CN111778263A - 用于疫苗中的登革热病毒嵌合式构建物的组合物及方法 - Google Patents

用于疫苗中的登革热病毒嵌合式构建物的组合物及方法 Download PDF

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CN111778263A
CN111778263A CN202010617988.1A CN202010617988A CN111778263A CN 111778263 A CN111778263 A CN 111778263A CN 202010617988 A CN202010617988 A CN 202010617988A CN 111778263 A CN111778263 A CN 111778263A
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丹·T·斯廷奇科姆
克莱尔·凯尼
理查德·M·凯尼
吉尔·A·利文格德
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Takeda Vaccines Inc
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Abstract

本文的实施方案报告了登革热病毒构建物和活减毒登革热病毒的组合物、用途及制造。一些实施方案涉及这样的组合物，其包括但不限于四价登革热病毒组合物。在某些实施方案中，组合物可包含登革热病毒的一种或多种血清型的构建物，诸如登革热‑1(DEN‑1)病毒、登革热‑2(DEN‑2)病毒、登革热‑3(DEN‑3)病毒或登革热‑4(DEN‑4)病毒的构建物。在其它实施方案中，本文所公开的构建物可组合于组合物中以产生对抗多于一种或多种登革热病毒构建物的疫苗，该构建物可以或可以不接续地在哺乳动物细胞内传代。

Description

用于疫苗中的登革热病毒嵌合式构建物的组合物及方法
本申请是申请日为2014年3月12日的PCT国际专利申请PCT/US2014/024603进入中国国家阶段的中国专利申请号201480028001.5、发明名称为“用于疫苗中的登革热病毒嵌合式构建物的组合物及方法”的分案申请。
优先权
本PCT申请要求于2013年3月15日提交的第61/800,204号美国临时申请的优先权。该申请通过援引以其整体并入本文以用于全部目的。
联邦资助研发
本文公开的一些实施方案以资助号R43 AI08429-01获得美国国家卫生研究院的部分支持。美国政府对实施本发明拥有某些权利。
技术领域
本文的实施方案报告了登革热病毒构建物的组合物、方法、用途和制造程序及其疫苗组合物。一些实施方案涉及这样的组合物，其包括但不限于嵌合式黄病毒构建物，该构建物单独地或组合其它构建物可用于疫苗组合物。在某些实施方案中，组合物可包含多于一种登革热病毒血清型的构建物，诸如登革热-1(DEN-1)病毒、登革热-2(DEN-2)病毒、登革热-3(DEN-3)病毒和/或登革热-4(DEN-4)病毒的构建物。在其它实施方案中，制造策略可改善重组活减毒嵌合式登革热疫苗(DENVax)病毒的安全性和遗传稳定性。某些实施方案包括至少一种活减毒登革热病毒与被确定在疫苗组合物中是安全且有效的登革热病毒嵌合式构建物的组合，其中该构建物已经在细胞培养中经历额外的传代。
背景技术
感染登革热病毒可能导致不同严重程度的疼痛发烧。迄今为止，已经鉴定四种登革热病毒血清型：登革热-1(DEN-1)、登革热-2(DEN-2)、登革热-3(DEN-3)和登革热-4(DEN-4)。登革热由登革热病毒感染而引起。未来可能找到其它血清型(例如DEN-5)。登革热病毒血清型1-4也可能引发出血性登革热(DHF)和登革热休克综合征(DSS)。感染的最严重后果(DHF和DSS)可能威胁生命。登革热病毒每年引发5千万至1亿例病弱型登革热病毒、500,000例DHF/DSS，以及多于20,000例死亡。迄今为止，没有有效的疫苗来抵御登革热，且没有药物来治疗该病。蚊虫防治努力未能有效地预防登革热在病区的爆发，或防止该病的进一步地理扩散。估计有35亿人受登革热病毒感染的威胁。此外，登革热病毒乃是病区(诸如亚洲、中南美洲及加勒比海地区)的旅行者发烧的主要病因。
全部四种登革热病毒血清型在全球热带地区均为地方性的，且构成全球热带地区对人类有最大威胁的蚊媒病毒。登革热病毒主要由埃及伊蚊(Aedes aegypti)蚊子传播给人类。感染一种登革热病毒血清型导致终生免于再受该血清型的重复感染，但无法免于另外减毒登革热病毒血清型之一的二次感染。实际上，先前感染一种登革热病毒血清型导致当二次感染不同血清型时重症(DHF/DSS)的风险增高。有效疫苗的开发代表了此全球性疾病的预防和控制的重要方法。多重免疫接种使得对登革热病毒地方性国家中的公共卫生努力以及旅行者两者进行完整疫苗覆盖变得困难。
发明内容
本文的实施方案涉及嵌合式登革热病毒构建物的组合物、方法及用途。在一些实施方案中，组合物可包含具有减毒登革热病毒骨架的嵌合式登革热病毒构建物，所述构建物带有来自至少另一个登革热病毒血清型的结构基因。其它实施方案涉及至少一种活减毒病毒与一种或多种嵌合式登革热病毒的组合。其它实施方案可包括嵌合式登革热病毒的组合物，其具有修饰的DEN-2骨架(例如PDK-53作为P1(1-代)的起始骨架以及传代变异性(在许可细胞系上体外传代和生长之后)如针对P2、P3、...、P8、...P10等所指示的)以及DEN-1、DEN-2、DEN-3或DEN-4的一种或多种结构组分。在其它实施方案中，生成免疫原性组合物，当引入个体时，该组合物在个体内产生针对一种或多种登革热病毒的免疫应答。因此，本文预期的构建物可在体外生成并传代，以及每一代各自提供预期用于药学上可接受的疫苗组合物的减毒登革热病毒。在某些实施方案中，活减毒病毒可为单独的活减毒登革热-2病毒或其与一种或多种嵌合式登革热病毒的组合。
在某些实施方案中，登革热病毒血清型的嵌合式登革热病毒构建物可包含7-代(P7)活减毒病毒或嵌合式病毒，其具有由SEQ ID NO：1、4、7及10确定的核酸序列或由SEQID NO：2、3、5、6、8、9、11及12指示的多肽序列。本文预期了本文所述的任何活减毒病毒的任何传代可用于免疫原性组合物以诱导针对呈示的登革热病毒(例如血清型1-4)的免疫应答。依据这些实施方案，根据所选的构建物，包含P-8分离的活减毒病毒的免疫原性组合物可被给予个体以诱导对抗一种或多种登革热病毒血清型的免疫原性应答。此外，活减毒病毒可与这些嵌合式病毒中的一种或多种相组合。针对在各次接续细胞传代(例如非洲绿猴肾Vero细胞产生，后文称：Vero细胞)中分离/产生的每一活减毒病毒，预期了上述情况。本文预期了能够产生登革热病毒的任何细胞系(例如GMP产生的细胞存库、经FDA或EMA批准的)以制造规模用于使该病毒构建物中任一种传代，或视情况，本文预期了随后用于对抗登革热病毒的疫苗或免疫原性组合物。
在其它实施方案中，本文预期的组合物可与对抗其它黄病毒的其它免疫原性组合物如西尼罗病毒、日本脑炎病毒或任何其它黄病毒嵌合式构建物和/或活减毒病毒相组合。在某些实施方案中，单一组合物可用以对抗多种黄病毒。
在某些实施方案中，本发明的免疫原性组合物可包含对抗DEN-1、DEN-2、DEN-3、和/或DEN-4中的一个或多个的单独的嵌合式登革热病毒，或其与活减毒登革热病毒组合物的组合。
在其它实施方案中，构建物可包括在病毒的结构区或非结构区具有适应性突变的构建物，该突变使生长或生产得到提高而不影响引入个体时病毒的减毒或安全性。在某些实施方案中，预期的嵌合式登革热病毒构建物中的任一种可包括具有特定突变的活减毒DEN-2病毒用作为骨架，其中活减毒DEN-2 PDK病毒还包含其它登革热病毒血清型的prM(前膜)和E(被膜)结构蛋白质中的一种或多种的结构蛋白质。此外，DEN-2骨架可包括额外的突变，从而当给药时在个体中使对预定组合物的免疫应答的产生得到提高，或增强该免疫应答(例如嵌合式登革热病毒2/1、2/3或2/4)。
在一些实施方案中，结构蛋白质基因可包括在DEN-2骨架上的DEN-1、DEN-2、DEN-3或DEN-4的prM和E基因，所述DEN-2骨架具有一个或两个作为活减毒登革热病毒的一部分的突变。举例而言，在某些实施方案中，登革热构建物可包括称为DENVax-1-A、DENVax-2-F、DENVax-3-F和DENVax-4-F(参见实施例部分)的那些构建物，其中DEN-2骨架具有来自DEN-2活减毒病毒的一个或多个突变(例如，在P1或其它先前传代的病毒或PDK-53中未找到的)，所述DEN-2活减毒病毒先前验证为安全有效地诱导免疫应答。本申请的DEN-2活减毒病毒是原先使用的DEN-2活减毒病毒的改良版本。本发明的嵌合式构建物可包括修饰的减毒DEN-2PDK-53骨架，其具有第二登革热病毒血清型的一种或多种结构蛋白质，其中所述结构蛋白质可包括额外的突变以增加对嵌合式构建物的免疫原性应答。在一些实施方案中，由减毒DEN-2 PDK-53获得的某些突变在与P1构建物不同的构建物中可以产生或可以不产生保守性氨基酸变化，这可导致期望的产生特征等。
在其它实施方案中，活减毒DEN-2基因组可用以生成登革热病毒血清型1(DEN-1)、登革热病毒血清型3(DEN-3)、及登革热病毒血清型4(DEN-4)的构建物，其中DEN-2病毒基因组的一种或多种结构蛋白质基因可分别被DEN-1、DEN-3或DEN-4的一种或多种结构蛋白质基因替换。在一些实施方案中，结构蛋白质可为第二登革热病毒的C、prM或E蛋白。在某些实施方案中，结构蛋白质基因包括DEN-1、DEN-3或DEN-4的prM和E基因。这些杂交病毒表达DEN-1、DEN-3或DEN-4的表面抗原，同时保留亲代减毒DEN-2的减毒表现型。
本文公开的构建物可包括使用减毒DEN-2病毒作为骨架的表达DEN-1、DEN-3或DEN-4的表面抗原的DEN-4、DEN-2、DEN-1及DEN-3的嵌合式构建物。
在某些实施方案中，本发明的组合物可以包括这样的组合物，其包含本文公开的单一嵌合式登革热病毒构建物和药学上可接受的载剂或赋形剂。或者，本发明的组合物可以包括这样的组合物，其包含两种或更多种、或三种或更多种的本文公开的嵌合式登革热病毒构建物和药学上可接受的载剂或赋形剂。依据这些实施方案，本文预期的一种或多种登革热病毒嵌合式构建物可与一种或多种活减毒登革热病毒相组合。在某些实施方案中，活减毒病毒可为活减毒DEN-2病毒，其中在NCR、NS1区或其它区中的额外的突变可增强免疫应答，增加改良的活减毒登革热病毒的病毒生长或其它改善。
在某些实施方案中，DENV-2疫苗的减毒基因座(即核苷酸5’NCR-57-T、NS1-53-Asp及NS3-250-Val)先前已经确定，而全部这些变化均由四种DENVax病毒的共用PDK-53病毒特异性遗传背景所分享。针对cGMP制造的DENVax种，小心地监控三个减毒基因座的基因序列以及这些疫苗候选物先前建立的体外和活体内减毒表现型。本报告描述了用以生成主病毒种(MVS)的策略以及其用于制造登革热病毒疫苗组合物的基因表征和表现型表征。这些MVS可用以制造临床材料并最终供应商业疫苗。
附图说明
下列附图形成本说明书的一部分，且包括该附图以进一步展示某些实施方案。一些实施方案可以通过单独参考这些附图中的一张或多张或者与所呈示的特定实施方案的详细说明的组合而得到更好地理解。
图1示出示例性图表，其反映出与先前生成的构建物和野生型登革热病毒相比较的本发明的示例性嵌合式构建物DEN-2/DEN-4。
图2示出示例性直方图，其比较了使用活减毒DEN-2骨架(带有额外的突变)和第二登革热病毒血清型作为结构组分替代登革热-2结构组分(例如DENVax-1 MVS)的各种反应。该图例示DENVax MVS的溶菌斑尺寸。野生型登革热病毒及先前公开的研究级疫苗候选物病毒被包括在内以用于对照和比较。该图例示与对照登革热病毒嵌合式构建物相比，登革热病毒构建物的改善的产生。
图3示出示例性直方图，其示出DENVax MVS(主病毒种)的温度敏感性。野生型登革热病毒及先前公开的研究级疫苗候选物病毒被包括在内以用于与MVS级比较。
图4示出示例性直方图，其示出与对照相比，在C6/36细胞中DENVax MVS的病毒生长。野生型登革热病毒及先前公开的研究级疫苗候选物病毒被包括在内以用于与DENVaxMVS比较。
图5A-5C示出在新生小鼠中的神经毒力的示例性图。多个实验的汇集结果总结了野生型(wt)DENV-2 16681病毒在使用104pfu病毒(A)ic激发的CDC-ICR(n＝72)和Taconic-ICR(n＝32)新生小鼠中的神经毒力。以104pfu(B)或103pfu(C)剂量在Taconic-ICR小鼠中测试DENVax MVS的神经毒力。指出在一个实验(n＝16)或两个汇集实验(n＝31或32)中每组测试的动物数。
图6示出示例性直方图，其例示DENVax MVS、WVS和BVS的溶菌斑尺寸。在感染后第9天测量在琼脂糖覆盖层下方的Vero细胞或LLC-MK2细胞中的病毒溶菌斑的平均溶菌斑直径±SD(误差柱)。野生型DENV及先前公开的研究级疫苗候选物病毒被包括在内以用于对照和比较。
图7示出示例性直方图，其例示在两个培养温度下于C6/36细胞中DENVax MVS、WVS和BVS的生长以证实在大规模制造之后保留这种体外减毒标记。
图8示出示例性直方图，其例示在C6/36细胞中DENVax MVS、WVS和BVS的受限制的生长。感染后七天在C6/36细胞中复制的病毒的平均滴度±SD(误差柱)。野生型登革热病毒及先前公开的研究级疫苗候选物病毒被包括在内以用于比较。
图9A-9B示出DENVax MVS在新生ICR小鼠中神经毒力的示例性数据图。(A)104pfu剂量的病毒的IC接种。(B)103pfu剂量的病毒的IC接种。
图10示出示例性图表，其比较了新活减毒病毒与先前生成的活减毒登革热病毒。
具体实施方式
定义
如本文所用，“一(a)”或“一(an)”可表示一项或多于一项。
如本文所用，“一个个体”或“多个个体”可包括但不限于，哺乳动物，诸如人类或哺乳动物(驯养的或野生的)，例如犬、猫、其它居家宠物(例如仓鼠、豚鼠、小鼠、大鼠)、雪貂、免、猪、马、牛、土拨鼠、野生啮齿类、或动物园动物。
如本文所用，术语“病毒嵌合体”、“嵌合式病毒”、“黄病毒嵌合体”及“嵌合式黄病毒”可表示这样的构建物，其包含登革热-2病毒的核苷酸序列的一部分且还包含非来自登革热-2病毒的核苷酸序列或来自不同黄病毒的核苷酸序列。“登革热嵌合体”包含至少两种不同的登革热病毒血清型，但非不同的黄病毒。因此，其它登革热病毒或黄病毒的实例包括但不限于，来自登革热-1病毒、登革热-3病毒、登革热-4病毒、西尼罗病毒、日本脑炎病毒、圣路易脑炎病毒、蜱媒脑炎病毒、黄热病毒、及其任何组合。
如本文所用，“核酸嵌合体”可表示本发明的构建物，其包含登革热-2病毒的核苷酸序列的一部分且进一步包含与登革热-2病毒的核苷酸序列不同来源的核苷酸序列。相应地，本文公开的任何嵌合式黄病毒或黄病毒嵌合体可认为是核酸嵌合体的实例。
如本文所用，“活减毒病毒”可表示野生型病毒，其经突变或选择而具有用于疫苗或其它免疫原性组合物的特征，其中一些特征可包括毒力降低、安全性、功效、或改善的生长等。
描述
于下列部分中，描述各种示例性组合物和方法以详细说明各种实施方案。对本领域技术人员显而易见的是，实施各种实施方案并不要求采用本文概述的全部或甚至某些特定细节，而可以通过常规实验修正浓度、时间及其它特定细节。在一些情况下，公知的方法或组分不包括于该描述中。
依据本发明的实施方案，可采用在本领域的技能范围之内的常规的分子生物学、蛋白质化学、微生物学及重组DNA技术。此类技术完整地阐述于参考文献中。参见，例如，Sambrook,Fritsch&Maniatis，Molecular Cloning:A Laboratory Manual,第二版，1989，Cold Spring Harbor Laboraoty Press,Cold Spring Harbor,N.Y.；Animal CellCulture,R.I.Freshney编辑，1986。
本文的实施方案涉及个别地或同时地于个体中诱导对抗一种或多种登革热病毒血清型的免疫应答的组合物、方法及用途。依据这些实施方案，生成减毒登革热病毒及核酸嵌合体，并用于本文公开的疫苗组合物。一些实施方案涉及修饰或突变的登革热构建物或嵌合体。其它实施方案涉及引入突变以修饰登革热病毒的结构蛋白质的氨基酸序列，其中该突变提高对病毒的免疫原性。
已经由在细胞培养中传代野生型病毒而发展出全部四种血清型的活减毒登革热病毒。这些是用于对抗黄病毒和特定的登革热病毒感染和/或疾病的免疫接种的最有希望的活减毒疫苗候选物种的一些候选物。这些疫苗候选物已经由其登革热血清型的组合、其传代的细胞系及其传代次数加以标示。因此，在PDK细胞中传代13次的登革热血清型1野生型病毒标示为DEN-1 PDK-13病毒。其它疫苗候选物为DEN-2 PDK-53、DEN-3 PGMK-30/FRhL-3(例如于初代绿猴肾细胞传代30次，接着于胎儿恒河猴肺细胞传代3次)和DEN-4 PDK-48。这四种候选物疫苗病毒分别由野生型亲代DEN-1 16007、DEN-2 16681、DEN-3 16562及DEN-4 1036病毒的组织培养传代而衍生。
在某些实施方案中，活减毒登革热-2 PDK-53疫苗病毒含有病毒混合物，并且群体含有各种核苷酸差异。在减毒突变的基因表征之后，某些减毒特性被概括出并被改造进cDNA传染性克隆。从此种传染性克隆转录RNA并引入Vero细胞作为新表征且衍生的PDK-53-Vero-DEN-2-P 1病毒的1-代(例如参考表1)。针对各个DEN血清型产生此类减毒病毒，但针对DEN-1、DEN-3及DEN-4，prM和E基因被改造进3种单独的cDNA传染性克隆，由此生成四种单独的PDK-53-Vero病毒(本文分别称为：PDK-53-Vero-DEN-2-P 1、PDK-53-Vero-DEN-1-P 1、PDK-53-Vero-DEN-3-P 1、以及PDK-53-Vero-DEN-4-P 1)。这些减毒疫苗病毒株于Vero细胞中传代10次(表1)，并且各个单独的世系当其适应于Vero细胞中生长时获得突变(表3)。本文某些实施方案涉及这些活减毒登革热病毒的衍生及用途。
先前使用这些减毒病毒的人类临床试验已经指出DEN-2 PDK-53在人类中具有最低感染剂量(50％溶菌斑形成单位或PFU的50％最低感染剂量)，具有强免疫原性，并且未引起明显的安全性担忧。DEN-1 PDK-13、DEN-3 PGMK-30/FRhL-3及DEN-4 PDK-48疫苗病毒候选物在人类中分别具有10,000、3500及150PFU的较高50％最低感染剂量。虽然使用一价DEN-2 PDK-53病毒或DEN-4 PDK-48病毒只需要一次免疫接种即可在人类个体中实现100％血清转化，但针对具有用于人类的两个最高感染剂量的DEN-1 PDK-13和DEN-3 PGMK-30/FRhL-3病毒，仍需要加强接种(booster)才能实现相同的血清转化率。
DEN-2 PDK-53病毒疫苗候选物也缩写为PDK-53，其具有与减毒相关的多种可测量的生物标记，包括温度敏感性、小溶菌斑尺寸、在蚊C6136细胞培养中的复制减少，在完好蚊体内的复制降低，对乳小鼠的神经毒力丧失以及在猴中的病毒血症发生率降低。候选物PDK-53疫苗的临床试验已经证实其在人类中的安全性和免疫原性。此外，PDK-53疫苗在人类疫苗接受者中诱导登革热病毒特异性T-细胞记忆反应。本文的一些实施方案描述了对在本文公开的嵌合式构建物中使用的DEN-2 PDK-53的改良。
具有登革热-2病毒骨架以及至少一个另一种登革热病毒血清型的结构蛋白质的免疫原性黄病毒嵌合体可用于制备登革热病毒嵌合体，并描述了用于产生登革热病毒嵌合体的方法。在药学上可接受的载剂中单独地或组合地提供了免疫原性登革热病毒嵌合体作为免疫原性组合物，从而最小化、抑制或使个体免疫于单独或组合的一种或多种血清型(例如登革热病毒血清型DEN-1、DEN-2、DEN-3及DEN-4)的感染。当组合时，免疫原性登革热病毒嵌合体可用作多价疫苗(例如二价、三价及四价)以赋予同时对抗多于一种的黄病毒物种或病毒株的感染的保护作用。在某些实施方案中，登革热病毒嵌合体组合于可用作对抗已知登革热病毒血清型的二价、三价或四价疫苗的免疫原性组合物，或通过包含编码一种或多种来自不同黄病毒的蛋白质的核酸而赋予对其它病原性黄病毒的免疫力。
在一些实施方案中，本文提供的无毒力免疫原性登革热病毒嵌合体含有：减毒登革热-2病毒(例如PDK-53)的非结构蛋白质基因，或其等同物；以及将要在个体内诱导对抗黄病毒的免疫原性的该黄病毒的结构蛋白质基因或其免疫原性部分中的一种或多种。例如，一些实施方案涉及这样的嵌合体，其具有：作为病毒骨架的减毒登革热-2病毒PDK-53基因组；以及PDK-53基因组的编码衣壳、前膜/膜、或被膜的一种或多种结构蛋白质基因或其组合，所述结构蛋白质基因或其组合被将要经保护以对抗诸如不同黄病毒或不同登革热病毒血清型的来自DEN-1、DEN-3或DEN-4或其它黄病毒的一种或多种相对应的结构蛋白质基因替换。依据这些实施方案，本文公开的核酸嵌合体可具有减毒登革热-2病毒的功能性质且为无毒力的，但表达DEN-1、DEN-3或DEN-4以及其它黄病毒的结构基因产物的抗原表位，并且为免疫原性的(例如在个体中诱导对基因产物的免疫应答)。然后，这些DNA构建物用于从传染性克隆转录RNA，此种RNA被引入Vero细胞内，再次产生处于P1的新后代。这些新的后代可与PDK-53区别(参见，例如P1-P10)。
在另一实施方案中，核酸嵌合体可为这样的核酸嵌合体，其具有(但不限于)编码来自减毒登革热-2病毒的非结构蛋白质的第一核苷酸序列以及编码来自单独登革热-4病毒或来自登革热-4病毒与另一种黄病毒组合的结构蛋白质的第二核苷酸序列。在其它实施方案中，减毒登革热-2病毒可为具有一种或多种突变氨基酸的疫苗株PDK-53(参见实施例)。这些额外的突变赋予了用作活减毒登革热-2或本文所述的嵌合式构建物的期望特性。一些实施方案包括第二登革热病毒的C、prM或E蛋白中的一种或多种的结构蛋白质。
其它方面包括嵌合式病毒可包括例如在对照PDK-53登革热-2基因组中的核苷酸和氨基酸取代、删除或插入以减少对靶标登革热病毒血清型的免疫原性应答的干扰。这些修饰可在结构蛋白质及非结构蛋白质中单独进行，或与本文公开的示例性修饰组合地进行，并且可通过使减毒病毒传代且获得用以诱导对抗一种或多种登革热病毒血清型的免疫应答的改良组合物而生成。
本文公开的某些实施方案提供了通过将所需的取代插入适当的骨架基因组中而使用重组技术制造本发明的嵌合式病毒的方法。本文的其它实施方案涉及使经确认的(例如安全且有效的)活减毒嵌合式病毒传代以获得额外的改良。在某些实施方案中，本文使用的登革热-2骨架可包括表3呈现的一个或多个突变。在其它实施方案中，本申请的登革热-登革热嵌合体可包括表3呈现的一个或多个突变。在其它实施方案中，登革热-登革热嵌合体可包括针对Den-2/Den-1、Den-2/Den-3或Den-2/Den-4的表3呈现的各种嵌合体的全部突变。预期了包括表3的构建物表示的活减毒病毒的药物组合物。例如，本文预期了使用表3呈现的嵌合体和活减毒登革热-2病毒的一-、二-、三-或四价组合物的用途。
在某些实施方案中，本文预期的活减毒DEN-2变异体可配制成药物组合物，其中该药物组合物可单独给予或与登革热-登革热嵌合体或登革热-黄病毒嵌合体组合地给予。在某些实施方案中，二价、三价或四价组合物可以单次施用或多次施用的形式给予至个体。
黄病毒嵌合体
登革热型1-4(DEN-1至DEN-4)为蚊媒黄病毒病原体。黄病毒基因组含有5’-非编码区(5’-NC)，接着为衣壳蛋白(C)编码区，接着为前膜/膜蛋白(prM)编码区，接着为被膜蛋白(E)编码区，接着为编码非结构蛋白(NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5)的区域及最后为3’非编码区(3’NC)。病毒结构蛋白质为C、prM及E，并且非结构蛋白质为NS1-NS5。结构蛋白质和非结构蛋白质翻译为单一多蛋白并由细胞和病毒蛋白酶处理。
黄病毒嵌合体可为通过融合来自登革热或黄病毒科病毒种的一种类型或血清型的非结构蛋白质基因与来自登革热病毒或黄病毒科病毒种的不同类型或血清型的蛋白质基因例如结构蛋白质基因而形成的构建物。或者，本发明的黄病毒嵌合体为通过如下方式而形成的构建物，即，融合来自登革热病毒或黄病毒科病毒种的一种类型或血清型的非结构蛋白质基因与指导选自其它登革热病毒血清型或其它黄病毒科病毒的多肽或蛋白质的合成的其它核苷酸序列。
在其它实施方案中，本文提供的无毒力免疫原性黄病毒嵌合体含有减毒登革热-2病毒的非结构蛋白质基因或其等同物，以及将要被赋予的免疫原性所针对的黄病毒的结构蛋白质基因中的一种或多种或其抗原部分。适当的黄病毒包括但不限于表1列举的那些。
用于构建嵌合体的其它适当的登革热病毒可为野生型毒力DEN-1 16007、DEN-216681、DEN-3 16562及DEN-4 1036以及减毒疫苗株DEN-1 PDK-13、DEN-2 PDK-53、DEN-3PMK-30/FRhL-3及DEN-4 PDK-48。预期了DEN-1、DEN-2、DEN-3及DEN-4野生型/减毒病毒对之间的基因差异，以及由病毒基因组编码的氨基酸序列的变化。
DEN-2 PDK-53的序列列表对应于DEN-2 PDK-53-V变异体，其中基因组核苷酸位置5270从A突变为T，并且多蛋白的氨基酸位置1725或NS3蛋白的氨基酸位置250含有缬氨酸残基。不含此种核苷酸突变的DEN-2 PDK-53，即DEN-2 PDK-53-E，与PDK-53-V的差异只在于这一个位置。DEN-2 PDK-53-E在核苷酸位置5270处具有A且在多蛋白氨基酸位置1725(即NS3蛋白氨基酸位置250)处具有谷氨酸根。应理解本文的实施方案包括修饰的PDK 53，其包括在单独的宿主细胞(例如Vero细胞，参考表1)中的一次或多次传代，其中生成本文预期的用于疫苗组合物的所需特性。
在某些实施方案中，嵌合体的标示可基于DEN-2病毒特异性传染性克隆修饰骨架及其它登革热病毒或其它黄病毒的结构基因(prM-E或C-prM-E)插入物。DEN-2为登革热-2骨架，接着是插入结构基因所来自的株。一个DEN-2骨架变异体在数字标示后的下个字母中得到反映。从中构建嵌合体的一个特定DEN-2骨架变异体由置于连字符后方的字母指示，亲代16681(P)，PDK-53-E(E)或PDK-53-V(V)；最末字母指示来自亲代(P)株或其疫苗衍生物(V)的C-prM-E结构基因或来自亲代(P)或其疫苗衍生物(V1)的prM-E结构基因。例如，DEN-2/1-VP表示该嵌合体包含减毒DEN-2 PDK-53V骨架(在NS3-250处包含缬氨酸)和来自野生型DEN-1 16007的C-prM-E基因；DEN-2/1-VV表示DEN-2 PDK-53V骨架具有登革热-1、DEN-1PDK-13的疫苗株；DEN-2/1-VP1表示DEN-2 PDK-53V骨架和来自野生型DEN-1 16007的prM-E基因；DEN-2/3-VP1表示DEN-2 PDK-53V骨架和来自野生型DEN-3 16562的prM-E基因；DEN-2/4VP1表示DEN-2 PDK-53V骨架和来自野生型DEN-4 1036的prM-E基因。本文公开的其它嵌合体以相同方式指示。
在一个实施方案中，本文公开的嵌合体含有减毒登革热-2病毒PDK-53基因组作为病毒骨架，其中PDK-53基因组的编码C、prM及E蛋白的结构蛋白质基因或其组合可以被来自登革热-1、登革热-3或登革热-4病毒以及任选地待被抵御的另一种黄病毒(诸如不同的黄病毒或不同的登革热病毒株)的相对应的结构蛋白质基因替代。
在非结构蛋白质区域，在非结构蛋白质NS1-53(基因组核苷酸位置2579)处发现Gly-至-Asp(野生型至PDK-53)突变；在非结构蛋白质NS2A-181(基因组核苷酸位置4018)处发现Leu-至-Phe(野生型-至-PDK-53)突变；在非结构蛋白质NS3-250(基因组核苷酸位置5270)处发现Glu-至-Val(野生型-至-PDK-53)突变；以及在非结构蛋白质NS4A-75(基因组核苷酸位置6599)处发现Gly-至-Ala(野生型-至-PDK-53)突变。本发明的活减毒DEN-2病毒还包括如表3的任何嵌合体或活减毒登革热-2病毒所呈现的突变。
PDK-53病毒株在基因组核苷酸5270处具有混合基因型。相当一部分(约29％)的病毒群体编码存在于野生型DEN-2 16681病毒的非突变NS3-250-Glu，而非NS3-250-Val突变。因两种基因变异体均为无毒力的，故此种突变在无毒力嵌合体中可为非必要的。
先前，发现减毒PDK-53病毒株的无毒力可促成在编码非结构蛋白质的核苷酸序列中以及在5’非编码区中的突变。例如，在NS1-53的单一突变、在NS1-53和5’NC-57的双突变，在NS1-53和NS3-250的双突变、以及在NS1-53、5’NC-57和NS3-250的三突变，导致DEN-2病毒的减毒。因此，在这些基因座处含有此类非保守氨基酸取代或核苷酸取代的任何登革热-2病毒的基因组可用作基础序列以衍生本文公开的修饰PDK-53病毒。根据需要，在5’非编码区的茎/环结构的茎中的另一突变会提供额外的无毒力表现型稳定性。对该区域的突变破坏对于病毒复制的重要的潜在二级结构。在DEN和委内瑞拉马脑炎病毒两者的这种短(长度只有6个核苷酸残基)茎结构中的单一突变破坏发夹结构的形成。在此茎结构的进一步突变降低了在此基因座反转的可能性，同时维持病毒活力。
本文公开的突变可通过本领域已知的任何种方法实现，包括但不限于，具有额外特征的天然存在或所选的克隆在感兴趣的细胞系(例如Vero细胞)中传代一次。本领域技术人员应理解如本文所述且本领域公知的毒力筛检试验可用于区别毒力的与无毒力的骨架结构。
黄病毒嵌合体的构建
本文所述的黄病毒嵌合体可通过如下制备，通过期望免疫力所针对的黄病毒的结构蛋白质基因中的一种或多种剪接入PDK-53登革热病毒基因组骨架；使用重组工程或通过本领域已知的其它方法来去除相对应的PDK-53基因并用登革热-1、登革热-3或登革热-4病毒基因或本领域已知的其它基因对其进行替换。
或者，使用序列表中提供的序列，使用已知的核酸合成技术可合成编码黄病毒蛋白质的核酸分子，并将其插入适当的载体中。因此，使用本领域技术人员已知的重组工程技术产生无毒力免疫原性病毒。
靶标基因可插入骨架中，其编码DEN-1、DEN-3、DEN-4或其它黄病毒的感兴趣的黄病毒结构蛋白质。将要插入的黄病毒基因可为编码C蛋白、PrM蛋白和/或E蛋白的基因。插入登革热-2骨架的序列可编码PrM和E结构蛋白质。插入登革热-2骨架的序列可编码C、prM及E结构蛋白质中的全部或一种。
可通过以下来评估含有编码其它黄病毒或登革热病毒血清型的结构蛋白质的核苷酸序列的适当嵌合体病毒或核酸嵌合体作为疫苗的有用性，根据指示无毒力的前述减毒表现型标记对其进行筛选，以及根据免疫原性对其进行筛选。可使用本领域技术人员已知的常规筛选程序，利用与黄病毒抗体或免疫反应性血清的体外或活体内反应性来评估抗原性和免疫原性。
登革热病毒疫苗
在某些实施方案中，嵌合体病毒和核酸嵌合体可提供可用作为免疫原或疫苗的活减毒病毒。一些实施方案包括展现对登革热-4病毒高免疫原性、同时不产生危险的致病或致命效应的嵌合体。
为了减少在个体中出现DHF/DSS，需要四价疫苗来提供对全部四种病毒血清型同时的免疫力。通过将本发明的活减毒登革热-2病毒与上述登革热-2/1、登革热-2/3及登革热-2/4嵌合体在适当的药学载剂内组合而制备四价疫苗，从而以多价疫苗形式给药。
本发明的嵌合式病毒或核酸嵌合体可包括在毒力或减毒DEN-2病毒骨架中的野生型或活减毒病毒的结构基因。例如，嵌合体可在任一DEN-2背景下表达野生型DEN-4 1036病毒的结构蛋白质基因、其候选物疫苗衍生物。
本文所述的嵌合体中使用的病毒可使用本领域已知的方式生长。然后，进行病毒溶菌斑滴定并计数溶菌斑以评估生长培养物的活力和表现型特性。野生型病毒可通过培养的细胞系来传代，从而衍生减毒候选物起始材料。
嵌合式传染性克隆可由可用的各种登革热血清型克隆构建。若需要，也可完成病毒特异性cDNA片段的克隆。通过逆转录酶-聚合酶链式反应(RT-PCR)使用各种引物从登革热病毒RNA扩增含有结构蛋白质或非结构蛋白质基因的cDNA片段。扩增的片段被克隆入其它中间克隆的裂解位置。然后，中间嵌合式登革热病毒克隆经测序以证实所插入的登革热病毒特异性cDNA的准确性。
通过将登革热血清型病毒的结构蛋白质和/或非结构蛋白质基因区插入载体内而构建的全基因组长度嵌合式质粒可使用本领域技术人员公知的重组技术获得。
核苷酸及氨基酸分析
在DEN-2 PDK-53疫苗病毒中的NS1-53突变对此种病毒的减毒表现型为重要的，因为DEN-2 16681病毒的NS1-53-Gly在至今为止已测序的几乎全部的黄病毒，包括蜱媒病毒，中保守。DEN-4疫苗病毒也可含有在NS1蛋白的位置253处的氨基酸突变。在DEN-4 PDK-48疫苗病毒中的Gln-至-His突变的这种基因座在全部四种野生登革热病毒血清型中均为Gln。此种Gln残基为黄病毒属内的登革热病毒所独有的。NS1蛋白为从黄病毒感染的细胞分泌的糖蛋白。其存在于感染的细胞的表面，并且NS1-特异性抗体存在于病毒感染的个体的血清中。已经报告用NS1蛋白免疫接种或用NS1特异性抗体被动免疫接种来保护动物。NS1蛋白可能参与早期病毒RNA复制。
出现于DEN-1、-2、-3及-4减毒株的NS2A、NS2B、NS4A及NS4B蛋白中的突变本质上为保守的。DEN-2和DEN-4疫苗病毒分别的NS4A-75和NS4A-95突变出现在登革热病毒中氨基酸保守的位点，但通常不出现在黄病毒中。
黄病毒NS3蛋白具有至少两种已认知的功能：病毒性蛋白酶以及RNA螺旋酶/NTPase。698-aa长度(DEN-2病毒)NS3蛋白含有氨基末端的丝氨酸蛋白酶结构域(NS3-51-His、-75-Asp、-135-Ser催化三联体)，接着为RNA螺旋酶/NTPase功能的序列基序(NS3-196-GAGKT(SEQ ID NO：147)、-284-DEAH、-459-GRIGR)。在DEN-1、DEN-2或DEN-3病毒的NS3蛋白中无一突变出现在已认知的基序中。在DEN-1 PDK-13病毒中的NS-510Tyr-至-Phe突变为保守的。因为野生型DEN-2、-3及-4病毒在此位置含有Phe，所以Tyr-至-Phe突变在DEN-1病毒的减毒方面不可能发挥作用。在DEN-1 PDK-13病毒中的NS3-182 Glu-至-Lys突变出现在大部分蚊媒黄病毒中保守为Asp或Glu的位置，并且在减毒方面可发挥一些作用。此种突变位于GAGKT螺旋酶基序上游15个氨基酸残基处。如先前报告所记，在DEN-2 16681病毒中的NS3-250-Glu在除黄热病病毒之外的全部蚊媒黄病毒中保守。
核酸探针与编码DEN-1、DEN-3及DEN-4病毒的核酸分子或其互补序列选择性地杂交。“选择性的”或“选择性地”表示序列不与其它核酸杂交以防止登革热病毒的充足检测。因此，在杂交核酸的设计中，选择性将取决于样本内存在的其它组分。杂交核酸应具有与其所杂交至的核酸片段至少70％的互补性。如本文用于描述核酸那样，术语“选择性地杂交”排除偶尔随机杂交的核酸，因而具有与“特异性杂交”相同的定义。本发明的选择性杂交核酸可具有与其所杂交至的序列片段至少70％、80％、85％、90％、95％、97％、98％及99％的互补性，优选85％或更高。
预期了选择性杂交至编码核酸或该核酸的互补链或相反链的序列、探针或引物。与核酸的特异性杂交可在核酸中的微小修饰或取代的情况下发生，只要保持功能性种类特异性杂交能力即可。“探针”表示可用作与互补核酸序列选择性杂交以用于其检测或扩增的探针或引物的核酸序列，所述探针的长度可由约5个核苷酸变化至100个核苷酸，或优选约10个核苷酸至50个核苷酸，或约18个核苷酸至24个核苷酸。
若用作引物，则该组合物优选包含至少两个杂交至靶标分子的不同区的核酸分子，以扩增期望的区。根据探针或引物的长度，靶标区可为70％互补碱基至完全互补之间，而仍在严格条件下杂交。例如，为了检测登革热病毒的存在，杂交核酸(探针或引物)与其所杂交的序列之间的互补程度至少足够区分与来自其它有机体的核酸的杂交。
编码DEN-4、DEN-3或DEN-1病毒的核酸序列(例如结构元件)可插入载体(诸如质粒)内，并在活有机体内重组表达(例如在登革热-2骨架内)，从而制造重组登革热病毒肽和/或多肽和/或病毒。
核酸检测方法
本发明提供了快速基因测试，其可诊断本文所述的各种疫苗病毒。本发明的该实施方案提高了从出现病毒血症的疫苗接种的人类的血清分离的病毒的分析，以及增加对使用候选物疫苗病毒免疫接种的非人灵长类动物中的病毒血症的表征。
这些序列包括诊断性TaqMan探针，其用于报告通过使用逆转录酶/聚合酶链式反应(RT/PCR)从病毒基因组RNA模板扩增的cDNA扩增子的检测，以及正向和反向扩增引物，所述扩增引物经设计以扩增cDNA扩增子，如下文所述。在某些情况下，扩增引物之一已经被设计成在扩增引物3’-末端处含有疫苗病毒特异性突变，这有效使得该测试对疫苗株甚至更具有特异性，因为只要病毒RNA模板含有该特异性突变，即可发生在靶标位点的引物延长以及随后的扩增。
可使用自动化的基于PCR的核酸序列检测系统，或用于核酸检测的其它已知技术。TaqMan测定是高度特异性且敏感的测定，其允许对来自样本核酸模板的PCR生成的扩增子的自动化实时可视化和定量。TaqMan可测定特定序列是否存在。在此测定中，正向和反向引物经设计而分别退火靶标突变位置的上游和下游。特异性检测子探针经设计而具有比任一扩增引物高约10℃的熔化温度，并且含有疫苗病毒特异性核苷酸突变或其补体(这取决于被检测的RT/PCR扩增子的链)，构成此测定的第三引物组分。
设计为特异性检测在疫苗病毒基因组之一中的突变基因座的探针将在探针中部含有疫苗特异性核苷酸。若病毒RNA模板为疫苗病毒特异性的，则此探针将在TaqMan测定中产生可检测的荧光。然而，来自野生型DEN病毒的基因组RNA模板将具有降低的探针杂交效率，原因在于单一核苷酸错配(在亲代病毒DEN病毒的情况下)，或可能多于一个错配(可能发生于其它野生型DEN病毒中)将不会产生显著的荧光。DNA聚合酶更可能从RT/PCR扩增子模板替换错配的探针，而非裂解错配的探针以释放报告子染料(TaqMan等位基因分型测定，Applied Biosystems)。
诊断性基因测试的一种策略是使用分子信标。分子信标策略也利用用于扩增子RT/PCR扩增的引物，并通过在探针末端含报告子和淬灭剂染料的探针来检测扩增子内部的特定序列。在此测定中，探针形成茎-环结构。分子信标测定采用与TaqMan测定所使用的不同淬灭剂和报告子染料。
药物组合物
本文的实施方案提供向个体给予化合物，所述化合物以适于活体内药学给药的生物相容形式。“适于活体内给药的生物相容形式”表示将要给予的活性剂(例如实施方案的药物化学品、蛋白质、基因)的形式，其中活性剂的治疗效果超过任何毒性效应。给予治疗上活性量的治疗组合物被定义为在实现期望结果所需的剂量和时间段方面有效的量。例如，治疗上活性量的化合物可依据多项因素而改变，诸如个体的疾病状态、年龄、性别及体重，以及抗体在个体中引发期望应答的能力。剂量方案可经调整以提供最佳治疗反应。
在一个实施方案中，可以常规方式给予化合物(例如所述实施方案的药物化学品、蛋白质、肽等)，例如皮下、静脉内、经口、吸入、皮内、经皮施用、阴道内施用、局部施用、鼻内或直肠给药。根据给药途径，活性化合物可含于保护缓冲剂(例如FTA、F127/海藻糖/白蛋白)中。在一个实施方案中，组合物可经口给予。在另一个实施方案中，组合物可静脉内给予。在一个实施方案中，组合物可于鼻内(诸如吸入)给予。在又一个实施方案中，组合物可使用无针系统(例如
Figure BDA0002561695400000191
)或其它皮内给药系统而皮内给予。
组合物可于适当载剂或稀释剂中与酶抑制剂共同给予施用于个体，或于适当载剂(诸如脂质体)中施用于个体。如本文所用的术语“药学上可接受的载剂”旨在包括稀释剂，诸如盐水和水性缓冲溶液。可能需要使用材料包被该化合物或与该化合物共同给予以防止其失活。活性剂也可经肠胃外或经腹膜内给予。分散液剂可在甘油、液体聚乙二醇及其混合物中以及在油中制备。在储存和使用的常见条件下，这些制剂可含有防腐剂(用于防止微生物的生长)或其它稳定配制剂(例如FTA)。
适于注射用途的药物组合物可利用本领域已知的手段给予。例如，可使用用于临时制备无菌注射溶液剂或分散液的无菌水性溶液(当为水溶性时)或分散液以及无菌粉末。在所有情况下，该组合物可为无菌的，且可为流体(其程度使得存在易于注射性(syringability))。其在制造和储存条件下可为稳定的，且可经保存而不受微生物(诸如细菌和真菌)的污染。药学上可接受的载剂可为溶剂或分散介质，包括例如水、乙醇、多元醇(例如甘油、丙二醇及液体聚乙二醇等)及其适当的混合物。可以例如通过使用包衣(诸如卵磷脂)，在分散液的情况下通过保持所需的粒径，以及通过使用界面活性剂，从而保持适当的流动性。微生物的预防可通过加热、将药剂暴露于清洁剂、辐照或添加多种抗菌剂或抗真菌剂而实现。
无菌注射溶液均可如下制备，通过将需要量的活性化合物(例如诱导对一种或多种登革热病毒血清型的免疫应答的化合物)与上文列举的成分之一或其组合(根据需要)并入适当的溶剂中，接着过滤灭菌。
当配制时，溶液将以与剂量配制剂相兼容的方式及以治疗有效量来给予。配制剂容易以多种剂型给予，诸如上述注射溶液类型。预期组合物尤其适用于肌肉内、皮下、皮内、鼻内及腹膜内给药。可寻求特定比例如1：1、1：2或其它比例(例如给定登革热病毒血清型的PFU)。
活性治疗剂可在混合物内以预定比例配制。可以针对给定情况(例如旅行前、登革热爆发时)以适当进度表给予单次剂量或多次剂量。
在另一实施方案中，鼻用溶液或喷雾剂、气溶胶或吸入剂可用于递送感兴趣的化合物。适用于其它给药方式的其它配制剂包括栓剂和阴道栓。
某些配制剂可包括赋形剂，例如药学等级的甘露糖醇、乳糖、淀粉、硬脂酸镁、糖精钠、纤维素、碳酸镁等。
药物组合物可使用保护活性成分免于从体内快速消除的载剂制备，诸如延时释放配制剂或包衣。此类载剂包括控释配制剂，诸如但不限于微胶囊递送系统、以及可生物降解、生物相容性聚合物，如乙烯乙酸乙烯酯、聚酐、聚乙醇酸、聚原酸酯、聚乳酸以及已知的其它物质。
药物组合物以有效抑制或缓解植入物的副作用和/或减少或预防排斥的量和频率给予。精确的治疗剂量和治疗持续时间可使用已知的测试方案或通过在本领域已知的模型系统中测试该组合物并由此外推而经验地确定。剂量也可随病况的严重程度而改变。药物组合物通常经配制和给予而发挥治疗有用的效果，同时最小化非期望的副作用。一般而言，约102至106PFU的剂量范围可初步给予，任选地随后根据情况在30天内或至多180天后进行第二次给药。在某些实施方案中，个体可接受本文公开的一价、二价、三价或四价组合物的双重给药，其中该组合物为单一组成混合物或具有不同登革热病毒血清型的预定组成。在一些实施方案中，DEN2/4嵌合体可以比其它登革热血清型(诸如活减毒登革热-1)更高的浓度存在。
对于任何特定个体，显然可根据个别需要随时间调整特定剂量方案。
在一个实施方案中，本文公开的组合物可经皮下或皮内给予于个体。
含有活减毒登革热病毒的药物组合物可给予至个体，特别是人类，例如通过皮下、肌肉内、鼻内、经口、局部、经皮、肠胃外、经胃肠、经支气管及经肺泡。局部给药经由包含治疗有效量的丝氨酸蛋白酶抑制剂的局部施用的乳膏、凝胶、洗剂等而实现。经皮给药通过施用能够使丝氨酸蛋白酶抑制剂穿透皮肤并进入血流的乳膏、洗剂、凝胶等而实现。此外，渗透泵可用于给药。所需剂量将随正在治疗的特定病况、给药方法及该分子从体内的清除速率而改变。
在本发明方法的某些实施方案中，个体可为哺乳动物，诸如人类或兽医动物和/或家畜或牲口或野生动物。
治疗方法
在本发明的一个实施方案中，提供了使用本文预期的活减毒和/或嵌合式病毒构建物的一价、二价、三价或四价配制剂，诱导对登革热病毒血清型的免疫应答的方法。
以下是本发明的一些具体实施方案。
1.核酸嵌合体，包含编码来自修饰的活减毒登革热-2病毒株PDK-53的非结构蛋白质的第一核苷酸序列，和编码至少一种来自登革热-1的结构蛋白质的第二核苷酸序列，其中所述活减毒登革热-2病毒还包含至少一种额外的突变，所述额外的突变包括核酸位置3823处的突变，其中NS2A基因的氨基酸位置116为亮氨酸替代异亮氨酸。
2.如上述方面1所述的核酸嵌合体，其中所述活减毒登革热-2病毒还包括位置4407处的突变，其中NS2B的氨基酸位置92为天冬氨酸替代谷氨酸。
3.如上述方面1所述的核酸嵌合体，其中所述活减毒登革热-2病毒还包括位置7148处的突变，其中NS4B的氨基酸位置108为异亮氨酸替代苏氨酸。
4.如上述方面1所述的核酸嵌合体，其中所述活减毒登革热-2病毒还包括位置7311处的突变。
5.如上述方面1所述的核酸嵌合体，其中所述第二核苷酸序列编码来自登革热-1的E蛋白，所述登革热-1还包含位置2384处的突变，其中所述E蛋白的氨基酸位置483为丙氨酸替代甘氨酸。
6.药物组合物，包含上述方面1至5中任一项所述的核酸嵌合体，以及药学上可接受的赋形剂。
7.药物组合物，包含由上述方面1至5中任一项所述的核酸嵌合体编码的多肽，以及药学上可接受的赋形剂。
8.在个体内诱导免疫应答的方法，包括给予药学上可接受量的上述方面6和7所述的组合物中的任一种，其中所述组合物在所述个体内诱导对登革热病毒的免疫应答。
9.载体，其编码上述方面1至5中任一项所述的核酸序列。
10.核酸序列，其包含编码修饰的活减毒登革热-2病毒株PDK-53的核酸序列，其中所述活减毒登革热-2病毒还包含至少一个额外的突变，所述额外的突变包括核酸位置6481处的突变，其中NS4A基因的氨基酸位置36为脯氨酸替代丙氨酸。
