGTTGC AGCCCATCCG GATTTATTCC AGAGCCAGCT TATATGGCCC 600TAATATTGGG CGGCCGAGGA AGAATGTCAT CGCCCTCCTA GATGGATTCA 650TGAAGGTGGC AGGAAGTACA GTAGATGCAG TTACCTGGCA ACATTGCTAC 700ATTGATGGCC GGGTGGTCAA GGTGATGGAC TTCCTGAAAA CTCGCCTGTT 750AGACACACTC TCTGACCAGA TTAGGAAAAT TCAGAAAGTG GTTAATACAT 800ACACTCCAGG AAAGAAGATT TGGCTTGAAG GTGTGGTGAC CACCTCAGCT 850GGAGGCACAA ACAATCTATC CGATTCCTAT GCTGCAGGAT TCTTATGGTT 900GAACACTTTA GGAATGCTGG CCAATCAGGG CATTGATGTC GTGATACGGC 950ACTCATTTTT TGACCATGGA TACAATCACC TCGTGGACCA GAATTTTAAC 1000CCATTACCAG ACTACTGGCT CTCTCTCCTC TACAAGCGCC TGATCGGCCC 1050CAAAGTCTTG GCTGTGCATG TGGCTGGGCT CCAGCGGAAG CCACGGCCTG 1100GCCGAGTGAT CCGGGACAAA CTAAGGATTT ATGCTCACTG CACAAACCAC 1150CACAACCACA ACTACGTTCG TGGGTCCATT ACACTTTTTA TCATCAACTT 1200GCATCGATCA AGAAAGAAAA TCAAGCTGGC TGGGACTCTC AGAGACAAGC 1250TGGTTCACCA GTACCTGCTG CAGCCCTATG GGCAGGAGGG CCTAAAGTCC 1300AAGTCAGTGC AACTGAATGG CCAGCCCTTA GTGATGGTGG ACGACGGGAC 1350CCTCCCAGAA TTGAAGCCCC GCCCCCTTCG GGCCGGCCGG ACATTGGTCA 1400TCCCTCCAGT CACCATGGGC TTTTTTGTGG TCAAGAATGT CAATGCTTTG 1450GCCTGCCGCT ACCGATAAGC TATCCTCACA CTCATGGCTA CCAGTGGGCC 1500TGCTGGGCTG CTTCCACTCC TCCACTCCAG TAGTATCCTC TGTTTTCAGA 1550CATCCTAGCA ACCAGCCCCT GCTGCCCCAT CCTGCTGGAA TCAACACAGA 1600CTTGCTCTCC AAAGAGACTA AATGTCATAG CGTGATCTTA GCCTAGGTAG 1650GCCACATCCA TCCCAAAGGA AAATGTAGAC ATCACCTGTA CCTATATAAG 1700GATAAAGGCA TGTGTATAGA GCAA 1724(2)SEQ ID NO:7信息
(i)序列特征:
(A)长度:480
(B)类型:氨基酸
(C)链型:单链
(D)拓扑结构:线性
(xi)序列描述: SEQ ID NO:7:Met Arg Val Leu Cys Ala Phe Pro Glu Ala Met Pro Ser Ser Asn
5 10 15Ser Arg Pro Pro Ala Cys Leu Ala Pro Gly Ala Leu Tyr Leu Ala
20 25 30Leu Leu Leu His Leu Ser Leu Ser Ser Gln Ala Gly Asp Arg Arg
35 40 45Pro Leu Pro Val Asp Arg Ala Ala Gly Leu Lys Glu Lys Thr Leu
50 55 60Ile Leu Leu Asp Val Ser Thr Lys Asn Pro Val Arg Thr Val Asn
65 70 75Glu Asn Phe Leu Ser Leu Gln Leu Asp Pro Ser Ile Ile His Asp
80 85 90Gly Trp Leu Asp Phe Leu Ser Ser Lys Arg Leu Val Thr Leu Ala
95 100 105Arg Gly Leu Ser Pro Ala Phe Leu Arg Phe Gly Gly Lys Arg Thr
110 115 120Asp Phe Leu Gln Phe Gln Asn Leu Arg Asn Pro Ala Lys Ser Arg
125 130 135Gly Gly Pro Gly Pro Asp Tyr Tyr Leu Lys Asn Tyr Glu Asp Glu
140 145 150Pro Asn Asn Tyr Arg Thr Met His Gly Arg Ala Val Asn Gly Ser
155 160 165Gln Leu Gly Lys Asp Tyr Ile Gln Leu Lys Ser Leu Leu Gln Pro
