CN1205226C - 一种小鼠白细胞介素-17受体样蛋白及其编码基因与应用 - Google Patents
一种小鼠白细胞介素-17受体样蛋白及其编码基因与应用 Download PDFInfo
- Publication number
- CN1205226C CN1205226C CN 02123541 CN02123541A CN1205226C CN 1205226 C CN1205226 C CN 1205226C CN 02123541 CN02123541 CN 02123541 CN 02123541 A CN02123541 A CN 02123541A CN 1205226 C CN1205226 C CN 1205226C
- Authority
- CN
- China
- Prior art keywords
- sequence
- ser
- 17rlm
- mil
- leu
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 29
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 16
- 101100071930 Mus musculus Il17ra gene Proteins 0.000 title abstract 6
- 125000000539 amino acid group Chemical group 0.000 claims abstract description 14
- 108091028043 Nucleic acid sequence Proteins 0.000 claims abstract description 6
- 230000004913 activation Effects 0.000 claims description 15
- 230000001404 mediated effect Effects 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 3
- 102000001712 STAT5 Transcription Factor Human genes 0.000 claims description 2
- 108010029477 STAT5 Transcription Factor Proteins 0.000 claims description 2
- 230000008034 disappearance Effects 0.000 claims description 2
- 239000012190 activator Substances 0.000 claims 1
- 239000003795 chemical substances by application Substances 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 5
- 230000004663 cell proliferation Effects 0.000 abstract description 4
- 102000013691 Interleukin-17 Human genes 0.000 description 26
- 108050003558 Interleukin-17 Proteins 0.000 description 26
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 23
- 210000004027 cell Anatomy 0.000 description 21
- 230000003834 intracellular effect Effects 0.000 description 16
- 230000014509 gene expression Effects 0.000 description 13
- 102000005962 receptors Human genes 0.000 description 13
- 108020003175 receptors Proteins 0.000 description 13
- 102000004127 Cytokines Human genes 0.000 description 9
- 108090000695 Cytokines Proteins 0.000 description 9
- 241000699666 Mus <mouse, genus> Species 0.000 description 9
- 108020004414 DNA Proteins 0.000 description 8
- 239000000370 acceptor Substances 0.000 description 8
- 230000000638 stimulation Effects 0.000 description 7
- 101150063267 STAT5B gene Proteins 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- 102000053460 interleukin-17 receptor activity proteins Human genes 0.000 description 5
- 108040001304 interleukin-17 receptor activity proteins Proteins 0.000 description 5
- 230000026731 phosphorylation Effects 0.000 description 5
- 238000006366 phosphorylation reaction Methods 0.000 description 5
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 5
- NHBKXEKEPDILRR-UHFFFAOYSA-N 2,3-bis(butanoylsulfanyl)propyl butanoate Chemical compound CCCC(=O)OCC(SC(=O)CCC)CSC(=O)CCC NHBKXEKEPDILRR-UHFFFAOYSA-N 0.000 description 4
- 102000039989 IL-17 family Human genes 0.000 description 4
- 108091069193 IL-17 family Proteins 0.000 description 4
- 102220369447 c.1352G>A Human genes 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 230000006870 function Effects 0.000 description 4
- 230000001939 inductive effect Effects 0.000 description 4
- 108020004999 messenger RNA Proteins 0.000 description 4
- 238000013508 migration Methods 0.000 description 4
- 230000005012 migration Effects 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 4
