CN1618969A - 哺乳动物细胞因子样多肽-10 - Google Patents
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Abstract
称为Zcyto10的哺乳动物细胞因子样多肽,编码它的多核苷酸,可特异结合该多肽的抗体,及可结合该抗体的抗独特型抗体。Zcyto10有助于促进伤口的愈合并利于刺激血小板的增殖。
Description
本申请是申请日为1998年11月25日的中国专利申请98812428.9的分案申请。
发明背景
激素和多肽生长因子调控多细胞生物的细胞增殖和分化。这些可扩散分子使细胞相互交流并一齐作用以形成细胞和器官,及修复和再生损伤组织。激素和生长因子的例子包括类固醇激素(如雌激素、睾丸素)、甲状旁腺激素、促卵泡激素、白细胞介素、血小板衍生生长因子(PDGF)、表皮生长因子(EGF)、粒细胞巨噬细胞集落刺激因子(GM-CSF)、促红细胞生成素(EPO)和降钙素。
激素和生长因子通过结合蛋白质影响细胞代谢。蛋白质可以是与细胞内信号传导途径有关的整合膜蛋白,诸如第二信使系统。其它类的蛋白质是可溶性分子。
尤其令人感兴趣的是细胞因子,即促进细胞增殖和/或分化的分子。细胞因子的例子包括促红细胞生成素(EPO),它刺激红细胞的发育;血小板生成素(TPO),它刺激巨核细胞谱系细胞的发育;及粒细胞集落刺激因子(G-CSF),它刺激嗜中性粒细胞的发育。这些细胞因子有助于恢复贫血症病人或接受癌症化疗病人体内正常的血细胞水平。这些细胞因子已经证实的体内活性说明了其它细胞因子、细胞因子兴奋剂及细胞因子拮抗物的巨大临床潜力和对它们的需求。
发明简述
本发明通过提供新多肽及相关组合物和方法来满足此需求。一方面,本发明提供了编码被称为Zcyto10之哺乳动物四α-螺旋细胞因子的分离多核苷酸。人Zcyto10多肽包括如SEQ ID NO:1和SEQ ID NO:2中所示之带起始甲硫氨酸的176个氨基酸的序列。现认为氨基酸残基1-24是信号序列,包含第25位残基(亮氨酸)至176位氨基酸残基(谷氨酸)、由SEQ ID NO:12所限定的氨基酸序列代表成熟Zcyto10多肽。
本发明的另一实施方案由SEQ ID NO:3和SEQ ID NO:4的序列所限定。SEQ ID NO:4的多肽包括151个氨基酸残基,其中第1-24位氨基酸含信号序列,成熟序列包括也由SEQ ID NO:13限定的第25位氨基酸残基(亮氨酸)至第151位氨基酸(谷氨酸)。另一活性变体包括SEQ ID NO:2中第33位氨基酸残基(半胱氨酸)至第176位氨基酸残基。该变体也由SEQ ID NO:26限定。
小鼠Zcyto10也是包括如SEQ ID NO:18和19所限定之176个氨基酸残基的多肽。小鼠Zcyto10具有从SEQ ID NO:19的第1位氨基酸残基甲硫氨酸延伸至并包括第24位氨基酸残基甘氨酸的信号序列。因而,成熟小鼠Zcyto10从SEQ ID NO:19的第25位氨基酸残基亮氨酸延伸至并包括第176位氨基酸残基亮氨酸,也由SEQ ID NO:20所限定。另一活性变体被认为自SEQ ID NO:19的第33位氨基酸半胱氨酸延伸至第176位氨基酸。该变体也由SEQ ID NO:25确定。在另外的实施方案中,该多肽进一步包括亲和标志。
SEQ ID NO:33和34表示小鼠Zcyto10的一种变体。该变体为154个氨基酸残基长,并具有从SEQ ID NO:34第1位氨基酸残基甲硫氨酸延伸至并包括第24位氨基酸残基甘氨酸的信号序列。因而,成熟序列从SEQ ID NO:34的第25位氨基酸残基亮氨酸延伸至并包括第154位氨基酸残基亮氨酸。成熟序列也用SEQ ID NO:35表示。
本发明的第二方面提供了表达载体,其中含(a)转录启动子;(b)编码Zcyto10多肽的DNA区段,和(c)转录终止子,其中启动子、DNA区段和终止子有效连接。
本发明的第三方面提供了已引入如上所述表达载体的人工培养真核或原核细胞,其中所说细胞表达该DNA区段所编码的多肽。
本发明的进一步方面提供了基本上由通过肽键连接之第一部分和第二部分组成的嵌合多肽。该嵌合多肽的第一部分基本上由以下组成:(a)如SEQ ID NO:2中所示的Zcyto10多肽;(b)SEQ ID NO:2、SEQ ID NO:4、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:25、SEQ ID NO:26、SEQ ID NO:34或SEQ ID NO:35的等位变体;和(c)与(a)或(b)至少90%相同的蛋白质多肽。嵌合多肽的第二部分基本上由诸如亲和标志之类的另一多肽组成。某实施方案中,亲和标志是免疫球蛋白Fc多肽。本发明还提供编码嵌合多肽的表达载体及被转染以产生嵌合多肽的宿主细胞。
本发明的另一方面提供了能特异结合如上所公开Zcyto10多肽的抗体,以及能中和抗Zcyto10多肽抗体的抗独特型抗体。
本发明的另一方面提供了含与药用可接受载体联合之纯化Zcyto10多肽的药物组合物。如本文所进一步论述的,在特征在于不适当细胞增殖、细胞分化或细胞因子产生的紊乱的预防和治疗中,这样的组合物可有助于调节细胞增殖、细胞分化或细胞因子产生。更特异的是,Zcyto10多肽可通过抑制细胞免疫应答而有助于自身免疫疾病的预防和治疗。可用Zcyto10治疗的自身免疫疾病包括IDDM、多发性硬化症、类风湿性关节炎等等。同样,本发明的Zcyto10多肽可有助于抑制癌细胞生长或增殖。
本发明的Zcyto10多肽还可刺激免疫系统以更好地抵御微生物或病毒感染。尤其是,可系统性服用Zcyto10以提高个体的血小板产量。此外,本发明的Zcyto10多肽可用于气管特异或气管支气管特异的应用中,诸如用于气管支气管上皮或其基底细胞的维持或伤口修复中,用于调节粘液产生或残渣的粘纤毛清除中或用于哮喘、支气管炎或气管支气管道其它疾病的治疗中。它还可增强伤口愈合及促进受影响组织的再生,尤其有助于牙周疾病的治疗。此外,Zcyto10多肽可用于治疗皮肤病,大体上有诸如牛皮癣、湿疹和干皮病。
本发明的另一实施方案涉及具Zcyto10多肽带表位部分之氨基酸序列的肽或多肽,该Zcyto10多肽具有上述氨基酸序列。尽管长达并包括上述本发明多肽整个氨基酸序列的任何长度带表位多肽也包括于本发明中,但具本发明Zcyto10多肽带表位部分的氨基酸序列的肽或多肽包括具至少9个,优选至少15个,更优选至少30-50个氨基酸的多肽部分。还要求保护的是任何与另一多肽或载体分子融合的这些多肽。这样的表位变体包括但不局限于SEQ ID NO:25-32。自Zcyto10这些带表位部分产生的抗体可用于从细胞培养基中纯化Zcyto10。
参照以下详述和附图,本发明的这些和其它方面将更加明显。
发明详述
此文所引用的所有参考文献均全部引用作为参考。
在详述本发明之前,定义以下术语有助于对其的理解:
术语“亲和标记”在此用于指能附着到第二种多肽上以利于第二种多肽的纯化和检测或者为第二种多肽与底物的结合提供位点的多肽片段。原则上,可得到抗体或其它特异结合剂的任何肽或蛋白质都可用作亲和标记。亲和标记包括多聚组氨酸片段、蛋白A[Nilsson等人,EMBO J.4:1075(1985);Nilsson等人,酶学方法,198:3(1991)]、谷胱甘肽S转移酶[Smith和Johnson,基因67:31(1988)]、谷氨酸-谷氨酸亲和标记[Grussenmeyer等人,美国国家科学院院报82:7952-4(1985)]、P物质、FlagTM肽[Hopp等人,生物技术学6:1204-1210(1988)]、链霉亲和素结合肽、或其它抗原性表位或结合结构域。通常参阅Ford等人,蛋白质表达和纯化2:95-107(1991)。编码亲和标记的DNA可从商品供应商处购得(如Pharmacia Biotech,Piscataway,NJ)。
术语“等位变体”在此用于指一个基因占据相同染色体位点的两种或多种变换形式中的任一种。等位变异是通过突变自然发生的,并可能导致种群内的表型多态性。基因突变可以是沉默的(所编码多肽无变化)或编码具有改变了的氨基酸序列的多肽。等位变体一词在本文中也用于指由基因的等位变体编码的蛋白质。
术语“氨基末端”和“羧基末端”在此用于表示多肽中的位置。在内容允许之处,这些术语用于表示参照某一多肽特定序列或部分的接近度或相对位置。例如,位于多肽中参照序列羧基端的某序列即是在参照序列羧基末端附近,但不必在完整多肽的羧基末端。
术语“互补物/反互补物对”表示在适当条件下形成非共价结合的稳定对的不相同部分。例如,生物素和抗生物素蛋白(或链霉亲和素)是互补物/反互补物对的典型成员。其它例子的互补物/反互补物对包括受体/配体对、抗体/抗原(或半抗原或表位)对、有义/反义多核苷酸对,等等。在需要随后解离的互补物/反互补物对中,优选互补物/反互补物对的结合亲和力小于109M-1。
术语“多核苷酸分子的互补物”是具有与参照序列反向互补的碱基序列的多核苷酸分子。例如,序列5′ATGCACGGG 3′与5′CCCGTGCAT3′是互补的。
术语“毗连序列”表示具有与另一多核苷酸相同或互补之邻接序列的多核苷酸。邻接序列被称为与所给多核苷酸序列的一段全部或该多核苷酸部分“重叠”。例如,多核苷酸序列5′-ATGGCTTAGCTT-3′的代表性毗连序列是5′-TAGCTTgagtct-3′和3′-gtcgacTACCGA-5′。
术语“简并核苷酸序列”表示包括一个或多个简并密码子(与编码多肽的参照多核苷酸分子相比)的核苷酸序列。简并密码子包括不同的核苷酸三联体,但编码相同的氨基酸残基(即,GAU和GAC三联体均编码天冬氨酸)。
术语“表达载体”用于表示含与用于转录的附加片段有效连接的目的多肽编码片段的线性或环状DNA分子。这些附加片段包括启动子和终止子序列,还可包括一个或多个复制起点、一个或多个选择标记、增强子、聚腺苷酸化信号,等等。表达载体通常来源于质粒或病毒DNA,或可能两者的成分都包括。
术语“分离”当用于多核苷酸分子时,是指多核苷酸分子已从天然遗传环境中脱离,并因此去掉了其它多余的或不想要的编码序列,处于一种适用于基因工程化蛋白生产系统的形式。这样的分离分子是那些脱离其天然环境的分子,包括cDNA和基因组克隆。本发明的分离DNA分子无通常相关的其它基因,但可包括天然存在的5′和3′不翻译区,诸如启动子和终止子。相关区域的鉴定对本领域常规技术人员来说是明显的[例如参阅,Dynan和Tijan,自然316:774-78(1985)]。
“分离”的多肽或蛋白质是处于其天然环境之外的条件下的多肽和蛋白质,例如从血液和动物组织中分离的多肽或蛋白质。在优选的形式中,分离多肽基本上无其它多肽,尤其是动物来源的其它多肽。优选提供高纯化形式的多肽,即纯度超过95%,更优选纯度超过99%。应用于本文中时,术语“分离的”不排除相同多肽以其它物理形式存在,如二聚体或者糖基化的或衍生的形式。
术语“有效连接”在指DNA片段时,表示片段被适当排列以使其功能与目的相一致,如转录起始于启动子并贯穿编码序列进行到终止子。
术语“正同系物(ortholog)”表示得自一个物种、为另一物种的多肽或蛋白质的功能对应物的多肽或蛋白质。正同系物之间的序列差异是物种形成的结果。
“副同系物(parolog)”是由一种生物体产生的相互区别但又结构相关的蛋白质。副同系物被认为是通过基因重复产生的。例如α-珠蛋白、β-珠蛋白和肌红蛋白互为副同系物。
“多核苷酸”为从5′末端读至3′末端的脱氧核糖核苷酸或核糖核苷酸碱基的单链或双链多聚体。多核苷酸包括RNA和DNA,可分离自天然来源,可体外合成,或结合天然分子与合成分子而制成。多核苷酸的大小表示为碱基对(缩写“bp”)、核苷酸(“nt”)或千碱基(“kb”)。只要内容允许,后两个术语可描述单链或双链的多核苷酸。当该术语用于指双链分子时,表示的是全长,应理解为等同于术语“碱基对”。本领域内技术人员将认识到双链多核苷酸的两条链长度可能稍有不同,其末端可能因酶切而成交错切口状;因而双链多核苷酸分子中的所有核苷酸不一定配对。这些未配对的末端长度一般不超过20nt。
“多肽”是天然产生的或合成的以肽键连接的氨基酸残基多聚体。不足约10个氨基酸残基的多肽通常称为“肽”。
术语“启动子”在此用其本领域公认的含义,表示含有可结合RNA聚合酶并起始转录的DNA序列的基因部分。启动子序列常发现位于基因的5′非编码区,但并不总是这样。
“蛋白质”是包含一条或多条多肽链的大分子。蛋白质也可含非肽成分,如糖基。糖和其它非肽取代基可由产生该蛋白质的细胞加在蛋白质上,且在不同细胞类型中各不相同。蛋白质在本文以其氨基酸骨架结构表示;取代基如糖基通常不特别指明,但仍可能存在。
术语“受体”表示可与生物活性分子(即配体)相结合并介导配体作用于细胞上的细胞相关蛋白。膜结合受体特征在于有多结构域的结构,包括胞外配体结合结构域及一般涉及信号转导的胞内效应子结构域。配体与受体结合导致了受体的构象转变,从而引起效应子结构域与细胞中其它分子间的相互作用。这一相互作用依次导致细胞代谢的改变。与受体-配体相互作用有关联的代谢活动包括基因转录、磷酸化、去磷酸化、环腺苷酸产量的提高、细胞钙的转移、膜脂的移动、细胞粘附、肌醇脂的水解和磷脂的水解。一般说来,受体可以是膜结合的、胞质的或核的;单体的(如促甲状腺素受体、β-肾上腺素能受体)或多聚体的(如PDGF受体、生长激素受体、IL-3受体、GM-CSF受体、G-CSF受体、促红细胞生成素受体和IL-6受体)。
术语“分泌信号序列”表示编码多肽(“分泌肽”)的DNA序列,所述多肽作为一个更大多肽的组分,可指导该更大的多肽穿过合成它的细胞的分泌途径。在通过分泌途径转运的过程中该更大的多肽通常被裂解以去除分泌肽。
术语“剪接变体”在此用于表示由基因转录出来的RNA的诸可互换形式。剪接变体一般通过利用转录RNA分子内(或较少情况下是分别转录的RNA分子之间)的可互换剪接位点而天然产生,并可导致由同一基因转录好几种mRNA。剪接变体可编码含不同氨基酸序列的多肽。术语剪接变体用在本文中还表示由基因转录出来的mRNA的剪接变体编码的蛋白质。
用不精确的分析方法(如,凝胶电泳)测定的多聚体分子量和长度应理解为近似值。当该值表达为“大约”X或“近似”X时,所说的X值应理解为精确至±10%。
Zcyto10的螺旋D中的保守氨基酸可用作鉴别新家族成员的工具。螺旋D是最高度保守的,与IL-10的螺旋D具有约32%的相同性。例如,逆转录聚合酶链式反应(RT-PCR)可用于扩增编码获自种种组织来源或细胞系之RNA保守区(以上结构域、区域或基元)的序列。尤其是,设计自Zcyto10序列的高度简并引物可用于此目的。
在本发明的优选实施方案中,分离的多核苷酸可在严谨条件下与SEQ ID NO:1、SEQ ID NO:3、SEQ ID NO:18、SEQ ID NO:33或其互补序列的相似大小区域杂交。一般说来,严谨条件选择为在确定的离子强度和pH下较该特异序列的热熔点(Tm)低约5℃。Tm是50%靶序列与完全匹配探针杂交的温度(在确定的离子强度和pH下)。一般的严谨条件是盐浓度约0.02M或pH低于7及温度至少约60℃。如前面所提及,本发明的分离多核苷酸包括DNA和RNA。用于分离DNA和RNA的方法在本领域是众所周知的。用盐酸胍提取随后以氯化铯梯度离心分离可制备总RNA[Chirgwin等人,生物化学18:52-94,(1979)]。poly(A)+RNA可用Aviv和Leder,美国国家科学院院报69:1408-1412(1972)的方法制备自总RNA。互补DNA(cDNA)可用已知方法制备自poly(A)+RNA。然后可用例如杂交或PCR鉴别和分离编码Zcyto10多肽的多核苷酸。
