CN1490056A - 针对hiv-1的免疫方法和组合物 - Google Patents
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Abstract
本发明提供了用于治疗有患HIV相关疾病危险或正患有HIV相关疾病的受试者的核酸分子。本发明包括两条初始的核酸分子ADVAXI和ADVAXII,以及加强牛痘核酸分子ADMVA2。本发明还包括用于治疗和/或防护HIV相关疾病的方法,对患有HIV相关疾病或有患HIV相关疾病危险的受试者接种的方法,以及包含本发明的核酸疫苗的试剂盒。
Description
发明领域
本发明涉及用于预防HIV-1感染的DNA疫苗,以及对有患HIV感染危险的受试者进行接种的方法。
发明背景
尽管针对HIV-1进行二十多的努力,但其全球流行一直困扰着人类。至今,大约2千2百万人死于AIDS,目前超过3千6百万人感染HIV-1。对发展中世界的影响不成比例,占总病例的95%。在5%接受抗逆转录病毒治疗的人当中,绝大部分由于副作用不能耐受现有药物,另一部分具有药物抗性病毒变体。尽管公共卫生事业的发展可有助于延缓HIV-1在某些地区的传播速度,但是很明显,防护性的疫苗将是解决全球问题的最令人满意的方法。
目前,世界上50%以上的新HIV-1感染是由HIV-1病毒的亚型C引起的。亚型C从印度和缅甸传到了中国,估计是通过在国家西南部的静脉注射吸毒者中间传播的。云南省特别严重,约占全中国HIV-1病例中的一半以上。根据云南省卫生局的资料,2000年该省IVDU中HIV-1感染流行率是29%,预计到2005年将达到40.7%。云南省的5个县(文山、红河、德洪、陵长和大理)的流行率最高,估计为50-70%。HIV-1亚型C也已传播到邻省,例如四川和广西,并引起很远的新疆的许多感染。
DNA接种,或遗传免疫是疫苗学上有希望的新策略。先前设计针对HIV-1的疫苗的尝试表明常规方法如蛋白质/亚基或失活病毒对逆转录病毒的感染不起作用。另外,广泛认为活的减毒疫苗在用于对抗HIV-1时有不能接受的危险。因此,用核酸免疫是新的、及时的想法。
发明简述
本发明提供了针对HIV-1的DNA疫苗,以及最大实用性和效率的接种方法学策略。本发明的预防疫苗方案包括用包含两种新DNA载体的接种物初始免疫,然后用表达相应HIV-1蛋白质的修饰的牛痘Ankara(Modified Vaccinia Ankara)(MVA)重组核酸加强免疫。
第一方面,本发明的特征在于核酸载体,其是从商品化的质粒(pVAX1)(InvitrogenTM)开发而来的(图1),并通过插入另外的启动子修饰。在一个实施方案中,该核酸载体包含人延伸因子1α(hEF1α)启动子(pADVAX)(图2)
第二方面,本发明的特征在于包含两种HIV-1基因的核酸载体,其中每种HIV-1基因由不同的启动子控制。在更具体的实施方案中,本发明的核酸载体包含pCMV启动子和人延伸因子1α(hEF1α)启动子。在另一个实施方案中,HIV-1基因包含env和gag。在更具体的实施方案中,对核酸载体进行密码子优化以在人类受试者中表达。在另一更具体的实施方案中,该核酸载体进一步包含tPA前导序列:(MDAMKRGLCCVLLLCGAVFVSAR)(SEQ ID NO:1)。在更具体的实施方案中,核酸载体包含密码子优化的HIV-1 env基因和tPA前导序列,其包含SEQID NO:7的序列和编码SEQ ID NO:8的氨基酸序列。在另一更具体的实施方案中,核酸载体包含密码子优化的HIV-1 gag基因和tPA前导序列,其包含SEQ ID NO:9的核酸序列,编码SEQ ID NO:10的氨基酸序列。在另一实施方案中,核酸载体包含密码子优化的HIV-1 tPA-env和tPA-gag基因,其具有SEQ ID NO:15的序列(ADVAXI)。
在另一实施方案中,HIV-1基因包含pol、nef和tat。在更具体的实施方案中,对核酸载体进行密码子优化以在人类受试者中表达。在一个实施方案中,核酸载体包含密码子优化的HIV-1 pol和tPA序列,其包含SEQ ID NO:11的序列(其编码SEQ ID NO:12的氨基酸序列)。在另一具体的实施方案中,核酸载体包含密码子优化的HIV-1 nef-tat融合基因序列和tPA前导序列,其包含SEQ ID NO:13的序列(编码SEQ ID NO:14的氨基酸序列)。在另一实施方案中,核酸载体包含密码子优化的HIV-1tPA-pol和tPA-nef-tat基因,其具有SEQ ID NO:16的序列(ADVAXII)。
第三方面,本发明的特征在于包含两种核酸载体的接种物,其中每种载体包含两个启动子,每个启动子控制一种或多种HIV-1基因的表达。在更具体的实施方案中,一种核酸载体包含pCMV启动子和hEF1α启动子,以及HIV-1基因gag和nev,另一种核酸载体包含pCMV启动子和hEF1α启动子,以及HIV-1基因pol、nef和tat。在另一个更具体的实施方案中,一种核酸载体包含pCMV启动子和hEF1α启动子,以及HIV-1基因gag-pol和nev,另一种核酸载体包含pCMV启动子和hEF1α启动子,以及HIV-1基因nef和tat。在更具体的实施方案中,接种物包含一种核酸载体(其包含密码子优化的HIV-1 tPA-env和tPAa-gag基因,具有SEQ IDNO:15的序列(ADVAXI))和另一种核酸载体(其包含密码子优化的HIV-1tPA-pol和tPA-nef-tat基因,具有SEQ ID NO:16的序列(ADVAXII))。
第四方面,本发明的特征在于包含两条插入到MVA DelIII区的核酸序列的修饰的牛痘Ankara(MVA)载体,其中每条核酸序列受单独的启动子控制。在更具体的实施方案中,第一种修饰的载体包含表达三种HIV-1序列,env和gal-pol融合序列的修饰的MVA载体,其中env序列是编码SEQ ID NO:18氨基酸序列的的tPA-δV2 env(SEQ ID NO:17);融合基因序列tPA-gag-pol包含编码SEQ ID NO:20的氨基酸序列(ADMVA1)的SEQID NO:19的序列。
第五方面,本发明的特征在于另一种修饰的载体,其包含表达五种HIV-1融合基因序列:env、gal-pol和nef-tat的修饰的DNA。更具体而言,该修饰的MVA载体包含编码氨基酸序列SEQ ID NO:18的tPA-δV2 env(SEQ ID NO:17);编码SEQ ID NO:20的氨基酸序列的tPA-gag-pol(SEQID NO:19),以及编码SEQ ID NO:22的氨基酸序列(ADMVA2)的tPA-nef-tatSEQ ID NO:21。
第六方面,本发明的特征在于针对HIV-1对人类受试者进行接种的方法,其包含给予DNA疫苗,该疫苗包含一种含有ADVAXI(SEQ ID NO:15)的核酸及另一种含有ADVAXII(SEQ ID NO:16)的核酸。在一个实施方案中,用ADVAXI和II初步免疫后,用表达相应HVI-1蛋白质的修饰的牛痘Ankara(ADMVA2)载体进行加强免疫。
第七方面,本发明的特征在于用于给予本发明的一种或多种核酸的试剂盒,其用于治疗有患HIV相关疾病危险或患有HIV相关疾病的人类受试者,其包含初始DNA疫苗ADVAXI和ADVAXII中的一种或两种(单独或联合使用),以及用于制备或传递疫苗的试剂、缓冲液或溶液。该试剂盒还包括使用试剂盒中组分进行接种或治疗HIV相关疾病的用法说明。该试剂盒还可包括进行对本发明的核酸给药的仪器和装置,例如注射器和针。在本发明的一个实施方案中,该试剂盒包括两种初始DNA疫苗ADVAXI和ADVAXII,单独或联合使用,以及加强疫苗ADMVA2,和用于制备或传递该疫苗的试剂、缓冲液或溶液,及使用该试剂盒的说明。
本发明的其他特征和好处在发明详述、附图和实施例中很明显。
附图简述
图1是pVAX1的简图。
图2是pADVAX(包括人延伸因子1α(hEF1α)作为另一启动子的修饰的pVAX1)的简图。
图3是为ADVAXII而进行的对pol基因的修饰的简要示意图。PR=蛋白酶,RT=逆转录酶,IN=整合酶。蛋白酶中的缺失(DTGA)包含野生型基因的25-28位氨基酸。在逆转录酶中的点实变(M到G)是在野生型基因的184位。对用pVAX1-tPA-实变的pol(A)和单独的pVAX1(B)转染的293T细胞的上清液进行Western印迹。未断裂的tPA-pol是110KD。
图4是对用nef构建体:单独的载体、nef、tPA-nef和tpa-nef-tat转染的293T细胞上MHC-I类的表达的流式细胞术分析结果。
图5是ADVAXI和ADVAXII的简图。
图6是对来源于肽混合物的Env和Gag的IFN-γELISpol反应。肽是有10处重叠的20聚体。每种混合物包括12种肽,除GagA-I是以前鉴定的BALB/c小鼠上CTL表位的特异的9聚体(AMGMLKDTI)(SEQ ID NO:2)。Env P1包含24-144位的氨基酸,Env P4包含403-573位的氨基酸,GagP3包含251-380位的氨基酸,Gag A-1包含217-225位的氨基酸。
图7显示了用不同剂量的ADVAXI接种的小鼠中Env-和Gag-特异的IFN-γELESpot反应。
图8是从pLW7构建pZC1和pZC3的简图。
图9是从pLW22构建pZC22的简图。
图10.用不同的DNA疫苗接种的小鼠中血清抗Gag抗体。各组小鼠在0周、3周和6周用不同DNA疫苗IM(肌内注射)免疫。在第8周时收集每只小鼠的血清,每组混合在一起,并用ELISA分析抗Gag抗体反应。
图11.对来源于肽混合物的pol、Tat和Nef的IFN-γELISpot反应。肽是有11处重叠的15聚体,每种混合物包含25-30种肽。在我们修饰的基因中,Pol P2包含92-226位的氨基酸,Pol P4包含343-477位的氨基酸,Pol P7包含711-845位的氨基酸。Pol P8包含839-969位氨基酸,Nef P1和P2包括全部Nef蛋白。
图12.用不同剂量的ADVAXII接种的小鼠中Pol-和Nef-Tat-特异性IFN-γELISpot反应。如前所述,结果反映了对选择的肽混合物的免疫应答。
发明详述
在描述本发明的方法学、载体和组合物之前,应当理解本发明不限于特定的方法,或实验化合物以及所述的实验条件,因为该方法和条件可能改变。也应当理解的是此处所用的术语只是为了描述特定的实施方案,不是为了限制,因为本发明的范围只由所附的权利要求来限制。
在本说明书和所附的权利要求中所用的单数形式除非上下文另有明确规定,包括复数范围。因此例如,涉及到“方法”时包括一种或多种方法,和/或此处所述的和/或在阅读本公开内容后对本领域技术人员很明显的方法等。
除非另有规定,此处所用的技术和科学术语与本发明所属的技术领域的普通技术人员通常理解的意义相同。尽管任何与此处所述的方法和材料类似或等价的方法和材料都可用于本发明的实践或检验,但此处描述的是优选的方法和材料。此处提到的所有出版物此处引用用来参考,以公开和描述与出版物中涉及的方法和/或材料相关的方法和/或材料。
定义
短语“与HIV感染相关的疾病”或“HIV-1相关疾病”等,此处是指此HIV感染为特征的疾病状态。此类与HIV感染相关的疾病包括但不限于AIDS、卡波西肉瘤(Kaposi’s Sarcoma)、机会性感染例如由卡氏肺囊虫(Pneumocystis carinii)和结核分枝杆菌(Mycobacteriumtuberculosis)引起的感染;包括鹅口疮、毛状白斑和口腔溃疡的口腔损伤;全身淋巴结病;带状疱疹;血小板减少症;无菌性脑膜炎;神经系统疾病如弓形体病、隐球菌病(cyrptoco ccosis)、CMV感染、原发性CNS淋巴瘤和HIV相关的疾病;末梢神经病;癫痫发作和肌病。
有患HIV感染或HIV相关疾病“危险”的人是指其HIV感染的危险比群体的危险高的人。
术语“治疗上有效的剂量”是指因所给剂量而产生目的效果的剂量。确切的剂量取决于治疗的目的,并可由本领域技术人员利用已知的技术确定(例如参见Lieberman(1992)Pharmaceutical Dosage FormsVOl.1-3;Lolyd(1999)The Art,Science and Technology ofpharmaceutical Compounding;and Pickar(1999)Dosage Calculations)。对于本发明的DNA疫苗的治疗上有效的剂量是对个体HIV感染或AIDS治疗中获得成功的必需量,包括任何客观的和主观的标准,如HIV病毒抑制、与HIV感染和AIDS有关的病状的减少,或改善了病人的生理或心理健康。
发明概述
本发明涉及将编码病毒抗原的DNA直接注射入皮肤或肌肉的策略。然后局部细胞吸收该质粒并表达外源蛋白本身,基本上在原位产生疫苗免疫原。该方法经济实用。但更重要的是,该方法可在体内非常有效。
已知细胞介导的免疫(CMI)对控制HIV-1复制非常重要(Ogg等人,(1998)Science 279:2103-6;Schmitz等人,(1999)Science283:857-60;Jin等人,(1999)J.Exp.Med.189:991-8;McMichael等人,(2001)Nature 410:980-7)。也许由于涉及从头表达,看起来DNA接种可以针对CMI的生成产生更好的抗原呈递作用。在一项研究中,DNA接种可至少部分保护恒河猴免受致病性SHIV的实验性攻击(Barouch等,(2000)Science 290:486-92)。但是,在重组载体联合作为初始-加强免疫方案中,DNA接种甚至对刺激CMI和封闭SHIV的感染更有效(Robinson等,(1999)Nat.Med.5:526-34;Hanke等,(1999)Vaccine17:589-96;Hanke等,(1999)J.Virol.73:7524-32;Allen等,(2000)J.Immunol.164:4968-78;Amara等,(2001)Science 292:69-74;Barouch等,(2001)J.Virol.75:5151-8)。
在下述实验中,构建了两种独特的初始DNA疫苗,其包括两种双启动子载体:表达env和gag的ADVAXI,以及表达pol和nef-tat的ADVAXII(图5)。这两种疫苗所携带的全部五种HIV-1基因都在体外进行了全面的评估,以确保在独特的质粒载体中的表达能力和安全性。该实验还描述了表达相应HIV-1蛋白质(ADMVA2)并用于接种ADVAXI和II后加强免疫应答的修饰的Modified Vaccinia AnKara(修饰的牛痘Ankara)(MVA)载体。
给药
药物可接受的载体部分地由预给药的特定的组合物以及化合物给药的特定方法所决定。本发明包括包含核酸的药物组合物的传递。因此,存在许多该核酸药物组合物的合适制剂(例如参见RemingtonsPharmaceutical Science,17thed.1989)。可以任何常规的方式给药,例如,注射、口服、吸入法、经皮的应用或直肠给药。
适于非肠道给药(例如肌内的、皮内的和皮下途径)的制剂包括水性的和非水性等渗的无菌注射液,其可包括抗氧化剂、缓冲液、制菌剂,以及使制剂与受者血液等渗的溶质、水性和非水性的无菌悬液(其可包括悬浮剂、增溶剂、加厚剂、稳定剂和防腐剂)。在本发明的实践中,组合物可通过例如静脉注射、口腔、局部、腹膜内、膀胱内或鞘内给药。非肠道给药是优选的给药方法。制剂可以单位剂量或多剂量密封在容器(如安瓿和小瓶)内存在。
利用本领域已知的方法制备用于给药的纯化的疫苗溶液,包括过滤溶液来除菌、稀释溶液、添加佐剂和稳定溶液。可以将该疫苗冻干,制备干燥形式的针对HIV的疫苗,以便于运输和贮存。进一步地可将该疫苗制成初始疫苗ADVAXI和ADVAXII单独成联合形式、单独加强疫苗ADMVA2形式或可包括至少一种其它抗原的形式,只要所加的抗原不干扰初始或加强疫苗的效力、不另外或协同增加副作用和有害的反应即可。
试剂盒
本发明包括用于接种需要接种的对象的试剂盒。在一个实施方案中,本发明的试剂盒通常包括本发明的初始DNA疫苗,包括ADVAXI和ADVAXII(单独地或联合地)。在本发明的另一个实施方案中,试剂盒包括本发明的加强疫苗ADMVA2。在另一个实施方案中,试剂盒既包括单独或联合的初始疫苗ADVAXI和ADVAXII,也包括加强疫苗ADMVA2。试剂盒还包括给药本发明疫苗必需的试剂、溶液或缓冲液,以及医疗设备,如注射器和针,还有如何使用试剂盒中组分的用法说明。
DNA疫苗ADVAXI和II的构建
本发明的预防性疫苗方案包括用包含两种新DNA载体的接种物初始免疫,然后用表达相应HIV-1蛋白质的修饰的牛痘Ankara(MVA)重组体加强免疫。用于瞬时疫苗的基因衍生自分化体C(clade C)菌株:Circulating Recombinant Form007(循环重组型007)或HIVCHN.AD,其还包括分化体B(clade B)(在云南省内的优势亚型)中的小片段。
大多数针对HIV-1的疫苗研究涉及病毒的结构基因产物:Env、Gag和Pol,其为包含潜在重要的免疫原性表位的蛋白质。但是,越来越多的证据表明HIV-1调节蛋白质如Nef和Tat的免疫原性重要性(Allen等,(2000)Nature 407:386-90;Addo等,(2001)Pros.Natl.Acad.Sci.USA98:1781-6;Cafaro等,(1999)Nat.Med.5:643-50;Pauza等,(2000)Proc,Natl.Acad.Sci.USA 97:3515-9;Cafaro等,(2001)Vaccine19:2862-77),特别是针对CMI诱导的重要性。因为该基因产物在病毒生活周期的早期表达,因此它们可能是免疫控制HIV-1感染的关键物质。因此,结构基因和调节基因都包括在本发明策略中,并被设计为包含最多的免疫原性表位。
本发明的DNA疫苗以Invitrogen的商品化质粒pVAX1为基础(图1)。对该载体进行特别的设计以用于开发DNA疫苗,并构建该载体以符合美国食品与药品管理(FDA)的规定(Center for Biologics Evaluation andResearch,FDA,22 Dec 1996,Docket NO.96N-0400)。但是,通过插入另外的启动子对原始载体进行修饰。从商品化的载体pBudCE4.1(Invitrogen)中利用PCR扩增人延伸因子1α(hEF1α)启动子。将该启动子克隆到pVAX1的EcoR1/Not1位点,并用测序验证新构建体。其它人已对hEF1α启动子进行了完善的性质鉴定(Najjar等,(1999)Gene230:41-5;Nishimura等,(1999)Vaccine 18:675-80;Wallich等,(2001)Infect.Inmun.69:2130-6)。发现经过对pVAX1的这种改变产生的pADVAX(图2)允许另外基因插入片段的独立的高水平表达。pADVAX的双顺反子能力比利用内部核糖体进入位点或IRES获得的更有效(10-20倍)(Martinez-Salas(1999)Curr.Opin.Biotechnol.10:458-64)。Western印迹表明在pADVAX的双启动子的控制下的每一基因的蛋白质表达水平可以达到pVAX1中由CMV启动子单独驱动的水平。
在构建pADVAX载体后,准备用于插入的病毒基因。合成包含最适于哺乳动物表达的密码子的HIV-1 env和gag基因。密码子优化可以促进Rev/RRE-独立的核输出(Schneider等,(1997)J.Virol.71:4892-903;Kotsopoulou等,(2000)J.Virol.74:4839-52),并且一直发现其可加强病毒基因的表达。用重叠PCR将具有反映该理想密码子选择的序列的寡核苷酸(80-960聚体,有16-18处重复)联结起来。由此,基因表达提高100-1000倍(由ELISA或Western印迹测量)。
还通过掺入组织纤溶酶原激活剂(tPA)前导序列(MDAMKRGLCCVLLLCGAVFVSAR)(SEQ ID NO:1)、置换env的天然序列和补充gag基因对该基因进行进一步修饰。认为该序列通过协助蛋白质从内质网(ER)到高尔基体的运输而部分地增加表达(Haddad等,(1997)FEMS Immunol.Med.Microbiol.18:193-202;Li等(1999)Infect.Immun.67:4780-6;Weiss等,(1999)Vaccine 18:815-24;Qiu等,(2000)Virol.74:5997-6005)。通过该修饰,基因表达进一步被增加3-5倍。在利用针对env基因产物的多克隆抗体进行的Western印迹测量中,对通过密码子优化和加入tPA前导序列而得到的增加的env表达获得类似的结果(结果未列出)。该结果确定了利用基因修饰保存了Env功能。在涉及带有CD4/CCR5(HIV-1受体/共-受体)的Hela细胞的融合试验中,用tPA-优化的env载体转染的293T细胞能够融合形成合胞体(结果未列出)。
通过适当的所需的基因修饰,将两种HIV-1基因克隆到pADVAX中产生第一种疫苗ADVAXI。用Western印迹确证双顺子表达(结果未列出)。
再如上所述构建第二种疫苗,用重叠PCR联结密码子优化的寡核苷酸以合成pol、nef和tat。但是,采取另外的措施以确保体内应用的安全性。首先,在pol基因中包含蛋白酶(PR)活性位点的缺失。这可防止多肽的加工(Loeb等(1989)Nature 340:307-400),结果由Western印迹确证(图3)。采取了另外的预防措施,还在pol基因中的逆转录酶(RT)活性位点掺入了点突变(Wakefield等,(1992)J.Virol66:6806-12;Chao等,(1995)Nucleic Acid Res.23:803-10)。
为了能将全部3种基因掺入到一个基于pADVAX的载体中,利用重叠PCR产生nef-tat融合基因。结果两种基因序列都保持完整,因而在所得的融合蛋白质中保留了所有免疫原性表位。同前一样,将tPA前导序列加入到pol和nef-tat中。通过对相关载体转染的293T细胞裂解物和上清液进行Western印迹证实获得的表达和分泌效率的增加。所用的抗体是多克隆兔抗-Nef抗体(由Cecilia Cheny-Mayer博士提供)。
重要的是,同Pol一样,必须确保Nef-Tat融合蛋白质体内应用的安全性。已知Nef下调CD4和I类MHC的表面表达(Collins等,(1998)Nature 391:397-401;AiKen等,(1994)Cell 76:853-64;Collins等,(1999)Immunol.Rev.168:65-74),Tat具有免疫抑制作用(可能通过作为一般的反式激活蛋白起作用)(Goldstein,(1996)Nat.Med.2:960-4;Garber等,(1999)Curr.Opin.Immunol.11:460-5)。但是通过流式细胞术分析,我们证明tPA前导序列使Nef对I类MHC的表达的作用失效(图4)。
类似地,在Nef-Tat融合蛋白质中,Tat失去其反式激活的能力。这种现象在“MAGI”实验中很明显,该实验中用到经改造在功能性HIV-1 Tat存在下表达β-半乳糖苷酶基因的Hela细胞(Kimpton等,(1992)J.Virol.66:2232-9)。因此,在加入X-gal底物后,只有在Tat有活性时,细胞才转为蓝色(结果未列出)。因此可以推出,该疫苗产生的Nef-Tat融合蛋白质在体内不会有免疫抑制作用。实际上,既使忽略了通常的反式激活的危险,也已发现编码野生型HIV-1 tat的DNA在无免疫应答的个体中用作疫苗也是安全的(Calarota等,(1999)J.Immunol.163:2330-8)。
在ADVAXI和ADVAXII的构建和体外性质鉴定之后(如上所述),用ELISpot方法评估该载体的体内免疫原性,因为该方法快速、可重复、并对检测CD8+和CD4+T细胞活性灵敏。用5种不同的疫苗pVAX1-env、pVAX1-gag、pVAX1-env、pVAX1-gag、pVAX1(对照)和ADVAXI免疫BALB/c小鼠。将各组处死的小鼠的脾细胞合并,并测定用Env和Gag抗原特异性肽混合物体外再刺激过程中干扰素-γ(IFN-γ)。针对两种单启动子驱动的载体(pVAX1-env和pAVX1-gag)都产生强的免疫应答,每种在每一百万脾细胞中产生大约700个点形成细胞(spot-forming Cell,SFC)。由ADVAX1诱导的免疫应答相对明显,针对所检测的Env和Gag肽混合物产生大约600SFC/百万脾细胞(图6)。
总之,该细胞介导的免疫应答定向针对至少九种不同的表位,包括一种被发现对CD8+或CD4+T细胞特异的表位(数据未列出)。没有检测到在ADVAXI的两种基因产物间有免疫原性协同作用或干扰作用。剂量放大实验揭示了明显的剂量-反应效果(图7)。
作为HIV-1疫苗的修饰的牛痘Ankara