- 108090001005 Interleukin-6 Proteins 0.000 description 3
- 102000004889 Interleukin-6 Human genes 0.000 description 3
- 108090001007 Interleukin-8 Proteins 0.000 description 3
- 102000004890 Interleukin-8 Human genes 0.000 description 3
- 108060001084 Luciferase Proteins 0.000 description 3
- 239000005089 Luciferase Substances 0.000 description 3
- 108010057466 NF-kappa B Proteins 0.000 description 3
- 102000003945 NF-kappa B Human genes 0.000 description 3
- 108091000080 Phosphotransferase Proteins 0.000 description 3
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 3
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 102000014909 interleukin-1 receptor activity proteins Human genes 0.000 description 3
- 108040006732 interleukin-1 receptor activity proteins Proteins 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 102000020233 phosphotransferase Human genes 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 102220023256 rs387907547 Human genes 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- 102000015271 Intercellular Adhesion Molecule-1 Human genes 0.000 description 2
- 108010064593 Intercellular Adhesion Molecule-1 Proteins 0.000 description 2
- 102000003777 Interleukin-1 beta Human genes 0.000 description 2
- 108090000193 Interleukin-1 beta Proteins 0.000 description 2
- 108010017525 Interleukin-17 Receptors Proteins 0.000 description 2
- 101710186071 Interleukin-17 receptor B Proteins 0.000 description 2
- 102100035014 Interleukin-17 receptor B Human genes 0.000 description 2
- 102100033096 Interleukin-17D Human genes 0.000 description 2
- 108010066979 Interleukin-27 Proteins 0.000 description 2
- 241000699660 Mus musculus Species 0.000 description 2
- 108700026244 Open Reading Frames Proteins 0.000 description 2
- 101150099493 STAT3 gene Proteins 0.000 description 2
- 101150058731 STAT5A gene Proteins 0.000 description 2
- 210000001744 T-lymphocyte Anatomy 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- 230000008827 biological function Effects 0.000 description 2
- 102220369446 c.1274G>A Human genes 0.000 description 2
- 230000007783 downstream signaling Effects 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 239000013613 expression plasmid Substances 0.000 description 2
- 230000002607 hemopoietic effect Effects 0.000 description 2
- 238000001114 immunoprecipitation Methods 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 230000028709 inflammatory response Effects 0.000 description 2
- 210000003734 kidney Anatomy 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 230000000770 proinflammatory effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000004936 stimulating effect Effects 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 1
- 101100366888 Dictyostelium discoideum dstA gene Proteins 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 102000005698 Frizzled receptors Human genes 0.000 description 1
- 108010045438 Frizzled receptors Proteins 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102000004269 Granulocyte Colony-Stimulating Factor Human genes 0.000 description 1
- 108010017080 Granulocyte Colony-Stimulating Factor Proteins 0.000 description 1
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 description 1
