CN1236393A - 几丁质酶材料和方法 - Google Patents
几丁质酶材料和方法 Download PDFInfo
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- CN1236393A CN1236393A CN97197145A CN97197145A CN1236393A CN 1236393 A CN1236393 A CN 1236393A CN 97197145 A CN97197145 A CN 97197145A CN 97197145 A CN97197145 A CN 97197145A CN 1236393 A CN1236393 A CN 1236393A
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Abstract
本发明提供纯化的和分离的编码人几丁质酶的多核苷酸序列。还提供重组生产人几丁质酶产物的材料和方法,所述产物期望能用作治疗真菌性感染的产品或用于开发治疗真菌性感染的产品。
Description
这是1996年6月14日提交的美国申请序号08/663,618的部分继续。
发明领域
本发明总的来说涉及人几丁质酶,更确切的说是涉及纯化的和分离的编码人几丁质酶的多核苷酸、所述核苷酸所编码的几丁质酶产物、重组生产几丁质酶产物的材料和方法和对该几丁质酶专一性的抗体物质。
背景
几丁质是一种β-(1,4)-连接的N-乙酰氨基葡萄糖残基的线状同聚物。该多糖仅次于作为最丰富有机物质的纤维素。节肢动物的外骨骼是由几丁质组成的。另外,真菌和其它的寄生物在它们的外部细胞璧中含有几丁质,在细胞壁中几丁质行使重要的结构和保护作用。破坏真菌的细胞壁和膜已成为对付真菌和寄生物的有用的治疗方案。例如两性霉素和氟康唑通过作用于膜类固醇来行使它们的抗真菌活性。尽管有这些抗真菌疗法,但人类的真菌感染已日益成为威胁生命的疾病的主要原因。参见Geogopapadakou等,Trends Microbiol.,3:98-104(1995)。导致这些疾病的真菌种类和寄生物主要是:念珠菌属、曲霉属、隐球菌属、组织浆菌属、球孢子菌属和肺囊虫属。这些病原体对无免疫应答的个体,例如艾滋病病人、正在进行化疗的病人和免疫抑制器官移植病人特别危险。
几丁质能被几丁质酶降解。在植物、微生物和动物中发现几丁质酶。细菌几丁质帮助提供细胞生长的碳源。昆虫在每次蜕皮时产生几丁质酶来消化它们的角质层。在植物中,几丁质酶被认为为抵抗寄生性真菌提供保护作用。几丁质酶已经从许多细菌[例如粘质沙雷氏菌,Jones等,EMBO J,5:467-473(1996)]、植物[例如烟草,Heitz等,Mol.Gen.Genet.,245:246-254(1994)]和昆虫[例如黄蜂,Krishnan等,J.Biol.Chem.,269:20971-20976(1994)]物种中克隆出来。
已经在哺乳动物中鉴定出与细菌、昆虫和植物的几丁质酶具有低同源性(低于40%的氨基酸同一性)的几种蛋白质,例如人软骨gp-39(C-gp39)[Hakale等,J.Biol.Chem.,268:25803-25810(1993)]、人糖蛋白YKL-40[Johansen等,Eur.J.Cancer,31A:1437-1442(1995)]、来源于绵羊[DeSouza等,Endocrinology,136:2485-2496(1995)]、奶牛、人类的输卵管专一性的***诱导的蛋白质以及来源于激活的鼠巨噬细胞的分泌蛋白质[Chang等,基因库M94584]。然而,还没有报道这些蛋白质的几丁质-降解活性。这些蛋白质的功能是未知的,但是已经假设它们参与组织重建(remodeling)。Hakala等(见上述)报道:在来源于类风湿性关节炎病人的滑膜和软骨的样本中可检测到C-gp39,但对于正常人未检测到。Rechlies等,Arthritis Rheumatism,36(9 SUPPL.):S190(1993)报道了将C-gp39蛋白质定位于软骨浅层的一独特的细胞群体上。Johansen等(见上述)报道:测定YPL-40血清水平作为复发性乳腺癌病人的转移性疾病和存活的程度的有潜力的预后标记是有价值的。
Fscott等,Infect.Immun.,63:4770-4773(1995)在白细胞和人血清中证实了几丁质酶的活性。Overdijk等,Glycobiology,4:797-803(1994)描述了从人血清和大鼠肝脏中分离一种几丁质酶(4-甲基伞形基(lumbelliferyl)-四-乙酰壳四糖苷水解酶)。Renkema等,J.Biol.Chem.,270:2198-2202 (1995年二月)从高歇氏病病人的脾脏中制备出人壳三糖苷酶。他们的制备物显示有几丁质酶活性,并且该论文报道了该制备物的蛋白质组分的一小部分氨基酸序列(22个氨基末端残基和胰蛋白酶片段的21个残基)。人几丁质酶的功能也是未知的,但在此论文中提出了几丁质酶与高歇氏病的病理学关系。该小组最近发表的论文[Boot等,J.Biol.Chem.,270(44):26252-26256(1995年11月)],描述了编码显示有几丁质酶活性的产物的人巨噬细胞cDNA的克隆。该小组在1995年2月的论文中所报道的部分氨基酸序列与推断出的人巨噬细胞cDNA产物的氨基酸序列部分相吻合。另参见国际专利公布号WO96/40940。
考虑到日益增加的威胁生命的真菌感染在无免疫应答病人中的发病率,本领域现在需要鉴定和分离编码人几丁质酶的多核苷酸序列,来开发用于该酶的重组生产的材料和方法,并生产用于检测血浆中几丁质酶的试剂。
发明概述
本发明提供新型的纯化的和分离的编码人几丁质酶或其片段和其类似物的多核苷酸(即DNA和RNA,有义链和反义链);提供重组生产几丁质酶多肽、其片段和其类似物的方法;提供纯化的和分离的几丁质酶多肽片段和类似物;提供这些多肽片段和其片段及类似物的抗体;和包含这些多肽、片段、类似物或抗体的药用组合物。
具体提供的有:编码人几丁质酶氨基酸序列SEQ ID NO:2或4,特别是其1-445氨基酸的纯化分离的多核苷酸;包含SEQ ID NO:1或3的编码蛋白质的核苷酸、特别是SEQ ID NO:1的65-1402核苷酸或SEQ IDNO:3的90-1427核苷酸的DNA;包含编码SEQ ID NO:14或15的氨基酸序列的多核苷酸序列的纯化分离的多核苷酸;选自以下的编码人几丁质酶的纯化分离的多核苷酸:(a)包含或示于SEQ ID NO:1或示于SEQID NO:3中序列的蛋白质编码部分的双链DNA,(b)在严格条件下杂交到(a)的DNA非编码链上的DNA,和(c)如果没有遗传密码的丰余性,能在严格条件下杂交到(a)或(b)的DNA序列的非编码链上的DNA;包含这些DNA的载体,特别是其中DNA***纵性连接到表达控制DNA序列上的表达载体;用这些DNA以允许在所述的人几丁质酶宿主细胞中表达的方式稳定转化或转染的宿主细胞;生产人几丁质酶的方法,包括在营养培养基中培养这些宿主细胞,并从所述的宿主细胞或所述营养培养基中分离人几丁质酶;通过该方法生产的纯化分离的多肽;包含SEQID NO:2或4的人几丁质酶氨基酸序列、特别是其1-445氨基酸的纯化分离的多肽;缺乏成熟人几丁质酶从1到大约72个C末端氨基酸残基的人几丁质酶片段,特别是SEQ ID NO:14的人几丁质酶片段;SEQ IDNO:15的人几丁质酶类似物;产生与上述多肽的其中一个发生专一性反应的单克隆抗体的杂交瘤细胞系;和由这些杂交瘤产生的单克隆抗体。
本发明优选的DNA序列包括基因组序列和cDNA序列,还包括完全或部分化学合成的DNA序列。在SEQ ID NO:1和SEQ ID NO:3中显示了两个人类cDNA的核苷酸序列,它们编码人几丁质酶假定等位变异体,并包括5’和3’的非编码序列。本发明设想了这些DNA序列和在标准的严格条件下,或如果没有遗传密码的丰余性则杂交到其非编码链的DNA序列。本发明优选的DNA包括人几丁质酶编码区(对应于SEQID NO:1的2-1402核苷酸或SEQ ID NO:3的27-1427核苷酸);和无其信号序列的成熟的、分泌的人几丁质酶蛋白质的假定编码序列(SEQ IDNO:1的65-1402核苷酸,或SEQ ID NO:3的90-1427核苷酸)。
典型的严格杂交条件如下:在42℃下于50%甲酰胺中杂交,然后在60℃下于0.1×SSC、0.1%SDS中清洗。本领域技术人员知道:在这些条件下发生的变异是基于被杂交序列的长度和GC核苷酸碱基的含量。本领域的程序标准适合确定准确的杂交条件。参见Sambrook等,Molecular Cloning中的9.47-9.51,冷泉港实验室出版,冷泉港,纽约(1989)。
在SEQ ID NO:2和4中显示了人几丁质酶的两个氨基酸序列。SEQID NO:2序列由SEQ ID NO:1的核苷酸序列编码,SEQ IDNO:4由SEQID NO:3的核苷酸编码。除了上述的那些多核苷酸外,本发明优选的多核苷酸包括编码SEQ ID NO:2或SEQ ID NO:4的-21-445氨基酸的多核苷酸;和仅由于著名的遗传密码的简并性而不同于前面段落所描述的多核苷酸的多核苷酸。类似的,由于SEQ ID NO:2和4的21个氨基酸(位置-21至-1)包含一信号肽,该信号肽切割后产生成熟的人几丁质酶蛋白质,优选的多核苷酸包括编码包含SEQ ID NO:2或SEQ ID NO:4的1-445氨基酸的多肽的那些多核苷酸。
在本发明多核苷酸的用途中有作为杂交探针使用,来鉴定和分离编码人几丁质酶的基因组DNA;鉴定和分离编码与人几丁质酶同源的蛋白质的非-人类基因;鉴定与人几丁质酶具有相似性的人和非-人类蛋白质(包括那些可能参与组织重建的蛋白质);和鉴定那些表达人几丁质酶的细胞和表达该蛋白质的生物学条件。
在另一方面,本发明包括本发明DNA序列的生物复制物(即在体内或体外制得的分离的DNA序列的拷贝)。本发明提供自主复制重组构成物,例如:加入几丁质酶多核苷酸的质粒和病毒DNA载体,包括上面描述的任何一种DNA。优选的载体包括表达载体,在这些表达载体中加入的编码几丁质酶的cDNA在操纵上被连接到一内源或异源表达控制序列和转录终止子上。这些表达载体还可以包括在操纵上被连接到编码几丁质酶的DNA序列上的编码多肽的DNA序列,这些载体能被表达,以产生包含所需的多肽的融合蛋白。
按照本发明的另一方面,以一种允许所需的几丁质酶产物在其中表达的方式,用本发明的DNA序列稳定地转化或转染原核或真核宿主细胞。表达几丁质酶产物的宿主细胞可以有许多有用的用途。这些细胞为与几丁质酶发生特异性免疫反应的抗体物质的研制构成了一个有价值的免疫原来源。本发明的宿主细胞对大规模地生产几丁质酶的方法是有用的,其中细胞生长在合适的培养基中,并通过例如免疫亲合纯化,从细胞或这些细胞所生长的培养基中分离所需的多肽产物。
几丁质酶产物能从天然细胞来源中作为分离物获得或能化学合成,但最好是通过涉及本发明的原核或真核宿主细胞的重组方法来生产。本发明的几丁质酶产物可以是全长的多肽、其片段或其类似物。设想了具有在SEQ ID NO:2或SEQ ID NO:4中显示的部分或全部的氨基酸序列的几丁质酶产物。在SEQ ID NO:14中显示了缺乏成熟蛋白质的C-末端72个氨基酸残基的一优选片段。已确认:这72个末端残基对几丁质酶的酶活性是不重要的。实施例5说明了该C-末端片段的产生;在氨基酸373的密码子后引入一终止密码子,导致产生一大约39KD的重组几丁质酶片段,这个片段与全长的重组人几丁质酶相比,保留有相似的专一性活性。
类似物可包括其中的一个或多个特定(即自然编码的)氨基酸缺失或取代、或其中增加一个或多个非特异性氨基酸的几丁质酶类似物:(1)没有丧失一项或多项酶活性或几丁质酶特有的免疫学特征;或(2)特异性地丧失几丁质酶的独特生物学活性。实施例3说明了这一类似物(SEQ ID NO:15)的产生,其中用丝氨酸取代370位置上的脯氨酸,并在其中已删除了C-末端的72个氨基酸残基。哺乳动物宿主细胞的应用还期望提供翻译后修饰(例如肉豆蔻酰化、糖基化、截短、脂化以及酪氨酸、丝氨酸或苏氨酸磷酸化),这些修饰对于赋于本发明的重组表达产物以最佳生物学活性可能是需要的。
与几丁质酶结合的蛋白质或其它分子能用来调节它的活性。本发明还包括抗体物质(例如:单克隆和多克隆抗体、单链抗体、嵌合抗体、CDR-移植抗体等等)和其它对几丁质酶特异性的结合蛋白质。用从血浆、重组几丁质酶、几丁质酶变异体或表达这些产物的细胞中分离出来的几丁质酶可以鉴定专一性地与几丁质酶结合的蛋白质或其它分子(例如小分子)。同样,结合蛋白质可再用于免疫组合物和纯化几丁质酶的组合物,而且可用来用已知的免疫学程序检测或定量分析体液和组织样本中的几丁质酶。还设想了对几丁质酶-专一性抗体物质特异性的抗个体基因型抗体。
通过本发明DNA和氨基酸序列的公开内容带来的信息的科学价值是显而易见的。作为一系列的例子,对几丁质酶cDNA序列的了解使通过DNA/DNA杂交来进行的分离成为可能,所述DNA/DNA杂交为编码几丁质酶的基因组DNA序列与如启动子、操纵子等等的几丁质酶表达控制调节序列杂交。用本发明的DNA序列,同样期望在本领域严格性标准的条件下进行的DNA/DNA杂交步骤允许分离这样的DNA,所述DNA编码人几丁质酶的等位变异体、具有几丁质酶的一项或多项生物化学和/或免疫学性质的其它结构上相关的人蛋白质、以及与几丁质酶同源的非人类物种的蛋白质。本发明所提供的DNA序列的信息还使得通过用同源重组或“剔除”策略来生产不能表达功能性几丁质酶、过度表达几丁质酶、或表达变异几丁质酶的啮齿类成为可能。本发明的多核苷酸当合适标记后,可用于杂交检验,以检测细胞合成几丁质酶的能力。本发明的多核苷酸也可以是用于鉴定导致疾病状态的几丁质酶基因座遗传改变的诊断方法的基础。本发明还能通过正常表达几丁质酶的细胞,得到与调节几丁质酶表达有关的反义多核苷酸。
本发明设想了对哺乳动物受治疗者、特别是人类给予本发明的几丁质酶制剂,来减轻由含几丁质寄生物例如真菌引起的病态。真菌感染(真菌病)如念珠菌病、曲霉病、球孢子菌病、芽生菌病、类球孢子菌病、组织浆菌病、球孢子菌病、产色真菌病、孢子丝菌病、毛霉菌病和皮癣菌病,可以能表现为急性或慢性疾病。病原性真菌在无免疫应答的个体中能引起严重的、通常是致命的疾病。正进行化疗的癌症病人、免疫抑制个体和HIV-感染个体易受念珠菌属、曲霉属、卡氏肺囊虫和其它真菌引起的真菌病的感染。两性霉素B和氟康唑对真菌感染是有用的疗法,但这些药物带有的毒性引起严重的不良副作用,这样就限制了它们的应用。尽管用两性霉素B治疗,全身性念珠菌病的死亡率仍高于50%。因此,需要能在体内抑制真菌生长的药物;并且这些产品能作为单独的药物使用或与目前已认可的、传统的抗-真菌化合物联用。因为生长中的真菌需要几丁质合成来维持生存,重组人几丁质酶引起的抑制能用于限制真菌的体内感染。下面在实施例8-14中说明真菌感染的动物模型,并在本领域已有描述。
本发明特别设想了用于治疗对真菌感染敏感的或受真菌感染的哺乳动物的方法中的几丁质酶组合物,该治疗方法包括给予哺乳动物足以补充内源性几丁质酶活性的量的几丁质酶。本发明设想:几丁质酶能与其它传统抗真菌药物一起给予,这些传统药物包括:两性霉素B和结构相关的化合物制霉菌素及游霉素;5’-氟胞嘧啶;吡咯衍生物例如:氟康唑、酮康唑、克霉唑、双氯苯咪唑、氯苯甲氧咪唑、布康唑、奥昔康唑、硫康唑、特康唑、依他康唑和噻康唑;烯丙胺-硫代氨甲酸盐,例如发癣退、纳呋替芬和特比萘芬;灰黄霉素;环己吡酮氨乙醇;氯丙炔碘;十一烯酸;和苯甲酸。[参见例如,Goodman和Gilman,The Pharmacological Basis of Therapeutics,第9版,McGraw-Hill,纽约(1996)]。几丁质酶可以提高这些传统的抗真菌剂的效力,这可能是通过甚至在单独的几丁质酶对抑制真菌生长无效的环境中,几丁质酶也能使得酵母对这些药物的活性更敏感来实现的。通过降低传统抗真菌剂行使所要求的治疗作用的需要量,几丁质酶能允许药物以较低的毒性水平使用。例如,Davis和Pope,Nature,273:235-236(1978)报道:对曲霉属-感染的小鼠给予和通常无效剂量的抗真菌药联用的真菌酶(mycolases)(降解真菌细胞壁的酶),提供协同有效的治疗。注意到:真菌几丁质酶和地衣多糖酶的组合对破坏真菌细胞璧比两种酶单独使用更有效。