170 175 180Ile Arg Ile Tyr Ser Arg Ala Ser Leu Tyr Gly Pro Asn Ile Gly
185 190 195Arg Pro Arg Lys Asn Val Ile Ala Leu Leu Asp Gly Phe Met Lys
200 205 210Val Ala Gly Ser Thr Val Asp Ala Val Thr Trp Gln His Cys Tyr
215 220 225Ile Asp Gly Arg Val Val Lys Val Met Asp Phe Leu Lys Thr Arg
230 235 240Leu Leu Asp Thr Leu Ser Asp Gln Ile Arg Lys Ile Gln Lys Val
245 250 255Val Asn Thr Tyr Thr Pro Gly Lys Lys Ile Trp Leu Glu Gly Val
260 265 270Val Thr Thr Ser Ala Gly Gly Thr Asn Asn Leu Ser Asp Ser Tyr
275 280 285Ala Ala Gly Phe Leu Trp Leu Asn Thr Leu Gly Met Leu Ala Asn
290 295 300Gln Gly Ile Asp Val Val Ile Arg His Ser Phe Phe Asp His Gly
305 310 315Tyr Asn His Leu Val Asp Gln Asn Phe Asn Pro Leu Pro Asp Tyr
320 325 330Trp Leu Ser Leu Leu Tyr Lys Arg Leu Ile Gly Pro Lys Val Leu
335 340 345Ala Val His Val Ala Gly Leu Gln Arg Lys Pro Arg Pro Gly Arg
350 355 360Val Ile Arg Asp Lys Leu Arg Ile Tyr Ala His Cys Thr Asn His
365 370 375His Asn His Asn Tyr Val Arg Gly Ser Ile Thr Leu Phe Ile Ile
380 385 390Asn Leu His Arg Ser Arg Lys Lys Ile Lys Leu Ala Gly Thr Leu
395 400 405Arg Asp Lys Leu VaL His Gln Tyr Leu Leu Gln Pro Tyr Gly Gln
410 415 420Glu Gly Leu Lys Ser Lys Ser Val Gln Leu Asn Gly Gln Pro Leu
425 430 435Val Met Val Asp Asp Gly Thr Leu Pro Glu Leu Lys Pro Arg Pro
440 445 450Leu Arg Ala Gly Arg Thr Leu Val Ile Pro Pro Val Thr Met Gly
455 460 465Phe Phe Val Val Lys Asn Val Asn Ala Leu Ala Cys Arg Tyr Arg
470 475 480(2)SEQ ID NO:8信息
(i)序列特征:
(A)长度:351
(B)类型:氨基酸
(C)链型:单链
(D)拓扑结构:线性
(xi)序列描述:SEQ ID NO:8:GTTCGGCAGA GGATCATGTC TGATGTACAG AGACATTGTC CGGAGTGATG 50TTGCCTTGGA CAAGCAGAAA GGCTGTAAGA TTGGCCAGCA CCCTGATGTC 100ATGCTGGAGC TCCAGAGAGA GAAGGCATCC AGACTGTCTG GTTCTTCTGA 150AGGAGCAATA CTCCAATACT TACAGTAACC TCATATTAAC AGGTCTCTAG 200ACAAACTTTA TAACTTTGCT GATTGCTCTG GACTCCACCT GATATTTGCT 250CTAAATGCAC TGCGTCGTAA TCCCAATAAC TCCTGGAACA GTTCTAGTGC 300CCTGAGCCTG TTGAAGTACA GTGCCAGCAA AAAGTACAAC ATTTCTTGGG 350A 351(2)SEQ ID NO:9信息
(i)序列特征:
(A)长度:543
(B)类型:氨基酸
(C)链型:单链
(D)拓扑结构:线性
(xi)序列描述: SEQ ID NO:9:Met Leu Leu Arg Ser Lys Pro Ala Leu Pro Pro Pro Leu Met Leu Leu
5 10 15Leu Leu Gly Pro Leu Gly Pro Leu Ser Pro Gly Ala Leu Pro Arg Pro
20 25 30Ala Gln Ala Gln Asp Val Val Asp Leu Asp Phe Phe Thr Gln Glu Pro
35 40 45Leu His Leu Val Ser Pro Ser Phe Leu Ser Val Thr Ile Asp Ala Asn