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 description 1
- 101001050288 Homo sapiens Transcription factor Jun Proteins 0.000 description 1
- 108010042653 IgA receptor Proteins 0.000 description 1
- 102000003816 Interleukin-13 Human genes 0.000 description 1
- 108090000176 Interleukin-13 Proteins 0.000 description 1
- 102000004554 Interleukin-17 Receptors Human genes 0.000 description 1
- 102100035018 Interleukin-17 receptor A Human genes 0.000 description 1
- 108090000978 Interleukin-4 Proteins 0.000 description 1
- 102000004388 Interleukin-4 Human genes 0.000 description 1
- 108010002616 Interleukin-5 Proteins 0.000 description 1
- 102000000743 Interleukin-5 Human genes 0.000 description 1
- 101150009057 JAK2 gene Proteins 0.000 description 1
- 206010023126 Jaundice Diseases 0.000 description 1
- 108700027649 Mitogen-Activated Protein Kinase 3 Proteins 0.000 description 1
- 102100024193 Mitogen-activated protein kinase 1 Human genes 0.000 description 1
- 108700015928 Mitogen-activated protein kinase 13 Proteins 0.000 description 1
- 102100024192 Mitogen-activated protein kinase 3 Human genes 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 101001019603 Mus musculus Interleukin-17 receptor B Proteins 0.000 description 1
- 102000010168 Myeloid Differentiation Factor 88 Human genes 0.000 description 1
- 108010077432 Myeloid Differentiation Factor 88 Proteins 0.000 description 1
- 238000000636 Northern blotting Methods 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 102100034014 Prolyl 3-hydroxylase 3 Human genes 0.000 description 1
- 206010038389 Renal cancer Diseases 0.000 description 1
- 208000006265 Renal cell carcinoma Diseases 0.000 description 1
- 108010043943 Starch Phosphorylase Proteins 0.000 description 1
- 102100023132 Transcription factor Jun Human genes 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 210000000577 adipose tissue Anatomy 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 102220369445 c.668T>C Human genes 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- LEVWYRKDKASIDU-IMJSIDKUSA-N cystine group Chemical group C([C@@H](C(=O)O)N)SSC[C@@H](C(=O)O)N LEVWYRKDKASIDU-IMJSIDKUSA-N 0.000 description 1
- 230000003229 cytophilic effect Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000006471 dimerization reaction Methods 0.000 description 1
- XEYBRNLFEZDVAW-ARSRFYASSA-N dinoprostone Chemical compound CCCCC[C@H](O)\C=C\[C@H]1[C@H](O)CC(=O)[C@@H]1C\C=C/CCCC(O)=O XEYBRNLFEZDVAW-ARSRFYASSA-N 0.000 description 1
- 229960002986 dinoprostone Drugs 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000003328 fibroblastic effect Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000011132 hemopoiesis Effects 0.000 description 1
- 239000000710 homodimer Substances 0.000 description 1
- 231100000652 hormesis Toxicity 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 238000012744 immunostaining Methods 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 230000000394 mitotic effect Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 210000002220 organoid Anatomy 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 108010068338 p38 Mitogen-Activated Protein Kinases Proteins 0.000 description 1
- XEYBRNLFEZDVAW-UHFFFAOYSA-N prostaglandin E2 Natural products CCCCCC(O)C=CC1C(O)CC(=O)C1CC=CCCCC(O)=O XEYBRNLFEZDVAW-UHFFFAOYSA-N 0.000 description 1