此外,本发明的多核苷酸可用DNA合成仪合成。通常所选方法是亚磷酰胺方法。如果诸如基因或基因片段合成之类应用需要化学合成的双链DNA,那么分别制备各互补链。短基因(60-80bp)的生产在技术上是简单的,可通过合成互补链然后将其退火而完成。然而,对于较长基因(>300bp)的生产,必须实行特殊的策略,因为在DNA化学合成中各循环的偶联效率很少能达到100%。为了克服这一问题,合成的基因(双链)由长度为20-100个核苷酸的单链片段以模块形式装配成。参阅Glick,Bernard R.和Jack J.Pasternak,分子生物技术学,重组DNA的原理及应用(ASM出版社,华盛顿特区,1994),Itakura,K等人,合成寡核苷酸的合成和用途,生物化学年鉴(Annu.Rev.Biochem.)53:323-356(1984),和Climie,S.等人,胸苷酸合成酶基因的化学合成,美国国家科学院院报87:633-637(1990)。
本领域技术人员将认识到,公开于SEQ ID NO:1,2,3和4中的序列代表了人的两个等位基因,而SEQ ID NO:18,19,33和34代表了小鼠的两个等位基因。可按标准步骤探测来自不同个体的cDNA或基因组文库而克隆这些序列的其它等位变体。可按标准步骤通过探测来自不同个体的cDNA或基因组文库而克隆该序列的等位变体。SEQ IDNO:1中所示DNA序列的等位变体,包括那些含沉默突变的变体及其中突变导致氨基酸序列改变的变体,均在本发明范围内,作为SEQ IDNO:2等位变体的蛋白质也一样。产生自其它方式剪接mRNA的保留了Zcyto10多肽特性的cDNA,也包括于本发明范围内,由这些cDNA和mRNA所编码的多肽也一样。按本领域已知标准步骤通过探测来自不同个体或组织的cDNA或基因组文库,可克隆这些序列的等位变体和剪接变体。
本发明进一步提供了来自其它物种的相应蛋白质和多核苷酸(“物种正同系物”)。目前特别感兴趣的是来自其它哺乳动物的Zcyto10多肽,包括鼠的、猪的、绵羊的、牛的、狗的、猫的、马的及其它灵长类的Zcyto10多肽。可将本发明提供的信息和组合物与常规克隆技术联合起来克隆人Zcyto10蛋白质的物种正同系物。例如,可用获自表达该蛋白质的组织或细胞类型的mRNA克隆cDNA。可用按本文公开序列设计的探针,通过探测Northern印迹鉴定mRNA的合适来源。随后从阳性组织或细胞系的mRNA制备文库。然后可用各种方法分离编码蛋白质的cDNA,如用人或小鼠整个或部分cDNA进行探测,或用基于公开序列的一套或多套简并探针进行探测。cDNA也可用聚合酶链式反应或PCR(Mullis等人,美国专利第4,683,202号)方法,使用按本文公开序列设计的引物进行克隆。在另外的方法中,可利用cDNA文库转化或转染宿主细胞,并用该蛋白质的抗体检测目的cDNA的表达。相似技术也可用于基因组克隆的分离上。正如使用和要求专利保护的,“编码多肽的分离多核苷酸,所说的多核苷酸由SEQ ID NO:2、4、12、13、19、20、25、26、34和35表示”这一措词意指包括这些多肽的所有等位变体和物种正同系物。
本发明还提供了与SEQ ID NO:2的蛋白质多肽和其物种正同系物基本相同的分离蛋白质多肽。“分离的”意指存在于其天然环境之外的条件下的蛋白质或多肽,如脱离自血液和动物组织。在一优选形式中,分离的多肽基本上无其它多肽,尤其是动物来源的其它多肽。优选提供的多肽是高度纯化形式的,即纯度高于95%,更优选纯度超过99%。本文所用的“基本相同”一词意指多肽与SEQ ID NO:2、4、12、13、19、20、25、26、34和35所示序列或其物种正同系物有50%、优选60%、更优选至少80%相同。这样的多肽优选与SEQ ID NO:2或其物种正同系物有至少90%相同,最优选95%或更多相同。序列相同性百分率可用常规方法测定。参阅,例如,Altschul等人,Bull.Math.Bio.48:603-616(1986)和Henikoff和Henikoff,美国国家科学院院报89:10915-10919(1992)。简短地说,就是用缺口开放罚分为10、缺口延伸罚分为1及如表1所示(氨基酸用标准单字母符号表示)Henikoff和Henikoff(同上)的“blossom62”评分矩阵,将两个氨基酸序列进行比对,以使比对评分最佳。相同性百分率计算如下:
表1
A R N D C Q E G H I L K M F P S T W Y V
A 4
R -1 5
N -2 0 6
D -2 -2 1 6
C 0 -3 -3 -3 9
Q -1 1 0 0 -3 5
E -1 0 0 2 -4 2 5
G 0 -2 0 -1 -3 -2 -2 6
H -2 0 1 -1 -3 0 0 -2 8
I -1 -3 -3 -3 -1 -3 -3 -4 -3 4
L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4
K -1 2 0 -1 -3 1 1 -2 -1 -3 -2 5
M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5
F -2 -3 -3 -3 -2 -3 -3 -3 -1 0 0 -3 0 6
P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4 7
S 1 -1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4
T 0 -1 0 -1 -1 -1 -1 -2 -2 -1 -1 -1 -1 -2 -1 1 5
W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4 -3 -2 11
Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2 -2 2 7
V 0 -3 -3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 4
利用如上所公开的比率通过相似方法测定多核苷酸分子间的序列相同性。
变体Zcyto10多肽或基本相同的蛋白质和多肽特征是具一个或多个氨基酸取代、删除或添加。这些改变优选较小性质的改变,即保守氨基酸取代(见表2)和其它不会显著影响蛋白质或多肽折叠或活性的取代;小的删除,一般约1-30个氨基酸的删除;及小的氨基或羧基端延伸,诸如氨基端甲硫氨酸残基、高达约20-25个残基长的小连接肽,或利于纯化的小延伸(亲和标记),诸如多聚组氨酸片段、蛋白A[Nilsson等人,EMBO J.4:1075(1985);Nilsson等人,酶学方法,198:3(1991)]、谷胱甘肽S转移醇[Smith和Johnson,基因67:31(1988)],或其它抗原性表位或结合结构域。通常参阅Ford等人,蛋白质表达和纯化2:95-107(1991)。编码亲和标记的DNA可购自商品供应商处(如,Pharmacia Biotech,Piscataway,NJ)。
表2
保守氨基酸取代
碱性的: 精氨酸
赖氨酸
组氨酸
酸性的: 谷氨酸
天冬氨酸
极性的: 谷氨酰胺
天冬酰胺
疏水的: 亮氨酸
异亮氨酸
缬氨酸
芳香族的: 苯丙氨酸
色氨酸
酪氨酸
小的: 甘氨酸
丙氨酸
丝氨酸
苏氨酸
甲硫氨酸
本发明进一步提供了各种其它多肽融合体(及含一个或多个多肽融合体的相关多聚化蛋白质)。例如,可按美国专利第5,155,027和5,567,584号所公开的将Zcyto10多肽制备成与二聚化蛋白质的融合体。在该方面的优选二聚化蛋白质包括免疫球蛋白恒定区结构域。免疫球蛋白-Zcyto10多肽融合体可于基因工程化细胞中表达(以产生各种多聚体Zcyto10类似物)。可将辅助结构域融合至Zcyto10多肽上,以将它们定向于特异细胞、组织、或大分子(如胶原)。例如,通过将多肽与特异结合靶细胞表面受体之配体融合,可将Zcyto10多肽或蛋白质定向于预定细胞类型。以此方式,可将多肽和蛋白质定向用于治疗或诊断目的。可将Zcyto10多肽与两个或多个部分融合,诸如用于纯化的亲和标记和定向结构域。多肽融合体还可包括一个或多个切割位点,尤其是在结构域之间的切割位点。参阅,Tuan等人,结缔组织研究(Connective Tissue Research)34:1-9(1996)。
本发明的蛋白质还可以包括非天然存在的氨基酸残基。非天然存在的氨基酸包括不局限于反式-3-甲基脯氨酸、2,4-甲撑脯氨酸、顺式-4-羟脯氨酸、反式-4-羟脯氨酸、N-甲基甘氨酸、别苏氨酸、甲基苏氨酸、羟乙基半胱氨酸、羟乙基高半胱氨酸、硝基谷氨酰胺、高谷氨酰胺、六氢吡啶羧酸、噻唑烷羧酸、脱氢脯氨酸、3-和4-甲基脯氨酸、3,3-二甲基脯氨酸、叔亮氨酸、正缬氨酸、2-氮杂苯丙氨酸(2-azaphenylalanine)、3-氮杂苯丙氨酸、4-氮杂苯丙氨酸和4-氟苯丙氨酸。本领域已知几种方法可用于将非天然存在氨基酸残基掺入蛋白质。例如,可利用一个体外系统,其中无义突变均用化学氨酰化抑制子tRNA进行抑制。合成氨基酸和氨酰tRNA的方法为本领域内已知。
本发明多肽中的必需氨基酸可按本领域内已知的方法,如定点诱变或丙氨酸扫描诱变进行鉴定[Cunningham和Wells,科学244:1081-1085(1989);Bass等人,美国国家科学院院报88:4498-4502(1991)]。在后一技术中,于分子中的每个残基处导入单个丙氨酸突变,检验所得突变分子的生物学活性(如配基结合和信号转导)以鉴定对分子活性比较关键的氨基酸残基。也可通过对诸如核磁共振、晶体学技术或光亲和标记之类的技术确定的晶体结构进行分析而测定配体-蛋白质相互作用的位点。参阅,例如,de Vos等人,科学255:306-312(1992);Smith等,分子生物学杂志224:899-904(1992);Wlodaver等,FEBS Lett.309:59-64(1992)。必需氨基酸也可从与相关蛋白质的同源性分析推断出。
可用已知诱变和筛选方法制备并检验多重氨基酸取代,如Reidhaar-Olson和Sauer所公开的方法(科学241:53-57,1988)或Bowie和Sauer所公开的方法(美国国家科学院院报86:2152-2156,1989)。简单地说,这些作者公开的方法是同时使多肽中的两个或多个位置随机化,选择功能性多肽,然后测序经诱变的多肽以确定每个位置处的容许取代范围。其它可用的方法包括噬菌体展示(如Lowman等,生物化学30:10832-10837(1991);Ladner等,美国专利号5,223,409;Huse,WIPO公开WO92/06204)和区域定向诱变(Derbyshire等,基因46:145,1986;Ner等,DNA7:127,1988)。
如上所公开的诱变方法可结合高容量筛选方法以检测宿主细胞中克隆的诱变蛋白质的活性。在这一点上优选的试验包括细胞增殖试验和基于生物传感器的配体结合试验,以下对这些试验有描述。编码活性蛋白或其部分(如配体结合片段)的诱变DNA分子可从宿主细胞中回收并用现代装置迅速测序。这些方法可快速测定目的多肽中各氨基酸残基的重要性,并可适用于未知结构的多肽。
用上述方法,本领域常规技术人员能制备与SEQ ID NO:2、4、12、13、19、20、25、26、34和35或其等位变体基本相同并保留野生型蛋白质特性的种种多肽。正如本文所表达和声称的,“SEQ ID NO:2表示的多肽”包括该多肽的所有等位变体和物种正同系物。
本发明的蛋白多肽,包括全长蛋白质、蛋白质片段(如配体结合片段)及融合多肽均可按常规技术在基因工程化宿主细胞中生产。合适的宿主细胞是那些能被外源DNA转化或转染并能培养生长的细胞类型,包括细菌、真菌细胞及培养的高等真核细胞。优选真核细胞,尤其是多细胞生物的培养细胞。操作克隆DNA分子和将外源DNA引入各种宿主细胞的技术公开于以下文献中:Sambrook等人,分子克隆:实验室手册,第二版(冷泉港实验室出版社,冷泉港,纽约,1989),及Ausubel等人,同上。
本发明的多核苷酸,通常为cDNA序列,编码上述多肽。编码本发明多肽的DNA序列由一系列密码子组成,多肽的每一氨基酸残基均由密码子编码,每个密码子由3个核苷酸组成。
氨基酸残基由如下相应密码子编码。
丙氨酸(Ala)由GCA、GCC、GCG或GCT编码;
半胱氨酸(Cys)由TGC或TGT编码;
天冬氨酸(Asp)由GAC或GAT编码;
谷氨酸(Glu)由GAA或GAG编码;
苯丙氨酸(Phe)由TTC或TTT编码;
甘氨酸(G1y)由GGA、GGC、GGG或GGT编码;
组氨酸(His)由CAC或CAT编码;
异亮氨酸由ATA、ATC或ATT编码;
赖氨酸(Lys)由AAA或AAG编码;
亮氨酸(Leu)由TTA、TTG、CTA、CTC、CTG或CTT编码;
甲硫氨酸(Met)由ATG编码;
天冬酰胺(Asn)由AAC或AAT编码;
脯氨酸(Pro)由CCA、CCC、CCG或CCT编码;
谷氨酰胺(Gln)由CAA或CAG编码;
精氨酸(Arg)由AGA、AGG、CGA、CGC、CGG或CGT编码;
丝氨酸(Ser)由AGC、AGT、TCA、TCC、TCG或TCT编码;
苏氨酸(Thr)由ACA、ACC、ACG或ACT编码;
缬氨酸(Val)由GTA、GTC、GTG或GTT编码;
色氨酸(Trp)由TGG编码;及
酪氨酸(Tyr)由TAC或TAT编码。
根据本发明可以认识到,当cDNA如上所述要求专利保护时,应理解为要求专利保护的是有义链、反义链及有义和反义链通过各自氢键一起退火形成的双链DNA。要求专利保护的还有编码本发明多肽的信使RNA(mRNA)及由上述cDNA所编码的mRNA。信使RNA(mRNA)用与上文指示的相同密码子编码多肽,只是每个胸苷(T)均被尿苷(U)所取代。
通常,在表达载体中,编码Zcyto7多肽的DNA序列与其表达所需的其它遗传元件有效连接,所述元件一般包括转录启动子和终止子。尽管本领域技术人员会认为在某些系统中选择标记可提供于分离载体上,且外源DNA的复制可通过整合进入宿主细胞基因组而提供,但载体通常还包含一个或多个选择标记及一个或多个复制起点。启动子、终止子、选择标记、载体及其它元件的选择是在本领域普通技术人员水平之内的常规设计问题。许多这些元件在文献中都有描述,并可购自商品供应商处。
为了指导Zcyto10多肽进入宿主细胞的分泌途径,可在表达载体中提供分泌信号序列(也称前导序列、前原序列或前序列)。分泌信号序列可以是该蛋白质本身的,或可来自另一分泌蛋白质(如t-PA)或从头合成。分泌信号序列以正确阅读框架与Zcyto10 DNA序列连接。尽管某些信号序列可位于目的DNA序列中的其它位置(参阅,如Welch等人,美国专利第5,037,743;Holland等人,美国专利第5,143,830),但分泌信号序列通常位于编码目的多肽之DNA序列的5′端。
