CN1216912C - 人白细胞介素-17受体样蛋白及其编码基因与应用 - Google Patents
人白细胞介素-17受体样蛋白及其编码基因与应用 Download PDFInfo
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Abstract
本发明公开了一种人白细胞介素-17受体样蛋白及其编码基因与应用。人白细胞介素-17受体样蛋白,它具有序列表中序列2的氨基酸残基序列或将序列2的氨基酸残基序列经过一个氨基酸残基的取代、缺失或添加且具有与序列2的氨基酸残基序列相同活性的由序列2衍生的蛋白质。人白细胞介素-17受体样蛋白的编码基因,是具有序列表中序列1的DNA序列。本发明的人白细胞介素-17受体样蛋白可以使Stat5因子活化、介导细胞增殖,并可望在促进或抑制精子形成的药物中得到应用。
Description
技术领域
本发明涉及一种人白细胞介素-17受体样蛋白及其编码基因与应用。
背景技术
白细胞介素是一类重要的细胞因子,可以通过与靶细胞膜表面的特异受体结合而介导广泛的生物学作用,诸如:细胞增殖、分化、造血调节、免疫及炎症应答等。白细胞介素17(IL-17/IL-17A)是IL-17家族第一个被克隆的T细胞来源的促炎症细胞因子。人IL-17通常以一个二硫键连接组成的同二聚体分泌性糖蛋白形式存在,分子质量为30~35kD。尽管IL-17只在局限的组织和细胞系表达,但却有广泛的生物学功能,尤其是在促炎症和造血功能方面较为突出,与IL-17的低亲和力受体一IL-17R广泛表达相一致。IL-17还能刺激多种细胞因子的产生,如:来源于巨嗜细胞的肿瘤坏死因子α,来源于成纤维细胞的IL-1β、IL-6、IL-8、ICAM-1以及来源于滑膜细胞的G-CSF、PGE2,因此可介导多种生物学作用。
尽管目前至少已克隆了7种IL-17家族成员,但有关该家族成员的受体研究却知之甚少。IL-17R是第一个被鉴定的IL-17受体家族成员,广泛表达于多种不同的组织。但是,IL-17与IL-17R的亲和力较弱,与IL-17具有的广泛的生物学功能并不匹配,并且IL-17R并不含有与先前已知蛋白相似的结构。IL-17Rh1/IL-17BR与IL-17R一样,并不含有与先前已知蛋白相似的结构域,但它们的胞外结构域具有保守的胱氨酸残基,胞内结构域含有与IL-17R相似的氨基酸残基序列,暗示它们可能具有相似的下游信号事件。研究结果表明:NF-κB是它们共同介导的下游信号分子。
曾报道,IL-17A和IL-17E能诱导核转录因子NF-κB的活化,另外也有研究结果表明IL-17A所诱导的下游信号事件包括活化胞外调控激酶(ERK-1/ERK-2),氮末端激酶(JNK,c-Jun),p38有丝分裂原激酶,Raf、Stats等下游信号分子。IL-17AR的敲基因小鼠研究表明,在肿瘤坏死因子-α结合因子-6(TRAF-6)缺陷的细胞系,IL-17诱导的下游信号事件明显阻断,提示TRAF-6对于IL-17刺激的信号是必要的。由于已知的几种IL-17受体成员的胞内结构域并不含有与Toll/IL-1R相似的结构,因此寻找和鉴定未知的IL-17家族成员的潜在受体显得尤其必要。
发明内容
本发明的目的是提供一种新的人白细胞介素-17受体样蛋白。
一种人白细胞介素-17受体样蛋白,它具有序列表中序列2的氨基酸残基序列或将序列2的氨基酸残基序列经过一个氨基酸残基的取代、缺失或添加且具有与序列2的氨基酸残基序列相同活性的由序列2衍生的蛋白质。
本发明的人白细胞介素-17受体样蛋白命名为hIL-17RLM-L,是一种I型单跨膜细胞因子受体,由739个氨基酸残基组成。hIL-17RLM-L氮末端含有一个17个氨基酸组成的信号肽,一个跨膜结构域和一个较长的胞浆结构域。此外其较长的胞浆结构域还含有一个潜在的SH3相互作用结构域和众多的酪氨酸磷酸化部位,在胞浆邻膜区含有一个Toll/IL-1受体样结构域-TIR(其它已知的IL-17受体样蛋白不含有TIR结构域)。
本发明采用多组织Northern Blot方法,检测了hIL-17RLM-L的mRNA组织表达分布,结果表明:hIL-17RLM-L mRNA主要在肾及睾丸表达,少量在心、脑、脾、子宫表达,并且主要表达在细胞膜及细胞浆部位。在肝、肺、外周血和胸腺几乎无表达。RT-PCR检测表明hIL-17RLM-L在大多数细胞系有表达。hIL-17RLM-L在生精过程中具有细胞表达特异性,主要在睾丸组织的生精细胞、支持细胞表达,而在曲细精管基底膜部位的成纤维细胞和睾丸支持组织部位的间质细胞染色阴性。在隐睾组织,生精过程中止,但是hIL-17RLM-L在曲细精管的生精细胞却有极强的染色。
本发明的第二个目的是提供编码人白细胞介素-17受体样蛋白的基因。
本发明所提供的人白细胞介素-17受体样蛋白的编码基因,是具有序列表中序列1的DNA序列,命名为hIL-17RLM-L。
本发明人白细胞介素-17受体样蛋白hIL-17RLM-L的编码基因由4477个碱基组成,位于人的3号染色体(3p21.1)上,由13个外显子横跨12万个碱基翻译成蛋白前体。
研究表明,本发明的人白细胞介素-17受体样蛋白可以使Stat5因子活化、介导细胞增殖,并可望在促进或抑制精子形成的药物中得到应用。
具体实施方式
实施例1、hIL-17RLM-L基因的克隆及表达
根据EST拼接和hIL-17RLM(595aa)的人类基因组结构,以及蛋白blast NCBI EST和trace数据库而预测到hIL-17RLM(595aa)存在一个更长的选择性剪切形式,命名为hIL-17RLM-L。