11.如上述方面10所述的核酸序列，其中所述活减毒登革热-2病毒还包括位置7156处的突变，其中NS4B的氨基酸位置111为苯丙氨酸替代亮氨酸。
12.如上述方面10所述的核酸序列，其中所述活减毒登革热-2病毒还包括位置8803处的突变，其中NS5的氨基酸位置412为缬氨酸替代异亮氨酸。
13.如上述方面1所述的核酸序列，其中所述活减毒登革热-2病毒还包括位置592处的突变，其中prM的氨基酸位置52为谷氨酸替代赖氨酸。
14.药物组合物，包含上述方面10至13中任一项所述的核酸嵌合体，以及药学上可接受的赋形剂。
15.药物组合物，包含由上述方面10至13中任一项所述的核酸嵌合体编码的多肽，以及药学上可接受的赋形剂。
16.在个体内诱导免疫应答的方法，包括给予药学上可接受量的上述方面14和15所述的组合物中的任一种，其中所述组合物在所述个体内诱导对登革热病毒的免疫应答。
17.载体，其编码上述方面10至13中任一项所述的核酸序列。
18.核酸嵌合体，包含编码来自修饰的活减毒登革热-2病毒株PDK-53的非结构蛋白质的第一核苷酸序列，和编码至少一种来自登革热-3的结构蛋白质的第二核苷酸序列，其中所述活减毒登革热-2病毒还包含至少一种额外的突变，所述额外的突变包括核酸位置6436处的突变，其中N42A基因的氨基酸位置23为天冬酰胺替代天冬氨酸。
19.如上述方面16所述的核酸嵌合体，其中所述活减毒登革热-2病毒还包括位置7620处的突变。
20.如上述方面16所述的核酸嵌合体，其中所述第二核苷酸序列编码来自登革热-3的E蛋白，所述登革热-3还包含位置1603处的突变，其中所述E蛋白的氨基酸位置223为丝氨酸替代苏氨酸。
21.药物组合物，包含上述方面18至20中任一项所述的核酸嵌合体，以及药学上可接受的赋形剂。
22.药物组合物，包含由上述方面18至20中任一项所述的核酸嵌合体编码的多肽，以及药学上可接受的赋形剂。
23.在个体内诱导免疫应答的方法，包括给予药学上可接受量的上述方面21和22所述的组合物中的任一种，其中所述组合物在所述个体内诱导对登革热病毒的免疫应答。
24.载体，其编码上述方面18至20中任一项所述的核酸序列。
25.核酸嵌合体，包含编码来自修饰的活减毒登革热-2病毒株PDK-53的非结构蛋白质的第一核苷酸序列，和编码至少一种来自登革热-4的结构蛋白质的第二核苷酸序列，其中所述活减毒登革热-2病毒还包含至少一个额外的突变，所述额外的突变包括核酸位置3674处的突变，其中NS2A基因的氨基酸位置66为甘氨酸替代天冬氨酸。
26.如上述方面22所述的核酸嵌合体，其中所述活减毒登革热-2病毒还包括位置3773处的突变，其中NS2A的氨基酸位置99为精氨酸替代赖氨酸。
27.如上述方面22所述的核酸嵌合体，其中所述活减毒登革热-2病毒还包括位置5391处的突变。
28.如上述方面22所述的核酸嵌合体，其中所述活减毒登革热-2病毒还包括位置6437处的突变，其中NS4A的氨基酸位置21为缬氨酸替代丙氨酸。
29.如上述方面22所述的核酸嵌合体，其中所述活减毒登革热-2病毒还包括位置7026处的突变。
30.如上述方面22所述的核酸嵌合体，其中所述活减毒登革热-2病毒还包括位置7538处的突变，其中NS4B的氨基酸位置238为苯丙氨酸替代丝氨酸。
31.如上述方面22所述的核酸嵌合体，其中所述活减毒登革热-2病毒还包括位置9750处的突变。
32.如上述方面22所述的核酸嵌合体，其中所述活减毒登革热-2病毒还包括位置225处的突变。
33.药物组合物，包含上述方面25至32中任一项所述的核酸嵌合体，以及药学上可接受的赋形剂。
34.药物组合物，包含由上述方面25至32中任一项所述的核酸嵌合体编码的多肽，以及药学上可接受的赋形剂。
35.在个体内诱导免疫应答的方法，包括给予药学上可接受量的上述方面33和34所述的组合物中的任一种，其中所述组合物在所述个体内诱导对登革热病毒的免疫应答。
36.载体，其编码上述方面25至32中任一项所述的核酸序列。
37.核酸序列，其包含以SEQ ID NO.1、7和10确定的核酸序列中的一种或多种。
38.如上述方面33所述的核酸，其中所述核酸嵌合体编码由SEQ ID NO.2、3、8、9、11或12表示的多肽。
39.免疫原性组合物，包含上述方面37所述的核酸序列或上述方面38所述的多肽中的一种或多种，以及药学上可接受的载剂。
40.如上述方面39所述的组合物，其还包含活减毒登革热-2病毒。
41.如上述方面40所述的组合物，其中所述活减毒登革热-2病毒以SEQ ID NO.4的核酸序列或SEQ ID NO.5和6中的一个或多个的氨基酸序列表示。
42.如上述方面40所述的组合物，其中所述组合物含有全部四种登革热病毒血清型四价组合物。
43.如上述方面39所述的组合物，还包含针对选自以下的黄病毒的免疫原性组合物：黄热病毒、蜱媒脑炎病毒、日本脑炎病毒、西尼罗病毒、丙型肝炎病毒、及其中两种或更多种的组合。
44.试剂盒，包括上述方面1至5、10至13、18至20或25至32中任一项所述的核酸序列中的至少一种；或上述方面6、7、14、15、21、22、33或34中任一项所述组合物中的至少一种，以及容器。
45.活减毒病毒，包含上述方面1至5、10至13、18至20或25至32中任一项所述的核酸序列中的一种或多种。
46.包括表3的核酸序列的核酸序列，或由包括表3的核酸序列的一种或多种核酸序列编码的多肽。
47.包括DENVax-1-A、DENVax-2-F、DENVax-3-F和DENVax-4-F中的一个或多个的核酸序列，或由包括DENVax-1-A、DENVax-2-F、DENVax-3-F和DENVax-4-F中的一个或多个的一种或多种核酸序列编码的多肽。
48.组合物，包含上述方面47所述的核酸序列中的一种或多种或由上述方面47所述核酸序列编码的多肽中的一种或多种，以及药学上可接受的载剂或赋形剂。
49.如上述方面48所述的组合物，其还包含药学上可接受的佐剂。
50.组合物，包含上述方面45所述的活减毒病毒中的一种或多种，以及药学上可接受的载剂。
51.如上述方面50所述的组合物，其中所述组合物包含能够在个体内诱导对抗全部四种登革热病毒血清型的免疫应答的四价登革热病毒组合物。
本发明的实施方案通过下列非限制性实施例而例示，所述实施例绝非视为对本发明的范围施加限制。相反地，显然应理解，在阅读本文的描述之后，本领域技术人员可以在不背离本发明的主旨或随附权利要求的范围的情况下，联想到可以采取多种其它实施方案、修改、及其等同物。
实施例
包括下列实施例以验证本文呈示的某些实施方案。本领域技术人员应理解下列实施例中公开的技术呈示了发现在本文公开的实施中运行良好的技术，因此可视为构成其实施的优选模式。然而，本领域技术人员鉴于本公开内容应认识到在不背离本文的主旨和范围的情况下可在所公开的特定实施方案中进行诸多变化，并且仍获得相同或类似的结果。
实施例1
在一些示例性方法中，公开了用于产生本文称为“主代病毒种(MVS)”的组合物。这些组合物可衍生自一种或多种活减毒登革热病毒，诸如DEN-1、DEN-2、DEN-3及DEN-4。在某些方法中，组合物可衍生自一种或多种活减毒登革热病毒，包括但不限于，本文公开的称为DENVax-1、DENVax-2、DENVax-3及DENVax-4的特定构建物。在其它示例性方法中，提供了用于生成和表征这些组合物的策略。在其它实施方案中，提供了四价登革热病毒配制剂以及这些配制剂的遗传学和表现型表征。
前主代DENVax病毒的制造及分析
进行某些程序以生成前主代登革热病毒种，诸如登革热病毒(例如DENVax)的一系列扩增和纯化。首先，通过将从全长重组DENVax cDNA转录的病毒RNA转染入核准生产的细胞(例如Vero细胞)而再衍生DENVax病毒，获得P1(1代)病毒种。来自登革热-1至登革热-4中每一种的四种P1病毒随后经扩增和溶菌斑纯化以获得候选前主代疫苗P7种(参见表1)。进行某些测试以分析登革热病毒的传代。例如，全长基因组测序证实全部四种P2(2代)种病毒在遗传学上与其同源祖先(经研究衍生的研究等级候选疫苗病毒)相同。原始溶菌斑表现型也保留在P2病毒中。从P2种分离登革热各个血清型(例如DENVax 1-4)的六种溶菌斑纯化病毒(P3 A-F)，并且每一分离的溶菌斑直接再进行溶菌斑纯化两次。每一病毒的第三次溶菌斑纯化(P5)于Vero细胞中扩增两次(P6 A-F和P7 A-F)，从而制造潜在前主代P7 DENVax种(表1)。
表1.DENVax病毒于WCB-Vero细胞中的cGMP再衍生的实例
Figure BDA0002561695400000271
Figure BDA0002561695400000281
进一步进行一些试验以表征P7 DENVax种，诸如分析P7种的基因组序列和溶菌斑表现型，并与P2种进行比较(表2)。P7病毒的溶菌斑表现型大致于类似P2种的溶菌斑表现型。在一些示例性实验中，监测病毒滴度。大部分P7种的病毒滴度超过6.0log pfu/ml，但5种病毒除外。在Vero细胞中经10次以上连续传代后，超过60种候选物疫苗病毒种的基因组测序确定在DEN-2 PDK-53基因载体的三个主要减毒决定子在NS1-53和NS3-250处并无回复事件，表明这2个基因座在候选物疫苗病毒种中是相当稳定的。这两个位点的从正测序和反测序生成的24种候选物株的全部序列色谱图均为同源的，而在NS1-53和NS3-250遗传基因座处明显没有任何微小核苷酸群体。与NS1和NS3位点相反，从多个连续传代的研究等级的疫苗病毒确定在5’NCR-57减毒基因座处的不同程度的回复，表明该基因座在细胞培养中经多次传代后可能不如NS-1和NS3稳定。因此，开发了敏感性错配扩增测定(TaqMAMA)以通过实时RT-PCR准确地测量5’NCR57基因座处的回复率。在一些研究中，通过TaqMAMA测量全部24种P7种的5’NCR57回复率。根据该测定中对每一病毒的输入病毒RNA浓度，TaqMAMA的敏感性极限为0.01％至0.07％的回复，这比通过共有基因组序列分析可检测的10-30％回复敏感性极限更加敏感。所得数据表明24种P7病毒中15种具有极少或无法检测的回复(<0.07％)，一种病毒(DENVax-3-D)具有几乎100％的回复，以及8种病毒(1种DENVax-1、1种DENVax-2、2种DENVax-3以及4种DENVax-4)具有0.08％至12.85％的部分回复(表2)。对于24种P7病毒中的16种进行全长基因组测序，并且如TaqMAMA所测量的，5’NCR57回复的水平低。全部测序的病毒保持另两种DENVax减毒决定子(NS1-53、NS3-250)，并且均具有在原始改造重组cDNA克隆中所不存在的额外突变(表2)。在一个示例性靶标疫苗组合物中，选择DENVax-1-A、DENVax-2-F、DENVax-3-F及DENVax-4-F作为每一血清型的靶标前主代种，因为其基因型和溶菌斑表现型最类似于原始设计的疫苗重组体。DENVax-1-A、DENVax-2-F及DENVax-4-F具有两种非同义突变，而DENVax-3-F则具有一种。证据表明在这4种前主代种中观察到的这些额外突变不会造成所述病毒的安全性忧虑或免疫原性改变。这些前主代种被进一步扩增以生成MVS(主代种，标示为P7，表1)。
本文提供的示例性方法使用来自克隆cDNA质粒的纯化体外转录病毒RNA作为纯来源以转染核准用于疫苗(vaccine-certified)的Vero细胞，从而生成疫苗病毒。将连续溶菌斑纯化和全基因组序列分析并入制造程序中以确保所制造的疫苗种具有最佳纯度和遗传稳定性。对DENVax的每一血清型制备六种克隆病毒作为潜在前主代种。通过基因组分析，包括TaqMAMA和完整基因组测序以及病毒溶菌斑表现型的表征，前主代种经选择以用于每一血清型(血清型1-4)的主代病毒种制备。所选的前主代种在5’NCR-57基因座具有无法检测的回复(<0.01％或<0.07％)，在其基因组中具有1种或2种氨基酸取代，以及保留先前观察到的小型溶菌斑表现型。
表2.前主代(P7)种的表征
Figure BDA0002561695400000291
Figure BDA0002561695400000301
实施例2
在一些示例性方法中，描述了主代病毒种、工作病毒种及散装病毒种(bulk virusseeds)的组合物及其遗传学和表现型表征。提供这些组合物用于临床材料的制造及最终商业疫苗供应。连续溶菌斑纯化及全基因组序列分析被并入制造过程中以确保疫苗种的组合物具有用于制造临床试验材料的最佳安全性和遗传稳定性。
MVS、WVS及BVS的产生及制造质量控制
在一些研究中，通过将前主代P7种于核准的Vero细胞中扩增而产生4种DENVax的MVS。在其它研究中，MVS用于细胞培养器中制备大量的WVS。此外，从WVS扩增DENVax的BVS原料，并配制成用于人类临床试验的四价药品混合物。产物释放的质量控制以一些示例性方法进行，包括但不限于，测试全部MVS、WVS及BVS的身份、感染滴度、无菌性、支原体以及于体外和活体内的外源因子。使用血清型特异性RT-PCR测定，全部的种均通过病毒身份测试，其显示对应其血清型的阳性扩增以及对于非同源血清型的阴性扩增(数据未显示)。在MVS、WVS或BVS原料中未检测到可检测的支原体或外源因子。
MVS、WVS及BVS的遗传分析
在某些示例性方法中，在从所选的前MVS(P7)生成MVS后，产生如上所选的株并对各病毒RNA再次测序。全长基因组测序显示DENVax-1的MVS与其前主代种相同，而WVS及随后的BVS获得在E-483和NS4B-108处的2个额外的取代(参见表2和3)。在E-483处的Ala取代表示在MVS中的部分基因型，但变成BVS中的主要基因型。DENVax-2和DENVax-3与其各自得前主代种相同(表2和3)。DENVax-2 MVS与其前主代种相同，以及WVS和BVS具有在NS4A-36和NS4B-111处的两种额外的突变。两种突变在WVS中均为部分基因型，并且在BVS中为主要基因型。DENVax-3的MVS再度与前主代种相同，但WVS和BVS含有在NS4A-23处的额外的氨基酸取代。DENVax-4 MVS在产生MVS期间获得在基因座NS2A-99处的额外的氨基酸突变(Lys至Lys/Arg混合基因型)(表3)。其WVS和BVS保留NS2A-99Lys/Arg混合基因型，并且BVS具有额外的NS4B-238Ser/Phe混合基因型。共有序列结果也证实MVS、WVS及BV保留在5’NCR-57、NS1-53及NS3-250基因座处的三种减毒基因决定子。通过TaqMAMA分析最不稳定的减毒基因座证实5’NCR-57回复率在MVS中﹤0.7％至0.13％，在WVS中≤0.07％，以及在BVS中﹤0.07％至0.21％。在5’NCR-57基因座处的3％回复被视为疫苗批次可接受的最大容许回复率(表3)。
表3.DENVax种中的核苷酸和氨基酸取代
Figure BDA0002561695400000321
Figure BDA0002561695400000331
全基因组序列分析显示在DENVax-4 MVS中发展出额外的氨基酸突变，而另外三个DENVax MVS批次保留其前主代种的共有基因组序列。总而言之，从P1种衍生至前主代(P7)种，在给定种中只出现1或2种非同义突变。从P1至MVS(P8)种，在任何给定DENVax种中鉴定2至7种核苷酸取代，这些取代中只有2至3种导致氨基酸改变。因此，出现微小改变。RNA病毒在其基因组复制中有出错趋势，因此在细胞传代期间黄病毒基因组的基因取代并非出乎意料的。在MSV中没有沉默突变在可能影响病毒复制的5’或3’NCR内。只有在DENVax-2中的prM-52Lys-Glu的改变以及在DENVax-4中的NS2A-66 Asp-Gly的取代是非保守性改变。DENVax-4的NS2A-66突变在DENV-2 PDK-53的非结构骨架部分中。虽然NS2A-66基因座在DENV-2的各种株中通常为Asp，但对DENV-4通常为Gly。可能的是，在DENVax-4中Asp至Gly的改变与DENVax-4在Vero细胞中的适应度相关。DENVax-2prM-52突变位于prM的C端部分，该部分从成熟病毒颗粒剪接掉。在一些示例性方法中，进行表现型表征以证实在MVS种中的突变均未显著改变疫苗的减毒表现型。
DENVax病毒证明在制造过程中的高遗传稳定性。位于5’NCR、NS1-53及NS3-250中的三种经定义的DENV-2 PDK-53减毒基因座在当DENVax从前主代株连续传代至散装疫苗制剂时于共有基因组序列中保持稳定。5’NCR-57基因座的高度敏感的TaqMAMA证明在登革热病毒血清型的MVS、WVS(P9/工作)以及BVS(疫苗的散装病毒种)中极少或无法检测的回复。DENVax BVS制剂(P10-等同物)的5’NCR57回复率显著低于出现在研究等级疫苗候选物在Vero细胞中经10次连续传代后的5’NCR57回复率(4-74％回复)。本文提供的DENVax种的大规模制造的策略产生遗传上稳定的疫苗病毒种，其保留候选物疫苗病毒中的减毒标记。
DENVax MVS的溶菌斑表现型
在一个示例性方法中，对DENVax MVS与野生型登革热病毒及其同源研究级嵌合式病毒于Vero细胞中的溶菌斑表现型进行比较(图2)。在Vero细胞中，DENVax-1、-2及-3的MVS所产生的溶菌斑均显著小于其野生型同源物，且极为类似于(在0.4mm差值内)其同源研究级病毒。DENVax-4 MVS也显著小于野生型DENV-4，但稍微大于(0.9mm差值)原始实验室衍生的D2/4-V嵌合体。
图2示出示例性直方图，其例示DENVax MVS比较对照野生型病毒及研究级疫苗候选物病毒的溶菌斑尺寸。在感染后第9天测量在琼脂糖覆盖层下方的Vero细胞中的病毒溶菌斑的平均溶菌斑直径(mm)±SD(误差柱)。包括以黑柱表示的野生型DEN病毒，以及以白柱表示的先前公开的研究级疫苗候选物病毒，以作为对照并与灰柱表示的DENVax主代疫苗种进行比较。
DENVax MVS的温度敏感性
在另一示例性方法中，在Vero细胞中对DENVax MVS测试温度敏感性且与其同源野生型和原始研究级嵌合式疫苗病毒进行比较。野生型(wt)DENV-3 16562并非温度敏感的。野生型登革热病毒血清型1和登革热血清型4在39℃为中等温度敏感的(在39℃的滴度比37℃低约1.0log10 pfu/ml，图3)。野生型登革热病毒血清型-2 16681为所测试的野生型登革热病毒中温度敏感性最高的，导致在39℃的滴度降低100倍。DENVax-1、2及3与其原始同源研究级嵌合式疫苗病毒是同样温度敏感的(图2)。这些DENVax株在39℃的滴度降低2.0至3.0log10 pfu/ml。DENVax-4也为温度敏感的，表现滴度降低5倍。然而，原始研究级D2/4-V表现滴度降低约10倍。最终稳定化DENVax-4 MVS含有F127(及其它已知使这些配制剂稳定的试剂(FTA))，F127已显示提升登革热病毒的热稳定性。在DENVax-4 MVS中存在F127可能促成Vero培养测定中病毒较不明显的温度敏感性。在单独的实验中，进一步评估在不存在F127的情况下MVS衍生的DENVax-4株的温度敏感性。为了从该株去除F127，从DENVax-4散装病毒制剂分离病毒RNA，并转染于Vero细胞中。在不存在F127的情况下于感染后第3天，此种DENVax-4病毒表现与D2/4V研究株具有相同的温度敏感性(滴度降低1.5log10 pfu/ml)(图3)。
图3示出示例性直方图，其例示DENVax MVS的温度敏感性。包括了野生型登革热病毒及先前公开的研究级疫苗候选物病毒，以进行比较。DENVax-4 MVS含有额外的F-127，其可掩盖此测定中病毒的温度敏感性结果。也包括了单独的实验，以分析在不存在F127的情况下的替代DENVax-4。在37℃或39℃于Vero细胞中复制的病毒的平均滴度±SD(误差柱)。
蚊C6/36细胞中的DENVax MVS复制
在一些示例性方法中，使DENVax MVS于C6/36细胞中生长以证实其体外减毒表现型的保留性，已知研究级嵌合式疫苗病毒保留了骨架DENV-2 PDK53病毒在这些蚊细胞中的减毒表现型。与野生型登革热病毒相比，在感染后第6天，DENVax-1、DENVax-2及DENVax-4MVS显示于C6/36细胞中显著的生长降低(至少3log10 pfu/ml降低)(图4)。与野生型DENVax-3 16562相比，DENVax-3 MSV也显示生长降低，但降低较不显著(1-2log10 pfu/ml降低)。然而，DENVax-3种批次的C6/36滴度类似于(在1log10 pfu/ml差值内)原始研究级嵌合式D2/3-V疫苗病毒的C6/36滴度。
图4示出示例性直方图，其绘制DENVax MVS(灰柱)于C6/36细胞中与野生型登革热(黑柱)和研究级疫苗病毒(白柱)相比的受限生长。感染后第6天，在C6/36细胞中复制的病毒的平均滴度±SD(误差柱)。
于全蚊的病毒感染、散播及传播率
在一些示例性方法中，将DENVax的感染率及散播率与其亲代野生型登革热病毒相比较。在某些示例性实验中，在埃及伊蚊(Ae.aegypti)中进行经口感染实验。感染血粉经反向滴定以测量病毒滴度，并且在表4中只包括了针对每一血清型的亲代登革热病毒与DENVax之间的在血粉中具有类似病毒滴度(小于1log10 pfu/ml差值)的实验，以进行比较。DENVax-1、DENVax-2及研究级D2 PDK-53-VV45R不会通过经口进食而使蚊子感染，这与其亲代病毒DENV-1 16007(44％感染)和DENV-2 16681(43.3％感染)有显著差异(p<0.0001)。因没有蚊子受DENVax-1和-2感染，故这两种疫苗病毒极少有或没有散播忧虑。尽管DENVax-4确实通过经口进食而感染一些蚊子(55只中的2只)，但感染率显著比其亲代野生型病毒DENV-4 1036(50只中的8只)更低(p<0.05)。在其中血粉病毒滴度为5.2±0.02log10 pfu/ml的两次实验中DENVax-3不感染任何蚊子(表4)，而在血粉病毒滴度为6.0log10 pfu/ml的单独的实验中，30只蚊子中只有一只被感染(数据未显示)。然而，野生型登革热病毒-3 16562在5.2log10 pfu/ml下也具有极低的感染率(8％)，而在使用6.2log10 pfu/ml的较高血粉病毒滴度的单独的实验中，感染率不增加(3％，30只蚊子中1只呈阳性，数据未显示)。虽然野生型(wt)登革热病毒-3和登革热病毒-4具有比野生型登革热病毒-1和登革热病毒-2显著更低的感染率，但在感染的蚊子中的平均病毒滴度相近(3.1至3.9log10 pfu/蚊)。相反，来自两种感染的蚊子的DENVax-4滴度均为最低的(0.7log10 pfu/蚊)，这比来自由野生型登革热血清型-4 1036感染的蚊子的滴度(3.9±1.5pfu/蚊)低1,000倍。
针对那些感染的蚊子，可以通过测定病毒是否存在于腿部来评估从中肠散播出。四种亲代DENV产生36.3％至62.5％的散播率，来自腿部的平均病毒滴度(以log10 pfu计)为0.9±0.3至2.2±0.7(排除阴性样本)。两种DENVax-4感染的蚊子均未产生向腿部的病毒散播(表4)。虽然在腿部中可检测到散播的病毒，但在经口感染的蚊子的唾液中并未检测到四种野生型登革热病毒，表明经口进食条件可能不足以敏感地测量这些DENV的传播率。因此，在其它示例性方法中，随后进行通过直接IT接种的高度严格的人工蚊子感染(表4)。除了DENVax-4之外，全部病毒(野生型和DENVax)均实现IT接种的埃及伊蚊的100％感染。DENVax-4接种物具有比另外三种病毒接种物略低的病毒滴度，但仍然成功地感染70％的接种的蚊子。尽管通过IT接种实现高的身体感染率，但全部四种DENVax病毒与野生型登革热病毒(43-87％，表4)相比表现出显著较低(p<0.005)或无法检测的传播率(0-10％)。DENVax病毒于经口进食后显示有极少至没有感染和散播，高度严格的IT结果证实这些DENVax病毒在埃及伊蚊中的低传播能力。
表4：于全蚊中的病毒感染、散播及传播率
Figure BDA0002561695400000371
Figure BDA0002561695400000381
载体胜任性是活减毒黄病毒疫苗病毒的重要安全性要素。先前，将研究级DENV-2PDK-53-VV45R病毒及野生型衍生物在埃及伊蚊中测试，并发现NS1-53-Asp减毒突变是受损的蚊复制的主要决定子。DENV-2 PDK-53疫苗的其它两个主要减毒基因座，即核苷酸5’NCR-57-T和NS3-250-Val，也表现出对在蚊中复制的一些抑制效应，由此对蚊载体胜任性提供了额外的冗余限制。本文描述的一些示例性方法用于测试全部四种DENVax株的蚊子经口和IT感染和复制。DENVax-1、-2和-3不会通过经口感染而感染任何埃及伊蚊(表4)。DENVax-4只感染3.6％的经口暴露的蚊子，该水平显著低于野生型DENV-4，在蚊体中的复制平均滴度低于野生型DENV-4感染的蚊子。出乎意料地，在感染的蚊子的腿部中检测到DENVax-4，表明DENVax-4无法在经口感染后从蚊子中肠散布。DENVax-1、-2及-4的感染率全部显著低于其野生型配对物，但DENVax-3与野生型DENV-3 16562之间的差异不显著，原因在于两种病毒的感染率极低。与在同样收集自泰国美索省的埃及伊蚊中评估的DENV的其它野生株相比，用于改造DENVax的亲代野生型登革热病毒株表现具有经口感染的较低的感染率和散播率。用于改造基于黄热病(YF)17D疫苗的ChimeriVax-DEN疫苗的野生型DENV-1 PUO359、DENV-2PUO218、DENV-3 PaH881/88及DENV-4 1288具有47-77％的感染率。相反地，YF 17D疫苗无法感染埃及伊蚊。虽然ChimeriVax株含有来自这些高度传染性的野生型DENV的prM-E，但ChimeriVax保留其YF 17D复制骨架的蚊减毒表现型。本文提供的结果也表明DENV-2 PDK-53骨架的蚊减毒在DENVax株中得以保持。此外，在本文所述的组合物中所包括的构建物中使用具有较低蚊感染性的野生型登革热病毒株，提供了额外的安全性特征。
经口感染结果表明DENVax具有最低蚊感染性和散播能力。此外，进行更敏感且更严格的IT感染实验以进一步分析DENVax由埃及伊蚊传播的潜力。IT结果证明全部四种DENVax病毒与其野生型配对物相比均具有无法检测的或最低的蚊传播潜力。DENVax传播在理论上可能发生，条件是(1)载体在具有足够病毒血症滴度的疫苗上供应以感染蚊中肠，(2)病毒能够在中肠上皮中复制且能够随后散播出中肠，以及(3)散播的病毒可在唾液腺中复制且吐出的唾液中有足量病毒以用于传播。尚未充分地建立感染蚊子所需要的人病毒血症的阀值，但在自然野生型DENV感染后人病毒血症可为106-108蚊感染剂量50(MID50)/ml。此种MID50基于使用稀释的人血浆直接IT接种蚊子。在非人灵长类中的DENVax分析表明DENVax免疫接种后，病毒血症滴度极低(低于2.4log10 pfu/ml)并持续2-7日。考虑到DENVax的低病毒血症水平和低的蚊感染、散播及传播能力，这些疫苗病毒在自然界中不可能由蚊子传播或导致病毒血症。
因此，建议任何血清型的任一代(P1-P10)可用于组合物以生成针对1、2、3或全部4种登革热病毒血清型的安全有效疫苗。
在乳鼠中的神经毒力
原始研究级疫苗病毒在保持于DVBD/CDC内部的新生ICR小鼠中针对神经毒力进行高度减毒。所有这些小鼠均存活于使用104pfu的每一疫苗病毒的ic(脑内)攻击。另一方面，野生型登革热病毒血清型-2 16681病毒在各种实验中导致这些CDC-ICR小鼠的62.5％-100％死亡率。在一些实验中，得自Taconic Labs的商用ICR小鼠(Taconic-ICR)用于研究在新生小鼠中的神经毒力。观察到新生Taconic-ICR小鼠比先前CDC-ICR小鼠对登革热病毒血清型2感染显著地更敏感。图5A总结使用104pfu病毒ic攻击的野生型登革热病毒血清型-216681对CDC-ICR克隆和Taconic-ICR新生小鼠的神经毒力。Taconic-ICR小鼠(32只小鼠中100％死亡率，平均存活时间为8.3±0.5天)比先前CDC-ICR小鼠(72只小鼠中91％死亡率，平均存活时间14.6±2.3天)对脑内登革热病毒血清型-2 11681攻击更敏感。
在其它示例性方法中，为了评估DENVax MVS的神经毒力，Taconic-ICR小鼠最初在一次(n＝16)或两次(n＝31-32)实验中被约104pfu剂量的野生型登革热病毒血清型-216681、D2 PDK-53VV45R、D2/3-V或DENVax 1-4病毒ic(脑内)攻击(图5B)。在此剂量，D2/3-V研究级病毒，以及DENVax-1和DENVax-3 MVS，表现出完全减毒神经毒力表现型(未发病或致死)。如所预期，发现野生型登革热病毒血清型-2为“致命的”，平均小鼠存活时间(AST)为8.3±0.8天。在这些登革热病毒血清型-2-敏感Taconic-ICR小鼠中，D2 PDK-53-VV45R研究级病毒导致81.3％死亡率。DENVax-2 MVS和DENVax-4 MVS于Taconic-ICR中的致命性一致，分别显示9.8±1.7、10.2±1.4及11.3±0.4天的AST值。
在一些示例性方法中，在低10倍的剂量(103pfu，图5C)下，将野生型登革热病毒血清型-2 16681病毒的神经毒力与D2 PDK-53 VV45R、DENVax-2 MVS和DENVax-4 MVS以及D2/4-V研究级病毒进行比较。在此较低攻击剂量下，野生型登革热病毒血清型-2保留一致的致命神经毒力表现型，AST为9.0±1.4天。其它4种病毒表现出中间神经毒力表现型，神经毒力程度为血清型特异性的。D2 PDK-53-VV45R病毒及其DENVax-2 MVS同源物显示显著的减毒(分别为32.3％存活且AST为13.1±3.8天，以及31.2％存活且AST为10.5±3.4天)。DENVax-4 MVS和研究级D2/4-V病毒针对神经毒力为高度减毒的(分别为81.3％存活且AST为18.8±5.8天，以及100％存活率)。结果表明DENVax-1和-3的MVS表现出神经毒力的完全减毒，而DENVax-2和-4MVS批次保留了十分类似于其同源研究级病毒疫苗候选物的减毒表现型。
图5A-5C示出示例性图，其例示使用包括野生型登革热病毒血清型-2和不同减毒登革热病毒的各种组合物于新生小鼠中测试的神经毒力。诸多实验的汇集总结了在使用104pfu病毒(A)脑内攻击的CDC-ICR(n＝72)和Taconic-ICR(n＝32)新生小鼠中野生型登革热病毒血清型-2 16681的神经毒力。使用104pfu(B)或103pfu(C)剂量在Taconic-ICR小鼠中测试DENVax MVS的神经毒力。指出了一次实验(n＝16)或两次汇集实验(n＝31或32)中每组测试动物的数量。
WVS和BVS的溶菌斑表现型
进行某些研究来比较WVS和BVS与MVS、野生型登革热病毒及其同源实验室衍生的研究级嵌合体在Vero细胞中的溶菌斑表现型(图6)。针对每一疫苗病毒从10个溶菌斑算出平均溶菌斑尺寸，但野生型DENV-1、-3和-4的数目减少。在Vero细胞中，DENVax-1、-2和-3的MVS病毒均产生显著小于其野生型同源物的溶菌斑，而十分类似(在0.4毫米差值内)于其同源研究级病毒。DENVax-4 MVS也显著小于野生型DENV-4，但略微(0.9毫米)大于原始实验室衍生的D2/4-V嵌合体。除了DENVax-2之外，DENVax-1、-3、-4的WVS和BVS与从其野生型同源物产生的溶菌斑尺寸相比保留了显著更小的溶菌斑尺寸。在Vero细胞中，DENVax-2 WVS和BVS产生的溶菌斑与野生型DENV-2病毒的溶菌斑类似，但当在LLC-MK2细胞中测试时，所有DENVax-2制造的种所产生的溶菌斑均略小于野生型DENV-2(1.4±0.4)的溶菌斑且类似于实验室衍生的D2 PDK-53-VV45R(1.0±0.3)(图6)。
病毒减毒的表现型标记(包括小溶菌斑表现型、温度敏感性、于蚊细胞中复制减少、由蚊子感染/散播/传播减少、以及于新生ICR小鼠中神经毒力降低)的评估是针对MVS原料的组合物进行评价的。结果表明全部DENVax保留与原始研究级疫苗病毒类似的预期减毒表现型。考虑到负责减毒的突变均在全部MVS、WVS和BV中保守，可预期减毒表现型保留于针对人临床试验制造的材料中。
图6示出示例性直方图，其例示DENVax MVS、WVS及BVS的溶菌斑尺寸。在感染后第9天测量在琼脂糖覆盖层下方的Vero细胞或LLC-MK2细胞中的病毒溶菌斑的平均溶菌斑直径±SD(误差柱)。包括野生型DENV及先前公开的研究级疫苗候选物病毒以用于对照和比较。
在蚊C6/36细胞中的病毒复制
先前研究证实研究级的基于PDK-53的嵌合式疫苗病毒保留骨架DENV-2 PDK53病毒在C6/36细胞中的减毒表现型。在一些示例性方法中，使DENVax MSV、WVS及BVS于C6/36细胞中生长以证实在大规模制造后其保留此种体外减毒标记。与野生型登革热病毒相比，除了DENVax-3之外，所制造的种在感染后第6天于C6/36细胞中显示显著的生长降低(至少3log10 pfu/ml降低)(图7)。DENV-3种与野生型DENV-3 16562相比也显示生长降低，但降低较不显著(1-2log10 PFU/ml降低)。然而，DENVax-3种批次的滴度类似(在1log10 PFU/ml差值内)于原始研究级嵌合式D2/3-V疫苗病毒。
图8示出示例性直方图，其绘制DENVax MVS、WVS及BVS于C6/36细胞中的受限的生长。病毒在感染后第7天于C6/36细胞中复制的平均滴度±SD(误差柱)。包括野生型登革热病毒及先前公开的研究级疫苗候选物病毒以进行比较。
在乳鼠中的神经毒力
进行额外的实验以分析在新生ICR小鼠中的神经毒力。在104PFU的颅内剂量下，对于野生型DENV-2 16681和D2 PDK-53-VV45R，ICR小鼠的存活率分别为0％和18.8％(图9A)，但CDC ICR小鼠对于野生型DENV-2 16681的存活率为约20％以及对于D2 PDK-53-VV45R的存活率为100％。在此研究中，DENVax-1和DENVax-3 MVS在104PFU剂量下对于小鼠为减毒的(100％存活率)，但DENVax-2和DENVax-4的MVS于超过104PFU的剂量下造成100％死亡率(图5A)。然而，当在103PFU病毒的剂量下测试时，DENVax-2(31.3％存活)和DENVax-4(81.3％存活)显示相对于野生型登革热病毒血清型-2 16681(0％存活)的神经毒力降低，并且其存活率分别类似于研究级疫苗病毒D2 PDK-53-VV45R(32.3％)和D2/4-V(100％)(图9B)。虽然本研究不包括野生型DENV-1、-3或-4用于比较，但先前研究工作证实通过脑内途径野生型DENV-1 16007于CDC-ICR小鼠中为减毒的，而野生型DENV-3 16562和DENV-4 1036对CDC-ICR小鼠有高毒力(0％存活)。可能的是，这3种野生型DENV在较为敏感的Taconic ICR小鼠中会表现出相似的或较高的毒力。因此，包括这些野生型登革热病毒以与其同源DENVaxMVS比较被视为信息不足。此研究表明全部4种DENVax MVS和原始实验室衍生的候选物疫苗病毒相对于野生型DENV-2 16681表现出相当的小鼠减毒表现型。
图9A-9B示出DENVax MVS于新生ICR小鼠中的神经毒力的数据的示例性图。(A)病毒于104PFU剂量下的IC接种。(B)病毒于103PFU的剂量下的IC接种。
测试全部DENVax种批次的身份、无菌性及不含不期望的试剂。全基因组序列分析显示一种额外的氨基酸突变出现在DENVax-4 MVS中，而其它3种DENVax MVS保留其前主代种的共有基因组序列。在WVS批次中，相对于其前主代种，DENVax-3获得额外的氨基酸突变以及其它三种血清型积累两种额外的氨基酸取代。全部4个BVS批次的基因组序列均与WVS批次相同。整体而言，从P2种至前主代(P7)种，在给定的种中只出现1或2种非沉默突变。前主代种与BCS(P10)种之间，只观察到1至2种核苷酸取代，均出现于NS2A、4A、或4B，除了导致残基E-483处的保守甘氨酸和丙氨酸的单一核苷酸改变之外。从P2至BVS(P10)种，在任何给定的DENVax种中确定共3至8种核苷酸取代，并且这些取代中只有2至4种导致氨基酸改变。在BVS中的沉默突变无一在5’-或3’-NCR区内，该区域可能影响病毒复制。这些结果表明DENVax病毒在制造期间是遗传上高度稳定的。位于5’NCR、NS1-53及NS3-250的三种定义的DENV-2 PDK-53减毒基因座在DENVax连续传代而生成BVS原料之后于共有基因组序列中保持不变。5’-NCR-57基因座的高度敏感性TaqMAMA显示在DENVax的MVS、WVS及BVS中的极少或无法检测的回复。在DENVax-2 BVS中鉴定0.21％的最高回复率。P10-等同BVS的回复率(<0.07％至0.21％)显著低于在于Vero细胞连续传代后于其它疫苗候选物中出现的回复率(P10的4-74％回复)。这表明，用于DENVax种的大规模制造的此种策略在候选物疫苗病毒中保持遗传稳定性和减毒标记的保留方面是成功的。
因为本文公开的MVS原料将用于进一步制造WVS和BVS批次，故对全部MVS或其等同代用原料进行全组的病毒减毒表现型评估，包括小溶菌斑表现型、温度敏感性、于蚊细胞中复制减少、于全蚊中感染/散播/传播降低、以及于新生ICR小鼠中神经毒力降低。对WVS和BVS原料，也进行溶菌斑尺寸、于蚊细胞中的感染力以验证其减毒。结果表示DENVax的4种血清型的全部MVS原料保留了预期的减毒表现型，诸如小溶菌斑、于C6/36细胞中复制减少、以及小鼠神经毒力降低，与原始实验室衍生的疫苗病毒相似(图6、8及9)。如图3和图7所示，除了DENVax-4之外，DENVax的所有其它3种MVS原料在39℃均为TS。
针对WVS和BVS原料，分析和证实两个减毒表现型，即小溶菌斑和于C6/36细胞中的受限复制。由于MVS与BVS之间极少有遗传变化，因此预期其会保留如同MVS一样的减毒表现型。除了本报告中描述的实验之外，已经测试所制造的DENVax于Ag129小鼠及非人灵长类动物中的安全性和免疫原性。
本文提供示例性方法以展示在cGMP下DENVax MVS、WVS及BVS原料的制造。BVS原料用于配制目前在人类临床试验评估中的四价DENVax。一些示例性方法中提供了独特的制造策略以优化所制造的MVS的遗传稳定性和安全性。由于DENVax的主减毒基因座先前已经良好表征，并开发高度敏感且可量化的SNP测定TaqMAMA以整合基因组序列及TaqMAMA以鉴定用于制造MVS的最佳前主代种。完整分析MVS的遗传学及表现型特性，以证实这些病毒保留疫苗安全性所期望的减毒。其可能是由前主代一路制造成BVS原料期间可有效分析全部主要减毒基因座的唯一活减毒病毒疫苗。本文提供的结果例示出策略上设计的活减毒疫苗在疫苗安全性方面的优点。
图10示出示例性表格，其比较新的活减毒疫苗与先前生成的活减毒登革热病毒。指出突变与对照病毒(例如16681)或其它活减毒登革热-2病毒不同。
材料及方法
病毒及细胞
DENV-1 16007、DENV-2 16681、DENV-3 16562及DENV-4 1034用作野生型(wt)DENV对照，其为四种重组DENVax疫苗候选物的亲代基因型病毒。先前制备并表征DENVax祖先研究级病毒，标示为D2/1-V、D2 PDK-53-VV45R、D2/3-V及D2/4-V。用于制备主代的Vero(非洲绿猴肾)细胞和用于疫苗生产的工作细胞库源自于美国典型菌种保藏中心(ATCC)CCL81细胞系，其已由世界卫生组织(WHO)表征用于疫苗制造(WCB-Vero细胞)。
从cDNA克隆衍生活重组DENVax病毒
为了在cGMP制造条件下再度衍生候选物疫苗病毒，使用先前改造的DENV传染性cDNA克隆，即pD2 PDK-53-VV45R、pD2/1-V、pD2/4-V、以及体外接合的含全基因组长度病毒cDNA的pD2/3-V，通过如前所述的体外转录来制备新鲜的病毒RNA转录本。简言之，经XbaI-线性化的DENV基因组cDNA经蛋白酶K处理，使用酚/氯仿萃取，以及在乙醇中沉淀以去除任何残余蛋白质，然后于转录之前悬浮于不含RNAase的Tris-EDTA缓冲液。遵照制造商推荐的方案，使用AmpliScribe T7高产率转录试剂盒(Epicentre Technologies)进行体外转录。在2小时转录反应期间将RNA A-帽类似物，即m7G(5’)ppp(5’)A(New England BioLabs)并入以将5’-端A-帽添加至RNA转录本。然后，使用DNase I处理样本以消化模板cDNA，接着是低pH酚/氯仿萃取以及乙醇沉积以去除残余DNA和蛋白质。将悬浮于不含RNase的水中的纯化RNA转录本分散于20μl等分部分内，并于-80℃储存，直至准备用于细胞转染。通过琼脂糖凝胶电泳分析RNA转录本的完整性和浓度。估计各20μl等分部分含有足量基因组长度病毒RNA来允许通过电穿孔而感染0.4-1×107生产核准的Vero细胞。
在Shantha Biotechnics的cGMP培养器中进行每一RNA转录本转染入WCB-Vero细胞。DENVax RNA转录本经解冻，与400μl Vero细胞悬浮液(1×107细胞/ml)混合，并转移到预先冷冻的无菌电穿孔试管(4mm间隙)以用于由Gene Pulser Xcell系统(BioRadLaboratories)进行电穿孔。每一样本以250V/∞Ohms/500μF施加脉冲一次，在室温孵育10-15分钟，转移至含30ml细胞生长培养基(含10％FBS的MEM)的75cm2烧瓶，并在36℃±1℃，5％CO2孵育6至11天。收获培养基，离心澄清、稳定化、并以小等分部分于低于-60℃储存。由转染所得的候选物疫苗原料(传代级别1称为P1)的病毒滴度通过在Vero细胞中的溶菌斑滴定测定来测定，并用于DENVax种的进一步繁殖。
DENVax病毒种的制造
P1病毒种用于通过设计用于确保制造批次的最佳遗传稳定性和安全性的策略来繁殖DENVax前主代、主代、工作及散装病毒批次。此策略包括三次连续溶菌斑纯化，以及于各种传代水平下的遗传分析，从而选择用于连续种生产的最佳克隆病毒群体(表1)。简言之，从转染的细胞收获的P1种通过以0.001的MOI感染Vero细胞而扩增一次，从而生成P2种。通过溶菌斑形态和完整病毒基因组测序来评估P2种原料的等分部分。遗传上证实的P2原料涂覆于如下溶菌斑滴定部分所描述的具有覆盖培养基(overlay medium)的Vero细胞单层上，从而生成完全分离的溶菌斑。在使用中性红可视化之后，分离4种血清型疫苗病毒各自的6个个别溶菌斑(溶菌斑克隆A至F)并混合入0.5ml培养基内(P3代)。6个溶菌斑悬浮液各自进行另两轮的溶菌斑纯化，导致在P4和P5代的两次和三次溶菌斑纯化病毒种。P5病毒通过两次连续Vero传代而扩增，以产生P7种原料。
进行使用如先前所公开的斑点测序和/或基于Taqman的错配扩增突变测定(TaqMAMA)的三种主要DENVax减毒基因座的遗传分析，以及进行溶菌斑表现型分析，从而筛检全部24种P7种。然后通过完整基因组测序进一步表征具有适当初始特性的种。作为这些分析的结果，每一DENVax血清型的6种(克隆A-F)P7种之一基于DENV-2 PDK-53减毒突变、最低基因组序列变化及预期溶菌斑表现型的存在而被选用为前主代种。每一所选的前主代种通过在多个175cm2烧瓶的Vero细胞内以0.001 MOI进行一次病毒传代而扩展成主代病毒种(MVS或P8)。除了DENVax-4 MVS之外，在感染后(pi)第8至10天收获主代病毒种。于感染后(pi)第6至10天收获MVS原料，离心澄清，通过添加蔗糖/磷酸盐/谷氨酸盐溶液(最终浓度分别为7.5％蔗糖，3.4mM磷酸二氢钾，7.2mM磷酸氢二钾，5.4mM谷氨酸一钠)以及0.95至1.90％FBS(最终浓度)而稳定化。DENVax-4 MVS以不同方式制备来优化其产率。简言之，多个烧瓶的细胞在0.1％已经验证可增加DENV病毒之热稳定性的F-127TM泊洛沙姆407的存在下(其它EO-PO嵌段共聚物已经经过评估且可于此处替换，参见公布的专利)，以0.001的MOI感染DENVax-4前主代种。于感染后6至10日收获感染培养基，使用17％FBS(终浓度)稳定化，汇集及冷冻。全部四种DENVax MVS原料以1ml液分储存于低于-60℃。
通过在0.001 MOI下，在MVS的Vero细胞培养中一次传代来制备DENVax工作病毒种(WVS)。程序类似于MVS的产生，但在多层细胞培养器(6360cm2)中培养。WVS原料通过10μM和0.45μM过滤器过滤，使用用于MVS的相同稳定剂稳定化，等分入30ml PETG瓶或2.0ml冷冻小瓶内，并低于-60℃储存。
在某些方法中，通过使用90mL稀释WVS以实现0.001 MOI感染融合Vero细胞的多个细胞培养器(各为6360cm2)而产生散装病毒种(BVS)。用于稀释MVS接种物的培养基含有0.1％F-127TM且不含血清。经1.5小时吸附后，用PBS洗涤细胞3次，将800ml的不含血清的DMEM培养基添加至每一细胞培养器，并将培养器于36(±1)℃于5(±0.5)％CO2孵育。孵育4天之后，收集培养基的小等分部分以进行无菌性测试。在感染后第5天至第10天之间收获病毒，即刻通过0.45μm孔径过滤器过滤，1L的每一澄清病毒汇集物通过添加500ml的3倍FTA缓冲液(最终浓度为15％海藻糖，1.0％