将外源DNA引入哺乳类宿主细胞的方法包括磷酸钙介导的转染[Wigler等人,细胞14:725(1978);Corsaro和Pearson,体细胞遗传学7:603,1981;Graham和Van der Eb,病毒学52:456(1973)],电穿孔[Neumann等人,EMBO J.1:841-845(1982)],DEAE-葡聚糖介导的转染[Ausubel等人编,分子生物学通用手册(John Wiley和Sons,Inc.,NY,1987)],及脂质体介导的转染[Hawley-Nelson等人,Focus 15:73(1993);Ciccarone等人,Focus 15:80(1993)]。培养的哺乳类细胞中重组多肽的生产公开于如下文献中:Levinson等人,美国专利第4,713,339号;Hagen等人,美国专利号4,784,950;Palmiter等人,美国专利号4,579,821;及Ringold,美国专利号4,656,134。合适的培养哺乳类细胞包括COS-1(ATCC NO.CRL1650)、COS-7(ATCC NO.CRL1651)、BHK(ATCC NO.CRL1632)、BHK570(ATCC NO.CRL10314)、293[ATCC NO.CRL1573;Graham等人,普通病毒学杂志(J.Gen.Virol)36:59-72(1977)]和中国仓鼠卵巢细胞(如CHO-K1;ATCC NO.CCL61)细胞系。另外的合适细胞系在本领域中是已知的,并可获自公共保藏单位,如美国典型培养物保藏中心,洛克菲勒,马里兰州。一般说来,优选强转录启动子,如来自SV-40或巨细胞病毒的启动子。参阅如,美国专利号4,956,288。其它合适启动子包括来自金属硫蛋白基因的启动子(美国专利号4,579,821和4,601,978)和腺病毒主要晚期启动子。
通常利用药物选择来选出已插入外源DNA的培养哺乳类细胞。这些细胞通常称为“转染子”。已在有选择剂存在的条件下培养并能将目的基因传给子代的细胞称为“稳定的转染子”。优选的选择标记是编码对抗生素新霉素的抗性的基因。选择在新霉素型药物(如G418等等)存在的条件下进行。选择系统也可用于提高目的基因的表达水平,此过程称为“扩增”。扩增通过如下过程进行:在低水平选择剂存在的条件下培养转染子,然后增加选择剂的量以选择出产生高水平引入基因之产物的细胞。优选的可扩增选择标记是二氢叶酸还原酶,它赋予对氨甲蝶呤的抗性。其它的药物抗性基因(如潮霉素抗性、广谱抗药性、嘌呤霉素乙酰转移酶)也可使用。导入改变表型的其它标记,如绿色荧光蛋白,或细胞表面蛋白(如CD4、CD8、MHC I类分子),胎盘碱性磷酸酶均可用来通过FACS分选术或磁珠分离技术之类的方法从未转染细胞中选出转染细胞。
其它高等真核细胞也可用作宿主,包括昆虫细胞、植物细胞和禽细胞。昆虫细胞的转化及其中外源多肽的生产已公开于Guarino等人的美国专利第5,162,222、Bang等人的美国专利第4,775,624和WIPO公开WO94/06463中。用发根农杆菌作载体在植物细胞中表达基因已由Sinkar等人作了回顾[生物科学杂志(J.Biosci.)(Bangalore)11:47-58(1987)]。昆虫细胞可用杆状病毒重组体[通常衍生自苜蓿银纹夜蛾核型多角体病毒(AcNPV)]感染。见King,L.A.和Possee,R.D.,杆状病毒表达系统:实验室指南(Chapman&Hall,伦敦);O′Reilly,D.R.等人,杆状病毒表达载体:实验室手册(牛津大学出版社,纽约,1994);以及Richardson,C.D.编辑,杆状病毒表达方案,分子生物学方法(Humana出版社,Totowa,NJ,1995)。第二种制备重组Zcyto10杆状病毒的方法应用了基于转座子的系统,该系统综述于Luckow,V.A.,等人,病毒学杂志67:4566-79,1993中。这一应用了转移载体的系统在Bac-to-BacTM试剂盒(LifeTechnologies,Rockyille,MD)中有售。此系统利用含Tn7转座子的转移载体pFastBaclTM(Life Technologies)以将编码Zcyto10多肽的DNA转移入作为被称为“杆粒”的大质粒保持于大肠杆菌中的杆状病毒基因组中。见Hill-Perkins,M.S.和Possee,R.D.,普通病毒学杂志71:971-6,(1990);Bonning,B.C.等人,普通病毒学杂志75:1551-6(1994);以及Chazenbalk,G.D.和Rapoport,B.,生物化学杂志270:1543-9(1995)。此外,转移载体可包含在所表达Zcyto10多肽的C-或N-末端融合了表位标记(如Glu-Glu表位标记)的编码DNA的框内融合体[Grussenmeyer,T.等人,美国国家科学院院报82:7952-4(1985)]。用本领域已知技术可将含Zcyto10的转移载体转化进入大肠杆菌,并筛选出含有指示为重组杆状病毒的间断LacZ基因的杆粒。用常规技术分离含重组杆状病毒基因组的杆粒DNA并用于转染草地夜蛾细胞(如Sf9细胞)。表达Zcyto10的重组病毒即可产生。用本领域常用方法制备重组病毒原种。
重组病毒可用于感染宿主细胞,通常是衍生自秋粘虫(草地夜蛾)的细胞系。总见,Glick和Pasternak,分子生物技术学:重组DNA的原理和应用,ASM出版社,华盛顿特区(1994)。另一合适细胞系是衍生自粉纹夜蛾的High Five OTM细胞系(Invitrogen)(美国专利号5,300,435)。生产商提供的无血清培养基可用于培养和维持细胞。合适的培养基对于Sf9细胞是Sf900 IITM(Life Technologies)或ESF 921TM(Expression Systems);对于粉纹夜蛾细胞是Ex-Cell0405TM(JRH Biosciences,Lenexa,KS)或Express Five OTM(LifeTechnologies)。细胞从约为2-5×105个细胞的接种密度生长至1-2×106个细胞密度时,以0.1至10(更通常为近3)的感染复数(MOI)加入重组病毒原种。所用程序常描述于实验室手册中(King,L.A.和Possee,R.D.,同上;O′Reilly,D.R.等,同上;Richardson,C.D.,同上)。随后可用本文所述方法从上清中纯化出Zcyto10多肽。
真菌细胞,包括酵母细胞,尤其是酵母属的细胞也可用于本发明中,如用于生产蛋白质片段或融合多肽。用外源DNA转化酵母细胞并从中产生重组多肽的方法公开于下述文件,例如,Kawasaki,美国专利号4,599,311;Kawasaki等,美国专利号4,931,373;Brake,美国专利号4,870,008;Welch等,美国专利号5,037,743;和Murray等,美国专利号4,845,075。通过由选择标记确定的表型,通常是药物抗性或在缺乏某种特殊养分(如亮氨酸)的条件下生长的能力,来选择出转化细胞。用于酵母中的优选载体系统是POT1载体系统,公开于Kawasaki等人的美国专利第4,931,373中,该系统可通过在含葡萄糖的培养基上生长而选择出转化细胞。用于酵母中的合适启动子和终止子包括来自糖酵解基因(见,如,Kawasaki,美国专利号4,599,311;Kingsman等人,美国专利号4,615,974;和Bitter,美国专利号4,977,092)和乙醇脱氢酶基因的启动子和终止子。也可参阅美国专利第4,990,446号;第5,063,154号;第5,139,936号和第4,661,454号。本领域已知用于其它酵母的转化系统,所述酵母包括:多形汉逊酵母、粟酒裂殖酵母、乳酸克鲁维酵母、脆壁克鲁维酵母、玉蜀黍黑粉菌、巴斯德毕赤酵母、甲醇毕赤酵母(Pichiamethanolica)、季也蒙毕赤酵母和麦芽糖假丝酵母(Candidamaltosa)。参阅,如Gleeson等,普通微生物学杂志132:3459-3465(1986)和Cregg,美国专利第4,882,279号。可根据McKnight等的美国专利第4,935,349号的方法利用曲霉属细胞。转化产黄枝顶孢(Acremonium chrysogenum)的方法公开于Sumino等人的美国专利第5,162,228号中。转化脉孢菌属的方法公开于Lambowitz的美国专利第4,486,533号。
用甲醇毕赤酵母作为宿主生产重组蛋白质的方法描述于WIPO公开WO97/17450、WO97/17451、WO98/02536、和WO98/02565中。用于转化甲醇毕赤酵母的DNA分子将通常制成双链环状质粒,优选在转化之前先线性化。为了在甲醇毕赤酵母中生产多肽,优选质粒中的启动子和终止子为甲醇毕赤酵母内基因,如甲醇毕赤酵母醇利用基因(AUG1或AUG2)的启动子和终止子。其它有用的启动子包括二羟基丙酮合成酶(DHAS)基因,甲酸脱氢酶(FMD)基因和过氧化氢酶(CAT)基因的启动子。为了便于DNA整合入宿主染色体,优选质粒的整个表达片段两侧均为宿主DNA序列。优选用于甲醇毕赤酵母的选择标记为甲醇毕赤酵母ADE2基因,它编码磷酸核糖基-5-氨基咪唑羧化酶(AIRC;EC4.1.1.21),此酶可使得ade2宿主细胞在缺乏腺嘌呤时生长。为了进行希望少用甲醇的大批量工业化生产,优选用其中两个甲醇利用基因(AUG1和AUG2)均缺失的宿主细胞。为了生产分泌型蛋白质,优选液泡蛋白酶基因(PEP4和PRB1)有缺陷的宿主细胞。可利用电穿孔促进含目的多肽编码DNA的质粒引入甲醇毕赤酵母细胞。优选用电穿孔方法转化甲醇毕赤酵母细胞,所用电场为指数级衰减的脉冲电场,电场强度为2.5-4.5kV/cm,优选约为3.75kV/cm,时间常数(τ)为1-40毫秒,最优选约20毫秒。
原核宿主细胞,包括大肠杆菌、芽孢杆菌属及其它属的菌株也可用作本发明的宿主细胞。转化这些宿主并表达克隆其中的外源DNA的技术为本领域内众所周知(见,如,Sambrook等,同上)。当在诸如大肠杆菌之类的细菌中表达Zcyto10多肽时,该多肽可保留在细胞质中(通常为不溶颗粒),或可由细菌分泌序列引导至周质空间中。在前一种情况中,将细胞裂解,回收颗粒并用如异硫氰酸胍或尿素变性。然后可通过稀释变性剂,如对尿素溶液透析,和结合使用还原型和氧化型谷胱甘肽,再对缓冲盐溶液透析,使变性多肽重新折迭和二聚化。在后一种情况中,多肽可从周质间隙中以可溶性有功能形式回收,即通过破碎细胞(如通过超声处理或渗透休克)以释放周质间隙的内容物并回收蛋白质而完成,这样可不必变性和再折叠。
转化的或转染的细胞根据常规程序培养于含有所选宿主细胞生长必需的营养物和其它成分的培养基中。各种合适的培养基,包括已知成分培养基和复合培养基,在本领域中都是已知的,并通常包括碳源、氮源、必需氨基酸、维生素和矿物质。如果需要,培养基中也可包含诸如生长因子或血清之类的组分。培养基通常将通过例如药物选择或缺少某种必需养分来选择含外源导入DNA的细胞,这种缺少的必需养分可通过表达载体上携带的或共转染进入宿主细胞的选择标记得到补充。在约25℃-35℃将甲醇毕赤酵母细胞培养于含足够碳源、氮源和微量养分的培养基中。用常规方法向液体培养物中提供足够通气量,诸如小摇瓶或发酵罐喷雾。甲醇毕赤酵母的优选培养基是YEPD(2%D-葡萄糖、2%BactoTM Peptone(Difco实验室,底特律,MI)、1%BactoTM酵母提取物(Difco实验室)、O.004%腺嘌呤和O.006%L-亮氨酸)。
在本发明的一方面,通过培养细胞产生新的蛋白质,该细胞被用于筛选此蛋白质的一个或多个受体,包括天然受体,以及天然配基的激动剂和拮抗剂。
蛋白质分离:
优选纯化本发明多肽至不低于80%纯度,更优选不低于90%的纯度,甚至更优选不小于95%的纯度,尤其优选药学上的纯度,即相对于杂质大分子,特别是其它蛋白质和核酸,纯度超过99.9%,且不含传染因子和致热因子。优选地,纯化多肽基本不含其它多肽,特别是其它动物源多肽。
表达的重组多肽(或嵌合多肽)可用分级分离和/或常规纯化方法和介质进行纯化。硫酸铵沉淀及酸或离液剂提取可用于样品的分级分离中。典型的纯化步骤可包括羟基磷灰石层析、尺寸排阻层析、FPLC和反相高效液相层析。合适的阴离子交换介质包括葡聚糖衍生物、琼脂糖、纤维素、聚丙烯酰胺、特殊性能二氧化硅,等等。PEI、DEAE、QAE和Q衍生物是优选的,特别优选DEAE Fast-Flow Sepharose(Pharmacia,Piscataway,NJ)。典型的层析介质包括那些苯基、丁基或辛基衍生的介质,如Phenyl-Sepharose FF(Pharmacia)、Toyopearl butyl 650(Toso Haas,Montgomeryville,PA)、Octyl-Sepharose(Pharmacia),等等;或聚丙烯树脂,如AmberchromCG71(Toso Haas)等。合适的固相支持体包括玻璃珠、硅基树脂、纤维素树脂、琼脂糖珠、交联琼脂糖珠、聚苯乙烯珠、交联聚丙烯酰胺树脂等等,它们在所要使用的条件下是不溶的。这些支持物可用反应基团修饰,使得蛋白质可通过氨基、羧基、巯基、羟基和/或碳水化合物部分附着于支持物上。偶联化学法的例子包括溴化氰活化、N-羟基琥珀酰亚胺活化、环氧化物活化、巯基活化、酰肼活化及用于碳二亚胺偶联化学法的羧基和氨基衍生物。这些及其它固相介质在本领域中是众所周知并广泛应用的,且可购自产品供应商处。将受体多肽结合到支持介质上的方法在本领域中是众所周知的。特定方法的选择是一个常规设计的问题,它部分是由所选支持物的特性决定的。参阅例如《亲和层析:原理和方法》(Pharmacia LKB Biotechnology,Uppsala,Sweden,1988)。
可利用它们的特性分离本发明的多肽。例如,固定化金属离子吸附(IMAC)层析可用于纯化富含组氨酸的蛋白质。简单说,凝胶先带上二价金属离子,形成螯合物(E.Sulkowski,生化动态3:1-7,1985)。富含组氨酸的蛋白质将以不同的亲和力吸附于该基质上(这取决于所用的金属离子),并可通过竞争性洗脱作用,降低pH,或用强螯合剂而洗脱。其它的纯化方法包括用凝集素亲和层析和离子交换层析纯化糖基化的蛋白质(酶学方法,182卷,“蛋白质纯化指南”,M.Deutscher,(编),529-539页,Acad出版社,San Diego,1990)。或者,可构建目的多肽与亲和标记(如多聚组氨酸、麦芽糖结合蛋白质、免疫球蛋白结构域)的融合体以有助于纯化。
用途
本发明多肽具有四螺旋束细胞因子的结构特性。如果一种蛋白质可溶并具有通过细胞表面受体作用而传导信号和调节细胞增殖的能力,通常将它描述为细胞因子。细胞因子有数种三级结构折叠分类,包括富半胱氨酸的二聚体(如胰岛素、PDGF)、β-三叶形折迭(如FGF、IL-1)、及全α四螺旋束。后者的特征在于具有独特上-上-下-下拓扑结构的4个螺旋,标记为A、B、C和D,其中两个自上而下环连接螺旋A和B以及螺旋C和D。参阅,例如,Manavalan等,蛋白质化学杂志11(3):321-31(1992)。有时将四螺旋束细胞因子进一步再分成短链(如IL-4、IL-2、GM-CSF)和长链(如TPO、生长激素、leptin、IL-10),其中后者通常具有较长的A和D螺旋和自上而下的环。今后我们可同义地使用术语“细胞因子”和“四螺旋束细胞因子”。Zcyto10的螺旋A包括氨基酸残基35(异亮氨酸)至氨基酸残基49(异亮氨酸),也由SEQ ID NO:14表示;螺旋B包括第91位氨基酸亮氨酸至第105位氨基酸苏氨酸,也由SEQ ID NO:15表示;螺旋C包括第112位氨基酸残基亮氨酸至第126位氨基酸残基半胱氨酸,也由SEQ ID NO:16表示;螺旋D包括第158位氨基酸残基缬氨酸至第172位氨基酸残基甲硫氨酸,也由SEQ ID NO:17表示。