根据预测的hIL-17RLM-L的cDNA序列,设计引物,将其分子克隆。以293T和睾丸的总RNA为模板,通过RT-PCR将其克隆到T-A载体,并测序。测序结果与预测的编码cDNA完全一致。其5’端未翻译区含有终止密码子及Kozak序列,长度为4477bp。编码一个由739个氨基酸组成的单跨膜糖蛋白。按照SignalP信号肽预测软件分析结果,其含有一个由17个氨基酸组成的信号肽:MAPWLQLCSVFFTVNAC。按照HMMTOP,SOSUI,TMHMM,Tmpred,TopPred 2等蛋白跨膜区预测软件分析结果,其跨膜区由23个氨基酸组成(I298-M320):IRAVAITVPLVVISAFATLFTVM。另外在由297个氨基酸组成的胞外区含有8个胱氨酸残基,9个潜在的N-糖基化位点和一个假想的IgG样结构域。其较长的胞浆结构域(519aa)还含有一个潜在的SH3相互作用结构域(脯氨酸富集区)和众多的酪氨酸磷酸化部位,在胞浆邻膜区含有一个Toll/IL-1受体样结构域(TIR)。因为在其它已知的IL-17受体样分子并不含有TIR结构域,因此暗示hIL-17RLM-L胞内区具有重要信号潜力。
为了进一步深入研究hIL-17RLM-L及其选择性剪切形式的生物学功能,首先制备了抗人IL-17RLM的兔多克隆抗血清,选择hIL-17RLM-L胞内区的一段多肽(M320-I457),该段多肽包含潜在预测的Toll/IL-1R/MyD88样结构域-TIR和IL-17受体样结构域,也包含hIL-17RLM-L潜在的酪氨酸磷酸化部位-Y329。将这段开放阅读筐亚克隆入pGEX4T-1原核表达载体,构建GST-hIL-17RLM-L融合蛋白表达载体,在大肠杆菌BL21中诱导表达。借助GST亲和层析柱-GSTrapFF,纯化了GST-hIL-17RLM-L融合蛋白。进而,对家兔实行加强注射,获得效价和特异性极高的抗hIL-17RLM-L兔多克隆抗血清。经测试,ELISA效价达1∶100000,Western blot效价达1∶5000-10000。
将hIL-17RLM-L构建于pcDNA3真核表达载体,以便其在真核细胞瞬时表达和稳定表达。将hIL-17RLM-L瞬时转染于Cos7细胞,检测其在胞内的表达和糖基化情况。hIL-17RLM-L在Cos7细胞的表达显示其分子量约为100kD。
对hIL-17RLM-L在细胞器的表达定位进行研究。将hIL-17RLM-L构建于EGFP-N1真核表达载体,将其瞬时转染Cos7细胞,结果显示其主要表达在细胞膜上。
实施例2、hIL-17RLM-L胞内结构域的功能——活化Stat5和介导细胞增殖
hIL-17RLM-L胞内区含有潜在的TIR结构域,IL-17R样结构域,SH3相互作用结构域和众多的酪氨酸磷酸化位点,提示其胞内区可能具有潜在的信号潜力。本发明的发明人构建了一系列嵌合受体,通过对hIL-17RLM-L胞内区人工二聚化,证实了其传递信号的能力。
发明人将EPOR的胞外区和跨膜区与hIL-17RLM的胞内区构建一嵌合受体,借助EPO的刺激,进而检测hIL-17RLM-L的胞内区传递信号的潜力。首先进行了荧光素酶报道分析,瞬时共转染指示的受体表达质粒,Stat5应答的荧光素酶报道质粒4FTKSLN,pRL-TK载体(内对照),转染36小时后,使用重组人EPO刺激或不刺激(PBS)约30分钟后,收获细胞,进行荧光素酶报道分析。结果显示:EPOR/hIL-17RLM-L嵌合受体与全长EPOR一样,在EPO的刺激作用下,能够介导Stat5的活化;并且当共转染Stat5的负显性突变体(dominant mutant)-Stat5 CYF时,EPOR/hIL-17RLM-L嵌合受体介导Stat5的活化被明显抑止;全长hIL-17RLM-L的过表达并不能介导Stat5的活化。这个结果暗示,hIL-17RLM-L的胞内区具有能够介导Stat5活化的潜力。
Stat5活化的一个重要标志是其在Jak激酶的作用下,Stat5的酪氨酸被磷酸化,然后转位入核,介导下游靶基因的表达。为了进一步检测hIL-17RLM-L的胞内区介导Stat5活化的潜力,采用免疫沉淀方法,检测EPOR/hIL-17RLM-L是否能够介导Stat5酪氨酸磷酸化。在Cos7细胞共转染嵌合受体-EPORextm/hIL-17RLM-Licd或全长EPOR或空载体(Mock),Jak2,stat5高纯度表达质粒。在EPO刺激前30分钟,培养基中补加NaV3O4以抑制内源性磷酸化酶的水解。使用重组人EPO刺激或不刺激(PBS)约30分钟后,收获细胞,制备总细胞裂解液,一部分用于免疫沉淀,另一部分直接用于Western blot以检测转染质粒的表达情况。结果表明:EPORextm/hIL-17RLM-Licd嵌合受体同EPOR一样,在EPO的刺激下,能够介导Stat5酪氨酸磷酸化;而在EPO未刺激组和空载体组,并未检测到Stat5酪氨酸磷酸化。这一结果进一步表明hIL-17RLM-L的胞内区能够介导Stat5酪氨酸磷酸化。
为了进一步证实hIL-17RLM-L胞内区具有介导Stat5活化的潜力,采用gel shiftassay以分析是否EPOR/hIL-17RLM-L嵌合受体能够介导细胞核抽提物Stat5-DNA结合复合体的形成,以证实其介导Stat5活化。具体做法是共转染指示的表达质粒于Cos7细胞,在EPO的作用后,制备细胞核抽提物,进行凝胶电泳阻滞分析实验。实验结果显示:EPORextm/hIL-17RLM-Licd嵌合受体同EPOR一样,在EPO的刺激下,能够介导Stat5-DNA结合复合体的形成;而在EPO未刺激组和空载体组,并未检测到Stat5-DNA结合复合体的形成。采用冰冷的未标记的特异性结合Stat5的DNA探针,能够竞争性抑止Stat5-DNA结合复合体的形成。这一结果表明:hIL-17RLM-L胞内区具有介导特异性Stat5-DNA结合复合体形成的特性。