- 210000002307 prostate Anatomy 0.000 description 1
- 238000000197 pyrolysis Methods 0.000 description 1
- 201000010174 renal carcinoma Diseases 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 102220004457 rs11567847 Human genes 0.000 description 1
- 102220023258 rs387907548 Human genes 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 210000002027 skeletal muscle Anatomy 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 210000002437 synoviocyte Anatomy 0.000 description 1
- 210000003684 theca cell Anatomy 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 238000003146 transient transfection Methods 0.000 description 1
- 230000017105 transposition Effects 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
Landscapes
- Peptides Or Proteins (AREA)
Abstract
本发明公开了一种小鼠白细胞介素-17受体样蛋白及其编码基因与应用,小鼠白细胞介素-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1-10个氨基酸残基的取代、缺失或添加且具有激活STAT5和介导细胞增殖功能的由序列2衍生的蛋白质。本发明的小鼠白细胞介素-17受体样蛋白可以使Stat5因子活化并能够介导细胞增殖。
Description
技术领域
本发明涉及一种白细胞介素-17受体样蛋白及其编码基因与应用,特别是涉及一种小鼠白细胞介素-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。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-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。
IL-17E/IL-25是一种结构与功能相似于IL-17A的促炎症细胞因子,主要表达于活化的TH2 T细胞。IL-17E能够诱导某些细胞因子的表达,包括IL-8,IL-4,IL-5,IL-13,ICAM-1和IL-6。同时,IL-17E还能通过其低亲和力受体IL-17ER/IL-17BR/IL-17Rh1介导转录因子NF-κB的活化。IL-17E在小鼠的增强表达能诱导小鼠生长延缓、黄疸、多器官炎症等疾病症状和一种TH2型的炎症应答。IL-17B和IL-17C是IL-17家族的另外两个细胞因子,与IL-17有27%的氨基酸一致性。IL-17B信使RNA表达在前列腺、小肠、胃。IL-17B和IL-17C能够诱导人急性单核白血病细胞系THP-1释放IL-1β和TNF-α。并能与THP-1细胞有一定的结合力。最近,Hzamas等分离和克隆了一种新的IL-17相似的细胞因子,命名为IL-17D/IL-27,该细胞因子优先表达于骨骼肌、脂肪组织和中枢神经组织,诱导内皮细胞分泌IL-6、IL-8和GM-CSF等细胞因子,并能抑制造血功能。
曾报道,IL-17A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小鼠白细胞介素-17受体样蛋白及其编码基因。
一种小鼠白细胞介素-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1-10个氨基酸残基的取代、缺失或添加且具有激活STAT5和介导细胞增殖功能的由序列2衍生的蛋白质。
本发明的小鼠白细胞介素-17受体样蛋白命名为mIL-17RLM-S,是一种单跨膜细胞因子受体,由594个氨基酸残基组成。按照NCBI Conserved Domain Database查询结果,预测到mIL-17RLM-S胞内区(V214-M282)与Toll/IL-1R结构域-TIR domain相似的氨基酸残基序列:VFLCYSNKDGQNHMNVVQCFAYFLQDFCGCEVALDLWEDFSLCREGQREWAIQKIHESQFIIVVCSKGM。TIR结构域是IL-1R,TollR和MyD88胞内重要的信号结构域,这暗示mIL-17RLM-S是一个重要的新的信号受体。
采用多组织Northern Blot方法,检测了mIL-17RLM-S的mRNA组织表达分布,结果表明:mIL-17RLM-S mRNA主要在小鼠肾、肺表达,在心、脑表达微弱。
本发明序列表中序列1的DNA序列编码序列2的蛋白质,由2134个碱基组成,位于小鼠14号染色体上,共有14个外显子组成。
研究表明,本发明的小鼠白细胞介素-17受体样蛋白具有使Stat5因子活化、介导细胞增殖等作用。
下面结合具体实施例对本发明做进一步说明。
具体实施方式
实施例1、mIL-17RLM-S基因的克隆及表达
通过蛋白BLAST NCBI小鼠EST数据库,拼接出一个完整开放阅读框序列(1782bpscomplete cDNA),其编码594个氨基酸的潜在受体。根据推测的小鼠mIL-17RLM-S基因序列,设计引物,将其分子克隆。以小鼠肾总RNA为模板,通过RT-PCR扩增出与预期大小一致的DNA条带-1800bps。将其亚克隆于pMD18 T-A克隆载体,酶切鉴定阳性克隆。测序结果表明:所得到的序列由2134个碱基组成,其完整开放阅读框编码594个氨基酸组成的受体。
对mIL-17RLM-S在细胞器的表达定位进行研究。将mIL-17RLM-S构建于EGFP-N1真核表达载体,将其瞬时转染于Cos7细胞,结果显示其主要表达在细胞膜上。为了进一步检测mIL-17RLM-S在细胞的内源性表达定位,进行了免疫荧光染色实验。借助mIL-17RLM-S兔多克隆抗血清,选择两种肾癌细胞系786-0和GRC-1,进行免疫染色研究,以检测mIL-17RLM-S在细胞的内源性表达定位。结果清楚表明mIL-17RLM-S主要定位在细胞膜。
实施例2、mIL-17RLM-S胞内结构域的功能——活化Stat5和介导细胞增殖