Figure BDA0002561695400000471
F-127TM泊洛沙姆407、于PBS中的0.1％人白蛋白USP，pH 7.4)而稳定化。将稳定化的病毒分配于1-L PETG瓶内，在低于-60℃冷冻储存，以用于随后的汇集和质量控制测试。具有高于105PFU/ml的病毒滴度和可接受水平的残余DNA的全部稳定化病毒收获物于32℃水浴中快速解冻，然后以无菌方式汇集并混合。全部汇集的单层BVS被分配于加标签的PETG容器内，并低于-60℃储存，直至进一步使用。
制造产品质量控制
测试MVS、WVS及BVS种的身份、无菌性及可检测的外源因子。每一疫苗原料的身份通过RT-PCR使用DENVax血清型特异性引物而证实。扩增的cDNA片段含有E/NS1嵌合式接合位点以允许鉴定四种DENVax血清型中的每一种。每一种(seed)于全部4种血清型特异性RT-PCR反应中测试以证实病毒身份和免于异源DENVax血清型的交叉污染。根据USP 71(美国药典第71节)进行无菌性测试。进行支原体测试。
全部使用在种制造期间收集的未经澄清的未经稳定化的DENVax收获物进行下列针对病毒污染的体外及活体内测试。收获的感染性培养基首先于36±1℃下使用DENV兔多克隆抗血清(Inviragen)中和以使DENV失活。针对体外试验，已中和的种接种在25cm2烧瓶内的三种指示剂细胞系(MRC5、VERO及MA104)中。埃可病毒(CPE对照)或腮腺炎病毒(血细胞吸附对照)分别用作阳性CPE或血细胞吸附对照。每天监测全部细胞的CPE，共计14天。14天结束后，移除培养上清液，以10ml豚鼠红血细胞(RBC)溶液(3mL的含0.5％豚鼠RBC的磷酸盐缓冲盐水，使用细胞培养基补充至10ml)置换。然后烧瓶于5±3℃孵育30分钟，接着于室温孵育30分钟。用PBS洗涤单层，并在10倍放大下观察任何星形RBC簇的存在，用于血细胞吸附。
在乳鼠、断奶后小鼠及豚鼠中进行外源因子的活体内测试。乳鼠经由腹膜内(ip)注射接种0.1ml或0.01ml(每一剂量组中有10只小鼠)DENV-抗血清中和种样本。类似地，10只断奶后小鼠腹膜内接种0.5ml或0.03ml样本。豚鼠(5只/组)各自腹膜内接种5.0ml。每天观察乳鼠的发病率及死亡率，总计接种后14天。在接种后，观察断奶后小鼠共28天，并且观察豚鼠共42天。若≥80％的接种动物于整个观察期保持健康，则该试验组满足可接受的标准。
还在鸡胚蛋中实施污染物的活体内测试。针对每一样本，10颗鸡胚蛋(9日龄)各自通过0.5mL的DENV抗血清中和样本接种至尿囊液内，并于35℃孵育3天。从这10颗蛋收获尿囊液，汇集，传代入10颗新鲜鸡胚蛋(10-11日龄；0.5ml/蛋)的尿囊液内，并于35℃再孵育3天。类似地，针对每一样本，10颗鸡胚蛋(6-7日龄)通过注射入卵黄囊内而各自接种0.5ml/蛋(DENVax-2一价BVS)或0.25ml/蛋(DENVax-1、DENVax-3及DENVax-4 BVS)，并于35℃孵育9天。得自这10颗蛋的卵黄囊经收获和汇集，并将10％悬浮液传代入10颗新鲜鸡胚蛋(6-7日龄；0.5ml/蛋)的卵黄囊内并于35℃再孵育9天。在3天孵育后观察在尿囊液中接种的蛋(包括初次接种及传代接种)的存活率。在4℃和25℃下使用鸡、豚鼠及人O型红细胞测试尿囊液的两种汇集物的红细胞凝集活性。在9天孵育后观察在卵黄囊内接种的蛋(初次接种及传代接种二者)的存活率。
病毒溶菌斑测定及免疫焦点测定
使用Vero细胞通过溶菌斑测定或免疫焦点测定而测量病毒滴度。如先前所述，在融合Vero细胞的6孔板内于双琼脂糖覆盖层中进行溶菌斑测定，并且其也用于评估DENVax种的溶菌斑表现型。为了精确比较，在同一实验中测量全部病毒的溶菌斑尺寸并比较。在感染后第9天使用中性红可视化后，测量每一病毒的多达10个完全分离的溶菌斑，以用于平均溶菌斑尺寸计算。对野生型DENV-1、-3及-4测量较少溶菌斑，其较大的溶菌斑尺寸通常不允许测量10个完全分离的溶菌斑。
因四价DENVax含有全部四种DENV血清型，故开发DENV血清型特异性免疫焦点测定来对四价配制剂中的每一DENVax组分进行定量。每一个别DENVax MVS的免疫焦点测定与溶菌斑测定进行比较以确保病毒滴定结果在两种测定之间相当。在感染连续稀释的病毒的融合Vero细胞的6孔板内进行免疫焦点测定。细胞上方覆盖有含0.7％高粘度羧甲基纤维素(Sigma)的平衡盐培养基(BSS/YE-LAH培养基)，并于37℃使用5％CO2孵育7天。去除覆盖层后，使用PBS洗涤三次细胞层，于-20℃使用80％冷丙酮固定30分钟，使用PBS洗涤一次，并于37℃使用含2.5％(w/v)非脂奶粉、0.5％Triton X-100、含0.05％Tween-20的PBS的封闭缓冲液封闭30分钟。封闭的细胞与稀释DENV血清型-特异性MAb、1F1(DENV-1)、3H5(DENV-2)、8A-1(DENV-3)、或1H10(DENV-4)在封闭缓冲液中于37℃一起孵育1小时或于4℃一起孵育过夜，用洗涤缓冲液(含0.05％Tween-20的PBS)洗涤三次，并于37℃与碱性磷酸镁-或辣根过氧化酶(HRP)-结合亲和力-纯山羊抗小鼠IgG(Jackson Immuno Research Laboratories)一起孵育45-60分钟。将板洗涤三次，然后添加适当底物(1-步NBT/BCIP)加碱性磷酸酶的抑制物(Pierce)或用于HRP的载体-VIP试剂盒(Vector Labs)以显色。当斑点完全显现后用水冲洗以中止显色。在光箱上直接可视化并计数染色免疫斑点。
基因序列
对MVS和WVS的全长基因组进行测序(参见下文)。简言之，通过使用QIAamp病毒RNA试剂盒(Qiagen)从DENVax种提取病毒RNA，并使用Titan One Tube RT-PCR试剂盒(RocheApplied Science,Inc.)扩增覆盖整个基因组的重迭cDNA片段。扩增的cDNA片段被凝胶纯化，然后使用BigDye Terminator v3.1循环测序试剂盒(Applied Biosystems)使用正向引物和反向引物测序。使用BigDye XTerminator纯化试剂盒(Applied Biosystems)清洁测序反应，并在DVBD/CDC下于3130x1Genetic分析仪(Applied Biosystems)上运行。使用Lasergene SeqMan软件(DNAStar Inc.)进行基因组分析及比较。
基于Taqman的错配扩增突变测定(TaqMAMA)
TaqMAMA为敏感定量单一核苷酸多形性测定，其开发以用于更精细地评估于减毒5’NC-57基因座的回复水平，并针对此研究而进一步优化。通过使用对野生型或疫苗5’NC-57区有特异性的两组引物/Taqman探针的TaqMAMA来分析来自MVS和WVS的经提取的病毒RNA。用来检测DENV-2野生型和疫苗序列的正向引物分别为D2-41-GC和D2-40-TT。每一正向引物的3’-端核苷酸匹配各病毒的特异性5’NCR-57核苷酸，而在每一引物中与3’-端核苷酸相邻的核苷酸与DENV-2病毒基因组序列不同，以增加错配效应。用于野生型组和疫苗组的反向引物CD-207和Taqman探针CD-169F是相同的。先前描述了引物和探针的序列以及循环条件。在含5μl病毒RNA模板、0.4μM每一引物以及0.2μM探针的25μl反应中，使用BioRadiScript RT-PCR(用于探针)试剂盒，以iQ5或CFX-95系统(BioRad)进行实时RT-PCR。针对每一样本进行各野生型特异性测定和疫苗特异性测定的重复三次反应。相对于针对每一病毒基因型准备的标准曲线来确定基因组拷贝数，其中RNA标准为从含每一基因型特异性cDNA的nt 1-2670的质粒衍生的转录本。此外，通过使用异源基因型引物/探针组测试每一RNA标准而证实测定的特异性，以确保于每个实验中最低的交叉反应性。结果报告为表现出回复的病毒基因组的百分比。先前，由于较高交叉反应背景(其限制此测定的输入RNA水平)，原始检测敏感性为约0.1％回复(辨别能力)。此后，测定进一步使用改良的实时PCR设备和反应试剂盒而优化，并且交叉反应性背景在显著更高的RNA模板输入水平(7-8log10拷贝)下显著降低。此种优化导致检测敏感性显著改善，低至0.01-0.07％回复。
在蚊C6/36细胞中的病毒复制及在哺乳动物Vero细胞中的温度敏感性
在C6/36蚊细胞(白纹伊蚊(Aedes albopictus))中评估四个DENVax MVS原料及野生型DENV-1、-2、-3和-4病毒的复制表现型。生长于6孔板的C6/36细胞以0.001 MOI一式两份感染每一病毒，并于5％CO2孵育器内于28℃与含2％FBS的4ml/孔DMEM培养基一起孵育。在感染后第6天收集每一病毒的培养上清液的小等分部分，与含40％FBS的等体积培养基混合，并于-80℃储存，直至准备用于病毒溶菌斑滴定。
在6孔板中于Vero细胞感染后第5天，通过比较39℃的病毒生长与37℃病毒的生长而进行温度敏感性。细胞在37℃细胞用每一病毒于0.001 MOI一式四份感染。在病毒吸附后，感染的培养物与含2％FBS的4ml/孔的DMEM培养基在两个单独的5％CO2孵育器内一起孵育，一个设定(一式两份的板)于37℃，而另一个设定于39℃。感染后第5天收集培养物上清液的等分部分(50μl)，与含40％FBS的等体积DMEM混合，并于-80℃储存，直到准备用于病毒溶菌斑滴定。使用NIST-示踪培养器校准温度计(-1至51℃；ERTCO)校准孵育器温度。
蚊感染、散播及传播
用于研究的埃及伊蚊得自源于靠近泰国美索(16’N,33’E)的村庄的于2002年建立的集落。从幼虫蜕变之后，成蚊被保持在28℃和16：8(亮：暗)光周期下，并自由地供给10％蔗糖溶液。5至7日龄的雌蚊用于感染血粉进食或胸内(IT)接种。收获后即刻(未经任何冷冻-解冻周期)使用新鲜培养的DENVax和野生型DENV的等分部分来制备用于经口感染的如下指示的病毒血粉。向剩余的病毒上清液添加FBS，至最终浓度为20％，并且等分部分储存于-80℃以用于未来病毒溶菌斑滴定和IT接种物实验。用于这些实验的新鲜制备的DENVax种在Vero细胞中从前主代种扩增，并被视为DENVax MVS等同物。
在经口感染当天以1：1比例混合新鲜病毒与去纤维蛋白化的鸡血(ColoradoSerum Company)以制备感染血粉。蚊子被糖缺乏过夜，然后使用Hemotek膜进食系统(Discovery Workshops)提供病毒：血液混合物，历时1小时。50μl等分部分的血粉保持在-80℃，以用于病毒剂量的回滴定。完全饱食的雌蚊在冷麻醉下分类并置于纸箱内，自由提供10％蔗糖溶液。纸箱置于28℃和16：8小时(亮：暗)光周期下。14天后，得自每一病毒组的25至30只蚊子经由暴露于三乙胺(
Figure BDA0002561695400000521
Carolina Biological Supply Company)而麻醉，并取一只后腿置于含10％FBS和5％青霉素/链霉素(分别为100U/ml和100μg/ml)的0.5ml DMEM中。将麻醉的蚊子的喙插入含2.5％FBS和25％蔗糖溶液的毛细管内，从而收集唾液。允许蚊子分泌唾液至少15分钟，然后将毛细管和蚊体置于含DMEM的单独的试管内。蚊体、腿部及唾液储存于-80℃，直到其被磨碎并测定传染性病毒。对于IT接种，蚊子被冷麻醉并于0.34μl接种物内与约50pfu病毒一起孵育。接种的蚊子在与前述相同的条件下保持7天。然后将蚊子麻醉，如前述那样收集唾液和蚊体。样本储存于-80℃，直到进一步处理。
为了处理样本以进行病毒滴定，将蚊体和腿部样本使用混合磨以24周期/秒历时4分钟与铜包BB(Crossman Corporation，NY)一起均化，然后以3,000×g离心3分钟而澄清。唾液样本于3,000×g离心3分钟以从毛细管中排出流体。通过溶菌斑测定检测蚊体和腿部均化物和唾液样本的10倍稀释液的传染性病毒的存在。从蚊体、腿部及唾液所得的结果分别用于测定感染率、散播率及传播率。
小鼠神经毒力
定时妊娠雌ICR小鼠得自Taconic Labs，并每天监测数次以确定胎鼠(puplitters)的大致出生时间。在给定的实验中，出生后约12-24小时，每种病毒使用两窝八头胎鼠(n＝16)，使用30号针头通过颅内(ic)接种含103至104pfu病毒的20μl稀释液进行攻击。攻击后每日至少3次监测动物，至少历时32天。在疾病的第一迹象(毛皮粗糙、驼背、体重减轻、活动异常、瘫痪或嗜睡)时，使用异氟醚气体通过致命性麻醉，接着颈椎脱位，从而使动物安乐死。安乐死的感染后天数表示动物的“生病/发病的时间”或“存活时间”。动物实验遵照DVBD/CDC IACUC-核准的动物方案进行。
主代种病毒的衍生
DENVax-1主代病毒种(MVS)
本文提供了嵌合式病毒基因组的核苷酸序列及翻译蛋白质的推定氨基酸序列。大部分prM-E基因(nt 457至-2379，下画线)为野生型(wt)DEN-116007病毒特异性的；剩余基因组为DEN-2 PDK-53病毒特异性的。标出与野生型病毒(D116007或D216681)不同的全部改造的取代物，以及在MVS中检测的额外突变(从改造的cDNA克隆的改变)。
基因组及蛋白质中包括的取代：
D1(prM-E)与D2骨架之间的接合位点：
a.MluI(nt 451-456)：改造的沉默突变，nt-453A-至-G
b.NgoMIV(nt 2380-2385)：改造的突变，nt-2381/2382TG-至-CC(导致E-482Val-至-Ala改变)
D2 PDK-53病毒骨架(从野生型D2 16681的改变)：全部为粗体
a.5’-非编码区(NCR)-57(nt-57C-至-T)：主要减毒基因座(红色)
b.NS1-53 Gly-至-Asp(nt-2579G-至-A)：主要减毒基因座(红色)
c.NS2A-181Leu-至-Phe(nt-4018C-至-T)
d.NS3-250 Glu-至-Val(nt-5270A-至-T)：主要减毒基因座(红色)
e.nt-5547(NS3基因)T-至-C沉默突变
f.NS4A-75Gly-至-Ala(nt-6599G-至-C)
*PDK-53的nt-8571C-至-T沉默突变未在疫苗病毒中被改造
DEN-1prM-E(从野生型D1 16007的改变)
a.改造的nt-1575T-至-C沉默突变用于去除天然XbaI位点
在疫苗种中发现的额外取代(与原始克隆有0.03％nt不同)
a.NS2A-116Ile-至-Leu(nt-3823A-至-C，粗体)
b.NS2B-92Glu-至-Asp(nt-4407A-至-T，粗体)
c.nt-7311A-至-G沉默突变(粗体)
DENVax-1 MVS的核酸序列(SEQ ID NO:13)：
Figure BDA0002561695400000551
Figure BDA0002561695400000561
Figure BDA0002561695400000571
Figure BDA0002561695400000581
Figure BDA0002561695400000591
Figure BDA0002561695400000601
Figure BDA0002561695400000611
DENVax-2主代病毒种(MVS)
本文提供重组病毒基因组的核苷酸序列及翻译蛋白质的推定氨基酸序列。改造的病毒基于D2 PDK-53病毒。标示出与野生型DEN-2 16681病毒(也是PDK-53的亲代病毒)不同的全部改造取代，以及于MVS中检测到的额外突变(从改造的cDNA克隆的改变)。
基因组和蛋白质中包括的取代：
D2 PDK-53病毒骨架(从野生型D2 16681的改变)：均为粗体
a.5’-非编码区(NCR)-57(nt-57C-至-T)：主要减毒基因座(红色)
b.prM-29Asp-至-Val(nt-524A-至-T)
c.nt-2055C-至-T(E基因)沉默突变
d.NS1-53 Gly-至-Asp(nt-2579G-至-A)：主要减毒基因座(红色)
e.NS2A-181Leu-至-Phe(nt-4018C-至-T)
f.NS3-250 Glu-至-Val(nt-5270A-至-T)：主要减毒基因座(红色)
g.nt-5547(NS3基因)T-至-C沉默突变
h.NS4A-75Gly-至-Ala(nt-6599G-至-C)
*在疫苗病毒中的PDK-53的nt-8571C-至-T沉默突变未改造。
改造的克隆标记(沉默突变)：
a.nt-900T-至-C沉默突变：感染克隆标记
在疫苗种中发现的额外取代(与原始克隆差异0.02％nt)
a.prM-52Lys-至-Glu(nt-592A-至-G)，粗体
b.NS5-412 Ile-至-Val(nt-8803A-至-G)，粗体
DENVax-2 MVS的核酸序列(SEQ ID NO:14)：
Figure BDA0002561695400000621
Figure BDA0002561695400000631
Figure BDA0002561695400000641
Figure BDA0002561695400000651
Figure BDA0002561695400000661
DENVax-3主代病毒种(MVS)
本文提供嵌合式病毒基因组的核苷酸序列及翻译蛋白质的推定氨基酸序列。大部分prM-E基因(nt-457至-2373，下画线)为野生型(wt)DEN-3 16562病毒特异性的；剩余核苷酸序列为DEN-2 PDK-53病毒特异性的。DEN-3病毒的E蛋白比DEN-2的E蛋白少两个氨基酸。因此，从NgoMIV起始的nt位置比原始DEN-2 PDK-53nt位置少6nt。标记出与野生型病毒(DEN-3 16562或DEN-2 16681)不同的全部改造取代以及额外的突变(从改造cDNA克隆的改变)。
基因组和蛋白质中包括的取代：
接合位点：
a.MluI(nt 451-456)：改造的沉默突变，nt-453A-至-G
b.NgoMIV(nt 2374-2379)：改造的突变，nt-2375/2376TG-至-CC(导致E-480Val-至-Ala改变)
D2 PDK-53病毒骨架(从野生型D2 16681的改变)：粗体
a.5’-非编码区(NCR)-57(nt-57C-至-T)：主要减毒基因座(红色)
b.NS1-53 Gly-至-Asp(nt-2573G-至-A)：主要减毒基因座(红色)
c.NS2A-181Leu-至-Phe(nt-4012C-至-T)
d.NS3-250 Glu-至-Val(nt-5264A-至-T)：主要减毒基因座(红色)
e.nt-5541(NS3基因)T-至-C沉默突变
f.NS4A-75Gly-至-Ala(nt-6593G-至-C)
*PDK-53的nt-8565C-至-T沉默突变在疫苗病毒中未经改造
在DEN-3prM-E中的改造突变(从野生型D3 16562的改变)
a.改造的nt-552C-至-T沉默突变：克隆标记
b.改造的E-345His-至-Leu(nt-1970A-至-T)用于在培养物中有效复制
在疫苗种中发现的额外的取代(与原始克隆差异0.02％nt)
a.E-223Thr-至-Ser突变(nt1603 A-至-T，粗体)
b.nt-7620A-至-G沉默突变(粗体)
DENVax-3 MVS的核酸序列(SEQ ID NO:15)：
Figure BDA0002561695400000671
Figure BDA0002561695400000681
Figure BDA0002561695400000691
Figure BDA0002561695400000701
Figure BDA0002561695400000711
Figure BDA0002561695400000721
DENVax-4主代病毒种(MVS)
嵌合式病毒基因组的核苷酸序列及翻译蛋白质的推定氨基酸序列。大部分prM-E基因(nt-457至-2379，下画线)为野生型(wt)DEN-4 1036病毒特异性的；剩余核苷酸序列为DEN-2 PDK-53病毒特异性的。标记出与野生型病毒(DEN-3 16562或DEN-2 16681)不同的全部改造取代，以及额外的突变(从改造cDNA克隆的改变)。
基因组和蛋白质中包括的取代：
接合位点：
a.MluI(nt 451-456)：改造的沉默突变，nt-453A-至-G
b.NgoMIV(nt 2380-2385)：改造的突变，nt-2381/2382TG-至-CC(导致E-482Val-至-Ala改变)
D2 PDK-53病毒骨架(从野生型D2 16681的改变)
a.5’-非编码区(NCR)-57(nt-57C-至-T)：主要减毒基因座(红色)
b.NS1-53 Gly-至-Asp(nt-2579G-至-A)：主要减毒基因座(红色)
c.NS2A-181Leu-至-Phe(nt-4018C-至-T，粗体)
d.NS3-250 Glu-至-Val(nt-5270A-至-T)：主要减毒基因座(红色)
e.nt-5547(NS3基因)T-至-C沉默突变(粗体)
f.NS4A-75Gly-至-Ala(nt-6599G-至-C，粗体)
*PDK-53的nt-8571C-至-T沉默突变在疫苗病毒中未经改造
在cDNA克隆中的改造的取代
a.改造的C-100Arg-至-Ser(nt-396A-至-C)：可改善于培养物中的病毒复制
b.改造的nt-1401A-至-G沉默突变
c.改造的E-364Ala-至-Val(nt-2027C-至-T)：可改善于培养物中的病毒复制
d.改造的E-447Met-至-Leu(nt-2275A-至-C)：可改善于培养物中的病毒复制
在疫苗种中发现的额外的取代(与原始克隆差异0.06％nt)
a.nt-225(C基因)A-至-T沉默突变(粗体)
b.NS2A-66Asp-至-Gly(nt-3674A-至-G)突变(粗体)
c.NS2A-99Lys-至-Lys/Arg混合物(nt-3773A-至-A/G混合，粗体)
d.nt-5391C-至-T(NS3基因)沉默突变(粗体)
e.NS4A-21Ala-至-Val(nt-6437C-至-T，粗体)
f.nt-7026T-至-C/T混合沉默突变(粗体)
g.nt-9750A-至-C沉默突变(粗体)
DEN Vax-4 MVS的核酸序列(SEQID NO:16):
Figure BDA0002561695400000741
Figure BDA0002561695400000751
Figure BDA0002561695400000761
Figure BDA0002561695400000771
Figure BDA0002561695400000781
Figure BDA0002561695400000791
Figure BDA0002561695400000801
序列表
<110> 武田疫苗股份有限公司
美国政府健康及人类服务部
<120> 用于疫苗中的登革热病毒嵌合式构建物的组合物及方法
<130> T08239WOCND1/ES
<150> US 61/800,204
<151> 2013-03-15
<150> PCT/2014/024603
<151> 2014-03-12
<150> CN 201480028001.5
<151> 2014-03-12
<160> 16
<170> PatentIn version 3.5
<210> 1
<211> 10723
<212> DNA
<213> 人工序列
<220>
<223> 登革热病毒血清型1,BVS
<400> 1
agttgttagt ctacgtggac cgacaaagac agattctttg agggagctaa gctcaatgta 60
gttctaacag ttttttaatt agagagcaga tctctgatga ataaccaacg gaaaaaggcg 120
aaaaacacgc ctttcaatat gctgaaacgc gagagaaacc gcgtgtcgac tgtgcaacag 180
ctgacaaaga gattctcact tggaatgctg cagggacgag gaccattaaa actgttcatg 240
gccctggtgg cgttccttcg tttcctaaca atcccaccaa cagcagggat attgaagaga 300
tggggaacaa ttaaaaaatc aaaagctatt aatgttttga gagggttcag gaaagagatt 360
ggaaggatgc tgaacatctt gaataggaga cgcagatctg caggcatgat cattatgctg 420
attccaacag tgatggcgtt ccatttaacc acgcgtgggg gagagccgca tatgatagtt 480
agcaagcagg aaagaggaaa gtcacttttg ttcaagacct ctgcaggtgt caacatgtgc 540
accctcattg cgatggattt gggagagttg tgtgaggaca cgatgaccta caaatgcccc 600
cggatcactg aggcggaacc agatgacgtt gactgttggt gcaatgccac ggacacatgg 660
gtgacctatg gaacgtgctc tcaaactggc gaacaccgac gagacaaacg ttccgtcgca 720
ttggccccac acgtggggct tggcctagaa acaagagccg aaacgtggat gtcctctgaa 780
ggtgcttgga aacagataca aaaagtagag acttgggctc tgagacatcc aggattcacg 840
gtgatagccc tttttctagc acatgccata ggaacatcca tcacccagaa agggatcatt 900
ttcattttgc tgatgctggt aacaccatct atggccatgc gatgcgtggg aataggcaac 960
agagacttcg tggaaggact gtcaggagca acatgggtgg atgtggtact ggagcatgga 1020
agttgcgtca ccaccatggc aaaaaacaaa ccaacactgg acattgaact cttgaagacg 1080
gaggtcacaa accctgcagt tctgcgtaaa ttgtgcattg aagctaaaat atcaaacacc 1140
accaccgatt cgagatgtcc aacacaagga gaagccacac tggtggaaga acaagacgcg 1200
aactttgtgt gccgacgaac gttcgtggac agaggctggg gcaatggctg tgggctattc 1260
ggaaaaggta gtctaataac gtgtgccaag tttaagtgtg tgacaaaact agaaggaaag 1320
atagttcaat atgaaaacct aaaatattca gtgatagtca ccgtccacac tggagatcag 1380
caccaggtgg gaaatgagac tacagaacat ggaacaactg caaccataac acctcaagct 1440
cctacgtcgg aaatacagct gaccgactac ggaaccctta cattagattg ttcacctagg 1500
acagggctag attttaacga gatggtgttg ctgacaatga aagaaagatc atggcttgtc 1560
cacaaacaat ggttcctaga cttaccactg ccttggacct ctggggcttc aacatcccaa 1620
gagacttgga acagacaaga tttactggtc acatttaaga cagctcatgc aaagaagcag 1680
gaagtagtcg tactaggatc acaagaagga gcaatgcaca ctgcgctgac tggagcgaca 1740
gaaatccaaa cgtcaggaac gacaacaatt ttcgcaggac acctaaaatg cagactaaaa 1800
atggacaaac taactttaaa agggatgtca tatgtgatgt gcacaggctc attcaagtta 1860
gagaaagaag tggctgagac ccagcatgga actgttctgg tgcaggttaa atatgaagga 1920
acagacgcac catgcaagat tcccttttcg acccaagatg agaaaggagc aacccagaat 1980
gggagattaa taacagccaa ccccatagtc actgacaaag aaaaaccagt caatattgag 2040
gcagaaccac cctttggtga gagctacatc gtggtaggag caggtgaaaa agctttgaaa 2100
ctaagctggt tcaagaaagg aagcagcata gggaaaatgt ttgaagcaac tgcccgagga 2160
gcacgaagga tggccattct gggagacacc gcatgggact tcggttctat aggaggagtg 2220
ttcacgtcta tgggaaaact ggtacaccag gtttttggaa ctgcatatgg agttttgttt 2280
agcggagttt cttggaccat gaaaatagga atagggattc tgctgacatg gctaggatta 2340
aattcaagga acacgtccct ttcgatgatg tgcatcgcag ccgccattgt gacactgtat 2400
ttgggagtca tggtgcaggc cgatagtggt tgcgttgtga gctggaaaaa caaagaactg 2460
aaatgtggca gtgggatttt catcacagac aacgtgcaca catggacaga acaatacaag 2520
ttccaaccag aatccccttc aaaactagct tcagctatcc agaaagccca tgaagaggac 2580
atttgtggaa tccgctcagt aacaagactg gagaatctga tgtggaaaca aataacacca 2640
gaattgaatc acattctatc agaaaatgag gtgaagttaa ctattatgac aggagacatc 2700
aaaggaatca tgcaggcagg aaaacgatct ctgcggcctc agcccactga gctgaagtat 2760
tcatggaaaa catggggcaa agcaaaaatg ctctctacag agtctcataa ccagaccttt 2820
ctcattgatg gccccgaaac agcagaatgc cccaacacaa atagagcttg gaattcgttg 2880
gaagttgaag actatggctt tggagtattc accaccaata tatggctaaa attgaaagaa 2940
aaacaggatg tattctgcga ctcaaaactc atgtcagcgg ccataaaaga caacagagcc 3000
gtccatgccg atatgggtta ttggatagaa agtgcactca atgacacatg gaagatagag 3060
aaagcctctt tcattgaagt taaaaactgc cactggccaa aatcacacac cctctggagc 3120
aatggagtgc tagaaagtga gatgataatt ccaaagaatc tcgctggacc agtgtctcaa 3180
cacaactata gaccaggcta ccatacacaa ataacaggac catggcatct aggtaagctt 3240
gagatggact ttgatttctg tgatggaaca acagtggtag tgactgagga ctgcggaaat 3300
agaggaccct ctttgagaac aaccactgcc tctggaaaac tcataacaga atggtgctgc 3360
cgatcttgca cattaccacc gctaagatac agaggtgagg atgggtgctg gtacgggatg 3420
gaaatcagac cattgaagga gaaagaagag aatttggtca actccttggt cacagctgga 3480
catgggcagg tcgacaactt ttcactagga gtcttgggaa tggcattgtt cctggaggaa 3540
atgcttagga cccgagtagg aacgaaacat gcaatactac tagttgcagt ttcttttgtg 3600
acattgatca cagggaacat gtcctttaga gacctgggaa gagtgatggt tatggtaggc 3660
gccactatga cggatgacat aggtatgggc gtgacttatc ttgccctact agcagccttc 3720
aaagtcagac caacttttgc agctggacta ctcttgagaa agctgacctc caaggaattg 3780
atgatgacta ctataggaat tgtactcctc tcccagagca ccctaccaga gaccattctt 3840
gagttgactg atgcgttagc cttaggcatg atggtcctca aaatggtgag aaatatggaa 3900
aagtatcaat tggcagtgac tatcatggct atcttgtgcg tcccaaacgc agtgatatta 3960
caaaacgcat ggaaagtgag ttgcacaata ttggcagtgg tgtccgtttc cccactgttc 4020
ttaacatcct cacagcaaaa aacagattgg ataccattag cattgacgat caaaggtctc 4080
aatccaacag ctatttttct aacaaccctc tcaagaacca gcaagaaaag gagctggcca 4140
ttaaatgagg ctatcatggc agtcgggatg gtgagcattt tagccagttc tctcctaaaa 4200
aatgatattc ccatgacagg accattagtg gctggagggc tcctcactgt gtgctacgtg 4260
ctcactggac gatcggccga tttggaactg gagagagcag ccgatgtcaa atgggaagac 4320
caggcagaga tatcaggaag cagtccaatc ctgtcaataa caatatcaga agatggtagc 4380
atgtcgataa aaaatgaaga ggaagatcaa acactgacca tactcattag aacaggattg 4440
ctggtgatct caggactttt tcctgtatca ataccaatca cggcagcagc atggtacctg 4500
tgggaagtga agaaacaacg ggccggagta ttgtgggatg ttccttcacc cccacccatg 4560
ggaaaggctg aactggaaga tggagcctat agaattaagc aaaaagggat tcttggatat 4620
tcccagatcg gagccggagt ttacaaagaa ggaacattcc atacaatgtg gcatgtcaca 4680
cgtggcgctg ttctaatgca taaaggaaag aggattgaac catcatgggc ggacgtcaag 4740
aaagacctaa tatcatatgg aggaggctgg aagttagaag gagaatggaa ggaaggagaa 4800
gaagtccagg tattggcact ggagcctgga aaaaatccaa gagccgtcca aacgaaacct 4860
ggtcttttca aaaccaacgc cggaacaata ggtgctgtat ctctggactt ttctcctgga 4920
acgtcaggat ctccaattat cgacaaaaaa ggaaaagttg tgggtcttta tggtaatggt 4980
gttgttacaa ggagtggagc atatgtgagt gctatagccc agactgaaaa aagcattgaa 5040
gacaacccag agatcgaaga tgacattttc cgaaagagaa gactgaccat catggacctc 5100
cacccaggag cgggaaagac gaagagatac cttccggcca tagtcagaga agctataaaa 5160
cggggtttga gaacattaat cttggccccc actagagttg tggcagctga aatggaggaa 5220
gcccttagag gacttccaat aagataccag accccagcca tcagagctgt gcacaccggg 5280
cgggagattg tggacctaat gtgtcatgcc acatttacca tgaggctgct atcaccagtt 5340
agagtgccaa actacaacct gattatcatg gacgaagccc atttcacaga cccagcaagt 5400
atagcagcta gaggatacat ctcaactcga gtggagatgg gtgaggcagc tgggattttt 5460
atgacagcca ctcccccggg aagcagagac ccatttcctc agagcaatgc accaatcata 5520
gatgaagaaa gagaaatccc tgaacgctcg tggaattccg gacatgaatg ggtcacggat 5580
tttaaaggga agactgtttg gttcgttcca agtataaaag caggaaatga tatagcagct 5640
tgcctgagga aaaatggaaa gaaagtgata caactcagta ggaagacctt tgattctgag 5700
tatgtcaaga ctagaaccaa tgattgggac ttcgtggtta caactgacat ttcagaaatg 5760
ggtgccaatt tcaaggctga gagggttata gaccccagac gctgcatgaa accagtcata 5820
ctaacagatg gtgaagagcg ggtgattctg gcaggaccta tgccagtgac ccactctagt 5880
gcagcacaaa gaagagggag aataggaaga aatccaaaaa atgagaatga ccagtacata 5940
tacatggggg aacctctgga aaatgatgaa gactgtgcac actggaaaga agctaaaatg 6000
ctcctagata acatcaacac gccagaagga atcattccta gcatgttcga accagagcgt 6060
gaaaaggtgg atgccattga tggcgaatac cgcttgagag gagaagcaag gaaaaccttt 6120
gtagacttaa tgagaagagg agacctacca gtctggttgg cctacagagt ggcagctgaa 6180
ggcatcaact acgcagacag aaggtggtgt tttgatggag tcaagaacaa ccaaatccta 6240
gaagaaaacg tggaagttga aatctggaca aaagaagggg aaaggaagaa attgaaaccc 6300
agatggttgg atgctaggat ctattctgac ccactggcgc taaaagaatt taaggaattt 6360
gcagccggaa gaaagtctct gaccctgaac ctaatcacag aaatgggtag gctcccaacc 6420
ttcatgactc agaaggcaag agacgcactg gacaacttag cagtgctgca cacggctgag 6480
gcaggtggaa gggcgtacaa ccatgctctc agtgaactgc cggagaccct ggagacattg 6540
cttttactga cacttctggc tacagtcacg ggagggatct ttttattctt gatgagcgca 6600
aggggcatag ggaagatgac cctgggaatg tgctgcataa tcacggctag catcctccta 6660
tggtacgcac aaatacagcc acactggata gcagcttcaa taatactgga gttttttctc 6720
atagttttgc ttattccaga acctgaaaaa cagagaacac cccaagacaa ccaactgacc 6780
tacgttgtca tagccatcct cacagtggtg gccgcaacca tggcaaacga gatgggtttc 6840
ctagaaaaaa cgaagaaaga tctcggattg ggaagcattg caacccagca acccgagagc 6900
aacatcctgg acatagatct acgtcctgca tcagcatgga cgctgtatgc cgtggccaca 6960
acatttgtta caccaatgtt gagacatagc attgaaaatt cctcagtgaa tgtgtcccta 7020
acagctatag ccaaccaagc cacagtgtta atgggtctcg ggaaaggatg gccattgtca 7080
aagatggaca tcggagttcc ccttctcgcc attggatgct actcacaagt caaccccata 7140
actctcatag cagctctttt cttattggta gcacattatg ccatcatagg gccaggactc 7200
caagcaaaag caaccagaga agctcagaaa agagcagcgg cgggcatcat gaaaaaccca 7260
actgtcgatg gaataacagt gattgaccta gatccaatac cttatgatcc gaagtttgaa 7320
aagcagttgg gacaagtaat gctcctagtc ctctgcgtga ctcaagtatt gatgatgagg 7380
actacatggg ctctgtgtga ggctttaacc ttagctaccg ggcccatctc cacattgtgg 7440
gaaggaaatc cagggaggtt ttggaacact accattgcgg tgtcaatggc taacattttt 7500
agagggagtt acttggccgg agctggactt ctcttttcta ttatgaagaa cacaaccaac 7560
acaagaaggg gaactggcaa cataggagag acgcttggag agaaatggaa aagccgattg 7620
aacgcattgg gaaaaagtga attccagatc tacaagaaaa gtggaatcca ggaagtggat 7680
agaaccttag caaaagaagg cattaaaaga ggagaaacgg accatcacgc tgtgtcgcga 7740
ggctcagcaa aactgagatg gttcgttgag agaaacatgg tcacaccaga agggaaagta 7800
gtggacctcg gttgtggcag aggaggctgg tcatactatt gtggaggact aaagaatgta 7860
agagaagtca aaggcctaac aaaaggagga ccaggacacg aagaacccat ccccatgtca 7920
acatatgggt ggaatctagt gcgtcttcaa agtggagttg acgttttctt catcccgcca 7980
gaaaagtgtg acacattatt gtgtgacata ggggagtcat caccaaatcc cacagtggaa 8040
gcaggacgaa cactcagagt ccttaactta gtagaaaatt ggttgaacaa caacactcaa 8100