为了进一步确定Stat5-DNA结合复合体的特异性和Stat5具体成分,分别借助抗Stat5ab,Stat5b,Stat1和Stat3特异性抗体,进行了Supershift分析实验。结果显示:抗Stat5ab或Stat5b能够显著超迁移这个DNA结合复合体,而Stat1或Stat3特异性抗体却并不能超迁移这个DNA结合复合体,暗示EPOR/hIL-17RLM-L嵌合受体所介导的Stat5-DNA结合复合体主要为Stat5b-DNA复合体。这一结果进一步表明了hIL-17RLM-L胞内区具有活化Stat5信号的潜力。
上述实验结果显示:hIL-17RLM-L具有活化Stat5的信号潜力。为了进一步鉴定是否细胞内稳定表达的EPORextm/hIL-17RLM-Licd嵌合受体在重组人红细胞生成素(rhEPO)的刺激下也具有介导Stat5活化的信号能力,建立了稳定表达细胞系,命名为稳定表达EPOR和EPORextm/hIL-17RLM-Licd的阳性细胞系Ba/F3,采用Northernblot和western blot方法鉴定具有稳定表达EPOR和EPORe xtm/hIL-17RLM-Licd嵌合受体的阳性细胞克隆。
对稳定表达EPOR和EPORextm/hIL-17RLM-Licd的阳性Ba/F3细胞系进行gelshift assay实验。结果表明:EPOR/hIL-17RLM-L与EPhIL-17RLM-LOR稳定表达细胞系一样,EPO能够以时间依赖方式诱导其细胞核抽提物Stat5-DNA结合复合体的形成;然而在野生型Ba/F3细胞系,却并未诱导其细胞核抽提物Stat5-DNA结合复合体的形成。进一步表明hIL-17RLM-L的胞内区具有活化Stat5的信号能力。
Supershift分析结果显示:采用抗Stat5ab或Stat5b,能够超迁移Stat5-DNA结合复合体,然而采用抗Stat5a或Stat1特异性抗体却并未超迁移Stat5-DNA结合复合体。这表明EPOR/hIL-17RLM-L嵌合受体主要介导Stat5b的活化,而不是Stat5a的活化。另外。使用冰冷的未标记Stat5 DNA特异性结合探针,能够特异地竞争性抑止Stat5-DNA结合复合体地形成,而使用无关的探针却不能竞争性抑止Stat5-DNA结合复合体地形成。这些结果充分证明了EPOR/hIL-17RLM-L嵌合受体主要介导Stat5b的活化,而不是Stat5a的活化。这一结果也与通过瞬时过量表达EPOR/hIL-17RLM-L在Cos7细胞所介导Stat5活化的结果相吻合。据此可以得出,在Cos7细胞瞬时过量表达和在Ba/F3细胞稳定表达EPORextm/hIL-17RLM-Licd嵌合受体都能够介导Stat5活化,表明hIL-17RLM-L胞内区具有活化转录因子Stat5的信号潜力。
通常,I型细胞因子受体应答其偶连配体具有传递细胞增殖的信号能力。借助[3H]-Thymidine整合,进行了细胞增殖分析实验。结果表明:EPORextm/hIL-17RLM-Licd嵌和受体稳定表达的Ba/F3细胞系和EPOR稳定表达的Ba/F3细胞系一样,能够应答EPO的刺激,出现细胞增殖现象,并且这种增殖效应为EPO浓度依赖的方式和刺激时间依赖的方式。这暗示hIL-17RLM-L的胞内结构域具有传递细胞增殖的信号潜力。
实施例3、hIL-17RLM-L与FGFRs的相互作用、共定位及对细胞分化的影响
免疫沉淀和Western blot结果显示:hIL-17RLM-L能够与爪蟾(Xenopus)mFGFR1或mFGFR2在瞬时共转染的Cos7细胞相互作用。但并未检测到hIL-17RLM-L与mFGFR3或mFGFR4的相互作用。
为了进一步确定hIL-17RLM与mFGFR1或mFGFR2的相互作用,进行了免疫染色实验,并进行Merge分析。Merge实验结果显示,hIL-17RLM-L与mFGFR2共定位于细胞膜和胞浆部位。
为了探讨是否hIL-17RLM-L与FGFR1或FGFR2在人体内源性组织中共定位表达。首先借助常规免疫组化的方法,检测hIL-17RLM-L、FGFR1和FGFR2在人体组织中的原位表达分布。结果表明:hIL-17RLM-L、FGFR1和FGFR2在人体肾和睾丸组织中具有非常相似的表达分布,表达在组织的相同细胞类型,并且主要表达在组织细胞膜及细胞浆部位。另外FGFR1在肾及睾丸组织中的表达明显强于FGFR2的表达。
为了进一步确定是否hIL-17RLM-L与FGFR1在人体某些组织共定位表达,使用人多组织阵列(tissue array),采用双重或三重免疫组化染色试剂盒,大量筛检了hIL-17RLM-L与FGFR1在人体多种组织中的表达分布,并进行merge分析。结果显示:在睾丸、肾组织,hIL-17RLM-L(绿光)和FGFR1(红光)具有极强的阳性免疫染色,并且具有非常相似的表达样式和细胞特异性。另外merge实验分析结果表明,hIL-17RLM-L和FGFR1明显共表达定位于睾丸组织的特异细胞类型。在肾组织和其它组织,尽管hIL-17RLM-L和FGFR1具有较强的阳性免疫染色和相似的表达分布,但是merge结果却并未显示hIL-17RL M-L和FGFR1的共定位表达分布。这表明hIL-17RLM-L和FGFR1的共定位表达分布具有组织特异性。也暗示hIL-17RLM-L与FGFR1在睾丸组织生精细胞中的特异性共定位表达很可能与精子的形成有关。
实施例4、hIL-17RLM-L抑制FGF介导的下游信号通路