人Zcyto10在Cys33和Cys126之间有分子内二硫键。预期其它4个半胱氨酸Cys80、Cys132、Cys81和Cys134形成Cys80-Cys132和Cys81-Cys134形式的两个分子内二硫键。预计对Zcyto10的结构稳定性比较重要的残基包括Cys33、Cys126、Cys80、Cys132、Cys81和Cys134。预期这些残基中任何一个突变成其它残基均会使Zcyto10功能失活。
Zcyto10的结构稳定性还取决于维持四个α-螺旋上的埋藏的疏水面。预计残基Ile42、Phe46、Ile49、Leu91、Val94、Phe95、Tyr98、Leu112、Phe116、Ile119、Leu123、Val158、Leu162、Leu165、Leu168、Leu169和Met172埋藏于蛋白质核心中,如果它们改变,则取代氨基酸残基必须是疏水氨基酸。
预期涉及Zcyto10与细胞表面受体结合的残基包括螺旋A和B间自上而下的环中的Asp57,和预计暴露于螺旋D表面的带电残基Lys160和Glu164。在蛋白质表面上,环AB和螺旋D区域上是含残基Ile62、Leu71、Ile167和Trp171的疏水表面块。这些残基可能与细胞表面受体上的疏水表面块相互作用。
本发明的人Zcyto10多肽与白介素-10(IL-10)有约28%的相同性。小鼠Zcyto10多肽与人IL-10具有约24%的相同性,与小鼠IL-10有约27%的相同性。人Zcyto10多肽与小鼠Zcyto10多肽有约76%的相同性。
小鼠Zcyto10的螺旋A包括SEQ ID NO:19的第35位氨基酸残基异亮氨酸至第49位氨基酸残基精氨酸,也用SEQ ID NO:21表示。小鼠Zcyto10的螺旋B包括SEQ ID NO:19的第91位氨基酸残基亮氨酸至第105位氨基酸残基苏氨酸,也用SEQ ID NO:22表示。小鼠Zcyto10的螺旋C包括SEQ ID NO:19的第112位氨基酸残基亮氨酸至第126位氨基酸残基半胱氨酸,也用SEQ ID NO:23表示。小鼠Zcyto10的螺旋D包括SEQ ID NO:19的第158位氨基酸残基缬氨酸至第172位氨基酸残基甲硫氨酸,也用SEQ ID NO:24表示。
IL-10是一种细胞因子,它可抑制其它细胞因子的产生、诱导活化B淋巴细胞增殖和分化、抑制HIV-1复制并对γ干扰素呈现拮抗效应。IL-10似乎以由180°旋转相关的两α-螺旋多肽区形成的二聚体形式存在。参阅,例如,Zdanov等,结构:3(6):591-601(1996)。已报道IL-10是活化Th2 T-细胞、B-细胞、角质细胞和单核细胞/巨噬细胞的产物,能调节Th1 T-细胞应答。可通过抑制Th1 T-细胞的细胞因子合成来完成这样的调节。参阅,例如,Hus等,Int.Immunol.4:563(1992)和D’Andrea等,实验医学杂志(J.Exp.Med.)178:1042(1992)。也已报道IL-10可抑制天然杀伤细胞和单核细胞/巨噬细胞的细胞因子合成。参阅,例如,Hus等(上文所引用)和Fiorentino等,免疫学杂志,146:3444(1991)。此外,已发现IL-10对胰岛素依赖型糖尿病具保护作用。
在相应于该新DNA之mRNA的组织分布分析中,观察到有单个转录物,大约为1.2Kb。利用Clontech多组织Northern,观察到在气管、胎盘、睾丸、皮肤、唾液腺、前列腺、甲状腺中明显有人转录物,而在胃和胰中表达较低。Zcyto10表达于以下小鼠组织中:肾、骨骼肌、唾液腺、肝和皮肤。
Zcyto10表达的组织特异性显示,在气管和唾液腺、胃、胰和肌肉中Zcyto10可能是生长和/或维持因子;且在局部免疫应答中可能是重要的。而且,Zcyto10基因定位在染色体1q32.2上,这表明Zcyto10是生长/分化因子或如同IL-10一样在免疫应答调节中是重要的。
本发明还提供了可用于诊断应用中的试剂。含Zcyto 10 DNA或RNA或其亚序列的探针可用于测定Zcyto 10基因是否存在于染色体1上或是否发生了突变。
本发明还提供具显著治疗价值的试剂。Zcyto 10多肽(天然或重组形式)、其片段、它们的抗体和抗独特型抗体以及鉴定为与Zcyto 10多肽具结合亲和力的化合物,在异常生理或发育相关性疾病(包括异常增殖,如患癌或变性疾病或免疫性改变)的治疗中是有用的。
可纯化抗Zcyto 10多肽抗体并随后施用于病人。为了治疗的作用,这些试剂可与另外的活性或惰性成分相结合,如药学上可接受的载体或稀释液以及生理学上无害的稳定剂和赋形剂。这些混合物可经无菌过滤,并通过冷冻干燥置于试剂瓶中或保存于稳定的水制剂中以剂量形式放置。本发明还涉及抗体及其结合片段、或包括非补体结合形式在内的单链抗体的用途。
有效治疗所必需试剂的份量取决于许多不同的因素,包括施药方法、靶位点、病人的生理学状态及施用的其它药物。因此,应确定治疗剂量以使安全性和有效性达到最佳。一般说来,这些试剂的体外使用剂量能为其体内施用的有效量提供有用的指导。治疗特殊失调的有效剂量的动物试验将为人所用剂量提供进一步预兆性指示。施药方式包括口服、静脉内、腹膜内、肌肉、经皮用药或通过喷雾器或雾化器以喷雾形式施用入肺或气管。药学上可接受载体将包括水、盐水、一些缓冲液等。通常预期的剂量范围是1-1000μg/Kg体重/天。然而,剂量可以更高或更低,这可由本领域常规技术的内科医生确定。药物配方和剂量范围的完整讨论可参阅Remington′s PharmaceuticalScience,第18版(Mack Publishing Co.,Easton,Penn.,1996),及Goodman和Gilman的:治疗的药物学基础,第9版(Pergamon Press1996)。
基于核酸的治疗
如果哺乳动物的Zcyto 10基因突变或缺失,可将Zcyto 10基因引入该哺乳动物的细胞。在一实施方案中,将编码Zcyto 10多肽的基因在一病毒载体上引入体内。这样的载体包括减毒或缺陷型DNA病毒,诸如(但不局限于)单纯疮疹病毒(HSV)、乳头瘤病毒、非洲淋巴瘤病毒(EBV)、腺病毒、腺伴随病毒(AAV)等等。优选完全或几乎完全缺失病毒基因的缺陷型病毒。缺陷型病毒在引入细胞后无感染性。使用缺陷型病毒载体能够在特定的局部区域给与细胞,而不用担心此载体会感染其它细胞。特殊的载体例子包括,但不局限于,缺陷型疱疹病毒1(HSV1)载体[Kaplitt等,Molec.Cell.Neurosci.,2:320-330(1991)]、减毒的腺病毒载体,如由Stratford-Perricaudet等,临床研究杂志(J.Clin.Invest.),90:626-630(1992)所述的载体,及缺陷型腺伴随病毒载体[Samulski等,病毒学杂志,61:3096-3101(1987);Samulski等,病毒学杂志,63:3822-3828(1989)]。
在另一实施方案中,基因可在逆转录病毒载体中引入,如以下文献所述:Anderson等的美国专利第5,399,346号;Mann等,细胞,33:153(1983);Temin等的美国专利第4,650,764号;Temin等的美国专利第4,980,289号;Markowitz等,病毒学杂志,62:1120(1988);Temin等的美国专利第5,124,263号;国际专利公开号WO95/07358,由Dougherty等人于1995年3月16日公开;血液学,82:845(1993)。或者,载体可利用脂质体通过体内脂转染法而引入。合成的阳离子脂质可用来制备脂质体以用于标记编码基因的体内转染[Felgner等,美国国家科学院院报,84:7413-7417(1987);参阅Mackey等,美国国家科学院院报,85:8027-8031(1988)]。使用脂转染法将外源基因引入体内特殊器官具有某些实用的好处。脂质体对特定细胞的分子靶向性代表了优点的一方面。很显然,指导向特殊细胞的转染代表了优点的又一方面。也很清楚的是,在细胞异质性组织,如胰、肝、肾和脑中,向特殊细胞类型的定向转染尤其有利。为了靶向的目的可将脂类化学偶联上其它分子。靶向肽,如激素或神经递质,及如抗体之类的蛋白质,或非肽分子均可与脂质体化学偶联。这些脂质体也可以用喷雾器或雾化器的方法以喷雾形式施用于肺或气管。
将细胞从体内取出,然后将作为裸露DNA质粒的载体引入该细胞中,并随后将此转化细胞再植入体内,这样的操作是可能的。可按本领域已知技术将用于基因治疗的裸DNA载体引入目的宿主细胞中,如转染、电穿孔、显微注射、转导、细胞融合、DEAE葡聚糖、磷酸钙沉淀、基因枪的使用或DNA载体转运蛋白的使用[参阅,如Wu等,生物化学杂志267:963-967(1992);Wu等,生物化学杂志,263:14621-14624(1988)]。
Zcyto 10多肽也可用于制备可特异结合Zcyto 10多肽的抗体。这些抗体随后可用于制备抗独特型抗体。本文所用的“抗体”一词包括多克隆抗体、单克隆抗体、及其诸如F(ab)2和Fab片段之类的抗原结合片段,等等,其中包括基因工程抗体。若抗体与Zcyto 10多肽结合的Ka≥107/M,则此抗体被定义为可特异结合。本领域常规技术人员可轻易测定单克隆抗体的亲和力(参阅,例如,Scatchard,同上)。
制备多克隆和单克隆抗体的方法在本领域是众所周知的(参阅例如,Sambrook等,分子克隆:实验室手册(第二版)(冷泉港,纽约,1989);和Hurrell,J.G.R.,编辑,单克隆杂交瘤抗体:技术及应用(CRC Press,Inc.,Boca Raton,FL,1982),均在此引入作为参考)。本领域常规技术人员可明显看到,多克隆抗体可自各种温血动物,如马、牛、山羊、绵羊、狗、小鸡、兔子、小鼠和大鼠中制备。通过使用诸如弗氏完全或不完全佐剂之类的佐剂可提高Zcyto 10多肽的免疫原性。本领域技术熟练人员已知的各种试验均可用于检测能与Zcyto 10多肽特异结合的抗体。实例性试验详述于《抗体:实验室手册》,Harlow和Lane(编)(冷泉港实验室出版社,1988)。这些试验的代表性例子包括:合并免疫电泳、放射免疫分析、放射免疫沉淀法、酶联免疫吸附测定(ELISA)、斑点印迹试验、抑制或竞争试验及三明治试验。
抗Zcyto 10抗体可用于标记表达此蛋白质的细胞,用于亲和纯化,在诊断分析中用于测定可溶性蛋白质多肽的循环水平,并可作为拮抗剂以阻断体外和体内的配体结合和信号转导。
本发明的另一方面提供了含纯化Zcyto 10多肽与药用可接受载体结合的药物组合物。如本文将进一步论述的,这些组合物可用于在预防和治疗其特征为非正常细胞增殖、细胞分化或细胞因子产生的疾病中调节细胞增殖、细胞分化或细胞因子产生。此外,本发明的Zcyto 10多肽可用于气管特异或气管支气管特异的应用中,如气管支气管上皮或其基底细胞的维持或伤口修复、调节粘液产生或残渣的粘液性清除或治疗哮喘、支气管炎或气管支气管道的其它疾病。预计Zcyto 10多肽的施用剂量范围在与Zcyto 10-FC构建体所用相同的剂量至比其高100倍剂量之间,这取决于Zcyto 10多肽的稳定性。Zcyto 10的治疗剂量为5-5000μg/Kg/天。
本发明的Zcyto 10多肽高表达于唾液腺和气管中,通过Western印迹分析发现存在于唾液中。唾液腺合成和分泌具有不同生物学功能的许多蛋白质。这样的蛋白质有助于口腔的润滑(如粘蛋白和富脯氨酸蛋白质)、补充矿质(如statherin和离子性的富脯氨酸蛋白质)和消化(如淀粉酶、脂酶和蛋白酶)及指供抗微生物(如富脯氨酸蛋白质、溶菌酶、histatin和乳过氧化物酶)和粘膜成分维持(如粘蛋白)能力。此外,唾液是唾液腺所合成之生长因子的丰富来源。例如,已知唾液含表皮生长因子(EGF)、神经生长因子(NGF)、转化生长因子-α(TGF-α)、转化生长因子-β(TGF-β)、胰岛素、胰岛素类生长因子I和II(IGF-I和IGF-II)和成纤维细胞生长因子(FGF)。参阅,例如,Zelles等,J.Dental.Res.74(12):1826-32,1995。唾液腺合成生长因子的过程被认为是雄激素依赖型的,对于口腔及胃肠道的健康是必需的。
因而,Zcyto 10多肽、其兴奋剂或拮抗物在胃肠道或口腔再生中可能有治疗效用。为了证实本发明Zcyto多肽、兴奋剂或拮抗物有这样的能力,按本领域已知方法评估这些Zcyto 10多肽、兴奋剂或拮抗物降解淀粉的能力。通过干预Th1和Th2淋巴细胞的交叉调节及对诸如嗜酸性细胞、肥大细胞、嗜碱性细胞、嗜中性粒细胞和巨噬细胞之类其它炎性细胞介质的生长、分化和细胞因子产生的调节,Zcyto 10多肽、其兴奋剂或拮抗物可用于治疗哮喘及其它气管支气管道疾病。Zcyto 10多肽及其兴奋剂或拮抗物还可调节气管支气管道中肌肉的紧张性。
当掺入油膏或霜中时,Zcyto 10多肽还可用于治疗许多系统性或局部的皮肤病,例如通常为湿疹、牛皮癣或干皮病,或用于相关的皮肤保养。此外Zcyto 10多肽还可直接注射入肌肉以治疗老人、病人或长期卧床者的肌萎缩。
放射杂交绘图是用于构建哺乳动物染色体的高分辨率连续图谱的体细胞遗传技术[Cox等,科学250:245-250(1990)]。部分或完全清楚基因序列就可设计适于染色体放射杂交绘图板使用的PCR引物。覆盖整个人基因组的放射杂交绘图板,如Stanford G3 RH Panel和GeneBridge 4 RH Panel(Research Genetics,Inc.,HuntsVille,AL)可购买得到。这些板可基于PCR而快速地进行染色体定位及基因、序列标记位点(STS)和其它非多态性或多态性标志在目的区域内的排列。这包括直接建立新发现目的基因与原先图标标志间的成比例物理距离。确知基因位置在许多方面都是有用的,包括:1)确定一段序列是否为一已有毗连序列群的一部分,并得到诸如YAC-、BAC-或cDNA克隆之类多种形式的其它周围遗传序列,2)为显示与相同染色体区域连锁的遗传疾病提供可能的候选基因,及3)交叉参照模型生物,诸如小鼠,以协助确定特殊基因所具有的功能。
结果显示Zcyto 10基因绘图在WICGR放射杂交图谱上自人染色体1连锁群顶部的889.26cR-3000。近侧或远侧构架标记分别是DIS504和WI-9641(DIS2427)。利用周围标记将Zcyto 10基因定位于完整LDB染色体1图谱上的lq32.2区域(遗传定位数据库,南安普敦大学,WWW服务器:http://cedar.genetics.soton.ac.uk/public-html/)。许多基因已绘图于染色体1的1q32.2区域。尤其是,该区域内的突变已发现导致van der Woude综合症,和有时与腭裂相关的下唇畸形有关。因而,表达于唾液腺中的Zcyto 10基因可用于该综合症的基因治疗。若哺乳动物Zcyto 10基因突变或缺失,可将Zcyto10基因引入该哺乳动物的细胞中。
