CN1423695A - 反义寡核苷酸 - Google Patents
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Abstract
本发明提供了能有效抑制野生型COL1A1基因表达的反义DNA寡核苷酸。
Description
本发明涉及反义寡核苷酸及其在抑制I型原胶原表达中的用途。
胶原是一个密切相关蛋白的家族，具有三股螺旋蛋白结构。已经鉴定了许多胶原型(＞10)，其中I型原胶原(包括两条α1链和一条α2链)是骨、皮肤和键的主要成分。
多年来已经认识到许多病理状态是由于在瘢痕形成过程中产生过多胶原和过量的纤维组织造成的。例如，肝硬化包括两个步骤，其中正常肝组织首先被一种病毒或被乙醇或其它毒素破坏，接着在正常肝细胞再生前，过量的胶原纤维代替了被破坏细胞。特发性肺纤维化是一种正常肺组织逐渐被过量的胶原纤维代替的致命情况。进行性系统硬化症(硬皮病)是一种常常致命的疾病，皮肤和许多内脏器官由于过量的胶原纤维沉积而变为皮革样。在很多个体中，皮肤伤口或手术切口在肥大瘢痕和瘢痕疙瘩形成中伴随过量的胶原沉积，出现了美容问题，有时还有更严重的后果。而且，过量的瘢痕形成常常在正常个体外伤和手术操作后发生。在这些和相关的情况下，一种特异性抑制胶原合成的方法和组合物将非常有益。
PCT专利申请公开号WO94/11494公开了一种DNA或RNA寡核苷酸，包括5到200个核苷酸，它与突变型胶原核苷酸序列或正常野生型胶原核苷酸序列基本互补，能够抑制胶原基因表达。据称优选的寡核苷酸是反义寡核苷酸。WO94/11494的实施例描述了一系列DNA寡核苷酸，其中一些是反义的，这一系列寡核苷酸是根据人I型原胶原基因(COL1A1)的proα1链的外显子1的3’末端区域(从核苷酸198到222)和内含子1的起始两个核苷酸首先合成的。发现合成的寡核苷酸对人源内部缺失突变型COL1A1基因表达的抑制能力变化很大。然而没有证明该寡核苷酸抑制人野生型COL1A1基因表达的效力。由于人突变型和野生型COL1A1基因的RNA转录物的结构和构象很可能不同，不能必然认为能有效抑制突变型COL1A1基因表达的寡核苷酸也会是野生型COL1A1基因表达的有效抑制剂。
期望鉴定出能够抑制一种野生型COL1A1基因表达的反义DNA寡核苷酸。
因此根据本发明，提供了一种反义DNA寡核苷酸，包括18到25个核苷酸，它与SEQ ID NO：1中750位到3900位包括750位和3900位的核苷酸序列互补，其中SEQ ID NO：1包括编码包含根据SEQ ID NO：2的氨基酸序列的多肽的核苷酸序列，该寡核苷酸能够抑制该多肽在表达该多肽的细胞中的表达。
SEQ ID NO：1与EMBL注册号Z74615注册的核苷酸序列等同。SEQID NO：2是核苷酸序列SEQ ID NO：1编码的多肽的氨基酸序列。SEQ IDNO：1编码的多肽是野生型I型原胶原的proα1链的前体(“前原-α1(I)胶原”)。
根据本发明的反义DNA寡核苷酸包括18、19、20、21、22、23、24或25个核苷酸，优选长度为20个核苷酸。
优选反义DNA寡核苷酸与SEQ ID NO：1的下列区域之一的核苷酸序列互补，
区域1-750位到900位，包括750位和900位，
区域2-1200位到1300位，包括1200位和1300位，
区域3-1400位到1500位，包括1400位和1500位，
区域4-1450位到1550位，包括1450位和1550位，
区域5-1850位到2000位，包括1850位和2000位，
区域6-2500位到2600位，包括2500位和2600位，
区域7-2850位到2950位，包括2850位和2950位，
区域8-3800位到3900位，包括3800位和3900位。
特别优选的反义DNA寡核苷酸是与SEQ ID NO：1的区域2、4、6和8的核苷酸序列互补的寡核苷酸。
本发明的寡核苷酸可以通过任何本领域已知的合适方法制备。通过化学合成方法能非常方便地制备该寡核苷酸，例如，Tetrahedron Lett.，1991，32，30005-30008中描述的亚磷酰胺化学过程，用二硫化四乙基秋兰姆对乙腈进行硫化。
本发明的寡核苷酸具有优越性，因为它们抑制野生型COL1A1基因的表达。因此它们在治疗或预防由胶原纤维产生过多而引起的状态/紊乱中有用，如，肝硬化、肾、肝和心脏纤维化、硬皮病、肥大瘢痕和瘢痕疙瘩。
因此，本发明提供了一种根据本发明用于治疗的反义DNA寡核苷酸。
在另一个方面，本发明提供了根据本发明的反义DNA寡核苷酸在制备用于治疗的药物中的应用。
在本发明的说明书中，术语“治疗”也包括“预防”，除非有特别的相反指示。术语“治疗的”和“治疗地”应据此解释。
本发明进一步提供了一种治疗患有胶原紊乱或存在胶原紊乱风险患者的胶原紊乱，或降低胶原紊乱风险的方法，包括给患者施与治疗有效量的根据本发明的反义DNA寡核苷酸。
当然，对于上面提及的治疗用途中使用的剂量将根据所使用的寡核苷酸、给药方式、期望的治疗和紊乱指征而发生变化。有效的剂量是能够将细胞中产生的胶原蛋白量抑制在可消除或减少胶原蛋白产生伴发的症状或情况的水平。
根据本发明的寡核苷酸通常以药物组合物的形式给予，其中寡核苷酸与一种药学上可接受佐剂、稀释液或载体配制而成。
因而，本发明也提供了一种药物组合物，包括根据本发明的反义DNA寡核苷酸联合一种药学上可接受佐剂、稀释液或载体。
本发明进一步提供了一种本发明药物组合物的制备方法，包括将反义DNA寡核苷酸与一种药学上可接受佐剂、稀释液或载体混合。
本发明的药物组合物可以局部给予，如，以乳剂、洗剂或软膏的形式；或全身给予，如以药片、胶囊、糖浆、粉剂或粒剂的形式口服给药，或以无菌溶液或悬液的形式肠胃外途径给药。
现在将参考下列例证实施例来进一步解释本发明。
实施例
实施例1
寡核苷酸的合成
在PE生物系统394 DNA合成仪上，用TETD/乙腈硫化试剂采用亚磷酰胺化学过程进行1μm规模的硫代磷酸寡核苷酸合成。Poly-Pak TM II柱(Glen Research)纯化寡核苷酸，NAPTM 10柱(Amersham PharmaciaBiotech AB)除去盐分并用Dowex 50WX8-100离子交换树脂(Aldrich)进行离子交换。制备的12个反义DNA寡核苷酸(ASOs)具有下列序列(5’→3’)：
1.GGACGACCAGGTTTTCCAGC(SEQ ID NO：3)
2.GCAGCACCAGCAGGGCCAGG(SEQ ID NO：4)
3.GCCAGGAGCACCAGGTTCAC(SEQ ID NO：5)
4.CTTCCTCTCCAGCAGGGCCA(SEQ ID NO：6)
5.GCCTTGCCGGGCTCTCCAGC(SEQ ID NO：7)
6.CGGGAACACCTCGCTCTCCA(SEQ ID NO：8)
7.GCAGGACCGACAGCGCCAGG(SEQ ID NO：9)
8.TCCATCTTTGCCAGCAGGAC(SEQ ID NO：10)
9.GGTCCCTGAGCTCCAGCCTC(SEQ ID NO：11)
10.TTGGCCGTCAGCACCAGGG(SEQ ID NO：12)
11.TTTCTCGCCAGCAGGGCCAG(SEQ ID NO：13)
12.CTCGATCTGCTGGCTCAGGC(SEQ ID NO：14)
实施例2
细胞处理
人细胞系WI-26在含有10％胎牛血清的Dulbecco′s改良Eagle′s培养基(DMEM)中生长。将细胞铺在48-孔或6-孔平板(Costar，ComingInc.)上达到70-80％融合。24小时后，用预热的DMEM洗涤细胞2次，每孔加入含有5μg/ml脂转染试剂(Gibco BRL)或2.5μg/ml细胞转染试剂GSV(Glen Research Ltd)的0.35ml(对于48-孔试验)或1ml(6-孔试验)DMEM和200nM的寡核苷酸。37℃培养4-5小时后，用预热的DMEM洗涤细胞2次，0.35ml(48-孔平板)或1ml(6-孔平板)DMEM与20μg/ml抗坏血酸一起加入。细胞培养20小时后分析胶原水平。
实施例3
蛋白分析
试验最后，去除150μl培养基，用ELISA试剂盒(Amersham PharmaciaLtd)测定分泌的I型原胶原，结果以培养基中原胶原的纳克数/10,000细胞表示。为了校正细胞数量，用预热PBS洗涤平板，胰蛋白酶处理细胞，用Coulter自动计数器计算细胞数量。对于6-孔试验，细胞计数，用1mlTRI试剂(SIGMA Ltd)处理细胞，根据厂商说明提取蛋白和RNA。蛋白团在含蛋白酶抑制剂的1％SDS中重悬。100℃加热30-100μg细胞蛋白5分钟，接着在4-12％SDS聚丙烯酰胺凝胶中电泳。蛋白电转移至硝酸纤维素膜上，并与抗人I型胶原proα1(I)链(从Dr Larry Fisher，NIH，美国获得)相应合成肽的抗体杂交。用偶合HRP(Biorad Ltd)的抗-兔二抗检测proα1(I)带，ECL(Pierce Ltd)显影。用GAPDH的抗体(AdvancedImmunochemicals)处理膜，测定蛋白加样。用光密度测定法将蛋白加样标准化至GAPDH水平。
实施例4
RNA分析
用TRI试剂提取RNA，0.5％SDS中重悬最终的颗粒团。根据标准步骤(Sambrook，J.，Fritsch，E.F.and Maniatis，T.(1989)Molecular Cloning：ALaboratory Manual，2nd Ed.，Cold Spring Harbor Laboratory Press，冷泉港，美国)，总RNA的1到3μg在甲醛变性胶中电泳。RNA转移至Hybond-N膜(Amersham)上，使用T7聚合酶试剂盒(Amersham Pharmacia)与α1(I)cDNA放射性标记探针杂交24小时。洗涤后，滤光片曝光为X-线胶片，4-24小时后胶片显影。用光密度测定分析法分析α1(I)转录物(4.8kb&5.8kb)的放射自显影图象，用GAPDH转录物强度或作为内对照的28SrRNA的强度来校正RNA加样。
结果
下表I显示有关培养基中胶原水平或胶原mRNA水平的胶原抑制平均百分比(％)。在所使用的处理细胞试验中，培养基中测定的胶原抑制百分比与细胞内胶原mRNA水平的抑制百分比之间有很好的相关性。
表I
| ASO | 胶原抑制平均百分比％ |
| GGACGACCAGGTTTTCCAGC(SEQ ID NO：3) | 50 |
| GCAGCACCAGCAGGGCCAGG(SEQ ID NO：4) | 50-80 |
| GCCAGGAGCACCAGGTTCAC(SEQ ID NO：5) | 50 |