因此,本发明设想了几丁质酶在制备用于预防性或治疗性治疗真菌性感染的药物上的应用,并进一步设想了几丁质酶在制备用于与其它抗真菌剂共同给药的药物的应用。
本发明设想的治疗/药用组合物包括几丁质酶和生理上可接受的稀释液或载体,并还包括其它抗真菌剂。所指示的剂量应足以补充内源性几丁质酶的活性。对于一般的剂量考虑参见Remington:TheScience and Practice of Pharmacy,第19版,Mack出版公司,Easton,PA(1995)。剂量可在大约1μg/kg至100mg/kg体重之间变化,最好在大约0.1至大约20mg几丁质酶/kg体重之间的变化。根据所治疗的感染,本发明的治疗组合物能通过不同的途径给药,包括通过皮下、肌内、静脉内、肺内、经皮、鞘内、局部、口服或栓剂给药。
本发明还设想:在高歇氏病中或在炎症部位(例如在类风湿性关节炎中)几丁质酶的过度表达可能对胞外基质有有害作用,并且在这些疾病背景中,几丁质酶活性的抑制剂能例如通过降低重建(remodeling)或破坏胞外基质提供治疗益处。
从巨噬细胞cDNA文库中分离本发明的人几丁质酶cDNA。已知巨噬细胞与类风湿性关节炎损害有密切联系[Feldman等,Cell,85:307-310(1996)],如TNF-α的巨噬细胞产物与疾病进程有关。已在类风湿性关节炎病人的滑液和软骨中检测到与人几丁质酶C-gp39同源的蛋白质。尽管人几丁质酶的天然底物可能是来源于致病生物的几丁质,但该酶也可以对形成天然胞外基质的内源大分子显示活性。例如,已提示:胞外基质的重要成分透明质酸含有几丁质寡聚体的核心。[Semino等,Proc.Nat’l Acad.Sci.,93:4548-4553(1996);Varki,Proc.Nat’l Acad.Sci.,93:4523-4525(1996)]。因此,在如类风湿性关节炎的疾病中,几丁质酶可能参与胞外基质的降解。通过在从关节炎的关节中分离出的滑液中测定几丁质酶水平和/或几丁质酶用药的效果或几丁质酶的抑制作用,来确定几丁质酶的作用。通过酶分析或利用抗体能测定内源性几丁质酶的水平。可以在用几丁质酶处理之前和之后,测定滑液的粘度;与几丁质有关的粘度的降低应与内源性几丁质酶底物相一致。因此调节几丁质酶的活性能调节类风湿性关节炎的关节损坏进程。
本发明还设想了筛选几丁质酶活性抑制剂的方法,这些抑制剂能以在前面的段落所描述的方式使用。筛选样本来鉴定抑制几丁质酶的药物的方法报道在例如1995年12月21日公开的WO95/34678中。
进一步设想了测定几丁质酶内源水平的方法,例如用于诊断高歇氏病的诊断。Hollak等,J.Clin.Invest,93:1288-1292(1994)报道:血浆几丁质酶的水平是高歇氏病的诊断标记。期望本发明的重组蛋白比从人体纯化而来的制剂更有用,后者存在产量和被其它杂质或传染性因子污染的有关问题。
详细描述
考虑下面的解说性实施例将能明白本发明的其它方面和优点。实施例1描述了从人巨噬细胞cDNA库中分离人几丁质酶cDNA克隆。实施例2说明了在不同的人组织中几丁质酶基因表达的模式。实施例3描述了原核细胞中人几丁质酶基因的重组表达和所产生的酶的纯化。实施例4提供了在酵母中重组生产人几丁质酶的方案。实施例5描述了在哺乳动物细胞中人几丁质酶基因的重组表达和所产生的蛋白质的纯化。实施例6描述了通过肽合成或重组生产的方法来生产人几丁质酶多肽类似物。实施例7提供了生产专一性与人几丁质酶发生免疫反应的单克隆抗体的方案。实施例8描述了用于测定几丁质酶催化活性的分析。实施例9说明了人几丁质酶的体外抗真菌活性的测定。实施例10说明了人几丁质酶在小鼠模型中的体内抗真菌活性的测定,实施例11-14说明了侵染性曲霉病、弥散性念珠菌病、念珠菌性眼炎和念珠菌性心内膜炎的兔模型。
实施例1
几丁质酶cDNA克隆的分离
如Tjoelker等,Nature374:549-552(1995)中所描述的,从得自外周血单核细胞的巨噬细胞中制备cDNA文库。从该文库中随机选择克隆,并从单个克隆中纯化质粒DNA。在自动测序仪(373型,应用生物***,Foster,CA)上测定源于每个克隆的DNA末端大约300-500碱基的序列,这里用了引物JHSP6,它能杂交到与质粒载体pRc/CMV(Invitrogen,San Diego,CA)邻近cDNA克隆位点处。JHSP6:5’-GACACTATAGAATAGGGA-3’ (SEQ ID NO:5)
将这些cDNA克隆的核苷酸和推断的氨基酸序列与核苷酸和肽序列的数据库中的序列进行比较,来确定它们与已知基因的相似性。用Altschul等,J.Mol.Biol.,215:403-410(1990)的序列对比算法,由国立生物技术信息中心的BLAST网络服务来进行序列比较。克隆MO-911显示与几种不同的序列显著的同源性,包括巨噬细胞分泌蛋白质YM-1前体(基因库登记号M94584);人软骨gp-39(Hakala等,见上述);来源于绵羊、奶牛和人类的输卵管糖蛋白(Desouza等,见上述);和来源于寄生物(盘尾属,基因库登记号V10422)、黄蜂(Chelonus,基因登记号U10422)、植物(烟草,基因库登记号X77111)和细菌(沙雷氏菌属,基因库登记号Z36295)的几丁质酶;与所发现的克隆MO-911基因具有最高同源性的是编码具有几丁质酶同源性、但不显示几丁质酶活性的蛋白质的哺乳动物的基因。
克隆pMO-218(于1996年6月7日依照布达佩斯条约保藏于美国典型培养物保藏中心,12301Parklawn Drive,Rockville,MD20852,USA,保藏号为98077)的DNA序列示于SEQ ID NO:1中,而编码的氨基酸序列示于SEQ ID NO:2中。MO-218似乎包括人几丁质酶cDNA的整个编码区(SEQ ID NO:1的2-1404核苷酸),它包括21个氨基酸假定信号序列,后面接着445个编码的氨基酸(SEQ ID NO:2中1-445残基)。假定信号序列后的22个氨基酸与在Renbeme等(见上述)所报道的纯化的人壳三糖苷酶的氨基末端序列精确匹配。Renkema等也描述了来源于人壳三糖苷酶的胰蛋白酶片段的一个21个氨基酸序列,该序列准确地对应于MO-218(SEQ ID NO:2)的157-177残基。Boot等(见上述)报道了包含一基本等同于MO-218的编码序列的人壳三糖苷酶cDNA的克隆。由于在5’-末端额外的14个核苷酸和在编码区的核苷酸330位置上改变一个核苷酸,使MO-218序列不同于Boot等的序列。
为了确认MO-218的确包含该cDNA的整个编码区,制备了32P-标记的探针P-1(TGGGATCATCAGCAGGACCATGAAACCTGCCCAGGCCACAGACCGCACCAT,SEQ ID NO:6),它对应于MO-218(SEQ IDNO:1)的2-52核苷酸的互补链。设计探针P-1以与在5’-末端至少和MO-218一样长的克隆杂交。该探针在40%甲酰胺和杂交缓冲液中(5×SSPE、10×Denhardt’s、100μg/ml变性***哈鱼鱼精DNA和2%SDS)与上述人巨噬细胞cDNA文库的一部分(大约30,000个克隆)于42℃杂交过夜。清洗滤膜并挑选杂交上的三个克隆进行测序/分析。将最长的克隆命名为pMO-13B(于1996年6月7日依照布达佩斯条约保藏于美国典型培养物保藏中心,12301 Parklawn Drive,Rockville,MD 20852,美国,保藏号为98078)。pMO-13B的DNA序列示于SEQ ID NO:3中,并且编码的氨基酸序列示于SEQ ID NO:4中。与MO-218相比,这个克隆在5’末端包含25个额外的核苷酸;另外,MO-13B(SEQ ID NO:3)在核苷酸330(对应于MO-218的核苷酸305,SEQ ID NO:1)上含有一核苷酸置换,该置换将成熟蛋白的80位置上的编码氨基酸由甘氨酸(在SEQ IDNO:2中)改变为丝氨酸(在SEQ ID NO:4中)
实施例2
人组织中几丁质酶基因的表达模式
进行RNA印迹分析以鉴定表达人几丁质酶的组织。在标准严格条件下(根据Clontech实验手册),将一个多个人组织的RNA印迹与MO-218的整个编码区杂交。在肺组织(+++)和卵巢(+++)中观察到了最大程度的杂交,在胸腺和胎盘中观察到较低水平的杂交(+)。对于肺、卵巢和胸腺,杂交mRNA的大小是2.0kb,它正好对应于克隆cDNA的大小(1.6kb或包括polyA尾的1.8kb)。杂交的胎盘mRNA的大小小得多,是1.3kb。在脾脏、***、睾丸、小肠、结肠、外周血白细胞、心脏、大脑、肝脏、骨骼肌、肾脏或胰腺中未观察到几丁质酶杂交。肺中几丁质酶的表达与针对含有几丁质的致病生物的保护作用相符合,因为肺代表真菌病原体的最初侵入途径。
实施例3
在细菌细胞中生产重组人几丁质酶
工程改造MO-218的成熟编码区,以在大肠杆菌中作为C-末端截短的类似物表达。用PCR来产生用于表达的DNA片段,所用的引物对应于MO-218几丁质酶cDNA的65-88核苷酸,该引物以起始蛋氨酸密码子和一个XbaⅠ限制性内切酶位点(5’TACATCTAGAATTATGGCAAAACTGGTCTGCTACTTCACC-3’,SEQ ID NO:7)开头,下游引物编码MO-218的核苷酸1163-1183,后面接一终止密码子和一HindⅢ位点(5’-AGATCTAACCTTAGGTGCCTGAAGACAAGTATGG-3’,SEQ ID NO:8)。下游引物在碱基25上含有一腺嘌呤,而M-218序列在对应的核苷酸位置上含有一鸟嘌呤。因此,所产生的DNA片段在对应于MO-218序列的1172核苷酸位置上含有一胸腺嘧啶,而不是胞嘧啶,示于SEQ IDNO:15中的编码的几丁质酶片段也是一类似物,它在成熟氨基酸位置370上含有丝氨酸,而不是MO-218所编码的脯氨酸。用XbaⅠ和HindⅢ消化所得到的DNA片段,并克隆到质粒pAraBAD中(它还被命名为pAraCB)。
如下制备质粒pAraCB。修饰质粒pUC19,以包含一个***糖启动子并因此包含AKAP79编码序列。从作为EcoRⅠ/XbaⅠ片段的鼠伤寒沙门氏菌的***糖操纵子BAD,通过PCR扩增该***糖启动子[Wilcox等,Gene,34:123-128(1985);Wilcox等,Cene,18:157-163(1982)]和araC基因,施用引物araC-2(SEQ ID NO:9)和arab-1(SEQ ID NO:10):
araC-2 TACAGAATTCTTATTCACATCCGGCCCTG SEQID NO:9
arab-1 TACATCTAGACTCCATCCAGAAAAACAGGTATGGSEQ ID NO:10引物araC-2为araC基因产物编码一个EcoRⅠ位点(下划线)和终止密码子(斜体)。引物arab-1编码假定核糖体结合域(斜体)和一个XbaⅠ限制性位点(下划线)。用这些引物进行PCR产生一个1.2kb的片段,将该片段用EcoRⅠ和XbaⅠ消化,并克隆到已用同样的两种酶消化过的pUC19上(New Englang Biolabs,Beverly,MA)。所产生的质粒被命名为araCB,它包含一个多接头区(SEQ ID NO:11),在5’-末端的侧翼是一XbaⅠ限制性位点(下划线处),在3’-末端侧翼是一HindⅢ位点(斜体)。
AraCB多接头 SEQ ID NO:11
TCTAGAGTCGACCTGCAGGCATGCAAGCTT
用***糖诱导含有所产生的表达质粒(pAraMO218)的转化体,并在37℃生长。这些转化体产生包含39kDa蛋白质的包涵体,这种蛋白质是几丁质酶的截短形式(被工程改造成包含373个氨基酸而不是445个氨基酸)。该几丁质酶片段含有四个半胱氨酸残基,而全长的几丁质酶含有10个半胱氨酸残基。从大肠杆菌培养物中分离出包涵体,并在SDS-PAGE上电泳。将39kDa带转移到PVDF膜上,并测定氨基末端序列。该材料的大部分(大约三分之二)包含对应于人几丁质酶氨基末端的序列。剩下的材料相当于污染性大肠杆菌蛋白质膜孔蛋白。这种大肠杆菌的重组几丁质酶制剂能用于生产多克隆和单克隆抗体(下面在实施例7中说明)。
当将含有Ara-几丁质酶表达质粒的转化体在25℃中培养时,未观察到包涵体,并且重组产物的表达从总细胞蛋白质的大约10%下降到大约1%。然而,在25℃生产的该材料显示几丁质酶催化活性。
实施例4
在酵母细胞中生产重组人几丁质酶
在酵母中重组表达人几丁质酶和纯化产生的重组蛋白质的典型方案如下。或用PCR,或用设计成编码一融合多肽的接头寡核苷酸,将人几丁质酶的编码区工程改造到载体中,以便在酿酒酵母中表达,其中所述融合多肽包含分泌介导前导序列,并与对应于天然分子氨基末端的人几丁质酶编码区融合。分泌信号肽包括例如:SUC2或具有功能性信号肽酶切割位点的相当的前导序列、或前α因子原或显示KEX2的切割位点的前肽或信号肽和原区或间隔区构成的其它复合前导序列。通过寡核苷酸合成或通过PCR能得到编码该信号序列的DNA。用含有α交配因子基因的1-20核苷酸的引物和与该基因的255-235核苷酸互补的引物,通过PCR来获取编码前α因子原前导序列的DNA[Kurjan和Herskowitz,Cell,30:933-943(1982)]。将前α原前导序列编码序列和人几丁质酶编码序列片段连接到含有酵母乙醇脱氢酶(ADH2)启动子的质粒上,使得该启动子指导融合蛋白的表达。如Rose和Broach所描述的,[Meth Enz,185:234-279,D.Goeddel编辑,Academic Press,Inc.,San Diego,CA(1990)],该载体还包括该克隆位点下游的一ADH2转录终止子、酵母“2-微米”复制起点、一选择性标记(例如TRP1、CUP1或LEU2(或LEU2-d)或其它相当的基因)、酵母REP1和REP2基因、大肠杆菌β内酰胺酶基因和大肠杆菌复制起点。β内酰胺酶和TRP1基因分别为在细菌和酵母中提供选择。REP1和REP2基因编码参与质粒拷贝数目复制的蛋白质。
或者,可以选择几丁质酶编码区中的其它融合点。该编码区的截短物能用于增加产物的同质性、提高专一性活性或改变底物专一性。
用已知的方法,例如乙酸锂处理[Stearns等,Meth.Enz.,见上述,第280-297页]或用等同的方法,将前面段落所描述的DNA构成物转化到酵母细胞中。耗尽生长培养基中的葡萄糖时诱导ADH2启动子[Price等,Gene,55:287(1987)]。前α原序列或其它前导序列影响融合多肽的分泌,使成熟的人几丁质酶肽从细胞中释放出来。通过信号肽酶,或在前α原去掉原区的情况下,通过KEX2蛋白酶,加工该信号肽前导序列[Bitter等,Proc.Natl.Acad.Sci.USA,81:5330-5334(1984)]。
几丁质酶在它的成熟氨基酸序列中包含两个二碱基序列,它们位于位置107-108(Lys-Arg)和209-210(Arg-Lys),在分泌过程中这两个序列能被KEX2蛋白酶水解修剪。为了稳定和/或增加从细胞中分泌的产物的水平,可以使这些序列突变,以去掉潜在蛋白水解位点,如Gillis等所示[Behring Inst.Mitt.,No.83:1-7(1988)],或在KEX2缺陷的宿主细胞中表达没有二碱基修饰的几丁质酶。或通过筛选包含突变体的非-KEX2蛋白酶,或通过利用基因取代/基因破坏技术操纵基因组KEX2基因座[Orr-Weaver等,Proc.Natl,Acad.Sci,USA,78:6354-6358(1981)],来得到这些宿主细胞。
用包含替代的启动子PRB1、GAL4、TPI或其它合适的含有强启动子的片段或通过融合到一系列前导序列上的相似的载体,可从酿酒酵母中分泌出重组几丁质酶。
其它非酿酒酵母的合适表达宿主包括乳酸克鲁维酵母(Kluyceromyces lactis)、粟酒裂殖酵母(Schizosaccharomyces pombe)、巴斯德毕赤酵母(Pichia pastoris)和汉逊酵母属或曲霉属的成员。这些真菌的类似重组表达***包括这些有机体和它们的合适的自主复制载体[例如Falcone等,Plasmid,15:248-252(1988)]或繁殖整合表达盒(multiply。这些***还依赖于上述类型的信号序列或前导序列,以介导分泌到培养基中。