ttttgcataa aggttctcaa cccatatatg ccctcagtca tagaaaaaat ggaagcacta 8160
caaaggaaat atggaggagc cttagtgagg aatccactct cacgaaactc cacacatgag 8220
atgtactggg tatccaatgc ttccgggaac atagtgtcat cagtgaacat gatttcaagg 8280
atgttgatca acagatttac aatgagatac aagaaagcca cttacgagcc ggatgttgac 8340
ctcggaagcg gaacccgtaa catcgggatt gaaagtgaga taccaaacct agatataatt 8400
gggaaaagaa tagaaaaaat aaagcaagag catgaaacat catggcacta tgaccaagac 8460
cacccataca aaacgtgggc ataccatggt agctatgaaa caaaacagac tggatcagca 8520
tcatccatgg tcaacggagt ggtcaggctg ctgacaaaac cttgggacgt cgtccccatg 8580
gtgacacaga tggcaatgac agacacgact ccatttggac aacagcgcgt ttttaaagag 8640
aaagtggaca cgagaaccca agaaccgaaa gaaggcacga agaaactaat gaaaataaca 8700
gcagagtggc tttggaaaga attagggaag aaaaagacac ccaggatgtg caccagagaa 8760
gaattcacaa gaaaggtgag aagcaatgca gccttggggg ccatattcac tgatgagaac 8820
aagtggaagt cggcacgtga ggctgttgaa gatagtaggt tttgggagct ggttgacaag 8880
gaaaggaatc tccatcttga aggaaagtgt gaaacatgtg tgtacaacat gatgggaaaa 8940
agagagaaga agctagggga attcggcaag gcaaaaggca gcagagccat atggtacatg 9000
tggcttggag cacgcttctt agagtttgaa gccctaggat tcttaaatga agatcactgg 9060
ttctccagag agaactccct gagtggagtg gaaggagaag ggctgcacaa gctaggttac 9120
attctaagag acgtgagcaa gaaagaggga ggagcaatgt atgccgatga caccgcagga 9180
tgggatacaa gaatcacact agaagaccta aaaaatgaag aaatggtaac aaaccacatg 9240
gaaggagaac acaagaaact agccgaggcc attttcaaac taacgtacca aaacaaggtg 9300
gtgcgtgtgc aaagaccaac accaagaggc acagtaatgg acatcatatc gagaagagac 9360
caaagaggta gtggacaagt tggcacctat ggactcaata ctttcaccaa tatggaagcc 9420
caactaatca gacagatgga gggagaagga gtctttaaaa gcattcagca cctaacaatc 9480
acagaagaaa tcgctgtgca aaactggtta gcaagagtgg ggcgcgaaag gttatcaaga 9540
atggccatca gtggagatga ttgtgttgtg aaacctttag atgacaggtt cgcaagcgct 9600
ttaacagctc taaatgacat gggaaagatt aggaaagaca tacaacaatg ggaaccttca 9660
agaggatgga atgattggac acaagtgccc ttctgttcac accatttcca tgagttaatc 9720
atgaaagacg gtcgcgtact cgttgttcca tgtagaaacc aagatgaact gattggcaga 9780
gcccgaatct cccaaggagc agggtggtct ttgcgggaga cggcctgttt ggggaagtct 9840
tacgcccaaa tgtggagctt gatgtacttc cacagacgcg acctcaggct ggcggcaaat 9900
gctatttgct cggcagtacc atcacattgg gttccaacaa gtcgaacaac ctggtccata 9960
catgctaaac atgaatggat gacaacggaa gacatgctga cagtctggaa cagggtgtgg 10020
attcaagaaa acccatggat ggaagacaaa actccagtgg aatcatggga ggaaatccca 10080
tacttgggga aaagagaaga ccaatggtgc ggctcattga ttgggttaac aagcagggcc 10140
acctgggcaa agaacatcca agcagcaata aatcaagtta gatcccttat aggcaatgaa 10200
gaatacacag attacatgcc atccatgaaa agattcagaa gagaagagga agaagcagga 10260
gttctgtggt agaaagcaaa actaacatga aacaaggcta gaagtcaggt cggattaagc 10320
catagtacgg aaaaaactat gctacctgtg agccccgtcc aaggacgtta aaagaagtca 10380
ggccatcata aatgccatag cttgagtaaa ctatgcagcc tgtagctcca cctgagaagg 10440
tgtaaaaaat ccgggaggcc acaaaccatg gaagctgtac gcatggcgta gtggactagc 10500
ggttagagga gacccctccc ttacaaatcg cagcaacaat gggggcccaa ggcgagatga 10560
agctgtagtc tcgctggaag gactagaggt tagaggagac ccccccgaaa caaaaaacag 10620
catattgacg ctgggaaaga ccagagatcc tgctgtctcc tcagcatcat tccaggcaca 10680
gaacgccaga aaatggaatg gtgctgttga atcaacaggt tct 10723
<210> 2
<211> 3391
<212> PRT
<213> 人工序列
<220>
<223> 登革热病毒血清型1, BVS
<400> 2
Met Asn Asn Gln Arg Lys Lys Ala Lys Asn Thr Pro Phe Asn Met Leu
1 5 10 15
Lys Arg Glu Arg Asn Arg Val Ser Thr Val Gln Gln Leu Thr Lys Arg
20 25 30
Phe Ser Leu Gly Met Leu Gln Gly Arg Gly Pro Leu Lys Leu Phe Met
35 40 45
Ala Leu Val Ala Phe Leu Arg Phe Leu Thr Ile Pro Pro Thr Ala Gly
50 55 60
Ile Leu Lys Arg Trp Gly Thr Ile Lys Lys Ser Lys Ala Ile Asn Val
65 70 75 80
Leu Arg Gly Phe Arg Lys Glu Ile Gly Arg Met Leu Asn Ile Leu Asn
85 90 95
Arg Arg Arg Arg Ser Ala Gly Met Ile Ile Met Leu Ile Pro Thr Val
100 105 110
Met Ala Phe His Leu Thr Thr Arg Gly Gly Glu Pro His Met Ile Val
115 120 125
Ser Lys Gln Glu Arg Gly Lys Ser Leu Leu Phe Lys Thr Ser Ala Gly
130 135 140
Val Asn Met Cys Thr Leu Ile Ala Met Asp Leu Gly Glu Leu Cys Glu
145 150 155 160
Asp Thr Met Thr Tyr Lys Cys Pro Arg Ile Thr Glu Ala Glu Pro Asp
165 170 175
Asp Val Asp Cys Trp Cys Asn Ala Thr Asp Thr Trp Val Thr Tyr Gly
180 185 190
Thr Cys Ser Gln Thr Gly Glu His Arg Arg Asp Lys Arg Ser Val Ala
195 200 205
Leu Ala Pro His Val Gly Leu Gly Leu Glu Thr Arg Ala Glu Thr Trp
210 215 220
Met Ser Ser Glu Gly Ala Trp Lys Gln Ile Gln Lys Val Glu Thr Trp
225 230 235 240
Ala Leu Arg His Pro Gly Phe Thr Val Ile Ala Leu Phe Leu Ala His
245 250 255
Ala Ile Gly Thr Ser Ile Thr Gln Lys Gly Ile Ile Phe Ile Leu Leu
260 265 270
Met Leu Val Thr Pro Ser Met Ala Met Arg Cys Val Gly Ile Gly Asn
275 280 285
Arg Asp Phe Val Glu Gly Leu Ser Gly Ala Thr Trp Val Asp Val Val
290 295 300
Leu Glu His Gly Ser Cys Val Thr Thr Met Ala Lys Asn Lys Pro Thr
305 310 315 320
Leu Asp Ile Glu Leu Leu Lys Thr Glu Val Thr Asn Pro Ala Val Leu
325 330 335
Arg Lys Leu Cys Ile Glu Ala Lys Ile Ser Asn Thr Thr Thr Asp Ser
340 345 350
Arg Cys Pro Thr Gln Gly Glu Ala Thr Leu Val Glu Glu Gln Asp Ala
355 360 365
Asn Phe Val Cys Arg Arg Thr Phe Val Asp Arg Gly Trp Gly Asn Gly
370 375 380
Cys Gly Leu Phe Gly Lys Gly Ser Leu Ile Thr Cys Ala Lys Phe Lys
385 390 395 400
Cys Val Thr Lys Leu Glu Gly Lys Ile Val Gln Tyr Glu Asn Leu Lys
405 410 415
Tyr Ser Val Ile Val Thr Val His Thr Gly Asp Gln His Gln Val Gly
420 425 430
Asn Glu Thr Thr Glu His Gly Thr Thr Ala Thr Ile Thr Pro Gln Ala
435 440 445
Pro Thr Ser Glu Ile Gln Leu Thr Asp Tyr Gly Thr Leu Thr Leu Asp
450 455 460
Cys Ser Pro Arg Thr Gly Leu Asp Phe Asn Glu Met Val Leu Leu Thr
465 470 475 480
Met Lys Glu Arg Ser Trp Leu Val His Lys Gln Trp Phe Leu Asp Leu
485 490 495
Pro Leu Pro Trp Thr Ser Gly Ala Ser Thr Ser Gln Glu Thr Trp Asn
500 505 510
Arg Gln Asp Leu Leu Val Thr Phe Lys Thr Ala His Ala Lys Lys Gln
515 520 525
Glu Val Val Val Leu Gly Ser Gln Glu Gly Ala Met His Thr Ala Leu
530 535 540
Thr Gly Ala Thr Glu Ile Gln Thr Ser Gly Thr Thr Thr Ile Phe Ala
545 550 555 560
Gly His Leu Lys Cys Arg Leu Lys Met Asp Lys Leu Thr Leu Lys Gly
565 570 575
Met Ser Tyr Val Met Cys Thr Gly Ser Phe Lys Leu Glu Lys Glu Val
580 585 590
Ala Glu Thr Gln His Gly Thr Val Leu Val Gln Val Lys Tyr Glu Gly
595 600 605
Thr Asp Ala Pro Cys Lys Ile Pro Phe Ser Thr Gln Asp Glu Lys Gly
610 615 620
Ala Thr Gln Asn Gly Arg Leu Ile Thr Ala Asn Pro Ile Val Thr Asp
625 630 635 640
Lys Glu Lys Pro Val Asn Ile Glu Ala Glu Pro Pro Phe Gly Glu Ser
645 650 655
Tyr Ile Val Val Gly Ala Gly Glu Lys Ala Leu Lys Leu Ser Trp Phe
660 665 670
Lys Lys Gly Ser Ser Ile Gly Lys Met Phe Glu Ala Thr Ala Arg Gly
675 680 685
Ala Arg Arg Met Ala Ile Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser
690 695 700
Ile Gly Gly Val Phe Thr Ser Met Gly Lys Leu Val His Gln Val Phe
705 710 715 720
Gly Thr Ala Tyr Gly Val Leu Phe Ser Gly Val Ser Trp Thr Met Lys
725 730 735
Ile Gly Ile Gly Ile Leu Leu Thr Trp Leu Gly Leu Asn Ser Arg Asn
740 745 750
Thr Ser Leu Ser Met Met Cys Ile Ala Ala Ala Ile Val Thr Leu Tyr
755 760 765
Leu Gly Val Met Val Gln Ala Asp Ser Gly Cys Val Val Ser Trp Lys
770 775 780
Asn Lys Glu Leu Lys Cys Gly Ser Gly Ile Phe Ile Thr Asp Asn Val
785 790 795 800
His Thr Trp Thr Glu Gln Tyr Lys Phe Gln Pro Glu Ser Pro Ser Lys
805 810 815
Leu Ala Ser Ala Ile Gln Lys Ala His Glu Glu Asp Ile Cys Gly Ile
820 825 830
Arg Ser Val Thr Arg Leu Glu Asn Leu Met Trp Lys Gln Ile Thr Pro
835 840 845
Glu Leu Asn His Ile Leu Ser Glu Asn Glu Val Lys Leu Thr Ile Met
850 855 860
Thr Gly Asp Ile Lys Gly Ile Met Gln Ala Gly Lys Arg Ser Leu Arg
865 870 875 880
Pro Gln Pro Thr Glu Leu Lys Tyr Ser Trp Lys Thr Trp Gly Lys Ala
885 890 895
Lys Met Leu Ser Thr Glu Ser His Asn Gln Thr Phe Leu Ile Asp Gly
900 905 910
Pro Glu Thr Ala Glu Cys Pro Asn Thr Asn Arg Ala Trp Asn Ser Leu
915 920 925
Glu Val Glu Asp Tyr Gly Phe Gly Val Phe Thr Thr Asn Ile Trp Leu
930 935 940
Lys Leu Lys Glu Lys Gln Asp Val Phe Cys Asp Ser Lys Leu Met Ser
945 950 955 960
Ala Ala Ile Lys Asp Asn Arg Ala Val His Ala Asp Met Gly Tyr Trp
965 970 975
Ile Glu Ser Ala Leu Asn Asp Thr Trp Lys Ile Glu Lys Ala Ser Phe
980 985 990
Ile Glu Val Lys Asn Cys His Trp Pro Lys Ser His Thr Leu Trp Ser
995 1000 1005
Asn Gly Val Leu Glu Ser Glu Met Ile Ile Pro Lys Asn Leu Ala
1010 1015 1020
Gly Pro Val Ser Gln His Asn Tyr Arg Pro Gly Tyr His Thr Gln
1025 1030 1035
Ile Thr Gly Pro Trp His Leu Gly Lys Leu Glu Met Asp Phe Asp
1040 1045 1050
Phe Cys Asp Gly Thr Thr Val Val Val Thr Glu Asp Cys Gly Asn
1055 1060 1065
Arg Gly Pro Ser Leu Arg Thr Thr Thr Ala Ser Gly Lys Leu Ile
1070 1075 1080
Thr Glu Trp Cys Cys Arg Ser Cys Thr Leu Pro Pro Leu Arg Tyr
1085 1090 1095
Arg Gly Glu Asp Gly Cys Trp Tyr Gly Met Glu Ile Arg Pro Leu
1100 1105 1110
Lys Glu Lys Glu Glu Asn Leu Val Asn Ser Leu Val Thr Ala Gly
1115 1120 1125
His Gly Gln Val Asp Asn Phe Ser Leu Gly Val Leu Gly Met Ala
1130 1135 1140
Leu Phe Leu Glu Glu Met Leu Arg Thr Arg Val Gly Thr Lys His
1145 1150 1155
Ala Ile Leu Leu Val Ala Val Ser Phe Val Thr Leu Ile Thr Gly
1160 1165 1170
Asn Met Ser Phe Arg Asp Leu Gly Arg Val Met Val Met Val Gly
1175 1180 1185
Ala Thr Met Thr Asp Asp Ile Gly Met Gly Val Thr Tyr Leu Ala
1190 1195 1200
Leu Leu Ala Ala Phe Lys Val Arg Pro Thr Phe Ala Ala Gly Leu
1205 1210 1215
Leu Leu Arg Lys Leu Thr Ser Lys Glu Leu Met Met Thr Thr Ile
1220 1225 1230
Gly Ile Val Leu Leu Ser Gln Ser Thr Leu Pro Glu Thr Ile Leu
1235 1240 1245
Glu Leu Thr Asp Ala Leu Ala Leu Gly Met Met Val Leu Lys Met
1250 1255 1260
Val Arg Asn Met Glu Lys Tyr Gln Leu Ala Val Thr Ile Met Ala
1265 1270 1275
Ile Leu Cys Val Pro Asn Ala Val Ile Leu Gln Asn Ala Trp Lys
1280 1285 1290
Val Ser Cys Thr Ile Leu Ala Val Val Ser Val Ser Pro Leu Phe
1295 1300 1305
Leu Thr Ser Ser Gln Gln Lys Thr Asp Trp Ile Pro Leu Ala Leu
1310 1315 1320
Thr Ile Lys Gly Leu Asn Pro Thr Ala Ile Phe Leu Thr Thr Leu
1325 1330 1335
Ser Arg Thr Ser Lys Lys Arg Ser Trp Pro Leu Asn Glu Ala Ile
1340 1345 1350
Met Ala Val Gly Met Val Ser Ile Leu Ala Ser Ser Leu Leu Lys
1355 1360 1365
Asn Asp Ile Pro Met Thr Gly Pro Leu Val Ala Gly Gly Leu Leu
1370 1375 1380
Thr Val Cys Tyr Val Leu Thr Gly Arg Ser Ala Asp Leu Glu Leu
1385 1390 1395
Glu Arg Ala Ala Asp Val Lys Trp Glu Asp Gln Ala Glu Ile Ser
1400 1405 1410
Gly Ser Ser Pro Ile Leu Ser Ile Thr Ile Ser Glu Asp Gly Ser
1415 1420 1425
Met Ser Ile Lys Asn Glu Glu Glu Asp Gln Thr Leu Thr Ile Leu
1430 1435 1440
Ile Arg Thr Gly Leu Leu Val Ile Ser Gly Leu Phe Pro Val Ser
1445 1450 1455
Ile Pro Ile Thr Ala Ala Ala Trp Tyr Leu Trp Glu Val Lys Lys
1460 1465 1470
Gln Arg Ala Gly Val Leu Trp Asp Val Pro Ser Pro Pro Pro Met
1475 1480 1485
Gly Lys Ala Glu Leu Glu Asp Gly Ala Tyr Arg Ile Lys Gln Lys
1490 1495 1500
Gly Ile Leu Gly Tyr Ser Gln Ile Gly Ala Gly Val Tyr Lys Glu
1505 1510 1515
Gly Thr Phe His Thr Met Trp His Val Thr Arg Gly Ala Val Leu
1520 1525 1530
Met His Lys Gly Lys Arg Ile Glu Pro Ser Trp Ala Asp Val Lys
1535 1540 1545
Lys Asp Leu Ile Ser Tyr Gly Gly Gly Trp Lys Leu Glu Gly Glu
1550 1555 1560
Trp Lys Glu Gly Glu Glu Val Gln Val Leu Ala Leu Glu Pro Gly
1565 1570 1575
Lys Asn Pro Arg Ala Val Gln Thr Lys Pro Gly Leu Phe Lys Thr
1580 1585 1590
Asn Ala Gly Thr Ile Gly Ala Val Ser Leu Asp Phe Ser Pro Gly
1595 1600 1605
Thr Ser Gly Ser Pro Ile Ile Asp Lys Lys Gly Lys Val Val Gly
1610 1615 1620
Leu Tyr Gly Asn Gly Val Val Thr Arg Ser Gly Ala Tyr Val Ser
1625 1630 1635
Ala Ile Ala Gln Thr Glu Lys Ser Ile Glu Asp Asn Pro Glu Ile
1640 1645 1650
Glu Asp Asp Ile Phe Arg Lys Arg Arg Leu Thr Ile Met Asp Leu
1655 1660 1665
His Pro Gly Ala Gly Lys Thr Lys Arg Tyr Leu Pro Ala Ile Val
1670 1675 1680
Arg Glu Ala Ile Lys Arg Gly Leu Arg Thr Leu Ile Leu Ala Pro
1685 1690 1695
Thr Arg Val Val Ala Ala Glu Met Glu Glu Ala Leu Arg Gly Leu
1700 1705 1710
Pro Ile Arg Tyr Gln Thr Pro Ala Ile Arg Ala Val His Thr Gly
1715 1720 1725
Arg Glu Ile Val Asp Leu Met Cys His Ala Thr Phe Thr Met Arg
1730 1735 1740
Leu Leu Ser Pro Val Arg Val Pro Asn Tyr Asn Leu Ile Ile Met
1745 1750 1755
Asp Glu Ala His Phe Thr Asp Pro Ala Ser Ile Ala Ala Arg Gly
1760 1765 1770
Tyr Ile Ser Thr Arg Val Glu Met Gly Glu Ala Ala Gly Ile Phe
1775 1780 1785
Met Thr Ala Thr Pro Pro Gly Ser Arg Asp Pro Phe Pro Gln Ser
1790 1795 1800
Asn Ala Pro Ile Ile Asp Glu Glu Arg Glu Ile Pro Glu Arg Ser
1805 1810 1815
Trp Asn Ser Gly His Glu Trp Val Thr Asp Phe Lys Gly Lys Thr
1820 1825 1830
Val Trp Phe Val Pro Ser Ile Lys Ala Gly Asn Asp Ile Ala Ala
1835 1840 1845
Cys Leu Arg Lys Asn Gly Lys Lys Val Ile Gln Leu Ser Arg Lys
1850 1855 1860
Thr Phe Asp Ser Glu Tyr Val Lys Thr Arg Thr Asn Asp Trp Asp
1865 1870 1875
Phe Val Val Thr Thr Asp Ile Ser Glu Met Gly Ala Asn Phe Lys
1880 1885 1890
Ala Glu Arg Val Ile Asp Pro Arg Arg Cys Met Lys Pro Val Ile
1895 1900 1905
Leu Thr Asp Gly Glu Glu Arg Val Ile Leu Ala Gly Pro Met Pro
1910 1915 1920
Val Thr His Ser Ser Ala Ala Gln Arg Arg Gly Arg Ile Gly Arg
1925 1930 1935
Asn Pro Lys Asn Glu Asn Asp Gln Tyr Ile Tyr Met Gly Glu Pro
1940 1945 1950
Leu Glu Asn Asp Glu Asp Cys Ala His Trp Lys Glu Ala Lys Met
1955 1960 1965
Leu Leu Asp Asn Ile Asn Thr Pro Glu Gly Ile Ile Pro Ser Met
1970 1975 1980
Phe Glu Pro Glu Arg Glu Lys Val Asp Ala Ile Asp Gly Glu Tyr
1985 1990 1995
Arg Leu Arg Gly Glu Ala Arg Lys Thr Phe Val Asp Leu Met Arg
2000 2005 2010
Arg Gly Asp Leu Pro Val Trp Leu Ala Tyr Arg Val Ala Ala Glu
2015 2020 2025
Gly Ile Asn Tyr Ala Asp Arg Arg Trp Cys Phe Asp Gly Val Lys
2030 2035 2040
Asn Asn Gln Ile Leu Glu Glu Asn Val Glu Val Glu Ile Trp Thr
2045 2050 2055
Lys Glu Gly Glu Arg Lys Lys Leu Lys Pro Arg Trp Leu Asp Ala
2060 2065 2070
Arg Ile Tyr Ser Asp Pro Leu Ala Leu Lys Glu Phe Lys Glu Phe
2075 2080 2085
Ala Ala Gly Arg Lys Ser Leu Thr Leu Asn Leu Ile Thr Glu Met
2090 2095 2100
Gly Arg Leu Pro Thr Phe Met Thr Gln Lys Ala Arg Asp Ala Leu
2105 2110 2115
Asp Asn Leu Ala Val Leu His Thr Ala Glu Ala Gly Gly Arg Ala
2120 2125 2130
Tyr Asn His Ala Leu Ser Glu Leu Pro Glu Thr Leu Glu Thr Leu
2135 2140 2145
Leu Leu Leu Thr Leu Leu Ala Thr Val Thr Gly Gly Ile Phe Leu
2150 2155 2160
Phe Leu Met Ser Ala Arg Gly Ile Gly Lys Met Thr Leu Gly Met
2165 2170 2175
Cys Cys Ile Ile Thr Ala Ser Ile Leu Leu Trp Tyr Ala Gln Ile
2180 2185 2190
Gln Pro His Trp Ile Ala Ala Ser Ile Ile Leu Glu Phe Phe Leu
2195 2200 2205
Ile Val Leu Leu Ile Pro Glu Pro Glu Lys Gln Arg Thr Pro Gln
2210 2215 2220
Asp Asn Gln Leu Thr Tyr Val Val Ile Ala Ile Leu Thr Val Val
2225 2230 2235
Ala Ala Thr Met Ala Asn Glu Met Gly Phe Leu Glu Lys Thr Lys
2240 2245 2250
Lys Asp Leu Gly Leu Gly Ser Ile Ala Thr Gln Gln Pro Glu Ser
2255 2260 2265
Asn Ile Leu Asp Ile Asp Leu Arg Pro Ala Ser Ala Trp Thr Leu
2270 2275 2280
Tyr Ala Val Ala Thr Thr Phe Val Thr Pro Met Leu Arg His Ser
2285 2290 2295
Ile Glu Asn Ser Ser Val Asn Val Ser Leu Thr Ala Ile Ala Asn
2300 2305 2310
Gln Ala Thr Val Leu Met Gly Leu Gly Lys Gly Trp Pro Leu Ser
2315 2320 2325
Lys Met Asp Ile Gly Val Pro Leu Leu Ala Ile Gly Cys Tyr Ser
2330 2335 2340
Gln Val Asn Pro Ile Thr Leu Ile Ala Ala Leu Phe Leu Leu Val
2345 2350 2355
Ala His Tyr Ala Ile Ile Gly Pro Gly Leu Gln Ala Lys Ala Thr
2360 2365 2370
Arg Glu Ala Gln Lys Arg Ala Ala Ala Gly Ile Met Lys Asn Pro
2375 2380 2385
Thr Val Asp Gly Ile Thr Val Ile Asp Leu Asp Pro Ile Pro Tyr
2390 2395 2400
Asp Pro Lys Phe Glu Lys Gln Leu Gly Gln Val Met Leu Leu Val
2405 2410 2415
Leu Cys Val Thr Gln Val Leu Met Met Arg Thr Thr Trp Ala Leu
2420 2425 2430
Cys Glu Ala Leu Thr Leu Ala Thr Gly Pro Ile Ser Thr Leu Trp
2435 2440 2445
Glu Gly Asn Pro Gly Arg Phe Trp Asn Thr Thr Ile Ala Val Ser
2450 2455 2460
Met Ala Asn Ile Phe Arg Gly Ser Tyr Leu Ala Gly Ala Gly Leu
2465 2470 2475
Leu Phe Ser Ile Met Lys Asn Thr Thr Asn Thr Arg Arg Gly Thr
2480 2485 2490
Gly Asn Ile Gly Glu Thr Leu Gly Glu Lys Trp Lys Ser Arg Leu
2495 2500 2505
Asn Ala Leu Gly Lys Ser Glu Phe Gln Ile Tyr Lys Lys Ser Gly
2510 2515 2520
Ile Gln Glu Val Asp Arg Thr Leu Ala Lys Glu Gly Ile Lys Arg
2525 2530 2535
Gly Glu Thr Asp His His Ala Val Ser Arg Gly Ser Ala Lys Leu
2540 2545 2550
Arg Trp Phe Val Glu Arg Asn Met Val Thr Pro Glu Gly Lys Val
2555 2560 2565
Val Asp Leu Gly Cys Gly Arg Gly Gly Trp Ser Tyr Tyr Cys Gly
2570 2575 2580
Gly Leu Lys Asn Val Arg Glu Val Lys Gly Leu Thr Lys Gly Gly
2585 2590 2595
Pro Gly His Glu Glu Pro Ile Pro Met Ser Thr Tyr Gly Trp Asn
2600 2605 2610
Leu Val Arg Leu Gln Ser Gly Val Asp Val Phe Phe Ile Pro Pro
2615 2620 2625
Glu Lys Cys Asp Thr Leu Leu Cys Asp Ile Gly Glu Ser Ser Pro
2630 2635 2640
Asn Pro Thr Val Glu Ala Gly Arg Thr Leu Arg Val Leu Asn Leu
2645 2650 2655
Val Glu Asn Trp Leu Asn Asn Asn Thr Gln Phe Cys Ile Lys Val
2660 2665 2670
Leu Asn Pro Tyr Met Pro Ser Val Ile Glu Lys Met Glu Ala Leu
2675 2680 2685
Gln Arg Lys Tyr Gly Gly Ala Leu Val Arg Asn Pro Leu Ser Arg
2690 2695 2700
Asn Ser Thr His Glu Met Tyr Trp Val Ser Asn Ala Ser Gly Asn
2705 2710 2715
Ile Val Ser Ser Val Asn Met Ile Ser Arg Met Leu Ile Asn Arg
2720 2725 2730
Phe Thr Met Arg Tyr Lys Lys Ala Thr Tyr Glu Pro Asp Val Asp
2735 2740 2745
Leu Gly Ser Gly Thr Arg Asn Ile Gly Ile Glu Ser Glu Ile Pro
2750 2755 2760
Asn Leu Asp Ile Ile Gly Lys Arg Ile Glu Lys Ile Lys Gln Glu
2765 2770 2775
His Glu Thr Ser Trp His Tyr Asp Gln Asp His Pro Tyr Lys Thr
2780 2785 2790
Trp Ala Tyr His Gly Ser Tyr Glu Thr Lys Gln Thr Gly Ser Ala
2795 2800 2805
Ser Ser Met Val Asn Gly Val Val Arg Leu Leu Thr Lys Pro Trp
2810 2815 2820
Asp Val Val Pro Met Val Thr Gln Met Ala Met Thr Asp Thr Thr
2825 2830 2835
Pro Phe Gly Gln Gln Arg Val Phe Lys Glu Lys Val Asp Thr Arg
2840 2845 2850
Thr Gln Glu Pro Lys Glu Gly Thr Lys Lys Leu Met Lys Ile Thr
2855 2860 2865
Ala Glu Trp Leu Trp Lys Glu Leu Gly Lys Lys Lys Thr Pro Arg
2870 2875 2880
Met Cys Thr Arg Glu Glu Phe Thr Arg Lys Val Arg Ser Asn Ala
2885 2890 2895
Ala Leu Gly Ala Ile Phe Thr Asp Glu Asn Lys Trp Lys Ser Ala
2900 2905 2910
Arg Glu Ala Val Glu Asp Ser Arg Phe Trp Glu Leu Val Asp Lys
2915 2920 2925
Glu Arg Asn Leu His Leu Glu Gly Lys Cys Glu Thr Cys Val Tyr
2930 2935 2940
Asn Met Met Gly Lys Arg Glu Lys Lys Leu Gly Glu Phe Gly Lys
2945 2950 2955
Ala Lys Gly Ser Arg Ala Ile Trp Tyr Met Trp Leu Gly Ala Arg
2960 2965 2970
Phe Leu Glu Phe Glu Ala Leu Gly Phe Leu Asn Glu Asp His Trp
2975 2980 2985
Phe Ser Arg Glu Asn Ser Leu Ser Gly Val Glu Gly Glu Gly Leu
2990 2995 3000
His Lys Leu Gly Tyr Ile Leu Arg Asp Val Ser Lys Lys Glu Gly
3005 3010 3015
Gly Ala Met Tyr Ala Asp Asp Thr Ala Gly Trp Asp Thr Arg Ile
3020 3025 3030
Thr Leu Glu Asp Leu Lys Asn Glu Glu Met Val Thr Asn His Met
3035 3040 3045
Glu Gly Glu His Lys Lys Leu Ala Glu Ala Ile Phe Lys Leu Thr
3050 3055 3060
Tyr Gln Asn Lys Val Val Arg Val Gln Arg Pro Thr Pro Arg Gly
3065 3070 3075
Thr Val Met Asp Ile Ile Ser Arg Arg Asp Gln Arg Gly Ser Gly
3080 3085 3090
Gln Val Gly Thr Tyr Gly Leu Asn Thr Phe Thr Asn Met Glu Ala
3095 3100 3105
Gln Leu Ile Arg Gln Met Glu Gly Glu Gly Val Phe Lys Ser Ile
3110 3115 3120
Gln His Leu Thr Ile Thr Glu Glu Ile Ala Val Gln Asn Trp Leu
3125 3130 3135
Ala Arg Val Gly Arg Glu Arg Leu Ser Arg Met Ala Ile Ser Gly
3140 3145 3150
Asp Asp Cys Val Val Lys Pro Leu Asp Asp Arg Phe Ala Ser Ala
3155 3160 3165
Leu Thr Ala Leu Asn Asp Met Gly Lys Ile Arg Lys Asp Ile Gln
3170 3175 3180
Gln Trp Glu Pro Ser Arg Gly Trp Asn Asp Trp Thr Gln Val Pro
3185 3190 3195
Phe Cys Ser His His Phe His Glu Leu Ile Met Lys Asp Gly Arg
3200 3205 3210
Val Leu Val Val Pro Cys Arg Asn Gln Asp Glu Leu Ile Gly Arg
3215 3220 3225
Ala Arg Ile Ser Gln Gly Ala Gly Trp Ser Leu Arg Glu Thr Ala
3230 3235 3240
Cys Leu Gly Lys Ser Tyr Ala Gln Met Trp Ser Leu Met Tyr Phe
3245 3250 3255
His Arg Arg Asp Leu Arg Leu Ala Ala Asn Ala Ile Cys Ser Ala
3260 3265 3270
Val Pro Ser His Trp Val Pro Thr Ser Arg Thr Thr Trp Ser Ile
3275 3280 3285
His Ala Lys His Glu Trp Met Thr Thr Glu Asp Met Leu Thr Val
3290 3295 3300
Trp Asn Arg Val Trp Ile Gln Glu Asn Pro Trp Met Glu Asp Lys
3305 3310 3315
Thr Pro Val Glu Ser Trp Glu Glu Ile Pro Tyr Leu Gly Lys Arg
3320 3325 3330