有资料表明:FGF介导的下游Ras-Raf-MEK-MAPK信号通路受Sprouty家族成员的负调节。在早期斑马鱼胚胎发育过程中,由于FGF3,FGF8,spouty2,sprouty4与zSef/zIL-17RLM-L为一协同表达组。因而,我们假设hIL-17RLM-L有可能同sprouty家族成员一样,具有在高等生物抑制FGF介导的下游信号通路及相应的某些生物学功能。
实验中,首先构建了EGFP-tagged hIL-17RLM-L(WT),EGFP-hIL-17RLM-Lecdtm(ΔC)(含有hIL-17RLM-L的胞外结构域和跨膜结构域),EGFP-hIL-17RLM-Ltmcyd(ΔN)(含有hIL-17RLM-L的胞内结构域和跨膜结构域)和EGFP-hIL-17RLM-L(DN:Δ327-333)(缺失含有潜在酪氨酸磷酸化部位的一段基因序列)真核表达质粒。经DNA测序证实和western blot检测其在细胞的过表达正确。
在大鼠嗜铬神经细胞瘤细胞系-PC12细胞过表达上述真核表达质粒,转染36小时后,使用碱性成纤维细胞生长因子(FGF2)刺激细胞约72小时后,荧光显微镜拍照和计数细胞分化的百分率。结果显示:野生型hIL-17RLM-L能够显著抑制FGF2诱导PC12细胞的分化。然而,野生型全长hIL-17RLM对FGF2诱导PC12细胞分化的这种抑制作用能够被EGFP-hIL-17RLM-Lecdtm(ΔC)逆转,但并不能够被EGFP-hIL-17RLM-Ltmcyd(ΔN)和EGFP-hIL-17RLM-L(DN:Δ327-333)所逆转。这一结果暗示:hIL-17RLM-L的胞内结构域对与hIL-17RLM-L对FGF2诱导PC12细胞分化的抑制作用是必需的,然而hIL-17RLM-L的胞外结构域以及hIL-17RLM-L的胞内区潜在酪氨酸磷酸化部位却并不需要。这一结果也表明:hIL-17RLM-L的胞内结构域是对FGF2诱导PC12细胞分化抑制作用的功能结构域。
序列表
<160>2
<210>1
<211>4477
<212>DNA
<213>人属人种(Homo sapiens)
<400>1
gcggccgccg cggccaccgc ccactcgggg ctggccagcg gcgggcggcc ggggcgcaga 60
gaacggcctg gctgggcgag cgcacggcca tggccccgtg gctgcagctc tgctccgtct 120
tctttacggt caacgcctgc ctcaacggct cgcagctggc tgtggccgct ggcgggtccg 180
gccgcgcgcg gggcgccgac acctgtggct ggaggggagt ggggccagcc agcagaaaca 240
gtgggctgta caacatcacc ttcaaatatg acaattgtac cacctacttg aatccagtgg 300
ggaagcatgt gattgctgac gcccagaata tcaccatcag ccagtatgct tgccatgacc 360
aagtggcagt caccattctt tggtccccag gggccctcgg catcgaattc ctgaaaggat 420
ttcgggtaat actggaggag ctgaagtcgg agggaagaca gtgccaacaa ctgattctaa 480
aggatccgaa gcagctcaac agtagcttca aaagaactgg aatggaatct caacctttcc 540
tgaatatgaa atttgaaacg gattatttcg taaaggttgt cccttttcct tccattaaaa 600
acgaaagcaa ttaccaccct ttcttcttta gaacccgagc ctgtgacctg ttgttacagc 660
cggacaatct agcttgtaaa cccttctgga agcctcggaa cctgaacatc agccagcatg 720
gctcggacat gcaggtgtcc ttcgaccacg caccgcacaa cttcggcttc cgtttcttct 780
atcttcacta caagctcaag cacgaaggac ctttcaagcg aaagacctgt gagcaggagc 840
aaactacaga gatgaccagc tgcctccttc aaaatgtttc tccaggggat tatataattg 900
agctggtgga tgacactaac acaacaagaa aagtgatgca ttatgcctta aagccagtgc 960
actccccgtg ggccgggccc atcagagccg tggccatcac agtgccactg gtagtcatat 1020
cggcattcgc gacgctcttc actgtgatgt gccgcaagaa gcaacaagaa aatatatatt 1080
cacatttaga tgaagagagc tctgagtctt ccacatacac tgcagcactc ccaagagaga 1140
ggctccggcc gcggccgaag gtctttctct gctattccag taaagatggc cagaatcaca 1200
tgaatgtcgt ccagtgtttc gcctacttcc tccaggactt ctgtggctgt gaggtggctc 1260
tggacctgtg ggaagacttc agcctctgta gagaagggca gagagaatgg gtcatccaga 1320