通过例如用来从细菌和哺乳动物细胞上清液中纯化几丁质酶的方法,从酵母生长培养基中纯化分泌的重组人几丁质酶。(参见以上的实施例3和以下的实施例5)。
或者,可以在酿酒酵母细胞的细胞质或类似的宿主中表达成熟形式的重组人几丁质产物,并从裂解后的宿主细胞中纯化得到。该蛋白质能在纯化作用期间重新折叠,以获得合适水平的专一性活性。
实施例5
在哺乳动物细胞中生产重组人几丁质酶
A.在COS细胞中表达
分离两种都包含全长人几丁质酶cDNA3’至pRc/CMV的CMV启动子的MO-218克隆和MO-13B克隆。按照下面来制备对应于在上面实施例3中的细菌细胞中表达的相同C-末端片段的第三种质粒。通过PCR扩增质粒MO-218,用寡核苷酸218-1和互补的下游引物T-END作为引物,寡核苷酸218-1(CGCAAGCTTGAGAGCTCCGAACCGCCACATGGTGCGGTCTGTGGCCTGGG,SEQ ID NO:12)包含一个HindⅢ位点和MO-218几丁质酶cDNA SEQ ID NO:1的2-23核苷酸,而下游引物T-END(GACTCTAGACTAGGTGCCTGAAGGCAAGTATG,SEQ ID NO:13)含有MO-218的1164-1183核苷酸、一终止密码子和一XbaⅠ位点。将扩增的DNA用电泳法纯化,用XbaⅠ和HindⅢ消化,并克隆到已用同样的限制性内切酶切割的pRC/CMV载体(Invitrogen,San Diego,CA)上。在373型(应用生物***,Foster city,CA)上测定产物克隆的接点序列,它编码示于SEQ ID NO:14中的假定的工程改造蛋白质序列。
用DEAE转染方法将三种质粒全部瞬时转染到COS细胞中。[参见例如,Sambrook等,Molecular Cloning:alaboratory Manual,第二版,冷泉港,纽约:冷泉港实验室(1989)]。在37℃培养三天后,如以下实施例8所描述的,测定细胞的培养基的几丁质酶体外活性。每一培养物具有显著的几丁质酶活性(600-800mU/ml/min),在每个构成物中观察到类似的量。
重组人几丁质酶的纯化如下。用pRc/CMV-MO-13B质粒转化COS细胞5天后,收获来源于培养物的条件培养基,并用等体积的水稀释。将已稀释的条件培养基加到Q-Sepharose快速柱中(Pharmacia Biotech,Uppsale,瑞典),该柱已在25mM Tris、10mM氯化钠、1mM EDTA,pH8.0中预平衡。大约95%的几丁质酶活性流过柱子,并不结合到色谱柱上。将Q-Sepharose柱流出物调节至1.2M的硫酸铵,并加入到丁基-Sepharose4快速柱上(Pharmaca),该柱已在25mM Tris、1.2M硫酸铵、1mM EDTA,pH8.0中预平衡。用溶于25mM Tris、pH8.0中的1.2M-0M的硫酸铵反梯度洗脱蛋白质。于280nm处对应于几丁质酶活性峰的一单个吸收峰在低盐处洗脱下来。通过SDS-PAGE确定,该材料的纯度高于85%,并含有大约60%的几丁质酶活性。用10,000 MWCO膜(Ultrafree 10K,Millipore Corp.,Bedford,MA)浓缩该蛋白质,并将缓冲液改变成20mM Tris、150mM氯化钠、pH8.0。然后如下面的实施例8和9所描述的,测定该制备物的体外酶活性和抗真菌活性。该重组制备物具有90nm/min每克蛋白质的壳三糖苷酶活性。
B.在CHO细胞中表达
通过从pRc/CMV/MO-13B切下含全长几丁质酶基因的1.77kbHindⅢ/XbaⅠ片段,并将片段与HindⅢ/XbaⅠ消化过的pDEF1连接,以产生质粒pDEF1/CTN.1,将几丁质酶基因***到pDEF1中(pDEF1的构成已在1997年5月1日提交的美国申请序号08/847,218的实施例4中描述,通过引用结合到本文中)。含有几丁质酶基因的1.77kb的HindⅢ/XbaⅠ片段也被连接到用HindⅢ/XbaⅠ消化过的pHDEF1上,以产生质粒pHDEF1/CTN.1。质粒pHDEF1和pDEF1相同,除了以下两点区别:(1)在pHDEF1中,有一条源于pCEP4(Invitrogen Carlsbad)、包含一潮霉素抗性基因的2kb的EheⅠ/SalⅠ片段,该片段取代了pDEF1的19bp的PmeⅠ/SalⅠ片段;(2)在pHDEF1中,二氢叶酸还原酶(DHFR)基因的表达是受一缩短的SV40启动子控制的,该启动子包含在一120bp的NheⅠ/Asp718片段上,该片段取代了pDEF1的212bpNheⅠ/Asp718片段。用寡核苷酸94-26(5’-TGATACGGTACCGCCCCATGGCTGACTA-3’SEQ ID NO:16)(它含有一新的天冬氨酸位点)和94-27(5’-GCAAGTTTGGCGCGAAATCG-3’SEQ ID NO:17)作引物,用pDC1(已在1997年5月1日提交的美国申请序列号08/847,218的实施例4中描述)的带有SV40-DHFR盒的DNA作模板,首先扩增一条171bp的PCR片段,以制备120bp的NheⅠ/Asp718片段,然后用NheⅠ和Asp718消化这条171bp的PCR片段。
如在1997年5月1日提交的美国申请序号08/874,218的实施例5中描述的,用质粒pDEF1/CTN.1转染DHFR-阴性的中国仓鼠卵巢(CHO)细胞系DG44。还用质粒pHDEF1/CTN.1转染CHO细胞系DG44,接着用下面的修改程序来选择。在含有800mg/升的潮霉素(Calbiochem,SanDiego)和还含有次黄嘌呤和胸苷(这使培养基对DHFR基因没有选择性)的培养基(补充有2-10%透析的FBS的DMEM/F-12)中,先仅选择细胞对潮霉素的抗性。在选择对潮霉素基因具有抗性的转染子后,通过将细胞培养在缺乏次黄嘌呤和胸苷的培养基中,来进一步选择表达DHFR基因的细胞。然后,在最初含有10nM、接着20nM、最后50nM氨甲蝶呤的培养基中选择DHFR-阳性和潮霉素-抗性的CHO细胞,这就导致选择表达高水平几丁质酶的细胞。
按下列步骤纯化含有过度表达的重组人几丁质酶(rH-几丁质酶)的pHDEF1/CTN.1转染的CHO细胞的上清液。为准备阴离子交换色谱,将上清液用20mM Tris pH7.0(缓冲液A)按1∶3稀释。将填充有Q-Sephrose快速树脂(Pharmacia生物技术公司,Piscataway,NJ)的阴离子交换柱用缓冲液A平衡,并按每一升树脂15L稀释的上清液的量进样。在Q-Sepharose柱的流出液中收集到rH-几丁质酶,并调节到5%聚乙二醇(PEG)400(Mallinckrodt Baker Inc.,Phillipsburg,NJ)、30mM乙酸钠pH4.3,准备做阳离子交换色谱。用30mM乙酸钠、5% PEG 400 pH4.3(缓冲液B)平衡装填有CM-Sepharose快速树脂((Pharmacia生物技术公司,Piscataway,NJ)的阳离子交换柱。以每ml树脂1mg的量,将rH-几丁质酶样品加入到CM-Sepharose柱中,用40mM Tris、5%PEG400,pH7.5(缓冲液C)中从柱中洗脱rH-几丁质酶。然后通过将(NH4)2SO4增加到1.5M,准备rH-几丁质酶样品,以便做疏水作用色谱。用含有1.5M(NH4)2SO4的20mM Tris、5%PEG400,pH7.5(缓冲液D)平衡填充有Macro-Prep Methyl HlC填料(Bio-Red实验室,Hercules,CA)的色谱柱。将rH-几丁质酶样品以1mg每ml树脂的量加入到Macro-Prep Methyl柱上。用含有1.1M(NH4)2SO4的缓冲液D洗涤该柱,并用含0.2M(NH4)2SO4的缓冲液D洗脱rH-几丁质酶。用膜过滤法将纯化的洗脱液转变成150mMNaCl、20mMTris,pH7.0(缓冲液E)中。
实施例6
生产人几丁质酶类似物和片段
如在前面的实施例中描述的那些重组技术可以用来制备人几丁质酶多肽类似物或片段。更确切地说,用熟知的技术,例如点定诱变和聚合酶链式反应,来对编码人几丁质酶的多核苷酸进行修饰,使其编码感兴趣的多肽类似物。也可以通过例如去掉所述多核苷酸编码序列的合适部分,制备C-末端和N-末端缺失。通常参见Sambrook等,见上述,第15章。重组表达被修饰的核苷酸,并按照前面的实施例所描述的,来纯化所述重组多肽类似物或片段。
例如,通过与其它几丁质酶的同源性和通过用丙氨酸取代天然人几丁质酶氨基酸残基,鉴定对人几丁质酶活性起关键作用的残基。半胱氨酸对于蛋白质功能的完整性通常是关键的,因为它们具有形成二硫键的能力并限制二级结构。为确认人几丁质酶的任何一个半胱氨酸是否对酶活性至关重要,将每个半胱氨酸单独地诱变成丝氨酸。
如前面实施例3和5所描述的,通过在氨基酸373的密码子后引入一终止密码子,来制备成熟人几丁质酶蛋白质的一39kDa的C-末端截短片段。这条39kDa的片段缺乏成熟蛋白质的72个C-末端氨基酸,包括6个半胱氨酸,与全长重组人几丁质酶相比,该片段仍保留有类似的专一性酶活性。这个结果表明:丢失掉的包括6个半胱氨酸的72个C-末端残基不是酶活性所需要的。
可制作进一步的C-末端缺失,可以例如通过用核酸外切酶Ⅲ消化实施例3中描述的截短人几丁质酶编码序列的3’-末端不同时间,然后在全部三个阅读框架中将缩短的编码序列连接到质粒DNA的编码终止密码子上。用同样的方式通过消化该编码序列的5’-末端,然后将消化过的片段连接到含有一启动子序列和紧接该启动子位点上游的一起始蛋氨酸的质粒上,来制备N-末端缺失。这些N-末端缺失类似物或片段也可以作为融合蛋白表达。
或者,利用本领域的已知技术,通过全部或部分化学肽合成,也可以制备人几丁质酶多肽类似物[参见例如,Clark-Lewis等,IL-8的合成,J.Biol.Chem.,266:23128-34(1991);Clark-Lewis等,IL-3的合成,Science,231:134-139(1986);和Dawson等,通过连接合成,Science,266:776-779(1994)]。这些合成方法还允许选择性地引入新型的、非天然的氨基酸和其它化学修饰。
利用本领域认可的技术,如在下面的实施例8-14所描述的技术,来分析人几丁质酶多肽类似物的生物学活性,包括酶活性、抗真菌活性和胞外基质重建活性。
实施例7
制备针对人几丁质酶的单克隆抗体
下面两个方案(多次攻击免疫或单次注射免疫)可以用来生产针对人几丁质酶的单克隆抗体。在第一个方案中,用如在实施例3-6中任何一例中所述得到的重组几丁质酶(例如,10-20μg乳化在弗氏完全佐剂中)定期注射小鼠。对该小鼠给予最后的溶于PBS的人几丁质酶的预融合物加强免疫,四天后处死小鼠并取出它的脾脏。将该脾脏放在10ml无血清PRMI1640中,通过碾碎处于浸没在无血清PRMI1640中的两个玻璃显微镜载玻片的冰冻两端之间的脾脏,来形成单细胞悬浮液,无血清PRMI1640补充有2mML-谷酰胺、1mM丙酮酸钠、100单位/ml的青霉素和100μg/ml的链霉素(RPMI)(Gibco,加拿大)。将细胞悬浮液通过无菌的70目Nitex细胞渗透器(Becton Dickinson,Parsippany,新泽西)过滤,并通过在200g离心5分钟来清洗两次,并将沉淀物重新悬浮在20ml无血清的RPMI中。以同样的方式制备取自于三只初次试验的Balb/c小鼠的脾细胞,并用来作为对照。将在融合前在含有11%胎儿牛血清(FBS)(Hyclone实验室,公司,Logan,Utah)的PRMI中保存三天的对数期的NS-1骨髓瘤细胞在200g下离心5分钟,并如在前面段落描述的,将沉淀物清洗两次。
将1×108脾脏细胞与2.0×107NS-1细胞混合并离心,然后吸出上清液。轻轻敲打试管,使细胞沉淀离开原位,并在1分钟以内边搅拌边加入1ml37℃的PEG 1500(50%在75mM Hepe中,pH8.0)(BoehringerMannheim),接着在7分钟内加入7ml无血清的RPMI。加入另外的8mLRPMI并在200g中离心细胞10分钟。丢弃上清液后,将细胞沉淀重新悬浮在含有15%FBS、100μM次黄嘌呤钠、0.4μM氨基喋呤、16μM胸苷(HAT)(Gibco)、25单位/ml IL-6(Boehringer Mannheim)和1.5×106脾细胞/ml的200mlRPMI中,并平板接种到10Corning平底96-孔组织培养板上(Corning,Corning纽约)。
在融合后的第2、4和6天,从融合板的孔中取出100μl的培养基,并用新的培养基更换。在第8天用ELISA筛选融合,按照下面的描述测试结合到人几丁质酶上的小鼠IgG的存在。在37℃下用稀释在25mMTris,pH7.5中的100ng/孔的人几丁质酶包被Immulon 4板(Dynatch,Cambridge,MA)2小时。吸出包被溶液并加入200μl/孔的封闭溶液[稀释在CMF-PBS中的0.5%鱼皮明胶(Sigma)],在37℃下保温30分钟。用含有0.05%吐温20(PBST)的PBS清洗板三次,并加入50μl的培养上清液。在37℃下保温30分钟后,按上述清洗,加入50μl的以1∶3500稀释在PBST中的缀合山羊抗小鼠IgG(fc)的辣根过氧化物酶(JacksonImmunoResearch,West Grove,Pennsylvania)。按上述保温板,用PBST清洗四次,并加入100μl底物,该底物含有pH4.5 100mM柠檬酸盐中1mg/ml的对苯二胺(Sigma)和0.1μg/ml30%的H2O2。5分钟后通过加入50μl15%H2SO4来中止显色反应。在读板仪上(Dynatech)读出A490。通过稀释到96孔板中并在5天后观察记录每孔中的集落数,将选择出来的融合细胞克隆二次。用Isostrip***(Boechringer Mannheim,Indiannpolis,IN)做杂交瘤产生的单克隆抗体的统计数字象征性图表。
或者,可实施利用单次注射的脾脏内免疫的第二种方案,通常依照Spitz,Methods Enzymol.,121:33-41(1986)进行。将该动物的脾脏暴露,并注射如在实施例3-6任何一例中得到的重组人几丁质酶(例如:10-20μg以大约0.02%-0.04%的浓度存在于PBS中,带有或不带有铝佐剂),之后将该脾脏重新放回腹膜腔中,并将该动物紧密缝合上。三天后,处死小鼠并取出它的脾脏。制备脾脏细胞的悬浮液,用补充有3%胎牛血清(FCS)的RPMI 1640清洗两次,并重悬浮在25ml同样的培养基中。收集处于对数生长期的骨髓瘤细胞(NS-O),清洗一次并以3∶1或2∶1(脾脏细胞∶骨髓瘤细胞)的比例加入到50ml试管中的脾脏细胞悬浮液中。在大约450g(1500rpm)下将该混合物沉淀,吸出上清液,拍打试管使沉淀物松散。通过在一分钟内加入1ml的聚乙二醇(PEG)1500并持续搅拌,来进行融合。将该混合液再保温1分钟,然后在1分钟内加入1ml的温RPMI(30-37℃),接着在3分钟内加入5mlPRMI,并另外的3分钟林子加入另外的10ml RPMI。将细胞悬浮液离心并重新悬浮在大约200mlHAT选择性培养基中,该培养基由补充有100U/ml青霉素、100μg/链霉素、20%FCS、100mM次黄嘌呤、0.4mM氨基蝶呤和16mM胸苷的RPMI1640构成。将细胞悬浮液以1ml体积分加到组织培养板中,并在37℃下,在具有5%CO2-95%空气的湿氛围下培养8-10天。并每2-3天根据细胞生长,吸出上清液并用每孔1ml HAT培养基来饲养细胞。筛选铺满孔中的上清液以寻找专一性抗体,并将阳性孔克隆。
实施例8
重组几丁质酶的催化活性
用McIlvain缓冲液(Hollak等supra,)中的荧光底物4-甲基伞形基-β-D-N,N’,N”-三乙酰壳三糖(4MU-壳三糖,Sigma Chemical,圣路易斯,MO),来测定壳三糖苷酶(几丁质酶)的活性。将10μl的重组产物样品与总体积100μl中的10μl的牛血清白蛋白(10mg/ml)、15μl荧光底物(2.71mM)和65μl缓冲液(0.1M柠檬酸、0.2M磷酸钠、pH5.2)混合。将反应物在37℃下保温15分钟,然后通过加入2ml的0.3M甘氨酸/NaOH缓冲液(pH10.6)中止反应。用荧光计(SLM-AMINCO仪器公司,Rochester,纽约)于450nm处监测荧光酶切产物4-甲基伞形酮。为得到标准曲线,将几种底物浓度与过量的细胞几丁质酶反应,以保证底物完全酶切。将已知量的4-MU与来自于荧光计的荧光信号联系起来,用线性回归确定标准曲线。在该分析中用稀释的纯化重组几丁质酶所产生的信号来内推在反应时间内该酶酶切底物的nm量。这个数值除以蛋白质的浓度,得到nm/min每mg蛋白质(由A280确定并计算克分子消光系数)。