Glu Asp Gln Trp Cys Gly Ser Leu Ile Gly Leu Thr Ser Arg Ala
3335 3340 3345
Thr Trp Ala Lys Asn Ile Gln Ala Ala Ile Asn Gln Val Arg Ser
3350 3355 3360
Leu Ile Gly Asn Glu Glu Tyr Thr Asp Tyr Met Pro Ser Met Lys
3365 3370 3375
Arg Phe Arg Arg Glu Glu Glu Glu Ala Gly Val Leu Trp
3380 3385 3390
<210> 3
<211> 3391
<212> PRT
<213> 人工序列
<220>
<223> 登革热病毒血清型1, MVS
<400> 3
Met Asn Asn Gln Arg Lys Lys Ala Lys Asn Thr Pro Phe Asn Met Leu
1 5 10 15
Lys Arg Glu Arg Asn Arg Val Ser Thr Val Gln Gln Leu Thr Lys Arg
20 25 30
Phe Ser Leu Gly Met Leu Gln Gly Arg Gly Pro Leu Lys Leu Phe Met
35 40 45
Ala Leu Val Ala Phe Leu Arg Phe Leu Thr Ile Pro Pro Thr Ala Gly
50 55 60
Ile Leu Lys Arg Trp Gly Thr Ile Lys Lys Ser Lys Ala Ile Asn Val
65 70 75 80
Leu Arg Gly Phe Arg Lys Glu Ile Gly Arg Met Leu Asn Ile Leu Asn
85 90 95
Arg Arg Arg Arg Ser Ala Gly Met Ile Ile Met Leu Ile Pro Thr Val
100 105 110
Met Ala Phe His Leu Thr Thr Arg Gly Gly Glu Pro His Met Ile Val
115 120 125
Ser Lys Gln Glu Arg Gly Lys Ser Leu Leu Phe Lys Thr Ser Ala Gly
130 135 140
Val Asn Met Cys Thr Leu Ile Ala Met Asp Leu Gly Glu Leu Cys Glu
145 150 155 160
Asp Thr Met Thr Tyr Lys Cys Pro Arg Ile Thr Glu Ala Glu Pro Asp
165 170 175
Asp Val Asp Cys Trp Cys Asn Ala Thr Asp Thr Trp Val Thr Tyr Gly
180 185 190
Thr Cys Ser Gln Thr Gly Glu His Arg Arg Asp Lys Arg Ser Val Ala
195 200 205
Leu Ala Pro His Val Gly Leu Gly Leu Glu Thr Arg Ala Glu Thr Trp
210 215 220
Met Ser Ser Glu Gly Ala Trp Lys Gln Ile Gln Lys Val Glu Thr Trp
225 230 235 240
Ala Leu Arg His Pro Gly Phe Thr Val Ile Ala Leu Phe Leu Ala His
245 250 255
Ala Ile Gly Thr Ser Ile Thr Gln Lys Gly Ile Ile Phe Ile Leu Leu
260 265 270
Met Leu Val Thr Pro Ser Met Ala Met Arg Cys Val Gly Ile Gly Asn
275 280 285
Arg Asp Phe Val Glu Gly Leu Ser Gly Ala Thr Trp Val Asp Val Val
290 295 300
Leu Glu His Gly Ser Cys Val Thr Thr Met Ala Lys Asn Lys Pro Thr
305 310 315 320
Leu Asp Ile Glu Leu Leu Lys Thr Glu Val Thr Asn Pro Ala Val Leu
325 330 335
Arg Lys Leu Cys Ile Glu Ala Lys Ile Ser Asn Thr Thr Thr Asp Ser
340 345 350
Arg Cys Pro Thr Gln Gly Glu Ala Thr Leu Val Glu Glu Gln Asp Ala
355 360 365
Asn Phe Val Cys Arg Arg Thr Phe Val Asp Arg Gly Trp Gly Asn Gly
370 375 380
Cys Gly Leu Phe Gly Lys Gly Ser Leu Ile Thr Cys Ala Lys Phe Lys
385 390 395 400
Cys Val Thr Lys Leu Glu Gly Lys Ile Val Gln Tyr Glu Asn Leu Lys
405 410 415
Tyr Ser Val Ile Val Thr Val His Thr Gly Asp Gln His Gln Val Gly
420 425 430
Asn Glu Thr Thr Glu His Gly Thr Thr Ala Thr Ile Thr Pro Gln Ala
435 440 445
Pro Thr Ser Glu Ile Gln Leu Thr Asp Tyr Gly Thr Leu Thr Leu Asp
450 455 460
Cys Ser Pro Arg Thr Gly Leu Asp Phe Asn Glu Met Val Leu Leu Thr
465 470 475 480
Met Lys Glu Arg Ser Trp Leu Val His Lys Gln Trp Phe Leu Asp Leu
485 490 495
Pro Leu Pro Trp Thr Ser Gly Ala Ser Thr Ser Gln Glu Thr Trp Asn
500 505 510
Arg Gln Asp Leu Leu Val Thr Phe Lys Thr Ala His Ala Lys Lys Gln
515 520 525
Glu Val Val Val Leu Gly Ser Gln Glu Gly Ala Met His Thr Ala Leu
530 535 540
Thr Gly Ala Thr Glu Ile Gln Thr Ser Gly Thr Thr Thr Ile Phe Ala
545 550 555 560
Gly His Leu Lys Cys Arg Leu Lys Met Asp Lys Leu Thr Leu Lys Gly
565 570 575
Met Ser Tyr Val Met Cys Thr Gly Ser Phe Lys Leu Glu Lys Glu Val
580 585 590
Ala Glu Thr Gln His Gly Thr Val Leu Val Gln Val Lys Tyr Glu Gly
595 600 605
Thr Asp Ala Pro Cys Lys Ile Pro Phe Ser Thr Gln Asp Glu Lys Gly
610 615 620
Ala Thr Gln Asn Gly Arg Leu Ile Thr Ala Asn Pro Ile Val Thr Asp
625 630 635 640
Lys Glu Lys Pro Val Asn Ile Glu Ala Glu Pro Pro Phe Gly Glu Ser
645 650 655
Tyr Ile Val Val Gly Ala Gly Glu Lys Ala Leu Lys Leu Ser Trp Phe
660 665 670
Lys Lys Gly Ser Ser Ile Gly Lys Met Phe Glu Ala Thr Ala Arg Gly
675 680 685
Ala Arg Arg Met Ala Ile Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser
690 695 700
Ile Gly Gly Val Phe Thr Ser Met Gly Lys Leu Val His Gln Val Phe
705 710 715 720
Gly Thr Ala Tyr Gly Val Leu Phe Ser Gly Val Ser Trp Thr Met Lys
725 730 735
Ile Gly Ile Gly Ile Leu Leu Thr Trp Leu Gly Leu Asn Ser Arg Asn
740 745 750
Thr Ser Leu Ser Met Met Cys Ile Ala Ala Gly Ile Val Thr Leu Tyr
755 760 765
Leu Gly Val Met Val Gln Ala Asp Ser Gly Cys Val Val Ser Trp Lys
770 775 780
Asn Lys Glu Leu Lys Cys Gly Ser Gly Ile Phe Ile Thr Asp Asn Val
785 790 795 800
His Thr Trp Thr Glu Gln Tyr Lys Phe Gln Pro Glu Ser Pro Ser Lys
805 810 815
Leu Ala Ser Ala Ile Gln Lys Ala His Glu Glu Asp Ile Cys Gly Ile
820 825 830
Arg Ser Val Thr Arg Leu Glu Asn Leu Met Trp Lys Gln Ile Thr Pro
835 840 845
Glu Leu Asn His Ile Leu Ser Glu Asn Glu Val Lys Leu Thr Ile Met
850 855 860
Thr Gly Asp Ile Lys Gly Ile Met Gln Ala Gly Lys Arg Ser Leu Arg
865 870 875 880
Pro Gln Pro Thr Glu Leu Lys Tyr Ser Trp Lys Thr Trp Gly Lys Ala
885 890 895
Lys Met Leu Ser Thr Glu Ser His Asn Gln Thr Phe Leu Ile Asp Gly
900 905 910
Pro Glu Thr Ala Glu Cys Pro Asn Thr Asn Arg Ala Trp Asn Ser Leu
915 920 925
Glu Val Glu Asp Tyr Gly Phe Gly Val Phe Thr Thr Asn Ile Trp Leu
930 935 940
Lys Leu Lys Glu Lys Gln Asp Val Phe Cys Asp Ser Lys Leu Met Ser
945 950 955 960
Ala Ala Ile Lys Asp Asn Arg Ala Val His Ala Asp Met Gly Tyr Trp
965 970 975
Ile Glu Ser Ala Leu Asn Asp Thr Trp Lys Ile Glu Lys Ala Ser Phe
980 985 990
Ile Glu Val Lys Asn Cys His Trp Pro Lys Ser His Thr Leu Trp Ser
995 1000 1005
Asn Gly Val Leu Glu Ser Glu Met Ile Ile Pro Lys Asn Leu Ala
1010 1015 1020
Gly Pro Val Ser Gln His Asn Tyr Arg Pro Gly Tyr His Thr Gln
1025 1030 1035
Ile Thr Gly Pro Trp His Leu Gly Lys Leu Glu Met Asp Phe Asp
1040 1045 1050
Phe Cys Asp Gly Thr Thr Val Val Val Thr Glu Asp Cys Gly Asn
1055 1060 1065
Arg Gly Pro Ser Leu Arg Thr Thr Thr Ala Ser Gly Lys Leu Ile
1070 1075 1080
Thr Glu Trp Cys Cys Arg Ser Cys Thr Leu Pro Pro Leu Arg Tyr
1085 1090 1095
Arg Gly Glu Asp Gly Cys Trp Tyr Gly Met Glu Ile Arg Pro Leu
1100 1105 1110
Lys Glu Lys Glu Glu Asn Leu Val Asn Ser Leu Val Thr Ala Gly
1115 1120 1125
His Gly Gln Val Asp Asn Phe Ser Leu Gly Val Leu Gly Met Ala
1130 1135 1140
Leu Phe Leu Glu Glu Met Leu Arg Thr Arg Val Gly Thr Lys His
1145 1150 1155
Ala Ile Leu Leu Val Ala Val Ser Phe Val Thr Leu Ile Thr Gly
1160 1165 1170
Asn Met Ser Phe Arg Asp Leu Gly Arg Val Met Val Met Val Gly
1175 1180 1185
Ala Thr Met Thr Asp Asp Ile Gly Met Gly Val Thr Tyr Leu Ala
1190 1195 1200
Leu Leu Ala Ala Phe Lys Val Arg Pro Thr Phe Ala Ala Gly Leu
1205 1210 1215
Leu Leu Arg Lys Leu Thr Ser Lys Glu Leu Met Met Thr Thr Ile
1220 1225 1230
Gly Ile Val Leu Leu Ser Gln Ser Thr Leu Pro Glu Thr Ile Leu
1235 1240 1245
Glu Leu Thr Asp Ala Leu Ala Leu Gly Met Met Val Leu Lys Met
1250 1255 1260
Val Arg Asn Met Glu Lys Tyr Gln Leu Ala Val Thr Ile Met Ala
1265 1270 1275
Ile Leu Cys Val Pro Asn Ala Val Ile Leu Gln Asn Ala Trp Lys
1280 1285 1290
Val Ser Cys Thr Ile Leu Ala Val Val Ser Val Ser Pro Leu Phe
1295 1300 1305
Leu Thr Ser Ser Gln Gln Lys Thr Asp Trp Ile Pro Leu Ala Leu
1310 1315 1320
Thr Ile Lys Gly Leu Asn Pro Thr Ala Ile Phe Leu Thr Thr Leu
1325 1330 1335
Ser Arg Thr Ser Lys Lys Arg Ser Trp Pro Leu Asn Glu Ala Ile
1340 1345 1350
Met Ala Val Gly Met Val Ser Ile Leu Ala Ser Ser Leu Leu Lys
1355 1360 1365
Asn Asp Ile Pro Met Thr Gly Pro Leu Val Ala Gly Gly Leu Leu
1370 1375 1380
Thr Val Cys Tyr Val Leu Thr Gly Arg Ser Ala Asp Leu Glu Leu
1385 1390 1395
Glu Arg Ala Ala Asp Val Lys Trp Glu Asp Gln Ala Glu Ile Ser
1400 1405 1410
Gly Ser Ser Pro Ile Leu Ser Ile Thr Ile Ser Glu Asp Gly Ser
1415 1420 1425
Met Ser Ile Lys Asn Glu Glu Glu Asp Gln Thr Leu Thr Ile Leu
1430 1435 1440
Ile Arg Thr Gly Leu Leu Val Ile Ser Gly Leu Phe Pro Val Ser
1445 1450 1455
Ile Pro Ile Thr Ala Ala Ala Trp Tyr Leu Trp Glu Val Lys Lys
1460 1465 1470
Gln Arg Ala Gly Val Leu Trp Asp Val Pro Ser Pro Pro Pro Met
1475 1480 1485
Gly Lys Ala Glu Leu Glu Asp Gly Ala Tyr Arg Ile Lys Gln Lys
1490 1495 1500
Gly Ile Leu Gly Tyr Ser Gln Ile Gly Ala Gly Val Tyr Lys Glu
1505 1510 1515
Gly Thr Phe His Thr Met Trp His Val Thr Arg Gly Ala Val Leu
1520 1525 1530
Met His Lys Gly Lys Arg Ile Glu Pro Ser Trp Ala Asp Val Lys
1535 1540 1545
Lys Asp Leu Ile Ser Tyr Gly Gly Gly Trp Lys Leu Glu Gly Glu
1550 1555 1560
Trp Lys Glu Gly Glu Glu Val Gln Val Leu Ala Leu Glu Pro Gly
1565 1570 1575
Lys Asn Pro Arg Ala Val Gln Thr Lys Pro Gly Leu Phe Lys Thr
1580 1585 1590
Asn Ala Gly Thr Ile Gly Ala Val Ser Leu Asp Phe Ser Pro Gly
1595 1600 1605
Thr Ser Gly Ser Pro Ile Ile Asp Lys Lys Gly Lys Val Val Gly
1610 1615 1620
Leu Tyr Gly Asn Gly Val Val Thr Arg Ser Gly Ala Tyr Val Ser
1625 1630 1635
Ala Ile Ala Gln Thr Glu Lys Ser Ile Glu Asp Asn Pro Glu Ile
1640 1645 1650
Glu Asp Asp Ile Phe Arg Lys Arg Arg Leu Thr Ile Met Asp Leu
1655 1660 1665
His Pro Gly Ala Gly Lys Thr Lys Arg Tyr Leu Pro Ala Ile Val
1670 1675 1680
Arg Glu Ala Ile Lys Arg Gly Leu Arg Thr Leu Ile Leu Ala Pro
1685 1690 1695
Thr Arg Val Val Ala Ala Glu Met Glu Glu Ala Leu Arg Gly Leu
1700 1705 1710
Pro Ile Arg Tyr Gln Thr Pro Ala Ile Arg Ala Val His Thr Gly
1715 1720 1725
Arg Glu Ile Val Asp Leu Met Cys His Ala Thr Phe Thr Met Arg
1730 1735 1740
Leu Leu Ser Pro Val Arg Val Pro Asn Tyr Asn Leu Ile Ile Met
1745 1750 1755
Asp Glu Ala His Phe Thr Asp Pro Ala Ser Ile Ala Ala Arg Gly
1760 1765 1770
Tyr Ile Ser Thr Arg Val Glu Met Gly Glu Ala Ala Gly Ile Phe
1775 1780 1785
Met Thr Ala Thr Pro Pro Gly Ser Arg Asp Pro Phe Pro Gln Ser
1790 1795 1800
Asn Ala Pro Ile Ile Asp Glu Glu Arg Glu Ile Pro Glu Arg Ser
1805 1810 1815
Trp Asn Ser Gly His Glu Trp Val Thr Asp Phe Lys Gly Lys Thr
1820 1825 1830
Val Trp Phe Val Pro Ser Ile Lys Ala Gly Asn Asp Ile Ala Ala
1835 1840 1845
Cys Leu Arg Lys Asn Gly Lys Lys Val Ile Gln Leu Ser Arg Lys
1850 1855 1860
Thr Phe Asp Ser Glu Tyr Val Lys Thr Arg Thr Asn Asp Trp Asp
1865 1870 1875
Phe Val Val Thr Thr Asp Ile Ser Glu Met Gly Ala Asn Phe Lys
1880 1885 1890
Ala Glu Arg Val Ile Asp Pro Arg Arg Cys Met Lys Pro Val Ile
1895 1900 1905
Leu Thr Asp Gly Glu Glu Arg Val Ile Leu Ala Gly Pro Met Pro
1910 1915 1920
Val Thr His Ser Ser Ala Ala Gln Arg Arg Gly Arg Ile Gly Arg
1925 1930 1935
Asn Pro Lys Asn Glu Asn Asp Gln Tyr Ile Tyr Met Gly Glu Pro
1940 1945 1950
Leu Glu Asn Asp Glu Asp Cys Ala His Trp Lys Glu Ala Lys Met
1955 1960 1965
Leu Leu Asp Asn Ile Asn Thr Pro Glu Gly Ile Ile Pro Ser Met
1970 1975 1980
Phe Glu Pro Glu Arg Glu Lys Val Asp Ala Ile Asp Gly Glu Tyr
1985 1990 1995
Arg Leu Arg Gly Glu Ala Arg Lys Thr Phe Val Asp Leu Met Arg
2000 2005 2010
Arg Gly Asp Leu Pro Val Trp Leu Ala Tyr Arg Val Ala Ala Glu
2015 2020 2025
Gly Ile Asn Tyr Ala Asp Arg Arg Trp Cys Phe Asp Gly Val Lys
2030 2035 2040
Asn Asn Gln Ile Leu Glu Glu Asn Val Glu Val Glu Ile Trp Thr
2045 2050 2055
Lys Glu Gly Glu Arg Lys Lys Leu Lys Pro Arg Trp Leu Asp Ala
2060 2065 2070
Arg Ile Tyr Ser Asp Pro Leu Ala Leu Lys Glu Phe Lys Glu Phe
2075 2080 2085
Ala Ala Gly Arg Lys Ser Leu Thr Leu Asn Leu Ile Thr Glu Met
2090 2095 2100
Gly Arg Leu Pro Thr Phe Met Thr Gln Lys Ala Arg Asp Ala Leu
2105 2110 2115
Asp Asn Leu Ala Val Leu His Thr Ala Glu Ala Gly Gly Arg Ala
2120 2125 2130
Tyr Asn His Ala Leu Ser Glu Leu Pro Glu Thr Leu Glu Thr Leu
2135 2140 2145
Leu Leu Leu Thr Leu Leu Ala Thr Val Thr Gly Gly Ile Phe Leu
2150 2155 2160
Phe Leu Met Ser Ala Arg Gly Ile Gly Lys Met Thr Leu Gly Met
2165 2170 2175
Cys Cys Ile Ile Thr Ala Ser Ile Leu Leu Trp Tyr Ala Gln Ile
2180 2185 2190
Gln Pro His Trp Ile Ala Ala Ser Ile Ile Leu Glu Phe Phe Leu
2195 2200 2205
Ile Val Leu Leu Ile Pro Glu Pro Glu Lys Gln Arg Thr Pro Gln
2210 2215 2220
Asp Asn Gln Leu Thr Tyr Val Val Ile Ala Ile Leu Thr Val Val
2225 2230 2235
Ala Ala Thr Met Ala Asn Glu Met Gly Phe Leu Glu Lys Thr Lys
2240 2245 2250
Lys Asp Leu Gly Leu Gly Ser Ile Ala Thr Gln Gln Pro Glu Ser
2255 2260 2265
Asn Ile Leu Asp Ile Asp Leu Arg Pro Ala Ser Ala Trp Thr Leu
2270 2275 2280
Tyr Ala Val Ala Thr Thr Phe Val Thr Pro Met Leu Arg His Ser
2285 2290 2295
Ile Glu Asn Ser Ser Val Asn Val Ser Leu Thr Ala Ile Ala Asn
2300 2305 2310
Gln Ala Thr Val Leu Met Gly Leu Gly Lys Gly Trp Pro Leu Ser
2315 2320 2325
Lys Met Asp Ile Gly Val Pro Leu Leu Ala Ile Gly Cys Tyr Ser
2330 2335 2340
Gln Val Asn Pro Ile Thr Leu Thr Ala Ala Leu Phe Leu Leu Val
2345 2350 2355
Ala His Tyr Ala Ile Ile Gly Pro Gly Leu Gln Ala Lys Ala Thr
2360 2365 2370
Arg Glu Ala Gln Lys Arg Ala Ala Ala Gly Ile Met Lys Asn Pro
2375 2380 2385
Thr Val Asp Gly Ile Thr Val Ile Asp Leu Asp Pro Ile Pro Tyr
2390 2395 2400
Asp Pro Lys Phe Glu Lys Gln Leu Gly Gln Val Met Leu Leu Val
2405 2410 2415
Leu Cys Val Thr Gln Val Leu Met Met Arg Thr Thr Trp Ala Leu
2420 2425 2430
Cys Glu Ala Leu Thr Leu Ala Thr Gly Pro Ile Ser Thr Leu Trp
2435 2440 2445
Glu Gly Asn Pro Gly Arg Phe Trp Asn Thr Thr Ile Ala Val Ser
2450 2455 2460
Met Ala Asn Ile Phe Arg Gly Ser Tyr Leu Ala Gly Ala Gly Leu
2465 2470 2475
Leu Phe Ser Ile Met Lys Asn Thr Thr Asn Thr Arg Arg Gly Thr
2480 2485 2490
Gly Asn Ile Gly Glu Thr Leu Gly Glu Lys Trp Lys Ser Arg Leu
2495 2500 2505
Asn Ala Leu Gly Lys Ser Glu Phe Gln Ile Tyr Lys Lys Ser Gly
2510 2515 2520
Ile Gln Glu Val Asp Arg Thr Leu Ala Lys Glu Gly Ile Lys Arg
2525 2530 2535
Gly Glu Thr Asp His His Ala Val Ser Arg Gly Ser Ala Lys Leu
2540 2545 2550
Arg Trp Phe Val Glu Arg Asn Met Val Thr Pro Glu Gly Lys Val
2555 2560 2565
Val Asp Leu Gly Cys Gly Arg Gly Gly Trp Ser Tyr Tyr Cys Gly
2570 2575 2580
Gly Leu Lys Asn Val Arg Glu Val Lys Gly Leu Thr Lys Gly Gly
2585 2590 2595
Pro Gly His Glu Glu Pro Ile Pro Met Ser Thr Tyr Gly Trp Asn
2600 2605 2610
Leu Val Arg Leu Gln Ser Gly Val Asp Val Phe Phe Ile Pro Pro
2615 2620 2625
Glu Lys Cys Asp Thr Leu Leu Cys Asp Ile Gly Glu Ser Ser Pro
2630 2635 2640
Asn Pro Thr Val Glu Ala Gly Arg Thr Leu Arg Val Leu Asn Leu
2645 2650 2655
Val Glu Asn Trp Leu Asn Asn Asn Thr Gln Phe Cys Ile Lys Val
2660 2665 2670
Leu Asn Pro Tyr Met Pro Ser Val Ile Glu Lys Met Glu Ala Leu
2675 2680 2685
Gln Arg Lys Tyr Gly Gly Ala Leu Val Arg Asn Pro Leu Ser Arg
2690 2695 2700
Asn Ser Thr His Glu Met Tyr Trp Val Ser Asn Ala Ser Gly Asn
2705 2710 2715
Ile Val Ser Ser Val Asn Met Ile Ser Arg Met Leu Ile Asn Arg
2720 2725 2730
Phe Thr Met Arg Tyr Lys Lys Ala Thr Tyr Glu Pro Asp Val Asp
2735 2740 2745
Leu Gly Ser Gly Thr Arg Asn Ile Gly Ile Glu Ser Glu Ile Pro
2750 2755 2760
Asn Leu Asp Ile Ile Gly Lys Arg Ile Glu Lys Ile Lys Gln Glu
2765 2770 2775
His Glu Thr Ser Trp His Tyr Asp Gln Asp His Pro Tyr Lys Thr
2780 2785 2790
Trp Ala Tyr His Gly Ser Tyr Glu Thr Lys Gln Thr Gly Ser Ala
2795 2800 2805
Ser Ser Met Val Asn Gly Val Val Arg Leu Leu Thr Lys Pro Trp
2810 2815 2820
Asp Val Val Pro Met Val Thr Gln Met Ala Met Thr Asp Thr Thr
2825 2830 2835
Pro Phe Gly Gln Gln Arg Val Phe Lys Glu Lys Val Asp Thr Arg
2840 2845 2850
Thr Gln Glu Pro Lys Glu Gly Thr Lys Lys Leu Met Lys Ile Thr
2855 2860 2865
Ala Glu Trp Leu Trp Lys Glu Leu Gly Lys Lys Lys Thr Pro Arg
2870 2875 2880
Met Cys Thr Arg Glu Glu Phe Thr Arg Lys Val Arg Ser Asn Ala
2885 2890 2895
Ala Leu Gly Ala Ile Phe Thr Asp Glu Asn Lys Trp Lys Ser Ala
2900 2905 2910
Arg Glu Ala Val Glu Asp Ser Arg Phe Trp Glu Leu Val Asp Lys
2915 2920 2925
Glu Arg Asn Leu His Leu Glu Gly Lys Cys Glu Thr Cys Val Tyr
2930 2935 2940
Asn Met Met Gly Lys Arg Glu Lys Lys Leu Gly Glu Phe Gly Lys
2945 2950 2955
Ala Lys Gly Ser Arg Ala Ile Trp Tyr Met Trp Leu Gly Ala Arg
2960 2965 2970
Phe Leu Glu Phe Glu Ala Leu Gly Phe Leu Asn Glu Asp His Trp
2975 2980 2985
Phe Ser Arg Glu Asn Ser Leu Ser Gly Val Glu Gly Glu Gly Leu
2990 2995 3000
His Lys Leu Gly Tyr Ile Leu Arg Asp Val Ser Lys Lys Glu Gly
3005 3010 3015
Gly Ala Met Tyr Ala Asp Asp Thr Ala Gly Trp Asp Thr Arg Ile
3020 3025 3030
Thr Leu Glu Asp Leu Lys Asn Glu Glu Met Val Thr Asn His Met
3035 3040 3045
Glu Gly Glu His Lys Lys Leu Ala Glu Ala Ile Phe Lys Leu Thr
3050 3055 3060
Tyr Gln Asn Lys Val Val Arg Val Gln Arg Pro Thr Pro Arg Gly
3065 3070 3075
Thr Val Met Asp Ile Ile Ser Arg Arg Asp Gln Arg Gly Ser Gly
3080 3085 3090
Gln Val Gly Thr Tyr Gly Leu Asn Thr Phe Thr Asn Met Glu Ala
3095 3100 3105
Gln Leu Ile Arg Gln Met Glu Gly Glu Gly Val Phe Lys Ser Ile
3110 3115 3120
Gln His Leu Thr Ile Thr Glu Glu Ile Ala Val Gln Asn Trp Leu
3125 3130 3135
Ala Arg Val Gly Arg Glu Arg Leu Ser Arg Met Ala Ile Ser Gly
3140 3145 3150
Asp Asp Cys Val Val Lys Pro Leu Asp Asp Arg Phe Ala Ser Ala
3155 3160 3165
Leu Thr Ala Leu Asn Asp Met Gly Lys Ile Arg Lys Asp Ile Gln
3170 3175 3180
Gln Trp Glu Pro Ser Arg Gly Trp Asn Asp Trp Thr Gln Val Pro
3185 3190 3195
Phe Cys Ser His His Phe His Glu Leu Ile Met Lys Asp Gly Arg
3200 3205 3210
Val Leu Val Val Pro Cys Arg Asn Gln Asp Glu Leu Ile Gly Arg
3215 3220 3225
Ala Arg Ile Ser Gln Gly Ala Gly Trp Ser Leu Arg Glu Thr Ala
3230 3235 3240
Cys Leu Gly Lys Ser Tyr Ala Gln Met Trp Ser Leu Met Tyr Phe
3245 3250 3255
His Arg Arg Asp Leu Arg Leu Ala Ala Asn Ala Ile Cys Ser Ala
3260 3265 3270
Val Pro Ser His Trp Val Pro Thr Ser Arg Thr Thr Trp Ser Ile
3275 3280 3285
His Ala Lys His Glu Trp Met Thr Thr Glu Asp Met Leu Thr Val
3290 3295 3300
Trp Asn Arg Val Trp Ile Gln Glu Asn Pro Trp Met Glu Asp Lys
3305 3310 3315
Thr Pro Val Glu Ser Trp Glu Glu Ile Pro Tyr Leu Gly Lys Arg
3320 3325 3330
Glu Asp Gln Trp Cys Gly Ser Leu Ile Gly Leu Thr Ser Arg Ala
3335 3340 3345
Thr Trp Ala Lys Asn Ile Gln Ala Ala Ile Asn Gln Val Arg Ser
3350 3355 3360
Leu Ile Gly Asn Glu Glu Tyr Thr Asp Tyr Met Pro Ser Met Lys
3365 3370 3375
Arg Phe Arg Arg Glu Glu Glu Glu Ala Gly Val Leu Trp
3380 3385 3390
<210> 4
<211> 10723
<212> DNA
<213> 人工序列
<220>
<223> 登革热病毒血清型2, BVS
<400> 4
agttgttagt ctacgtggac cgacaaagac agattctttg agggagctaa gctcaatgta 60
gttctaacag ttttttaatt agagagcaga tctctgatga ataaccaacg gaaaaaggcg 120
aaaaacacgc ctttcaatat gctgaaacgc gagagaaacc gcgtgtcgac tgtgcaacag 180
ctgacaaaga gattctcact tggaatgctg cagggacgag gaccattaaa actgttcatg 240
gccctggtgg cgttccttcg tttcctaaca atcccaccaa cagcagggat attgaagaga 300
tggggaacaa ttaaaaaatc aaaagctatt aatgttttga gagggttcag gaaagagatt 360
ggaaggatgc tgaacatctt gaataggaga cgcagatctg caggcatgat cattatgctg 420
attccaacag tgatggcgtt ccatttaacc acacgtaacg gagaaccaca catgatcgtc 480
agcagacaag agaaagggaa aagtcttctg tttaaaacag aggttggcgt gaacatgtgt 540
accctcatgg ccatggacct tggtgaattg tgtgaagaca caatcacgta cgagtgtccc 600
cttctcaggc agaatgagcc agaagacata gactgttggt gcaactctac gtccacgtgg 660
gtaacttatg ggacgtgtac caccatggga gaacatagaa gagaaaaaag atcagtggca 720
ctcgttccac atgtgggaat gggactggag acacgaactg aaacatggat gtcatcagaa 780
ggggcctgga aacatgtcca gagaattgaa acttggatct tgagacatcc aggcttcacc 840
atgatggcag caatcctggc atacaccata ggaacgacac atttccaaag agccctgatc 900
ttcatcttac tgacagctgt cactccttca atgacaatgc gttgcatagg aatgtcaaat 960
agagactttg tggaaggggt ttcaggagga agctgggttg acatagtctt agaacatgga 1020
agctgtgtga cgacgatggc aaaaaacaaa ccaacattgg attttgaact gataaaaaca 1080
gaagccaaac agcctgccac cctaaggaag tactgtatag aggcaaagct aaccaacaca 1140
acaacagaat ctcgctgccc aacacaaggg gaacccagcc taaatgaaga gcaggacaaa 1200
aggttcgtct gcaaacactc catggtagac agaggatggg gaaatggatg tggactattt 1260
ggaaagggag gcattgtgac ctgtgctatg ttcagatgca aaaagaacat ggaaggaaaa 1320
gttgtgcaac cagaaaactt ggaatacacc attgtgataa cacctcactc aggggaagag 1380
catgcagtcg gaaatgacac aggaaaacat ggcaaggaaa tcaaaataac accacagagt 1440
tccatcacag aagcagaatt gacaggttat ggcactgtca caatggagtg ctctccaaga 1500
acgggcctcg acttcaatga gatggtgttg ctgcagatgg aaaataaagc ttggctggtg 1560
cacaggcaat ggttcctaga cctgccgtta ccatggttgc ccggagcgga cacacaaggg 1620
tcaaattgga tacagaaaga gacattggtc actttcaaaa atccccatgc gaagaaacag 1680
gatgttgttg ttttaggatc ccaagaaggg gccatgcaca cagcacttac aggggccaca 1740
gaaatccaaa tgtcatcagg aaacttactc ttcacaggac atctcaagtg caggctgaga 1800
atggacaagc tacagctcaa aggaatgtca tactctatgt gcacaggaaa gtttaaagtt 1860
gtgaaggaaa tagcagaaac acaacatgga acaatagtta tcagagtgca atatgaaggg 1920
gacggctctc catgcaagat cccttttgag ataatggatt tggaaaaaag acatgtctta 1980
ggtcgcctga ttacagtcaa cccaattgtg acagaaaaag atagcccagt caacatagaa 2040
gcagaacctc catttggaga cagctacatc atcataggag tagagccggg acaactgaag 2100