本发明的发明人构建了一系列嵌合受体,通过对mIL-17RLM-S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mIL-17RLM-S的胞内区构建一嵌合受体,借助EPO的刺激,进而检测mIL-17RLM-S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/mIL-17RLM-S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/mIL-17RLM-S嵌合受体介导Stat5的活化被明显抑止;全长mIL-17RLM-S的过表达并不能介导Stat5的活化。这个结果暗示,mIL-17RLM-S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mIL-17RLM-S的胞内区介导Stat5活化的潜力,采用免疫沉淀方法,检测EPOR/mIL-17RLM-S是否能够介导Stat5酪氨酸磷酸化。向Cos7细胞共转染嵌合受体-EPORextm/mIL-17RLM-Sicd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/mIL-17RLM-Sicd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mIL-17RLM-S的胞内区能够介导Stat5酪氨酸磷酸化。
为了进一步证实mIL-17RLM-S胞内区具有介导Stat5活化的潜力,采用gel shiftassay以分析是否EPOR/mIL-17RLM-S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/mIL-17RLM-Sicd嵌合受体同EPOR一样,在EPO的刺激下,能够介导Stat5一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:mIL-17RLM-S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/mIL-17RLM-S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mIL-17RLM-S胞内区具有活化Stat5信号的潜力。
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/mIL-17RLM-S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结合复合体的形成。这些结果充分证明了EPOR/mIL-17RLM-S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/mIL-17RLM-S在Cos7细胞所介导Stat5活化的结果相吻合。据此可以得出,mIL-17RLM-S胞内区具有活化转录因子Stat5的信号潜力。
实施例3、mIL-17RLM-S胞内结构域介导细胞增殖
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-Sicd嵌和受体稳定表达的细胞系和EPOR稳定表达的细胞系一样,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mIL-17RLM-S的胞内结构域具有传递细胞增殖的信号潜力。
序列表
<160>2
<210>1
<211>2134
<212>DNA
<213>小鼠属小鼠种BALB/c品系(Mus musculus strain BALB/c)
<400>1
agctcccagg ctcagtactg atgtgccgtg gctttcctat ccaagaagag acagcactct 60
ccttctgtag catctgggat ttgaaaccta agctaagtcc atcaagtgga cctggctttc 120
tgctttcctg ggctttgcct ggccctggaa ttatgtctca gtgacagtgt ggagtgcggg 180
tcttttgtag gtggtggggt ggacagttct ctacaccatg tgtgtctttg ttccctttta 240
gggaatggaa tctcagcctt tcctgaatat gaaatttgag acggattact ttgtaaagat 300
tgtccctttc ccttccatta aaaatgaaag caattaccat cccttcttct tcagaacacg 360
ggcctgtgac ctgttgttac aacctgacaa cttggcctgt aagcctttct ggaagcctcg 420
aaacctgaat atcagccagc atggttctga catgcacgtg tccttcgacc atgccccgca 480
gaacttcggc ttccgtggct tccatgttct ctataagctc aagcacgaag gccccttcag 540
gcggaggact tgcaggcagg accagaatac agagacaacc agctgcctcc tccaaaacgt 600
ttctccaggg gactatatca ttgagctggt ggatgacagc aacaccacca ggaaagctgc 660
tcagtatgtg gtgaagtcag tgcagtctcc ctgggctgga cccatcagag ctgtggccat 720
cactgtgcct ctggttgtca tatctgcgtt cgcaaccctg ttcactgtga tgtgcagaaa 780
gaagcaacaa gaaaatatat attcacattt agatgaagaa agcccggagt cgtccacata 840
cgctgcggct ctccccagag acaggctccg gcctcagccc aaggtcttcc tctgctactc 900
caataaagat ggccagaatc acatgaacgt ggtccagtgt ttcgcctatt tcctgcaaga 960
tttctgtggc tgtgaggtgg ctctggactt gtgggaagat ttcagcctct gcagagaggg 1020
gcagagagaa tgggccattc agaagatcca cgagtcccag ttcatcattg tcgtgtgctc 1080
caaaggcatg aagtactttg tagataagaa gaacttcaga cacaaaggag gcagccgcgg 1140
cgaggcgcaa ggcgagttct tcctggtggc cgtggcagcc attgctgaga agctccgtca 1200
ggccaagcag agctcatctg ccgcactgcg caagttcatc gccgtctact tcgattattc 1260
ctgtgaaggg gatgtaccct gcagcctgga cctgagcacc aagtacaagc tcatggacca 1320
ccttcctgag ctctgtgccc atctgcactc aggagagcag gaggtgctgg gtcagcaccc 1380
aggccacagc agcagaagga actacttccg gagcaaatcg ggccgctccc tgtatgttgc 1440
catttgcaac atgcaccagt ttattgatga ggagcctgac tggtttgaga agcagtttat 1500
acccttccaa catccccctg tgcgctacca ggagccagtc ctggagaaat ttgactcagg 1560
cttggtttta aatgatgtca taagcaaacc agggccagag agtgacttct gtcggaaagt 1620
cgaggcttgt gtacttgggg ccgctgggcc agccgactct tattcatacc tggagagtca 1680