本发明的另一方面涉及与SEQ ID NO:1,3,18和33所列多核苷酸的区段互补的反义多核苷酸组合物。可设计这样的合成反义寡核苷酸以结合编码Zcyto 10多肽的mRNA并抑制该mRNA的翻译。这样的反义寡核苷酸可用于抑制细胞培养物或被试者中Zcyto 10多肽编码基因的表达。
本发明还提供了在诊断应用中有用的试剂。例如,Zcyto 10基因、含Zcyto 10 DNA或RNA或其亚序列的探针可用于检测Zcyto 10基因是否存在于染色体1上或是否发生了突变。在Zcyto 10基因位置上的可检测染色体畸变包括但不局限于非整倍体、基因拷贝数改变、插入、删除、限制酶切位点改变和重排。通过分子遗传学技术,诸如限制性片段长度多态性(RFLP)分析、应用PCR技术的短串联重复序列(STR)分析及本领域已知的其它遗传连锁分析技术[Sambrook等,同上;Ausubel等,同上;Marian,A.J.,Chest,108:255-265,(1995)],用本发明的多核苷酸可检测这样的畸变。
本领域的那些技术熟练人员将认识到公开于SEQ ID NO:2、4、12、13、19、20、25、26、34和35中的序列代表了人和小鼠Zcyto 10基因和多肽的单个等位基因,并认识到会存在等位变异和其它剪接方式。按标准步骤通过对来自不同个体的cDNA或基因组文库进行探测可克隆等位变体。SEQ ID NO:1、3、18和33中所示DNA序列的等位变体,包括含沉默突变的变体和突变导致氨基酸序列改变的变体,均在本发明范围内。
Zcyto 10序列在SEQ ID NO:1之3’非翻泽区内位置706、813、855和906处具有7个信使不稳定性基元。用放线菌酮处理表达Zcyto10的细胞可削弱这种信使不稳定性。参阅Shaw,G.等,细胞46:659-667(1986)。此外,可在基因上改变或除去富AT的3’非翻译区以进一步提高信使稳定性。
使用Zcyto 10促进伤口愈合
实施例4的数据显示Zcyto 10在伤口愈合中起作用。因此,Zcyto10可应用于伤口或烧伤中促进伤口愈合。Zcyto 10可以1-100μg/Kg体重的剂量系统性地施用。Zcyto 10还可通过含1ng-1mg Zcyto 10/g药膏或软膏的药膏或软膏方式应用于伤口上。参阅Remington’sPharmaceutical Sciences,第18版(Mark Publishing Co.,Easton,Penn.,1996)。应每天将Zcyto 10用于清洁伤口上直到伤口愈合。
利用Zcyto 10增加血小板数
正如在以下实施例7中可见的,我们已发现Zcyto 10可用于增加血小板数。这对于由于化疗或放疗而血小板减少的癌症病人尤其重要。Zcyto 10可与药用可接受载体一起在治疗上施用。
本发明进一步由以下非限制实施例进行描述。
实施例1
Zcyto 10的克隆
Zcyto10×1(较长形式)和Zcyto10×2(较短形式)的全长序列通过以下步骤阐明:进行3’RACE,并将产生的两片段测序(SEQ ID NO:10和SEQ ID NO:11),然后用计算机将SEQ ID NO:5中所示的est序列与来自两个3’race的片段的重叠序列人工拼接在一起。
将寡核苷酸zc15907(SEQ ID NO:6)设计成恰位于Zcyto 10推定甲硫氨酸上游区域(5’)。将另一寡核苷酸zc15906(SEQ IN NO:7)设计在更远的下游,恰位于信号序列切割位点的上游区域。将这些寡核苷酸用于人气管marathon cDNA的3’RACE反应中。ZC15907用于第一3’race反应中,zc15906用于嵌套式3’race反应中。以购自Clontech的人气管mRNA开始,用Marathon cDNA扩增试剂盒(Clontech,Palo Alto,CA)按制造商说明书制备MARATHON cDNA。
按Marathon cDNA扩增试剂盒中制造商的说明书进行PCR反应,在热循环参数上有一些修改。用于第一PCR反应中的循环参数是:
94℃1分钟30秒,1次
94℃15秒、68℃1分钟,30次
72℃7分钟,1次
用于嵌套式PCR反应中的循环参数是:94℃1分钟30秒1次,94℃15秒68℃1分钟20秒,30次,72℃7分钟1次。
将产生的产物进行1.2%琼脂糖凝胶(Gibco琼脂糖)电泳,可见两主带,大约相距80bp。将这些带切出并按制造商说明书用QIAEXTM树脂(Qiagen)进行凝胶纯化。然后将这些片段测序,可识别Zcyto 10的全长序列。
实施例2
RNA印迹分析
对人的多组织印迹I、II、III和RNA主点印迹(Master DotBlot)(Clontech)进行探测,以确定Zcyto 10的组织分布。用est序列(SEQ ID NO:5bp 100-145)设计45-mer反义寡核苷酸(SEQ IDNO:9)并用于探测。
用T4多核苷酸激酶(Gibco-BRL)将15pm的SEQ ID NO:9进行32P末端标记。标记反应液含2μl 5X激酶反应缓冲液(Gibco-BRL)、1μl T4激酶、15pm SEQ ID NO:9、1μL 6000Ci/mmol 32Pγ-ATP(Amersham)和水至10μl。反应液37℃温育30分钟。用NucTrapProbe Purification Column(Stratagene)除去未掺入的放射性。多组织RNA印迹和人RNA主印迹(Clontech)在10ml ExpressHyb中50℃预杂交3小时,其中含1mg鲑精DNA和0.3mg人cotl DNA(Gibco-BRL),二者均煮沸3分钟,冰置2分钟然后加入ExpressHyb中。在50℃杂交过夜。起始洗涤条件如下:2×SSC、0.1%SDS室温40分钟,其中多次换洗涤液,然后1×SSC、0.1%SDS在64℃(Tm-10)30分钟。印迹随后对胶片曝光两天。
RNA印迹中Zcyto 10的表达显示在气管中约有1.2kb的带,在胃中有弱的1.5kb带,在胰中有这两种大小的较弱带。斑点印迹显示在气管、唾液腺、胎盘、睾丸、皮肤、前列腺、肾上腺和甲状腺中存在Zcyto 10。
在小鼠中,Zcyto 10发现存在于肾、骨骼肌、唾液腺、肝和皮肤内。
实施例3
Zcyto 10的染色体分布和定位
用可购买到的“Stanford G3放射杂交绘图板”(Reseach Genetics,Inc.,Hantsville,AL)将Zcyto 10绘图于第1号染色体上。该“Stanford G3 RH板”包含来自整个人基因组之83个放射杂交克隆中每一个的可PCR DNA,加上两个对照DNA(RM供体和A3受体)。一个公众可使用的WWW服务器(http://shgc-www.stanford.edu)可用于标记的染色体定位。
为了用“Stanford G3 RH板”给Zcyto10绘图,在可PCR的96孔微滴定板(Stratagene,La Jolla,CA)上建立20μl的反应体系,将其用于“RoboCycler Gradient96”热循环仪(Stratagene)上。85个PCR反应中的每一个组成如下:2μl 10x KlenTaq PCR反应缓冲液(CLONTECH Laboratories,Inc.,Palo Alto,CA)、1.6μl dNTP混合液(每种2.5mM,PERKIN-ELMER,Foster City,CA)、1μl有义引物(SEO ID NO:6,5’ATT CCT AGC TCC TGT GGT CTC CAG 3’)、1μl反义引物(SEQ ID No:8,5’TCC CAA ATT GAG TGT CTT CAG T3’)、2μl“Redi Load”(Research Genetics,Inc.,-Huntsville,AL)、0.4μl 50x Advantage KlenTaq Polymerase Mix(ClontechLaborabories,Inc.)、来自单个杂交克隆或对照的25ng DNA,用ddH2O补充至总体积20μl。用等量矿物油覆盖和密封反应液。PCR循环条件如下:开始的第一个循环为95℃变性5分钟,然后35个循环是95℃变性1分钟、66℃退火1分钟再72℃延伸1分钟,最后一个循环是72℃延伸7分钟。在2%琼脂糖凝胶(Life Technologies,Gaithersburg,MD)上电泳分离反应产物。
结果显示,Zcyto 10与构架标记SHGC-36215连锁,具大于10的LOD值,且距离标记为14.67CR-10000。借助于周围标记,将Zcyto10定位于完整LDB 1号染色体图谱的1q32.2区域(The GeneticLocation Database,南安普敦大学,WWW服务器:http://cedar.genetics.soton.ac.uk/public_html/)。
实施例4
利用Zcyto 10促进伤口愈合
本研究中使用了正常成熟雌性Balb/C小鼠。将它们以12小时光亮-黑暗的循环置于动物护理室中,在研究期间给水并以实验室啮齿类随意食水方式喂养。它们从外科手术的那天起被分别关入笼中。
在外科手术当日,用在无菌(0.2μ-过滤)磷酸缓冲盐溶液(PBS)中的104mg/kg氯胺酮(Vetalar,Aveco Inc.,Ft.Dodge,IA)加7mg/kg甲苯噻嗪(Rompun,Mobey Corp.,Shawnee,KS)通过腹膜内注射麻醉动物。剪掉背部毛发并用NAIR(Carter-Wallace,NewYork,NY)使皮肤脱毛,然后以水清洗。将100% aloe vera凝胶用于对抗由于NAIR处理所致的碱性灼伤,然后将动物置于循环水加热台上直到皮肤及周围毛皮干燥。
然后用metofane(Pittman Moore,Mundelein,NJ)麻醉动物并用70%酒精擦拭脱毛的背部。在胸腰椎水平穿过脊柱旁区域上方的皮肤和肉膜作4个切口,每个0.5平方厘米。用粘性的半透性封闭敷科BIOCLUSIVE(Johnson & Johnson,Arlington,TX)覆盖伤口和周围脱毛皮肤。为了稍后评估闭合参数,通过BIOCLUSIVE将切口边缘描到醋酸透明纸上。
用Qiagen RNeasy Midi Kit在不同时间点(7小时、15小时和24小时)处理对照皮肤和受伤皮肤。简短地说,将皮肤(对照和受伤区域)称重并于恰当体积裂解缓冲液(RLT)中匀浆。旋转裂解物以去除组织残渣并将等体积70%乙醇加入;混匀后上柱。样品旋转5分钟并用3.8mlRW1缓冲液洗1次,然后用RPE洗两次(各2.5ml)。用无RNase水洗脱总RNA。用实时PCR(Perkin Elmer ABI Prisn 7700 SequenceDetector)检测皮肤样品的表达水平。
实验设计为一无模板对照、一套标准和皮肤样品。用小鼠肾总RNA作标准曲线。三套皮肤总RNA(25ng)用于此实验中:7小时(对照和受伤的);15小时(对照和受伤的)、24小时(对照和受伤的)。每一样品在7700序列检测仪上通过一步逆转录PCR重复三次进行。该实验中使用了内置式(in-house)正向引物SEQ ID NO:36、反向引物SEQ IDNO:37和Perkin Elmer’s Taq Man probe(ZG-7-FAM)。一步逆转录PCR的条件如下:(逆转录步骤)48℃30分钟,(40个循环的PCR步骤)95℃10分钟,95℃15秒,60℃1分钟。
对照皮肤样品中在7小时和15小时时Zcyto10的表达水平类似,分别为2.46ng/ml和2.61ng/ml。24小时的对照皮肤样品中Zcyto10的表达水平是0。7小时时受伤皮肤的Zcyto10表达水平是5.17ng/ml(与对照样品的相比提高了2倍以上)。15小时时受伤皮肤的Zcyto 10表达水平是14.45ng/ml(与对照样品相比增加了5.5倍)。24小时时受伤皮肤的Zcyto 10表达水平是5.89ng/ml。重复实验还包括阴性对照(酵母tRNA),显示有相似倾向,酵母tRNA的结果接近0。结果显示扩增确实存在且为小鼠特异性。
这些数据表明Zcyto10在伤口修复中起作用,因为受伤组织的Zcyto10表达水平较对照样品的高且其随时间增加和减少。因此,Zcyto10可应用于受伤处以促进伤口愈合。
实施例5
转基因小鼠
生产在清蛋白或金属硫蛋白启动子控制下表达Zcyto10的转基因小鼠。出生时,数只小鼠具有发亮的外观,运动有限。这些小鼠的皮肤紧绷并有皱纹,其中数个还在下唇上有细丝类毛发。鼻孔和嘴区域、四肢和尾部有些肿。
使用清蛋白启动子的一只转基因小鼠存活3天,它严重地生长延迟。无耳朵发育,而足趾的发育延缓。当其在第1、2或3天濒死时处死所有动物。收集尾和肝样品并将其原位固定于10%中性福尔马林中并包埋在石蜡中,切成3μm的切片并用H&E染色。具此表型的所有小鼠均为转基因小鼠且有从低到高的Zcyto10表达。
在除皮肤外的大部分组织中观察不到显著的改变。表达Zcyto10的幼鼠,尤其是具高Zcyto10表达水平的那些小鼠的皮肤倾向于较无表达幼鼠的皮肤厚。这些幼鼠的颗粒层与无表达幼鼠相比厚度减少,棘层由于细胞层增加和/或细胞直径增加而较厚。
除了表皮中的改变外,具中等Zcyto10表达的一只小鼠的真皮被粘性物质病灶性地适度扩张。
实施例6
从细胞培养基纯化Zcyto10
用阳离子交换层析和大小排阻层析两步方法自细胞培养基中分离CHO细胞产生的Zcyto10。
A.阳离子交换层析步骤
所用材料
用具共价键合磺丙基(SP)的一种TOYOPEARL离子交换树脂,SP-650M阳离子交换树脂填充的2.2cm直径(D)×6cm高(H)柱(AMICON)。
收集来自已被含Zcyto质粒转染之幼仓鼠肾(BHK)细胞的15升培养基。用2N HCl将培养基pH调至pH5。用50mM乙酸钠(NaAc,pH5.0)平衡上述填充柱。以约8ml/分钟20个柱体积(CV)/小时的速率将培养基装入柱子。上柱完成后用10个柱体积的50mM NaAc,pH5.0洗柱。然后用50mM NaAc,pH5.0中的20柱体积NaCl梯度洗脱柱中的物质。NaCl梯度为0→0.5M NaCl。这将培养基中的物质从15升浓缩至170ml。
用截流5000的旋转离心浓缩器(Millipore,Inc.Bedford,MA)将产生的170ml收获物进一步浓缩至约5ml。
B.大小排阻(S-100)凝胶过滤步骤
所用材料
1.6cm(直径)×93cm(高)柱
S-100凝胶(Pharmacia,Piscataway,NJ)
随后将5ml收获液加到上述含S-100凝胶的柱子上。该柱子已用5X磷酸缓冲盐溶液平衡至柱pH约为7.0。用1×PBS以1.5ml/分钟的流速将Zcyto10与污染物分离。以2ml量收集各组分。通过以考马斯兰染色的十二烷基硫酸钠(SDS)聚丙烯酰胺凝胶电泳确定,在洗脱开始约90分钟时Zcyto10多肽洗下到组分52-64中。凝胶显示在约14KDa的预期分子量处有一条带。
实施例7
小鼠Zcyto10的克隆