ctcaactggt ttaagaaagg aagttctatc ggccaaatgt ttgagacaac aatgaggggg 2160
gcgaagagaa tggccatttt aggtgacaca gcctgggatt ttggatcctt gggaggagtg 2220
tttacatcta taggaaaggc tctccaccaa gtctttggag caatctatgg agctgccttc 2280
agtggggttt catggactat gaaaatcctc ataggagtca ttatcacatg gataggaatg 2340
aattcacgca gcacctcact gtctgtgaca ctagtattgg tgggaattgt gacactgtat 2400
ttgggagtca tggtgcaggc cgatagtggt tgcgttgtga gctggaaaaa caaagaactg 2460
aaatgtggca gtgggatttt catcacagac aacgtgcaca catggacaga acaatacaag 2520
ttccaaccag aatccccttc aaaactagct tcagctatcc agaaagccca tgaagaggac 2580
atttgtggaa tccgctcagt aacaagactg gagaatctga tgtggaaaca aataacacca 2640
gaattgaatc acattctatc agaaaatgag gtgaagttaa ctattatgac aggagacatc 2700
aaaggaatca tgcaggcagg aaaacgatct ctgcggcctc agcccactga gctgaagtat 2760
tcatggaaaa catggggcaa agcaaaaatg ctctctacag agtctcataa ccagaccttt 2820
ctcattgatg gccccgaaac agcagaatgc cccaacacaa atagagcttg gaattcgttg 2880
gaagttgaag actatggctt tggagtattc accaccaata tatggctaaa attgaaagaa 2940
aaacaggatg tattctgcga ctcaaaactc atgtcagcgg ccataaaaga caacagagcc 3000
gtccatgccg atatgggtta ttggatagaa agtgcactca atgacacatg gaagatagag 3060
aaagcctctt tcattgaagt taaaaactgc cactggccaa aatcacacac cctctggagc 3120
aatggagtgc tagaaagtga gatgataatt ccaaagaatc tcgctggacc agtgtctcaa 3180
cacaactata gaccaggcta ccatacacaa ataacaggac catggcatct aggtaagctt 3240
gagatggact ttgatttctg tgatggaaca acagtggtag tgactgagga ctgcggaaat 3300
agaggaccct ctttgagaac aaccactgcc tctggaaaac tcataacaga atggtgctgc 3360
cgatcttgca cattaccacc gctaagatac agaggtgagg atgggtgctg gtacgggatg 3420
gaaatcagac cattgaagga gaaagaagag aatttggtca actccttggt cacagctgga 3480
catgggcagg tcgacaactt ttcactagga gtcttgggaa tggcattgtt cctggaggaa 3540
atgcttagga cccgagtagg aacgaaacat gcaatactac tagttgcagt ttcttttgtg 3600
acattgatca cagggaacat gtcctttaga gacctgggaa gagtgatggt tatggtaggc 3660
gccactatga cggatgacat aggtatgggc gtgacttatc ttgccctact agcagccttc 3720
aaagtcagac caacttttgc agctggacta ctcttgagaa agctgacctc caaggaattg 3780
atgatgacta ctataggaat tgtactcctc tcccagagca ccataccaga gaccattctt 3840
gagttgactg atgcgttagc cttaggcatg atggtcctca aaatggtgag aaatatggaa 3900
aagtatcaat tggcagtgac tatcatggct atcttgtgcg tcccaaacgc agtgatatta 3960
caaaacgcat ggaaagtgag ttgcacaata ttggcagtgg tgtccgtttc cccactgttc 4020
ttaacatcct cacagcaaaa aacagattgg ataccattag cattgacgat caaaggtctc 4080
aatccaacag ctatttttct aacaaccctc tcaagaacca gcaagaaaag gagctggcca 4140
ttaaatgagg ctatcatggc agtcgggatg gtgagcattt tagccagttc tctcctaaaa 4200
aatgatattc ccatgacagg accattagtg gctggagggc tcctcactgt gtgctacgtg 4260
ctcactggac gatcggccga tttggaactg gagagagcag ccgatgtcaa atgggaagac 4320
caggcagaga tatcaggaag cagtccaatc ctgtcaataa caatatcaga agatggtagc 4380
atgtcgataa aaaatgaaga ggaagaacaa acactgacca tactcattag aacaggattg 4440
ctggtgatct caggactttt tcctgtatca ataccaatca cggcagcagc atggtacctg 4500
tgggaagtga agaaacaacg ggccggagta ttgtgggatg ttccttcacc cccacccatg 4560
ggaaaggctg aactggaaga tggagcctat agaattaagc aaaaagggat tcttggatat 4620
tcccagatcg gagccggagt ttacaaagaa ggaacattcc atacaatgtg gcatgtcaca 4680
cgtggcgctg ttctaatgca taaaggaaag aggattgaac catcatgggc ggacgtcaag 4740
aaagacctaa tatcatatgg aggaggctgg aagttagaag gagaatggaa ggaaggagaa 4800
gaagtccagg tattggcact ggagcctgga aaaaatccaa gagccgtcca aacgaaacct 4860
ggtcttttca aaaccaacgc cggaacaata ggtgctgtat ctctggactt ttctcctgga 4920
acgtcaggat ctccaattat cgacaaaaaa ggaaaagttg tgggtcttta tggtaatggt 4980
gttgttacaa ggagtggagc atatgtgagt gctatagccc agactgaaaa aagcattgaa 5040
gacaacccag agatcgaaga tgacattttc cgaaagagaa gactgaccat catggacctc 5100
cacccaggag cgggaaagac gaagagatac cttccggcca tagtcagaga agctataaaa 5160
cggggtttga gaacattaat cttggccccc actagagttg tggcagctga aatggaggaa 5220
gcccttagag gacttccaat aagataccag accccagcca tcagagctgt gcacaccggg 5280
cgggagattg tggacctaat gtgtcatgcc acatttacca tgaggctgct atcaccagtt 5340
agagtgccaa actacaacct gattatcatg gacgaagccc atttcacaga cccagcaagt 5400
atagcagcta gaggatacat ctcaactcga gtggagatgg gtgaggcagc tgggattttt 5460
atgacagcca ctcccccggg aagcagagac ccatttcctc agagcaatgc accaatcata 5520
gatgaagaaa gagaaatccc tgaacgctcg tggaattccg gacatgaatg ggtcacggat 5580
tttaaaggga agactgtttg gttcgttcca agtataaaag caggaaatga tatagcagct 5640
tgcctgagga aaaatggaaa gaaagtgata caactcagta ggaagacctt tgattctgag 5700
tatgtcaaga ctagaaccaa tgattgggac ttcgtggtta caactgacat ttcagaaatg 5760
ggtgccaatt tcaaggctga gagggttata gaccccagac gctgcatgaa accagtcata 5820
ctaacagatg gtgaagagcg ggtgattctg gcaggaccta tgccagtgac ccactctagt 5880
gcagcacaaa gaagagggag aataggaaga aatccaaaaa atgagaatga ccagtacata 5940
tacatggggg aacctctgga aaatgatgaa gactgtgcac actggaaaga agctaaaatg 6000
ctcctagata acatcaacac gccagaagga atcattccta gcatgttcga accagagcgt 6060
gaaaaggtgg atgccattga tggcgaatac cgcttgagag gagaagcaag gaaaaccttt 6120
gtagacttaa tgagaagagg agacctacca gtctggttgg cctacagagt ggcagctgaa 6180
ggcatcaact acgcagacag aaggtggtgt tttgatggag tcaagaacaa ccaaatccta 6240
gaagaaaacg tggaagttga aatctggaca aaagaagggg aaaggaagaa attgaaaccc 6300
agatggttgg atgctaggat ctattctgac ccactggcgc taaaagaatt taaggaattt 6360
gcagccggaa gaaagtctct gaccctgaac ctaatcacag aaatgggtag gctcccaacc 6420
ttcatgactc agaaggcaag agacgcactg gacaacttag cagtgctgca cacggctgag 6480
ccaggtggaa gggcgtacaa ccatgctctc agtgaactgc cggagaccct ggagacattg 6540
cttttactga cacttctggc tacagtcacg ggagggatct ttttattctt gatgagcgca 6600
aggggcatag ggaagatgac cctgggaatg tgctgcataa tcacggctag catcctccta 6660
tggtacgcac aaatacagcc acactggata gcagcttcaa taatactgga gttttttctc 6720
atagttttgc ttattccaga acctgaaaaa cagagaacac cccaagacaa ccaactgacc 6780
tacgttgtca tagccatcct cacagtggtg gccgcaacca tggcaaacga gatgggtttc 6840
ctagaaaaaa cgaagaaaga tctcggattg ggaagcattg caacccagca acccgagagc 6900
aacatcctgg acatagatct acgtcctgca tcagcatgga cgctgtatgc cgtggccaca 6960
acatttgtta caccaatgtt gagacatagc attgaaaatt cctcagtgaa tgtgtcccta 7020
acagctatag ccaaccaagc cacagtgtta atgggtctcg ggaaaggatg gccattgtca 7080
aagatggaca tcggagttcc ccttctcgcc attggatgct actcacaagt caaccccata 7140
actctcacag cagctttttt cttattggta gcacattatg ccatcatagg gccaggactc 7200
caagcaaaag caaccagaga agctcagaaa agagcagcgg cgggcatcat gaaaaaccca 7260
actgtcgatg gaataacagt gattgaccta gatccaatac cttatgatcc aaagtttgaa 7320
aagcagttgg gacaagtaat gctcctagtc ctctgcgtga ctcaagtatt gatgatgagg 7380
actacatggg ctctgtgtga ggctttaacc ttagctaccg ggcccatctc cacattgtgg 7440
gaaggaaatc cagggaggtt ttggaacact accattgcgg tgtcaatggc taacattttt 7500
agagggagtt acttggccgg agctggactt ctcttttcta ttatgaagaa cacaaccaac 7560
acaagaaggg gaactggcaa cataggagag acgcttggag agaaatggaa aagccgattg 7620
aacgcattgg gaaaaagtga attccagatc tacaagaaaa gtggaatcca ggaagtggat 7680
agaaccttag caaaagaagg cattaaaaga ggagaaacgg accatcacgc tgtgtcgcga 7740
ggctcagcaa aactgagatg gttcgttgag agaaacatgg tcacaccaga agggaaagta 7800
gtggacctcg gttgtggcag aggaggctgg tcatactatt gtggaggact aaagaatgta 7860
agagaagtca aaggcctaac aaaaggagga ccaggacacg aagaacccat ccccatgtca 7920
acatatgggt ggaatctagt gcgtcttcaa agtggagttg acgttttctt catcccgcca 7980
gaaaagtgtg acacattatt gtgtgacata ggggagtcat caccaaatcc cacagtggaa 8040
gcaggacgaa cactcagagt ccttaactta gtagaaaatt ggttgaacaa caacactcaa 8100
ttttgcataa aggttctcaa cccatatatg ccctcagtca tagaaaaaat ggaagcacta 8160
caaaggaaat atggaggagc cttagtgagg aatccactct cacgaaactc cacacatgag 8220
atgtactggg tatccaatgc ttccgggaac atagtgtcat cagtgaacat gatttcaagg 8280
atgttgatca acagatttac aatgagatac aagaaagcca cttacgagcc ggatgttgac 8340
ctcggaagcg gaacccgtaa catcgggatt gaaagtgaga taccaaacct agatataatt 8400
gggaaaagaa tagaaaaaat aaagcaagag catgaaacat catggcacta tgaccaagac 8460
cacccataca aaacgtgggc ataccatggt agctatgaaa caaaacagac tggatcagca 8520
tcatccatgg tcaacggagt ggtcaggctg ctgacaaaac cttgggacgt cgtccccatg 8580
gtgacacaga tggcaatgac agacacgact ccatttggac aacagcgcgt ttttaaagag 8640
aaagtggaca cgagaaccca agaaccgaaa gaaggcacga agaaactaat gaaaataaca 8700
gcagagtggc tttggaaaga attagggaag aaaaagacac ccaggatgtg caccagagaa 8760
gaattcacaa gaaaggtgag aagcaatgca gccttggggg ccgtattcac tgatgagaac 8820
aagtggaagt cggcacgtga ggctgttgaa gatagtaggt tttgggagct ggttgacaag 8880
gaaaggaatc tccatcttga aggaaagtgt gaaacatgtg tgtacaacat gatgggaaaa 8940
agagagaaga agctagggga attcggcaag gcaaaaggca gcagagccat atggtacatg 9000
tggcttggag cacgcttctt agagtttgaa gccctaggat tcttaaatga agatcactgg 9060
ttctccagag agaactccct gagtggagtg gaaggagaag ggctgcacaa gctaggttac 9120
attctaagag acgtgagcaa gaaagaggga ggagcaatgt atgccgatga caccgcagga 9180
tgggatacaa gaatcacact agaagaccta aaaaatgaag aaatggtaac aaaccacatg 9240
gaaggagaac acaagaaact agccgaggcc attttcaaac taacgtacca aaacaaggtg 9300
gtgcgtgtgc aaagaccaac accaagaggc acagtaatgg acatcatatc gagaagagac 9360
caaagaggta gtggacaagt tggcacctat ggactcaata ctttcaccaa tatggaagcc 9420
caactaatca gacagatgga gggagaagga gtctttaaaa gcattcagca cctaacaatc 9480
acagaagaaa tcgctgtgca aaactggtta gcaagagtgg ggcgcgaaag gttatcaaga 9540
atggccatca gtggagatga ttgtgttgtg aaacctttag atgacaggtt cgcaagcgct 9600
ttaacagctc taaatgacat gggaaagatt aggaaagaca tacaacaatg ggaaccttca 9660
agaggatgga atgattggac acaagtgccc ttctgttcac accatttcca tgagttaatc 9720
atgaaagacg gtcgcgtact cgttgttcca tgtagaaacc aagatgaact gattggcaga 9780
gcccgaatct cccaaggagc agggtggtct ttgcgggaga cggcctgttt ggggaagtct 9840
tacgcccaaa tgtggagctt gatgtacttc cacagacgcg acctcaggct ggcggcaaat 9900
gctatttgct cggcagtacc atcacattgg gttccaacaa gtcgaacaac ctggtccata 9960
catgctaaac atgaatggat gacaacggaa gacatgctga cagtctggaa cagggtgtgg 10020
attcaagaaa acccatggat ggaagacaaa actccagtgg aatcatggga ggaaatccca 10080
tacttgggga aaagagaaga ccaatggtgc ggctcattga ttgggttaac aagcagggcc 10140
acctgggcaa agaacatcca agcagcaata aatcaagtta gatcccttat aggcaatgaa 10200
gaatacacag attacatgcc atccatgaaa agattcagaa gagaagagga agaagcagga 10260
gttctgtggt agaaagcaaa actaacatga aacaaggcta gaagtcaggt cggattaagc 10320
catagtacgg aaaaaactat gctacctgtg agccccgtcc aaggacgtta aaagaagtca 10380
ggccatcata aatgccatag cttgagtaaa ctatgcagcc tgtagctcca cctgagaagg 10440
tgtaaaaaat ccgggaggcc acaaaccatg gaagctgtac gcatggcgta gtggactagc 10500
ggttagagga gacccctccc ttacaaatcg cagcaacaat gggggcccaa ggcgagatga 10560
agctgtagtc tcgctggaag gactagaggt tagaggagac ccccccgaaa caaaaaacag 10620
catattgacg ctgggaaaga ccagagatcc tgctgtctcc tcagcatcat tccaggcaca 10680
gaacgccaga aaatggaatg gtgctgttga atcaacaggt tct 10723
<210> 5
<211> 3391
<212> PRT
<213> 人工序列
<220>
<223> 登革热病毒血清型2, BVS
<400> 5
Met Asn Asn Gln Arg Lys Lys Ala Lys Asn Thr Pro Phe Asn Met Leu
1 5 10 15
Lys Arg Glu Arg Asn Arg Val Ser Thr Val Gln Gln Leu Thr Lys Arg
20 25 30
Phe Ser Leu Gly Met Leu Gln Gly Arg Gly Pro Leu Lys Leu Phe Met
35 40 45
Ala Leu Val Ala Phe Leu Arg Phe Leu Thr Ile Pro Pro Thr Ala Gly
50 55 60
Ile Leu Lys Arg Trp Gly Thr Ile Lys Lys Ser Lys Ala Ile Asn Val
65 70 75 80
Leu Arg Gly Phe Arg Lys Glu Ile Gly Arg Met Leu Asn Ile Leu Asn
85 90 95
Arg Arg Arg Arg Ser Ala Gly Met Ile Ile Met Leu Ile Pro Thr Val
100 105 110
Met Ala Phe His Leu Thr Thr Arg Asn Gly Glu Pro His Met Ile Val
115 120 125
Ser Arg Gln Glu Lys Gly Lys Ser Leu Leu Phe Lys Thr Glu Val Gly
130 135 140
Val Asn Met Cys Thr Leu Met Ala Met Asp Leu Gly Glu Leu Cys Glu
145 150 155 160
Asp Thr Ile Thr Tyr Glu Cys Pro Leu Leu Arg Gln Asn Glu Pro Glu
165 170 175
Asp Ile Asp Cys Trp Cys Asn Ser Thr Ser Thr Trp Val Thr Tyr Gly
180 185 190
Thr Cys Thr Thr Met Gly Glu His Arg Arg Glu Lys Arg Ser Val Ala
195 200 205
Leu Val Pro His Val Gly Met Gly Leu Glu Thr Arg Thr Glu Thr Trp
210 215 220
Met Ser Ser Glu Gly Ala Trp Lys His Val Gln Arg Ile Glu Thr Trp
225 230 235 240
Ile Leu Arg His Pro Gly Phe Thr Met Met Ala Ala Ile Leu Ala Tyr
245 250 255
Thr Ile Gly Thr Thr His Phe Gln Arg Ala Leu Ile Phe Ile Leu Leu
260 265 270
Thr Ala Val Thr Pro Ser Met Thr Met Arg Cys Ile Gly Met Ser Asn
275 280 285
Arg Asp Phe Val Glu Gly Val Ser Gly Gly Ser Trp Val Asp Ile Val
290 295 300
Leu Glu His Gly Ser Cys Val Thr Thr Met Ala Lys Asn Lys Pro Thr
305 310 315 320
Leu Asp Phe Glu Leu Ile Lys Thr Glu Ala Lys Gln Pro Ala Thr Leu
325 330 335
Arg Lys Tyr Cys Ile Glu Ala Lys Leu Thr Asn Thr Thr Thr Glu Ser
340 345 350
Arg Cys Pro Thr Gln Gly Glu Pro Ser Leu Asn Glu Glu Gln Asp Lys
355 360 365
Arg Phe Val Cys Lys His Ser Met Val Asp Arg Gly Trp Gly Asn Gly
370 375 380
Cys Gly Leu Phe Gly Lys Gly Gly Ile Val Thr Cys Ala Met Phe Arg
385 390 395 400
Cys Lys Lys Asn Met Glu Gly Lys Val Val Gln Pro Glu Asn Leu Glu
405 410 415
Tyr Thr Ile Val Ile Thr Pro His Ser Gly Glu Glu His Ala Val Gly
420 425 430
Asn Asp Thr Gly Lys His Gly Lys Glu Ile Lys Ile Thr Pro Gln Ser
435 440 445
Ser Ile Thr Glu Ala Glu Leu Thr Gly Tyr Gly Thr Val Thr Met Glu
450 455 460
Cys Ser Pro Arg Thr Gly Leu Asp Phe Asn Glu Met Val Leu Leu Gln
465 470 475 480
Met Glu Asn Lys Ala Trp Leu Val His Arg Gln Trp Phe Leu Asp Leu
485 490 495
Pro Leu Pro Trp Leu Pro Gly Ala Asp Thr Gln Gly Ser Asn Trp Ile
500 505 510
Gln Lys Glu Thr Leu Val Thr Phe Lys Asn Pro His Ala Lys Lys Gln
515 520 525
Asp Val Val Val Leu Gly Ser Gln Glu Gly Ala Met His Thr Ala Leu
530 535 540
Thr Gly Ala Thr Glu Ile Gln Met Ser Ser Gly Asn Leu Leu Phe Thr
545 550 555 560
Gly His Leu Lys Cys Arg Leu Arg Met Asp Lys Leu Gln Leu Lys Gly
565 570 575
Met Ser Tyr Ser Met Cys Thr Gly Lys Phe Lys Val Val Lys Glu Ile
580 585 590
Ala Glu Thr Gln His Gly Thr Ile Val Ile Arg Val Gln Tyr Glu Gly
595 600 605
Asp Gly Ser Pro Cys Lys Ile Pro Phe Glu Ile Met Asp Leu Glu Lys
610 615 620
Arg His Val Leu Gly Arg Leu Ile Thr Val Asn Pro Ile Val Thr Glu
625 630 635 640
Lys Asp Ser Pro Val Asn Ile Glu Ala Glu Pro Pro Phe Gly Asp Ser
645 650 655
Tyr Ile Ile Ile Gly Val Glu Pro Gly Gln Leu Lys Leu Asn Trp Phe
660 665 670
Lys Lys Gly Ser Ser Ile Gly Gln Met Phe Glu Thr Thr Met Arg Gly
675 680 685
Ala Lys Arg Met Ala Ile Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser
690 695 700
Leu Gly Gly Val Phe Thr Ser Ile Gly Lys Ala Leu His Gln Val Phe
705 710 715 720
Gly Ala Ile Tyr Gly Ala Ala Phe Ser Gly Val Ser Trp Thr Met Lys
725 730 735
Ile Leu Ile Gly Val Ile Ile Thr Trp Ile Gly Met Asn Ser Arg Ser
740 745 750
Thr Ser Leu Ser Val Thr Leu Val Leu Val Gly Ile Val Thr Leu Tyr
755 760 765
Leu Gly Val Met Val Gln Ala Asp Ser Gly Cys Val Val Ser Trp Lys
770 775 780
Asn Lys Glu Leu Lys Cys Gly Ser Gly Ile Phe Ile Thr Asp Asn Val
785 790 795 800
His Thr Trp Thr Glu Gln Tyr Lys Phe Gln Pro Glu Ser Pro Ser Lys
805 810 815
Leu Ala Ser Ala Ile Gln Lys Ala His Glu Glu Asp Ile Cys Gly Ile
820 825 830
Arg Ser Val Thr Arg Leu Glu Asn Leu Met Trp Lys Gln Ile Thr Pro
835 840 845
Glu Leu Asn His Ile Leu Ser Glu Asn Glu Val Lys Leu Thr Ile Met
850 855 860
Thr Gly Asp Ile Lys Gly Ile Met Gln Ala Gly Lys Arg Ser Leu Arg
865 870 875 880
Pro Gln Pro Thr Glu Leu Lys Tyr Ser Trp Lys Thr Trp Gly Lys Ala
885 890 895
Lys Met Leu Ser Thr Glu Ser His Asn Gln Thr Phe Leu Ile Asp Gly
900 905 910
Pro Glu Thr Ala Glu Cys Pro Asn Thr Asn Arg Ala Trp Asn Ser Leu
915 920 925
Glu Val Glu Asp Tyr Gly Phe Gly Val Phe Thr Thr Asn Ile Trp Leu
930 935 940
Lys Leu Lys Glu Lys Gln Asp Val Phe Cys Asp Ser Lys Leu Met Ser
945 950 955 960
Ala Ala Ile Lys Asp Asn Arg Ala Val His Ala Asp Met Gly Tyr Trp
965 970 975
Ile Glu Ser Ala Leu Asn Asp Thr Trp Lys Ile Glu Lys Ala Ser Phe
980 985 990
Ile Glu Val Lys Asn Cys His Trp Pro Lys Ser His Thr Leu Trp Ser
995 1000 1005
Asn Gly Val Leu Glu Ser Glu Met Ile Ile Pro Lys Asn Leu Ala
1010 1015 1020
Gly Pro Val Ser Gln His Asn Tyr Arg Pro Gly Tyr His Thr Gln
1025 1030 1035
Ile Thr Gly Pro Trp His Leu Gly Lys Leu Glu Met Asp Phe Asp
1040 1045 1050
Phe Cys Asp Gly Thr Thr Val Val Val Thr Glu Asp Cys Gly Asn
1055 1060 1065
Arg Gly Pro Ser Leu Arg Thr Thr Thr Ala Ser Gly Lys Leu Ile
1070 1075 1080
Thr Glu Trp Cys Cys Arg Ser Cys Thr Leu Pro Pro Leu Arg Tyr
1085 1090 1095
Arg Gly Glu Asp Gly Cys Trp Tyr Gly Met Glu Ile Arg Pro Leu
1100 1105 1110
Lys Glu Lys Glu Glu Asn Leu Val Asn Ser Leu Val Thr Ala Gly
1115 1120 1125
His Gly Gln Val Asp Asn Phe Ser Leu Gly Val Leu Gly Met Ala
1130 1135 1140
Leu Phe Leu Glu Glu Met Leu Arg Thr Arg Val Gly Thr Lys His
1145 1150 1155
Ala Ile Leu Leu Val Ala Val Ser Phe Val Thr Leu Ile Thr Gly
1160 1165 1170
Asn Met Ser Phe Arg Asp Leu Gly Arg Val Met Val Met Val Gly
1175 1180 1185
Ala Thr Met Thr Asp Asp Ile Gly Met Gly Val Thr Tyr Leu Ala
1190 1195 1200
Leu Leu Ala Ala Phe Lys Val Arg Pro Thr Phe Ala Ala Gly Leu
1205 1210 1215
Leu Leu Arg Lys Leu Thr Ser Lys Glu Leu Met Met Thr Thr Ile
1220 1225 1230
Gly Ile Val Leu Leu Ser Gln Ser Thr Ile Pro Glu Thr Ile Leu
1235 1240 1245
Glu Leu Thr Asp Ala Leu Ala Leu Gly Met Met Val Leu Lys Met
1250 1255 1260
Val Arg Asn Met Glu Lys Tyr Gln Leu Ala Val Thr Ile Met Ala
1265 1270 1275
Ile Leu Cys Val Pro Asn Ala Val Ile Leu Gln Asn Ala Trp Lys
1280 1285 1290
Val Ser Cys Thr Ile Leu Ala Val Val Ser Val Ser Pro Leu Phe
1295 1300 1305
Leu Thr Ser Ser Gln Gln Lys Thr Asp Trp Ile Pro Leu Ala Leu
1310 1315 1320
Thr Ile Lys Gly Leu Asn Pro Thr Ala Ile Phe Leu Thr Thr Leu
1325 1330 1335
Ser Arg Thr Ser Lys Lys Arg Ser Trp Pro Leu Asn Glu Ala Ile
1340 1345 1350
Met Ala Val Gly Met Val Ser Ile Leu Ala Ser Ser Leu Leu Lys
1355 1360 1365
Asn Asp Ile Pro Met Thr Gly Pro Leu Val Ala Gly Gly Leu Leu
1370 1375 1380
Thr Val Cys Tyr Val Leu Thr Gly Arg Ser Ala Asp Leu Glu Leu
1385 1390 1395
Glu Arg Ala Ala Asp Val Lys Trp Glu Asp Gln Ala Glu Ile Ser
1400 1405 1410
Gly Ser Ser Pro Ile Leu Ser Ile Thr Ile Ser Glu Asp Gly Ser
1415 1420 1425
Met Ser Ile Lys Asn Glu Glu Glu Glu Gln Thr Leu Thr Ile Leu
1430 1435 1440
Ile Arg Thr Gly Leu Leu Val Ile Ser Gly Leu Phe Pro Val Ser
1445 1450 1455
Ile Pro Ile Thr Ala Ala Ala Trp Tyr Leu Trp Glu Val Lys Lys
1460 1465 1470
Gln Arg Ala Gly Val Leu Trp Asp Val Pro Ser Pro Pro Pro Met
1475 1480 1485
Gly Lys Ala Glu Leu Glu Asp Gly Ala Tyr Arg Ile Lys Gln Lys
1490 1495 1500
Gly Ile Leu Gly Tyr Ser Gln Ile Gly Ala Gly Val Tyr Lys Glu
1505 1510 1515
Gly Thr Phe His Thr Met Trp His Val Thr Arg Gly Ala Val Leu
1520 1525 1530
Met His Lys Gly Lys Arg Ile Glu Pro Ser Trp Ala Asp Val Lys
1535 1540 1545
Lys Asp Leu Ile Ser Tyr Gly Gly Gly Trp Lys Leu Glu Gly Glu
1550 1555 1560
Trp Lys Glu Gly Glu Glu Val Gln Val Leu Ala Leu Glu Pro Gly
1565 1570 1575
Lys Asn Pro Arg Ala Val Gln Thr Lys Pro Gly Leu Phe Lys Thr
1580 1585 1590
Asn Ala Gly Thr Ile Gly Ala Val Ser Leu Asp Phe Ser Pro Gly
1595 1600 1605
Thr Ser Gly Ser Pro Ile Ile Asp Lys Lys Gly Lys Val Val Gly
1610 1615 1620
Leu Tyr Gly Asn Gly Val Val Thr Arg Ser Gly Ala Tyr Val Ser
1625 1630 1635
Ala Ile Ala Gln Thr Glu Lys Ser Ile Glu Asp Asn Pro Glu Ile
1640 1645 1650
Glu Asp Asp Ile Phe Arg Lys Arg Arg Leu Thr Ile Met Asp Leu
1655 1660 1665
His Pro Gly Ala Gly Lys Thr Lys Arg Tyr Leu Pro Ala Ile Val
1670 1675 1680
Arg Glu Ala Ile Lys Arg Gly Leu Arg Thr Leu Ile Leu Ala Pro
1685 1690 1695
Thr Arg Val Val Ala Ala Glu Met Glu Glu Ala Leu Arg Gly Leu
1700 1705 1710
Pro Ile Arg Tyr Gln Thr Pro Ala Ile Arg Ala Val His Thr Gly
1715 1720 1725
Arg Glu Ile Val Asp Leu Met Cys His Ala Thr Phe Thr Met Arg
1730 1735 1740
Leu Leu Ser Pro Val Arg Val Pro Asn Tyr Asn Leu Ile Ile Met
1745 1750 1755
Asp Glu Ala His Phe Thr Asp Pro Ala Ser Ile Ala Ala Arg Gly
1760 1765 1770
Tyr Ile Ser Thr Arg Val Glu Met Gly Glu Ala Ala Gly Ile Phe
1775 1780 1785
Met Thr Ala Thr Pro Pro Gly Ser Arg Asp Pro Phe Pro Gln Ser
1790 1795 1800
Asn Ala Pro Ile Ile Asp Glu Glu Arg Glu Ile Pro Glu Arg Ser
1805 1810 1815
Trp Asn Ser Gly His Glu Trp Val Thr Asp Phe Lys Gly Lys Thr
1820 1825 1830
Val Trp Phe Val Pro Ser Ile Lys Ala Gly Asn Asp Ile Ala Ala
1835 1840 1845
Cys Leu Arg Lys Asn Gly Lys Lys Val Ile Gln Leu Ser Arg Lys
1850 1855 1860
Thr Phe Asp Ser Glu Tyr Val Lys Thr Arg Thr Asn Asp Trp Asp
1865 1870 1875
Phe Val Val Thr Thr Asp Ile Ser Glu Met Gly Ala Asn Phe Lys
1880 1885 1890
Ala Glu Arg Val Ile Asp Pro Arg Arg Cys Met Lys Pro Val Ile
1895 1900 1905
Leu Thr Asp Gly Glu Glu Arg Val Ile Leu Ala Gly Pro Met Pro