gcatgtaggc ctggaccaag acactgaggc ccagccctcc tgtgatagtg cccctgcctt 1740
gcagcccctg ttacacgcag tgaaagctgg cagtccctca gagatgccac gggactcagg 1800
catatatgat tcttctgtac cctcatcaga gctctctctg cctctgatgg agggactctc 1860
cccggatcag atagaaacat cttctctgac cgagagtgta tcttcctcct ctggcctagg 1920
tgaggaggac ccccctaccc tcccttccaa gctctttgcc tctggggtgt ccagagaaca 1980
tggttgccac agccacactg acgaactgca agcgcttgct cctttgtaag gactcggaag 2040
agtctaagca tcgccacttt agctgctgat ctctctggct ccccagttca cctctgtggt 2100
tgtgcagcct acttggagct gaaggcgcac acgg 2134
<210>2
<211>594
<212>PRT
<213>小鼠属小鼠种BALB/c品系(Mus musculus strain BALB/c)
<400>2
Met Glu Ser Gln Pro Phe Leu Asn Met Lys Phe Glu Thr Asp Tyr
1 5 10 15
Phe Val Lys Ile Val Pro Phe Pro Ser Ile Lys Asn Glu Ser Asn
20 25 30
Tyr His Pro Phe Phe Phe Arg Thr Arg Ala Cys Asp Leu Leu Leu
35 40 45
Gln Pro Asp Asn Leu Ala Cys Lys Pro Phe Trp Lys Pro Arg Asn
50 55 60
Leu Asn Ile Ser Gln His Gly Ser Asp Met His Val Ser Phe Asp
65 70 75
His Ala Pro Gln Asn Phe Gly Phe Arg Gly Phe His Val Leu Tyr
80 85 90
Lys Leu Lys His Glu Gly Pro Phe Arg Arg Arg Thr Cys Arg Gln
95 100 105
Asp Gln Asn Thr Glu Thr Thr Ser Cys Leu Leu Gln Asn Val Ser
110 115 120
Pro Gly Asp Tyr Ile Ile Glu Leu Val Asp Asp Ser Asn Thr Thr
125 130 135
Arg Lys Ala Ala Gln Tyr Val Val Lys Ser Val Gln Ser Pro Trp
140 145 150
Ala Gly Pro Ile Arg Ala Val Ala Ile Thr Val Pro Leu Val Val
155 160 165
Ile Ser Ala Phe Ala Thr Leu Phe Thr Val Met Cys Arg Lys Lys
170 175 180
Gln Gln Glu Asn Ile Tyr Ser His Leu Asp Glu Glu Ser Pro Glu
185 190 195
Ser Ser Thr Tyr Ala Ala Ala Leu Pro Arg Asp Arg Leu Arg Pro
200 205 210
Gln Pro Lys Val Phe Leu Cys Tyr Ser Asn Lys Asp Gly Gln Asn
215 220 225
His Met Asn Val Val Gln Cys Phe Ala Tyr Phe Leu Gln Asp Phe
230 235 240
Cys Gly Cys Glu Val Ala Leu Asp Leu Trp Glu Asp Phe Ser Leu
245 250 255
Cys Arg Glu Gly Gln Arg Glu Trp Ala Ile Gln Lys Ile His Glu
260 265 270
Ser Gln Phe Ile Ile Val Val Cys Ser Lys Gly Met Lys Tyr Phe
275 280 285
Val Asp Lys Lys Asn Phe Arg His Lys Gly Gly Ser Arg Gly Glu
290 295 300
Ala Gln Gly Glu Phe Phe Leu Val Ala Val Ala Ala Ile Ala Glu
305 310 315
Lys Leu Arg Gln Ala Lys Gln Ser Ser Ser Ala Ala Leu Arg Lys
320 325 330
Phe Ile Ala Val Tyr Phe Asp Tyr Ser Cys Glu Gly Asp Val Pro
335 340 345
Cys Ser Leu Asp Leu Ser Thr Lys Tyr Lys Leu Met Asp His Leu
350 355 360
Pro Glu Leu Cys Ala His Leu His Ser Gly Glu Gln Glu Val Leu
365 370 375
Gly Gln His Pro Gly His Ser Ser Arg Arg Asn Tyr Phe Arg Ser
380 385 390
Lys Ser Gly Arg Ser Leu Tyr Val Ala Ile Cys Asn Met His Gln
395 400 405
Phe Ile Asp Glu Glu Pro Asp Trp Phe Glu Lys Gln Phe Ile Pro
410 415 420
Phe Gln His Pro Pro Val Arg Tyr Gln Glu Pro Val Leu Glu Lys
425 430 435
Phe Asp Ser Gly Leu Val Leu Asn Asp Val Ile Ser Lys Pro Gly
440 445 450
Pro Glu Ser Asp Phe Cys Arg Lys Val Glu Ala Cys Val Leu Gly
455 460 465
Ala Ala Gly Pro Ala Asp Ser Tyr Ser Tyr Leu Glu Ser Gln His
470 475 480
Val Gly Leu Asp Gln Asp Thr Glu Ala Gln Pro Ser Cys Asp Ser
485 490 495
Ala Pro Ala Leu Gln Pro Leu Leu His Ala Val Lys Ala Gly Ser
500 505 510
Pro Ser Glu Met Pro Arg Asp Ser Gly Ile Tyr Asp Ser Ser Val
515 520 525
Pro Ser Ser Glu Leu Ser Leu Pro Leu Met Glu Gly Leu Ser Pro
530 535 540
Asp Gln Ile Glu Thr Ser Ser Leu Thr Glu Ser Val Ser Ser Ser
545 550 555