如在实施例5A中描述的、在COS细胞中生产的重组人几丁质酶的壳三糖苷活性测定为90nm/min每克蛋白质。
实施例9
重组几丁质酶的体外抗真菌活性
在初步实验中,检测重组人几丁质酶在体外对真菌生长的抑制作用。两个真菌白色念珠菌和烟曲霉是无免疫应答病人的严重的病原体。念珠菌属和曲霉属都于37℃下在RPMI生长培养基中生长到大约10,000-50,000菌落形成单位(CFU)每毫升。将重组人几丁质酶(如在实施例5A所描述的在COS细胞中生产的)以0、2.8、11.25、或45μg/ml加入到培养物中。24小时后,用培养物的浊度估计真菌的生长。在该分析的非生理条件下,所有培养物似乎以类似的速度生长,与几丁质酶的浓度无关。然而,测试的真菌浓度比在体内真菌感染期间观察到的真菌负荷量高得多。在不同的条件下,例如用较低的真菌负荷量、或当人几丁质酶与其它抗真菌剂结合测试时,可得到不同的结果。还期望几丁质酶在生理条件下在体内更有效。
在另外的实验中,在琼脂糖扩散分析中、在依照国立临床实验标准委员会的肉汤分析中、以及在依照Selitrennikoff,Antimicrob.AgentsChemother.,23:757-765(1983)的细胞壁抑制分析中,评价重组人几丁质酶(如在实施例5A所描述的在COS细胞中生产的)的抗真菌活性。
在琼脂扩散中,将大约1×106细胞/ml的白色念珠菌(ATCC no.90028)接种到1.5%的琼脂上(用2-(N-吗啉代)丙烷磺酸(MOPS),pH7.0缓冲的RPMI1640培养基)。将含有50μg样品(A:重组人几丁质酶,B:缓冲液对照,C:对照蛋白质,D:具有几丁质酶活性的细菌溶胞物,或已知的抗真菌剂)的圆片铺上琼脂,测定生长抑制带。结果如下面表1所示。
表1
a样品:A=按照实施例5A从COS细胞制备出的rH-几丁质酶,存在于150mMNaCl、1mM EDTA、20mM Tris,pH7.5中;B=缓冲液(150mM NaCl、1mM EDTA、20mM Tris,pH7.5);C=无活性蛋白质PAF-AH(由2mg/ml贮液稀释的);D=具有几丁质酶活性的粘质沙雷氏菌溶胞物(SIGMA #C-7809)。b生长评价符号解释:(+)=正常生长,未观察到生长抑制;(-)=抑制真菌生长,观察到抑制带。c样品D刺激烟曲霉的生长。
样品a(50μg/圆片) | 白色念珠菌生长b | 烟曲霉生长 |
A:rH-几丁质酶 | + | + |
B:缓冲液对照 | + | + |
C:对照蛋白质 | + | + |
D:具有几丁质酶活性的细菌溶胞物 | + | +c |
两性霉素B(400ng/圆片) | - | - |
在肉汤分析中,将50μg/ml的样品(A:重组人几丁质酶,B:缓冲液对照,C:对照蛋白质,D:具有几丁质酶活性的细菌溶胞物,或已知的抗真菌剂)与一定浓度的测试真菌有机体加入到用MOPS缓冲的pH7.0RPMI1640培养基中。将样品在35℃下保温,在120rpm下摇动48小时,然后通过测定该悬浮液的浊度来评价生长。被试验的真菌的大致浓度如下:2.5×104细胞/ml的白色念珠菌(ATCC no.90028);5×104细胞/ml多烯抗性白色念珠菌(ATCC no.38247);1×104细胞/ml的烟曲霉(ATCC no.16424);1×104细胞/ml的粗糙脉孢菌(ATCC no.18889);和1×104细胞/ml的酿酒酵母(ATCC no.26108)。结果如下面表2所示。
表2
a样品:A=按照实施例5A从COS细胞制备出的rH-几丁质酶,存在于150mMNaCl、1mM EDTA、20mM Tris,pH7.5中;B=缓冲液(150mM NaCl、1mM EDTA、20mM Tris,pH7.5);C=无活性蛋白质PAF-AH(由2mg/ml贮液稀释的);D=具有几丁质酶活性的粘质沙雷氏菌溶胞物(SIGMA #C-7809)。b生长评价符号解释:0=没有细菌生长;1=生长值为对照的25%;2=生长值为对照的50%;3=生长值为对照的75%;4=生长值等于对照;5=生长值比对照大。cIC90:化合物抑制至少90%的有机体生长的最低浓度;相当于至少评分为0。dIC50:化合物抑制至少50%的有机体生长的最低浓度;相当于至少评分为2。
样品a(50μg/ml) | 白色念珠菌生长b | 多烯抗性-白体生长 | 烟曲霉生长 | 粗糙脉孢菌生长 | 酿酒酵母生长 |
A:rH-几丁质酶 | 2 | 2 | 3 | 4 | 2 |
B:缓冲液对照 | 2 | 2 | 4 | 4 | 2 |
C:对照蛋白质 | 4 | 3 | 4 | 4 | 4 |
D:具有几丁质酶活性的细菌溶胞体 | 4 | 4 | 4 | 4 | 4 |
两性霉素B的IC90 c | 0.5μg/ml | >16μg/ml | 2μg/ml | 0.5μg/ml | 1μg/ml |
氟康唑的IC50 d | 0.5μg/ml | 16μg/ml | NA | 8μg/ml | 4μg/ml |
5-氟胞嘧啶的IC50 | 2μg/ml | 0.06μg/ml | 16μg/ml | >64μg/ml | 0.125μg/ml |
双氯苯咪唑的IC50 | NA | 2μg/ml | 0.5μg/ml | NA | NA |
基本按照Selitrennikoff(见上述)所述,用包埋在琼脂(Vogel培养基N,7.5%山梨醇、1.5%蔗糖、10μg/ml烟酰胺和1%琼脂)中、在37℃下培育72小时的1.5×105原生质体/ml的接种物,进行鉴定真菌细胞璧生物合成抑制剂的os-l全细胞分析。在含有50μg的样品(A:重组几丁质酶,B:缓冲液对照,C:对照蛋白质,D:具有几丁质酶活性的细菌溶胞物或已知的抗真菌剂)的圆片铺上琼脂培养基后,监测培养物的生长和形态变化。os-l细胞是粗糙脉孢菌的突变菌株,该菌株当培养在37℃的某些条件下时,作为没有细胞璧的原生质体生长,但当温度转变到大约22℃时,在合适的条件下该菌株再生组细胞璧。抑制生长的样品被看作真菌生长抑制剂;阻碍细胞璧再生,但不杀伤细胞的样品被看作是细胞璧专一性抑制剂。结果示于下面表3。
表3
a样品:A=按照实施例5A从COS细胞制备出的rH-几丁质酶,存在于150mMNaCl、1mM EDTA、20mM Tris,pH7.5中;B=缓冲液(150mM NaCl、1mM EDTA、20mM Tris,pH7.5);C=无活性蛋白质PAF-AH(由2mg/ml贮液稀释的);D=具有几丁质酶活性的粘质沙雷氏菌溶胞物(SIGMA #C-7809)。b真菌生长评价符号解释:(+)=正常生长,未观察到生长抑制;(-)=抑制真菌生长,观察到抑制区带。c细胞璧再生评价符号解释:(+)=正常细胞璧再生;(-)=细胞璧再生被抑制。c受抑制细璧再生距圆片中心的半径测定。e受抑制的生长距圆片中心的半径测定。
样品a(50μg/圆片) | 细胞生长/形态学b | 细胞璧再生c |
A:rH-几丁质酶 | +原生质体 | -10mmd |
B:缓冲液对照 | +原生质体 | -5mmd |
C:对照蛋白质 | +菌丝 | + |
D:具有几丁质酶活性的细菌溶胞体 | +原生质体 | -7mmd |
Nikkomycin Z(1μg/圆片) | +原生质体 | -30mmd |
两性霉素B(400μg/圆片) | -细胞碎片 | +10mme |
这些分析结果显示:在os-l全细胞分析中,几丁质酶样品是细胞璧专一性抑制剂;在液体培养基分析中,几丁质酶样品是温和的抗真菌剂。
实施例10
重组几丁质酶在小鼠体内抗真菌活性
如下测定重组人几丁质酶在小鼠中的药物代谢动力学。对6-8周龄雌性的Balb/c小鼠用0.5mg/kg、5.0mg/kg和50mg/kg的重组人几丁质酶,在尾部静脉进行静脉内注射。对于每一剂量,在注射后的0.01、0.25、1.8和24小时,对小鼠进行末端取血(terminally bleed)(每一剂量每一时间点用两只动物)。然后分析血清样品的几丁质酶活性和浓度。结果如下面表4所示。
表4
AUC:曲线下时间趋向无限的峰面积Vss:分配稳态的体积cL:清除率MRT:总体平均滞留时间Cmax:峰值血清浓度
剂量(mg/kg) | AUC(μg/ml/h) | Vss(ml/Kg) | cL(ml/h/kg) | MRT(h) | 半衰期(h) | Cmax(μg) |
0.5 | 31.24 | 12.03 | 16.01 | 0.75 | 0.74 | 22.30 |
5.0 | 278.50 | 13.61 | 17.95 | 0.76 | 1.38 | 162.84 |
50.0 | 2505.83 | 52.92 | 19.95 | 2.65 | 2.33 | 1179.19 |
已经开发了测定抗真菌化合物的效力的几种动物模型[参见Louie等,Infect.Immun.,62:2761-2772,1994;Kinsman等,Antimicrobial Agentsand Chemotherapy,37:1243-1246,1993;Nakajima等,AntimicrobialAgents and Chemotherapy 39:1517-1521,1995;Tonetti等,Eur.J.Immunol.,25:1559-1565(1995);Dennin和Stevens,Antimicrob.AgentsChemother.,35:1329-1333(1991);还参见Stevens,J.Mycol.Med.,6(增刊Ⅰ):7-10(1996)]。简要地说,用所述真菌感染动物宿主,对动物给予不同剂量的几丁质酶,随时间测定它们的存活情况。用几丁质酶作为唯一的治疗剂来进行实验,或用传统抗真菌剂的组合物例如两性霉素B和氟康唑进行试验,来确定几丁质酶是否提高这些化合物的效力。确切地说,通过用10×106CUF白色念珠菌进行腹膜内或静脉内攻击,在小鼠中得到急性全身性念珠菌病。在攻击之前或之后的1-5小时给予治疗剂,并在5天后确定存活动物的数目。另外,可以处死小鼠并在特定的器官中测定真菌负荷量,所述器官例如:大脑、肾脏、肺、肝脏和脾脏。或者,用较低剂量的真菌,例如曲霉属(8-10×106CUF)或念珠菌属(1×106CUF)对小鼠进行攻击,在这种情况下可以在更长的时间点,例如,45天测定存活。可以通过持续治疗一周或更长,例如11天,并跟踪动物几周以上,例如18天到一个月,来评价单独的几丁质酶或与其它抗真菌药联用的几丁质酶的长效杀真菌/抑制真菌的活性。有效的抗真菌剂提高动物的长期存活率并降低血液和器官中的真菌负荷量。
实施例11
侵袭性曲霉病兔模型中几丁质酶的体内活性
在免疫抑制的侵袭性霉菌病兔模型中,评价单独的或与其它传统抗真菌剂联用的几丁质酶的效力,该模型在过去10年中已用于评价一系列抗真菌疗法。参见例如,Andriole等,Clin.Infect.Dis.,14(增刊Ⅰ):S134-S138(1992)。一般按照Patterson等,Antimicrob.Agents Chemother.,37:2307-2310(1993)或George等,J.Infect.Dis.,168:692-698(1993)进行研究。简要地说,在第一天将兔静脉内注射环磷酰胺(200mg),使得它们白细胞减少,接着在该试验过程中每天皮下注射醋酸去炎松(10mg)。在第二天,在免疫抑制24小时后,静脉内给予大约106(致死性攻击)或大约105(亚致死性攻击)烟曲霉分生孢子,对该动物进行攻击。在攻击后24小时或攻击前48小时(用于预防)开始抗真菌治疗(单独的几丁质酶或与其它传统的抗真菌剂例如:两性霉素B、氟康唑、或5-氟胞嘧啶联用),并持续治疗5-6天或直到死亡。传统的抗真菌剂的典型剂量是1.5或0.5mg/kg静脉内注射两性霉素B、60或120mg/kg/天口服氟康唑和100mg/kg/天口服5-氟胞嘧啶。对照兔不用任何抗真菌剂处理。
在尸体解剖或死亡时,对肝脏、脾脏、肾脏、肺和大脑进行半定量真菌培养物和组织病理检查。还可进行心脏、尿和血液培养。不时地取血样,分析血细胞计数并用ELISA分析循环的曲霉属碳水化合物抗原。以下面三个终点评价试验药物的治疗作用:降低死亡率、减少从靶器官培养的曲霉属有机体的数目(真菌负荷量)以及降低循环曲霉属抗原的水平。有效的抗真菌剂能降低死亡率和/或真菌负荷量。
或者,在这个模型中通常按照Chilvers等,Mycopathologia,108:163-71(1989)所述,来评价肺曲霉病,在这个模型中,用气管内滴入烟曲霉分生孢子进行攻击免疫抑制兔,接着在攻击后第1、2、4、7和10天进行支气管肺泡灌洗;对灌洗液进行真菌培养、几丁质分析、白细胞计数和组织病理学,以确定肺中的感染负荷。有效的真菌药物降低感染负荷或肺中的炎症。
实施例12
弥散性念珠菌病的兔模型中几丁质酶的体内活性
在弥散性念珠病兔模型中,通常按照Rouse等,Antimicrob.AgentsChemother.,36:56-58(1992)评价单独的或与其它传统抗真菌剂联用的几丁质酶的效力。用大约3×106白色念珠菌芽生孢子来全身性感染新西兰白兔。在用念珠菌攻击后48小时(或在攻击前用于预防)开始抗真菌治疗,并持续例如四天。处死存活的动物,在具有附着赘生物的大动脉瓣上、肺、肾脏、和脾脏进行真菌培养。还可在这些和其它器官例如:肝脏、大脑和心脏上进行真菌培养和组织病理学检查。也可以对尿和血液培养物做同样的实验。确定抗真菌治疗法对死亡率和循环的或组织的真菌负荷量的作用。
Bayer等,Antimicrob.Agents Chemother.,19:179-184(1991),在此论文中,用大约5×108CFU白色念珠菌对兔进行腹膜内接种。在腹膜内接种后四天从每只动物得到含盐的腹膜吸出物并将其培养,将真菌培养吸出物呈阳性的动物随机地分为对照组或治疗组。在攻击7天后,开始抗真菌治疗。用间接检眼镜检查评价所有兔的眼睛,因为弥散性念珠菌病能导致念珠菌性眼炎。在开始治疗后第7、11和14天处死动物,并检查它们的腹腔以寻找腹膜炎和腹膜内脓肿形成的证据。检查眼睛是否有肉眼可见的损害。将取自腹膜脓肿、所有其它可见的脓肿、肾脏、肝脏、脾脏、和眼结构的组织样本进行称重,在脑心脏输注肉汤中进行匀浆,连续稀释并培养,来测定CFU/克组织。也将肾脏脓肿和腹膜脓肿在10%的中性甲醛中固定,并进行组织病理学检查。将切片用高碘酸-席夫试剂染色,以确定真菌负荷量和真菌形态学。评价被试验的药物对提高存活率和降低真菌负荷的效果。
实施例13
真菌性眼炎兔模型中的几丁质酶体内活性
在念珠菌眼内炎兔模型中,通常按照Park等,Antimicrob.AgentsChemother.,39:958-963(1995)所述,评价单独的或与其它传统抗真菌剂结合的几丁质酶的效力。简要地说,用大约1,000CFU白色念珠菌对2-2.5kg的新西兰白兔玻璃体内(intravitreal)接种进行感染。接种的5天后用间接检眼镜检查确认眼炎,眼炎被定义为中度至严重的玻璃体模糊,并且高于50%的视网膜和脉络膜脉管***部分或全部的暗化(obscuration)。按标准给玻璃体浊度评分,可给基底外观评分并通过基底照相记载。按照下面的处理条件对兔进行随机化处理:单独用几丁质酶处理2-4周、用几丁质酶与其它传统抗真菌剂(例如两性霉素B、氟康唑或5-氟胞嘧啶)的组合物处理2-4周、或不处理(对照)。传统抗真菌剂的典型剂量是80mg/kg/天口服氟康唑和每12小时100mg/kg的口服5-氟胞嘧啶。