50 55 60Leu Ala Thr Asp Pro Arg Phe Leu Ile Leu Leu Gly Ser Pro Lys Leu65 70 75 80Arg Thr Leu Ala Arg Gly Leu Ser Pro Ala Tyr Leu Arg Phe Gly Gly
85 90 95Thr Lys Thr Asp Phe Leu Ile Phe Asp Pro Lys Lys Glu Ser Thr Phe
100 105 110Glu Glu Arg Ser Tyr Trp Gln Ser Gln Val Asn Gln Asp Ile Cys Lys
115 120 125Tyr Gly Ser Ile Pro Pro Asp Val Glu Glu Lys Leu Arg Leu Glu Trp
130 135 140Pro Tyr Gln Glu Gln Leu Leu Leu Arg Glu His Tyr Gln Lys Lys Phe145 150 155 160Lys Asn Ser Thr Tyr Ser Arg Ser Ser Val Asp Val Leu Tyr Thr Phe
165 170 175Ala Asn Cys Ser Gly Leu Asp Leu Ile Phe Gly Leu Asn Ala Leu Leu
180 185 190Arg Thr Ala Asp Leu Gln Trp Asn Ser Ser Asn Ala Gln Leu Leu Leu
195 200 205Asp Tyr Cys Ser Ser Lys Gly Tyr Asn Ile Ser Trp Glu Leu Gly Asn
210 215 220Glu Pro Asn Ser Phe Leu Lys Lys Ala Asp Ile Phe Ile Asn Gly Ser225 230 235 240Gln Leu Gly Glu Asp Tyr Ile Gln Leu His Lys Leu Leu Arg Lys Ser
245 250 255Thr Phe Lys Asn Ala Lys Leu Tyr Gly Pro Asp Val Gly Gln Pro Arg
260 265 270Arg Lys Thr Ala Lys Met Leu Lys Ser Phe Leu Lys Ala Gly Gly Glu
275 280 285Val Ile Asp Ser Val Thr Trp His His Tyr Tyr Leu Asn Gly Arg Thr
290 295 300Ala Thr Arg Glu Asp Phe Leu Asn Pro Asp Val Leu Asp Ile Phe Ile305 310 315 320Ser Ser Val Gln Lys Val Phe Gln Val Val Glu Ser Thr Arg Pro Gly
325 330 335Lys Lys Val Trp Leu Gly Glu Thr Ser Ser Ala Tyr Gly Gly Gly Ala
340 345 350Pro Leu Leu Ser Asp Thr Phe Ala Ala Gly Phe Met Trp Leu Asp Lys
355 360 365Leu Gly Leu Ser Ala Arg Met Gly Ile Glu Val Val Met Arg Gln Val
370 375 380Phe Phe Gly Ala Gly Asn Tyr His Leu Val Asp Glu Asn Phe Asp Pro385 390 395 400Leu Pro Asp Tyr Trp Leu Ser Leu Leu Phe Lys Lys Leu Val Gly Thr
405 410 415Lys Val Leu Met Ala Ser Val Gln Gly Ser Lys Arg Arg Lys Leu Arg
420 425 430Val Tyr Leu His Cys Thr Asn Thr Asp Asn Pro Arg Tyr Lys Glu Gly
435 440 445Asp Leu Thr Leu Tyr Ala Ile Asn Leu His Asn Val Thr Lys Tyr Leu
450 455 460Arg Leu Pro Tyr Pro Phe Ser Asn Lys Gln Val Asp Lys Tyr Leu Leu465 470 475 480Arg Pro Leu Gly Pro His Gly Leu Leu Ser Lys Ser Val Gln Leu Asn
485 490 495Gly Leu Thr Leu Lys Met Val Asp Asp Gln Thr Leu Pro Pro Leu Met
500 505 510Glu Lys Pro Leu Arg Pro G1y Ser Ser Leu Gly Leu Pro Ala Phe Ser
515 520 525Tyr Ser Phe Phe Val Ile Arg Asn Ala Lys Val Ala Ala Cys Ile
530 535 540(2)SEQ ID NO:10信息
(i)序列特征:
(A)长度:23