| CTTCCTCTCCAGCAGGGCCA(SEQ ID NO：6) | 50-60 |
| GCCTTGCCGGGCTCTCCAGC(SEQ ID NO：7) | 50 |
| CGGGAACACCTCGCTCTCCA(SEQ ID NO：8) | 50 |
| GCAGGACCGACAGCGCCAGG(SEQ ID NO：9) | 50 |
| TCCATCTTTGCCAGCAGGAC(SEQ ID NO：10) | 50 |
| GGTCCCTGAGCTCCAGCCTC(SEQ ID NO：11) | 50 |
| TTGGCCGTCAGCACCAGGG(SEQ ID NO：12) | 50-80 |
| TTTCTCGCCAGCAGGGCCAG(SEQ ID NO：13) | 50-70 |
| CTCGATCTGCTGGCTCAGGC(SEQ ID NO：14) | 50-80 |
序列表
序列表<110>阿斯特拉曾尼卡有限公司(AstraZeneca AB)<120>反义寡核苷酸<130>Z70611<140><141><160>14<170>PatentIn Ver.2.1<210>1<211>6728<212>DNA<213>人(Homo sapiens)<220><221>CDS<222>(120)..(4511)<220><221>信号肽<222>(120)..(185)<300><308>EMBL/Z74615<309>1996-07-01<400>1agcagacggg agtttctcct cggggtcgga gcaggaggca cgcggagtgt gaggccacgc 60atgagcggac gctaaccccc tccccagcca caaagagtct acatgtctag ggtctagac 119atg ttc agc ttt gtg gac ctc cgg ctc ctg ctc ctc tta gcg gcc acc 167Met Phe Ser Phe Val Asp Leu Arg Leu Leu Leu Leu Leu Ala Ala Thr1 5 10 15gcc ctc ctg acg cac ggc caa gag gaa ggc caa gtc gag ggc caa gac 215Ala Leu Leu Thr His Gly Gln Glu Glu Gly Gln Val Glu Gly Gln Asp
20 25 30gaa gac atc cca cca atc acc tgc gta cag aac ggc ctc agg tac cat 263Glu Asp Ile Pro Pro Ile Thr Cys Val Gln Asn Gly Leu Arg Tyr His
35 40 45gac cga gac gtg tgg aaa ccc gag ccc tgc cgg atc tgc gtc tgc gac 311Asp Arg Asp Val Trp Lys Pro Glu Pro Cys Arg Ile Cys Val Cys Asp
50 55 60aac ggc aag gtg ttg tgc gat gac gtg atc tgt gac gag acc aag aac 359Asn Gly Lys Val Leu Cys Asp Asp Val Ile Cys Asp Glu Thr Lys Asn65 70 75 80tgc ccc ggc gcc gaa gtc ccc gag ggc gag tgc tgt ccc gtc tgc ccc 407Cys Pro Gly Ala Glu Val Pro Glu Gly Glu Cys Cys Pro Val Cys Pro
85 90 95gac ggc tca gag tca ccc acc gac caa gaa acc acc ggc gtc gag gga 455Asp Gly Ser Glu Ser Pro Thr Asp Gln Glu Thr Thr Gly Val Glu Gly
100 105 110ccc aag gga gac act ggc ccc cga ggc cca agg gga ccc gca ggc ccc 503Pro Lys Gly Asp Thr Gly Pro Arg Gly Pro Arg Gly Pro Ala Gly Pro
115 120 125cct ggc cga gat ggc atc cct gga cag cct gga ctt ccc gga ccc ccc 551Pro Gly Arg Asp Gly Ile Pro Gly Gln Pro Gly Leu Pro Gly Pro Pro
130 135 140gga ccc ccc gga cct ccc gga ccc cct ggc ctc gga gga aac ttt gct 599Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Leu Gly Gly Asn Phe Ala145 150 155 160ccc cag ctg tct tat ggc tat gat gag aaa tca acc gga gga att tcc 647Pro Gln Leu Ser Tyr Gly Tyr Asp Glu Lys Ser Thr Gly Gly Ile Ser
165 170 175gtg cct ggc ccc atg ggt ccc tct ggt cct cgt ggt ctc cct ggc ccc 695Val Pro Gly Pro Met Gly Pro Ser Gly Pro Arg Gly Leu Pro Gly Pro
180 185 190cct ggt gca cct ggt ccc caa ggc ttc caa ggt ccc cct ggt gag cct 743Pro Gly Ala Pro Gly Pro Gln Gly Phe Gln Gly Pro Pro Gly Glu Pro
195 200 205ggc gag cct gga gct tca ggt ccc atg ggt ccc cga ggt ccc cca ggt 791Gly Glu Pro Gly Ala Ser Gly Pro Met Gly Pro Arg Gly Pro Pro Gly
210 215 220ccc cct gga aag aat gga gat gat ggg gaa gct gga aaa cct ggt cgt 839Pro Pro Gly Lys Asn Gly Asp Asp Gly Glu Ala Gly Lys Pro Gly Arg225 230 235 240cct ggt gag cgt ggg cct cct ggg cct cag ggt gct cga gga ttg ccc 887Pro Gly Glu Arg Gly Pro Pro Gly Pro Gln Gly Ala Arg Gly Leu Pro
245 250 255gga aca gct ggc ctc cct gga atg aag gga cac aga ggt ttc agt ggt 935Gly Thr Ala Gly Leu Pro Gly Met Lys Gly His Arg Gly Phe Ser Gly
260 265 270ttg gat ggt gcc aag gga gat gct ggt cct gct ggt cct aag ggt gag 983Leu Asp Gly Ala Lys Gly Asp Ala Gly Pro Ala Gly Pro Lys Gly Glu
275 280 285cct ggc agc cct ggt gaa aat gga gct cct ggt cag atg ggc ccc cgt 1031Pro Gly Ser Pro Gly Glu Asn Gly Ala Pro Gly Gln Met Gly Pro Arg
290 295 300ggc ctg cct ggt gag aga ggt cgc cct gga gcc cct ggc cct gct ggt 1079Gly Leu Pro Gly Glu Arg Gly Arg Pro Gly Ala Pro Gly Pro Ala Gly305 310 315 320gct cgt gga aat gat ggt gct act ggt gct gcc ggg ccc cct ggt ccc 1127Ala Arg Gly Asn Asp Gly Ala Thr Gly Ala Ala Gly Pro Pro Gly Pro
325 330 335acc ggc ccc gct ggt cct cct ggc ttc cct ggt gct gtt ggt gct aag 1175Thr Gly Pro Ala Gly Pro Pro Gly Phe Pro Gly Ala Val Gly Ala Lys
340 345 350ggt gaa gct ggt ccc caa ggg ccc cga ggc tct gaa ggt ccc cag ggt 1223Gly Glu Ala Gly Pro Gln Gly Pro Arg Gly Ser Glu Gly Pro Gln Gly
355 360 365gtg cgt ggt gag cct ggc ccc cct ggc cct gct ggt gct gct ggc cct 1271Val Arg Gly Glu Pro Gly Pro Pro Gly Pro Ala Gly Ala Ala Gly Pro
370 375 380gct gga aac cct ggt gct gat gga cag cct ggt gct aaa ggt gcc aat 1319Ala Gly Asn Pro Gly Ala Asp Gly Gln Pro Gly Ala Lys Gly Ala Asn385 390 395 400ggt gct cct ggt att gct ggt gct cct ggc ttc cct ggt gcc cga ggc 1367Gly Ala Pro Gly Ile Ala Gly Ala Pro Gly Phe Pro Gly Ala Arg Gly