5’MARATHON RACETM(Clontech,Palo Alto,CA)的PCR引物为附着于MARATHONTM AP1衔接物的SEQ ID NO:38,它与附着于AP2MARATHONTM衔接物的SEQ ID NO:39嵌套,3’MARATHON RACETM的引物为附着于MARATHON RACETM AP1衔接物的SEQ ID NO:40,它与附着于MARATHON RACETM AP2衔接物的SEQ ID NO:41嵌套,对小鼠皮肤MARATHON RACETM cDNA进行5’和3’PCR。将来自这些反应的数个片段进行凝胶纯化和测序,可阐明小鼠Zcyto10的全长编码序列及一些5’和3’UTR序列。发现两小鼠Zcyto10变体,称为SEQ ID NO:18以及19和SEQ ID NO:33和34。物引物SEQ ID NO:42和43 PCR扩增这些克隆。
实施例8
通过腺病毒将Zcyto10施用于正常小鼠
通过含Zcyto10基因的腺病毒施用Zcyto10。如下所述有三组小鼠。将腺病毒从静脉内注射入C57B1/6雄性和雌性小鼠。在处死前3天所有小鼠接受含有溴脱氧尿苷(Brdu)的饮用水。这样可通过组织学方法检测细胞增殖。检测参数包括体重改变、全血细胞计数、血清化学、组织学、器官重量及通过BrdU检测的细胞增殖。
实验设计
第一组 Zcyto 10×1(SEQ ID NO:18)/pAC-CMV/AdV
1×1011颗粒/剂
(在第21天处死9只雌性、9只雄性小鼠)
(在第11天处死2只雌性、2只雄性小鼠)
总数=22只小鼠
第二组 空CMV/AdV对照
1×1011粒/剂
(在第21天处死10只雌性、10只雄性小鼠)
(在第11天处死2只雌性、2只雄性小鼠)
总数=24只小鼠
第三组 不经处理
(5只雌性、5只雄性小鼠)
总数=10
结果
最引人注目的结果是,与空腺病毒对照相比,在用Zcyto10-腺病毒处理的雄性和雌性小鼠中观察到血小板计数显著增加。在雄性小鼠中这伴随着血细胞比容降低及脾和肝重增加。在雄性中胸腺重量也减少。与此形成对照的是,与空病毒对照相比,Zcyto10腺病毒处理的雌性小鼠显示白细胞计数显著增加,主要是淋巴细胞和中性粒细胞计数增加。
这些结果表明Zcyto10处理影响血细胞生成,但除了血小板计数增加这一效果在两性都有之外,其它影响是性别特异性的。
其它影响包括以下结果:
在被处理组中雌性的葡萄糖水平降低,而雄性葡萄糖水平显示无显著变化。
在被处理组的雄性和雌性中血尿氮(BUN)均提高。
在被处理组中雌性的碱性磷酸酶较高,而雄性显示无显著变化。
被处理组的雄性和雌性中血小板计数均较高。
在被处理组中雌性的总白细胞计数(WBC)较高而雄性显示无显著变化。
序列表
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Leu Gly Ser Cys Val Ile Thr Ala Asn Leu Gln Ala Ile Gln Lys Glu
30 35 40 45
ttt tct gag att cgg gat agt gtg caa gct gaa gat aca aat att gac 253
Phe Ser Glu Ile Arg Asp Ser Val Gln Ala Glu Asp Thr Asn Ile Asp
50 55 60
atc aga att tta agg acg act gag tct ttg aaa gac ata aag tct ttg 301
Ile Arg Ile Leu Arg Thr Thr Glu Ser Leu Lys Asp Ile Lys Ser Leu
65 70 75
gat agg tgc tgc ttc ctt cgt cat cta gtg aga ttc tat ctg gac agg 349
Asp Arg Cys Cys Phe Leu Arg His Leu Val Arg Phe Tyr Leu Asp Arg
80 85 90
gta ttc aaa gtc tac cag acc cct gac cac cat acc ctg aga aag atc 397
Val Phe Lys Val Tyr Gln Thr Pro Asp His His Thr Leu Arg Lys Ile
95 100 105
agc agc ctc gcc aac tcc ttt ctt atc atc aag aag gac ctc tca gtc 445
Ser Ser Leu Ala Asn Ser Phe Leu Ile Ile Lys Lys Asp Leu Ser Val
110 115 120 125
tgt cat tct cac atg gca tgt cat tgt ggg gaa gaa gca atg gag aaa 493
Cys His Ser His Met Ala Cys His Cys Gly Glu Glu Ala Met Glu Lys
130 135 140
tac aac caa att ctg agt cac ttc ata gag ttg gaa ctt cag gca gcg 541
Tyr Asn Gln Ile Leu Ser His Phe Ile Glu Leu Glu Leu Gln Ala Ala
145 150 155
gtg gta aag gct ttg gga gaa cta ggc att ctt ctg aga tgg atg gag 589
Val Val Lys Ala Leu Gly Glu Leu Gly Ile Leu Leu Arg Trp Met Glu
160 165 170
gag atg cta tagatgaaag tggagaggct gctgagaaca ctcctgtcca 638
Glu Met Leu
175
agaatctcag acctcagcac catgaagaca tggccccagg tgctggcatt tctactcaag 698
agttccagtc ctcagcacca cgaagatggc ctcaaaccac cacccctttg tgatataact 758
tagtgctagc tatgtgtata ttatttctac attattggct cccttatgtg aatgccttca 818
tgtgtc 824
<210>19
<211>176
<212>PRT
<213>Mus musculus
<400>19
Met Lys Gly Phe Gly Leu Ala Phe Gly Leu Phe Ser Ala Val Gly Phe
1 5 10 15
Leu Leu Trp Thr Pro Leu Thr Gly Leu Lys Thr Leu His Leu Gly Ser
20 25 30
Cys Val Ile Thr Ala Asn Leu Gln Ala Ile Gln Lys Glu Phe Ser Glu
35 40 45
Ile Arg Asp Ser Val Gln Ala Glu Asp Thr Asn Ile Asp Ile Arg Ile
50 55 60
Leu Arg Thr Thr Glu Ser Leu Lys Asp Ile Lys Ser Leu Asp Arg Cys
65 70 75 80
Cys Phe Leu Arg His Leu Val Arg Phe Tyr Leu Asp Arg Val Phe Lys
85 90 95
Val Tyr Gln Thr Pro Asp His His Thr Leu Arg Lys Ile Ser Ser Leu
100 105 110
Ala Asn Ser Phe Leu Ile Ile Lys Lys Asp Leu Ser Val Cys His Ser
115 120 125
His Met Ala Cys His Cys Gly Glu Glu Ala Met Glu Lys Tyr Asn Gln
130 135 140
Ile Leu Ser His Phe Ile Glu Leu Glu Leu Gln Ala Ala Val Val Lys
145 150 155 160
Ala Leu Gly Glu Leu Gly Ile Leu Leu Arg Trp Met Glu Glu Met Leu
165 170 175
<210>20
<211>152
<212>PRT
<213>Mus musculus
<400>20
Leu Lys Thr Leu His Leu Gly Ser Cys Val Ile Thr Ala Asn Leu Gln
1 5 10 15
Ala Ile Gln Lys Glu Phe Ser Glu Ile Arg Asp Ser Val Gln Ala Glu
20 25 30
Asp Thr Asn Ile Asp Ile Arg Ile Leu Arg Thr Thr Glu Ser Leu Lys
35 40 45
Asp Ile Lys Ser Leu Asp Arg Cys Cys Phe Leu Arg His Leu Val Arg
50 55 60
Phe Tyr Leu Asp Arg Val Phe Lys Val Tyr Gln Thr Pro Asp His His
65 70 75 80
Thr Leu Arg Lys Ile Ser Ser Leu Ala Asn Ser Phe Leu Ile Ile Lys
85 90 95
Lys Asp Leu Ser Val Cys His Ser His Met Ala Cys His Cys Gly Glu
100 105 110
Glu Ala Met Glu Lys Tyr Ash Gln Ile Leu Ser His Phe Ile Glu Leu
115 120 125
Glu Leu Gln Ala Ala Val Val Lys Ala Leu Gly Glu Leu Gly Ile Leu
130 135 140
Leu Arg Trp Met Glu Glu Met Leu
145 150
<210>21
<211>16
<212>PRT
<213>Mus musculus
<400>21
Ile Thr Ala Asn Leu Gln Ala Ile Gln Lys Glu Phe Ser Glu Ile Arg
1 5 10 15
<210>22
<211>15
<212>PRT
<213>Mus musculus
<400>22
Leu Asp Arg Val Phe Lys Val Tyr Gln Thr Pro Asp His His Thr
1 5 10 15
<210>23
<211>15
<212>PRT
<213>Mus musculus
<400>23
Leu Ala Asn Ser Phe Leu Ile Ile Lys Lys Asp Leu Ser Val Cys
1 5 10 15
<210>24
<211>15
<212>PRT
<213>Mus muculus
<400>24
Val Val Lys Ala Leu Gly Glu Leu Gly Ile Leu Leu Arg Trp Met
1 5 10 15
<210>25
<211>144
<212>PRT
<213>Mus muculus
<400>25
Cys Val Ile Thr Ala Asn Leu Gln Ala Ile Gln Lys Glu Phe Ser Glu
1 5 10 15
Ile Arg Asp Ser Val Gln Ala Glu Asp Thr Asn Ile Asp Ile Arg Ile
20 25 30
Leu Arg Thr Thr Glu Ser Leu Lys Asp Ile Lys Ser Leu Asp Arg Cys
35 40 45
Cys Phe Leu Arg His Leu Val Arg Phe Tyr Leu Asp Arg Val Phe Lys
50 55 60
Val Tyr Gln Thr Pro Asp His His Thr Leu Arg Lys Ile Ser Ser Leu
65 70 75 80
Ala Asn Ser Phe Leu Ile Ile Lys Lys Asp Leu Ser Val Cys His Ser
85 90 95
His Met Ala Cys His Cys Gly Glu Glu Ala Met Glu Lys Tyr Asn Gln
100 105 110
Ile Leu Ser His Phe Ile Glu Leu Glu Leu Gln Ala Ala Val Val Lys
115 120 125
Ala Leu Gly Glu Leu Gly Ile Leu Leu Arg Trp Met Glu Glu Met Leu
130 135 140
<210>26
<211>144
<212>PRT
<213>人
<400>26
Cys Val Ile Ala Thr Asn Leu Gln Glu Ile Arg Asn Gly Phe Ser Asp
1 5 10 15
Ile Arg Gly Ser Val Gln Ala Lys Asp Gly Asn Ile Asp Ile Arg Ile
20 25 30
Leu Arg Arg Thr Glu Ser Leu Gln Asp Thr Lys Pro Ala Asn Arg Cys
35 40 45
Cys Leu Leu Arg His Leu Leu Arg Leu Tyr Leu Asp Arg Val Phe Lys
50 55 60
Asn Tyr Gln Thr Pro Asp His Tyr Thr Leu Arg Lys Ile Ser Ser Leu
65 70 75 80
Ala Asn Ser Phe Leu Thr Ile Lys Lys Asp Leu Arg Leu Cys His Ala
85 90 95
His Met Thr Cys His Cys Gly Glu Glu Ala Met Lys Lys Tyr Ser Gln
100 105 110
Ile Leu Ser His Phe Glu Lys Leu Glu Pro Gln Ala Ala Val Val Lys
115 120 125
Ala Leu Gly Glu Leu Asp Ile Leu Leu Gln Trp Met Glu Glu Thr Glu
130 135 140
<210>27
<211>38
<212>PRT
<213>人
<400>27
Cys Gly Glu Glu Ala Met Lys Lys Tyr Ser Gln Ile Leu Ser His Phe
1 5 10 15
Glu Lys Leu Glu Pro Gln Ala Ala Val Val Lys Ala Leu Gly Glu Leu
20 25 30
Asp Ile Leu Leu Gln Trp
35
<210>28
<211>71
<212>PRT
<213>人
<400>28
Ile Ala Thr Asn Leu Gln Glu Ile Arg Asn Gly Phe Ser Asp Ile Arg
1 5 10 15