目前痘苗病毒是在哺乳动物细胞的细胞质中表达基因的唯一的活载体。重组痘苗病毒已经作为科学工具用于研究防护包括AIDS的特异性感染性疾病所需的免疫应答的类型(Girard(1990)CaccerDetect.Prev.14:411-3;Haynes(1996)The Lancet 348:933-7;Moss(1996)Pros.Natl.Acad.Sci.USA 93:11341-8)。由于痘苗病毒对人有感染性,因此使用活痘苗病毒时所关心的主要问题是它们的安全性。不能在无免疫应答的病人(例如带HIV的患者、血癌患者或正接受化疗的患者)中使用常规的痘苗病毒。为此,已经开发了几种高度减毒的痘苗病毒菌株用作天花疫苗(Paoletti(1996)Proc.Natl.Acad.Sci.USA93:11349-53)。尽管不再需要这些减毒的病毒进行针对天花的免疫,但是它们在人类中的早期应用为指导筛选合适的AIDS疫苗的菌株提供了重要的安全信息。
三种高度减毒的和感染性的基于痘病毒的载体,包括NYVAC、金丝雀痘(Canarypox)(ALVAC)和修饰的牛痘Ankara(MVA),可用于作为人和兽医药物中重组疫苗的定向应用(例如参见,Moss等,(1996)Adv.Exp.Med.Biol.397:7-13)。MVA菌株已应用于大量的疫苗试验和超过120000人的初始免疫的临床实践。甚至在高危患者接受初始免疫时,也没有发现与其使用相关的副作用(Mayr等,(1978)ZBL Bakt Hyg.1Abt.Orig.B 167:375-90)。
MVA是宿主范围限制的和高度减毒的痘苗病毒菌株。该MVA菌株已经在鸡胚胎成纤维细胞(CEF)中传代超过570次,由于其基因组中包含六个相对于牛痘WR菌株的重大缺失,MVA菌株已经失去了在大多数哺乳动物细胞系中繁殖的能力(Altenburger等,(1989)Arch.Virol.105:15-27;Meyer等,(1991)J.Gen.Virol.71:1031-8)。该缺失位于病毒基因组两末端的附近,其中的一个缺失影响55K及32K的人类宿主范围基因。进一步分析表明病毒减毒部分由于大约三分之二的宿主范围基因的缺失引起。MVA菌株在禽类细胞中生长良好,但在测试的人类和大多数其它哺乳动物细胞中不能增殖。然而,MVA DNA的复制看起来正常,并在人类细胞中合成早期和晚期的病毒蛋白(Sutter等,(1992)Proc.Natl.Acad.Sci.USA 89:10847-51)。
已经证明高度减毒和复制缺陷的重组MVA的免疫原性和保护效力比许多常规痘苗病毒(VV)强。利用感染复数(Multiplicity ofinfection,MOI)为1,该高度减毒株MVA唯一代表能诱导大量IFNα/β(其具有抗病毒作用)的VV。用五种熟知的常规VV菌株以及中国VV菌株TienTan(VVTT)作为重组疫苗不能诱导白细胞IFN(Buttner等,(1995)Vet,Immunol.46:237-50)。在小动物中,表达HA-NP基因的重组MVA不但诱导血清IgG抗体、粘膜IgA抗体和强CTL反应,也保护免疫的小鼠的肺免受流感病毒的侵袭,甚至在口服免疫之后也能如此(Bender等,(1996)J.Virol.70:6418-24)。最重要的是,用SIV/SHIV重组MVA免疫的恒河猴比用SIV重组NYCBH-VV免疫的恒河猴更有可能成为长期不发展者(nonprogressor)(Hirsch等,(1996)70:3741-52;Amara等,(2001)Science 292:69)。这些恒河猴,就象HIV-1感染的人类长期不发展者一样,具有低水平的、伴随持续病毒复制限制的初始血浆病毒血(症),其与维持正常淋巴细胞亚群和完整的淋巴样结构相关。该结果和先前的有关MVA在人体中的安全性的数据一起暗示了重组MVA在人类AIDS的预防性接种中的重要用途。目前,还没有HIV重组MVA被构建或用于人类HIV-1接种。
原始的MVA插入载体pLW22和pLW7分别靶向MVA基因组的Del II区和Del III区,其从NIH的B.Moss处获得。基于该两种惠赠的载体,产生两种插入载体pZC22和pZC1,其同样靶向Del II区和Del III区。载体pZC1经pLW7修饰而来(图8)。该新插入载体pZC1被构建递送两种外源基因到MVA基因组的Del III区,而不是递送一种外源基因。由于pZC1带有两种不同的启动子,此处不存在潜在启动子竞争的问题。因此,pZC1可以递送env和gag-pol到MVA的Del III区,每种在单独的不同牛痘启动子控制之下,但在同一插入盒中。由于已经证实包膜免疫染色是灵敏的和可靠的,所以可以用env作为替代标志物去筛选gag-pol的存在,其本身很难检测。通过这种方法,对包膜染色呈阳性的细胞也具有整合到基因组上的gag-pol基因。经过多轮富集之后,可以通过western印记确证gag-pol的表达,其比原位免疫染色更灵敏。
如下所述,将HIV-1 env和gag-pol都插入到pZC1中单独的启动子下。通过同源重组将env-gag-pol pZC1插入到野生型MVA的Del III区。通过利用抗Env抗体的免疫染色鉴定重组的env-gag-pol MVA,并通过利用Western印记分析检测Gag-pol的表达进一步确证。因此,两种基因都在双启动子的构建体中进行表达。已经通过利用抗Env抗体的富集/选择进一步增殖了重组的env-gag-pol MVA菌株(“ADMVA1”)。
另一种插入载体(pLW22)经修饰产生pZC22(图9),其递送外源基因至MVA基因组的Del II区。修饰去除了报告基因。Del II区在DelIII区上游超过120kbp处。理论上,利用pZC1和pZC22载体都可以将多HIV-1基因重组进单一的MVA基因组。
为了将5种HIV-1基因用于DNA接种,构建了称为ADMVA2的第2代ADMVA。将HIV-1 nef-tat基因插入pZC22,并经同源重组将该nef-tat pZC22引入噬斑纯化的ADMVA1的Del II区。通过利用抗Env和抗Nef抗体的双重免疫染色鉴定该重组的ADMVA2。已经通过利用抗Env和抗Nef抗体的选择噬斑纯化了该重组的ADMVA2。
用富集的ADMVA2评估感染后的细胞中五种HIV-1基因产物的表达。已经通过western印记分析确证了全部5种基因的有效表达。另外,已经确定全部5种基因都可从ADMVA2基因组DNA中扩增。序列分析已经确证了插入基因的身份。
ADMVA2的感染力可以达到108-109TCID50/mL,并且病毒很容易地以1∶10的比例扩增。ADMVA2在体外传代6次仍然稳定。除鸡胚胎成纤维细胞外,ADMVA2还可感染人细胞。
实施例
提供下列实施例,为本领域普通技术人员提供如何制造和使用本发明的测定、筛选和治疗方法的完整的公开和描述,并不想限制本发明人认为的其发明的范围。已经努力确保所用数据(例如量、温度等)的准确性,但是,应考虑到某些实验错误和误差。除非另有说明,份数是指重量份数,分子量是指平均分子量,温度是摄氏度,压力是或接近大气压。
实施例1.方案:直接ELISA测量免疫动物中抗-HIV-1gag抗体
Gag蛋白质-样品-抗小鼠IgG.ALP。用100μl溶于0.1M NaHCO3(pH9.6)溶液中的Gag蛋白质(0.5μg/孔)4℃过夜包被培养板(Immulon-2,Dynex Technologies,Chantilly,VA或Costar EIA/RIA高结合的96孔板9018,Corning Inc.,Corning,NY)。用200μl的PBS洗一次板,加入含5%脱脂奶粉、0.5%6BSA的PBS室温封闭1-2小时。将在封闭缓冲液中的无菌稀释的动物血清或对照加入到培养板中,并在室温温育1小时。用含有0.05%Tween-20的PBS将板洗涤4次。加入碱性磷酸酶标记的山羊抗小鼠IgG(Pharmingen BD,AKP多克隆山羊抗小鼠Ig,Cat#:554000,lot:M061790),1∶10000稀释:10ml封闭缓冲液中加入1μl的缀合物,并在室温温育板30分钟。用AmpliQ洗涤缓冲液洗4次板。AmpliQ的使用方法如下:在每孔中加入100μl的新配制的底物(50μl溶液A+50μl溶液B),室温作用15分钟。用AmpliQ终止溶液终止反应,并在15分钟内在490nm处读板(AmpliQ:DAKO Diagnostics Ltd.)。
实施例2.竞争性gp120-sCD4 ELISA方案
抗gp120Abs-gp120-sCD4.IgG-抗人IgG.ALP。用溶于0.1M NaHCO3(pH8.6)溶液中的抗gp120抗体室温过夜包被培养板。用PBS将板洗涤2次,加入含有4%脱脂奶粉、0.5%BSA的PBS室温封闭1小时。混合封闭液,并加入gp120上清,室温作用1小时。用含有Tween-20的PBS将板洗涤4次。加入用封闭缓冲液无菌稀释的sCD4或对照,并在室温温育板1小时。用含有Tween-20的PBS将板洗涤4次。加入固定浓度的sCD4-人IgG,并在室温温育板1小时。用含有Tween-20的PBS将板洗涤4次。加入碱性磷酸酶标记的抗人IgG,并在室温温育板30分钟。用AMPAK洗涤缓冲液将板洗涤4次。按如下的AMPAK操作方法进行:加入100μl的新配制的底物作用20分钟,然后加入100μl的扩增液作用10分钟。用50μl 0.5M的H2SO4终止反应,在492nm处读板。
实施例3.小鼠IFN-γELISpot试验
第1天。将ELISpot过滤板进行如下预包被:加入用包被缓冲液1∶50稀释的捕捉抗体(如小鼠IFN-γ)(例如在5ml包被缓冲液中加入125μl抗体)。在每孔中放置100μl的捕捉抗体/包被缓冲液,加盖,然后放在4℃温育过夜。
第2天。收获细胞,用PBST接种4块板。每孔用R10(200μl/孔)37℃封闭2小时。根据特定板的计划加入细胞。加入肽,并在CO2培养箱中37℃温育过夜。
第3天。用PBST洗板5次,然后,每孔加入100μl在1%BSA中1∶60稀释的检测抗体。将板4℃温育过夜。
第4天。用PBST洗板4次,然后在每孔加入100μl在1%BSA中1∶60稀释的SAP。将板室温温育2小时,用PBST洗涤4次,然后用ddH2O洗涤1次。每孔中加入100μl底物,然后室温避光温育大约15分钟或直到完全形成。用自来水洗板,彻底干燥,数点。
实施例4.体内免疫原性评估
用ELISpot试验如上所述评估针对ADVAXI和II的CMI反应(也参见Hanke等,(1998)J.Gen.Virol 79:83-90;Carvalho等,(2001)J.Immunol.Methods 252:207-18;Tobery等,(2001)J.Immunol.Methods254:59-66;Novitsky等,(2001)J.Virol.75:9210-28,该出版物此处全文引用以供参考)。
开始用ADVAXI的GLP级贮存物(Aldevron,Fargo,ND)免疫6-8周龄的雌性BALB/c小鼠。在0周、3周和6周IM给药200μg的疫苗。总共5组、每组6只小鼠,每组进行如下接种:p VAX1-nev,p VAX1-gag,p VAX1-nev+p VAX1-gag,p VAX1(对照)和ADVAXI。肽代表如下的特异表位:已知诱发CD4+细胞介导的反应的Env34(VPVWKEAKTTLFCASDAKAY)(SEQ ID NO:3),诱发CD8+细胞介导的反应的Env43(RNVSSDGTYNETYNEIKNCS)(SEQ ID NO:4),诱发CD4+细胞介导的反应的Gag26(TSNPPIPVGDIYKRWIILGL)(SEQ ID NO:5)和诱发CD8+细胞介导的反应的Gag A-I(AMQMLKDTI)(SEQ ID NO:6或2)。
第3次注射两周后处死小鼠。合并每组的脾细胞,用ELISpot测定在用Env和Gag抗原特异性肽混合物(NIH AIDS研究和参考试剂)体外再刺激过程中其分泌干扰素-γ(IFN-γ)的能力。此时,只有来自异源株(HIV96ZM65.8,目录号3993)的Gag肽。类似地,在ELISpot测定时我们没有整套的同源Env肽(HIVCHN.AD,目录号4974,80%完整)。但是,结果表明针对两种单启动子驱动的载体(p VAX1-nev和p VAX1-gag)都有强的免疫应答,每种产生大约700个点形成细胞(spot-formingcells,SFC)/百万脾细胞。由ADVAXI诱导的免疫应答也很明显,大约产生特异性针对所检测的Env和Gag肽混合物的600SFC/百万脾细胞。可断定地,针对p VAX1对照的反应为零,由于脾细胞混合物中缺失CD8+细胞,没有检测到针对GAG A-I的ELISpot反应(图6)。
总之,该细胞介导的免疫应答定向针对至少9种不同的表位,包括被发现特异性针对CD8+或CD4+T细胞的表位(数据未列出)。没有检测到在ADVAXI的两种基因产物之间有免疫原性协同作用和干扰作用的证据。剂量放大试验表明了明显的剂量-反应效果(图7)。对于至少一种表位(Env34),发现150μg时的ELISpot反应量是5μg时的大约7倍。但是,该剂量-反应趋势对所有检测的表位都是正确的,不管是特异性针对CD4+或CD8+细胞介导的反应。
实施例5.临床前体内免疫原性评估
下列数据表明ADVAXI的体内体液免疫原性。在小鼠试验中,在最后(第三次)免疫后两周收集血清样品,用ELISA测试抗-Gag抗体。尽管在用p VAX1-gag接种的小鼠中的滴度最高,在用ADVAXI免疫组中也有较大的滴度,其与接受p VAX1-env+p VAX1-gag的动物中显示的反应相当(图10)。通过western印记也表明在ADVAXI组中收集的血清样本中有针对Env的抗体反应(数据未列或定量)。
用ADVAXII进行了类似的体内研究。具体地,用ADVAXII的GLP-级贮存物(Aldevron,Fargo,ND)免疫6-8周龄的雌性BALB/c小鼠。在0周、3周和6周IM注射200μg疫苗。每组5只、共5组小鼠,每组进行如下接种:p VAX1-pol、p VAX1-nef-tat、p VAX1-pol+pVAX1-nef-tat、p VAX1(对照)和ADVAXII(只有双启动子载体)。第三次注射后两周处死小鼠。然后合并每组的脾细胞,并用ELISpot分析其在用Pol、Tat和Nef衍生的肽混合物体外再刺激过程中分泌干扰素-γ的能力。注意的是在分析中没有分化体C。此处的抗原特异性肽是基于分化体B的共有序列的15聚体(NIH AIDS研究和参考试剂计划:Tat目录号5138,Nef目录号5189,和Pol目录号6208)。但是,和ADVAXI试验一样,我们发现单基因载体和双启动子疫苗都有很好的反应。例如,针对Pol混合物的反应最好的是单独的p VAX1-pol(300-800SFC/百万脾细胞,取决于混合物)。对于p VAX1-pol+p VAX1-nef-tat,结果为180-500SFC/百万脾细胞,对于ADVAXII,其反应为180-600SFC/百万脾细胞。针对Tat混合物,对于p VAX1-nef-tat,其反应大约为180SFC,对于p VAX1-pol+p VAX1-nef-tat和ADVAXII都是大约100SFC。利用Nef混合物,对于pVAX1-nef-tat,其反应为30-200SFC,对于p VAX1-pol+p VAX1-nef-tat和ADVAXII都是20-150SFC(图11)。
我们又用ADVAXII进行了剂量放大研究,表明了明显的剂量-反应效果(图12)。在0周、3周和6周给小鼠IM注射5μg、10μg、50μg、100μg或150μg的DNA。在第8周处死小鼠,合并脾细胞,用衍生自分化体B共有序列的肽体外再刺激(再次重申,没有分化体C试剂)。Pol的反应随剂量的升高而升高,从10μg的250-450SFC到100μg的500-700SFC。(150μg时的反应与100μg时的类似)。Nef的反应范围为大约20SFC至大约200SFC,Tat的反应在大约25至大约100SFC之间。
进行初始-加强疫苗方案的全部计划如下:将ADVAXI和ADVAXII联合作为初始免疫组分,重组的MVA载体组成加强免疫的组分。因此将ADVAXI+II一起给药作为联合接种物进行体外试验。在0周、3周和6周对各组小鼠IM注射5μg、10μg、50μg、100μg或150μg的ADVAXI+II。对照组注射pVAX1-gag、pVAX1-env、pVAX1-pol和pVAX1-nef-tat各50μg的混合物。在最后的免疫之后两周,处死小鼠,将每组的脾细胞合并,用ELISpot测定干扰素-γ的释放。用包含CD4+和CD8+T细胞表位的Env、Gag和Pol特异性肽进行体外再刺激,同样用自体亚型CTat和Nef序列进行体外再刺激。如在单独的ADVAXI和ADVAXII试验中看到的结果一样,联合接种物也对两种载体显示比较好的反应。用ADVAXI+II接种的小鼠针对所有试验的肽(肽混合物)具有抗原特异性反应,并且该反应是以剂量依赖的方式诱导的(表1)。引人注意的是,在该试验中发现用亚型C衍生的Nef和Tat肽混合物体外再刺激的脾细胞上的反应特别强烈,相反,在ADVAXII试验中发现的反应较轻微。这种差异可能是由于异体和自体蛋白质之间的差异造成的。因此,我们相信,在ADVAXII中的nef-tat融合基因实际上可以诱导非常有效的免疫应答。另外,联合接种试验表明用干扰素-γELISpot测量在不同的抗原特异性反应中没有检测到干扰。因此我们可得到结论:可以认为ADVAXI+II可以进一步临床开发为针对HIV-1的初步-加强疫苗策略中的初步免疫的组分。
表1.针对联合接种物和对照的抗原特异性干扰素-γELISpot反应
疫苗 IFN-γ点形成细胞(SFC)/106脾细胞
(剂量) Gag 26 Gag A-I Env 34 Env T-I Pol 223 Pol YLI Pol VGI Tat pool Nef pool1 Nef pool2
pVAX1-gag+
pVAX1-env+
pVAX1-pol+
pVAX1-nef-tat 120 150 350 700 70 500 500 110 400 300
(每种50μg)
ADVAXI+II 112 252 400 700 117 700 830 120 225 370
(150μg+150μg)
ADVAXI+II 70 210 400 600 100 550 500 70 200 330
(100μg+100μg)
ADVAXI+II 30 100 220 500 84 400 420 50 140 160
(50μg+50μg)
ADVAXI+II 30 60 180 250 70 275 375 30 110 140
(10μg+10μg)
ADVAXI+II 10 30 110 180 60 250 370 20 70 140
(5μg+5μg)注:针对两种细胞输入水平归一化每百万脾细胞的点数,并根据双孔将每种样品和抗原进行平均。
Gag 26、Env 34和Pol 223包含CD4表位,而Gag A-I、Env T-I、Pol YLI和Pol VGI包含CD8
表位。用于Tat和Nef的肽混合物衍生自自体亚型C序列。
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Chen,Zhiwei
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20<210>4<211>20<212>PRT<213>人工序列<220><223>片段<400>4Arg Asn Val Ser Ser Asp Gly Thr Tyr Asn Glu Thr Tyr Asn Glu Ile1 5 10 15Lys Asn Cys Ser
20<210>5<211>20<212>PRT<213>人工序列<220><223>片段<400>5Thr Ser Asn Pro Pro Ile Pro Val Gly Asp Ile Tyr Lys Arg Trp lle1 5 10 15Ile Leu Gly Leu
20<210>6<211>9<212>PRT<213>人工序列<220><223>片段<400>6Ala Met Gln Met Leu Lys Asp Thr Ile1 5<210>7<211>2520<212>DNA<213>人(Homo sapiens)<400>7atggatgcaa tgaagagagg gctctgctgt gtgctgctgc tgtgtggagc agtcttcgtt 60agcgccgccg agaacttgtg ggtgaccgtg tactacggcg tgcccgtgtg gaaggaggcc 120aagaccaccc tgttctgcgc ctccgacgcc aaggcctacg agaaggaggt gcacaacgtg 180tgggccaccc acgcctgcgt gcccaccgac cccaaccccc aggagatggt gttggagaac 240gtgaccgaga acttcaacat gtggaagaac gacatggtga accagatgca cgaggacgtc 300atcagcttgt gggaccagag cctgaagccc tgcgtgaagt tgacccccct gtgcgtgacc 360ttggagtgca ggaacgtgag cagcaacggc acctacaacg agacctacaa cgagatcaag 420aactgctcct tcaacgccac caccgtgttg agggacagga agcagaccgt gtacgccctg 480ttctacaggc tggacatcgt gcccctgaac aagaagaact ccagcgagaa ctccagcgag 540tactacaggt tgatcaactg caacacctcc gccatcaccc aggcctgccc caaggtgacc 600ttcgacccca tccccatcca ctactgcacc cccgccggct acgccatcct gaagtgcaac 660gacaagacct tcaacggcac cggcccctgc cacaacgtga gcaccgtgca gtgcacccac 720ggcatcaagc ccgtggtgtc cacccagctg ctgttgaacg gcagcctggc cgagagggag 780atcatcatca ggtccgagaa cctgaccaac aacgtgaaga ccatcatcgt gcacctgaac 840cagtccgtgg agatcgtgtg caccaggccc aacaacaaca ccaggaagag catcaggatc 900ggccccggcc agaccttcta cgccaccggc gacatcatcg gcgacatcag gcaggcccac 960tgcaacatca gcaaggacaa gtggaaggag accttgcaga gggtgggcaa gaagttggcc 1020gagcacttcc ccaacaagac catcgagttc gcctcctcct ccggcggcga cctggagatc 1080accacccaca gcttcaactg caggggcgag ttcttctact gcaacacctc cagcctgttc 1140aacggcacct acatgcccaa cggcaccgag ggcaactcca gctccatcat caccatcccc 1200tgcaggatca agcagatcat caacatgtgg caggaggtgg gccgcgccat gtacgccccc 1260cccatcgagg gcaacatcac ctgcaagtcc aacatcaccg gcctgctgtt ggtgcgcgac 1320ggcggcaagg agaccaacga caccgagacc ttcaggcccg gcggcggcga catgagggac 1380aactggagga gcgagttgta caagtacaag gtggtggaga tcaagccctt gggcatcgcc 1440cccaccgccg ccaagaggag ggtggtggag agggagaaga gggccgtggg catcggcgcc 1500gtgttcctgg gcttcctggg cgccgccggc agcaccatgg gcgccgccag catcaccctg 1560accgtgcagg cccgccagct gctgagcggc atcgtgcagc agcagagcaa cctgctgcgc 1620gccatcgagg cccagcagca cctgctgcag ctgaccgtgt ggggcatcaa gcagctgcag 1680acccgcgttc tggccatcga gcgctacctg aaggaccagc agctgctggg catctggggc 1740tgcagcggca agctgatctg caccaccgcc gtgcactgga acagcagctg gagcaaccgc 1800agccaggagg agatctggaa caacatgacc tggatgcagt gggaccgcga gatcagcaac 1860tacaccaaca ccatctaccg cctgctggag gacagccaga accagcagga gcgcaacgag 1920aaggacctgc tggccctgga caactggaag aacctgtgga gctggttcga catcaccaac 1980tggctgtggt acatccgcat cttcatcatg atcgtgggcg gcctgatcgg cctgcgcatc 2040atcttcgccg tgctgagcat cgtgaaccgc gtgcgccagg gctacagccc cctgagcttc 2100cagaccctga cccccaaccc cggcggcccc gaccgcctgg gccgcatcga ggaggagggc 2160ggcgagcagg acaagaaccg cagcatccgc ctggtgaacg gcttcctggc cctggcctgg 2220gacgacctgc gcaacctgtg ccgcttcagc taccacctgc tgcgcgacct gctgctgatc 2280gtggcccgca tcgtggagct gctgggccgc cgcggctggg aggccctgcg ctactggtgg 2340aacctgctga agtactgggt gcaggagctg aagaacagcg ccgtgagcct gctgaacgcc 2400accgccatcg ccgtggccga gggcaccgac cgcgtgatcg aggtggtgca gggcgcctac 2460cgcgccatcc tgcacatccc ccgccgcatc cgccagggct tcgaggccgc cctgcagtaa 2520<210>8<211>839<212>PRT<213>人<400>8Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly1 5 10 15Ala Val Phe Val Ser Ala Ala Glu Asn Leu Trp Val Thr Val Tyr Tyr