在治疗后的2和4周,通过间接检眼镜检查、定量真菌培养和组织病理学评价治疗效果。对于定量真菌培养,将眼睛切成碎片并称重,将每个样本的已称重部分匀浆,并将其在35℃下、在5-10%CO2中的布鲁氏杆菌琼脂-5%马血培养板中培养48小时。在铺板前也可用无菌的盐水将已匀浆的样品稀释10或100倍。对菌落计数,并在从被测量的样品部分产生的生长的基础上,计算眼睛中的CFU。依照整个眼内的真菌负荷的降低来评价治疗效果。对于组织病理学,摘出代表性眼睛、以***固定、包埋在塑料中、并切成5μm的切片。将切片用苏木精-曙红或Gomori六胺银盐染色剂染色,并用光学显微镜检查炎症、纤维状组织和真菌成分。评价抗真菌剂对降低死亡率、降低真菌负荷或降低与真菌感染有关的炎症的作用。
或者,通常按照Jain等,Doc.Ophthalmol.,69:227-235(1988)所述使用曲霉性眼炎的兔模型。简要地说,用大约40个烟曲霉孢子接种在新西兰白兔的一支眼睛中。其对侧的(对照)眼睛接受相似但无菌的接种物。用试验的药物(单独的几丁质酶、或与其它试剂联用的几丁质酶)治疗后,可通过临床外观、视网膜电图波形、间接检眼镜、定量真菌培养和组织病理学来评价兔的眼睛。在接种后的3-7天中通常产生临床症状明显的眼炎。
实施例14
真菌性心内膜炎的兔模型中的几丁质酶的体内活性
通常依照Witt和Bayer等,Antimicrob.Agents Chemother,35:2481-2485(1991)所述,也参见Longman等,Rev.Infect.Dis.,12(增刊3):S294-298(1990),评价念珠菌属心内膜炎的兔模型中单独的或与其它传统抗真菌剂联用的几丁质酶的效力。在新西兰白兔中通过安插跨主动脉瓣的无菌聚乙烯导管(内径,0.86mm),来产生无菌血栓性心内膜炎,该导管在研究期间内保留在该位置上。在导管***术后48小时,通过静脉内注射大约2×107白色念珠菌芽生孢子建立感染性心内膜炎。或者,可使用***滑念珠菌(C.Parapsilosis)。或在真菌性攻击前24小时或攻击后24-60小时开始抗真菌治疗(几丁质酶或与其它传统抗真菌剂联用的几丁质酶)。每天进行治疗,持续9或12天。传统抗真菌剂的典型剂量是1mg/kg/天静脉内注射两性霉素B、50mg/kg/天或100mg/kg/天静脉内或腹膜内注射氟康唑。对照兔不给予抗真菌试剂。处死动物时,取出心脏并确认放置导管的位置。从每只动物中取出心脏赘生物,合并、称重并在1ml无菌盐水中匀浆。连续稀释匀浆物,并在35℃下将其定量培养在酵母葡萄糖钾琼脂(porassium dextrose agar)中48小时。在平均赘生物重量的基础上含有低于2 log10 CFU/克被视为培养-阴性赘生物。
预计熟知该项技术的人会想到上述发明的许多修改和变更。相应地,仅在所附的权利要求书中出现限制才限制本发明。
序列表(1)一般资料:(ⅰ)申请人:ICOS CORPORATION(ⅱ)发明名称:几丁质酶材料和方法(ⅲ)序列数:17(ⅳ)通信地址:
(A)收信人:Marshall,O’Toole,Gerstein,Murray & Borun
(B)街道:6300 Sears Tower,233 South Wacker Drive
(C)城市:芝加哥
(D)州:依里诺
(E)国家:美国
(F)邮政编码:60606-6402(ⅴ)计算机可读形式:
(A)媒体类型:软盘
(B)计算机:IBM PC兼容机
(C)操作***:PC-DOS/MS-DOS
(D)S软件:PatentIn Release#1.0,版本#1.25(ⅵ)当前申请数据:
(A)申请号:
(B)申请日:
(C)分类:(ⅶ)先前申请数据:
(A)申请号:US 08/663,618
(B)申请日:14-JUN-1996(ⅷ)代理律师/代理人资料:
(A)姓名:Rin-Laures,Li-Hsien
(B)登记号:33,547
(C)参考/档案号:27866/33994(ⅸ)电讯资料:
(A)电话:312/474-6300
(B)传真:312/474-0448
(C)电报:25-3856(2)SEQ ID NO:1的信息:(ⅰ)序列特征:
(A)长度:1636个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑学:线性(ⅱ)分子类型:cDNA(ⅸ)特征:
(A)名称/关键词:CDS
(B)位置:2..1399(ⅸ)特征:
(A)名称/关键词:成熟肽
(B)位置:65..1399(ⅹⅰ)序列描述:SEQ ID NO:1:C ATG GTG CGG TCT GTG GCC TGG GCA GGT TTC ATG GTC CTG CTG ATG 46Met Val Arg Ser Val Ala Trp Ala Gly Phe Met Val Leu Leu Met-21 -20 -15 -10ATC CCA TGG GGC TCT GCT GCA AAA CTG GTC TGC TAC TTC ACC AAC TGG 94Ile Pro Trp Gly Ser Ala Ala Lys Leu Val Cys Tyr Phe Thr Asn Trp
-5 1 5 10GCC CAG TAC AGA CAG GGG GAG GCT CGC TTC CTG CCC AAG GAC TTG GAC 142Ala Gln Tyr Arg Gln Gly Glu Ala Arg Phe Leu Pro Lys Asp Leu Asp
15 20 25CCC AGC CTT TGC ACC CAC CTC ATC TAC GCC TTC GCT GGC ATG ACC AAC 190Pro Ser Leu Cys Thr His Leu Ile Tyr Ala Phe Ala Gly Met Thr Asn
30 35 40CAC CAG CTG AGC ACC ACT GAG TGG AAT GAC GAG ACT CTC TAC CAG GAG 238His Gln Leu Ser Thr Thr Glu Trp Asn Asp Glu Thr Leu Tyr Gln Glu
45 50 55TTC AAT GGC CTG AAG AAG ATG AAT CCC AAG CTG AAG ACC CTG TTA GCC 286Phe Asn Gly Leu Lys Lys Met Asn Pro Lys Leu Lys Thr Leu Leu Ala
60 65 70ATC GGA GGC TGG AAT TTC GGC ACT CAG AAG TTC ACA GAT ATG GTA GCC 334Ile Gly Gly Trp Asn Phe Gly Thr Gln Lys Phe Thr Asp Met Val Ala75 80 85 90ACG GCC AAC AAC CGT CAG ACC TTT GTC AAC TCG GCC ATC AGG TTT CTG 382Thr Ala Asn Asn Arg Gln Thr Phe Val Asn Ser Ala Ile Arg Phe Leu
95 100 105CGC AAA TAC AGC TTT GAC GGC CTT GAC CTT GAC TGG GAG TAC CCA GGA 430Arg Lys Tyr Ser Phe Asp Gly Leu Asp Leu Asp Trp Glu Tyr Pro Gly
110 115 120AGC CAG GGG AGC CCT GCC GTA GAC AAG GAG CGC TTC ACA ACC CTG GTA 478Ser Gln Gly Ser Pro Ala Val Asp Lys Glu Arg Phe Thr Thr Leu Val
125 130 135CAG GAC TTG GCC AAT GCC TTC CAG CAG GAA GCC CAG ACC TCA GGG AAG 526Gln Asp Leu Ala Asn Ala Phe Gln Gln Glu Ala Gln Thr Ser Gly Lys
140 145 150GAA CGC CTT CTT CTG AGT GCA GCG GTT CCA GCT GGG CAG ACC TAT GTG 574Glu Arg Leu Leu Leu Ser Ala Ala Val Pro Ala Gly Gln Thr Tyr Val155 160 165 170GAT GCT GGA TAC GAG GTG GAC AAA ATC GCC CAG AAC CTG GAT TTT GTC 622Asp Ala Gly Tyr Glu Val Asp Lys Ile Ala Gln Asn Leu Asp Phe Val
175 180 185AAC CTT ATG GCC TAC GAC TTC CAT GGC TCT TGG GAG AAG GTC ACG GGA 670Asn Leu Met Ala Tyr Asp Phe His Gly Ser Trp Glu Lys Val Thr Gly
190 195 200CAT AAC AGC CCC CTC TAC AAG AGG CAA GAA GAG AGT GGT GCA GCA GCC 718His Asn Ser Pro Leu Tyr Lys Arg Gln Glu Glu Ser Gly Ala Ala Ala
205 210 215AGC CTC AAC GTG GAT GCT GCT GTG CAA CAG TGG CTG CAG AAG GGG ACC 766Ser Leu Asn Val Asp Ala Ala Val Gln Gln Trp Leu Gln Lys Gly Thr
220 225 230CCT GCC AGC AAG CTG ATC CTT GGC ATG CCT ACC TAC GGA CGC TCC TTC 814Pro Ala Ser Lys Leu Ile Leu Gly Met Pro Thr Tyr Gly Arg Ser Phe235 240 245 250ACA CTG GCC TCC TCA TCA GAC ACC AGA GTG GGG GCC CCA GCC ACA GGG 862Thr Leu Ala Ser Ser Ser Asp Thr Arg Val Gly Ala Pro Ala Thr Gly
255 260 265TCT GGC ACT CCA GGC CCC TTC ACC AAG GAA GGA GGG ATG CTG GCC TAC 910Ser Gly Thr Pro Gly Pro Phe Thr Lys Glu Gly Gly Met Leu Ala Tyr
270 275 280TAT GAA GTC TGC TCC TGG AAG GGG GCC ACC AAA CAG AGA ATC CAG GAT 958Tyr Glu Val Cys Ser Trp Lys Gly Ala Thr Lys Gln Arg Ile Gln Asp
285 290 295CAG AAG GTG CCC TAC ATC TTC CGG GAC AAC CAG TGG GTG GGC TTT GAT 1006Gln Lys Val Pro Tyr Ile Phe Arg Asp Asn Gln Trp Val Gly Phe Asp
300 305 310GAT GTG GAG AGC TTC AAA ACC AAG GTC AGC TAT CTG AAG CAG AAG GGA 1054Asp Val Glu Ser Phe Lys Thr Lys Val Ser Tyr Leu Lys Gln Lys Gly315 320 325 330CTG GGC GGG GCC ATG GTC TGG GCA CTG GAC TTA GAT GAC TTT GCC GGC 1102Leu Gly Gly Ala Met Val Trp Ala Leu Asp Leu Asp Asp Phe Ala Gly
335 340 345TTC TCC TGC AAC CAG GGC CGA TAC CCC CTC ATC CAG ACG CTA CGG CAG 1150Phe Ser Cys Asn Gln Gly Arg Tyr Pro Leu Ile Gln Thr Leu Arg Gln
350 355 360GAA CTG AGT CTT CCA TAC TTG CCT TCA GGC ACC CCA GAG CTT GAA GTT 1198Glu Leu Ser Leu Pro Tyr Leu Pro Ser Gly Thr Pro Glu Leu Glu Val
365 370 375CCA AAA CCA GGT CAG CCC TCT GAA CCT GAG CAT GGC CCC AGC CCT GGA 1246Pro Lys Pro Gly Gln Pro Ser Glu Pro Glu His Gly Pro Ser Pro Gly
380 385 390CAA GAC ACG TTC TGC CAG GGC AAA GCT GAT GGG GTC TAT CCC AAT CCT 1294Gln Asp Thr Phe Cys Gln Gly Lys Ala Asp Gly Leu Tyr Pro Asn Pro395 400 405 410CGG GAA CGG TCC AGC TTC TAC AGC TGT GCA GCG GGG CGG CTG TTC CAG 1342Arg Glu Arg Ser Ser Phe Tyr Ser Cys Ala Ala Gly Arg Leu Phe Gln
415 420 425CAA AGC TGC CCG ACA GGC CTG GTG TTC AGC AAC TCC TGC AAA TGC TGC 1390Gln Ser Cys Pro Thr Gly Leu Val Phe Ser Asn Ser Cys Lys Cys Cys
430 435 440ACC TGG AAT TGAGTCGCTA AAGCCCCTCC AGTCCCAGCT TTGAGGCTGG 1439Thr Trp Asn
445GCCCAGGATC ACTCTACAGC CTGCCTCCTG GGTTTTCCCT GGGGGCCGCA ATCTGGCTCC 1499TGCAGGCCTT TCTGTGGTCT TCCTTTATCC AGGCTTTCTG CTCTCAGCCT TGCCTTCCTT 1559TTTTCTGGGT CTCCTGGGCT GCCCCTTTCA CTTGCAAAAT AAATCTTTGG TTTGTGCCCC 1619TCTTCCCAAA AAAAAAA 1636(2)SEQ ID NO:2的信息:(ⅰ)序列特征:
(A)长度:466个氨基酸s
(B)类型:氨基酸
(D)拓扑学:线性(ⅱ)分子类型:蛋白质(ⅹⅰ)序列描述:SEQ ID NO:2:Met Val Arg Ser Val Ala Trp Ala Gly Phe Met Val Leu Leu Met Ile-21 -20 -15 -10Pro Trp Gly Ser Ala Ala Lys Leu Val Cys Tyr Phe Thr Asn Trp Ala-5 1 5 10Gln Tyr Arg Gln Gly Glu Ala Arg Phe Leu Pro Lys Asp Leu Asp Pro
15 20 25Ser Leu Cys Thr His Leu Ile Tyr Ala Phe Ala Gly Met Thr Asn His
30 35 40Gln Leu Ser Thr Thr Glu Trp Asn Asp Glu Thr Leu Tyr Gln Glu Phe
45 50 55Asn Gly Leu Lys Lys Met Asn Pro Lys Leu Lys Thr Leu Leu Ala Ile60 65 70 75Gly Gly Trp Asn Phe Gly Thr Gln Lys Phe Thr Asp Met Val Ala Thr
80 85 90Ala Asn Asn Arg Gln Thr Phe Val Asn Ser Ala Ile Arg Phe Leu Arg
95 100 105Lys Tyr Ser Phe Asp Gly Leu Asp Leu Asp Trp Glu Tyr Pro Gly Ser