Gly Ser Val Gln Ala Lys Asp Gly Asn Ile Asp Ile Arg Ile Leu Arg
20 25 30
Arg Thr Glu Ser Leu Gln Asp Thr Lys Pro Ala Asn Arg Cys Cys Leu
35 40 45
Leu Arg His Leu Leu Arg Leu Tyr Leu Asp Arg Val Phe Lys Asn Tyr
50 55 60
Gln Thr Pro Asp His Tyr Thr
65 70
<210>29
<211>92
<212>PRT
<213>人
<400>29
Ile Ala Thr Asn Leu Gln Glu Ile Arg Asn Gly Phe Ser Asp Ile Arg
1 5 10 15
Gly Ser Val Gln Ala Lys Asp Gly Asn Ile Asp Ile Arg Ile Leu Arg
20 25 30
Arg Thr Glu Ser Leu Gln Asp Thr Lys Pro Ala Asn Arg Cys Cys Leu
35 40 45
Leu Arg His Leu Leu Arg Leu Tyr Leu Asp Arg Val Phe Lys Asn Tyr
50 55 60
Gln Thr Pro Asp His Tyr Thr Leu Arg Lys Ile Ser Ser Leu Ala Asn
65 70 75 80
Ser Phe Leu Thr Ile Lys Lys Asp Leu Arg Leu Cys
85 90
<210>30
<211>82
<212>PRT
<213>人
<400>30
Leu Asp Arg Val Phe Lys Asn Tyr Gln Thr Pro Asp His Tyr Thr Leu
1 5 10 15
Arg Lys Ile Ser Ser Leu Ala Asn Ser Phe Leu Thr Ile Lys Lys Asp
20 25 30
Leu Arg Leu Cys His Ala His Met Thr Cys His Cys Gly Glu Glu Ala
35 40 45
Met Lys Lys Tyr Ser Gln Ile Leu Ser His Phe Glu Lys Leu Glu Pro
50 55 60
Gln Ala Ala Val Val Lys Ala Leu Gly Glu Leu Asp Ile Leu Leu Gln
65 70 75 80
Trp Met
<210>31
<211>36
<212>PRT
<213>人
<400>31
Leu Asp Arg Val Phe Lys Asn Tyr Gln Thr Pro Asp His Tyr Thr Leu
1 5 10 15
Arg Lys Ile Ser Ser Leu Ala Asn Ser Phe Leu Thr Ile Lys Lys Asp
20 25 30
Leu Arg Leu Cys
35
<210>32
<211>61
<212>PRT
<213>人
<400>32
Leu Ala Asn Ser Phe Leu Thr Ile Lys Lys Asp Leu Arg Leu Cys His
1 5 10 15
Ala His Met Thr Cys His Cys Gly Glu Glu Ala Met Lys Lys Tyr Ser
20 25 30
Gln Ile Leu Ser His Phe Glu Lys Leu Glu Pro Gln Ala Ala Val Val
35 40 45
Lys Ala Leu Gly Glu Leu Asp Ile Leu Leu Gln Trp Met
50 55 60
<210>33
<211>756
<212>DNA
<213>Mus musculus
<220>
<221>CDS
<222>(71)...(532)
<400>33
tgggagacat cgatagccct gattgatctc tttgaatttt cgcttctggt ctccaggatc 60
taggtgtaag atg aaa ggc ttt ggt ctt gcc ttt gga ctg ttc tcc gct 109
Met Lys Gly Phe Gly Leu Ala Phe Gly Leu Phe Ser Ala
1 5 10
gtg ggt ttt ctt ctc tgg act cct tta act ggg ctc aag acc ctc cat 157
Val Gly Phe Leu Leu Trp Thr Pro Leu Thr Gly Leu Lys Thr Leu His
15 20 25
ttg gga agc tgt gtg att act gca aac cta cag gca ata caa aag gaa 205
Leu Gly Ser Cys Val Ile Thr Ala Asn Leu Gln Ala Ile Gln Lys Glu
30 35 40 45
ttt tct gag att cgg gat agt gtg tct ttg gat agg tgc tgc ttc ctt 253
Phe Ser Glu Ile Arg Asp Ser Val Ser Leu Asp Arg Cys Cys Phe Leu
50 55 60
cgt cat cta gtg aga ttc tat ctg gac agg gta ttc aaa gtc tac cag 301
Arg His Leu Val Arg Phe Tyr Leu Asp Arg Val Phe Lys Val Tyr Gln
65 70 75
acc cct gac cac cat acc ctg aga aag atc agc agc ctc gcc aac tcc 349
Thr Pro Asp His His Thr Leu Arg Lys Ile Ser Ser Leu Ala Asn Ser
80 85 90
ttt ctt atc atc aag aag gac ctc tca gtc tgt cat tct cac atg gca 397
Phe Leu Ile Ile Lys Lys Asp Leu Ser Val Cys His Ser His Met Ala
95 100 105
tgt cat tgt ggg gaa gaa gca atg gag aaa tac aac caa att ctg agt 445
Cys His Cys Gly Glu Glu Ala Met Glu Lys Tyr Asn Gln Ile Leu Ser
110 115 120 125
cac ttc ata gag ttg gaa ctt cag gca gcg gtg gta aag gct ttg gga 493
His Phe Ile Glu Leu Glu Leu Gln Ala Ala Val Val Lys Ala Leu Gly
130 135 140
gaa cta ggc att ctt ctg aga tgg atg gag gag atg cta tagatgaaag 542
Glu Leu Gly Ile Leu Leu Arg Trp Met Glu Glu Met Leu
145 150
tggataggct gctgagaaca ctcctgtcca agaatctcag acctcagcac catgaagaca 602
tggccccagg tgctggcatt tctactcaag agttccagtc ctcagcacca cgaagatggc 662
ctcaaaccac cacccctttg tgatataact tagtgctagc tatgtgtata ttatttctac 722
attattggct cccttatgtg aatgccttca tgtg 756
<210>34
<211>154
<212>PRT
<213>Mus musculus
<400>34
Met Lys Gly Phe Gly Leu Ala Phe Gly Leu Phe Ser Ala Val Gly Phe
1 5 10 15
Leu Leu Trp Thr Pro Leu Thr Gly Leu Lys Thr Leu His Leu Gly Ser
20 25 30
Cys Val Ile Thr Ala Asn Leu Gln Ala Ile Gln Lys Glu Phe Ser Glu
35 40 45
Ile Arg Asp Ser Val Ser Leu Asp Arg Cys Cys Phe Leu Arg His Leu
50 55 60
Val Arg Phe Tyr Leu Asp Arg Val Phe Lys Val Tyr Gln Thr Pro Asp
65 70 75 80
His His Thr Leu Arg Lys Ile Ser Ser Leu Ala Asn Ser Phe Leu Ile
85 90 95
Ile Lys Lys Asp Leu Ser Val Cys His Ser His Met Ala Cys His Cys
100 105 110
Gly Glu Glu Ala Met Glu Lys Tyr Asn Gln Ile Leu Ser His Phe Ile
115 120 125
Glu Leu Glu Leu Gln Ala Ala Val Val Lys Ala Leu Gly Glu Leu Gly
130 135 140
Ile Leu Leu Arg Trp Met Glu Glu Met Leu
145 150
<210>35
<211>130
<212>PRT
<213>Mus musculus
<400>35
Leu Lys Thr Leu His Leu Gly Ser Cys Val Ile Thr Ala Asn Leu Gln
1 5 10 15
Ala Ile Gln Lys Glu Phe Ser Glu Ile Arg Asp Ser Val Ser Leu Asp
20 25 30
Arg Cys Cys Phe Leu Arg His Leu Val Arg Phe Tyr Leu Asp Arg Val
35 40 45
Phe Lys Val Tyr Gln Thr Pro Asp His His Thr Leu Arg Lys Ile Ser
50 55 60
Ser Leu Ala Asn Ser Phe Leu Ile Ile Lys Lys Asp Leu Ser Val Cys
65 70 75 80
His Ser His Met Ala Cys His Cys Gly Glu Glu Ala Met Glu Lys Tyr
85 90 95
Asn Gln Ile Leu Ser His Phe Ile Glu Leu Glu Leu Gln Ala Ala Val
100 105 110
Val Lys Ala Leu Gly Glu Leu Gly Ile Leu Leu Arg Trp Met Glu Glu
115 120 125
Met Leu
130
<210>36
<211>27
<212>DNA
<213>人
<400>36
agattctatc tggacagggt attcaaa 27
<210>37
<211>17
<212>DNA
<213>人
<400>37
gcgaggctga tctttct 17
<210>38
<211>25
<212>DNA
<213>Mus musculis
<400>38
tggcgaggct gctgatcttt ctcag 25
<210>39
<211>25
<212>DNA
<213>Mus musculis
<400>39
ctttatgtct ttcaaagact cagtc 25
<210>40
<211>26
<212>DNA
<213>Mus musculis
<400>40
catcagaatt ttaaggacga ctgagt 26
<210>41
<211>25
<212>DNA
<213>Mus musculis
<400>41
ggtggtcagg ggtctggtag acttt 25
<210>42
<211>23
<212>DNA
<213>Mus musculis
<400>42
ggtgcatatt cctggtggct aga 23
<210>43
<211>25
<212>DNA
<213>Mus musculis
<400>43
attgcagtgt aagggaatac agaga 25
Claims (7)
1.编码多肽的分离多核苷酸,所说的多肽与SEQ ID No:4或SEQ ID No:13的多肽有至少90%相同。
2.权利要求1的分离多核苷酸,其中所述多核苷酸编码含有SEQ ID No:4或SEQ ID No:13之氨基酸序列的多肽。
3.权利要求1的分离多核苷酸,其中所说的多核苷酸为SEQ IDNo:3。
4.与选自SEQ ID No:4或SEQ ID No:13多肽至少90%相同的分离多肽。
5.权利要求4的分离多肽,其中所说的多肽选自SEQ ID No:4和SEQ ID No:13。
6.可选择性地结合多肽表位部分的抗体,所述多肽选自:SEQ IDNo:4和SEQ ID No:13。
7.可与权利要求6之抗体结合的抗独特型抗体。
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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US97915697A | 1997-11-26 | 1997-11-26 | |
US08/979,156 | 1997-11-26 |
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CNB988124289A Division CN1247780C (zh) | 1997-11-26 | 1998-11-25 | 哺乳动物细胞因子样多肽-10 |
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Publication Number | Publication Date |
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CN1618969A true CN1618969A (zh) | 2005-05-25 |
Family
ID=25526742
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CNA2004100877931A Pending CN1618969A (zh) | 1997-11-26 | 1998-11-25 | 哺乳动物细胞因子样多肽-10 |