20 25 30Gly Val Pro Val Trp Lys Glu Ala Lys Thr Thr Leu Phe Cys Ala Ser
35 40 45Asp Ala Lys Ala Tyr Glu Lys Glu Val His Asn Val Trp Ala Thr His
50 55 60Ala Cys Val Pro Thr Asp Pro Asn Pro Gln Glu Met Val Leu Glu Asn65 70 75 80Val Thr Glu Asn Phe Asn Met Trp Lys Asn Asp Met Val Asn Gln Met
85 90 95His Glu Asp Val Ile Ser Leu Trp Asp Gln Ser Leu Lys Pro Cys Val
100 105 110Lys Leu Thr Pro Leu Cys Val Thr Leu Glu Cys Arg Asn Val Ser Ser
115 120 125Asn Gly Thr Tyr Asn Glu Thr Tyr Asn Glu Ile Lys Asn Cys Ser Phe
130 135 140Asn Ala Thr Thr Val Leu Arg Asp Arg Lys Gln Thr Val Tyr Ala Leu145 150 155 160Phe Tyr Arg Leu Asp Ile Val Pro Leu Asn Lys Lys Asn Ser Ser Glu
165 170 175Asn Ser Ser Glu Tyr Tyr Arg Leu Ile Asn Cys Asn Thr Ser Ala Ile
180 185 190Thr Gln Ala Cys Pro Lys Val Thr Phe Asp Pro Ile Pro Ile His Tyr
195 200 205Cys Thr Pro Ala Gly Tyr Ala Ile Leu Lys Cys Asn Asp Lys Thr Phe
210 215 220Asn Gly Thr Gly Pro Cys His Asn Val Ser Thr Val Gln Cys Thr His225 230 235 240Gly Ile Lys Pro Val Val Ser Thr Gln Leu Leu Leu Asn Gly Ser Leu
245 250 255Ala Glu Arg Glu Ile Ile Ile Arg Ser Glu Asn Leu Thr Asn Asn Val
260 265 270Lys Thr Ile Ile Val His Leu Asn Gln Ser Val Glu Ile Val Cys Thr
275 280 285Arg Pro Asn Asn Asn Thr Arg Lys Ser Ile Arg Ile Gly Pro Gly Gln
290 295 300Thr Phe Tyr Ala Thr Gly Asp Ile Ile Gly Asp Ile Arg Gln Ala His305 310 315 320Cys Asn Ile Ser Lys Asp Lys Trp Lys Glu Thr Leu Gln Arg Val Gly
325 330 335Lys Lys Leu Ala Glu His Phe Pro Asn Lys Thr Ile Glu Phe Ala Ser
340 345 350Ser Ser Gly Gly Asp Leu Glu Ile Thr Thr His Ser Phe Asn Cys Arg
355 360 365Gly Glu Phe Phe Tyr Cys Asn Thr Ser Ser Leu Phe Asn Gly Thr Tyr
370 375 380Met Pro Asn Gly Thr Glu Gly Asn Ser Ser Ser Ile Ile Thr Ile Pro385 390 395 400Cys Arg Ile Lys Gln Ile Ile Asn Met Trp Gln Glu Val Gly Arg Ala
405 410 415Met Tyr Ala Pro Pro Ile Glu Gly Asn Ile Thr Cys Lys Ser Asn Ile
420 425 430Thr Gly Leu Leu Leu Val Arg Asp Gly Gly Lys Glu Thr Asn Asp Thr
435 440 445Glu Thr Phe Arg Pro Gly Gly Gly Asp Met Arg Asp Asn Trp Arg Ser
450 455 460Glu Leu Tyr Lys Tyr Lys Val Val Glu Ile Lys Pro Leu Gly Ile Ala465 470 475 480Pro Thr Ala Ala Lys Arg Arg Val Val Glu Arg Glu Lys Arg Ala Val
485 490 495Gly Ile Gly Ala Val Phe Leu Gly Phe Leu Gly Ala Ala Gly Ser Thr
500 505 510Met Gly Ala Ala Ser Ile Thr Leu Thr Val Gln Ala Arg Gln Leu Leu
515 520 525Ser Gly Ile Val Gln Gln Gln Ser Asn Leu Leu Arg Ala Ile Glu Ala
530 535 540Gln Gln His Leu Leu Gln Leu Thr Val Trp Gly Ile Lys Gln Leu Gln545 550 555 560Thr Arg Val Leu Ala Ile Glu Arg Tyr Leu Lys Asp Gln Gln Leu Leu
565 570 575Gly Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys Thr Thr Ala Val His
580 585 590Trp Asn Ser Ser Trp Ser Asn Arg Ser Gln Glu Glu Ile Trp Asn Asn
595 600 605Met Thr Trp Met Gln Trp Asp Arg Glu Ile Ser Asn Tyr Thr Asn Thr
610 615 620Ile Tyr Arg Leu Leu Glu Asp Ser Gln Asn Gln Gln Glu Arg Asn Glu625 630 635 640Lys Asp Leu Leu Ala Leu Asp Asn Trp Lys Asn Leu Trp Ser Trp Phe
645 650 655Asp Ile Thr Asn Trp Leu Trp Tyr Ile Arg Ile Phe Ile Met Ile Val
660 665 670Gly Gly Leu Ile Gly Leu Arg Ile Ile Phe Ala Val Leu Ser Ile Val
675 680 685Asn Arg Val Arg Gln Gly Tyr Ser Pro Leu Ser Phe Gln Thr Leu Thr
690 695 700Pro Asn Pro Gly Gly Pro Asp Arg Leu Gly Arg Ile Glu Glu Glu Gly705 710 715 720Gly Glu Gln Asp Lys Asn Arg Ser Ile Arg Leu Val Asn Gly Phe Leu
725 730 735Ala Leu Ala Trp Asp Asp Leu Arg Asn Leu Cys Arg Phe Ser Tyr His
740 745 750Leu Leu Arg Asp Leu Leu Leu Ile Val Ala Arg Ile Val Glu Leu Leu
755 760 765Gly Arg Arg Gly Trp Glu Ala Leu Arg Tyr Trp Trp Asn Leu Leu Lys
770 775 780Tyr Trp Val Gln Glu Leu Lys Asn Ser Ala Val Ser Leu Leu Asn Ala785 790 795 800Thr Ala Ile Ala Val Ala Glu Gly Thr Asp Arg Val Ile Glu Val Val
805 810 815Gln Gly Ala Tyr Arg Ala Ile Leu His Ile Pro Arg Arg Ile Arg Gln
820 825 830Gly Phe Glu Ala Ala Leu Gln
835<210>9<211>1545<212>DNA<213>人<400>9atggacgcca tgaagcgcgg cctgtgctgc gtgctgctgc tgtgcggcgc cgtgttcgtg 60agcgcccgca tgggcgcccg cgccagcatc ctgcgcggcg gcaagctgga caagtgggag 120aagatccgcc tgcgccccgg cggcaagaag cactacatgc tgaagcacct ggtgtgggcc 180agccgcgagc tggagcgctt cgccctgaac cccggcctgc tggagaccag cgagggctgc 240aagcagatca tcaagcagct gcagcccgcc ctgcagaccg gcaccgagga gctgcgcagc 300ctgttcaaca ccgtggccac cctgtactgc gtgcacgagg gcatcgagat ccgcgacacc 360aaggaggccc tggacaagat cgaggaggag cagaacaaga tccagcagaa gacccagcag 420gccaagaagg ccgacgagaa ggtgagccag aactacccca tcgtgcagaa cctgcagggc 480cagatggtgc accaggccat ctcccccagg accttgaacg cctgggtgaa ggtgatcgag 540gagaaggcct tcagccccga ggtgatcccc atgttcaccg ccttgtccga gggcgccacc 600ccccaggact tgaacaccat gttgaacacc gtgggcggcc accaggccgc catgcagatg 660ttgaaggaca ccatcaacga ggaggccgcc gagtgggaca gggtgcaccc cgtgcacgcc 720ggccccatcg cccccggcca gatgagggag cccaggggca gcgacatcgc cggcaccacc 780agcaccctgc agggccagat cgcctggatg accagcaacc cccccgtgcc cgtgggcgag 840atctacaaga ggtggatcat cctgggcttg aacaagatcg tgaggatgta cagccccgtg 900agcatcttgg acatcaagca gggccccaag gagcccttca gggactacgt ggaccgcttc 960ttcaagacct tgagggccga gcaggccacc caggacgtga agaactggat gaccgacacc 1020ttgttggtgc agaacgccaa ccccgactgc aagaccatct tgagggcctt gggccccggc 1080gcctccttgg aggagatgat gaccgcctgc cagggcgtgg gcggccccag ccacaaggcc 1140agggtgttgg ccgaggccat gagccaggcc aacggcacca tcctgatgca gaggagcaac 1200ttcaagggct ccaagaggat cgtgaagtgc ttcgactgcg gcaaggaggg ccacatcgcc 1260aggaactgca gggcccccag gaagaagggc tgctggaagt gcggcaagga gggccaccag 1320atgaaggact gcaccgagag gcaggccaac ttcttgggca agatctggcc ctcccacaag 1380ggcaggcccg gcaacttcct gcagagcagg cccgagccca ccgccccccc cgccgagagc 1440ttcaggttcg aggagaccac ccccgccccc aagcaggagc ccaaggacag ggagcccttg 1500acctccctga agtccctgtt cggcagcgac cccttgtccc agtaa 1545<210>10<211>514<2L2>PRT<213>人<400>10Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly1 5 10 15Ala Val Phe Val Ser Ala Arg Met Gly Ala Arg Ala Ser Ile Leu Arg
20 25 30Gly Gly Lys Leu Asp Lys Trp Glu Lys Ile Arg Leu Arg Pro Gly Gly
35 40 45Lys Lys His Tyr Met Leu Lys His Leu Val Trp Ala Ser Arg Glu Leu
50 55 60Glu Arg Phe Ala Leu Asn Pro Gly Leu Leu Glu Thr Ser Glu Gly Cys65 70 75 80Lys Gln Ile Ile Lys Gln Leu Gln Pro Ala Leu Gln Thr Gly Thr Glu
85 90 95Glu Leu Arg Ser Leu Phe Asn Thr Val Ala Thr Leu Tyr Cys Val His
100 105 110Glu Gly Ile Glu Ile Arg Asp Thr Lys Glu Ala Leu Asp Lys Ile Glu
115 120 125Glu Glu Gln Asn Lys Ile Gln Gln Lys Thr Gln Gln Ala Lys Lys Ala
130 135 140Asp Glu Lys Val Ser Gln Asn Tyr Pro Ile Val Gln Asn Leu Gln Gly145 150 155 160Gln Met Val His Gln Ala Ile Ser Pro Arg Thr Leu Asn Ala Trp Val
165 170 175Lys Val Ile Glu Glu Lys Ala Phe Ser Pro Glu Val Ile Pro Met Phe
180 185 190Thr Ala Leu Ser Glu Gly Ala Thr Pro Gln Asp Leu Asn Thr Met Leu
195 200 205Asn Thr Val Gly Gly His Gln Ala Ala Met Gln Met Leu Lys Asp Thr
210 215 220Ile Asn Glu Glu Ala Ala Glu Trp Asp Arg Val His Pro Val His Ala225 230 235 240Gly Pro Ile Ala Pro Gly Gln Met Arg Glu Pro Arg Gly Ser Asp Ile
245 250 255Ala Gly Thr Thr Ser Thr Leu Gln Gly Gln Ile Ala Trp Met Thr Ser
260 265 270Asn Pro Pro Val Pro Val Gly Glu Ile Tyr Lys Arg Trp Ile Ile Leu
275 280 285Gly Leu Asn Lys Ile Val Arg Met Tyr Ser Pro Val Ser Ile Leu Asp
290 295 300Ile Lys Gln Gly Pro Lys Glu Pro Phe Arg Asp Tyr Val Asp Arg Phe305 310 315 320Phe Lys Thr Leu Arg Ala Glu Gln Ala Thr Gln Asp Val Lys Asn Trp
325 330 335Met Thr Asp Thr Leu Leu Val Gln Asn Ala Asn Pro Asp Cys Lys Thr
340 345 350Ile Leu Arg Ala Leu Gly Pro Gly Ala SerLeu Glu Glu Met Met Thr
355 360 365Ala Cys Gln Gly Val Gly Gly Pro Ser His Lys Ala Arg Val Leu Ala
370 375 380Glu Ala Met Ser Gln Ala Asn Gly Thr Ile Leu Met Gln Arg Ser Asn385 390 395 400Phe Lys Gly Ser Lys Arg Ile Val Lys Cys Phe Asn Cys Gly Lys Glu
405 410 415Gly His Ile Ala Arg Asn Cys Arg Ala Pro Arg Lys Lys Gly Cys Trp
420 425 430Lys Cys Gly Lys Glu Gly His Gln Met Lys Asp Cys Thr Glu Arg Gln
435 440 445Ala Asn Phe Leu Gly Lys Ile Trp Pro Ser His Lys Gly Arg Pro Gly
450 455 460Asn Phe Leu Gln Ser Arg Pro Glu Pro Thr Ala Pro Pro Ala Glu Ser465 470 475 480Phe Arg Phe Glu Glu Thr Thr Pro Ala Pro Lys Gln Glu Pro Lys Asp
485 490 495Arg Glu Pro Leu Thr Ser Leu Lys Ser Leu Phe Gly Ser Asp Pro Leu
500 505 510Ser Gln<210>11<211>2901<212>DNA<213>人<400>11atggacgcca tgaagcgcgg cctgtgctgc gtgctgctgc tgtgcggcgc cgtgttcgtg 60agcgcccgcc cccagatcac cctgtggcag cgccccctgg tgtccatccg cgtgggcggc 120cagatcaagg aggccctgct ggacgacacc gtgttggagg aggtgaactt gcccggcaag 180tggaagccca agatgatcgg cggcatcggc ggcttcatca aggtgaggca gtacgaccag 240atccccatcg agatctgcgg caagaaggcc atcggcaccg tgttggtggg ccccaccccc 300gtgaacatca tcggcaggaa catgttgacc cagctgggct gcaccctgaa cttccccatc 360agccccatcg agaccatccc cgtgaagctg aagcccggca tggacggccc ccgcgtgaag 420cagtggcccc tgaccgagga gaagatcaag gccctgaccg ccatctgcga cgagatggag 480aaggagggca agatcaccaa gatcggcccc gagaacccct acaacacccc cgtgttcgcc 540atcaagaaga aggacagcac caagtggcgc aagctggtgg acttccgcga gctgaacaag 600cgcacccagg acttctggga ggtgcagctg ggcatccccc accccgccgg cctgaagaag 660aagaagtccg tgaccgtgct ggacgtgggc gacgcctact tctccgtgcc cctgtacgag 720gacttccgca agtacaccgc cttcaccatc cccagcatca acaacgagac ccccggcatc 780cgctaccagt acaacgtgct gccccagggc tggaagggct cccccgccat cttccagtgc 840agcatgacca agatcctgga gcccttccgg gcccagaacc ccgagatcgt gatctaccag 900tacggcgacg acctgtacgt gggctccgac ctggagatcg gccagcaccg cgccaagatc 960gaggagttgc gcgagcacct gctgaagtgg ggcttcacca cccccgacaa gaagcaccag 1020aaggagcccc ccttcctgtg gatgggctac gagctgcacc ccgacaagtg gaccgtgcag 1080cccatccagc tgcccgagaa ggacagctgg accgtgaacg acatccagaa gctggtgggc 1140aagctgaact gggccagcca gatctacccc ggcatcaagg tgcgccagct gtgcaagctg 1200ctgcgcggcg ccaaggccct gaccgacatc atccccctga ccgaggaggc cgagctggag 1260ctggccgaga accgcgagat cctgaaggag cccgtgcacg gcgcctacta cgacccctcc 1320aaggacctga tcgccgagat ccagaagcag ggccaggacc agtggaccta ccagatctac 1380caggagccct tcaagaacct gaagaccggc aagtacgcca agatgcgcac cgcccacacc 1440aacgacgtga agcagctgac cgaggccgtg cagaagatct ccatggagag catcgtgatc 1500tggggcaaga tccccaagtt ccgcctgccc atccagaagg agacctggga gacccgctgg 1560accgcctact ggcaggccac ctggatcccc gagtgggagt tcgtgaacac cccccccctg 1620gtgaagctgt ggtaccagct ggagaaggac cccatcgccg gcgtggagac cttctacgtg 1680gacggcgccg ccaaccgcga gaccaagatg ggcaaggccg gctacgtgac cgaccgcggc 1740cgccagaaga tcgtgtccct gaccgagacc accaaccaga agaccgagct gcaggccatc 1800tgcctggcct tgcaggactc cggctccgag gtgaacatcg tgaccgactc ccagtacgcc 1860ctgggcatca tccaggccca gcccgacaag agcgagtccg agctggtgaa ccagatcatc 1920gagcagctga tcaagaagga gcgcgtgtac ctgtcctggg tgcccgccca caagggcatc 1980ggcggcaacg agcaggtgga caagctggtg agcaacggca tccgcaaggt gctgttcctg 2040gacggcatcg acaaggccca ggaggagcac gagaagtacc acagcaactg gcgcgccatg 2100gccagcgact tcaacctgcc ccccatcgtg gccaaggaga tcgtggccag ctgcgaccag 2160tgccagctga agggcgaggc catgcacggc caggtggact gcagccccgg catctggcag 2220ctggactgca cccacctgga gggcaagatc atcctggtgg ccgtgcacgt ggccagcggc 2280tacatcgagg ccgaggtgat ccccgccgag accggccagg agaccgccta cttcatcctg 2340aagctggccg gccgctggcc cgtgaagatc atccacaccg acaacggcag caacttcacc 2400agcgccgccg tgaaggccgc ctgctggtgg gccggcatcc agcaggagtt cggcatcccc 2460tacaaccccc agagccaggg cgtggtggag tccatgaaca aggagctgaa gaagatcatc 2520ggccaggtgc gcgaccaggc cgagcacctg aagaccgccg tgcagatggc cgtgttcatc 2580cacaacttca agcgcaaggg cggcatcggc ggctacagcg ccggcgagcg catcatcgac 2640atcatcgcca ccgacatcca gaccaaggag ctgcagaagc agatcatcaa gatccagaac 2700ttccgcgtgt actaccgcga cagccgcgac cccatctgga agggccccgc caagctgctg 2760tggaagggcg agggcgccgt ggtgatccag gacaacagcg acatcaaggt ggtgccccgc 2820cgcaaggcca agatcatcaa ggactacggc aagcagatgg ccggcgccga ctgcgtggcc 2880agccgccagg acgaggacta g 2901<210>12<211>966<212>PRT<213>人<400>12Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly1 5 10 15Ala Val Phe Val Ser Ala Arg Pro Gln Ile Thr Leu Trp Gln Arg Pro