110 115 120Gln Gly Ser Pro Ala Val Asp Lys Glu Arg Phe Thr Thr Leu Val Gln
125 130 135Asp Leu Ala Asn Ala Phe Gln Gln Glu Ala Gln Thr Ser Gly Lys Glu140 145 150 155Arg Leu Leu Leu Ser Ala Ala Val Pro Ala Gly Gln Thr Tyr Val Asp
160 165 170Ala Gly Tyr Glu Val Asp Lys Ile Ala Gln Asn Leu Asp Phe Val Asn
175 180 185Leu Met Ala Tyr Asp Phe His Gly Ser Trp Glu Lys Val Thr Gly His
190 195 200Asn Ser Pro Leu Tyr Lys Arg Gln Glu Glu Ser Gly Ala Ala Ala Ser
205 210 215Leu Asn Val Asp Ala Ala Val Gln Gln Trp Leu Gln Lys Gly Thr Pro220 225 230 235Ala Ser Lys Leu Ile Leu Gly Mer Pro Thr Tyr Gly Arg Ser Phe Thr
240 245 250Leu Ala Ser Ser Ser Asp Thr Arg Val Gly Ala Pro Ala Thr Gly Ser
255 260 265Gly Thr Pro Gly Pro Phe Thr Lys Glu Gly Gly Met Leu Ala Tyr Tyr
270 275 280Glu Val Cys Ser Trp Lys Gly Ala Thr Lys Gln Arg Ile Gln Asp Gln
285 290 295Lys Val Pro Tyr Ile Phe Arg Asp Asn Gln Trp Val Gly Phe Asp Asp300 305 310 315Val Glu Ser Phe Lys Thr Lys Val Ser Tyr Leu Lys Gln Lys Gly Leu
320 325 330Gly Gly Ala Met Val Trp Ala Leu Asp Leu Asp Asp Phe Ala Gly Phe
335 340 345Ser Cys Asn Gln Gly Arg Tyr Pro Leu Ile Gln Thr Leu Arg Gln Glu
350 355 360Leu Ser Leu Pro Tyr Leu Pro Ser Gly Thr Pro Glu Leu Glu Val Pro
365 370 375Lys Pro Gly Gln Pro Ser Glu Pro Glu His Gly Pro Ser Pro Gly Gln380 385 390 395Asp Thr Phe Cys Gln Gly Lys Ala Asp Gly Leu Tyr Pro Asn Pro Arg
400 405 410Glu Arg Ser Ser Phe Tyr Ser Cys Ala Ala Gly Arg Leu Phe Gln Gln
415 420 425Ser Cys Pro Thr Gly Leu Val Phe Ser Asn Ser Cys Lys Cys Cys Thr
430 435 440Trp Asn
445(2)SEQ ID NO:3的信息:(ⅰ)序列特征:
(A)长度:1656个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑学:线性(ⅱ)分子类型:cDNA(ⅸ)特征:
(A)名称/关键词:CDS
(B)位置:27..1424(ⅸ)特征:
(A)名称/关键词:成熟肽
(B)位置:90..1424(ⅹⅰ)序列描述:SEQ ID NO:3:GCTGCAGCCT GCCGCTGAGC TGCATC ATG GTG CGG TCT GTG GCC TGG GCA GGT 53
Met Val Arg Ser Val Ala Trp Ala Gly
-21 -20 -15TTC ATG GTC CTG CTG ATG ATC CCA TGG GGC TCT GCT GCA AAA CTG GTC 101Phe Met Val Leu Leu Met Ile Pro Trp Gly Ser Ala Ala Lys Leu Val
-10 -5 1TGC TAC TTC ACC AAC TGG GCC CAG TAC AGA CAG GGG GAG GCT CGC TTC 149Cys Tyr Phe Thr Asn Trp Ala Gln Tyr Arg Gln Gly Glu Ala Arg Phe5 10 15 20CTG CCC AAG GAC TTG GAC CCC AGC CTT TGC ACC CAC CTC ATC TAC GCC 197Leu Pro Lys Asp Leu Asp Pro Ser Leu Cys Thr His Leu Ile Tyr Ala
25 30 35TTC GCT GGC ATG ACC AAC CAC CAG CTG AGC ACC ACT GAG TGG AAT GAC 245Phe Ala Gly Met Thr Asn His Gln Leu Ser Thr Thr Glu Trp Asn Asp
40 45 50GAG ACT CTC TAC CAG GAG TTC AAT GGC CTG AAG AAG ATG AAT CCC AAG 293Glu Thr Leu Tyr Gln Glu Phe Asn Gly Leu Lys Lys Met Asn Pro Lys
55 60 65CTG AAG ACC CTG TTA GCC ATC GGA GGC TGG AAT TTC AGC ACT CAG AAG 341Leu Lys Thr Leu Leu Ala Ile Gly Gly Trp Asn Phe Ser Thr Gln Lys
70 75 80TTC ACA GAT ATG GTA GCC ACG GCC AAC AAC CGT CAG ACC TTT GTC AAC 389Phe Thr Asp Met Val Ala Thr Ala Asn Asn Arg Gln Thr Phe Val Asn85 90 95 100TCG GCC ATC AGG TTT CTG CGC AAA TAC AGC TTT GAC GGC CTT GAC CTT 437Ser Ala Ile Arg Phe Leu Arg Lys Tyr Ser Phe Asp Gly Leu Asp Leu
105 110 115GAC TGG GAG TAC CCA GGA AGC CAG GGG AGC CCT GCC GTA GAC AAG GAG 485Asp Trp Glu Tyr Pro Gly Ser Gln Gly Ser Pro Ala Val Asp Lys Glu
120 125 130CGC TTC ACA ACC CTG GTA CAG GAC TTG GCC AAT GCC TTC CAG CAG GAA 533Arg Phe Thr Thr Leu Val Gln Asp Leu Ala Asn Ala Phe Gln Gln Glu
135 140 145GCC CAG ACC TCA GGG AAG GAA CGC CTT CTT CTG AGT GCA GCG GTT CCA 581Ala Gln Thr Ser Gly Lys Glu Arg Leu Leu Leu Ser Ala Ala Val Pro
150 155 160GCT GGG CAG ACC TAT GTG GAT GCT GGA TAC GAG GTG GAC AAA ATC GCC 629Ala Gly Gln Thr Tyr Val Asp Ala Gly Tyr Glu Val Asp Lys Ile Ala165 170 175 180CAG AAC CTG GAT TTT GTC AAC CTT ATG GCC TAC GAC TTC CAT GGC TCT 677Gln Asn Leu Asp Phe Val Asn Leu Met Ala Tyr Asp Phe His Gly Ser
185 190 195TGG GAG AAG GTC ACG GGA CAT AAC AGC CCC CTC TAC AAG AGG CAA GAA 725Trp Glu Lys Val Thr Gly His Asn Ser Pro Leu Tyr Lys Arg Gln Glu
200 205 210GAG AGT GGT GCA GCA GCC AGC CTC AAC GTG GAT GCT GCT GTG CAA CAG 773Glu Ser Gly Ala Ala Ala Ser Leu Asn Val Asp Ala Ala Val Gln Gln
215 220 225TGG CTG CAG AAG GGG ACC CCT GCC AGC AAG CTG ATC CTT GGC ATG CCT 821Trp Leu Gln Lys Gly Thr Pro Ala Ser Lys Leu Ile Leu Gly Met Pro
230 235 240ACC TAC GGA CGC TCC TTC ACA CTG GCC TCC TCA TCA GAC ACC AGA GTG 869Thr Tyr Gly Arg Ser Phe Thr Leu Ala Ser Ser Ser Asp Thr Arg Val245 250 255 260GGG GCC CCA GCC ACA GGG TCT GGC ACT CCA GGC CCC TTC ACC AAG GAA 917Gly Ala Pro Ala Thr Gly Ser Gly Thr Pro Gly Pro Phe Thr Lys Glu
265 270 275GGA GGG ATG CTG GCC TAC TAT GAA GTC TGC TCC TGG AAG GGG GCC ACC 965Gly Gly Met Leu Ala Tyr Tyr Glu Val Cys Ser Trp Lys Gly Ala Thr
280 285 290AAA CAG AGA ATC CAG GAT CAG AAG GTG CCC TAC ATC TTC CGG GAC AAC 1013Lys Gln Arg Ile Gln Asp Gln Lys Val Pro Tyr Ile Phe Arg Asp Asn
295 300 305CAG TGG GTG GGC TTT GAT GAT GTG GAG AGC TTC AAA ACC AAG GTC AGC 1061Gln Trp Val Gly Phe Asp Asp Val Glu Ser Phe Lys Thr Lys Val Ser
310 315 320TAT CTG AAG CAG AAG GGA CTG GGC GGG GCC ATG GTC TGG GCA CTG GAC 1109Tyr Leu Lys Gln Lys Gly Leu Gly Gly Ala Met Val Trp Ala Leu Asp325 330 335 340TTA GAT GAC TTT GCC GGC TTC TCC TGC AAC CAG GGC CGA TAC CCC CTC 1157Leu Asp Asp Phe Ala Gly Phe Ser Cys Asn Gln Gly Arg Tyr Pro Leu
345 350 355ATC CAG ACG CTA CGG CAG GAA CTG AGT CTT CCA TAC TTG CCT TCA GGC 1205Ile Gln Thr Leu Arg Gln Glu Leu Ser Leu Pro Tyr Leu Pro Ser Gly
360 365 370ACC CCA GAG CTT GAA GTT CCA AAA CCA GGT CAG CCC TCT GAA CCT GAG 1253Thr Pro Glu Leu Glu Val Pro Lys Pro Gly Gln Pro Ser Glu Pro Glu
375 380 385CAT GGC CCC AGC CCT GGA CAA GAC ACG TTC TGC CAG GGC AAA GCT GAT 1301His Gly Pro Ser Pro Gly Gln Asp Thr Phe Cys Gln Gly Lys Ala Asp
390 395 400GGG CTC TAT CCC AAT CCT CGG GAA CGG TCC AGC TTC TAC AGC TGT GCA 1349Gly Leu Tyr Pro Asn Pro Arg Glu Arg Ser Ser Phe Tyr Ser Cys Ala405 410 415 420GCG GGG CGG CTG TTC CAG CAA AGC TGC CCG ACA GGC CTG GTG TTC AGC 1397Ala Gly Arg Leu Phe Gln Gln Ser Cys Pro Thr Gly Leu Val Phe Ser
425 430 435AAC TCC TGC AAA TGC TGC ACC TGG AAT TGAGTCGCTA AAGCCCCTCC 1444Asn Ser Cys Lys Cys Cys Thr Trp Asn
440 445AGTCCCAGCT TTGAGGCTGG GCCCAGGATC ACTCTACAGC CTGCCTCCTG GGTTTTCCCT 1504GGGGGCCGCA ATCTGGCTCC TGCAGGCCTT TCTGTGGTCT TCCTTTATCC AGGCTTTCTG 1564CTCTCAGCCT TGCCTTCCTT TTTTCTGGGT CTCCTGGGCT GCCCCTTTCA CTTGCAAAAT 1624AAATCTTTGG TTTGTGCCCC TCAAAAAAAA AA 1656(2)SEQ ID NO:4的信息:(ⅰ)序列特征:
(A)长度:466个氨基酸s
(B)类型:氨基酸
(D)拓扑学:线性(ⅱ)分子类型:蛋白质(ⅹⅰ)序列描述:SEQ ID NO:4:Met Val Arg Ser Val Ala Trp Ala Gly Phe Met Val Leu Leu Met Ile-21 -20 -15 -10Pro Trp Gly Ser Ala Ala Lys Leu Val Cys Tyr Phe Thr Asn Trp Ala-5 1 5 10Gln Tyr Arg Gln Gly Glu Ala Arg Phe Leu Pro Lys Asp Leu Asp Pro
15 20 25Ser Leu Cys Thr His Leu Ile Tyr Ala Phe Ala Gly Met Thr Asn His
30 35 40Gln Leu Ser Thr Thr Glu Trp Asn Asp Glu Thr Leu Tyr Gln Glu Phe
45 50 55Asn Gly Leu Lys Lys Met Asn Pro Lys Leu Lys Thr Leu Leu Ala Ile60 65 70 75Gly Gly Trp Asn Phe Ser Thr Gln Lys Phe Thr Asp Met Val Ala Thr
80 85 90Ala Asn Asn Arg Gln Thr Phe Val Asn Ser Ala Ile Arg Phe Leu Arg
95 100 105Lys Tyr Ser Phe Asp Gly Leu Asp Leu Asp Trp Glu Tyr Pro Gly Ser