405 410 415ccc tct gga ccc cag ggc ccc ggc ggc cct cct ggt ccc aag ggt aac 1415Pro Ser Gly Pro Gln Gly Pro Gly Gly Pro Pro Gly Pro Lys Gly Asn
420 425 430agc ggt gaa cct ggt gct cct ggc agc aaa gga gac act ggt gct aag 1463Ser Gly Glu Pro Gly Ala Pro Gly Ser Lys Gly Asp Thr Gly Ala Lys
435 440 445gga gag cct ggc cct gtt ggt gtt caa gga ccc cct ggc cot gct gga 1511Gly Glu Pro Gly Pro Val Gly Val Gln Gly Pro Pro Gly Pro Ala Gly
450 455 460gag gaa gga aag cga gga gct cga ggt gaa ccc gga ccc act ggc ctg 1559Glu Glu Gly Lys Arg Gly Ala Arg Gly Glu Pro Gly Pro Thr Gly Leu465 470 475 480ccc gga ccc cct ggc gag cgt ggt gga cct ggt agc cgt ggt ttc cct 1607Pro Gly Pro Pro Gly Glu Arg Gly Gly Pro Gly Ser Arg Gly Phe Pro
485 490 495ggc gca gat ggt gtt gct ggt ccc aag ggt ccc gct ggt gaa cgt ggt 1655Gly Ala Asp Gly Val Ala Gly Pro Lys Gly Pro Ala Gly Glu Arg Gly
500 505 510tct cct ggc ccc gct ggc ccc aaa gga tct cct ggt gaa gct ggt cgt 1703Ser Pro Gly Pro Ala Gly Pro Lys Gly Ser Pro Gly Glu Ala Gly Arg
515 520 525ccc ggt gaa gct ggt ctg cct ggt gcc aag ggt ctg act gga agc cct 1751Pro Gly Glu Ala Gly Leu Pro Gly Ala Lys Gly Leu Thr Gly Ser Pro
530 535 540ggc agc cct ggt cct gat ggc aaa act ggc ccc cct ggt ccc gcc ggt 1799Gly Ser Pro Gly Pro Asp Gly Lys Thr Gly Pro Pro Gly Pro Ala Gly545 550 555 560caa gat ggt cgc ccc gga ccc cca ggc cca cct ggt gcc cgt ggt cag 1847Gln Asp Gly Arg Pro Gly Pro Pro Gly Pro Pro Gly Ala Arg Gly Gln
565 570 575gct ggt gtg atg gga ttc cct gga cct aaa ggt gct gct gga gag ccc 1895Ala Gly Val Met Gly Phe Pro Gly Pro Lys Gly Ala Ala Gly Glu Pro
580 585 590ggc aag gct gga gag cga ggt gtt ccc gga ccc cct ggc gct gtc ggt 1943Gly Lys Ala Gly Glu Arg Gly Val Pro Gly Pro Pro Gly Ala Val Gly
595 600 605cct gct ggc aaa gat gga gag gct gga gct cag gga ccc cct ggc cct 1991Pro Ala Gly Lys Asp Gly Glu Ala Gly Ala Gln Gly Pro Pro Gly Pro
610 615 620gct ggt ccc gct ggc gag aga ggt gaa caa ggc cct gct ggc tcc ccc 2039Ala Gly Pro Ala Gly Glu Arg Gly Glu Gln Gly Pro Ala Gly Ser Pro625 630 635 640gga ttc cag ggt ctc cct ggt cct gct ggt cct cca ggt gaa gca ggc 2087Gly Phe Gln Gly Leu Pro Gly Pro Ala Gly Pro Pro Gly Glu Ala Gly
645 650 655aaa cct ggt gaa cag ggt gtt cct gga gac ctt ggc gcc cct ggc ccc 2135Lys Pro Gly Glu Gln Gly Val Pro Gly Asp Leu Gly Ala Pro Gly Pro
660 665 670tct gga gca aga ggc gag aga ggt ttc cct ggc gag cgt ggt gtg caa 2183Ser Gly Ala Arg Gly Glu Arg Gly Phe Pro Gly Glu Arg Gly Val Gln
675 680 685ggt ccc cct ggt cct gct gga ccc cga ggg gcc aac ggt gct ccc ggc 2231Gly Pro Pro Gly Pro Ala Gly Pro Arg Gly Ala Asn Gly Ala Pro Gly
690 695 700aac gat ggt gct aag ggt gat gct ggt gcc cct gga gct ccc ggt agc 2279Asn Asp Gly Ala Lys Gly Asp Ala Gly Ala Pro Gly Ala Pro Gly Ser705 710 715 720cag ggc gcc cct ggc ctt cag gga atg cct ggt gaa cgt ggt gca gct 2327Gln Gly Ala Pro Gly Leu Gln Gly Met Pro Gly Glu Arg Gly Ala Ala
725 730 735ggt ctt cca ggg cct aag ggt gac aga ggt gat gct ggt ccc aaa ggt 2375Gly Leu Pro Gly Pro Lys Gly Asp Arg Gly Asp Ala Gly Pro Lys Gly
740 745 750gct gat ggc tct cct ggc aaa gat ggc gtc cgt ggt ctg acc ggc ccc 2423Ala Asp Gly Ser Pro Gly Lys Asp Gly Val Arg Gly Leu Thr Gly Pro
755 760 765att ggt cct cct ggc cct gct ggt gcc cct ggt gac aag ggt gaa agt 2471Ile Gly Pro Pro Gly Pro Ala Gly Ala Pro Gly Asp Lys Gly Glu Ser
770 775 780ggt ccc agc ggc cct gct ggt ccc act gga gct cgt ggt gcc ccc gga 2519Gly Pro Ser Gly Pro Ala Gly Pro Thr Gly Ala Arg Gly Ala Pro Gly785 790 795 800gac cgt ggt gag cct ggt ccc ccc ggc cct gct ggc ttt gct ggc ccc 2567Asp Arg Gly Glu Pro Gly Pro Pro Gly Pro Ala Gly Phe Ala Gly Pro
805 810 815cct ggt gct gac ggc caa cct ggt gct aaa ggc gaa cct ggt gat gct 2615Pro Gly Ala Asp Gly Gln Pro Gly Ala Lys Gly Glu Pro Gly Asp Ala
820 825 830ggt gcc aaa ggc gat gct ggt ccc cct ggg cct gcc gga ccc gct gga 2663Gly Ala Lys Gly Asp Ala Gly Pro Pro Gly Pro Ala Gly Pro Ala Gly
835 840 845ccc cct ggc ccc att ggt aat gtt ggt gct cct gga gcc aaa ggt gct 2711Pro Pro Gly Pro Ile Gly Asn Val Gly Ala Pro Gly Ala Lys Gly Ala
850 855 860cgc ggc agc gct ggt ccc cct ggt gct act ggt ttc cct ggt gct gct 2759Arg Gly Ser Ala Gly Pro Pro Gly Ala Thr Gly Phe Pro Gly Ala Ala865 870 875 880ggc cga gtc ggt cct cct ggc ccc tct gga aat gct gga ccc cct ggc 2807Gly Arg Val Gly Pro Pro Gly Pro Ser Gly Asn Ala Gly Pro Pro Gly
885 890 895cct cct ggt cct gct ggc aaa gaa ggc ggc aaa ggt ccc cgt ggt gag 2855Pro Pro Gly Pro Ala Gly Lys Glu Gly Gly Lys Gly Pro Arg Gly Glu
900 905 910act ggc cct gct gga cgt cct ggt gaa gtt ggt ccc cct ggt ccc cct 2903Thr Gly Pro Ala Gly Arg Pro Gly Glu Val Gly Pro Pro Gly Pro Pro