20 25 30Leu Val Ser Ile Arg Val Gly Gly Gln Ile Lys Glu Ala Leu Leu Asp
35 40 45Asp Thr Val Leu Glu Glu Val Asn Leu Pro Gly Lys Trp Lys Pro Lys
50 55 60Met Ile Gly Gly Ile Gly Gly Phe Ile Lys Val Arg Gln Tyr Asp Gln65 70 75 80Ile Pro Ile Glu Ile Cys Gly Lys Lys Ala Ile Gly Thr Val Leu Val
85 90 95Gly Pro Thr Pro Val Asn Ile Ile Gly Arg Asn Met Leu Thr Gln Leu
100 105 110Gly Cys Thr Leu Asn Phe Pro Ile Ser Pro Ile Glu Thr Ile Pro Val
115 120 125Lys Leu Lys Pro Gly Met Asp Gly Pro Arg Val Lys Gln Trp Pro Leu
130 135 140Thr Glu Glu Lys Ile Lys Ala Leu Thr Ala Ile Cys Asp Glu Met Glu145 150 155 160Lys Glu Gly Lys Ile Thr Lys Ile Gly Pro Glu Asn Pro Tyr Asn Thr
165 170 175Pro Val Phe Ala Ile Lys Lys Lys Asp Ser Thr Lys Trp Arg Lys Leu
180 185 190Val Asp Phe Arg Glu Leu Asn Lys Arg Thr Gln Asp Phe Trp Glu Val
195 200 205Gln Leu Gly Ile Pro His Pro Ala Gly Leu Lys Lys Lys Lys Ser Val
210 215 220Thr Val Leu Asp Val Gly Asp Ala Tyr Phe Ser Val Pro Leu Tyr Glu225 230 235 240Asp Phe Arg Lys Tyr Thr Ala Phe Thr Ile Pro Ser Ile Asn Asn Glu
245 250 255Thr Pro Gly Ile Arg Tyr Gln Tyr Asn Val Leu Pro Gln Gly Trp Lys
260 265 270Gly Ser Pro Ala Ile Phe Gln Cys Ser Met Thr Lys Ile Leu Glu Pro
275 280 285Phe Arg Ala Gln Asn Pro Glu Ile Val Ile Tyr Gln Tyr Gly Asp Asp
290 295 300Leu Tyr Val Gly Ser Asp Leu Glu Ile Gly Gln His Arg Ala Lys Ile305 310 315 320Glu Glu Leu Arg Glu His Leu Leu Lys Trp Gly Phe Thr Thr Pro Asp
325 330 335Lys Lys His Gln Lys Glu Pro Pro Phe Leu Trp Met Gly Tyr Glu Leu
340 345 350His Pro Asp Lys Trp Thr Val Gln Pro Ile Gln Leu Pro Glu Lys Asp
355 360 365Ser Trp Thr Val Asn Asp Ile Gln Lys Leu Val Gly Lys Leu Asn Trp
370 375 380Ala Ser Gln Ile Tyr Pro Gly Ile Lys Val Arg Gln Leu Cys Lys Leu385 390 395 400Leu Arg Gly Ala Lys Ala Leu Thr Asp Ile Ile Pro Leu Thr Glu Glu
405 410 415Ala Glu Leu Glu Leu Ala Glu Asn Arg Glu Ile Leu Lys Glu Pro Val
420 425 430His Gly Ala Tyr Tyr Asp Pro Ser Lys Asp Leu Ile Ala Glu Ile Gln
435 440 445Lys Gln Gly Gln Asp Gln Trp Thr Tyr Gln Ile Tyr Gln Glu Pro Phe
450 455 460Lys Asn Leu Lys Thr Gly Lys Tyr Ala Lys Met Arg Thr Ala His Thr465 470 475 480Asn Asp Val Lys Gln Leu Thr Glu Ala Val Gln Lys Ile Ser Met Glu
485 490 495Ser Ile Val Ile Trp Gly Lys Ile Pro Lys Phe Arg Leu Pro Ile Gln
500 505 510Lys Glu Thr Trp Glu Thr Arg Trp Thr Ala Tyr Trp Gln Ala Thr Trp
515 520 525Ile Pro Glu Trp Glu Phe Val Asn Thr Pro Pro Leu Val Lys Leu Trp
530 535 540Tyr Gln Leu Glu Lys Asp Pro Ile Ala Gly Val Glu Thr Phe Tyr Val545 550 555 560Asp Gly Ala Ala Asn Arg Glu Thr Lys Met Gly Lys Ala Gly Tyr Val
565 570 575Thr Asp Arg Gly Arg Gln Lys Ile Val Ser Leu Thr Glu Thr Thr Asn
580 585 590Gln Lys Thr Glu Leu Gln Ala Ile Cys Leu Ala Leu Gln Asp Ser Gly
595 600 605Ser Glu Val Asn Ile Val Thr Asp Ser Gln Tyr Ala Leu Gly Ile Ile
610 615 620Gln Ala Gln Pro Asp Lys Ser Glu Ser Glu Leu Val Asn Gln Ile Ile625 630 635 640Glu Gln Leu Ile Lys Lys Glu Arg Val Tyr Leu Ser Trp Val Pro Ala
645 650 655His Lys Gly Ile Gly Gly Asn Glu Gln Val Asp Lys Leu Val Ser Asn
660 665 670Gly Ile Arg Lys Val Leu Phe Leu Asp Gly Ile Asp Lys Ala Gln Glu
675 680 685Glu His Glu Lys Tyr His Ser Asn Trp Arg Ala Met Ala Ser Asp Phe
690 695 700Asn Leu Pro Pro Ile Val Ala Lys Glu Ile Val Ala Ser Cys Asp Gln705 710 715 720Cys Gln Leu Lys Gly Glu Ala Met His Gly Gln Val Asp Cys Ser Pro
725 730 735Gly Ile Trp Gln Leu Asp Cys Thr His Leu Glu Gly Lys Ile Ile Leu
740 745 750Val Ala Val His Val Ala Ser Gly Tyr Ile Glu Ala Glu Val Ile Pro
755 760 765Ala Glu Thr Gly Gln Glu Thr Ala Tyr Phe Ile Leu Lys Leu Ala Gly
770 775 780Arg Trp Pro Val Lys Ile Ile His Thr Asp Asn Gly Ser Asn Phe Thr785 790 795 800Ser Ala Ala Val Lys Ala Ala Cys Trp Trp Ala Gly Ile Gln Gln Glu
805 810 815Phe Gly Ile Pro Tyr Asn Pro Gln Ser Gln Gly Val Val Glu Ser Met
820 825 830Asn Lys Glu Leu Lys Lys Ile Ile Gly Gln Val Arg Asp Gln Ala Glu
835 840 845His Leu Lys Thr Ala Val Gln Met Ala Val Phe Ile His Asn Phe Lys
850 855 860Arg Lys Gly Gly Ile Gly Gly Tyr Ser Ala Gly Glu Arg Ile Ile Asp865 870 875 880Ile Ile Ala Thr Asp Ile Gln Thr Lys Glu Leu Gln Lys Gln Ile Ile
885 890 895Lys Ile Gln Asn Phe Arg Val Tyr Tyr Arg Asp Ser Arg Asp Pro Ile
900 905 910Trp Lys Gly Pro Ala Lys Leu Leu Trp Lys Gly Glu Gly Ala Val Val
915 920 925Ile Gln Asp Asn Ser Asp Ile Lys Val Val Pro Arg Arg Lys Ala Lys
930 935 940Ile Ile Lys Asp Tyr Gly Lys Gln Met Ala Gly Ala Asp Cys Val Ala945 950 955 960Ser Arg Gln Asp Glu Asp
965<210>13<211>993<212>DNA<213>人<400>13atggacgcca tgaagcgcgg cctgtgctgc gtgctgctgc tgtgcggcgc cgtgttcgtg 60agcgcccgca tgggcggcaa gtggtccaag agcagcatcg tgggctggcc cgccatccgc 120gagcgcattc gccgcaccga gcccgccgcc gacggcgtgg gcgccgtgtc tcgcgacctg 180gagaagcatg gcgccatcac cagtaacaac accgccgaca ccaacgccga ctgcgcctgg 240ctggagaccc aggaggagga ggaggtgggc ttccccgtcc gcccccaggt gcccttgcgc 300cccatgacct tcaagggcgc cttggacctc agcttcttcc tgaaggagaa gggcggcctg 360gaggggttga tctacagcaa gaagaggcag gagatcctgg acttgtgggt ctaccacacc 420cagggctact tccccgactg gcacaactac acccccggcc ccggcgtccg cttccccctg 480accttcggct ggtgcttcaa gctggtgccc gtggaccccg gcgaggtgga ggaggccaac 540gagggcgaga acaactgctt gctgcacccc gtctgccagc acggcatgga cgacgagcac 600cgcgaggtgc tgaagtggaa gttcgacagc cagctggccc accgccacag ggcccgcgag 660ctgcacccgg agttctacaa ggactgcatg gagcccgtgg accccaacct ggagccctgg 720aaccaccccg gcagccagcc cgagaccgcc tgcaacaact gctactgcaa gcgctgcagc 780taccactgcc tggtgtgctt ccagaagaag ggcctgggca tcagctacgg ccgcaagaag 840cgccgccagc gccgcagcgc cccccccagc agcgaggacc accagaaccc catcagcaag 900cagcccctgc cccgcaccca gggcgacccc accggcagcg aggagagcaa gaagaaggtg 960gagagcaaga ccaagaccga ccccttcgac tag 993<210>14<211>330<212>PRT<213>人<400>14Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly1 5 10 15Ala Val Phe Val Ser Ala Arg Met Gly Gly Lys Trp Ser Lys Ser Ser
20 25 30Ile Val Gly Trp Pro Ala Ile Arg Glu Arg Ile Arg Arg Thr Glu Pro
35 40 45Ala Ala Asp Gly Val Gly Ala Val Ser Arg Asp Leu Glu Lys His Gly
50 55 60Ala Ile Thr Ser Asn Asn Thr Ala Asp Thr Asn Ala Asp Cys Ala Trp65 70 75 80Leu Glu Thr Gln Glu Glu Glu Glu Val Gly Phe Pro Val Arg Pro Gln
85 90 95Val Pro Leu Arg Pro Met Thr Phe Lys Gly Ala Leu Asp Leu Ser Phe
100 105 110Phe Leu Lys Glu Lys Gly Gly Leu Glu Gly Leu Ile Tyr Ser Lys Lys
115 120 125Arg Gln Glu Ile Leu Asp Leu Trp Val Tyr His Thr Gln Gly Tyr Phe
130 135 140Pro Asp Trp His Asn Tyr Thr Pro Gly Pro Gly Val Arg Phe Pro Leu145 150 155 160Thr Phe Gly Trp Cys Phe Lys Leu Val Pro Val Asp Pro Gly Glu Val
165 170 175Glu Glu Ala Asn Glu Gly Glu Asn Asn Cys Leu Leu His Pro Val Cys
180 185 190Gln His Gly Met Asp Asp Glu His Arg Glu Val Leu Lys Trp Lys Phe
195 200 205Asp Ser Gln Leu Ala His Arg His Arg Ala Arg Glu Leu His Pro Glu
210 215 220Phe Tyr Lys Asp Cys Met Glu Pro Val Asp Pro Asn Leu Glu Pro Trp225 230 235 240Asn His Pro Gly Ser Gln Pro Glu Thr Ala Cys Asn Asn Cys Tyr Cys
245 250 255Lys Arg Cys Ser Tyr His Cys Leu Val Cys Phe Gln Lys Lys Gly Leu
260 265 270Gly Ile Ser Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Ser Ala Pro
275 280 285Pro Ser Ser Glu Asp His Gln Asn Pro Ile Ser Lys Gln Pro Leu Pro
290 295 300Arg Thr Gln Gly Asp Pro Thr Gly Ser Glu Glu Ser Lys Lys Lys Val305 310 315 320Glu Ser Lys Thr Lys Thr Asp Pro Phe Asp
325 330<210>15<211>8186<212>DNA<213>人<400>15gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta 60atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata 120acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat 180aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga 240gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc 300ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt 360atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat 420gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag 480tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc 540aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga 600ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga 660aattaatacg actcactata gggagaccca agctggctag ccgccaccat ggatgcaatg 720aagagggggc tctgctgtgt gctgctgctg tgtggagcag tcttcgttag cgccgccgag 780aacttgtggg tgaccgtgta ctacggcgtg cccgtgtgga aggaggccaa gaccaccctg 840ttctgcgcct ccgacgccaa ggcctacgag aaggaggtgc acaacgtgtg ggccacccac 900gcctgcgtgc ccaccgaccc caacccccag gagatggtgt tggagaacgt gaccgagaac 960ttcaacatgt ggaagaacga catggtgaac cagatgcacg aggacgtcat cagcttgtgg 1020gaccagagcc tgaagccctg cgtgaagttg acccccctgt gcgtgacctt ggagtgcagg 1080aacgtgagca gcaacggcac ctacaacgag acctacaacg agatcaagaa ctgctccttc 1140aacgccacca ccgtgttgag ggacaggaag cagaccgtgt acgccctgtt ctacaggctg 1200gacatcgtgc ccctgaacaa gaagaactcc agcgagaact ccagcgagta ctacaggttg 1260atcaactgca acacctccgc catcacccag gcctgcccca aggtgacctt cgaccccatc 1320cccatccact actgcacccc cgccggctac gccatcctga agtgcaacga caagaccttc 1380aacggcaccg gcccctgcca caacgtgagc accgtgcagt gcacccacgg catcaagccc 1440gtggtgtcca cccagctgct gttgaacggc agcctggccg agagggagat catcatcagg 1500tccgagaacc tgaccaacaa cgtgaagacc atcatcgtgc acctgaacca gtccgtggag 1560atcgtgtgca ccaggcccaa caacaacacc aggaagagca tcaggatcgg ccccggccag 1620accttctacg ccaccggcga catcatcggc gacatcaggc aggcccactg caacatcagc 1680aaggacaagt ggaaggagac cttgcagagg gtgggcaaga agttggccga gcacttcccc 1740aacaagacca tcgagttcgc ctcctcctcc ggcggcgacc tggagatcac cacccacagc 1800ttcaactgca ggggcgagtt cttctactgc aacacctcca gcctgttcaa cggcacctac 1860atgcccaacg gcaccgaggg