110 115 120Gln Gly Ser Pro Ala Val Asp Lys Glu Arg Phe Thr Thr Leu Val Gln
125 130 135Asp Leu Ala Asn Ala Phe Gln Gln Glu Ala Gln Thr Ser Gly Lys Glu140 145 150 155Arg Leu Leu Leu Ser Ala Ala Val Pro Ala Gly Gln Thr Tyr Val Asp
160 165 170Ala Gly Tyr Glu Val Asp Lys Ile Ala Gln Asn Leu Asp Phe Val Asn
175 180 185Leu Met Ala Tyr Asp Phe His Gly Ser Trp Glu Lys Val Thr Gly His
190 195 200Asn Ser Pro Leu Tyr Lys Arg Gln Glu Glu Ser Gly Ala Ala Ala Ser
205 210 215Leu Asn Val Asp Ala Ala Val Gln Gln Trp Leu Gln Lys Gly Thr Pro220 225 230 235Ala Ser Lys Leu Ile Leu Gly Met Pro Thr Tyr Gly Arg Ser Phe Thr
240 245 250Leu Ala Ser Ser Ser Asp Thr Arg Val Gly Ala Pro Ala Thr Gly Ser
255 260 265Gly Thr Pro Gly Pro Phe Thr Lys Glu Gly Gly Met Leu Ala Tyr Tyr
270 275 280Glu Val Cys Ser Trp Lys Gly Ala Thr Lys Gln Arg Ile Gln Asp Gln
285 290 295Lys Val Pro Tyr Ile Phe Arg Asp Asn Gln Trp Val Gly Phe Asp Asp300 305 310 315Val Glu Ser Phe Lys Thr Lys Val Ser Tyr Leu Lys Gln Lys Gly Leu
320 325 330Gly Gly Ala Met Val Trp Ala Leu Asp Leu Asp Asp Phe Ala Gly Phe
335 340 345Ser Cys Asn Gln Gly Arg Tyr Pro Leu Ile Gln Thr Leu Arg Gln Glu
350 355 360Leu Ser Leu Pro Tyr Leu Pro Ser Gly Thr Pro Glu Leu Glu Val Pro
365 370 375Lys Pro Gly Gln Pro Ser Glu Pro Glu His Gly Pro Ser Pro Gly Gln380 385 390 395Asp Thr Phe Cys Gln Gly Lys Ala Asp Gly Leu Tyr Pro Asn Pro Arg
400 405 410Glu Arg Ser Ser Phe Tyr Ser Cys Ala Ala Gly Arg Leu Phe Gln Gln
415 420 425Ser Cys Pro Thr Gly Leu Val Phe Ser Asn Ser Cys Lys Cys Cys Thr
430 435 440Trp Asn
445(2)SEQ ID NO:5的信息:(ⅰ)序列特征:
(A)长度:18个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑学:线性(ⅱ)分子类型:cDNA(ⅹⅰ)序列描述:SEQ ID NO:5:GACACTATAG AATAGGGC 18(2)SEQ ID NO:6的信息:(ⅰ)序列特征:
(A)长度:51
(B)类型:核酸
(C)链型:单链
(D)拓扑学:线性(ⅱ)分子类型:cDNA(ⅹⅰ)序列描述:SEQ ID NO:6:TGGGATCATC AGCAGGACCA TGAAACCTGC CCAGGCCACA GACCGCACCA T 51(2)SEQ ID NO:7的信息:(ⅰ)序列特征:
(A)长度:40个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑学:线性(ⅱ)分子类型:cDNA(ⅹⅰ)序列描述:SEQ ID NO:7:TACATCTAGA ATTATGGCAA AACTGGTCTG CTACTTCACC 40(2)SEQ ID NO:8的信息:(ⅰ)序列特征:
(A)长度:34个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑学:线性(ⅱ)分子类型:cDNA(ⅹⅰ)序列描述:SEQ ID NO:8:AGATCTAACC TTAGGTGCCT GAAGACAAGT ATGG 34(2)SEQ ID NO:9的信息:(ⅰ)序列特征:
(A)长度:29个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑学:线性(ⅱ)分子类型:cDNA(ⅹⅰ)序列描述:SEQ ID NO:9的信息:TACAGAATTC TTATTCACAT CCGGCCCTG 29(2)SEQ ID NO:10:(ⅰ)序列特征:
(A)长度:34个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑学:线性(ⅱ)分子类型:cDNA(ⅹⅰ)序列描述:SEQ ID NO:10的信息:TACATCTAGA CTCCATCCAG AAAAACAGGT ATGG 34(2)SEQ ID NO:11的信息:(ⅰ)序列特征:
(A)长度:30个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑学:线性(ⅱ)分子类型:cDNA(ⅹⅰ)序列描述:SEQ ID NO:11:TCTAGAGTCG ACCTGCAGGC ATGCAAGCTT 30(2)SEQ ID NO:12的信息:(ⅰ)序列特征:
(A)长度:50个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑学:线性(ⅱ)分子类型:cDNA(ⅹⅰ)序列描述:SEQ ID NO:12:CGCAAGCTTG AGAGCTCCGT TCCGCCACAT GGTGCGGTCT GTGGCCTGGG 50(2)SEQ ID NO:13的信息:(ⅰ)序列特征:
(A)长度:32个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑学:线性(ⅱ)分子类型:cDNA(ⅹⅰ)序列描述:SEQ ID NO:13:GACTCTAGAC TAGGTGCCTG AAGGCAAGTA TG 32(2)SEQ ID NO:14的信息:(ⅰ)序列特征:
(A)长度:373个氨基酸s
(B)类型:氨基酸
(C)链型:单链
(D)拓扑学:线性(ⅱ)分子类型:肽(ⅹⅰ)序列描述:SEQ ID NO:14:Ala Lys Leu Val Cys Tyr Phe Thr Asn Trp Ala Gln Tyr Arg Gln Gly1 5 10 15Glu Ala Arg Phe Leu Pro Lys Asp Leu Asp Pro Ser Leu Cys Thr His
20 25 30Leu Ile Tyr Ala Phe Ala Gly Met Thr Asn His Gln Leu Ser Thr Thr
35 40 45Glu Trp Asn Asp Glu Thr Leu Tyr Gln Glu Phe Asn Gly Leu Lys Lys
50 55 60Met Asn Pro Lys Leu Lys Thr Leu Leu Ala Ile Gly Gly Trp Asn Phe65 70 75 80Gly Thr Gln Lys Phe Thr Asp Met Val Ala Thr Ala Asn Asn Arg Gln
85 90 95Thr Phe Val Asn Ser Ala Ile Arg Phe Leu Arg Lys Tyr Ser Phe Asp
100 105 110Gly Leu Asp Leu Asp Trp Glu Tyr Pro Gly Ser Gln Gly Ser Pro Ala
115 120 125Val Asp Lys Glu Arg Phe Thr Thr Leu Val Gln Asp Leu Ala Asn Ala
130 135 140Phe Gln Gln Glu Ala Gln Thr Ser Gly Lys Glu Arg Leu Leu Leu Ser145 150 155 160Ala Ala Val Pro Ala Gly Gln Thr Tyr Val Asp Ala Gly Tyr Glu Val
165 170 175Asp Lys Ile Ala Gln Asn Leu Asp Phe Val Asn Leu Met Ala Tyr Asp
180 185 190Phe His Gly Ser Trp Glu Lys Val Thr Gly His Asn Ser Pro Leu Tyr
195 200 205Lys Arg Gln Glu Glu Ser Gly Ala Ala Ala Ser Leu Asn Val Asp Ala
210 215 220Ala Val Gln Gln Trp Leu Gln Lys Gly Thr Pro Ala Ser Lys Leu Ile225 230 235 240Leu Gly Met Pro Thr Tyr Gly Arg Ser Phe Thr Leu Ala Ser Ser Ser
245 250 255Asp Thr Arg Val Gly Ala Pro Ala Thr Gly Ser Gly Thr Pro Gly Pro
260 265 270Phe Thr Lys Glu Gly Gly Met Leu Ala Tyr Tyr Glu Val Cys Ser Trp
275 280 285
Lys Gly Ala Thr Lys Gln Arg Ile Gln Asp Gln Lys Val Pro Tyr Ile
290 295 300
Phe Arg Asp Asn Gln Trp Val Gly Phe Asp Asp Val Glu Ser Phe Lys
305 310 315 320
Thr Lys Val Ser Tyr Leu Lys Gln Lys Gly Leu Gly Gly Ala Met Val
325 330 335
Trp Ala Leu Asp Leu Asp Asp Phe Ala Gly Phe Ser Cys Asn Gln Gly
340 345 350
Arg Tyr Pro Leu Ile Gln Thr Leu Arg Gln Glu Leu Ser Leu Pro Tyr
355 360 365
Leu Pro Ser Gly Thr
370(2)SEQ ID NO:15的信息:(ⅰ)序列特征:
(A)长度:373个氨基酸s
(B)类型:氨基酸
(C)链型:单链
(D)拓扑学:线性(ⅱ)分子类型:肽(ⅹⅰ)序列描述:SEQ ID NO:15:Ala Lys Leu Val Cys Tyr Phe Thr Asn Trp Ala Gln Tyr Arg Gln Gly1 5 10 15Glu Ala Arg Phe Leu Pro Lys Asp Leu Asp Pro Ser Leu Cys Thr His
20 25 30Leu Ile Tyr Ala Phe Ala Gly Met Thr Asn His Gln Leu Ser Thr Thr
35 40 45Glu Trp Asn Asp Glu Thr Leu Tyr Gln Glu Phe Asn Gly Leu Lys Lys
50 55 60Met Asn Pro Lys Leu Lys Thr Leu Leu Ala Ile Gly Gly Trp Asn Phe65 70 75 80Gly Thr Gln Lys Phe Thr Asp Met Val Ala Thr Ala Asn Asn Arg Gln
85 90 95Thr Phe Val Asn Ser Ala Ile Arg Phe Leu Arg Lys Tyr Ser Phe Asp
100 105 110Gly Leu Asp Leu Asp Trp Glu Tyr Pro Gly Ser Gln Gly Ser Pro Ala
115 120 125Val Asp Lys Glu Arg Phe Thr Thr Leu Val Gln Asp Leu Ala Asn Ala
130 135 140Phe Gln Gln Glu Ala Gln Thr Ser Gly Lys Glu Arg Leu Leu Leu Ser145 150 155 160Ala Ala Val Pro Ala Gly Gln Thr Tyr Val Asp Ala Gly Tyr Glu Val
165 170 175Asp Lys Ile Ala Gln Asn Leu Asp Phe Val Asn Leu Met Ala Tyr Asp
180 185 190Phe His Gly Ser Trp Glu Lys Val Thr Gly His Asn Ser Pro Leu Tyr
195 200 205Lys Arg Gln Glu Glu Ser Gly Ala Ala Ala Ser Leu Asn Val Asp Ala
210 215 220Ala Val Gln Gln Trp Leu Gln Lys Gly Thr Pro Ala Ser Lys Leu Ile225 230 235 240Leu Gly Met Pro Thr Tyr Gly Arg Ser Phe Thr Leu Ala Ser Ser Ser
245 250 255Asp Thr Arg Val Gly Ala Pro Ala Thr Gly Ser Gly Thr Pro Gly Pro
260 265 270Phe Thr Lys Glu Gly Gly Met Leu Ala Tyr Tyr Glu Val Cys Ser Trp
275 280 285Lys Gly Ala Thr Lys Gln Arg Ile Gln Asp Gln Lys Val Pro Tyr Ile
290 295 300Phe Arg Asp Asn Gln Trp Val Gly Phe Asp Asp Val Glu Ser Phe Lys305 310 315 320Thr Lys Val Ser Tyr Leu Lys Gln Lys Gly Leu Gly Gly Ala Met Val
325 330 335Trp Ala Leu Asp Leu Asp Asp Phe Ala Gly Phe Ser Cys Asn Gln Gly
340 345 350Arg Tyr Pro Leu Ile Gln Thr Leu Arg Gln Glu Leu Ser Leu Pro Tyr
355 360 365Leu Ser Ser Gly Thr
370(2)SEQ ID NO:16的信息:(ⅰ)序列特征:
(A)长度:28个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑学:线性(ⅱ)分子类型:其它核酸
(A)描述:/desc=″寡核苷酸引物″(ⅹⅰ)序列描述:SEQ ID NO:16:TGATACGGTA CCGCCCCATG GCTGACTA 28(2)SEQ ID NO:17的信息:(ⅰ)序列特征:
(A)长度:20个碱基对