1910 1915 1920
Val Thr His Ser Ser Ala Ala Gln Arg Arg Gly Arg Ile Gly Arg
1925 1930 1935
Asn Pro Lys Asn Glu Asn Asp Gln Tyr Ile Tyr Met Gly Glu Pro
1940 1945 1950
Leu Glu Asn Asp Glu Asp Cys Ala His Trp Lys Glu Ala Lys Met
1955 1960 1965
Leu Leu Asp Asn Ile Asn Thr Pro Glu Gly Ile Ile Pro Ser Met
1970 1975 1980
Phe Glu Pro Glu Arg Glu Lys Val Asp Ala Ile Asp Gly Glu Tyr
1985 1990 1995
Arg Leu Arg Gly Glu Ala Arg Lys Thr Phe Val Asp Leu Met Arg
2000 2005 2010
Arg Gly Asp Leu Pro Val Trp Leu Ala Tyr Arg Val Ala Ala Glu
2015 2020 2025
Gly Ile Asn Tyr Ala Asp Arg Arg Trp Cys Phe Asp Gly Val Lys
2030 2035 2040
Asn Asn Gln Ile Leu Glu Glu Asn Val Glu Val Glu Ile Trp Thr
2045 2050 2055
Lys Glu Gly Glu Arg Lys Lys Leu Lys Pro Arg Trp Leu Asp Ala
2060 2065 2070
Arg Ile Tyr Ser Asp Pro Leu Ala Leu Lys Glu Phe Lys Glu Phe
2075 2080 2085
Ala Ala Gly Arg Lys Ser Leu Thr Leu Asn Leu Ile Thr Glu Met
2090 2095 2100
Gly Arg Leu Pro Thr Phe Met Thr Gln Lys Ala Arg Asp Ala Leu
2105 2110 2115
Asp Asn Leu Ala Val Leu His Thr Ala Glu Pro Gly Gly Arg Ala
2120 2125 2130
Tyr Asn His Ala Leu Ser Glu Leu Pro Glu Thr Leu Glu Thr Leu
2135 2140 2145
Leu Leu Leu Thr Leu Leu Ala Thr Val Thr Gly Gly Ile Phe Leu
2150 2155 2160
Phe Leu Met Ser Ala Arg Gly Ile Gly Lys Met Thr Leu Gly Met
2165 2170 2175
Cys Cys Ile Ile Thr Ala Ser Ile Leu Leu Trp Tyr Ala Gln Ile
2180 2185 2190
Gln Pro His Trp Ile Ala Ala Ser Ile Ile Leu Glu Phe Phe Leu
2195 2200 2205
Ile Val Leu Leu Ile Pro Glu Pro Glu Lys Gln Arg Thr Pro Gln
2210 2215 2220
Asp Asn Gln Leu Thr Tyr Val Val Ile Ala Ile Leu Thr Val Val
2225 2230 2235
Ala Ala Thr Met Ala Asn Glu Met Gly Phe Leu Glu Lys Thr Lys
2240 2245 2250
Lys Asp Leu Gly Leu Gly Ser Ile Ala Thr Gln Gln Pro Glu Ser
2255 2260 2265
Asn Ile Leu Asp Ile Asp Leu Arg Pro Ala Ser Ala Trp Thr Leu
2270 2275 2280
Tyr Ala Val Ala Thr Thr Phe Val Thr Pro Met Leu Arg His Ser
2285 2290 2295
Ile Glu Asn Ser Ser Val Asn Val Ser Leu Thr Ala Ile Ala Asn
2300 2305 2310
Gln Ala Thr Val Leu Met Gly Leu Gly Lys Gly Trp Pro Leu Ser
2315 2320 2325
Lys Met Asp Ile Gly Val Pro Leu Leu Ala Ile Gly Cys Tyr Ser
2330 2335 2340
Gln Val Asn Pro Ile Thr Leu Thr Ala Ala Phe Phe Leu Leu Val
2345 2350 2355
Ala His Tyr Ala Ile Ile Gly Pro Gly Leu Gln Ala Lys Ala Thr
2360 2365 2370
Arg Glu Ala Gln Lys Arg Ala Ala Ala Gly Ile Met Lys Asn Pro
2375 2380 2385
Thr Val Asp Gly Ile Thr Val Ile Asp Leu Asp Pro Ile Pro Tyr
2390 2395 2400
Asp Pro Lys Phe Glu Lys Gln Leu Gly Gln Val Met Leu Leu Val
2405 2410 2415
Leu Cys Val Thr Gln Val Leu Met Met Arg Thr Thr Trp Ala Leu
2420 2425 2430
Cys Glu Ala Leu Thr Leu Ala Thr Gly Pro Ile Ser Thr Leu Trp
2435 2440 2445
Glu Gly Asn Pro Gly Arg Phe Trp Asn Thr Thr Ile Ala Val Ser
2450 2455 2460
Met Ala Asn Ile Phe Arg Gly Ser Tyr Leu Ala Gly Ala Gly Leu
2465 2470 2475
Leu Phe Ser Ile Met Lys Asn Thr Thr Asn Thr Arg Arg Gly Thr
2480 2485 2490
Gly Asn Ile Gly Glu Thr Leu Gly Glu Lys Trp Lys Ser Arg Leu
2495 2500 2505
Asn Ala Leu Gly Lys Ser Glu Phe Gln Ile Tyr Lys Lys Ser Gly
2510 2515 2520
Ile Gln Glu Val Asp Arg Thr Leu Ala Lys Glu Gly Ile Lys Arg
2525 2530 2535
Gly Glu Thr Asp His His Ala Val Ser Arg Gly Ser Ala Lys Leu
2540 2545 2550
Arg Trp Phe Val Glu Arg Asn Met Val Thr Pro Glu Gly Lys Val
2555 2560 2565
Val Asp Leu Gly Cys Gly Arg Gly Gly Trp Ser Tyr Tyr Cys Gly
2570 2575 2580
Gly Leu Lys Asn Val Arg Glu Val Lys Gly Leu Thr Lys Gly Gly
2585 2590 2595
Pro Gly His Glu Glu Pro Ile Pro Met Ser Thr Tyr Gly Trp Asn
2600 2605 2610
Leu Val Arg Leu Gln Ser Gly Val Asp Val Phe Phe Ile Pro Pro
2615 2620 2625
Glu Lys Cys Asp Thr Leu Leu Cys Asp Ile Gly Glu Ser Ser Pro
2630 2635 2640
Asn Pro Thr Val Glu Ala Gly Arg Thr Leu Arg Val Leu Asn Leu
2645 2650 2655
Val Glu Asn Trp Leu Asn Asn Asn Thr Gln Phe Cys Ile Lys Val
2660 2665 2670
Leu Asn Pro Tyr Met Pro Ser Val Ile Glu Lys Met Glu Ala Leu
2675 2680 2685
Gln Arg Lys Tyr Gly Gly Ala Leu Val Arg Asn Pro Leu Ser Arg
2690 2695 2700
Asn Ser Thr His Glu Met Tyr Trp Val Ser Asn Ala Ser Gly Asn
2705 2710 2715
Ile Val Ser Ser Val Asn Met Ile Ser Arg Met Leu Ile Asn Arg
2720 2725 2730
Phe Thr Met Arg Tyr Lys Lys Ala Thr Tyr Glu Pro Asp Val Asp
2735 2740 2745
Leu Gly Ser Gly Thr Arg Asn Ile Gly Ile Glu Ser Glu Ile Pro
2750 2755 2760
Asn Leu Asp Ile Ile Gly Lys Arg Ile Glu Lys Ile Lys Gln Glu
2765 2770 2775
His Glu Thr Ser Trp His Tyr Asp Gln Asp His Pro Tyr Lys Thr
2780 2785 2790
Trp Ala Tyr His Gly Ser Tyr Glu Thr Lys Gln Thr Gly Ser Ala
2795 2800 2805
Ser Ser Met Val Asn Gly Val Val Arg Leu Leu Thr Lys Pro Trp
2810 2815 2820
Asp Val Val Pro Met Val Thr Gln Met Ala Met Thr Asp Thr Thr
2825 2830 2835
Pro Phe Gly Gln Gln Arg Val Phe Lys Glu Lys Val Asp Thr Arg
2840 2845 2850
Thr Gln Glu Pro Lys Glu Gly Thr Lys Lys Leu Met Lys Ile Thr
2855 2860 2865
Ala Glu Trp Leu Trp Lys Glu Leu Gly Lys Lys Lys Thr Pro Arg
2870 2875 2880
Met Cys Thr Arg Glu Glu Phe Thr Arg Lys Val Arg Ser Asn Ala
2885 2890 2895
Ala Leu Gly Ala Val Phe Thr Asp Glu Asn Lys Trp Lys Ser Ala
2900 2905 2910
Arg Glu Ala Val Glu Asp Ser Arg Phe Trp Glu Leu Val Asp Lys
2915 2920 2925
Glu Arg Asn Leu His Leu Glu Gly Lys Cys Glu Thr Cys Val Tyr
2930 2935 2940
Asn Met Met Gly Lys Arg Glu Lys Lys Leu Gly Glu Phe Gly Lys
2945 2950 2955
Ala Lys Gly Ser Arg Ala Ile Trp Tyr Met Trp Leu Gly Ala Arg
2960 2965 2970
Phe Leu Glu Phe Glu Ala Leu Gly Phe Leu Asn Glu Asp His Trp
2975 2980 2985
Phe Ser Arg Glu Asn Ser Leu Ser Gly Val Glu Gly Glu Gly Leu
2990 2995 3000
His Lys Leu Gly Tyr Ile Leu Arg Asp Val Ser Lys Lys Glu Gly
3005 3010 3015
Gly Ala Met Tyr Ala Asp Asp Thr Ala Gly Trp Asp Thr Arg Ile
3020 3025 3030
Thr Leu Glu Asp Leu Lys Asn Glu Glu Met Val Thr Asn His Met
3035 3040 3045
Glu Gly Glu His Lys Lys Leu Ala Glu Ala Ile Phe Lys Leu Thr
3050 3055 3060
Tyr Gln Asn Lys Val Val Arg Val Gln Arg Pro Thr Pro Arg Gly
3065 3070 3075
Thr Val Met Asp Ile Ile Ser Arg Arg Asp Gln Arg Gly Ser Gly
3080 3085 3090
Gln Val Gly Thr Tyr Gly Leu Asn Thr Phe Thr Asn Met Glu Ala
3095 3100 3105
Gln Leu Ile Arg Gln Met Glu Gly Glu Gly Val Phe Lys Ser Ile
3110 3115 3120
Gln His Leu Thr Ile Thr Glu Glu Ile Ala Val Gln Asn Trp Leu
3125 3130 3135
Ala Arg Val Gly Arg Glu Arg Leu Ser Arg Met Ala Ile Ser Gly
3140 3145 3150
Asp Asp Cys Val Val Lys Pro Leu Asp Asp Arg Phe Ala Ser Ala
3155 3160 3165
Leu Thr Ala Leu Asn Asp Met Gly Lys Ile Arg Lys Asp Ile Gln
3170 3175 3180
Gln Trp Glu Pro Ser Arg Gly Trp Asn Asp Trp Thr Gln Val Pro
3185 3190 3195
Phe Cys Ser His His Phe His Glu Leu Ile Met Lys Asp Gly Arg
3200 3205 3210
Val Leu Val Val Pro Cys Arg Asn Gln Asp Glu Leu Ile Gly Arg
3215 3220 3225
Ala Arg Ile Ser Gln Gly Ala Gly Trp Ser Leu Arg Glu Thr Ala
3230 3235 3240
Cys Leu Gly Lys Ser Tyr Ala Gln Met Trp Ser Leu Met Tyr Phe
3245 3250 3255
His Arg Arg Asp Leu Arg Leu Ala Ala Asn Ala Ile Cys Ser Ala
3260 3265 3270
Val Pro Ser His Trp Val Pro Thr Ser Arg Thr Thr Trp Ser Ile
3275 3280 3285
His Ala Lys His Glu Trp Met Thr Thr Glu Asp Met Leu Thr Val
3290 3295 3300
Trp Asn Arg Val Trp Ile Gln Glu Asn Pro Trp Met Glu Asp Lys
3305 3310 3315
Thr Pro Val Glu Ser Trp Glu Glu Ile Pro Tyr Leu Gly Lys Arg
3320 3325 3330
Glu Asp Gln Trp Cys Gly Ser Leu Ile Gly Leu Thr Ser Arg Ala
3335 3340 3345
Thr Trp Ala Lys Asn Ile Gln Ala Ala Ile Asn Gln Val Arg Ser
3350 3355 3360
Leu Ile Gly Asn Glu Glu Tyr Thr Asp Tyr Met Pro Ser Met Lys
3365 3370 3375
Arg Phe Arg Arg Glu Glu Glu Glu Ala Gly Val Leu Trp
3380 3385 3390
<210> 6
<211> 3391
<212> PRT
<213> 人工序列
<220>
<223> 登革热病毒血清型2, MVS
<400> 6
Met Asn Asn Gln Arg Lys Lys Ala Lys Asn Thr Pro Phe Asn Met Leu
1 5 10 15
Lys Arg Glu Arg Asn Arg Val Ser Thr Val Gln Gln Leu Thr Lys Arg
20 25 30
Phe Ser Leu Gly Met Leu Gln Gly Arg Gly Pro Leu Lys Leu Phe Met
35 40 45
Ala Leu Val Ala Phe Leu Arg Phe Leu Thr Ile Pro Pro Thr Ala Gly
50 55 60
Ile Leu Lys Arg Trp Gly Thr Ile Lys Lys Ser Lys Ala Ile Asn Val
65 70 75 80
Leu Arg Gly Phe Arg Lys Glu Ile Gly Arg Met Leu Asn Ile Leu Asn
85 90 95
Arg Arg Arg Arg Ser Ala Gly Met Ile Ile Met Leu Ile Pro Thr Val
100 105 110
Met Ala Phe His Leu Thr Thr Arg Asn Gly Glu Pro His Met Ile Val
115 120 125
Ser Arg Gln Glu Lys Gly Lys Ser Leu Leu Phe Lys Thr Glu Val Gly
130 135 140
Val Asn Met Cys Thr Leu Met Ala Met Asp Leu Gly Glu Leu Cys Glu
145 150 155 160
Asp Thr Ile Thr Tyr Glu Cys Pro Leu Leu Arg Gln Asn Glu Pro Glu
165 170 175
Asp Ile Asp Cys Trp Cys Asn Ser Thr Ser Thr Trp Val Thr Tyr Gly
180 185 190
Thr Cys Thr Thr Met Gly Glu His Arg Arg Glu Lys Arg Ser Val Ala
195 200 205
Leu Val Pro His Val Gly Met Gly Leu Glu Thr Arg Thr Glu Thr Trp
210 215 220
Met Ser Ser Glu Gly Ala Trp Lys His Val Gln Arg Ile Glu Thr Trp
225 230 235 240
Ile Leu Arg His Pro Gly Phe Thr Met Met Ala Ala Ile Leu Ala Tyr
245 250 255
Thr Ile Gly Thr Thr His Phe Gln Arg Ala Leu Ile Phe Ile Leu Leu
260 265 270
Thr Ala Val Thr Pro Ser Met Thr Met Arg Cys Ile Gly Met Ser Asn
275 280 285
Arg Asp Phe Val Glu Gly Val Ser Gly Gly Ser Trp Val Asp Ile Val
290 295 300
Leu Glu His Gly Ser Cys Val Thr Thr Met Ala Lys Asn Lys Pro Thr
305 310 315 320
Leu Asp Phe Glu Leu Ile Lys Thr Glu Ala Lys Gln Pro Ala Thr Leu
325 330 335
Arg Lys Tyr Cys Ile Glu Ala Lys Leu Thr Asn Thr Thr Thr Glu Ser
340 345 350
Arg Cys Pro Thr Gln Gly Glu Pro Ser Leu Asn Glu Glu Gln Asp Lys
355 360 365
Arg Phe Val Cys Lys His Ser Met Val Asp Arg Gly Trp Gly Asn Gly
370 375 380
Cys Gly Leu Phe Gly Lys Gly Gly Ile Val Thr Cys Ala Met Phe Arg
385 390 395 400
Cys Lys Lys Asn Met Glu Gly Lys Val Val Gln Pro Glu Asn Leu Glu
405 410 415
Tyr Thr Ile Val Ile Thr Pro His Ser Gly Glu Glu His Ala Val Gly
420 425 430
Asn Asp Thr Gly Lys His Gly Lys Glu Ile Lys Ile Thr Pro Gln Ser
435 440 445
Ser Ile Thr Glu Ala Glu Leu Thr Gly Tyr Gly Thr Val Thr Met Glu
450 455 460
Cys Ser Pro Arg Thr Gly Leu Asp Phe Asn Glu Met Val Leu Leu Gln
465 470 475 480
Met Glu Asn Lys Ala Trp Leu Val His Arg Gln Trp Phe Leu Asp Leu
485 490 495
Pro Leu Pro Trp Leu Pro Gly Ala Asp Thr Gln Gly Ser Asn Trp Ile
500 505 510
Gln Lys Glu Thr Leu Val Thr Phe Lys Asn Pro His Ala Lys Lys Gln
515 520 525
Asp Val Val Val Leu Gly Ser Gln Glu Gly Ala Met His Thr Ala Leu
530 535 540
Thr Gly Ala Thr Glu Ile Gln Met Ser Ser Gly Asn Leu Leu Phe Thr
545 550 555 560
Gly His Leu Lys Cys Arg Leu Arg Met Asp Lys Leu Gln Leu Lys Gly
565 570 575
Met Ser Tyr Ser Met Cys Thr Gly Lys Phe Lys Val Val Lys Glu Ile
580 585 590
Ala Glu Thr Gln His Gly Thr Ile Val Ile Arg Val Gln Tyr Glu Gly
595 600 605
Asp Gly Ser Pro Cys Lys Ile Pro Phe Glu Ile Met Asp Leu Glu Lys
610 615 620
Arg His Val Leu Gly Arg Leu Ile Thr Val Asn Pro Ile Val Thr Glu
625 630 635 640
Lys Asp Ser Pro Val Asn Ile Glu Ala Glu Pro Pro Phe Gly Asp Ser
645 650 655
Tyr Ile Ile Ile Gly Val Glu Pro Gly Gln Leu Lys Leu Asn Trp Phe
660 665 670
Lys Lys Gly Ser Ser Ile Gly Gln Met Phe Glu Thr Thr Met Arg Gly
675 680 685
Ala Lys Arg Met Ala Ile Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser
690 695 700
Leu Gly Gly Val Phe Thr Ser Ile Gly Lys Ala Leu His Gln Val Phe
705 710 715 720
Gly Ala Ile Tyr Gly Ala Ala Phe Ser Gly Val Ser Trp Thr Met Lys
725 730 735
Ile Leu Ile Gly Val Ile Ile Thr Trp Ile Gly Met Asn Ser Arg Ser
740 745 750
Thr Ser Leu Ser Val Thr Leu Val Leu Val Gly Ile Val Thr Leu Tyr
755 760 765
Leu Gly Val Met Val Gln Ala Asp Ser Gly Cys Val Val Ser Trp Lys
770 775 780
Asn Lys Glu Leu Lys Cys Gly Ser Gly Ile Phe Ile Thr Asp Asn Val
785 790 795 800
His Thr Trp Thr Glu Gln Tyr Lys Phe Gln Pro Glu Ser Pro Ser Lys
805 810 815
Leu Ala Ser Ala Ile Gln Lys Ala His Glu Glu Asp Ile Cys Gly Ile
820 825 830
Arg Ser Val Thr Arg Leu Glu Asn Leu Met Trp Lys Gln Ile Thr Pro
835 840 845
Glu Leu Asn His Ile Leu Ser Glu Asn Glu Val Lys Leu Thr Ile Met
850 855 860
Thr Gly Asp Ile Lys Gly Ile Met Gln Ala Gly Lys Arg Ser Leu Arg
865 870 875 880
Pro Gln Pro Thr Glu Leu Lys Tyr Ser Trp Lys Thr Trp Gly Lys Ala
885 890 895
Lys Met Leu Ser Thr Glu Ser His Asn Gln Thr Phe Leu Ile Asp Gly
900 905 910
Pro Glu Thr Ala Glu Cys Pro Asn Thr Asn Arg Ala Trp Asn Ser Leu
915 920 925
Glu Val Glu Asp Tyr Gly Phe Gly Val Phe Thr Thr Asn Ile Trp Leu
930 935 940
Lys Leu Lys Glu Lys Gln Asp Val Phe Cys Asp Ser Lys Leu Met Ser
945 950 955 960
Ala Ala Ile Lys Asp Asn Arg Ala Val His Ala Asp Met Gly Tyr Trp
965 970 975
Ile Glu Ser Ala Leu Asn Asp Thr Trp Lys Ile Glu Lys Ala Ser Phe
980 985 990
Ile Glu Val Lys Asn Cys His Trp Pro Lys Ser His Thr Leu Trp Ser
995 1000 1005
Asn Gly Val Leu Glu Ser Glu Met Ile Ile Pro Lys Asn Leu Ala
1010 1015 1020
Gly Pro Val Ser Gln His Asn Tyr Arg Pro Gly Tyr His Thr Gln
1025 1030 1035
Ile Thr Gly Pro Trp His Leu Gly Lys Leu Glu Met Asp Phe Asp
1040 1045 1050
Phe Cys Asp Gly Thr Thr Val Val Val Thr Glu Asp Cys Gly Asn
1055 1060 1065
Arg Gly Pro Ser Leu Arg Thr Thr Thr Ala Ser Gly Lys Leu Ile
1070 1075 1080
Thr Glu Trp Cys Cys Arg Ser Cys Thr Leu Pro Pro Leu Arg Tyr
1085 1090 1095
Arg Gly Glu Asp Gly Cys Trp Tyr Gly Met Glu Ile Arg Pro Leu
1100 1105 1110
Lys Glu Lys Glu Glu Asn Leu Val Asn Ser Leu Val Thr Ala Gly
1115 1120 1125
His Gly Gln Val Asp Asn Phe Ser Leu Gly Val Leu Gly Met Ala
1130 1135 1140
Leu Phe Leu Glu Glu Met Leu Arg Thr Arg Val Gly Thr Lys His
1145 1150 1155
Ala Ile Leu Leu Val Ala Val Ser Phe Val Thr Leu Ile Thr Gly
1160 1165 1170
Asn Met Ser Phe Arg Asp Leu Gly Arg Val Met Val Met Val Gly
1175 1180 1185
Ala Thr Met Thr Asp Asp Ile Gly Met Gly Val Thr Tyr Leu Ala
1190 1195 1200
Leu Leu Ala Ala Phe Lys Val Arg Pro Thr Phe Ala Ala Gly Leu
1205 1210 1215
Leu Leu Arg Lys Leu Thr Ser Lys Glu Leu Met Met Thr Thr Ile
1220 1225 1230
Gly Ile Val Leu Leu Ser Gln Ser Thr Ile Pro Glu Thr Ile Leu
1235 1240 1245
Glu Leu Thr Asp Ala Leu Ala Leu Gly Met Met Val Leu Lys Met
1250 1255 1260
Val Arg Asn Met Glu Lys Tyr Gln Leu Ala Val Thr Ile Met Ala
1265 1270 1275
Ile Leu Cys Val Pro Asn Ala Val Ile Leu Gln Asn Ala Trp Lys
1280 1285 1290
Val Ser Cys Thr Ile Leu Ala Val Val Ser Val Ser Pro Leu Phe
1295 1300 1305
Leu Thr Ser Ser Gln Gln Lys Thr Asp Trp Ile Pro Leu Ala Leu
1310 1315 1320
Thr Ile Lys Gly Leu Asn Pro Thr Ala Ile Phe Leu Thr Thr Leu
1325 1330 1335
Ser Arg Thr Ser Lys Lys Arg Ser Trp Pro Leu Asn Glu Ala Ile
1340 1345 1350
Met Ala Val Gly Met Val Ser Ile Leu Ala Ser Ser Leu Leu Lys
1355 1360 1365
Asn Asp Ile Pro Met Thr Gly Pro Leu Val Ala Gly Gly Leu Leu
1370 1375 1380
Thr Val Cys Tyr Val Leu Thr Gly Arg Ser Ala Asp Leu Glu Leu
1385 1390 1395
Glu Arg Ala Ala Asp Val Lys Trp Glu Asp Gln Ala Glu Ile Ser
1400 1405 1410
Gly Ser Ser Pro Ile Leu Ser Ile Thr Ile Ser Glu Asp Gly Ser
1415 1420 1425
Met Ser Ile Lys Asn Glu Glu Glu Glu Gln Thr Leu Thr Ile Leu
1430 1435 1440
Ile Arg Thr Gly Leu Leu Val Ile Ser Gly Leu Phe Pro Val Ser
1445 1450 1455
Ile Pro Ile Thr Ala Ala Ala Trp Tyr Leu Trp Glu Val Lys Lys
1460 1465 1470
Gln Arg Ala Gly Val Leu Trp Asp Val Pro Ser Pro Pro Pro Met
1475 1480 1485
Gly Lys Ala Glu Leu Glu Asp Gly Ala Tyr Arg Ile Lys Gln Lys
1490 1495 1500
Gly Ile Leu Gly Tyr Ser Gln Ile Gly Ala Gly Val Tyr Lys Glu
1505 1510 1515
Gly Thr Phe His Thr Met Trp His Val Thr Arg Gly Ala Val Leu
1520 1525 1530
Met His Lys Gly Lys Arg Ile Glu Pro Ser Trp Ala Asp Val Lys
1535 1540 1545
Lys Asp Leu Ile Ser Tyr Gly Gly Gly Trp Lys Leu Glu Gly Glu
1550 1555 1560
Trp Lys Glu Gly Glu Glu Val Gln Val Leu Ala Leu Glu Pro Gly
1565 1570 1575
Lys Asn Pro Arg Ala Val Gln Thr Lys Pro Gly Leu Phe Lys Thr
1580 1585 1590
Asn Ala Gly Thr Ile Gly Ala Val Ser Leu Asp Phe Ser Pro Gly
1595 1600 1605
Thr Ser Gly Ser Pro Ile Ile Asp Lys Lys Gly Lys Val Val Gly
1610 1615 1620
Leu Tyr Gly Asn Gly Val Val Thr Arg Ser Gly Ala Tyr Val Ser
1625 1630 1635
Ala Ile Ala Gln Thr Glu Lys Ser Ile Glu Asp Asn Pro Glu Ile
1640 1645 1650
Glu Asp Asp Ile Phe Arg Lys Arg Arg Leu Thr Ile Met Asp Leu
1655 1660 1665
His Pro Gly Ala Gly Lys Thr Lys Arg Tyr Leu Pro Ala Ile Val
1670 1675 1680
Arg Glu Ala Ile Lys Arg Gly Leu Arg Thr Leu Ile Leu Ala Pro
1685 1690 1695
Thr Arg Val Val Ala Ala Glu Met Glu Glu Ala Leu Arg Gly Leu
1700 1705 1710
Pro Ile Arg Tyr Gln Thr Pro Ala Ile Arg Ala Val His Thr Gly
1715 1720 1725
Arg Glu Ile Val Asp Leu Met Cys His Ala Thr Phe Thr Met Arg
1730 1735 1740
Leu Leu Ser Pro Val Arg Val Pro Asn Tyr Asn Leu Ile Ile Met
1745 1750 1755
Asp Glu Ala His Phe Thr Asp Pro Ala Ser Ile Ala Ala Arg Gly
1760 1765 1770
Tyr Ile Ser Thr Arg Val Glu Met Gly Glu Ala Ala Gly Ile Phe
1775 1780 1785
Met Thr Ala Thr Pro Pro Gly Ser Arg Asp Pro Phe Pro Gln Ser
1790 1795 1800
Asn Ala Pro Ile Ile Asp Glu Glu Arg Glu Ile Pro Glu Arg Ser
1805 1810 1815
Trp Asn Ser Gly His Glu Trp Val Thr Asp Phe Lys Gly Lys Thr
1820 1825 1830
Val Trp Phe Val Pro Ser Ile Lys Ala Gly Asn Asp Ile Ala Ala
1835 1840 1845
Cys Leu Arg Lys Asn Gly Lys Lys Val Ile Gln Leu Ser Arg Lys
1850 1855 1860
Thr Phe Asp Ser Glu Tyr Val Lys Thr Arg Thr Asn Asp Trp Asp
1865 1870 1875
Phe Val Val Thr Thr Asp Ile Ser Glu Met Gly Ala Asn Phe Lys
1880 1885 1890
Ala Glu Arg Val Ile Asp Pro Arg Arg Cys Met Lys Pro Val Ile
1895 1900 1905
Leu Thr Asp Gly Glu Glu Arg Val Ile Leu Ala Gly Pro Met Pro
1910 1915 1920
Val Thr His Ser Ser Ala Ala Gln Arg Arg Gly Arg Ile Gly Arg
1925 1930 1935
Asn Pro Lys Asn Glu Asn Asp Gln Tyr Ile Tyr Met Gly Glu Pro
1940 1945 1950
Leu Glu Asn Asp Glu Asp Cys Ala His Trp Lys Glu Ala Lys Met
1955 1960 1965
Leu Leu Asp Asn Ile Asn Thr Pro Glu Gly Ile Ile Pro Ser Met
1970 1975 1980
Phe Glu Pro Glu Arg Glu Lys Val Asp Ala Ile Asp Gly Glu Tyr
1985 1990 1995
Arg Leu Arg Gly Glu Ala Arg Lys Thr Phe Val Asp Leu Met Arg
2000 2005 2010
Arg Gly Asp Leu Pro Val Trp Leu Ala Tyr Arg Val Ala Ala Glu
2015 2020 2025
Gly Ile Asn Tyr Ala Asp Arg Arg Trp Cys Phe Asp Gly Val Lys
2030 2035 2040
Asn Asn Gln Ile Leu Glu Glu Asn Val Glu Val Glu Ile Trp Thr
2045 2050 2055
Lys Glu Gly Glu Arg Lys Lys Leu Lys Pro Arg Trp Leu Asp Ala
2060 2065 2070
Arg Ile Tyr Ser Asp Pro Leu Ala Leu Lys Glu Phe Lys Glu Phe
2075 2080 2085
Ala Ala Gly Arg Lys Ser Leu Thr Leu Asn Leu Ile Thr Glu Met
2090 2095 2100
Gly Arg Leu Pro Thr Phe Met Thr Gln Lys Ala Arg Asp Ala Leu
2105 2110 2115
Asp Asn Leu Ala Val Leu His Thr Ala Glu Ala Gly Gly Arg Ala
2120 2125 2130
Tyr Asn His Ala Leu Ser Glu Leu Pro Glu Thr Leu Glu Thr Leu
2135 2140 2145
Leu Leu Leu Thr Leu Leu Ala Thr Val Thr Gly Gly Ile Phe Leu
2150 2155 2160
Phe Leu Met Ser Ala Arg Gly Ile Gly Lys Met Thr Leu Gly Met
2165 2170 2175
Cys Cys Ile Ile Thr Ala Ser Ile Leu Leu Trp Tyr Ala Gln Ile
2180 2185 2190
Gln Pro His Trp Ile Ala Ala Ser Ile Ile Leu Glu Phe Phe Leu
2195 2200 2205
Ile Val Leu Leu Ile Pro Glu Pro Glu Lys Gln Arg Thr Pro Gln
2210 2215 2220
Asp Asn Gln Leu Thr Tyr Val Val Ile Ala Ile Leu Thr Val Val
2225 2230 2235
Ala Ala Thr Met Ala Asn Glu Met Gly Phe Leu Glu Lys Thr Lys
2240 2245 2250
Lys Asp Leu Gly Leu Gly Ser Ile Ala Thr Gln Gln Pro Glu Ser
2255 2260 2265
Asn Ile Leu Asp Ile Asp Leu Arg Pro Ala Ser Ala Trp Thr Leu
2270 2275 2280
Tyr Ala Val Ala Thr Thr Phe Val Thr Pro Met Leu Arg His Ser
2285 2290 2295
Ile Glu Asn Ser Ser Val Asn Val Ser Leu Thr Ala Ile Ala Asn
2300 2305 2310
Gln Ala Thr Val Leu Met Gly Leu Gly Lys Gly Trp Pro Leu Ser
2315 2320 2325
Lys Met Asp Ile Gly Val Pro Leu Leu Ala Ile Gly Cys Tyr Ser
2330 2335 2340
Gln Val Asn Pro Ile Thr Leu Thr Ala Ala Leu Phe Leu Leu Val
2345 2350 2355
Ala His Tyr Ala Ile Ile Gly Pro Gly Leu Gln Ala Lys Ala Thr
2360 2365 2370
Arg Glu Ala Gln Lys Arg Ala Ala Ala Gly Ile Met Lys Asn Pro
2375 2380 2385
Thr Val Asp Gly Ile Thr Val Ile Asp Leu Asp Pro Ile Pro Tyr
2390 2395 2400
Asp Pro Lys Phe Glu Lys Gln Leu Gly Gln Val Met Leu Leu Val
2405 2410 2415
Leu Cys Val Thr Gln Val Leu Met Met Arg Thr Thr Trp Ala Leu
2420 2425 2430
Cys Glu Ala Leu Thr Leu Ala Thr Gly Pro Ile Ser Thr Leu Trp
2435 2440 2445
Glu Gly Asn Pro Gly Arg Phe Trp Asn Thr Thr Ile Ala Val Ser
2450 2455 2460
Met Ala Asn Ile Phe Arg Gly Ser Tyr Leu Ala Gly Ala Gly Leu
2465 2470 2475
Leu Phe Ser Ile Met Lys Asn Thr Thr Asn Thr Arg Arg Gly Thr