agatccacga gtcccagttc atcattgtgg tttgttccaa aggtatgaag tactttgtgg 1380
acaagaagaa ctacaaacac aaaggaggtg gccgaggctc ggggaaagga gagctcttcc 1440
tggtggcggt gtcagccatt gccgaaaagc tccgccaggc caagcagagt tcgtccgcgg 1500
cgctcagcaa gtttatcgcc gtctactttg attattcctg cgagggagac gtccccggta 1560
tcctagacct gagtaccaag tacagactca tggacaatct tcctcagctc tgttcccacc 1620
tgcactcccg agaccacggc ctccaggagc cggggcagca cacgcgacag ggcagcagaa 1680
ggaactactt ccggagcaag tcaggccggt ccctatacgt cgccatttgc aacatgcacc 1740
agtttattga cgaggagccc gactggttcg aaaagcagtt cgttcccttc catcctcctc 1800
cactgcgcta ccgggagcca gtcttggaga aatttgattc gggcttggtt ttaaatgatg 1860
tcatgtgcaa accagggcct gagagtgact tctgcctaaa ggtagaggcg gctgttcttg 1920
gggcaaccgg accagccgac tcccagcacg agagtcagca tgggggcctg gaccaagacg 1980
gggaggcccg gcctgccctt gacggtagcg ccgccctgca acccctgctg cacacggtga 2040
aagccggcag cccctcggac atgccgcggg actcaggcat ctatgactcg tctgtgccct 2100
catccgagct gtctctgcca ctgatggaag gactctcgac ggaccagaca gaaacgtctt 2160
ccctgacgga gagcgtgtcc tcctcttcag gcctgggtga ggaggaacct cctgcccttc 2220
cttccaagct cctctcttct gggtcatgca aagcagatct tggttgccgc agctacactg 2280
atgaactcca cgcggtcgcc cctttgtaac aaaacgaaag agtctaagca ttgccacttt 2340
agctgctgcc tccctctgat tccccagctc atctccctgg ttgcatggcc cacttggagc 2400
tgaggtctca tacaaggata tttggagtga aatgctggcc agtacttgtt ctcccttgcc 2460
ccaacccttt accggatatc ttgacaaact ctccaatttt ctaaaatgat atggagctct 2520
gaaaggcatg tccataaggt ctgacaacag cttgccaaat ttggttagtc cttggatcag 2580
agcctgttgt gggaggtagg gaggaaatat gtaaagaaaa acaggaagat acctgcacta 2640
atcattcaga cttcattgag ctctgcaaac tttgcctgtt tgctattggc taccttgatt 2700
tgaaatgctt tgtgaaaaaa ggcactttta acatcatagc cacagaaatc aagtgccagt 2760
ctatctggaa tccatgttgt attgcagata atgttctcat ttatttttga tgtagaattt 2820
acattgccat gggtgttaaa taagctttga gtcaaaagtc aagaaagtga ctgaatatac 2880
agtcaccttt tatgaaatga gtctctgtgt tactgggtgg catgactgat tgaggtgaag 2940
ctcacggggc caggctgacc gtcttgaccg ttccacttga gataggttgg tcatcgtgca 3000
gaaggcccca ggacctcagc acacacagcc tcctcttggt ctgagtaggc atcatgtggg 3060
ggccagatct gcctgctgtt tccatgggtt acatttactg tgctgtatct cagatgttgg 3120
tgtctggaag tttattctta agagactgct acccagctgg tctgtattat tggaagttgc 3180
agttcgtgct ttggttggcc ttctggtcta aagctgtgtc ctgaatatta gggatcacaa 3240
ttcactgaaa tacagcagtg tgtggaggtg atggccagtt aatctgctga actggttttg 3300
actaatgaca aacctctttt taagatggta gaatggaggt gatagtcaca aaagtaaatg 3360