915 920 925ggc cct gct ggc gag aaa gga tcc cct ggt gct gat ggt cct gct ggt 2951Gly Pro Ala Gly Glu Lys Gly Ser Pro Gly Ala Asp Gly Pro Ala Gly
930 935 940gct cct ggt act ccc ggg cct caa ggt att gct gga cag cgt ggt gtg 2999Ala Pro Gly Thr Pro Gly Pro Gln Gly Ile Ala Gly Gln Arg Gly Val945 950 955 960gtc ggc ctg cct ggt cag aga gga gag aga ggc ttc cct ggt ctt cct 3047Val Gly Leu Pro Gly Gln Arg Gly Glu Arg Gly Phe Pro Gly Leu Pro
965 970 975ggc ccc tct ggt gaa cct ggc aaa caa ggt ccc tct gga gca agt ggt 3095Gly Pro Ser Gly Glu Pro Gly Lys Gln Gly Pro Ser Gly Ala Ser Gly
980 985 990gaa cgt ggt ccc ccc ggt ccc atg ggc ccc cct gga ttg gct gga ccc 3143Glu Arg Gly Pro Pro Gly Pro Met Gly Pro Pro Gly Leu Ala Gly Pro
995 1000 1005cct ggt gaa tct gga cgt gag ggg gct cct gct gcc gaa ggt tcc cct 3191Pro Gly Glu Ser Gly Arg Glu Gly Ala Pro Ala Ala Glu Gly Ser Pro1010 1015 1020gga cga gac ggt tct cct ggc gcc aag ggt gac cgt ggt gag acc ggc 3239Gly Arg Asp Gly Ser Pro Gly Ala Lys Gly Asp Arg Gly Glu Thr Gly1025 1030 1035 1040ccc gct gga ccc cct ggt gct cct ggt gct cct ggt gcc cct ggc ccc 3287Pro Ala Gly Pro Pro Gly Ala Pro Gly Ala Pro Gly Ala Pro Gly Pro
1045 1050 1055gtt ggc cct gct ggc aag agt ggt gat cgt ggt gag act ggt cct gct 3335Val Gly Pro Ala Gly Lys Ser Gly Asp Arg Gly Glu Thr Gly Pro Ala
1060 1065 1070ggt ccc gcc ggt ccc gtc ggc ccc gtc ggc gcc cgt ggc ccc gcc gga 3383Gly Pro Ala Gly Pro Val Gly Pro Val Gly Ala Arg Gly Pro Ala Gly
1075 1080 1085ccc caa ggc ccc cgt ggt gac aag ggt gag aca ggc gaa cag ggc gac 3431Pro Gln Gly Pro Arg Gly Asp Lys Gly Glu Thr Gly Glu Gln Gly Asp1090 1095 1100aga ggc ata aag ggt cac cgt ggc ttc tct ggc ctc cag ggt ccc cct 3479Arg Gly Ile Lys Gly His Arg Gly Phe Ser Gly Leu Gln Gly Pro Pro1105 1110 1115 1120ggc cct cct ggc tct cct ggt gaa caa ggt ccc tct gga gcc tct ggt 3527Gly Pro Pro Gly Ser Pro Gly Glu Gln Gly Pro Ser Gly Ala Ser Gly
1125 1130 1135cct gct ggt ccc cga ggt ccc cct ggc tct gct ggt gct cct ggc aaa 3575Pro Ala Gly Pro Arg Gly Pro Pro Gly Ser Ala Gly Ala Pro Gly Lys
1140 1145 1150gat gga ctc aac ggt ctc cct ggc ccc att ggg ccc cct ggt cct cgc 3623Asp Gly Leu Asn Gly Leu Pro Gly Pro Ile Gly Pro Pro Gly Pro Arg
1155 1160 1165ggt cgc act ggt gat gct ggt cct gtt ggt ccc ccc ggc cct cct gga 3671Gly Arg Thr Gly Asp Ala Gly Pro Val Gly Pro Pro Gly Pro Pro Gly1170 1175 1180cct cct ggt ccc cct ggt cct ccc agc gct ggt ttc gac ttc agc ttc 3719Pro Pro Gly Pro Pro Gly Pro Pro Ser Ala Gly Phe Asp Phe Ser Phe1185 1190 1195 1200ctg ccc cag cca cct caa gag aag gct cac gat ggt ggc cgc tac tac 3767Leu Pro Gln Pro Pro Gln Glu Lys Ala His Asp Gly Gly Arg Tyr Tyr
1205 1210 1215cgg gct gat gat gcc aat gtg gtt cgt gac cgt gac ctc gag gtg gac 3815Arg Ala Asp Asp Ala Asn Val Val Arg Asp Arg Asp Leu Glu Val Asp
1220 1225 1230acc acc ctc aag agc ctg agc cag cag atc gag aac atc cgg agc cca 3863Thr Thr Leu Lys Ser Leu Ser Gln Gln Ile Glu Asn Ile Arg Ser Pro
1235 1240 1245gag gga agc cgc aag aac ccc gcc cgc acc tgc cgt gac ctc aag atg 3911Glu Gly Ser Arg Lys Asn Pro Ala Arg Thr Cys Arg Asp Leu Lys Met1250 1255 1260tgc cac tct gac tgg aag agt gga gag tac tgg att gac ccc aac caa 3959Cys His Ser Asp Trp Lys Ser Gly Glu Tyr Trp Ile Asp Pro Asn Gln1265 1270 1275 1280ggc tgc aac ctg gat gcc atc aaa gtc ttc tgc aac atg gag act ggt 4007Gly Cys Asn Leu Asp Ala Ile Lys Val Phe Cys Asn Met Glu Thr Gly
1285 1290 1295gag acc tgc gtg tac ccc act cag ccc agt gtg gcc cag aag aac tgg 4055Glu Thr Cys Val Tyr Pro Thr Gln Pro Ser Val Ala Gln Lys Asn Trp
1300 1305 1310tac atc agc aag aac ccc aag gac aag agg cat gtc tgg ttc ggc gag 4103Tyr Ile Ser Lys Asn Pro Lys Asp Lys Arg His Val Trp Phe Gly Glu
1315 1320 1325agc atg acc gat gga ttc cag ttc gag tat ggc ggc cag ggc tcc gac 4151Ser Met Thr Asp Gly Phe Gln Phe Glu Tyr Gly Gly Gln Gly Ser Asp1330 1335 1340cct gcc gat gtg gcc atc cag ctg acc ttc ctg cgc ctg atg tcc acc 4199Pro Ala Asp Val Ala Ile Gln Leu Thr Phe Leu Arg Leu Met Ser Thr1345 1350 1355 1360gag gcc tcc cag aac atc acc tac cac tgc aag aac agc gtg gcc tac 4247Glu Ala Ser Gln Asn Ile Thr Tyr His Cys Lys Asn Ser Val Ala Tyr
1365 1370 1375atg gac cag cag act ggc aac ctc aag aag gcc ctg ctc ctc aag ggc 4295Met Asp Gln Gln Thr Gly Asn Leu Lys Lys Ala Leu Leu Leu Lys Gly
1380 1385 1390tcc aac gag atc gag atc cgc gcc gag ggc aac agc cgc ttc acc tac 4343Ser Asn Glu Ile Glu Ile Arg Ala Glu Gly Asn Ser Arg Phe Thr Tyr