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(xi)序列描述: SEQ ID NO:10:GGAGAGCAAG TCTGTGTTGA TTC 23(2)SEQ ID NO:11信息
(i)序列特征:
(A)长度:22
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(xi)序列描述: SEQ ID NO:11:CACTGGTAGC CATGAGTGTG AG 22(2)SEQ ID NO:12信息
(i)序列特征:
(A)长度:22
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(xi)序列描述: SEQ ID NO:12:TTGGTCATCC CTCCAGTCAC CA 22(2)SEQ ID NO:13信息
(i)序列特征:
(A)长度:2
(B)类型:氨基酸
(C)链型:单链
(D)拓扑结构:线性
(xi)序列描述: SEQ ID NO:13Asp Glu(2)SEQ ID NO:14信息
(i)序列特征:
(A)长度:23
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(xi)序列描述: SEQ ID NO:14:CTTGCCTGTA GACAGAGCTG CAG 23(2)SEQ ID NO:15信息
(i)序列特征:
(A)长度:2396
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(xi)序列描述: SEQ ID NO:15TTTCTAGTTG CTTTTAGCCA ATGTCGGATC AGGTTTTTCA AGCGACAAAG 50AGATACTGAG ATCCTGGGCA GAGGACATCC TAGCTCGGTC AGATTTGGGC 100AGGCTCAAGT GACCAGTGTC TTAAGGCAGA AGGGAGTCGG GGTAGGGTCT 150GGCTGAACCC TCAACCGGGG CTTTTAACTC AGGGTCTAGT CCTGGCGCCA 200AATGGATGGG ACCTAGAAAA GGTGACAGAG TGCGCAGGAC ACCAGGAAGC 250TGGTCCCACC CCTGCGCGGC TCCCGGGCGC TCCCTCCCCA GGCCTCCGAG 300GATCTTGGAT TCTGGCCACC TCCGCACCCT TTGGATGGGT GTGGATGATT 350TCAAAAGTGG ACGTGACCGC GGCGGAGGGG AAAGCCAGCA CGGAAATGAA 400AGAGAGCGAG GAGGGGAGGG CGGGGAGGGG AGGGCGCTAG GGAGGGACTC 450CCGGGAGGGG TGGGAGGGAT GGAGCGCTGT GGGAGGGTAC TGAGTCCTGG 500CGCCAGAGGC GAAGCAGGAC CGGTTGCAGG GGGCTTGAGC CAGCGCGCCG 550GCTGCCCCAG CTCTCCCGGC AGCGGGCGGT CCAGCCAGGT GGGATGCTGA 600GGCTGCTGCT GCTGTGGCTC TGGGGGCCGC TCGGTGCCCT GGCCCAGGGC 650GCCCCCGCGG GGACCGCGCC GACCGACGAC GTGGTAGACT TGGAGTTTTA 700CACCAAGCGG CCGCTCCGAA GCGTGAGTCC CTCGTTCCTG TCCATCACCA 750TCGACGCCAG CCTGGCCACC GACCCGCGCT TCCTCACCTT CCTGGGCTCT 800CCAAGGCTCC GTGCTCTGGC TAGAGGCTTA TCTCCTGCAT ACTTGAGATT 850TGGCGGCACA AAGACTGACT TCCTTATTTT TGATCCGGAC AAGGAACCGA 900CTTCCGAAGA AAGAAGTTAC TGGAAATCTC AAGTCAACCA TGATATTTGC 950AGGTCTGAGC CGGTCTCTGC TGCGGTGTTG AGGAAACTCC AGGTGGAATG 1000GCCCTTCCAG GAGCTGTTGC TGCTCCGAGA GCAGTACCAA AAGGAGTTCA 1050AGAACAGCAC CTACTCAAGA AGCTCAGTGG ACATGCTCTA CAGTTTTGCC 1100AAGTGCTCGG GGTTAGACCT GATCTTTGGT CTAAATGCGT TACTACGAAC 1150CCCAGACTTA CGGTGGAACA GcTCCAACGC CCAGCTTCTC CTTGACTACT 1200GCTCTTCCAA GGGTTATAAC ATcTCCTGGG AACTGGGCAA TGAGCCCAAC 1250AGTTTcTGGA AGAAAGCTCA CATTCTCATC GATGGGTTGC AGTTAGGAGA 1300AGACTTTGTG GAGTTGCATA AACTTcTACA AAGGTCAGCT TTCCAAAATG 1350CAAAACTCTA TGGTCCTGAC ATCGGTCAGC CTCGAGGGAA GACAGTTAAA 