ttccattttt atgaatgact ttctacagag tttctatttc taaagaaaaa acaattgttc 3420
acatcccatc tgatgattag catgtgtgta atgaatgctg tcttggtctc ccctgtggaa 3480
acccttctcc ctgtgcctta gagcaggtgt gtacatctct cactaccttt ctcatgggtg 3540
ctgttagatt ttggcacccg ttttctcagc attcagccca gggaatgtgg ttttcacttc 3600
ttcgtcagat aagaccaaca tgaaggggta tgttgagaaa catcctgagg caaggtggga 3660
ggtgggatgg ggcaggactt tcccttccaa gcacatgcat ggcaggtggg gaaagggggg 3720
cttgcacccc tgctggaaag aaaaggtttg tgtatatttc tgatgcaaat gtcatactca 3780
ctgctctgta aaggcagctg gcagcttttt gggaaaagaa cgtgctcgtc tgttctctgg 3840
catcaagttt cttgcagctg ctctgaggga gagacagtga gctgcaagac tgcctcccca 3900
taacaacagg caactcagag aagagtcatt ttatgttgtt cctatggaat ctggaatgag 3960
tgcagagctc ctacccacac atgactgccc cgccatttca tcctaggcat tctgtgaagg 4020
agattggtta gtccaaactt gctaacatac gaaaattcac ttggaacatg atgagagatt 4080
tcttattgag gccaagagat gtttcctgtc ccagaggaac cattaggagt cgcttttagg 4140
gtattcagct ttgttcatga aataaggcat ctctgagaaa gtggccccag ggagagaatg 4200
gaggactggg aggagaagca ttaactgagc tccaagggtg tgtgggcaga gagcttgcta 4260
tgtgaactca ctccttaaga aaatggaaga gaaaaagaga gtgctagtta aaaaatcggg 4320
atgttttagt ttggatttag ggttttgata cttatgttga aatactaatg tttctgatca 4380
ataaaatcaa actcttaata taccgagtaa tgaaaccata gtgtgattgc ctcagaataa 4440
attgagaagt ccaaaaaaaa aaaaaaaaaa aaaaaaa 4477
<210>2
<211>739
<212>PRT
<213>人属人种(Homo sapiens)
<400>序列号
Met Ala Pro Trp Leu Gln Leu Cys Ser Val Phe Phe Thr Val Asn
1 5 10 15
Ala Cys Leu Asn Gly Ser Gln Leu Ala Val Ala Ala Gly Gly Ser
20 25 30
Gly Arg Ala Arg Gly Ala Asp Thr Cys Gly Trp Arg Gly Val Gly
35 40 45
Pro Ala Ser Arg Asn Ser Gly Leu Tyr Asn Ile Thr Phe Lys Tyr
50 55 60
Asp Asn Cys Thr Thr Tyr Leu Asn Pro Val Gly Lys His Val Ile
65 70 75
Ala Asp Ala Gln Asn Ile Thr Ile Ser Gln Tyr Ala Cys His Asp
80 85 90
Gln Val Ala Val Thr Ile Leu Trp Ser Pro Gly Ala Leu Gly Ile
95 100 105
Glu Phe Leu Lys Gly Phe Arg Val Ile Leu Glu Glu Leu Lys Ser
110 115 120
Glu Gly Arg Gln Cys Gln Gln Leu Ile Leu Lys Asp Pro Lys Gln
125 130 135
Leu Asn Ser Ser Phe Lys Arg Thr Gly Met Glu Ser Gln Pro Phe
140 145 150
Leu Asn Met Lys Phe Glu Thr Asp Tyr Phe Val Lys Val Val Pro
155 160 165
Phe Pro Ser Ile Lys Asn Glu Ser Asn Tyr His Pro Phe Phe Phe
170 175 180
Arg Thr Arg Ala Cys Asp Leu Leu Leu Gln Pro Asp Asn Leu Ala
185 190 195
Cys Lys Pro Phe Trp Lys Pro Arg Asn Leu Asn Ile Ser Gln His
200 205 210
Gly Ser Asp Met Gln Val Ser Phe Asp His Ala Pro His Asn Phe