1395 1400 1405agc gtc act gtc gat ggc tgc acg agt cac acc gga gcc tgg ggc aag 4391Ser Val Thr Val Asp Gly Cys Thr Ser His Thr Gly Ala Trp Gly Lys1410 1415 1420aca gtg att gaa tac aaa acc acc aag tcc tcc cgc ctg ccc atc atc 4439Thr Val Ile Glu Tyr Lys Thr Thr Lys Ser Ser Arg Leu Pro Ile Ile1425 1430 1435 1440gat gtg gcc ccc ttg gac gtt ggt gcc cca gac cag gaa ttc ggc ttc 4487Asp Val Ala Pro Leu Asp Val Gly Ala Pro Asp Gln Glu Phe Gly Phe
1445 1450 1455gac gtt ggc cct gtc tgc ttc ctg taaactccct ccatcccaac ctggctccct 4541Asp Val Gly Pro Val Cys Phe Leu
1460cccacccaac caactttccc cccaacccgg aaacagacaa gcaacccaaa ctgaaccccc 4601ccaaaagcca aaaaatggga gacaatttca catggacttt ggaaaatatt tttttccttt 4661gcattcatct ctcaaactta gtttttatct ttgaccaacc gaacatgacc aaaaaccaaa 4721agtgcattca accttaccaa aaaaaaaaaa aaaaaaaaaa gaataaataa ataagttttt 4781aaaaaaggaa gcttggtcca cttgcttgaa gacccatgcg ggggtaagtc cctttctgcc 4841cgttgggtta tgaaacccca atgctgccct ttctgctcct ttctccacac cccccttggc 4901ctcccctcca ctccttccca aatctgtctc cccagaagac acaggaaaca atgtattgtc 4961tgcccagcaa tcaaaggcaa tgctcaaaca cccaagtggc ccccaccctc agcccgctcc 5021tgcccgccca gcacccccag gccctgggga cctggggttc tcagactgcc aaagaagcct 5081tgccatctgg cgctcccatg gctcttgcaa catctcccct tcgtttttga gggggtcatg 5141ccgggggagc caccagcccc tcactgggtt cggaggagag tcaggaaggg ccacgacaaa 5201gcagaaacat cggatttggg gaacgcgtgt catcccttgt gccgcaggct gggcgggaga 5261gactgttctg ttctgttcct tgtgtaactg tgttgctgaa agactacctc gttcttgtct 5321tgatgtgtca ccggggcaac tgcctggggg cggggatggg ggcagggtgg aagcggctcc 5381ccatttttat accaaaggtg ctacatctat gtgatgggtg gggtggggag ggaatcactg 5441gtgctataga aattgagatg cccccccagg ccagcaaatg ttcctttttg ttcaaagtct 5501atttttattc cttgatattt tttctttctt tttttttttt tttgtggatg gggacttgtg 5561aatttttcta aaggtgctat ttaacatggg aggagagcgt gtgcgctcca gcccagcccg 5621ctgctcactt tccaccctct ctccacctgc ctctggcttc tcaggcctct gctctccgac 5681ctctctcctc tgaaaccctc ctccacagct gcagcccatc ctcccggctc cctcctagtc 5741tgtcctgcgt cctctgtccc cgggtttcag agacaacttc ccaaagcaca aagcagtttt 5801tccctagggg tgggaggaag caaaagactc tgtacctatt ttgtatgtgt ataataattt 5861gagatgtttt taattatttt gattgctgga ataaagcatg tggaaatgac ccaaacataa 5921tccgcagtgg cctcctaatt tccttctttg gagttggggg aggggtagac atggggaagg 5981ggccttgggg tgatgggctt gccttccatt cctgcccttt ccctccccac tattctcttc 6041tagatccctc cataacccca ctcccctttc tctcaccctt cttataccgc aaacctttct 6101acttcctctt tcattttcta ttcttgcaat ttccttgcac cttttccaaa tcctcttctc 6161ccctgcaata ccatacaggc aatccacgtg cacaacacac acacacactc ttcacatctg 6221gggttgtcca aacctcatac ccactcccct tcaagcccat ccactctcca ccccctggat 6281gccctgcact tggtggcggt gggatgctca tggatactgg gagggtgagg ggagtggaac 6341ccgtgaggag gacctggggg cctctccttg aactgacatg aagggtcatc tggcctctgc 6401tcccttctca cccacgctga cctcctgccg aaggagcaac gcaacaggag aggggtctgc 6461tgagcctggc gagggtctgg gagggaccag gaggaaggcg tgctccctgc tcgctgtcct 6521ggccctgggg gagtgaggga gacagacacc tgggagagct gtggggaagg cactcgcacc 6581gtgctcttgg gaaggaagga gacctggccc tgctcaccac ggactgggtg cctcgacctc 6641ctgaatcccc agaacacaac ccccctgggc tggggtggtc tggggaacca tcgtgccccc 6701gcctcccgcc tactcctttt taagctt 6728<210>2<211>1464<212>PRT<213>人(Homo sapiens)<400>2Met Phe Ser Phe Val Asp Leu Arg Leu Leu Leu Leu Leu Ala Ala Thr1 5 10 15Ala Leu Leu Thr His Gly Gln Glu Glu Gly Gln Val Glu Gly Gln Asp
20 25 30Glu Asp Ile Pro Pro Ile Thr Cys Val Gln Asn Gly Leu Arg Tyr His
35 40 45Asp Arg Asp Val Trp Lys Pro Glu Pro Cys Arg Ile Cys Val Cys Asp
50 55 60Asn Gly Lys Val Leu Cys Asp Asp Val Ile Cys Asp Glu Thr Lys Asn65 70 75 80Cys Pro Gly Ala Glu Val Pro Glu Gly Glu Cys Cys Pro Val Cys Pro
85 90 95Asp Gly Ser Glu Ser Pro Thr Asp Gln Glu Thr Thr Gly Val Glu Gly
100 105 110Pro Lys Gly Asp Thr Gly Pro Arg Gly Pro Arg Gly Pro Ala Gly Pro
115 120 125Pro Gly Arg Asp Gly Ile Pro Gly Gln Pro Gly Leu Pro Gly Pro Pro
130 135 140Gly Pro Pro Gly Pro Pro Gly Pro Pro Gly Leu Gly Gly Asn Phe Ala145 150 155 160Pro Gln Leu Ser Tyr Gly Tyr Asp Glu Lys Ser Thr Gly Gly Ile Ser
165 170 175Val Pro Gly Pro Met Gly Pro Ser Gly Pro Arg Gly Leu Pro Gly Pro
180 185 190Pro Gly Ala Pro Gly Pro Gln Gly Phe Gln Gly Pro Pro Gly Glu Pro
195 200 205Gly Glu Pro Gly Ala Ser Gly Pro Met Gly Pro Arg Gly Pro Pro Gly
210 215 220Pro Pro Gly Lys Asn Gly Asp Asp Gly Glu Ala Gly Lys Pro Gly Arg225 230 235 240Pro Gly Glu Arg Gly Pro Pro Gly Pro Gln Gly Ala Arg Gly Leu Pro
245 250 255Gly Thr Ala Gly Leu Pro Gly Met Lys Gly His Arg Gly Phe Ser Gly
260 265 270Leu Asp Gly Ala Lys Gly Asp Ala Gly Pro Ala Gly Pro Lys Gly Glu
275 280 285Pro Gly Ser Pro Gly Glu Asn Gly Ala Pro Gly Gln Met Gly Pro Arg
290 295 300Gly Leu Pro Gly Glu Arg Gly Arg Pro Gly Ala Pro Gly Pro Ala Gly305 310 315 320Ala Arg Gly Asn Asp Gly Ala Thr Gly Ala Ala Gly Pro Pro Gly Pro