caactccagc tccatcatca ccatcccctg caggatcaag 1920cagatcatca acatgtggca ggaggtgggc cgcgccatgt acgccccccc catcgagggc 1980aacatcacct gcaagtccaa catcaccggc ctgctgttgg tgcgcgacgg cggcaaggag 2040accaacgaca ccgagacctt caggcccggc ggcggcgaca tgagggacaa ctggaggagc 2100gagttgtaca agtacaaggt ggtggagatc aagcccttgg gcatcgcccc caccgccgcc 2160aagaggaggg tggtggagag ggagaagagg gccgtgggca tcggcgccgt gttcctgggc 2220ttcctgggcg ccgccggcag caccatgggc gccgccagca tcaccctgac cgtgcaggcc 2280cgccagctgc tgagcggcat cgtgcagcag cagagcaacc tgctgcgcgc catcgaggcc 2340cagcagcacc tgctgcagct gaccgtgtgg ggcatcaagc agctgcagac ccgcgttctg 2400gccatcgagc gctacctgaa ggaccagcag ctgctgggca tctggggctg cagcggcaag 2460ctgatctgca ccaccgccgt gcactggaac agcagctgga gcaaccgcag ccaggaggag 2520atctggaaca acatgacctg gatgcagtgg gaccgcgaga tcagcaacta caccaacacc 2580atctaccgcc tgctggagga cagccagaac cagcaggagc gcaacgagaa ggacctgctg 2640gccctggaca actggaagaa cctgtggagc tggttcgaca tcaccaactg gctgtggtac 2700atccgcatct tcatcatgat cgtgggcggc ctgatcggcc tgcgcatcat cttcgccgtg 2760ctgagcatcg tgaaccgcgt gcgccagggc tacagccccc tgagcttcca gaccctgacc 2820cccaaccccg gcggccccga ccgcctgggc cgcatcgagg aggagggcgg cgagcaggac 2880aagaaccgca gcatccgcct ggtgaacggc ttcctggccc tggcctggga cgacctgcgc 2940aacctgtgcc gcttcagcta ccacctgctg cgcgacctgc tgctgatcgt ggcccgcatc 3000gtggagctgc tgggccgccg cggctgggag gccctgcgct actggtggaa cctgctgaag 3060tactgggtgc aggagctgaa gaacagcgcc gtgagcctgc tgaacgccac cgccatcgcc 3120gtggccgagg gcaccgaccg cgtgatcgag gtggtgcagg gcgcctaccg cgccatcctg 3180cacatccccc gccgcatccg ccagggcttc gaggccgccc tgcagtaaga attccgtgag 3240gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg 3300gaggggtcgg cgattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg 3360atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag 3420tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg 3480tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg ccttgaatta 3540cttccacctg gctgcagtac gtgattcttg atcccgagct tcgggttgga agtgggtggg 3600agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt gaggcctggc 3660ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt 3720tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt tttttctggc 3780aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt tttggggccg 3840cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag 3900cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct ctggtgcctg 3960gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg tcggcaccag 4020ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca aaatggagga 4080cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg gcctttccgt 4140cctcagccgt cgcttcatgt gactccacgg agtaccgggc gccgtccagg cacctcgatt 4200agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt tatgcgatgg 4260agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac ttgatgtaat 4320tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag cctcagacag 4380tggttcaaag tttttttctt ccatttcagg tgtcgtgaag cggccgccgc caccatggac 4440gccatgaagc gcggcctgtg ctgcgtgctg ctgctgtgcg gcgccgtgtt cgtgagcgcc 4500cgcatgggcg cccgcgccag catcctgcgc ggcggcaagc tgggcaagtg ggagaagatc 4560cgcctgcgcc ccggcgacaa gaagcactac atgctgaagc acctggtgtg ggccagccgc 4620gagctggagc gcttcgccct gaaccccggc ctgctggaga ccagcgaggg ctgcaagcag 4680atcatcaagc agctgcagcc cgccctgcag accggcaccg aggagctgcg cagcctgttc 4740aacaccgtgg ccaccctgta ctgcgtgcac gagggcatcg agatccgcga caccaaggag 4800gccctggaca agatcgagga ggagcagaac aagatccagc agaagaccca gcaggccaag 4860aaggccgacg agaaggtgag ccagaactac cccatcgtgc agaacctgca gggccagatg 4920gtgcaccagg ccatctcccc caggaccttg aacgcctggg tgaaggtgat cgaggagaag 4980gccttcagcc ccgaggtgat ccccatgttc accgccttgt ccgagggcgc caccccccag 5040gacttgaaca ccatgttgaa caccgtgggc ggccaccagg ccgccatgca gatgttgaag 5100gacaccatca acgaggaggc cgccgagtgg gacagggtgc accccgtgca cgccggcccc 5160attgcccccg gccagatgag ggagcccagg ggcagcgaca tcgccggcac caccagcacc 5220ctgcagggcc agatcgcctg gatgaccagc aacccccccg tgcccgtggg cgagatctac 5280aagaggtgga tcatcctggg cttgaacaag atcgtgagga tgtacagccc cgtgagcatc 5340ttggacatca agcagggccc caaggagccc ttcagggact acgtggaccg cttcttcaag 5400accttgaggg ccgagcaggc cacccaggac gtgaagaact ggatgaccga caccttgttg 5460gtgcagaacg ccaaccccga ctgcaagacc atcttgaggg ccttgggccc cggcgcctcc 5520ttggaggaga tgatgaccgc ctgccagggc gtgggcggcc ccagccacaa ggccagggtg 5580ttggccgagg ccatgagcca ggccaacggc accatcctga tgcagaggag caacttcaag 5640ggctccaaga ggatcgtgaa gtgcttcaac tgcggcaagg agggccacat cgccaggaac 5700tgcagggccc ccaggaagaa gggctgctgg aagtgcggca aggagggcca ccagatgaag 5760gactgcaccg agaggcaggc caacttcttg ggcaagatct ggccctccca caagggcagg 5820cccggcaact tcctgcagag caggcccgag cccaccgccc cccccgccga gagcttcagg 5880ttcgaggaga ccacccccgc ccccaagcag gagcccaagg acagggagcc cttgacctcc 5940ctgaagtccc tgttcggcag cgaccccttg tcccagtaat ctagagggcc cgtttaaacc 6000cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc 6060gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa 6120attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac 6180agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg 6240gcttctactg ggcggtttta tggacagcaa gcgaaccgga attgccagct ggggcgccct 6300ctggtaaggt tgggaagccc tgcaaagtaa actggatggc tttcttgccg ccaaggatct 6360gatggcgcag gggatcaagc tctgatcaag agacaggatg aggatcgttt cgcatgattg 6420aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg 6480actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg 6540ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg 6600aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg 6660ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc 6720tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc 6780tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc 6840gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc 6900aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg 6960atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct 7020tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt 7080tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc 7140tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt 7200tcttctgaat tattaacgct tacaatttcc tgatgcggta ttttctcctt acgcatctgt 7260gcggtatttc acaccgcatc aggtggcact tttcggggaa atgtgcgcgg aacccctatt 7320tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa 7380atgcttcaat aatagcacgt gctaaaactt catttttaat ttaaaaggat ctaggtgaag 7440atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg 7500tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc 7560tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag 7620ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt 7680cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac 7740ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc 7800gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt 7860tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt 7920gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc 7980ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt 8040tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca 8100ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt 8160tgctggcctt ttgctcacat gttctt 8186<210>16<211>8017<212>DNA<213>人<400>16gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta 60atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata 120acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat 180aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga 240gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc 300ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt 360atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat 420gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag 480tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc 540aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga 600ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga 660aattaatacg actcactata gggagaccca agctggctag cgccgccacc atggacgcca 720tgaagcgcgg cctgtgctgc gtgctgctgc tgtgcggcgc cgtgttcgtg agcgcccgcc 780cccagatcac cctgtggcag cgccccctgg tgtccatccg cgtggggggc cagatcaagg 840aggccctgct ggacgacacc gtgttggagg aggtgaactt gcccggcaag tggaagccca 900agatgatcgg cggcatcggc ggcttcatca aggtgaggca gtacgaccag atccccatcg 960agatctgcgg caagaaggcc atcggcaccg tgttggtggg ccccaccccc gtgaacatca 1020tcggcaggaa catgttgacc cagctgggct gcaccctgaa cttccccatc agccccatcg 1080agaccatccc cgtgaagctg aagcccggca tggacggccc ccgcgtgaag cagtggcccc 1140tgaccgagga gaagatcaag gccctgaccg ccatctgcga cgagatggag aaggagggca 1200agatcaccaa gatcggcccc gagaacccct acaacacccc cgtgttcgcc atcaagaaga 1260aggacagcac caagtggcgc aagctggtgg acttccgcga gctgaacaag cgcacccagg 1320acttctggga ggtgcagctg ggcatccccc accccgccgg cctgaagaag aagaagtccg 1380tgaccgtgct ggacgtgggc gacgcctact tctccgtgcc cctgtacgag gacttccgca 1440agtacaccgc cttcaccatc cccagcatca acaacgagac ccccggcatc cgctaccagt 1500acaacgtgct gccccagggc tggaagggct cccccgccat cttccagtgc agcatgacca 1560agatcctgga gcccttccgg gcccagaacc ccgagatcgt gatctaccag tacggcgacg 1620acctgtacgt gggctccgac ctggagatcg gccagcaccg cgccaagatc gaggagttgc 1680gcgagcacct gctgaagtgg ggcttcacca cccccgacaa gaagcaccag aaggagcccc 1740ccttcctgtg gatgggctac gagctgcacc ccgacaagtg gaccgtgcag cccatccagc 1800tgcccgagaa ggacagctgg accgtgaacg acatccagaa gctggtgggc aagctgaact 1860gggccagcca gatctacccc ggcatcaagg tgcgccagct gtgcaagctg ctgcgcggcg 1920ccaaggccct gaccgacatc atccccctga ccgaggaggc cgagctggag ctggccgaga 1980accgcgagat cctgaaggag cccgtgcacg gcgcctacta cgacccctcc aaggacctga 2040tcgccgagat ccagaagcag ggccaggacc agtggaccta ccagatctac caggagccct 2100tcaagaacct gaagaccggc aagtacgcca agatgcgcac cgcccacacc aacgacgtga 2160agcagctgac cgaggccgtg cagaagatct ccatggagag catcgtgatc tggggcaaga 2220tccccaagtt ccgcctgccc atccagaagg agacctggga gacccgctgg accgcctact 2280ggcaggccac ctggatcccc gagtgggagt tcgtgaacac cccccccctg gtgaagctgt 2340ggtaccagct ggagaaggac cccatcgccg gcgtggagac cttctacgtg gacggcgccg 2400ccaaccgcga gaccaagatg ggcaaggccg gctacgtgac cgaccgcggc cgccagaaga 2460tcgtgtccct gaccgagacc accaaccaga agaccgagct gcaggccatc tgcctggcct 2520tgcaggactc cggctccgag gtgaacatcg tgaccgactc ccagtacgcc ctgggcatca 2580tccaggccca gcccgacaag agcgagtccg agctggtgaa ccagatcatc gagcagctga 2640tcaagaagga gcgcgtgtac ctgtcctggg tgcccgccca caagggcatc ggcggcaacg 2700agcaggtgga caagctggtg agcaacggca tccgcaaggt gctgttcctg gacggcatcg 2760acaaggccca ggaggagcac gagaagtacc acagcaactg gcgcgccatg gccagcgact 2820tcaacctgcc ccccatcgtg gccaaggaga tcgtggccag ctgcgaccag tgccagctga 2880agggcgaggc catgcacggc caggtggact gcagccccgg catctggcag ctggactgca 2940cccacctgga gggcaagatc atcctggtgg ccgtgcacgt ggccagcggc tacatcgagg 3000ccgaggtgat ccccgccgag accggccagg agaccgccta cttcatcctg aagctggccg 3060gccgctggcc cgtgaagatc atccacaccg acaacggcag caacttcacc agcgccgccg 3120tgaaggccgc ctgctggtgg gccggcatcc agcaggagtt cggcatcccc tacaaccccc 3180agagccaggg cgtggtggag tccatgaaca aggagctgaa gaagatcatc ggccaggtgc 3240gcgaccaggc cgagcacctg aagaccgccg tgcagatggc cgtgttcatc cacaacttca 3300agcgcaaggg cggcatcggc ggctacagcg ccggcgagcg catcatcgac atcatcgcca 