215 220 225
Gly Phe Arg Phe Phe Tyr Leu His Tyr Lys Leu Lys His Glu Gly
230 235 240
Pro Phe Lys Arg Lys Thr Cys Glu Gln Glu Gln Thr Thr Glu Met
245 250 255
Thr Ser Cys Leu Leu Gln Asn Val Ser Pro Gly Asp Tyr Ile Ile
260 265 270
Glu Leu Val Asp Asp Thr Asn Thr Thr Arg Lys Val Met His Tyr
275 280 285
Ala Leu Lys Pro Val His Ser Pro Trp Ala Gly Pro Ile Arg Ala
290 295 300
Val Ala Ile Thr Val Pro Leu Val Val Ile Ser Ala Phe Ala Thr
305 310 315
Leu Phe Thr Val Met Cys Arg Lys Lys Gln Gln Glu Asn Ile Tyr
320 325 330
Ser His Leu Asp Glu Glu Ser Ser Glu Ser Ser Thr Tyr Thr Ala
335 340 345
Ala Leu Pro Arg Glu Arg Leu Arg Pro Arg Pro Lys Val Phe Leu
350 355 360
Cys Tyr Ser Ser Lys Asp Gly Gln Asn His Met Asn Val Val Gln
365 370 375
Cys Phe Ala Tyr Phe Leu Gln Asp Phe Cys Gly Cys Glu Val Ala
380 385 390
Leu Asp Leu Trp Glu Asp Phe Ser Leu Cys Arg Glu Gly Gln Arg
395 400 405
Glu Trp Val Ile Gln Lys Ile His Glu Ser Gln Phe Ile Ile Val
410 415 420
Val Cys Ser Lys Gly Met Lys Tyr Phe Val Asp Lys Lys Asn Tyr
425 430 435
Lys His Lys Gly Gly Gly Arg Gly Ser Gly Lys Gly Glu Leu Phe
440 445 450
Leu Val Ala Val Ser Ala Ile Ala Glu Lys Leu Arg Gln Ala Lys
455 460 465
Gln Ser Ser Ser Ala Ala Leu Ser Lys Phe Ile Ala Val Tyr Phe
470 475 480
Asp Tyr Ser Cys Glu Gly Asp Val Pro Gly Ile Leu Asp Leu Ser
485 490 495
Thr Lys Tyr Arg Leu Met Asp Asn Leu Pro Gln Leu Cys Ser His
500 505 510
Leu His Ser Arg Asp His Gly Leu Gln Glu Pro Gly Gln His Thr
515 520 525
Arg Gln Gly Ser Arg Arg Asn Tyr Phe Arg Ser Lys Ser Gly Arg
530 535 540
Ser Leu Tyr Val Ala Ile Cys Asn Met His Gln Phe Ile Asp Glu
545 550 555
Glu Pro Asp Trp Phe Glu Lys Gln Phe Val Pro Phe His Pro Pro
560 565 570
Pro Leu Arg Tyr Arg Glu Pro Val Leu Glu Lys Phe Asp Ser Gly
575 580 585
Leu Val Leu Asn Asp Val Met Cys Lys Pro Gly Pro Glu Ser Asp
590 595 600
Phe Cys Leu Lys Val Glu Ala Ala Val Leu Gly Ala Thr Gly Pro
605 610 615
Ala Asp Ser Gln His Glu Ser Gln His Gly Gly Leu Asp Gln Asp
620 625 630
Gly Glu Ala Arg Pro Ala Leu Asp Gly Ser Ala Ala Leu Gln Pro
635 640 645
Leu Leu His Thr Val Lys Ala Gly Ser Pro Ser Asp Met Pro Arg
650 655 660
Asp Ser Gly Ile Tyr Asp Ser Ser Val Pro Ser Ser Glu Leu Ser
665 670 675
Leu Pro Leu Met Glu Gly Leu Ser Thr Asp Gln Thr Glu Thr Ser
680 685 690
Ser Leu Thr Glu Ser Val Ser Ser Ser Ser Gly Leu Gly Glu Glu
695 700 705
Glu Pro Pro Ala Leu Pro Ser Lys Leu Leu Ser Ser Gly Ser Cys
710 715 720
Lys Ala Asp Leu Gly Cys Arg Ser Tyr Thr Asp Glu Leu His Ala