325 330 335Thr Gly Pro Ala Gly Pro Pro Gly Phe Pro Gly Ala Val Gly Ala Lys
340 345 350Gly Glu Ala Gly Pro Gln Gly Pro Arg Gly Ser Glu Gly Pro Gln Gly
355 360 365Val Arg Gly Glu Pro Gly Pro Pro Gly Pro Ala Gly Ala Ala Gly Pro
370 375 380Ala Gly Asn Pro Gly Ala Asp Gly Gln Pro Gly Ala Lys Gly Ala Asn385 390 395 400Gly Ala Pro Gly Ile Ala Gly Ala Pro Gly Phe Pro Gly Ala Arg Gly
405 410 415Pro Ser Gly Pro Gln Gly Pro Gly Gly Pro Pro Gly Pro Lys Gly Asn
420 425 430Ser Gly Glu Pro Gly Ala Pro Gly Ser Lys Gly Asp Thr Gly Ala Lys
435 440 445Gly Glu Pro Gly Pro Val Gly Val Gln Gly Pro Pro Gly Pro Ala Gly
450 455 460Glu Glu Gly Lys Arg Gly Ala Arg Gly Glu Pro Gly Pro Thr Gly Leu465 470 475 480Pro Gly Pro Pro Gly Glu Arg Gly Gly Pro Gly Ser Arg Gly Phe Pro
485 490 495Gly Ala Asp Gly Val Ala Gly Pro Lys Gly Pro Ala Gly Glu Arg Gly
500 505 510Ser Pro Gly Pro Ala Gly Pro Lys Gly Ser Pro Gly Glu Ala Gly Arg
515 520 525Pro Gly Glu Ala Gly Leu Pro Gly Ala Lys Gly Leu Thr Gly Ser Pro
530 535 540Gly Ser Pro Gly Pro Asp Gly Lys Thr Gly Pro Pro Gly Pro Ala Gly545 550 555 560Gln Asp Gly Arg Pro Gly Pro Pro Gly Pro Pro Gly Ala Arg Gly Gln
565 570 575Ala Gly Val Met Gly Phe Pro Gly Pro Lys Gly Ala Ala Gly Glu Pro
580 585 590Gly Lys Ala Gly Glu Arg Gly Val Pro Gly Pro Pro Gly Ala Val Gly
595 600 605Pro Ala Gly Lys Asp Gly Glu Ala Gly Ala Gln Gly Pro Pro Gly Pro
610 615 620Ala Gly Pro Ala Gly Glu Arg Gly Glu Gln Gly Pro Ala Gly Ser Pro625 630 635 640Gly Phe Gln Gly Leu Pro Gly Pro Ala Gly Pro Pro Gly Glu Ala Gly
645 650 655Lys Pro Gly Glu Gln Gly Val Pro Gly Asp Leu Gly Ala Pro Gly Pro
660 665 670Ser Gly Ala Arg Gly Glu Arg Gly Phe Pro Gly Glu Arg Gly Val Gln
675 680 685Gly Pro Pro Gly Pro Ala Gly Pro Arg Gly Ala Asn Gly Ala Pro Gly
690 695 700Asn Asp Gly Ala Lys Gly Asp Ala Gly Ala Pro Gly Ala Pro Gly Ser705 710 715 720Gln Gly Ala Pro Gly Leu Gln Gly Met Pro Gly Glu Arg Gly Ala Ala
725 730 735Gly Leu Pro Gly Pro Lys Gly Asp Arg Gly Asp Ala Gly Pro Lys Gly
740 745 750Ala Asp Gly Ser Pro Gly Lys Asp Gly Val Arg Gly Leu Thr Gly Pro
755 760 765Ile Gly Pro Pro Gly Pro Ala Gly Ala Pro Gly Asp Lys Gly Glu Ser
770 775 780Gly Pro Ser Gly Pro Ala Gly Pro Thr Gly Ala Arg Gly Ala Pro Gly785 790 795 800Asp Arg Gly Glu Pro Gly Pro Pro Gly Pro Ala Gly Phe Ala Gly Pro
805 810 815Pro Gly Ala Asp Gly Gln Pro Gly Ala Lys Gly Glu Pro Gly Asp Ala
820 825 830Gly Ala Lys Gly Asp Ala Gly Pro Pro Gly Pro Ala Gly Pro Ala Gly
835 840 845Pro Pro Gly Pro Ile Gly Asn Val Gly Ala Pro Gly Ala Lys Gly Ala
850 855 860Arg Gly Ser Ala Gly Pro Pro Gly Ala Thr Gly Phe Pro Gly Ala Ala865 870 875 880Gly Arg Val Gly Pro Pro Gly Pro Ser Gly Asn Ala Gly Pro Pro Gly
885 890 895Pro Pro Gly Pro Ala Gly Lys Glu Gly Gly Lys Gly Pro Arg Gly Glu
900 905 910Thr Gly Pro Ala Gly Arg Pro Gly Glu Val Gly Pro Pro Gly Pro Pro
915 920 925Gly Pro Ala Gly Glu Lys Gly Ser Pro Gly Ala Asp Gly Pro Ala Gly
930 935 940Ala Pro Gly Thr Pro Gly Pro Gln Gly Ile Ala Gly Gln Arg Gly Val945 950 955 960Val Gly Leu Pro Gly Gln Arg Gly Glu Arg Gly Phe Pro Gly Leu Pro
965 970 975Gly Pro Ser Gly Glu Pro Gly Lys Gln Gly Pro Ser Gly Ala Ser Gly
980 985 990Glu Arg Gly Pro Pro Gly Pro Met Gly Pro Pro Gly Leu Ala Gly Pro
995 1000 1005Pro Gly Glu Ser Gly Arg Glu Gly Ala Pro Ala Ala Glu Gly Ser Pro1010 1015 1020Gly Arg Asp Gly Ser Pro Gly Ala Lys Gly Asp Arg Gly Glu Thr Gly025 1030 1035 1040Pro Ala Gly Pro Pro Gly Ala Pro Gly Ala Pro Gly Ala Pro Gly Pro
1045 1050 1055Val Gly Pro Ala Gly Lys Ser Gly Asp Arg Gly Glu Thr Gly Pro Ala
1060 1065 1070Gly Pro Ala Gly Pro Val Gly Pro Val Gly Ala Arg Gly Pro Ala Gly
1075 1080 1085Pro Gln Gly Pro Arg Gly Asp Lys Gly Glu Thr Gly Glu Gln Gly Asp1090 1095 1100Arg Gly Ile Lys Gly His Arg Gly Phe Ser Gly Leu Gln Gly Pro Pro105 1110 1115 1120Gly Pro Pro Gly Ser Pro Gly Glu Gln Gly Pro Ser Gly Ala Ser Gly
1125 1130 1135Pro Ala Gly Pro Arg Gly Pro Pro Gly Ser Ala Gly Ala Pro Gly Lys
1140 1145 1150Asp Gly Leu Asn Gly Leu Pro Gly Pro Ile Gly Pro Pro Gly Pro Arg
1155 1160 1165Gly Arg Thr Gly Asp Ala Gly Pro Val Gly Pro Pro Gly Pro Pro Gly1170 1175 1180Pro Pro Gly Pro Pro Gly Pro Pro Ser Ala Gly Phe Asp Phe Ser Phe185 1190 1195 1200Leu Pro Gln Pro Pro Gln Glu Lys Ala His Asp Gly Gly Arg Tyr Tyr
1205 1210 1215Arg Ala Asp Asp Ala Asn Val Val Arg Asp Arg Asp Leu Glu Val Asp
1220 1225 1230Thr Thr Leu Lys Ser Leu Ser Gln Gln Ile Glu Asn Ile Arg Ser Pro
1235 1240 1245Glu Gly Ser Arg Lys Asn Pro Ala Arg Thr Cys Arg Asp Leu Lys Met1250 1255 1260Cys His Ser Asp Trp Lys Ser Gly Glu Tyr Trp Ile Asp Pro Asn Gln265 1270 1275 1280Gly Cys Asn Leu Asp Ala Ile Lys Val Phe Cys Asn Met Glu Thr Gly