(B)类型:核酸
(C)链型:单链
(D)拓扑学:线性(ⅱ)分子类型:其它核酸
(A)描述:/desc=″引物″(ⅹⅰ)序列描述:SEQ ID N0:17:GCAAGTTTGG CGCGAAATCG 20
Claims (35)
1.纯化分离的多核苷酸,它编码SEQ ID NO:2的人几丁质酶氨基酸序列。
2.权利要求1的多核苷酸,它是DNA。
3.权利要求2的DNA,它包含SEQ ID NO:1的蛋白编码核苷酸。
4.纯化分离的多核苷酸,它编码SEQ ID NO:2的1-445氨基酸。
5.权利要求4的多核苷酸,它是DNA。
6.权利要求5的DNA,它包含SEQ ID NO:1的65-1402核苷酸。
7.纯化分离的多核苷酸,编码SEQ ID NO:4的人几丁质酶氨基酸序列。
8.权利要求7的多核苷酸,它是DNA。
9.权利要求8的DNA,它包含SEQ ID NO:3的蛋白编码核苷酸。
10.纯化分离的多核苷酸,它编码SEQ ID NO:4的1-445氨基酸。
11.权利要求10的多核苷酸,它是DNA。
12.权利要求11的DNA,它包含SEQ ID NO:3的90-1427核苷酸。
13.编码人几丁质酶的纯化分离的多核苷酸,选自:
(a)双链DNA,包含示于SEQ ID NO:3的序列的蛋白编码部分;
(b)在严格条件下杂交到(a)的DNA非编码链上的DNA;和
(c)如果没有遗传密码的丰余性,则在严格条件下能够杂交到(a)或(b)的DNA序列的非编码链上的DNA。
14.权利要求13的多核苷酸,它是DNA。
15.包含权利要求2、3、5、6、8、9、11、12或14的DNA的载体。
16.权利要求15的载体,它是表达载体,其中所述DNA***纵连接到表达控制DNA序列上。
17.稳定转化或转染的宿主细胞,它是以允许在所述宿主细胞中表达人几丁质酶的方式,用权利要求2、3、5、6、8、9、11、12或14的DNA进行转化或转染。
18.生产人几丁质酶的方法,包括在营养培养基中培养权利要求17的宿主细胞,并从所述宿主细胞或所述营养培养基中分离人几丁质酶。
19.用权利要求18的方法生产的分离纯化的多核苷酸。
20.纯化分离的多肽,它包含SEQ ID NO:2的人几丁质酶氨基酸序列。
21.纯化分离的多肽,它包含SEQ ID NO:4的人几丁质酶氨基酸序列。
22.纯化分离的多肽,它包含SEQ ID NO:2的人几丁质酶1-445氨基酸。
23.纯化分离的多肽,它包含SEQ ID NO:4的人几丁质酶1-445氨基酸。
24.人几丁质酶多肽片段,它缺乏成熟的人几丁质酶1至大约72个C末端氨基酸残基。
25.SEQ ID NO:14的人几丁质酶多肽片段。
26.纯化分离的多核苷酸,包含编码SEQ ID NO:14的氨基酸序列的多核苷酸序列。
27.权利要求26的多核苷酸,它是DNA。
28.SEQ ID NO:15的人几丁质酶多肽类似物。
29.纯化分离的多核苷酸,它编码SEQ ID NO:15的氨基酸序列。
30.杂交瘤细胞系,它生产与权利要求19、20、21、22、23或28的多肽发生特异性反应的单克隆抗体。
31.用权利要求30的杂交瘤生产的单克隆抗体。
32.药用组合物,包含权利要求19-25或权利要求28中的任何一项的多肽。
33.治疗真菌性感染的方法,包括对受治疗者给予治疗有效量的人几丁质酶。
34.权利要求33的方法,还包括对所述的受治疗者给予治疗有效量的非几丁质酶抗真菌剂。
35.降低非几丁质酶抗真菌剂在受治疗者中发挥抗真菌活性所需用量的方法,包括对所述的受治疗者给予人几丁质酶,其量足以有效提高所述的非几丁质酶抗真菌剂的抗真菌活性。
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US08/663,618 US20030017570A1 (en) | 1996-06-14 | 1996-06-14 | Chitinase materials and methods |
US08/663,618 | 1996-06-14 |
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CN1236393A true CN1236393A (zh) | 1999-11-24 |
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Application Number | Title | Priority Date | Filing Date |
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CN97197145A Pending CN1236393A (zh) | 1996-06-14 | 1997-06-16 | 几丁质酶材料和方法 |
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US (2) | US20030017570A1 (zh) |
EP (1) | EP0918870A1 (zh) |
JP (1) | JP2001510325A (zh) |
CN (1) | CN1236393A (zh) |
AU (1) | AU731203B2 (zh) |
BR (1) | BR9709721A (zh) |
CA (1) | CA2257829A1 (zh) |
CZ (1) | CZ403598A3 (zh) |
HU (1) | HUP0001644A3 (zh) |
IL (1) | IL127568A0 (zh) |
NO (1) | NO985806L (zh) |
PL (1) | PL337060A1 (zh) |
SK (1) | SK169798A3 (zh) |
WO (1) | WO1997047752A1 (zh) |
Cited By (1)
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CN109971741A (zh) * | 2019-03-27 | 2019-07-05 | 大连大学 | 低温几丁质酶基因chiC在乳酸克鲁维酵母中的表达纯化方法 |
Families Citing this family (10)
Publication number | Priority date | Publication date | Assignee | Title |
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US6200951B1 (en) | 1998-03-12 | 2001-03-13 | Icos Corporation | Chitinase chitin-binding fragments |
AU7741000A (en) * | 1999-09-30 | 2001-04-30 | Icos Corporation | Chitinase immunoglobulin fusion products |
US7632654B2 (en) * | 2002-04-29 | 2009-12-15 | Academisch Ziekenhuis Bij De Universiteit Van Amsterdam | Means and methods for detecting endoglycosidase activity |
FR2908654B1 (fr) * | 2006-11-20 | 2014-04-04 | Oreal | Utilisation cosmetique de proteines de type chitinase |
EP2245457A1 (en) * | 2008-01-23 | 2010-11-03 | Rigshospitalet | Classification of individuals suffering from cardiovascular diseases according to survival prognoses as found by measuring the levels of biomarker ykl-40 |
EP2192409B1 (en) * | 2008-11-26 | 2014-04-23 | Corning Incorporated | Label independent detection biosensor composition and methods thereof |
WO2011159865A2 (en) * | 2010-06-16 | 2011-12-22 | Yale University | Compositions and methods for using human ykl-40 to treat acute lung injury |
EP2746407A1 (en) * | 2012-12-18 | 2014-06-25 | EADS Deutschland GmbH | Novel Aspergillus sp. strain NBIMCC 8735 and method of producing chitinase utilizing the same |
PL235436B1 (pl) | 2014-11-28 | 2020-08-10 | Univ Jagielloński | Kompleks białkowy zawierający białko G i chitynazę A, sposób wyodrębniania z roztworu wodnego chitynazy A oraz jego zastosowania |
WO2018078626A1 (en) * | 2016-10-27 | 2018-05-03 | Gavish-Galilee Bio Applications, Ltd. | Combination therapies including human chitinase (chit1) for the treatment of systemic fungal infection |
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US5326561A (en) | 1992-12-15 | 1994-07-05 | Cornell Research Foundation, Inc. | Antifungal synergistic combination of enzyme fungicide and non-enzymatic fungicide and use thereof |
US5561051A (en) | 1994-06-14 | 1996-10-01 | American Cyanamid Company | Screen for inhibitors of chitinase |
US5928928A (en) | 1995-06-07 | 1999-07-27 | Universiteit Van Amsterdam | Human chitinase, its recombinant production, its use for decomposing chitin, its use in therapy or prophylaxis against infection diseases |
AU2593797A (en) | 1996-03-29 | 1997-10-22 | Human Genome Sciences, Inc. | Chitotriosidase |
-
1996
- 1996-06-14 US US08/663,618 patent/US20030017570A1/en not_active Abandoned
-
1997
- 1997-06-16 HU HU0001644A patent/HUP0001644A3/hu unknown
- 1997-06-16 AU AU33978/97A patent/AU731203B2/en not_active Ceased
- 1997-06-16 CZ CZ984035A patent/CZ403598A3/cs unknown
- 1997-06-16 BR BR9709721A patent/BR9709721A/pt not_active Application Discontinuation
- 1997-06-16 CA CA002257829A patent/CA2257829A1/en not_active Abandoned
- 1997-06-16 SK SK1697-98A patent/SK169798A3/sk unknown
- 1997-06-16 EP EP97930059A patent/EP0918870A1/en not_active Withdrawn
- 1997-06-16 CN CN97197145A patent/CN1236393A/zh active Pending
- 1997-06-16 WO PCT/US1997/010460 patent/WO1997047752A1/en not_active Application Discontinuation
- 1997-06-16 US US08/877,599 patent/US6372212B1/en not_active Expired - Fee Related
- 1997-06-16 IL IL12756897A patent/IL127568A0/xx unknown
- 1997-06-16 PL PL97337060A patent/PL337060A1/xx unknown
- 1997-06-16 JP JP50188998A patent/JP2001510325A/ja not_active Ceased
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1998
- 1998-12-11 NO NO985806A patent/NO985806L/no not_active Application Discontinuation
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109971741A (zh) * | 2019-03-27 | 2019-07-05 | 大连大学 | 低温几丁质酶基因chiC在乳酸克鲁维酵母中的表达纯化方法 |
Also Published As
Publication number | Publication date |
---|---|
CZ403598A3 (cs) | 1999-07-14 |
US6372212B1 (en) | 2002-04-16 |
EP0918870A1 (en) | 1999-06-02 |
NO985806D0 (no) | 1998-12-11 |
WO1997047752A1 (en) | 1997-12-18 |
BR9709721A (pt) | 1999-08-10 |
AU731203B2 (en) | 2001-03-29 |
IL127568A0 (en) | 1999-10-28 |
US20030017570A1 (en) | 2003-01-23 |
CA2257829A1 (en) | 1997-12-18 |
HUP0001644A2 (hu) | 2000-09-28 |
AU3397897A (en) | 1998-01-07 |
HUP0001644A3 (en) | 2002-09-30 |
PL337060A1 (en) | 2000-07-31 |
SK169798A3 (en) | 2000-03-13 |
NO985806L (no) | 1999-02-11 |
JP2001510325A (ja) | 2001-07-31 |
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