1400CTGCTGAGGA GTTTCCTGAA GGCTGGCGGA GAAGTGATCG ACTCTCTTAC 1450ATGGCATCAC TATTACTTGA ATGGACGCAT CGCTACCAAA GAAGATTTTC 1500TGAGCTCTGA TGCGCTGGAC ACTTTTATTC TCTCTGTGCA AAAAATTCTG 1550AAGGTCACTA AAGAGATCAC ACCTGGCAAG AAGGTCTGGT TGGGAGAGAC 1600GAGCTCAGCT TACGGTGGCG GTGCACCCTT GCTGTCCAAC ACCTTTGCAG 1650CTGGCTTTAT GTGGCTGGAT AAATTGGGCC TGTCAGCCCA GATGGGCATA 1700GAAGTCGTGA TGAGGCAGGT GTTCTTCGGA GCAGGCAACT ACCACTTAGT 1750GGATGAAAAC TTTGAGCCTT TACCTGATTA CTGGCTCTCT CTTCTGTTCA 1800AGAAACTGGT AGGTCCCAGG GTGTTACTGT CAAGAGTGAA AGGCCCAGAC 1850AGGAGCAAAC TCCGAGTGTA TCTCCACTGC ACTAACGTCT ATCACCCACG 1900ATATCAGGAA GGAGATCTAA CTCTGTATGT CCTGAACCTC CATAATGTCA 1950CCAAGC&CTT GAAGGTACCG CCTCCGTTGT TCAGGAAACC AGTGGATACG 2000TACCTTCTGA AGCCTTCGGG GCCGGATGGA TTACTTTCCA AATCTGTCCA 2050ACTGAACGGT CAAATTCTGA AGATGGTGGA TGAGCAGACC CTGCCAGCTT 2100TGACAGAAAA ACCTCTCCCC GCAGGAAGTG CACTAAGCCT GCCTGCCTTT 2150TCCTATGGTT TTTTTGTCAT AAGAAATGCC AAAATCGCTG CTTGTATATG 2200AAAATAAAAG GCATACGGTA CCCCTGAGAC AAAAGCCGAG GGGGGTGTTA 2250TTCATAAAAC AAAACCCTAG TTTAGGAGGC CACCTCCTTG CCGAGTTCCA 2300GAGCTTCGGG AGGGTGGGGT ACACTTCAGT ATTACATTCA GTGTGGTGTT 2350CTCTCTAAGA AGAATACTGC AGGTGGTGAC AGTTAATAGC ACTGTG 2396(2)SEQ ID NO:16信息
(i)序列特征:
(A)长度:22
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(xi)序列描述: SEQ,ID NO:16:GAGCAGCCAG GTGAGCCCAR GA 22(2)SEQ ID NO:17信息
(i)序列特征:
(A)长度:24
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(xi)序列描述: SEQ ID NO:17:TCAGRTGCAA GCRGCAACTT TGGC 24(2)SEQ ID NO:18信息
(i)序列特征:
(A)长度:21
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(xi)序列描述: SEQ ID NO:18:CACCCTGATG TCATGCTGGA G 21(2)SEQ ID NO:19信息
(i)序列特征:
(A) 长度:23
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(xi)序列描述: SEQ ID NO:19:CATCTAGGAG AGCAATGACG TTC 23(2)SEQ ID NO:20信息
(i)序列特征:
(A)长度:27
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(xi)序列描述: SEQ ID NO:20:CCATCCTAAT ACGACTCACT ATAGGGC 27(2)SEQ ID NO:2l信息
(i)序列特征:
(A)长度:23
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(xi)序列描述: SEQ ID NO:21:ACTCACTATA GGGCTCGAGC GGC 23(2)SEQ ID NO:22信息
(i)序列特征:
(A)长度: 15
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(xi)序列描述: SEQ ID NO:22:TTTTTTTTTT TTTTT 15(2)SEQ ID NO:23信息
(i)序列特征:
(A)长度:560
(B)类型:核酸
(C)链型:双链
(D)拓扑结构:线性