3360ccgacatcca gaccaaggag ctgcagaagc agatcatcaa gatccagaac ttccgcgtgt 3420actaccgcga cagccgcgac cccatctgga agggccccgc caagctgctg tggaagggcg 3480agggcgccgt ggtgatccag gacaacagcg acatcaaggt ggtgccccgc cgcaaggcca 3540agatcatcaa ggactacggc aagcagatgg ccggcgccga ctgcgtggcc agccgccagg 3600acgaggacta ggaattccgt gaggctccgg tgcccgtcag tgggcagagc gcacatcgcc 3660cacagtcccc gagaagttgg ggggaggggt cggcaattga accggtgcct agagaaggtg 3720gcgcggggta aactgggaaa gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg 3780gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc 3840cgccagaaca caggtaagtg ccgtgtgtgg ttcccgcggg cctggcctct ttacgggtta 3900tggcccttgc gtgccttgaa ttacttccac ctggctgcag tacgtgattc ttgatcccga 3960gcttcgggtt ggaagtgggt gggagagttc gaggccttgc gcttaaggag ccccttcgcc 4020tcgtgcttga gttgaggcct ggcctgggcg ctggggccgc cgcgtgcgaa tctggtggca 4080ccttcgcgcc tgtctcgctg ctttcgataa gtctctagcc atttaaaatt tttgatgacc 4140tgctgcgacg ctttttttct ggcaagatag tcttgtaaat gcgggccaag atctgcacac 4200tggtatttcg gtttttgggg ccgcgggcgg cgacggggcc cgtgcgtccc agcgcacatg 4260ttcggcgagg cggggcctgc gagcgcggcc accgagaatc ggacgggggt agtctcaagc 4320tggccggcct gctctggtgc ctggcctcgc gccgccgtgt atcgccccgc cctgggcggc 4380aaggctggcc cggtcggcac cagttgcgtg agcggaaaga tggccgcttc ccggccctgc 4440tgcagggagc tcaaaatgga ggacgcggcg ctcgggagag cgggcgggtg agtcacccac 4500acaaaggaaa agggcctttc cgtcctcagc cgtcgcttca tgtgactcca cggagtaccg 4560ggcgccgtcc aggcacctcg attagttctc gagcttttgg agtacgtcgt ctttaggttg 4620gggggagggg ttttatgcga tggagtttcc ccacactgag tgggtggaga ctgaagttag 4680gccagcttgg cacttgatgt aattctcctt ggaatttgcc ctttttgagt ttggatcttg 4740gttcattctc aagcctcaga cagtggttca aagttttttt cttccatttc aggtgtcgtg 4800aagcggccgc cgccaccatg gacgccatga agcgcggcct gtgctgcgtg ctgctgctgt 4860gcggcgccgt gttcgtgagc gcccgcatgg gcggcaagtg gtccaagagc agcatcgtag 4920gctggcccgc catccgcgag cgcatccgcc gcaccgagcc cgccgccgac ggcgtgggcg 4980ccgtgtctcg cgacctggag aagcatggcg ccatcaccag taacaacacc gccgacacca 5040acgccgactg cgcctggctg gagacccagg aggaggagga ggtgggcttc cccgtccgcc 5100cccaggtgcc cttgcgcccc atgaccttca agggcgcctt ggacctcagc ttcttcctga 5160aggagaaggg cggcctggag gggttgatct acagccagaa gaggcaggag atcctggact 5220tgtgggtcta ccacacccag ggctacttcc ccgactggca caactacacc cccggccccg 5280gcgtccgctt ccccctgacc ttcggctggt gcttcaagct ggtgcccgtg gaccccggcg 5340aggtggagga ggccaacgag ggcgagaaca actgcttgct gcaccccgtc tgccagcacg 5400gcatggacga cgagcaccgc gaggtgctga agtggaagtt cgacagccag ctggcccacc 5460gccacagggc ccgcgagctg cacccggagt tctacaagga ctgcatggag cccgtggacc 5520ccaacctgga gccctggaac caccccggca gccagcccga gaccgcctgc aacaactgct 5580actacaagcg ctgcagctac cactgcctgg tgtgcttcca gaagaagggc ctgggcatca 5640gctacggccg caagaagcgc cgccagcgcc gtagcgcccc ccccagcagc gaggaccacc 5700agaaccccat cagcaagcag cccctgcccc gcacccaggg cgaccccacc ggcagcgagg 5760agagcaagaa gaaggtggag agcaagacca agaccgaccc cttcgactag tctagagggc 5820ccgtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgttt 5880gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat 5940aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg 6000tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcgg 6060tgggctctat ggcttctact gggcggtttt atggacagca agcgaaccgg aattgccagc 6120tggggcgccc tctggtaagg ttgggaagcc ctgcaaagta aactggatgg ctttcttgcc 6180gccaaggatc tgatggcgca ggggatcaag ctctgatcaa gagacaggat gaggatcgtt 6240tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg tggagaggct 6300attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg tgttccggct 6360gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg ccctgaatga 6420actgcaagac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc cttgcgcagc 6480tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg aagtgccggg 6540gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca tggctgatgc 6600aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc aagcgaaaca 6660tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg atgatctgga 6720cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg cgagcatgcc 6780cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata tcatggtgga 6840aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg accgctatca 6900ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat gggctgaccg 6960cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct 7020tcttgacgag ttcttctgaa ttattaacgc ttacaatttc ctgatgcggt attttctcct 7080tacgcatctg tgcggtattt cacaccgcat caggtggcac ttttcgggga aatgtgcgcg 7140gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat 7200aaccctgata aatgcttcaa taatagcacg tgctaaaact tcatttttaa tttaaaagga 7260tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt 7320tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc 7380tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc 7440cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac 7500caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac 7560cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt 7620cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct 7680gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat 7740acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt 7800atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg 7860cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt 7920gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt 7980tcctggcctt ttgctggcct tttgctcaca tgttctt 8017<210>17<211>2442<212>DNA<213>人<400>17atgagagtga cggggatcag gaagaactat cagcatttat ggagatgggg caccatgctc 60cttgggatgt tgatgatctg tagtgctgca gaaaacttgt gggtcacagt ctattatggg 120gtacctgtgt ggaaagaagc caaaactact ctattctgtg cgtcagatgc taaagcatat 180gagaaagaag tgcataatgt ctgggctaca catgcctgtg tacccacaga ccccaaccca 240caagaaatgg ttttggaaaa tgtaacagaa aattttaaca tgtggaaaaa tgacatggtg 300aatcagatgc atgaggatgt aatcagttta tgggatcaaa gcctaaagcc atgtgtaaag 360ttgaccccac tctgtgtcac tttagaatgt agaaatgtta gcagtaatgg tacctacaat 420gagacctaca atgagatcaa aaattgctct ttcggggccg ggtattatag attaataaat 480tgtaatacct cagccataac acaagcctgt ccaaaggtca cttttgatcc aattcctata 540cactattgca ctccagctgg ttatgcgatt ctaaagtgta atgataagac attcaatgga 600acaggaccat gccataatgt tagtacagta caatgtacac atggaattaa gccagtggta 660tcaactcaac tactgttaaa tggtagccta gcagaaagag agataataat tagatctgaa 720aatctgacaa acaatgtcaa aacaataata gtacatctta atcaatctgt agaaattgta 780tgtacaagac ccaacaataa tacaagaaaa agtataagga taggaccagg acaaacattc 840tatgcaacag gagacataat aggagatata agacaagcac attgtaacat tagtaaagat 900aaatggaatg aaactttaca aagggtaggt aaaaaattag cagaacactt ccctaataaa 960acaatagaat ttgcatcatc ctcaggaggg gacctagaaa ttacaacaca tagctttaat 1020tgtagaggag aatttttcta ttgtaataca tcaatcctgt ttaatggtac atacatgcct 1080aatggtacag aaggtaattc aagctcaatc atcacaatcc catgcagaat aaagcaaatt 1140ataaacatgt ggcaggaggt aggacgagca atgtatgccc ctcccattga gggaaacata 1200acatgtaaat caaatatcac aggactacta ttggtacgtg atggaggaaa agagacaaat 1260gatacagaga cattcagacc tggaggagga gatatgaggg acaattggag aagtgaatta 1320tataaatata aagtggtaga aattaagcca ttgggaatag cacccactgc agcaaaaagg 1380agagtggtgg agagagaaaa aagagcagtg ggaataggag ctgtgttcct tgggttcttg 1440ggagcagcag gaagcactat gggcgcggcg tcaataacgc tgacggtaca gtccagacaa 1500ttgttgtctg gtatagtgca acagcaaagc aatttgctga gggctataga ggcgcaacag 1560catctgttgc aactcacggt ctggggcatt aagcagctcc agacaagagt cctggctata 1620gaaagatacc taaaggatca acagctccta gggatttggg gctgctctgg aaaactcatc 1680tgcactactg ctgtacattg gaactccagt tggagtaaca gatctcaaga agagatttgg 1740aataacatga cttggatgca gtgggataga gaaattagta attacacaaa cacaatatac 1800aggttgcttg aagactcgca aaaccagcag gaaagaaatg aaaaagattt actagcattg 1860gacaattgga aaaatctatg gagttggttt gacataacaa attggctgtg gtatataaga 1920atattcataa tgatagtagg aggcttgata ggtttaagaa taatttttgc tgtgctctct 1980atagtgaata gagttaggca gggatactca cctttgtcgt ttcagaccct taccccgaac 2040ccagggggac ccgacaggct cggaagaatc gaagaagaag gtggagagca agacaaaaac 2100agatccattc gattagtgaa cggattctta gcacttgcct gggacgacct gcggaacctg 2160tgccgcttca gctaccacct cttgagagac ttactcttga ttgtagcaag gattgtggaa 2220cttctgggac gcagggggtg ggaagccctc agatattggt ggaatctcct gaagtattgg 2280gttcaggaac taaagaatag tgctgttagt ttgctcaatg ccacagctat agcagtagct 2340gaggggacag atagggttat agaagtagta caaggagctt atagagctat tctccacata 2400cctagaagaa taagacaggg ctttgaagca gctttgcaat aa 2442<210>18<211>813<212>PRT<213>人<400>18Met Arg Val Thr Gly Ile Arg Lys Asn Tyr Gln His Leu Trp Arg Trp1 5 10 15Gly Thr Met Leu Leu Gly Met Leu Met Ile Cys Ser Ala Ala Glu Asn
20 25 30Leu Trp Val Thr Val Tyr Tyr Gly Val Pro Val Trp Lys Glu Ala Lys
35 40 45Thr Thr Leu Phe Cys Ala Ser Asp Ala Lys Ala Tyr Glu Lys Glu Val
50 55 60His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn Pro65 70 75 80Gln Glu Met Val Leu Glu Asn Val Thr Glu Asn Phe Asn Met Trp Lys
85 90 95Asn Asp Met Val Asn Gln Met His Glu Asp Val Ile Ser Leu Trp Asp
100 105 110Gln Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu
115 120 125Glu Cys Arg Asn Val Ser Ser Asn Gly Thr Tyr Asn Glu Thr Tyr Asn
130 135 140Glu Ile Lys Asn Cys Ser Phe Gly Ala Gly Tyr Tyr Arg Leu Ile Asn145 150 155 160Cys Asn Thr Ser Ala Ile Thr Gln Ala Cys Pro Lys Val Thr Phe Asp
165 170 175Pro Ile Pro Ile His Tyr Cys Thr Pro Ala Gly Tyr Ala Ile Leu Lys
180 185 190Cys Asn Asp Lys Thr Phe Asn Gly Thr Gly Pro Cys His Asn Val Ser
195 200 205Thr Val Gln Cys Thr His Gly Ile Lys Pro Val Val Ser Thr Gln Leu
210 215 220Leu Leu Asn Gly Ser Leu Ala Glu Arg Glu Ile Ile Ile Arg Ser Glu225 230 235 240Asn Leu Thr Asn Asn Val Lys Thr Ile Ile Val His Leu Asn Gln Ser
245 250 255Val Glu Ile Val Cys Thr Arg Pro Asn Asn Asn Thr Arg Lys Ser Ile
260 265 270Arg Ile Gly Pro GlyGln Thr Phe Tyr Ala Thr Gly Asp Ile Ile Gly
275 280 285Asp Ile Arg Gln Ala His Cys Asn Ile Ser Lys Asp Lys Trp Asn Glu
290 295 300Thr Leu Gln Arg Val Gly Lys Lys Leu Ala Glu His Phe Pro Asn Lys305 310 315 320Thr Ile Glu Phe Ala Ser Ser Ser Gly Gly Asp Leu Glu Ile Thr Thr
325 330 335His Ser Phe Asn Cys Arg Gly Glu Phe Phe Tyr Cys Asn Thr Ser Ile
340 345 350Leu Phe Asn Gly Thr Tyr Met Pro Asn Gly Thr Glu Gly Asn Ser Ser
355 360 365Ser Ile Ile Thr Ile Pro Cys Arg Ile Lys Gln Ile Ile Asn Met Trp
370 375 380Gln Glu Val Gly Arg Ala Met Tyr Ala Pro Pro Ile Glu Gly Asn Ile385 390 395 400Thr Cys Lys Ser Asn Ile Thr Gly Leu Leu Leu Val Arg Asp Gly Gly
405 4l0 415Lys Glu Thr Asn Asp Thr Glu Thr Phe Arg Pro Gly Gly Gly Asp Met
420 425 430Arg Asp Asn Trp Arg Ser Glu Leu Tyr Lys Tyr Lys Val Val Glu Ile
435 440 445Lys Pro Leu Gly Ile Ala Pro Thr Ala Ala Lys Arg Arg Val Val Glu
450 455 460Arg Glu Lys Arg Ala Val Gly Ile Gly Ala Val Phe Leu Gly Phe Leu465 470 475 480Gly Ala Ala Gly Ser Thr Met Gly Ala Ala Ser Ile Thr Leu Thr Val
485 490 495Gln Ser Arg Gln Leu Leu Ser Gly Ile Val Gln Gln Gln Ser Asn Leu
500 505 510Leu Arg Ala Ile Glu Ala Gln Gln His Leu Leu Gln Leu Thr Val Trp
515 520 525Gly Ile Lys Gln Leu Gln Thr Arg Val Leu Ala Ile Glu Arg Tyr Leu
530 535 540Lys Asp Gln Gln Leu Leu Gly Ile Trp Gly Cys Ser Gly Lys Leu Ile545 550 555 560Cys Thr Thr Ala Val His Trp Asn Ser Ser Trp Ser Asn Arg Ser Gln