Ser Gly Leu Gly Glu Glu Asp Pro Pro Thr Leu Pro Ser Lys Leu
560 565 570
Phe Ala Ser Gly Val Ser Arg Glu His Gly Cys His Ser His Thr
575 580 585
Asp Glu Leu Gln Ala Leu Ala Pro Leu
590 594
Claims (6)
1、一种小鼠白细胞介素-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1-10个氨基酸残基的取代、缺失或添加且具有激活STAT5和介导细胞增殖功能的由序列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、编码权利要求1所述蛋白的基因。
4、根据权利要求3所述的基因,其特征在于:所述小鼠白细胞介素-17受体样蛋白的编码基因是序列表中序列1的DNA序列。
5、权利要求1所述的小鼠白细胞介素-17受体样蛋白在制备Stat5因子活化剂中的应用。
6、权利要求1所述的小鼠白细胞介素-17受体样蛋白在制备介导细胞增殖剂中的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN 02123541 CN1205226C (zh) | 2002-07-01 | 2002-07-01 | 一种小鼠白细胞介素-17受体样蛋白及其编码基因与应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN 02123541 CN1205226C (zh) | 2002-07-01 | 2002-07-01 | 一种小鼠白细胞介素-17受体样蛋白及其编码基因与应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN1465593A CN1465593A (zh) | 2004-01-07 |
CN1205226C true CN1205226C (zh) | 2005-06-08 |
Family
ID=34142364
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN 02123541 Expired - Fee Related CN1205226C (zh) | 2002-07-01 | 2002-07-01 | 一种小鼠白细胞介素-17受体样蛋白及其编码基因与应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN1205226C (zh) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
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 |
-
2002
- 2002-07-01 CN CN 02123541 patent/CN1205226C/zh not_active Expired - Fee Related
Also Published As
Publication number | Publication date |
---|---|
CN1465593A (zh) | 2004-01-07 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Tsai et al. | Grouper interleukin-12, linked by an ancient disulfide-bond architecture, exhibits cytokine and chemokine activities | |
Xu et al. | The first non-mammalian CXCR5 in a teleost fish: molecular cloning and expression analysis in grass carp (Ctenopharyngodon idella) | |
CN1399681A (zh) | 在细胞培养物中增强细胞因子产生的方法 | |
CN1205226C (zh) | 一种小鼠白细胞介素-17受体样蛋白及其编码基因与应用 | |
CN1216912C (zh) | 人白细胞介素-17受体样蛋白及其编码基因与应用 | |
CN1211402C (zh) | 小鼠白细胞介素-17受体样蛋白的新用途 | |
CN1211401C (zh) | 一种人白细胞介素-17受体样蛋白及其编码基因与应用 | |
CN1341660A (zh) | 一种新的多肽——人白细胞介素受体-配体(il-17b和il-17br)32.56和编码这种多肽的多核苷酸 | |
CN1274827C (zh) | 一种癌基因及其编码蛋白与该基因的转基因细胞系 | |
CN1524958A (zh) | 一种血管紧张素ⅱ相关基因及其编码蛋白与应用 | |
CN1757651A (zh) | 一种白介素17受体及其编码基因与应用 | |
CN1618808A (zh) | 肝癌相关蛋白及其编码序列和用途 | |
CN1322737A (zh) | 一种新的多肽——人Stat2蛋白10和编码这种多肽的多核苷酸 | |
CN1708585A (zh) | Gpr54敲除哺乳动物以及利用它们的筛选方法 | |
CN1322825A (zh) | 一种新的多肽——超氧化物歧化酶11和编码这种多肽的多核苷酸 | |
CN1322830A (zh) | 一种新的多肽——人粘着斑激酶(fak)13和编码这种多肽的多核苷酸 | |
Xu et al. | Research article The first non-mammalian CXCR5 in a teleost fish: molecular cloning and expression analysis in grass carp (Ctenopharyngodon idella) | |
CN1475500A (zh) | 新型人髓系分化标志物,其编码序列及用途 | |
CN1477193A (zh) | 新型人Rab GTP酶,其编码序列及用途 | |
CN1320607A (zh) | 一种新的多肽——层粘连蛋白19和编码这种多肽的多核苷酸 | |
CN1315452A (zh) | 一种新的多肽——人形成素蛋白12和编码这种多肽的多核苷酸 | |
CN1526728A (zh) | 人肿瘤生成及转移促进受体分子及其编码基因与应用 | |
CN1352007A (zh) | 一种新的多肽——细胞因子受体9.57和编码这种多肽的多核苷酸 | |
CN1352097A (zh) | 一种新的多肽——人层粘连蛋白b2链15.95和编码这种多肽的多核苷酸 | |
CN1314408A (zh) | 一种新的多肽——人dna错配修复基因蛋白9和编码这种多肽的多核苷酸 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C14 | Grant of patent or utility model | ||
GR01 | Patent grant | ||
C19 | Lapse of patent right due to non-payment of the annual fee | ||
CF01 | Termination of patent right due to non-payment of annual fee |