(xi)序列描述: SEQ ID NO:23GGCACGAGGC TAGTGGAGAG ACTGACAAGC AGTCAGCTCA GCGGTCACAA 50TACTGTGTGA CAGGAGCTGA GATCCAAGAA GTACTGGGTC CTGTGGGAGC 100ACCCCTGACT TGAAGGACAA GTCAGTGCAA CTGAATGGCC AGCCCTTAGT 150GATGGTGGAC GACGGGACCC TCCCAGAATT GAAGCCCCGC CCCCTTCGGG 200CCGGCCGGAC ATTGGTCATC CCTCCAGTCA CCATGGGCTT TTTTGTGGTC 250AAGAATGTCA ATGCTTTGGC CTGCCGCTAC CGATAAGCTA TCCTCACACT 300CATGGCTACC AGTGGGCCTG CTGGGCTGCT TCCACTCCTC CACTCCAGTA 350GTATCCTCTG TTTTCAGACA TCCTAGCAAC CAGCCCCTGC TGCCCCATCC 400TGCTGGAATC AACACAGACT TGCTCTCCAA AGAGACTAAA TGTCATAGCG 450TGATCTTAGC CTAGGTAGGC CACATCCATC CCAAAGGAAA ATGTAGACAT 500CACCTGTACC TATATAAGGA TAAAGGCATG TGTATAGAGC AAAAAAAAAA 550AAAAAAAAAA 560(2)SEQ ID NO:24信息
(i)序列特征:
(A)长度:1721
(B)类型:核酸
(C)链型:双链
(D)拓扑结构:线性
(xi)序列描述: SEQ ID NO:24:CTAGAGCTTT CGACTCTCCG CTGCGCGGCA GCTGGCGGGG GGAGCAGCCA GGTGAGCCCA 60AGATGCTGCT GCGCTCGAAG CCTGCGCTGC CGCCGCCGCT GATGCTGCTG CTCCTGGGGC 120CGCTGGGTCC CCTCTCCCCT GGCGCCCTGC CCCGACCTGC GCAAGCACAG GACGTCGTGG 180ACCTGGACTT CTTCACCCAG GAGCCGCTGC ACCTGGTGAG CCCCTCGTTC CTGTCCGTCA 240CCATTGACGC CAACCTGGCC ACGGACCCGC GGTTCCTCAT CCTCCTGGGT TCTCCAAAGC 300TTCGTACCTT GGCCAGAGGC TTGTCTCCTG CGTACCTGAG GTTTGGTGGC ACCAAGACAG 360ACTTCCTAAT TTTCGATCCC AAGAAGGAAT CAACCTTTGA AGAGAGAAGT TACTGGCAAT 420CTCAAGTCAA CCAGGATATT TGCAAATATG GATCCATCCC TCCTGATGTG GAGGAGAAGT 480TACGGTTGGA ATGGCCCTAC CAGGAGCAAT TGCTACTCCG AGAACACTAC CAGAAAAAGT 540TCAAGAACAG CACCTACTCA AGAAGCTCTG TAGATGTGCT ATACACTTTT GCAAACTGCT 600CAGGACTGGA CTTGATCTTT GGCCTAAATG CGTTATTAAG AACAGCAGAT TTGCAGTGGA 660ACAGTTCTAA TGCTCAGTTG CTCCTGGACT ACTGCTCTTC CAAGGGGTAT AACATTTCTT 720GGGAACTAGG CAATGAACCT AACAGTTTCC TTAAGAAGGC TGATATTTTC ATCAATGGGT 780CGCAGTTAGG AGAAGATTAT ATTCAATTGC ATAAACTTCT AAGAAAGTCC ACCTTCAAAA 840ATGCAAAACT CTATGGTCCT GATGTTGGTC AGCCTCGAAG AAAGACGGCT AAGATGCTGA 900AGAGCTTCCT GAAGGCTGGT GGAGAAGTGA TTGATTCAGT TACATGGCAT CACTACTATT 960TGAATGGACG GACTGCTACC AGGGAAGATT TTCTAAACCC TGATGTATTG GACATTTTTA 1020TTTCATCTGT GCAAAAAGTT TTCCAGGTGG TTGAGAGCAC CAGGCCTGGC AAGAAGGTCT 1080GGTTAGGAGA AACAAGCTCT GCATATGGAG GCGGAGCGCC CTTGCTATCC GACACCTTTG 1140CAGCTGGCTT TATGTGGCTG GATAAATTGG GCCTGTCAGC CCGAATGGGA ATAGAAGTGG 1200TGATGAGGCA AGTATTCTTT GGAGCAGGAA ACTACCATTT AGTGGATGAA AACTTCGATC 1260CTTTACCTGA TTATTGGCTA TCTCTTCTGT TCAAGAAATT GGTGGGCACC AAGGTGTTAA 1320TGGCAAGCGT GCAAGGTTCA AAGAGAAGGA AGCTTCGAGT ATACCTTCAT TGCACAAACA 1380CTGACAATCC AAGGTATAAA GAAGGAGATT