565 570 575Glu Glu Ile Trp Asn Asn Met Thr Trp Met Gln Trp Asp Arg Glu Ile
580 585 590Ser Asn Tyr Thr Asn Thr Ile Tyr Arg Leu Leu Glu Asp Ser Gln Asn
595 600 605Gln Gln Glu Arg Asn Glu Lys Asp Leu Leu Ala Leu Asp Asn Trp Lys
610 615 620Asn Leu Trp Ser Trp Phe Asp Ile Thr Asn Trp Leu Trp Tyr Ile Arg625 630 635 640Ile Phe Ile Met Ile Val Gly Gly Leu Ile Gly Leu Arg Ile Ile Phe
645 650 655Ala Val Leu Ser Ile Val Asn Arg Val Arg Gln Gly Tyr Ser Pro Leu
660 665 670Ser Phe Gln Thr Leu Thr Pro Asn Pro Gly Gly Pro Asp Arg Leu Gly
675 680 685Arg Ile Glu Glu Glu Gly Gly Glu Gln Asp Lys Asn Arg Ser Ile Arg
690 695 700Leu Val Asn Gly Phe Leu Ala Leu Ala Trp Asp Asp Leu Arg Asn Leu705 710 715 720Cys Arg Phe Ser Tyr His Leu Leu Arg Asp Leu Leu Leu Ile Val Ala
725 730 735Arg Ile Val Glu Leu Leu Gly Arg Arg Gly Trp Glu Ala Leu Arg Tyr
740 745 750Trp Trp Asn Leu Leu Lys Tyr Trp Val Gln Glu Leu Lys Asn Ser Ala
755 760 765Val Ser Leu Leu Asn Ala Thr Ala Ile Ala Val Ala Glu Gly Thr Asp
770 775 780Arg Val Ile Glu Val Val Gln Gly Ala Tyr Arg Ala Ile Leu His Ile785 790 795 800Pro Arg Arg Ile Arg Gln Gly Phe Glu Ala Ala Leu Gln
805 810<210>19<211>4404<212>DNA<213>人<400>19gccgccacca tggacgccat gaagcgcggc ctgtgctgcg tgctgctgct gtgcggcgcc 60gtgttcgtga gcgcccgcat gggtgcgaga gcgtcaatat taagaggggg aaaattagat 120aaatgggaaa aaattaggtt aaggccaggg ggaaagaaac actatatgct aaaacacata 180gtatgggcaa gcagagagct ggaaagattt gcacttaacc ctggcctttt agagacatca 240gaaggctgta aacaaataat aaaacagcta caaccagctc ttcagacagg aacagaggaa 300cttagatcat tattcaacac agtagcaact ctctattgtg tacatgcagg gatagaagta 360cgagacacca aagaagcctt agacaagata gaggaagaac aaaacaaaat gcagcaaaaa 420acacagcagg caaaagaggc tgacgggaag gtcagtcaaa attatcctat agtgcagaat 480ctccaagggc aaatggtgca ccaggccata tcacctagaa ctttgaatgc atgggtaaaa 540gtaatagagg aaaaggcttt tagcccagag gtaataccca tgtttacagc attatcagaa 600ggagccaccc cacaagattt aaacaccatg ttaaatacag tagggggaca tcaggcagcc 660atgcaaatgt taaaagatac tatcaatgaa gaggctgcag aatgggatag agtacatcca 720gtacatgcag ggcctattgc accaggccaa atgagagaac caaggggaag tgacatagca 780ggaactacta gtacccttca gggacaaata gcatggatga cgagtaaccc acctgttcca 840gtgggagaaa tctataaaag atggataatt ctggggttaa ataaaatagt aagaatgtat 900agccctgtga gcattttgga cataaaacaa gggccaaagg aaccctttag agactatgta 960gaccggttct ttaaaacttt aagagctgaa caagctacac aagatgtaaa aaattggatg 1020acagacacct tgttggtcca aaatgcgaac ccagattgta agaccatttt aagagcatta 1080ggaccagggg cttcattaga agagatgatg acagcatgtc agggagtggg aggacctaac 1140cacaaagcaa gagtgttggc tgaggcaatg agccaagcaa acggtaccat actgatgcag 1200agaagcaatt ttaaaggctc taaaagaatt gttaaatgtt tcaactgtgg caaggaaggg 1260cacatagcca gaaattgcag ggcccctagg aaaaaaggct gttggaaatg tggaaaggaa 1320ggacaccaaa tgaaagactg cactgaaagg caggctaatt ttttagggaa aatttggcct 1380ccccacaagg ggaggccagg gaatttcctc cagagcagac cagagccaac agccccacca 1440gcagagagct tcgggttcga ggagacaacc ccagctccga agcaggagcc gaaagacagg 1500gaacccttaa cttccctcaa atcactcttt ggcagcgacc ccttgtctca acctcaaatc 1560actctttggc agcgacccct tgtctcaata agagtagggg gccagataaa agaggctctc 1620ttagatgata cagtattaga agaagtaaat ttgccaggaa aatggaaacc aaaaatgata 1680ggaggaattg gaggttttat caaagtaaga caatatgatc aaatacctat agaaatttgt 1740ggaaaaaagg ctataggtac agtattagtg ggacccacac ctgtcaacat aattggaaga 1800aatatgttga ctcaacttgg atgcacacta aattttccaa tcagtcccat tgaaactata 1860ccagtaaaat taaagccagg aatggatggc ccaagggtta aacaatggcc attgacagaa 1920gagaaaataa aagcattaac agcaatttgt gatgaaatgg agaaggaagg aaaaattaca 1980aaaattgggc ctgaaaatcc atataacact ccagtatttg ccataaaaaa gaaggacagt 2040actaagtgga gaaaattagt agatttcagg gaactcaata aaagaactca agatttttgg 2100gaagttcaat taggaatacc acacccagca gggttaaaaa agaaaaaatc agtgacagta 2160ctggatgtgg gggatgcata tttttcagtt cctttatatg aagacttcag aaaatatact 2220gcattcacca tacctagtat aaacaatgaa acaccaggga tcaggtatca atataatgtg 2280cttccacagg gatggaaggg atcaccagca atattccagt gtagcatggc aaaaatctta 2340gagcccttta gggcacaaaa tccagaaata gtcatctatc aatatggcga tgacttgtat 2400gtaggatctg acttagagat agggcaacat agagcaaaaa tagaggagtt aagagaacat 2460ctgttaaagt ggggatttac cacaccagac aagaaacatc agaaagaacc tccatttctt 2520tggatggggt atgaactcca tcctgacaaa tggacagtac agcctataca gctgccagaa 2580aaggatagct ggactgtcaa tgatatacag aagttagtgg gaaaattaaa ctgggcaagt 2640cagatttacc caggaattaa agtaaggcaa ctttgtaaac tccttagggg ggccaaagca 2700ctaacagaca taataccact aactgaagaa gcagaattgg agttggcaga aaacagggaa 2760attctaaaag aaccagtaca tggagcatat tatgacccat caaaagactt gatagctgaa 2820atacagaaac aggggcagga ccaatggaca tatcaaattt accaagaacc attcaaaaat 2880ctgaaaacag ggaaatatgc aaaaatgagg actgcccaca ctaatgatgt aaaacagtta 2940acagaggctg tgcagaaaat atccatggaa agcatagtaa tatggggaaa aattcctaaa 3000tttaggttac ccatcccaaa agaaacctgg gagacacggt ggacagccta ttggcaagcc 3060acctggattc ctgagtggga atttgttaat acccctccct tagtaaaatt atggtaccag 3120ctggagaaag atcccatagc aggagtagaa actttctatg tagatggagc agctaatagg 3180gaaactaaaa tgggaaaagc agggtatgtt actgacagag gaaggcagaa aattgtgtct 3240ctaactgaaa caacaaatca gaagactgaa ttgcaagcaa tttgtctagc tttgcaagat 3300tcaggatcag aagtaaatat agtaacagat tcacagtatg cattaggaat cattcaagca 3360caaccagata agagtgagtc agagttagtt aaccaaataa tagaacaatt aataaaaaag 3420gaaagggtct acctgtcgtg ggtaccagca cataaaggaa ttggaggaaa tgaacaagta 3480gataaattag taagtaatgg aatcaggaaa gtgctatttc tagatggaat agataaagct 3540caagaagagc atgaaaagta tcacagcaat tggagagcaa tggctagtga ctttaatctg 3600ccacccatag tagcaaaaga aatagtagct agctgtgatc aatgtcagct aaaaggggaa 3660gccatgcatg gacaagtaga ctgtagtcca gggatatggc aattagattg tacacattta 3720gaaggaaaaa tcatcctggt agcagtccat gtagccagtg gctacataga agcagaagtt 3780atcccagcag aaacaggaca agaaacagca tactttatac taaaattagc aggaagatgg 3840ccagtcaaaa taatacatac agacaatggt agcaatttca ccagtgctgc agttaaggca 3900gcctgttggt gggcaggtat ccaacaggaa tttggaattc cctacaatcc ccaaagtcag 3960ggagtagtag aatccatgaa taaagaatta aagaaaatta tagggcaggt aagagatcaa 4020gctgagcacc ttaagacagc agtacaaatg gcagtattca ttcacaattt taaaagaaaa 4080ggggggattg gggggtacag tgcaggggaa agaataatag acataatagc aacagacata 4140caaactaaag aattacaaaa acaaattata aaaattcaaa attttcgggt ttattacaga 4200gacagcagag accccatttg gaaaggacca gccaaactac tctggaaagg tgaaggggca 4260gtagtaatac aagataatag tgacataaag gtagtaccaa ggaggaaagc aaaaatcatt 4320aagggctatg gaaaacagat ggcaggtgct gattgtgtgg caagtagaca ggatgaagat 4380tagtaatttt ttatgcggcc gcta 4404<210>20<211>1457<212>PRT<213>人<400>20Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly1 5 10 15Ala Val Phe Val Ser Ala Arg Met Gly Ala Arg Ala Ser Ile Leu Arg
20 25 30Gly Gly Lys Leu Asp Lys Trp Glu Lys Ile Arg Leu Arg Pro Gly Gly
35 40 45Lys Lys His Tyr Met Leu Lys His Ile Val Trp Ala Ser Arg Glu Leu
50 55 60Glu Arg Phe Ala Leu Asn Pro Gly Leu Leu Glu Thr Set Glu Gly Cys65 70 75 80Lys Gln Ile Ile Lys Gln Leu Gln Pro Ala Leu Gln Thr Gly Thr Glu
85 90 95Glu Leu Arg Ser Leu Phe Asn Thr Val Ala Thr Leu Tyr Cys Val His
100 105 110Ala Gly Ile Glu Val Arg Asp Thr Lys Glu Ala Leu Asp Lys Ile Glu
115 120 125Glu Glu Gln Asn Lys Met Gln Gln Lys Thr Gln Gln Ala Lys Glu Ala
130 135 140Asp Gly Lys Val Ser Gln Asn Tyr Pro Ile Val Gln Asn Leu Gln Gly145 150 155 160Gln Met Val His Gln Ala Ile Ser Pro Arg Thr Leu Asn Ala Trp Val
165 170 175Lys Val Ile Glu Glu Lys Ala Phe Ser Pro Glu Val Ile Pro Met Phe
180 185 190Thr Ala Leu Ser Glu Gly Ala Thr Pro Gln Asp Leu Asn Thr Met Leu
195 200 205Asn Thr Val Gly Gly His Gln Ala Ala Met Gln Met Leu Lys Asp Thr
210 215 220Ile Asn Glu Glu Ala Ala Glu Trp Asp Arg Val His Pro Val His Ala225 230 235 240Gly Pro Ile Ala Pro Gly Gln Met Arg Glu Pro Arg Gly Ser Asp Ile
245 250 255Ala Gly Thr Thr Ser Thr Leu Gln Gly Gln Ile Ala Trp Met Thr Ser
260 265 270Asn Pro Pro Val Pro Val Gly Glu Ile Tyr Lys Arg Trp Ile Ile Leu
275 280 285Gly Leu Asn Lys Ile Val Arg Met Tyr Ser Pro Val Ser Ile Leu Asp
290 295 300Ile Lys Gln Gly Pro Lys Glu Pro Phe Arg Asp Tyr Val Asp Arg Phe305 310 315 320Phe Lys Thr Leu Arg Ala Glu Gln Ala Thr Gln Asp Val Lys Asn Trp
325 330 335Met Thr Asp Thr Leu Leu Val Gln Asn Ala Asn Pro Asp Cys Lys Thr
340 345 350Ile Leu Arg Ala Leu Gly Pro Gly Ala Ser Leu Glu Glu Met Met Thr
355 360 365Ala Cys Gln Gly Val Gly Gly Pro Asn His Lys Ala Arg Val Leu Ala
370 375 380Glu Ala Met Ser Gln Ala Asn Gly Thr Ile Leu Met Gln Arg Ser Asn385 390 395 400Phe Lys Gly Ser Lys Arg Ile Val Lys Cys Phe Asn Cys Gly Lys Glu
405 410 415Gly His Ile Ala Arg Asn Cys Arg Ala Pro Arg Lys Lys Gly Cys Trp
420 425 430Lys Cys Gly Lys Glu Gly His Gln Met Lys Asp Cys Thr Glu Arg Gln
435 440 445Ala Asn Phe Leu Gly Lys Ile Trp Pro Pro His Lys Gly Arg Pro Gly
450 455 460Asn Phe Leu Gln Ser Arg Pro Glu Pro Thr Ala Pro Pro Ala Glu Ser465 470 475 480Phe Gly Phe Glu Glu Thr Thr Pro Ala Pro Lys Gln Glu Pro Lys Asp
485 490 495Arg Glu Pro Leu Thr Ser Leu Lys Ser Leu Phe Gly Ser Asp Pr0 Leu
500 505 510Ser Gln Pro Gln Ile Thr Leu Trp Gln Arg Pro Leu Val Ser Ile Arg
515 520 525Val Gly Gly Gln Ile Lys Glu Ala Leu Leu Asp Asp Thr Val Leu Glu
530 535 540Glu Val Asn Leu Pro Gly Lys Trp Lys Pro Lys Met Ile Gly Gly Ile545 550 555 560Gly Gly Phe Ile Lys Val Arg Gln Tyr Asp Gln Ile Pro Ile Glu Ile
565 570 575Cys Gly Lys Lys Ala Ile Gly Thr Val Leu Val Gly Pro Thr Pro Val
580 585 590Asn Ile Ile Gly Arg Asn Met Leu Thr Gln Leu Gly Cys Thr Leu Asn
595 600 605Phe Pro Ile Ser Pro Ile Glu Thr Ile Pro Val Lys Leu Lys Pro Gly
610 615 620Met Asp Gly Pro Arg Val Lys Gln Trp Pro Leu Thr Glu Glu Lys Ile625 630 635 640Lys Ala Leu Thr Ala Ile Cys Asp Glu Met Glu Lys Glu Gly Lys Ile
645 650 655Thr Lys Ile Gly Pro Glu Asn Pro Tyr Asn Thr Pro Val Phe Ala Ile
660 665 670Lys Lys Lys Asp Ser Thr Lys Trp Arg Lys Leu Val Asp Phe Arg Glu
675 680 685Leu Asn Lys Arg Thr Gln Asp Phe Trp Glu Val Gln Leu Gly Ile Pro
690 695 700His Pro Ala Gly Leu Lys Lys Lys Lys Ser Val Thr Val Leu Asp Val705 710 715 720Gly Asp Ala Tyr Phe Ser Val Pro Leu Tyr Glu Asp Phe Arg Lys Tyr
725 730 735Thr Ala Phe Thr Ile Pro Ser Ile Asn Asn Glu Thr Pro Gly Ile Arg
740 745 750Tyr Gln Tyr Asn Val Leu Pro Gln Gly Trp Lys Gly Ser Pro Ala Ile
755 760 765Phe Gln Cys Ser Met Ala Lys Ile Leu Glu Pro Phe Arg Ala Gln Asn
770 775 780Pro Glu Ile Val Ile Tyr Gln Tyr Gly Asp Asp Leu Tyr Val Gly Ser785 790 795 800Asp Leu Glu Ile Gly Gln His Arg Ala Lys Ile Glu Glu Leu Arg Glu
805 810 815His Leu Leu Lys Trp Gly Phe Thr Thr Pro Asp Lys Lys His Gln Lys
820 825 830Glu Pro Pro Phe Leu Trp Met Gly Tyr Glu Leu His Pro Asp Lys Trp
835 840 845Thr Val Gln Pro Ile Gln Leu Pro Glu Lys Asp Ser Trp Thr Val Asn
850 855 860Asp Ile Gln Lys Leu Val Gly Lys Leu Asn Trp Ala Ser Gln Ile Tyr865 870 875 880Pro Gly Ile Lys Val Arg Gln Leu Cys Lys Leu Leu Arg Gly Ala Lys
885 890 895Ala Leu Thr Asp Ile Ile Pro Leu Thr Glu Glu Ala Glu Leu Glu Leu
900 905 910Ala Glu Asn Arg Glu Ile Leu Lys Glu Pro Val His Gly Ala Tyr Tyr
915 920 925Asp Pro Ser Lys Asp Leu Ile Ala Glu Ile Gln Lys Gln Gly Gln Asp
930 935 940Gln Trp Thr Tyr Gln Ile Tyr Gln Glu Pro Phe Lys Asn Leu Lys Thr945 950 955 960Gly Lys Tyr Ala Lys Met Arg Thr Ala His Thr Asn Asp Val Lys Gln