725 730 735
Val Ala Pro Leu
739
Claims (7)
1、一种人白细胞介素-17受体样蛋白,它具有序列表中序列2的氨基酸残基序列或将序列2的氨基酸残基序列经过一个氨基酸残基的取代、缺失或添加且具有与序列2的氨基酸残基序列相同活性的由序列2衍生的蛋白质。
2、根据权利要求1所述的蛋白质,其特征在于:所述蛋白质具有序列表中序列2的氨基酸残基序列。
3、具有序列表中序列1 DNA序列的人白细胞介素-17受体样蛋白的编码基因。
4、权利要求1所述的人白细胞介素-17受体样蛋白在体外使Stat5因子活化中的应用。
5、权利要求1所述的人白细胞介素-17受体样蛋白在体外介导细胞增殖中的应用。
6、权利要求1所述的人白细胞介素-17受体样蛋白在制造促进或抑制精子形成的药物中的作用。
7、权利要求1所述的人白细胞介素-17受体样蛋白在体外抑制高等生物FGF介导的下游信号通路中的应用。
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
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CN021234477A CN1216912C (zh) | 2002-06-28 | 2002-06-28 | 人白细胞介素-17受体样蛋白及其编码基因与应用 |
US10/608,449 US7141390B2 (en) | 2002-06-28 | 2003-06-30 | Polynucleotide encoding human interleukin-17 receptor like molecule |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN021234477A CN1216912C (zh) | 2002-06-28 | 2002-06-28 | 人白细胞介素-17受体样蛋白及其编码基因与应用 |
Publications (2)
Publication Number | Publication Date |
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CN1463982A CN1463982A (zh) | 2003-12-31 |
CN1216912C true CN1216912C (zh) | 2005-08-31 |
Family
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CN021234477A Expired - Fee Related CN1216912C (zh) | 2002-06-28 | 2002-06-28 | 人白细胞介素-17受体样蛋白及其编码基因与应用 |
Country Status (2)
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US (1) | US7141390B2 (zh) |
CN (1) | CN1216912C (zh) |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
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US7629325B2 (en) * | 2004-10-11 | 2009-12-08 | Technion Research & Development Foundation Ltd. | Human Sef isoforms and methods of using same for cancer diagnosis and gene therapy |
US20060079444A1 (en) * | 2004-10-11 | 2006-04-13 | Dina Ron | Human Sef isoforms and methods of using same for cancer gene therapy |
CN100391974C (zh) * | 2006-01-09 | 2008-06-04 | 浙江理工大学 | 一种重组胶原蛋白及其合成和表达纯化方法 |
CN106660048B (zh) | 2014-06-19 | 2019-07-05 | 阿科尼生物系统公司 | 分子分析系统及其应用 |
-
2002
- 2002-06-28 CN CN021234477A patent/CN1216912C/zh not_active Expired - Fee Related
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2003
- 2003-06-30 US US10/608,449 patent/US7141390B2/en not_active Expired - Fee Related
Also Published As
Publication number | Publication date |
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US20040265834A1 (en) | 2004-12-30 |
US7141390B2 (en) | 2006-11-28 |
CN1463982A (zh) | 2003-12-31 |
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