TAACTCTGTA TGCCATAAAC CTCCATAACG 1440TCACCAAGTA CTTGCGGTTA CCCTATCCTT TTTCTAACAA GCAAGTGGAT AAATACCTTC 1500TAAGACCTTT GGGACCTCAT GGATTACTTT CCAAATCTGT CCAACTCAAT GGTCTAACTC 1560TAAAGATGGT GGATGATCAA ACCTTGCCAC CTTTAATGGA AAAACCTCTC CGGCCAGGAA 1620GTTCACTGGG CTTGCCAGCT TTCTCATATA GTTTTTTTGT GATAAGAAAT GCCAAAGTTG 1680CTGCTTGCAT CTGAAAATAA AATATACTAG TCCTGACACT G 1721(2)SEQ ID NO:25信息
(i)序列特征:
(A)长度:45
(B)类型:核酸
(C)链型:单链
(D)拓扑结构:线性
(xi)序列描述: SEQ ID NO:24:CTTACTTGTC ATCGTCGTCC TTGTAGTCTC GGTAGCGGCA GGCCA 45
Claims (21)
1.一种分离的核酸,它包括能与SEQ ID NO:1,4,6或其部分杂交的多核苷酸,杂交条件是:68℃6×SSC,1%SDS,5×Denharts,10%硫酸葡聚糖,100ug/ml鲑精DNA,32p标记的探针,用3×SSC,0.1%SDS在68℃洗涤。
2.一种分离的核酸,当用DNA序列分析软件包的Bestfit程序分析时,它包括的多核苷酸至少与SEQ ID NO 1、4、6或其部分有60%的一致性,该软件包由Wisconsin大学的Genetic Computer Group(GCG)开发(间隙产生补偿-50,间隙扩展补偿-3)。
3.权利要求2的分离的核酸,其中所述多核苷酸如SEQ ID NO 1、4、6中所示或是其部分。
4 一种分离的核酸,它包括编码多肽的多核苷酸,当用DNA序列分析软件包的Bestfit程序分析时,该多肽至少与SEQ ID NO 3、5、7或其部分有60%的同源性,该软件包由Wisconsin大学的GeneticComputer Group(GCG)开发(间隙产生补偿-50,间隙扩展补偿-3)。
5 一种重组蛋白质,它包括由权利要求1的多核苷酸编码的多肽。
6 一种重组蛋白质,它包括由权利要求2的多核苷酸编码的多肽。
7 一种重组蛋白质,它包括由权利要求3的多核苷酸编码的多肽。
8 一种重组蛋白质,它包括由权利要求4的多核苷酸编码的多肽。
9 一种重组蛋白质,当用DNA序列分析软件包的Bestfit程序分析时,它包括的多肽至少与SEQ ID NO 3、5、7或其部分有60%的同源性,该软件包由Wisconsin大学的Genetic Computer Group(GCG)开发(间隙产生补偿-50,间隙扩展补偿-3)。
10 权利要求9的重组蛋白质,其中所述多肽如SEQ ID NO 3、5、7中所示或是其部分。
11 一种核酸构建体,它包括权利要求1的分离的核酸。
12 一种核酸构建体,它包括权利要求2的分离的核酸。
13 一种核酸构建体,它包括权利要求3的分离的核酸。
14 一种核酸构建体,它包括权利要求4的分离的核酸。
15 一种宿主细胞,它包括权利要求11的核酸构建体。
16 一种宿主细胞,它包括权利要求12的核酸构建体。
17 一种宿主细胞,它包括权利要求13的核酸构建体。
18 一种宿主细胞,它包括权利要求14的核酸构建体。
19 一种反义寡核苷酸,它包括在生理条件下,至少有10个碱基能与下列序列在体内杂交的多核苷酸或多核苷酸类似物:
(1〕多核苷酸链的一部分,当用DNA序列分析软件包的Bestfit程序分析时,它编码的多肽至少与SEQ ID NO 3、5、7或其部分有60%的同源性,该软件包由Wisconsin大学的Genetic ComputerGroup(GCG)开发(间隙产生补偿-50,间隙扩展补偿-3);
〔2〕多核苷酸链的一部分,当用DNA序列分析软件包的Bestfit程序分析时,它至少与SEQ ID NO 1、4、6或其部分有60%的一致性,该软件包由Wisconsin大学的Genetic Computer Group(GCG)开发(间隙产生补偿-50,间隙扩展补偿-3)。
20 一种包括权利要求19的反义寡核苷酸的核酶和核酶序列。
21 一种反义核酸构建体,它包括一启动子序列和指导反义RNA序列合成的多核苷酸序列,反义RNA序列至少有10个碱基能在生理条件下与下列序列在体内杂交:
(1)多核苷酸链的一部分,当用DNA序列分析软件包的Bestfit程序分析时,它编码的多肽至少与SEQ ID NO 3、5、7或其部分有60%的同源性,该软件包由Wisconsin大学的Genetic ComputerGroup(GCG)开发(间隙产生补偿-50,间隙扩展补偿-3);
(2)多核苷酸链的一部分,当用DNA序列分析软件包的Bestfit程序分析时,它至少与SEQ ID NO 1、4、6或其部分有60%的一致性,该软件包由Wisconsin大学的Genetic Computer Group(GCG)开发(间隙产生补偿-50,间隙扩展补偿-3)。