1285 1290 1295Glu Thr Cys Val Tyr Pro Thr Gln Pro Ser Val Ala Gln Lys Asn Trp
1300 1305 1310Tyr Ile Ser Lys Asn Pro Lys Asp Lys Arg His Val Trp Phe Gly Glu
1315 1320 1325Ser Met Thr Asp Gly Phe Gln Phe Glu Tyr Gly Gly Gln Gly Ser Asp1330 1335 1340Pro Ala Asp Val Ala Ile Gln Leu Thr Phe Leu Arg Leu Met Ser Thr345 1350 1355 1360Glu Ala Ser Gln Asn Ile Thr Tyr His Cys Lys Asn Ser Val Ala Tyr
1365 1370 1375Met Asp Gln Gln Thr Gly Asn Leu Lys Lys Ala Leu Leu Leu Lys Gly
1380 1385 1390Ser Asn Glu Ile Glu Ile Arg Ala Glu Gly Asn Ser Arg Phe Thr Tyr
1395 1400 1405Ser Val Thr Val Asp Gly Cys Thr Ser His Thr Gly Ala Trp Gly Lys1410 1415 1420Thr Val Ile Glu Tyr Lys Thr Thr Lys Ser Ser Arg Leu Pro Ile Ile425 1430 1435 1440Asp Val Ala Pro Leu Asp Val Gly Ala Pro Asp Gln Glu Phe Gly Phe
1445 1450 1455Asp Val Gly Pro Val Cys Phe Leu
1460<210>3<211>20<212>DNA<213>人(Homo sapiens)<220><223>反义寡核苷酸<400>3ggacgaccag gttttccagc 20<210>4<211>20<212>DNA<213>人(Homo sapiens)<220><223>反义寡核苷酸<400>4gcagcaccag cagggccagg 20<210>5<211>20<212>DNA<213>人(Homo sapiens)<220><223>反义寡核苷酸<400>5gccaggagca ccaggttcac 20<210>6<211>20<212>DNA<213>人(Homo sapiens)<220><223>反义寡核苷酸<400>6cttcctctcc agcagggcca 20<210>7<211>20<212>DNA<213>人(Homo sapiens)<220><223>反义寡核苷酸<400>7gccttgccgg gctctccagc 20<210>8<211>20<212>DNA<213>人(Homo sapiens)<220><223>反义寡核苷酸<400>8cgggaacacc tcgctctcca 20<210>9<211>20<212>DNA<213>人(Homo sapiens)<220><223>反义寡核苷酸<400>9gcaggaccga cagcgccagg 20<210>10<211>20<212>DNA<213>人(Homo sapiens)<220><223>反义寡核苷酸<400>10tccatctttg ccagcaggac 20<210>11<211>20<212>DNA<213>人(Homo sapiens)<220><223>反义寡核苷酸<400>11ggtccctgag ctccagcctc 20<210>12<211>19<212>DNA<213>人(Homo sapiens)<220><223>反义寡核苷酸<400>12ttggccgtca gcaccaggg 19<210>13<211>20<212>DNA<213>人(Homo sapiens)<220><223>反义寡核苷酸<400>13tttctcgcca gcagggccag 20<210>14<211>20<212>DNA<213>人(Homo sapiens)<220><223>反义寡核苷酸<400>14ctcgatctgc tggctcaggc 20
Claims (15)
1一种反义DNA寡核苷酸，包括18到25个核苷酸，它与SEQ IDNO：1中750位到3900位包括750位和3900位的核苷酸序列互补，其中SEQ ID NO：1包括编码包含根据SEQ ID NO：2的氨基酸序列的多肽的核苷酸序列，该寡核苷酸能够抑制该多肽在表达该多肽的细胞中的表达。
2.根据权利要求1的寡核苷酸，它与SEQ ID NO：1中750位到900位包括750位和900位的核苷酸序列互补。
3.根据权利要求1的寡核苷酸，它与SEQ ID NO：1中1200位到1300位包括1200位和1300位的核苷酸序列互补。
4.根据权利要求1的寡核苷酸，它与SEQ ID NO：1中1400位到1500位包括1400位和1500位的核苷酸序列互补。
5.根据权利要求1的寡核苷酸，它与SEQ ID NO：1中1450位到1550位包括1450位和1550位的核苷酸序列互补。
6.根据权利要求1的寡核苷酸，它与SEQ ID NO：1中1850位到2000位包括1850位和2000位的核苷酸序列互补。
7.根据权利要求1的寡核苷酸，它与SEQ ID NO：1中2500位到2600位包括2500位和2600位的核苷酸序列互补。
8.根据权利要求1的寡核苷酸，它与SEQ ID NO：1中2850位到2950位包括2850位和2950位的核苷酸序列互补。
9.根据权利要求1的寡核苷酸，它与SEQ ID NO：1中3800位到3900位包括3800位和3900位的核苷酸序列互补。
10.根据权利要求1的寡核苷酸，它选自SEQ ID NO：3、SEQ IDNO：4、SEQ ID NO：5、SEQ ID NO：6、SEQ ID NO：7、SEQ ID NO：8、SEQID NO：9、SEQ ID NO：10、SEQ ID NO：11、SEQ ID NO：12、SEQ ID NO：13和SEQ ID NO：14。
11.一种药物组合物，包括权利要求1至10任一项限定的寡核苷酸与药学上可接受的佐剂、稀释液或载体。
12.一种权利要求11的药物组合物的制备方法，包括将权利要求1至10任一项限定的寡核苷酸与药学上可接受的佐剂、稀释液或载体混合。
13.根据权利要求1至10任一项的寡核苷酸用于治疗。
14.根据权利要求1至10任一项的寡核苷酸在制备用于治疗的药物中的应用。
15.一种治疗患有胶原紊乱或存在胶原紊乱风险患者的胶原紊乱，或降低胶原紊乱风险的方法，包括给患者施与治疗有效量的权利要求1至10任一项限定的寡核苷酸或权利要求11限定的药物组合物。
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB9929487.8 | 1999-12-15 | ||
| GBGB9929487.8A GB9929487D0 (en) | 1999-12-15 | 1999-12-15 | Antisense oligonucleotides |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1423695A true CN1423695A (zh) | 2003-06-11 |
Family
ID=10866254
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN00817282A Pending CN1423695A (zh) | 1999-12-15 | 2000-12-12 | 反义寡核苷酸 |
Country Status (17)
| Country | Link |
|---|---|
| US (1) | US7173122B2 (zh) |
| EP (3) | EP1244781B1 (zh) |
| JP (1) | JP2003516752A (zh) |
| KR (1) | KR20020064936A (zh) |
| CN (1) | CN1423695A (zh) |
| AT (2) | ATE410510T1 (zh) |
| AU (1) | AU2529701A (zh) |
| BR (1) | BR0016347A (zh) |
| CA (1) | CA2390674A1 (zh) |
| DE (2) | DE60040495D1 (zh) |
| GB (1) | GB9929487D0 (zh) |
| IL (1) | IL149640A0 (zh) |
| MX (1) | MXPA02005871A (zh) |
| NO (1) | NO20022799L (zh) |
| NZ (1) | NZ519008A (zh) |
| WO (1) | WO2001044455A2 (zh) |
| ZA (1) | ZA200203878B (zh) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111378657A (zh) * | 2018-12-28 | 2020-07-07 | 苏州瑞博生物技术有限公司 | 抑制COL1A1基因表达的siRNA、含有该siRNA的药物组合物及其用途 |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7396823B2 (en) * | 1999-12-15 | 2008-07-08 | Nath Rahul K | Therapy inhalation involving antisense oligonucleotides for treating idiopathic pulmonary fibrosis |
Family Cites Families (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6265157B1 (en) * | 1991-12-03 | 2001-07-24 | Allegheny University Of The Health Sciences | Compositions and methods for detecting altered COL1A1 gene sequences |