965 970 975Leu Thr Glu Ala Val Gln Lys Ile Ser Met Glu Ser Ile Val Ile Trp
980 985 990Gly Lys Ile Pro Lys Phe Arg Leu Pro Ile Pro Lys Glu Thr Trp Glu
995 1000 1005Thr Arg Trp Thr Ala Tyr Trp Gln Ala Thr Trp Ile Pro Glu Trp Glu
1010 1015 1020Phe Val Asn Thr Pro Pro Leu Val Lys Leu Trp Tyr Gln Leu Glu Lys1025 1030 1035 1040Asp Pro Ile Ala Gly Val Glu Thr Phe Tyr Val Asp Gly Ala Ala Asn
1045 1050 1055Arg Glu Thr Lys Met Gly Lys Ala Gly Tyr Val Thr Asp Arg Gly Arg
1060 1065 1070Gln Lys Ile Val Ser Leu Thr Glu Thr Thr Asn Gln Lys Thr Glu Leu
1075 1080 1085Gln Ala Ile Cys Leu Ala Leu Gln Asp Ser Gly Ser Glu Val Asn Ile
1090 1095 1100Val Thr Asp Ser Gln Tyr Ala Leu Gly Ile Ile Gln Ala Gln Pro Asp1105 1110 1115 1120Lys Ser Glu Ser Glu Leu Val Asn Gln Ile Ile Glu Gln Leu Ile Lys
1125 1130 1135Lys Glu Arg Val Tyr Leu Ser Trp Val Pro Ala His Lys Gly Ile Gly
1140 1145 1150Gly Asn Glu Gln Val Asp Lys Leu Val Ser Asn Gly Ile Arg Lys Val
1155 1160 1165Leu Phe Leu Asp Gly Ile Asp Lys Ala Gln Glu Glu His Glu Lys Tyr
1170 1175 1180His Ser Asn Trp Arg Ala Met Ala Ser Asp Phe Asn Leu Pro Pro Ile1185 1190 1195 1200Val Ala Lys Glu Ile Val Ala Ser Cys Asp Gln Cys Gln Leu Lys Gly
1205 1210 1215Glu Ala Met His Gly Gln Val Asp Cys Ser Pro Gly Ile Trp Gln Leu
1220 1225 1230Asp Cys Thr His Leu Glu Gly Lys Ile Ile Leu Val Ala Val His Val
1235 1240 1245Ala Ser Gly Tyr Ile Glu Ala Glu Val Ile Pro Ala Glu Thr Gly Gln
1250 1255 1260Glu Thr Ala Tyr Phe Ile Leu Lys Leu Ala Gly Arg Trp Pro Val Lys1265 1270 1275 1280Ile Ile His Thr Asp Asn Gly Ser Asn Phe Thr Ser Ala Ala Val Lys
1285 1290 1295Ala Ala Cys Trp Trp Ala Gly Ile Gln Gln Glu Phe Gly Ile Pro Tyr
1300 1305 1310Asn Pro Gln Ser Gln Gly Val Val Glu Ser Met Asn Lys Glu Leu Lys
1315 1320 1325Lys Ile Ile Gly Gln Val Arg Asp Gln Ala Glu His Leu Lys Thr Ala
1330 1335 1340Val Gln Met Ala Val Phe Ile His Asn Phe Lys Arg Lys Gly Gly Ile1345 1350 1355 1360Gly Gly Tyr Ser Ala Gly Glu Arg Ile Ile Asp Ile Ile Ala Thr Asp
1365 1370 1375Ile Gln Thr Lys Glu Leu Gln Lys Gln Ile Ile Lys Ile Gln Asn Phe
1380 1385 1390Arg Val Tyr Tyr Arg Asp Ser Arg Asp Pro Ile Trp Lys Gly Pro Ala
1395 1400 1405Lys Leu Leu Trp Lys Gly Glu Gly Ala Val Val Ile Gln Asp Asn Ser
1410 1415 1420Asp Ile Lys Val Val Pro Arg Arg Lys Ala Lys Ile Ile Lys Gly Tyr1425 1430 1435 1440Gly Lys Gln Met Ala Gly Ala Asp Cys Val Ala Ser Arg Gln Asp Glu
1445 1450 1455Asp<210>21<211>1011<212>DNA<213>人<400>21gccgccacca tggacgccat gaagcgcggc ctgtgctgcg tgctgctgct gtgcggcgcc 60gtgttcgtga gcgcccgcat ggggggcaag tggtcaaaaa gtagcatagt tggatggcct 120gctataagag aaagaataag acgaactgaa ccagcagcag atggggtggg agcagtatct 180cgagacctgg aaaaacatgg agcaatcaca agtaacaaca cagcagatac taatgctgat 240tgtgcctggc tagaaacaca agaggaggag gaggtgggtt ttccagtcag acctcaggta 300cccttaagac caatgacttt taagggagca ttggatctca gcttcttttt aaaagaaaag 360gggggactgg aagggttaat ttactctaag aaaaggcaag agatccttga tttgtgggtc 420tatcacacac aaggctactt ccctgactgg cacaactaca caccaggacc aggggtcaga 480ttcccactga cttttgggtg gtgcttcaag ctagtaccag ttgacccagg ggaagtggaa 540gaggccaatg aaggagaaaa caactgtttg ctacaccctg tctgccagca tggaatggat 600gatgaacaca gagaagtatt aaagtggaag tttgacagtc aactagcaca cagacacagg 660gcccgcgagc tacatccgga gttttacaaa gactgcatgg agccagtaga tcctaaccta 720gagccctgga accatccagg aagtcagcct gaaactgctt gcaataactg ttattgtaaa 780cgctgtagct accattgtct agtttgcttt cagaaaaaag gcttaggcat ttcctatggc 840aggaagaagc ggagacagcg acgaagcgct cctccaagca gtgaggatca tcaaaatcct 900atatcaaagc agcccttacc ccgaacccag ggggacccga caggctcgga agaatcgaag 960aagaaggtgg agagcaagac aaaaacagat ccattcgatt agtaattttt t 1011<210>22<211>330<212>PRT<213>人<400>22Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly1 5 10 15Ala Val Phe Val Ser Ala Arg Met Gly Gly Lys Trp Ser Lys Ser Ser
20 25 30Ile Val Gly Trp Pro Ala Ile Arg Glu Arg Ile Arg Arg Thr Glu Pro
35 40 45Ala Ala Asp Gly Val Gly Ala Val Ser Arg Asp Leu Glu Lys His Gly
50 55 60Ala Ile Thr Ser Asn Asn Thr Ala Asp Thr Asn Ala Asp Cys Ala Trp65 70 75 80Leu Glu Thr Gln Glu Glu Glu Glu Val Gly Phe Pro Val Arg Pro Gln
85 90 95Val Pro Leu Arg Pro Met Thr Phe Lys Gly Ala Leu Asp Leu Ser Phe
100 105 110Phe Leu Lys Glu Lys Gly Gly Leu Glu Gly Leu Ile Tyr Ser Lys Lys
115 120 125Arg Gln Glu Ile Leu Asp Leu Trp Val Tyr His Thr Gln Gly Tyr Phe
130 135 140Pro Asp Trp His Asn Tyr Thr Pro Gly Pro Gly Val Arg Phe Pro Leu145 150 155 160Thr Phe Gly Trp Cys Phe Lys Leu Val Pro Val Asp Pro Gly Glu Val
165 170 175Glu Glu Ala Asn Glu Gly Glu Asn Asn Cys Leu Leu His Pro Val Cys
180 185 190Gln His Gly Met Asp Asp Glu His Arg Glu Val Leu Lys Trp Lys Phe
195 200 205Asp Ser Gln Leu Ala His Arg His Arg Ala Arg Glu Leu His Pro Glu
210 215 220Phe Tyr Lys Asp Cys Met Glu Pro Val Asp Pro Asn Leu Glu Pro Trp225 230 235 240Asn His Pro Gly Ser Gln Pro Glu Thr Ala Cys Asn Asn Cys Tyr Cys
245 250 255Lys Arg Cys Ser Tyr His Cys Leu Val Cys Phe Gln Lys Lys Gly Leu
260 265 270Gly Ile Ser Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Ser Ala Pro
275 280 285Pro Ser Ser Glu Asp His Gln Asn Pro Ile Ser Lys Gln Pro Leu Pro
290 295 300Arg Thr Gln Gly Asp Pro Thr Gly Ser Glu Glu Ser Lys Lys Lys Val305 310 315 320Glu Ser Lys Thr Lys Thr Asp Pro Phe Asp
325 330
Claims (36)
1.包含两个启动子的核酸载体。
2.权利要求1的核酸载体,其中启动子是pCMV和人延伸因子1α(hEF1α)。
3.权利要求2的核酸载体,其包含至少两种核酸序列,其中每种在不同启动子的控制之下。
4.权利要求3的核酸载体,其中核酸序列是HIV-1核酸序列。
5.权利要求4的核酸载体,其中HIV-1核酸序列是env和gag。
6.权利要求5的核酸载体,其中HIV-1核酸序列进一步包含异源前导序列。
7.权利要求6的核酸载体,其中前导序列是tPA(SEQ ID NO:1)。
8.权利要求5的核酸载体,其中HIV-1序列是tPA-env(SEQ ID NO:7)。
9.权利要求5的核酸载体,其中HIV-1序列是tPA-gag(SEQ ID NO:9)。
10.权利要求4的核酸载体,其包含SEQ ID NO:15的核酸序列。
11.包含权利要求10的核酸载体的DNA疫苗。
12.权利要求4的核酸载体,其中HIV-1核酸序列是pol、nef和tat或其融合序列。
13.权利要求12的核酸载体,其中HIV-1核酸序列进一步包含异源前导序列。
14.权利要求13的核酸载体,其中前导序列是tPA(SEQ ID NO:1)。
15.权利要求12的核酸载体,其中HIV-1 pol序列是SEQ ID NO:11的序列。
16.权利要求5的核酸载体,其中nef和tat是包含SEQ ID NO:13序列的融合nef-tat序列。
17.权利要求12的核酸载体,其包含SEQ ID NO:16的核酸序列。
18.包含权利要求17的核酸载体的DNA疫苗。
19.包含两种核酸载体的组合物,其中每种载体包含两个启动子,每一个启动子都控制一种HIV-1基因的表达。
20.权利要求19的组合物,其中一种核酸载体包含pCMV启动子和hEF1α启动子,以及HIV-1基因gag和env,另一种核酸载体包含pCMV启动子和hEF1α启动子,以及HIV基因pol、nef和tat。
21.权利要求20的组合物,其包含SEQ ID NO:15和16。
22.修饰的牛痘Ankara(MVA)载体,其包含至少三种插入到MVADelIII区的核酸序列,其中每一种核酸序列都在单独的启动子控制之下。
23.权利要求22的修饰的MVA载体,其中核酸序列是HIV-1序列env、gag和pol或其融合序列。
24.权利要求23的修饰的MVA载体,其中核酸序列是SEQ ID NO:17和SEQ ID NO:19。
25.权利要求24的修饰的MVA载体,其进一步包含HIV-1 nef-tat融合序列。
26.权利要求25的修饰的MVA载体,其中HIV nef-tat序列是SEQ IDNO:21。
27.治疗有患HIV相关疾病危险的人类受试者的方法,其包含给予包含含SEQ ID NO:15的一种核酸和含SEQ ID NO:16的另一种核酸的核酸。
28.权利要求27的方法,其进一步包含给予包含SEQ ID NO:17、19和21的修饰的牛痘Ankara载体。
29.权利要求27的方法,其进一步包含给予权利要求26的修饰的牛痘Ankara载体。
30.防护有患HIV-1相关疾病危险的人类受试者的方法,其包含给予包含含SEQ ID NO:15的一种核酸和含SEQ ID NO:16的另一种核酸的DNA疫苗。
31.权利要求30的方法,其进一步包含给予包含SEQ ID NO:17、19和21的修饰的牛痘Ankara载体。
32.权利要求30的方法,其进一步包含给予权利要求26的修饰的牛痘Ankara载体。
33.改善患HIV-1相关疾病的受试者中HIV-1相关疾病的方法,其包含给予包含含SEQ ID NO:15的一种核酸和含SEQ ID NO:16的另一种核酸的DNA疫苗。
34.权利要求33的方法,其进一步包含给予包含SEQ ID NO:17、19和21的修饰的牛痘Ankara载体(ADMVA2)。
35.权利要求33的方法,其进一步包含给予权利要求26的修饰的牛痘Ankara载体。
36.用于对人类受试者提供针对HIV-1感染的防护的试剂盒,其包含包括SEQ ID NO:15的序列的一种核酸载体、包括SEQ ID NO:16的序列的另一种核酸载体和包括SEQ ID NO:17、19和21的加强牛痘疫苗以及进一步包含使用说明。
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CN113913393A (zh) * | 2021-06-07 | 2022-01-11 | 中国农业科学院哈尔滨兽医研究所(中国动物卫生与流行病学中心哈尔滨分中心) | 新型冠状病毒肺炎的重组新城疫病毒疫苗 |
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EP1789438B1 (en) | 2004-08-27 | 2015-04-15 | THE GOVERNMENT OF THE UNITED STATES OF AMERICA, as represented by THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES | Recombinant MVA viruses expressing modified env, gag and pol genes from HIV clade A/G, clade B, and clade C |
TWI311152B (en) * | 2004-09-17 | 2009-06-21 | Boehringer Ingelheim Rcv Gmbh & Co K | Host-vector system for antibiotic-free cole1 plasmid propagation |
ES2281252B1 (es) | 2005-07-27 | 2009-02-16 | Consejo Superior De Investigaciones Cientificas | Vectores recombinantes basados en el virus modificado de ankara (mva) como vacunas preventivas y terapeuticas contra el sida. |
JPWO2007102326A1 (ja) * | 2006-03-07 | 2009-07-23 | 公立大学法人横浜市立大学 | アデノウイルス5型/35型ベクターとワクシニアウイルスmvaベクターとの併用による強力な免疫誘導 |
EP2579892A2 (en) | 2010-06-09 | 2013-04-17 | Vaccine Technologies, Incorporated | Therapeutic immunization in hiv infected subjects receiving stable antiretroviral treatment |
RU2609769C2 (ru) | 2011-04-06 | 2017-02-02 | Биоваксим Лимитед | Фармацевтическая композиция для профилактики и(или) лечения заболеваний вич у людей |
CN102961743A (zh) * | 2012-12-20 | 2013-03-13 | 中国农业科学院哈尔滨兽医研究所 | 表达鸡毒支原体TM1蛋白的重组新城疫LaSota弱毒疫苗株 |
KR102395493B1 (ko) * | 2013-11-14 | 2022-05-09 | 더 트러스티스 오브 더 유니버시티 오브 펜실바니아 | Hiv-1 env dna 백신과 단백질 부스터 |
WO2016065252A2 (en) * | 2014-10-24 | 2016-04-28 | International Aids Vaccine Initiative | Native trimeric env immunogen design |
EP3310368A4 (en) * | 2015-06-19 | 2018-12-19 | Seracare Life Sciences Inc. | Sindbis control virus |
EP3841108A4 (en) * | 2018-08-23 | 2022-04-27 | Zion Medical B.V. | PHARMACEUTICAL COMPOSITIONS COMPRISING INTEGRATION-PROMOTING PEPTIDES |
JP2023527146A (ja) * | 2020-05-19 | 2023-06-27 | ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア | 免疫応答を誘導するためのコンジュゲートポリペプチドおよびワクチン |
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Publication number | Priority date | Publication date | Assignee | Title |
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US5747324A (en) * | 1988-06-10 | 1998-05-05 | Therion Biologics Corporation | Self-assembled, defective, non-self-propagating lentivirus particles |
WO2002072754A2 (en) * | 2001-03-08 | 2002-09-19 | The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Mva expressing modified hiv envelope, gag, and pol genes |
AU9456201A (en) * | 2000-09-15 | 2002-03-26 | Merck & Co Inc | Enhanced first generation adenovirus vaccines expressing codon optimized hiv1-gag, pol, nef and modifications |
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2002
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2003
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- 2003-10-17 AU AU2003286486A patent/AU2003286486A1/en not_active Abandoned
- 2003-10-17 EP EP03777686A patent/EP1570067A4/en not_active Withdrawn
- 2003-10-17 CN CNA2003801060851A patent/CN1726285A/zh active Pending
- 2003-10-17 WO PCT/US2003/033112 patent/WO2004035006A2/en not_active Application Discontinuation
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108778236A (zh) * | 2015-12-03 | 2018-11-09 | Dna精华股份有限公司 | 食品、饮料、化妆品和药物制剂中的寡核苷酸 |
CN113913393A (zh) * | 2021-06-07 | 2022-01-11 | 中国农业科学院哈尔滨兽医研究所(中国动物卫生与流行病学中心哈尔滨分中心) | 新型冠状病毒肺炎的重组新城疫病毒疫苗 |
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EP1570067A2 (en) | 2005-09-07 |
WO2004035006A2 (en) | 2004-04-29 |
CN1726285A (zh) | 2006-01-25 |
ZA200504021B (en) | 2006-09-27 |
EP1570067A4 (en) | 2007-10-03 |
AU2003286486A8 (en) | 2004-05-04 |
WO2004035006A3 (en) | 2005-06-16 |
AU2003286486A1 (en) | 2004-05-04 |
US20090227658A1 (en) | 2009-09-10 |
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