CNB988124289A Expired - Lifetime CN1247780C (zh) | 1997-11-26 | 1998-11-25 | 哺乳动物细胞因子样多肽-10 |
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CNB988124289A Expired - Lifetime CN1247780C (zh) | 1997-11-26 | 1998-11-25 | 哺乳动物细胞因子样多肽-10 |
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EP (3) | EP1032671B1 (zh) |
JP (2) | JP5191619B2 (zh) |
KR (1) | KR100574387B1 (zh) |
CN (2) | CN1618969A (zh) |
AT (1) | ATE269902T1 (zh) |
AU (1) | AU739420B2 (zh) |
BR (1) | BR9814904A (zh) |
CA (1) | CA2312000C (zh) |
CZ (1) | CZ300849B6 (zh) |
DE (1) | DE69824755T2 (zh) |
DK (1) | DK1032671T3 (zh) |
EA (2) | EA010741B1 (zh) |
ES (1) | ES2218872T3 (zh) |
HU (1) | HU227703B1 (zh) |
IL (2) | IL136076A0 (zh) |
NO (2) | NO326561B1 (zh) |
NZ (1) | NZ504751A (zh) |
PL (2) | PL198687B1 (zh) |
UA (2) | UA73275C2 (zh) |
WO (1) | WO1999027103A1 (zh) |
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US5945511A (en) * | 1997-02-20 | 1999-08-31 | Zymogenetics, Inc. | Class II cytokine receptor |
JP2003535037A (ja) | 1999-12-23 | 2003-11-25 | ザイモジェネティクス,インコーポレイティド | 炎症を治療する方法 |
US6610286B2 (en) | 1999-12-23 | 2003-08-26 | Zymogenetics, Inc. | Method for treating inflammation using soluble receptors to interleukin-20 |
UA84830C2 (uk) | 1999-12-23 | 2008-12-10 | Займодженетікс, Інк. | Розчинний рецептор інтерлейкіну-20 |
AU2001290524A1 (en) | 2000-08-08 | 2002-02-18 | Zymogenetics Inc. | Soluble zcytor 11 cytokine receptors |
ATE333888T1 (de) | 2000-09-15 | 2006-08-15 | Zymogenetics Inc | Verwendung eines polypeptids, welches die extrazelluläre domäne von il-20ra und il-20rb enthält, zur behandlung von entzündungen |
CA2429044A1 (en) * | 2000-10-20 | 2002-05-02 | Zymogenetics, Inc. | Secreted alpha-helical cytokine-like protein zlmda24 |
WO2002058724A2 (en) * | 2001-01-26 | 2002-08-01 | Eli Lilly And Company | Use of lp82 to treat body weight disorders |
MXPA03007653A (es) * | 2001-02-28 | 2003-12-04 | Lilly Co Eli | Uso de lp82 para tratar trastornos hematopoyeticos. |
WO2003035096A1 (en) * | 2001-10-22 | 2003-05-01 | Eli Lilly And Company | Soluble proteins that inhibit cytokine signal transduction pathways |
JP4532902B2 (ja) | 2001-12-17 | 2010-08-25 | ザイモジェネティクス,インコーポレイティド | 子宮頸部癌の治療方法 |
WO2004085476A2 (en) * | 2003-03-24 | 2004-10-07 | Zymogenetics, Inc. | Anti-il-22ra antibodies and binding partners and methods of using in inflammation |
WO2005014028A1 (en) * | 2003-08-08 | 2005-02-17 | Novo Nordisk A/S | Interleukin-20 for treating and diagnosing conditions associated with neovascularisation |
ES2334140T3 (es) | 2003-11-21 | 2010-03-05 | Zymogenetics, Inc. | Anticuerpos anti-il-20 y componentes de union y procedimiento de uso en antiinflamacion. |
ATE506433T1 (de) | 2003-12-19 | 2011-05-15 | Novo Nordisk As | Prozessierung von peptiden und proteinen |
ATE550041T1 (de) | 2004-01-21 | 2012-04-15 | Novo Nordisk Healthcare Ag | Transglutaminase-vermittelte konjugation von peptiden |
WO2005087810A2 (en) | 2004-03-08 | 2005-09-22 | Zymogenetics, Inc. | Dimeric fusion proteins and materials and methods for producing them |
EP1812476B1 (en) | 2004-10-22 | 2010-07-21 | ZymoGenetics, Inc. | Anti-il-22ra antibodies and binding partners and methods of using in inflammation |
EP1856156A2 (en) | 2005-02-08 | 2007-11-21 | ZymoGenetics, Inc. | Anti-il-20, anti-il-22 and anti-il-22ra antibodies and binding partners and methods of using in inflammation |
RU2618868C2 (ru) | 2008-05-27 | 2017-05-11 | Дако Денмарк А/С | Композиции и способы определения хромосомных аберраций с новыми буферами для гибридизации |
EP2297203A1 (en) | 2008-06-30 | 2011-03-23 | Novo Nordisk A/S | Anti-human interleukin-20 antibodies |
WO2010042634A1 (en) * | 2008-10-07 | 2010-04-15 | National Cheng Kung University | Use of il-20 antagonists for treating rheumatoid arthritis and osteoporosis |
US9303287B2 (en) | 2009-02-26 | 2016-04-05 | Dako Denmark A/S | Compositions and methods for RNA hybridization applications |
US8454956B2 (en) | 2009-08-31 | 2013-06-04 | National Cheng Kung University | Methods for treating rheumatoid arthritis and osteoporosis with anti-IL-20 antibodies |
US10662465B2 (en) | 2011-09-30 | 2020-05-26 | Agilent Technologies, Inc. | Hybridization compositions and methods using formamide |
EP3252173A1 (en) | 2011-10-21 | 2017-12-06 | Dako Denmark A/S | Hybridization compositions and methods |
JP5976849B2 (ja) * | 2012-03-05 | 2016-08-24 | シージーン アイエヌシー | PTO切断及び伸長アッセイによるターゲット核酸配列でのヌクレオチド変異検出{DetectionofNucleotideVariationonTargetNucleicAcidSequencebyPTOCleavageandExtensionAssay} |
WO2013164440A1 (en) | 2012-05-03 | 2013-11-07 | Novo Nordisk A/S | Methods related to treatment of inflammatory diseases and disorders |
US9221904B2 (en) | 2012-07-19 | 2015-12-29 | National Cheng Kung University | Treatment of osteoarthritis using IL-20 antagonists |
US8852588B2 (en) | 2012-08-07 | 2014-10-07 | National Cheng Kung University | Treating allergic airway disorders using anti-IL-20 receptor antibodies |
US8603470B1 (en) | 2012-08-07 | 2013-12-10 | National Cheng Kung University | Use of IL-20 antagonists for treating liver diseases |
EP3464357A1 (en) | 2016-05-24 | 2019-04-10 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Methods and pharmaceutical compositions for the treatment of pulmonary bacterial infections |
CN113248567B (zh) * | 2021-02-10 | 2023-02-03 | 渤海大学 | 一种苦味受体阻滞肽及其应用 |
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JP2001500369A (ja) * | 1996-08-30 | 2001-01-16 | ヒューマン ジノーム サイエンシーズ,インコーポレイテッド | インターロイキン―19 |
JP2003535037A (ja) * | 1999-12-23 | 2003-11-25 | ザイモジェネティクス,インコーポレイティド | 炎症を治療する方法 |
-
1998
- 1998-11-25 CN CNA2004100877931A patent/CN1618969A/zh active Pending
- 1998-11-25 WO PCT/US1998/025228 patent/WO1999027103A1/en active IP Right Grant
- 1998-11-25 IL IL13607698A patent/IL136076A0/xx active IP Right Grant
- 1998-11-25 PL PL378071A patent/PL198687B1/pl unknown
- 1998-11-25 KR KR1020007005732A patent/KR100574387B1/ko not_active IP Right Cessation
- 1998-11-25 EP EP98960493A patent/EP1032671B1/en not_active Expired - Lifetime
- 1998-11-25 CZ CZ20001910A patent/CZ300849B6/cs not_active IP Right Cessation
- 1998-11-25 EA EA200401501A patent/EA010741B1/ru not_active IP Right Cessation
- 1998-11-25 AU AU16077/99A patent/AU739420B2/en not_active Ceased
- 1998-11-25 NZ NZ504751A patent/NZ504751A/en not_active IP Right Cessation
- 1998-11-25 DE DE69824755T patent/DE69824755T2/de not_active Expired - Lifetime
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2007
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