| WO1993011149A1 (en) * | 1991-12-03 | 1993-06-10 | Thomas Jefferson University | Methods of detecting a genetic predisposition for osteoporosis |
| CA2148687A1 (en) * | 1992-11-09 | 1994-05-26 | Darwin Prockop | Antisense oligonucleotides to inhibit expression of mutated and wild type genes for collagen |
| CA2159195A1 (en) * | 1993-03-26 | 1994-10-13 | Darwin J. Prockop | Use of a col 1a1 mini-gene construct to inhibit collagen synthesis |
| DE19508923A1 (de) * | 1995-03-13 | 1996-09-19 | Hoechst Ag | Phosphonomonoesternukleinsäuren, Verfahren zu ihrer Herstellung und ihre Verwendung |
| ATE206131T1 (de) * | 1995-03-13 | 2001-10-15 | Aventis Pharma Gmbh | Phosphonomonoesternnukleinsäuren, verfahren zu ihrer herstellung und ihre verwendung |
| EP0736608A1 (de) * | 1995-04-08 | 1996-10-09 | Roche Diagnostics GmbH | Verfahren zur spezifischen Vervielfältigung und zum Nachweis von DNA bzw. RNA |
| EP0856579A1 (en) * | 1997-01-31 | 1998-08-05 | BIOGNOSTIK GESELLSCHAFT FÜR BIOMOLEKULARE DIAGNOSTIK mbH | An antisense oligonucleotide preparation method |
| US6238921B1 (en) * | 1998-03-26 | 2001-05-29 | Isis Pharmaceuticals, Inc. | Antisense oligonucleotide modulation of human mdm2 expression |
| US6007995A (en) * | 1998-06-26 | 1999-12-28 | Isis Pharmaceuticals Inc. | Antisense inhibition of TNFR1 expression |
| BR0015507A (pt) * | 1999-11-12 | 2002-10-22 | Fibrogen Inc | Gelatinas e colágenos animais |
-
1999
- 1999-12-15 GB GBGB9929487.8A patent/GB9929487D0/en not_active Ceased
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2000
- 2000-12-12 JP JP2001545532A patent/JP2003516752A/ja active Pending
- 2000-12-12 NZ NZ519008A patent/NZ519008A/en unknown
- 2000-12-12 WO PCT/GB2000/004741 patent/WO2001044455A2/en active IP Right Grant
- 2000-12-12 BR BR0016347-3A patent/BR0016347A/pt not_active Application Discontinuation
- 2000-12-12 EP EP00988966A patent/EP1244781B1/en not_active Expired - Lifetime
- 2000-12-12 CA CA002390674A patent/CA2390674A1/en not_active Abandoned
- 2000-12-12 AT AT05024183T patent/ATE410510T1/de not_active IP Right Cessation
- 2000-12-12 AT AT00988966T patent/ATE309338T1/de not_active IP Right Cessation
- 2000-12-12 DE DE60040495T patent/DE60040495D1/de not_active Expired - Lifetime
- 2000-12-12 IL IL14964000A patent/IL149640A0/xx unknown
- 2000-12-12 EP EP08017586A patent/EP2025752A3/en not_active Withdrawn
- 2000-12-12 MX MXPA02005871A patent/MXPA02005871A/es unknown
- 2000-12-12 KR KR1020027007591A patent/KR20020064936A/ko not_active Application Discontinuation
- 2000-12-12 CN CN00817282A patent/CN1423695A/zh active Pending
- 2000-12-12 US US10/149,352 patent/US7173122B2/en not_active Expired - Fee Related
- 2000-12-12 DE DE60023943T patent/DE60023943T2/de not_active Expired - Lifetime
- 2000-12-12 AU AU25297/01A patent/AU2529701A/en not_active Abandoned
- 2000-12-12 EP EP05024183A patent/EP1698695B1/en not_active Expired - Lifetime
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2002
- 2002-05-15 ZA ZA200203878A patent/ZA200203878B/xx unknown
- 2002-06-12 NO NO20022799A patent/NO20022799L/no not_active Application Discontinuation
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111378657A (zh) * | 2018-12-28 | 2020-07-07 | 苏州瑞博生物技术有限公司 | 抑制COL1A1基因表达的siRNA、含有该siRNA的药物组合物及其用途 |
| CN111378657B (zh) * | 2018-12-28 | 2024-03-15 | 苏州瑞博生物技术股份有限公司 | 抑制COL1A1基因表达的siRNA、含有该siRNA的药物组合物及其用途 |
Also Published As
| Publication number | Publication date |
|---|---|
| EP1698695A2 (en) | 2006-09-06 |
| WO2001044455A3 (en) | 2002-01-10 |
| EP2025752A2 (en) | 2009-02-18 |
| KR20020064936A (ko) | 2002-08-10 |
| US7173122B2 (en) | 2007-02-06 |
| CA2390674A1 (en) | 2001-06-21 |
| ZA200203878B (en) | 2003-08-15 |
| DE60023943D1 (de) | 2005-12-15 |
| IL149640A0 (en) | 2002-11-10 |
| NO20022799D0 (no) | 2002-06-12 |
| BR0016347A (pt) | 2002-09-10 |
| NO20022799L (no) | 2002-06-12 |
| EP1244781B1 (en) | 2005-11-09 |
| NZ519008A (en) | 2004-04-30 |
| AU2529701A (en) | 2001-06-25 |
| DE60040495D1 (de) | 2008-11-20 |
| GB9929487D0 (en) | 2000-02-09 |
| ATE309338T1 (de) | 2005-11-15 |
| MXPA02005871A (es) | 2002-10-23 |
| ATE410510T1 (de) | 2008-10-15 |
| JP2003516752A (ja) | 2003-05-20 |
| EP1244781A2 (en) | 2002-10-02 |
| EP2025752A3 (en) | 2009-08-12 |
| EP1698695B1 (en) | 2008-10-08 |
| WO2001044455A2 (en) | 2001-06-21 |
| DE60023943T2 (de) | 2006-08-03 |
| EP1698695A3 (en) | 2007-02-14 |
| US20030105050A1 (en) | 2003-06-05 |
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