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EP1157118A4 (en) * | 1999-03-01 | 2002-07-17 | Insight Strategy & Marketing | POLYNUCLEOTID ENCODING A POLYPEPTIDE WITH HEPARANASE ACTIVITY AND ITS EXPRESSION IN GENETICALLY MODIFIED CELLS |
EP1214423A1 (en) * | 1999-09-23 | 2002-06-19 | MERCK PATENT GmbH | Heparanase-2, a member of the heparanase protein family |
CA2393855A1 (en) * | 1999-12-22 | 2001-06-28 | Oxford Glycosciences (Uk) Ltd. | Homologues of human heparanase and splice variants thereof |
KR20020062375A (ko) * | 1999-12-23 | 2002-07-25 | 쉐링 악티엔게젤샤프트 | 인간 헤파라나제-관련 폴리펩티드 및 핵산 |
GB0014447D0 (en) * | 2000-06-13 | 2000-08-09 | Smithkline Beecham Plc | Novel compounds |
AU2001287610A1 (en) * | 2000-07-12 | 2002-01-21 | Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw | A second human heparanase, and splice variants thereof, with a predominant expression in skeletal muscle, heart and pancreas |
IL162276A0 (en) * | 2004-06-01 | 2005-11-20 | Hadasit Med Res Service | Nucleic acid molecules as heparanase potent inhibitors, compositions and methods of use thereof |
FI20085308A0 (fi) * | 2008-04-11 | 2008-04-11 | Polysackaridforskning I Uppsal | Heparanaasittomia ihmiskuntaan kuulumattomia nisäkkäitä |
JP5622451B2 (ja) * | 2010-06-21 | 2014-11-12 | 小川香料株式会社 | 茶エキス |
JP6321506B2 (ja) * | 2014-09-22 | 2018-05-09 | 株式会社 資生堂 | ヘパラナーゼ阻害剤による美白方法及び美白効果を有する物質の評価方法 |
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US6177545B1 (en) * | 1997-09-02 | 2001-01-23 | Insight Strategy & Marketing Ltd. | Heparanase specific molecular probes and their use in research and medical applications |
CA2307830A1 (en) * | 1997-10-28 | 1999-05-06 | The Australian National University | Isolated nucleic acid molecule encoding mammalian endoglucuronidase and uses therefor |
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HUP0300901A2 (hu) | 2003-07-28 |
KR20020028906A (ko) | 2002-04-17 |
CA2377498A1 (en) | 2001-01-04 |
IL146527A0 (en) | 2002-07-25 |
MXPA01011708A (es) | 2003-09-10 |
NO20015526L (no) | 2001-12-18 |
NO20015526D0 (no) | 2001-11-12 |
EP1212341A4 (en) | 2002-11-27 |
WO2001000643A3 (en) | 2005-02-24 |
WO2001000643A2 (en) | 2001-01-04 |
AU777343B2 (en) | 2004-10-14 |
EP1212341A1 (en) | 2002-06-12 |
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JP2003503070A (ja) | 2003-01-28 |
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