2480 2485 2490
Gly Asn Ile Gly Glu Thr Leu Gly Glu Lys Trp Lys Ser Arg Leu
2495 2500 2505
Asn Ala Leu Gly Lys Ser Glu Phe Gln Ile Tyr Lys Lys Ser Gly
2510 2515 2520
Ile Gln Glu Val Asp Arg Thr Leu Ala Lys Glu Gly Ile Lys Arg
2525 2530 2535
Gly Glu Thr Asp His His Ala Val Ser Arg Gly Ser Ala Lys Leu
2540 2545 2550
Arg Trp Phe Val Glu Arg Asn Met Val Thr Pro Glu Gly Lys Val
2555 2560 2565
Val Asp Leu Gly Cys Gly Arg Gly Gly Trp Ser Tyr Tyr Cys Gly
2570 2575 2580
Gly Leu Lys Asn Val Arg Glu Val Lys Gly Leu Thr Lys Gly Gly
2585 2590 2595
Pro Gly His Glu Glu Pro Ile Pro Met Ser Thr Tyr Gly Trp Asn
2600 2605 2610
Leu Val Arg Leu Gln Ser Gly Val Asp Val Phe Phe Ile Pro Pro
2615 2620 2625
Glu Lys Cys Asp Thr Leu Leu Cys Asp Ile Gly Glu Ser Ser Pro
2630 2635 2640
Asn Pro Thr Val Glu Ala Gly Arg Thr Leu Arg Val Leu Asn Leu
2645 2650 2655
Val Glu Asn Trp Leu Asn Asn Asn Thr Gln Phe Cys Ile Lys Val
2660 2665 2670
Leu Asn Pro Tyr Met Pro Ser Val Ile Glu Lys Met Glu Ala Leu
2675 2680 2685
Gln Arg Lys Tyr Gly Gly Ala Leu Val Arg Asn Pro Leu Ser Arg
2690 2695 2700
Asn Ser Thr His Glu Met Tyr Trp Val Ser Asn Ala Ser Gly Asn
2705 2710 2715
Ile Val Ser Ser Val Asn Met Ile Ser Arg Met Leu Ile Asn Arg
2720 2725 2730
Phe Thr Met Arg Tyr Lys Lys Ala Thr Tyr Glu Pro Asp Val Asp
2735 2740 2745
Leu Gly Ser Gly Thr Arg Asn Ile Gly Ile Glu Ser Glu Ile Pro
2750 2755 2760
Asn Leu Asp Ile Ile Gly Lys Arg Ile Glu Lys Ile Lys Gln Glu
2765 2770 2775
His Glu Thr Ser Trp His Tyr Asp Gln Asp His Pro Tyr Lys Thr
2780 2785 2790
Trp Ala Tyr His Gly Ser Tyr Glu Thr Lys Gln Thr Gly Ser Ala
2795 2800 2805
Ser Ser Met Val Asn Gly Val Val Arg Leu Leu Thr Lys Pro Trp
2810 2815 2820
Asp Val Val Pro Met Val Thr Gln Met Ala Met Thr Asp Thr Thr
2825 2830 2835
Pro Phe Gly Gln Gln Arg Val Phe Lys Glu Lys Val Asp Thr Arg
2840 2845 2850
Thr Gln Glu Pro Lys Glu Gly Thr Lys Lys Leu Met Lys Ile Thr
2855 2860 2865
Ala Glu Trp Leu Trp Lys Glu Leu Gly Lys Lys Lys Thr Pro Arg
2870 2875 2880
Met Cys Thr Arg Glu Glu Phe Thr Arg Lys Val Arg Ser Asn Ala
2885 2890 2895
Ala Leu Gly Ala Val Phe Thr Asp Glu Asn Lys Trp Lys Ser Ala
2900 2905 2910
Arg Glu Ala Val Glu Asp Ser Arg Phe Trp Glu Leu Val Asp Lys
2915 2920 2925
Glu Arg Asn Leu His Leu Glu Gly Lys Cys Glu Thr Cys Val Tyr
2930 2935 2940
Asn Met Met Gly Lys Arg Glu Lys Lys Leu Gly Glu Phe Gly Lys
2945 2950 2955
Ala Lys Gly Ser Arg Ala Ile Trp Tyr Met Trp Leu Gly Ala Arg
2960 2965 2970
Phe Leu Glu Phe Glu Ala Leu Gly Phe Leu Asn Glu Asp His Trp
2975 2980 2985
Phe Ser Arg Glu Asn Ser Leu Ser Gly Val Glu Gly Glu Gly Leu
2990 2995 3000
His Lys Leu Gly Tyr Ile Leu Arg Asp Val Ser Lys Lys Glu Gly
3005 3010 3015
Gly Ala Met Tyr Ala Asp Asp Thr Ala Gly Trp Asp Thr Arg Ile
3020 3025 3030
Thr Leu Glu Asp Leu Lys Asn Glu Glu Met Val Thr Asn His Met
3035 3040 3045
Glu Gly Glu His Lys Lys Leu Ala Glu Ala Ile Phe Lys Leu Thr
3050 3055 3060
Tyr Gln Asn Lys Val Val Arg Val Gln Arg Pro Thr Pro Arg Gly
3065 3070 3075
Thr Val Met Asp Ile Ile Ser Arg Arg Asp Gln Arg Gly Ser Gly
3080 3085 3090
Gln Val Gly Thr Tyr Gly Leu Asn Thr Phe Thr Asn Met Glu Ala
3095 3100 3105
Gln Leu Ile Arg Gln Met Glu Gly Glu Gly Val Phe Lys Ser Ile
3110 3115 3120
Gln His Leu Thr Ile Thr Glu Glu Ile Ala Val Gln Asn Trp Leu
3125 3130 3135
Ala Arg Val Gly Arg Glu Arg Leu Ser Arg Met Ala Ile Ser Gly
3140 3145 3150
Asp Asp Cys Val Val Lys Pro Leu Asp Asp Arg Phe Ala Ser Ala
3155 3160 3165
Leu Thr Ala Leu Asn Asp Met Gly Lys Ile Arg Lys Asp Ile Gln
3170 3175 3180
Gln Trp Glu Pro Ser Arg Gly Trp Asn Asp Trp Thr Gln Val Pro
3185 3190 3195
Phe Cys Ser His His Phe His Glu Leu Ile Met Lys Asp Gly Arg
3200 3205 3210
Val Leu Val Val Pro Cys Arg Asn Gln Asp Glu Leu Ile Gly Arg
3215 3220 3225
Ala Arg Ile Ser Gln Gly Ala Gly Trp Ser Leu Arg Glu Thr Ala
3230 3235 3240
Cys Leu Gly Lys Ser Tyr Ala Gln Met Trp Ser Leu Met Tyr Phe
3245 3250 3255
His Arg Arg Asp Leu Arg Leu Ala Ala Asn Ala Ile Cys Ser Ala
3260 3265 3270
Val Pro Ser His Trp Val Pro Thr Ser Arg Thr Thr Trp Ser Ile
3275 3280 3285
His Ala Lys His Glu Trp Met Thr Thr Glu Asp Met Leu Thr Val
3290 3295 3300
Trp Asn Arg Val Trp Ile Gln Glu Asn Pro Trp Met Glu Asp Lys
3305 3310 3315
Thr Pro Val Glu Ser Trp Glu Glu Ile Pro Tyr Leu Gly Lys Arg
3320 3325 3330
Glu Asp Gln Trp Cys Gly Ser Leu Ile Gly Leu Thr Ser Arg Ala
3335 3340 3345
Thr Trp Ala Lys Asn Ile Gln Ala Ala Ile Asn Gln Val Arg Ser
3350 3355 3360
Leu Ile Gly Asn Glu Glu Tyr Thr Asp Tyr Met Pro Ser Met Lys
3365 3370 3375
Arg Phe Arg Arg Glu Glu Glu Glu Ala Gly Val Leu Trp
3380 3385 3390
<210> 7
<211> 10717
<212> DNA
<213> 人工序列
<220>
<223> 登革热病毒血清型3, BVS
<400> 7
agttgttagt ctacgtggac cgacaaagac agattctttg agggagctaa gctcaatgta 60
gttctaacag ttttttaatt agagagcaga tctctgatga ataaccaacg gaaaaaggcg 120
aaaaacacgc ctttcaatat gctgaaacgc gagagaaacc gcgtgtcgac tgtgcaacag 180
ctgacaaaga gattctcact tggaatgctg cagggacgag gaccattaaa actgttcatg 240
gccctggtgg cgttccttcg tttcctaaca atcccaccaa cagcagggat attgaagaga 300
tggggaacaa ttaaaaaatc aaaagctatt aatgttttga gagggttcag gaaagagatt 360
ggaaggatgc tgaacatctt gaataggaga cgcagatctg caggcatgat cattatgctg 420
attccaacag tgatggcgtt ccatttaacc acgcgtgatg gagagccgcg catgattgtg 480
gggaagaatg aaagaggaaa atccctactt ttcaagacag cctctggaat caacatgtgc 540
acactcatag ctatggatct gggagagatg tgtgatgaca cggtcactta caaatgcccc 600
cacattaccg aagtggagcc tgaagacatt gactgctggt gcaaccttac atcgacatgg 660
gtgacttatg gaacatgcaa tcaagctgga gagcatagac gcgataagag atcagtggcg 720
ttagctcccc atgttggcat gggactggac acacgcactc aaacctggat gtcggctgaa 780
ggagcttgga gacaagtcga gaaggtagag acatgggccc ttaggcaccc agggtttacc 840
atactagccc tatttcttgc ccattacata ggcacttcct tgacccagaa agtggttatt 900
tttatactat taatgctggt taccccatcc atgacaatga gatgtgtagg agtaggaaac 960
agagattttg tggaaggcct atcgggagct acgtgggttg acgtggtgct cgagcacggt 1020
gggtgtgtga ctaccatggc taagaacaag cccacgctgg acatagagct tcagaagacc 1080
gaggccaccc aactggcgac cctaaggaag ctatgcattg agggaaaaat taccaacata 1140
acaaccgact caagatgtcc cacccaaggg gaagcgattt tacctgagga gcaggaccag 1200
aactacgtgt gtaagcatac atacgtggac agaggctggg gaaacggttg tggtttgttt 1260
ggcaagggaa gcttggtgac atgcgcgaaa tttcaatgtt tagaatcaat agagggaaaa 1320
gtggtgcaac atgagaacct caaatacacc gtcatcatca cagtgcacac aggagaccaa 1380
caccaggtgg gaaatgaaac gcagggagtc acggctgaga taacacccca ggcatcaacc 1440
gctgaagcca ttttacctga atatggaacc ctcgggctag aatgctcacc acggacaggt 1500
ttggatttca atgaaatgat ctcattgaca atgaagaaca aagcatggat ggtacataga 1560
caatggttct ttgacttacc cctaccatgg acatcaggag cttcagcaga aacaccaact 1620
tggaacagga aagagcttct tgtgacattt aaaaatgcac atgcaaaaaa gcaagaagta 1680
gttgttcttg gatcacaaga gggagcaatg catacagcac tgacaggagc tacagagatc 1740
caaacctcag gaggcacaag tatctttgcg gggcacttaa aatgtagact caagatggac 1800
aaattggaac tcaaggggat gagctatgca atgtgcttga gtagctttgt gttgaagaaa 1860
gaagtctccg aaacgcagca tgggacaata ctcattaagg ttgagtacaa aggggaagat 1920
gcaccctgca agattccttt ctccacggag gatggacaag gaaaagctct caatggcaga 1980
ctgatcacag ccaatccagt ggtgaccaag aaggaggagc ctgtcaacat tgaggctgaa 2040
cctccttttg gagaaagtaa catagtaatt ggaattggag acaaagccct gaaaatcaac 2100
tggtacaaga agggaagctc gattgggaag atgttcgagg ccactgccag aggtgcaagg 2160
cgcatggcca tcttgggaga cacagcctgg gactttggat cagtgggtgg tgttttgaat 2220
tcattaggga aaatggtcca ccaaatattt gggagtgctt acacagccct atttggtgga 2280
gtctcctgga tgatgaaaat tggaataggt gtcctcttaa cctggatagg gttgaactca 2340
aaaaatactt ctatgtcatt ttcatgcatc gcggccggca ttgtgacact gtatttggga 2400
gtcatggtgc aggccgatag tggttgcgtt gtgagctgga aaaacaaaga actgaaatgt 2460
ggcagtggga ttttcatcac agacaacgtg cacacatgga cagaacaata caagttccaa 2520
ccagaatccc cttcaaaact agcttcagct atccagaaag cccatgaaga ggacatttgt 2580
ggaatccgct cagtaacaag actggagaat ctgatgtgga aacaaataac accagaattg 2640
aatcacattc tatcagaaaa tgaggtgaag ttaactatta tgacaggaga catcaaagga 2700
atcatgcagg caggaaaacg atctctgcgg cctcagccca ctgagctgaa gtattcatgg 2760
aaaacatggg gcaaagcaaa aatgctctct acagagtctc ataaccagac ctttctcatt 2820
gatggccccg aaacagcaga atgccccaac acaaatagag cttggaattc gttggaagtt 2880
gaagactatg gctttggagt attcaccacc aatatatggc taaaattgaa agaaaaacag 2940
gatgtattct gcgactcaaa actcatgtca gcggccataa aagacaacag agccgtccat 3000
gccgatatgg gttattggat agaaagtgca ctcaatgaca catggaagat agagaaagcc 3060
tctttcattg aagttaaaaa ctgccactgg ccaaaatcac acaccctctg gagcaatgga 3120
gtgctagaaa gtgagatgat aattccaaag aatctcgctg gaccagtgtc tcaacacaac 3180
tatagaccag gctaccatac acaaataaca ggaccatggc atctaggtaa gcttgagatg 3240
gactttgatt tctgtgatgg aacaacagtg gtagtgactg aggactgcgg aaatagagga 3300
ccctctttga gaacaaccac tgcctctgga aaactcataa cagaatggtg ctgccgatct 3360
tgcacattac caccgctaag atacagaggt gaggatgggt gctggtacgg gatggaaatc 3420
agaccattga aggagaaaga agagaatttg gtcaactcct tggtcacagc tggacatggg 3480
caggtcgaca acttttcact aggagtcttg ggaatggcat tgttcctgga ggaaatgctt 3540
aggacccgag taggaacgaa acatgcaata ctactagttg cagtttcttt tgtgacattg 3600
atcacaggga acatgtcctt tagagacctg ggaagagtga tggttatggt aggcgccact 3660
atgacggatg acataggtat gggcgtgact tatcttgccc tactagcagc cttcaaagtc 3720
agaccaactt ttgcagctgg actactcttg agaaagctga cctccaagga attgatgatg 3780
actactatag gaattgtact cctctcccag agcaccatac cagagaccat tcttgagttg 3840
actgatgcgt tagccttagg catgatggtc ctcaaaatgg tgagaaatat ggaaaagtat 3900
caattggcag tgactatcat ggctatcttg tgcgtcccaa acgcagtgat attacaaaac 3960
gcatggaaag tgagttgcac aatattggca gtggtgtccg tttccccact gttcttaaca 4020
tcctcacagc aaaaaacaga ttggatacca ttagcattga cgatcaaagg tctcaatcca 4080
acagctattt ttctaacaac cctctcaaga accagcaaga aaaggagctg gccattaaat 4140
gaggctatca tggcagtcgg gatggtgagc attttagcca gttctctcct aaaaaatgat 4200
attcccatga caggaccatt agtggctgga gggctcctca ctgtgtgcta cgtgctcact 4260
ggacgatcgg ccgatttgga actggagaga gcagccgatg tcaaatggga agaccaggca 4320
gagatatcag gaagcagtcc aatcctgtca ataacaatat cagaagatgg tagcatgtcg 4380
ataaaaaatg aagaggaaga acaaacactg accatactca ttagaacagg attgctggtg 4440
atctcaggac tttttcctgt atcaatacca atcacggcag cagcatggta cctgtgggaa 4500
gtgaagaaac aacgggccgg agtattgtgg gatgttcctt cacccccacc catgggaaag 4560
gctgaactgg aagatggagc ctatagaatt aagcaaaaag ggattcttgg atattcccag 4620
atcggagccg gagtttacaa agaaggaaca ttccatacaa tgtggcatgt cacacgtggc 4680
gctgttctaa tgcataaagg aaagaggatt gaaccatcat gggcggacgt caagaaagac 4740
ctaatatcat atggaggagg ctggaagtta gaaggagaat ggaaggaagg agaagaagtc 4800
caggtattgg cactggagcc tggaaaaaat ccaagagccg tccaaacgaa acctggtctt 4860
ttcaaaacca acgccggaac aataggtgct gtatctctgg acttttctcc tggaacgtca 4920
ggatctccaa ttatcgacaa aaaaggaaaa gttgtgggtc tttatggtaa tggtgttgtt 4980
acaaggagtg gagcatatgt gagtgctata gcccagactg aaaaaagcat tgaagacaac 5040
ccagagatcg aagatgacat tttccgaaag agaagactga ccatcatgga cctccaccca 5100
ggagcgggaa agacgaagag ataccttccg gccatagtca gagaagctat aaaacggggt 5160
ttgagaacat taatcttggc ccccactaga gttgtggcag ctgaaatgga ggaagccctt 5220
agaggacttc caataagata ccagacccca gccatcagag ctgtgcacac cgggcgggag 5280
attgtggacc taatgtgtca tgccacattt accatgaggc tgctatcacc agttagagtg 5340
ccaaactaca acctgattat catggacgaa gcccatttca cagacccagc aagtatagca 5400
gctagaggat acatctcaac tcgagtggag atgggtgagg cagctgggat ttttatgaca 5460
gccactcccc cgggaagcag agacccattt cctcagagca atgcaccaat catagatgaa 5520
gaaagagaaa tccctgaacg ctcgtggaat tccggacatg aatgggtcac ggattttaaa 5580
gggaagactg tttggttcgt tccaagtata aaagcaggaa atgatatagc agcttgcctg 5640
aggaaaaatg gaaagaaagt gatacaactc agtaggaaga cctttgattc tgagtatgtc 5700
aagactagaa ccaatgattg ggacttcgtg gttacaactg acatttcaga aatgggtgcc 5760
aatttcaagg ctgagagggt tatagacccc agacgctgca tgaaaccagt catactaaca 5820
gatggtgaag agcgggtgat tctggcagga cctatgccag tgacccactc tagtgcagca 5880
caaagaagag ggagaatagg aagaaatcca aaaaatgaga atgaccagta catatacatg 5940
ggggaacctc tggaaaatga tgaagactgt gcacactgga aagaagctaa aatgctccta 6000
gataacatca acacgccaga aggaatcatt cctagcatgt tcgaaccaga gcgtgaaaag 6060
gtggatgcca ttgatggcga ataccgcttg agaggagaag caaggaaaac ctttgtagac 6120
ttaatgagaa gaggagacct accagtctgg ttggcctaca gagtggcagc tgaaggcatc 6180
aactacgcag acagaaggtg gtgttttgat ggagtcaaga acaaccaaat cctagaagaa 6240
aacgtggaag ttgaaatctg gacaaaagaa ggggaaagga agaaattgaa acccagatgg 6300
ttggatgcta ggatctattc tgacccactg gcgctaaaag aatttaagga atttgcagcc 6360
ggaagaaagt ctctgaccct gaacctaatc acagaaatgg gtaggctccc aaccttcatg 6420
actcagaagg caagaaacgc actggacaac ttagcagtgc tgcacacggc tgaggcaggt 6480
ggaagggcgt acaaccatgc tctcagtgaa ctgccggaga ccctggagac attgctttta 6540
ctgacacttc tggctacagt cacgggaggg atctttttat tcttgatgag cgcaaggggc 6600
atagggaaga tgaccctggg aatgtgctgc ataatcacgg ctagcatcct cctatggtac 6660
gcacaaatac agccacactg gatagcagct tcaataatac tggagttttt tctcatagtt 6720
ttgcttattc cagaacctga aaaacagaga acaccccaag acaaccaact gacctacgtt 6780
gtcatagcca tcctcacagt ggtggccgca accatggcaa acgagatggg tttcctagaa 6840
aaaacgaaga aagatctcgg attgggaagc attgcaaccc agcaacccga gagcaacatc 6900
ctggacatag atctacgtcc tgcatcagca tggacgctgt atgccgtggc cacaacattt 6960
gttacaccaa tgttgagaca tagcattgaa aattcctcag tgaatgtgtc cctaacagct 7020
atagccaacc aagccacagt gttaatgggt ctcgggaaag gatggccatt gtcaaagatg 7080
gacatcggag ttccccttct cgccattgga tgctactcac aagtcaaccc cataactctc 7140
acagcagctc ttttcttatt ggtagcacat tatgccatca tagggccagg actccaagca 7200
aaagcaacca gagaagctca gaaaagagca gcggcgggca tcatgaaaaa cccaactgtc 7260
gatggaataa cagtgattga cctagatcca ataccttatg atccaaagtt tgaaaagcag 7320
ttgggacaag taatgctcct agtcctctgc gtgactcaag tattgatgat gaggactaca 7380
tgggctctgt gtgaggcttt aaccttagct accgggccca tctccacatt gtgggaagga 7440
aatccaggga ggttttggaa cactaccatt gcggtgtcaa tggctaacat ttttagaggg 7500
agttacttgg ccggagctgg acttctcttt tctattatga agaacacaac caacacaaga 7560
aggggaactg gcaacatagg agagacgctt ggagagaaat ggaaaagccg attgaacgcg 7620
ttgggaaaaa gtgaattcca gatctacaag aaaagtggaa tccaggaagt ggatagaacc 7680
ttagcaaaag aaggcattaa aagaggagaa acggaccatc acgctgtgtc gcgaggctca 7740
gcaaaactga gatggttcgt tgagagaaac atggtcacac cagaagggaa agtagtggac 7800
ctcggttgtg gcagaggagg ctggtcatac tattgtggag gactaaagaa tgtaagagaa 7860
gtcaaaggcc taacaaaagg aggaccagga cacgaagaac ccatccccat gtcaacatat 7920
gggtggaatc tagtgcgtct tcaaagtgga gttgacgttt tcttcatccc gccagaaaag 7980
tgtgacacat tattgtgtga cataggggag tcatcaccaa atcccacagt ggaagcagga 8040
cgaacactca gagtccttaa cttagtagaa aattggttga acaacaacac tcaattttgc 8100
ataaaggttc tcaacccata tatgccctca gtcatagaaa aaatggaagc actacaaagg 8160
aaatatggag gagccttagt gaggaatcca ctctcacgaa actccacaca tgagatgtac 8220
tgggtatcca atgcttccgg gaacatagtg tcatcagtga acatgatttc aaggatgttg 8280
atcaacagat ttacaatgag atacaagaaa gccacttacg agccggatgt tgacctcgga 8340
agcggaaccc gtaacatcgg gattgaaagt gagataccaa acctagatat aattgggaaa 8400
agaatagaaa aaataaagca agagcatgaa acatcatggc actatgacca agaccaccca 8460
tacaaaacgt gggcatacca tggtagctat gaaacaaaac agactggatc agcatcatcc 8520
atggtcaacg gagtggtcag gctgctgaca aaaccttggg acgtcgtccc catggtgaca 8580
cagatggcaa tgacagacac gactccattt ggacaacagc gcgtttttaa agagaaagtg 8640
gacacgagaa cccaagaacc gaaagaaggc acgaagaaac taatgaaaat aacagcagag 8700
tggctttgga aagaattagg gaagaaaaag acacccagga tgtgcaccag agaagaattc 8760
acaagaaagg tgagaagcaa tgcagccttg ggggccatat tcactgatga gaacaagtgg 8820
aagtcggcac gtgaggctgt tgaagatagt aggttttggg agctggttga caaggaaagg 8880
aatctccatc ttgaaggaaa gtgtgaaaca tgtgtgtaca acatgatggg aaaaagagag 8940
aagaagctag gggaattcgg caaggcaaaa ggcagcagag ccatatggta catgtggctt 9000
ggagcacgct tcttagagtt tgaagcccta ggattcttaa atgaagatca ctggttctcc 9060
agagagaact ccctgagtgg agtggaagga gaagggctgc acaagctagg ttacattcta 9120
agagacgtga gcaagaaaga gggaggagca atgtatgccg atgacaccgc aggatgggat 9180
acaagaatca cactagaaga cctaaaaaat gaagaaatgg taacaaacca catggaagga 9240
gaacacaaga aactagccga ggccattttc aaactaacgt accaaaacaa ggtggtgcgt 9300
gtgcaaagac caacaccaag aggcacagta atggacatca tatcgagaag agaccaaaga 9360
ggtagtggac aagttggcac ctatggactc aatactttca ccaatatgga agcccaacta 9420
atcagacaga tggagggaga aggagtcttt aaaagcattc agcacctaac aatcacagaa 9480
gaaatcgctg tgcaaaactg gttagcaaga gtggggcgcg aaaggttatc aagaatggcc 9540
atcagtggag atgattgtgt tgtgaaacct ttagatgaca ggttcgcaag cgctttaaca 9600
gctctaaatg acatgggaaa gattaggaaa gacatacaac aatgggaacc ttcaagagga 9660
tggaatgatt ggacacaagt gcccttctgt tcacaccatt tccatgagtt aatcatgaaa 9720
gacggtcgcg tactcgttgt tccatgtaga aaccaagatg aactgattgg cagagcccga 9780
atctcccaag gagcagggtg gtctttgcgg gagacggcct gtttggggaa gtcttacgcc 9840
caaatgtgga gcttgatgta cttccacaga cgcgacctca ggctggcggc aaatgctatt 9900
tgctcggcag taccatcaca ttgggttcca acaagtcgaa caacctggtc catacatgct 9960
aaacatgaat ggatgacaac ggaagacatg ctgacagtct ggaacagggt gtggattcaa 10020
gaaaacccat ggatggaaga caaaactcca gtggaatcat gggaggaaat cccatacttg 10080
gggaaaagag aagaccaatg gtgcggctca ttgattgggt taacaagcag ggccacctgg 10140
gcaaagaaca tccaagcagc aataaatcaa gttagatccc ttataggcaa tgaagaatac 10200
acagattaca tgccatccat gaaaagattc agaagagaag aggaagaagc aggagttctg 10260
tggtagaaag caaaactaac atgaaacaag gctagaagtc aggtcggatt aagccatagt 10320
acggaaaaaa ctatgctacc tgtgagcccc gtccaaggac gttaaaagaa gtcaggccat 10380
cataaatgcc atagcttgag taaactatgc agcctgtagc tccacctgag aaggtgtaaa 10440
aaatccggga ggccacaaac catggaagct gtacgcatgg cgtagtggac tagcggttag 10500
aggagacccc tcccttacaa atcgcagcaa caatgggggc ccaaggcgag atgaagctgt 10560
agtctcgctg gaaggactag aggttagagg agaccccccc gaaacaaaaa acagcatatt 10620
gacgctggga aagaccagag atcctgctgt ctcctcagca tcattccagg cacagaacgc 10680
cagaaaatgg aatggtgctg ttgaatcaac aggttct 10717
<210> 8
<211> 3389
<212> PRT
<213> 人工序列
<220>
<223> 登革热病毒血清型3, BVS
<400> 8
Met Asn Asn Gln Arg Lys Lys Ala Lys Asn Thr Pro Phe Asn Met Leu
1 5 10 15
Lys Arg Glu Arg Asn Arg Val Ser Thr Val Gln Gln Leu Thr Lys Arg
20 25 30
Phe Ser Leu Gly Met Leu Gln Gly Arg Gly Pro Leu Lys Leu Phe Met
35 40 45
Ala Leu Val Ala Phe Leu Arg Phe Leu Thr Ile Pro Pro Thr Ala Gly
50 55 60
Ile Leu Lys Arg Trp Gly Thr Ile Lys Lys Ser Lys Ala Ile Asn Val
65 70 75 80
Leu Arg Gly Phe Arg Lys Glu Ile Gly Arg Met Leu Asn Ile Leu Asn
85 90 95
Arg Arg Arg Arg Ser Ala Gly Met Ile Ile Met Leu Ile Pro Thr Val
100 105 110
Met Ala Phe His Leu Thr Thr Arg Asp Gly Glu Pro Arg Met Ile Val
115 120 125
Gly Lys Asn Glu Arg Gly Lys Ser Leu Leu Phe Lys Thr Ala Ser Gly
130 135 140
Ile Asn Met Cys Thr Leu Ile Ala Met Asp Leu Gly Glu Met Cys Asp
145 150 155 160
Asp Thr Val Thr Tyr Lys Cys Pro His Ile Thr Glu Val Glu Pro Glu
165 170 175
Asp Ile Asp Cys Trp Cys Asn Leu Thr Ser Thr Trp Val Thr Tyr Gly
180 185 190
Thr Cys Asn Gln Ala Gly Glu His Arg Arg Asp Lys Arg Ser Val Ala
195 200 205
Leu Ala Pro His Val Gly Met Gly Leu Asp Thr Arg Thr Gln Thr Trp
210 215 220
Met Ser Ala Glu Gly Ala Trp Arg Gln Val Glu Lys Val Glu Thr Trp
225 230 235 240
Ala Leu Arg His Pro Gly Phe Thr Ile Leu Ala Leu Phe Leu Ala His
245 250 255
Tyr Ile Gly Thr Ser Leu Thr Gln Lys Val Val Ile Phe Ile Leu Leu
260 265 270
Met Leu Val Thr Pro Ser Met Thr Met Arg Cys Val Gly Val Gly Asn
275 280 285
Arg Asp Phe Val Glu Gly Leu Ser Gly Ala Thr Trp Val Asp Val Val
290 295 300
Leu Glu His Gly Gly Cys Val Thr Thr Met Ala Lys Asn Lys Pro Thr
305 310 315 320
Leu Asp Ile Glu Leu Gln Lys Thr Glu Ala Thr Gln Leu Ala Thr Leu
325 330 335
Arg Lys Leu Cys Ile Glu Gly Lys Ile Thr Asn Ile Thr Thr Asp Ser
340 345 350
Arg Cys Pro Thr Gln Gly Glu Ala Ile Leu Pro Glu Glu Gln Asp Gln
355 360 365
Asn Tyr Val Cys Lys His Thr Tyr Val Asp Arg Gly Trp Gly Asn Gly
370 375 380
Cys Gly Leu Phe Gly Lys Gly Ser Leu Val Thr Cys Ala Lys Phe Gln
385 390 395 400
Cys Leu Glu Ser Ile Glu Gly Lys Val Val Gln His Glu Asn Leu Lys
405 410 415
Tyr Thr Val Ile Ile Thr Val His Thr Gly Asp Gln His Gln Val Gly
420 425 430
Asn Glu Thr Gln Gly Val Thr Ala Glu Ile Thr Pro Gln Ala Ser Thr
435 440 445
Ala Glu Ala Ile Leu Pro Glu Tyr Gly Thr Leu Gly Leu Glu Cys Ser
450 455 460
Pro Arg Thr Gly Leu Asp Phe Asn Glu Met Ile Ser Leu Thr Met Lys
465 470 475 480
Asn Lys Ala Trp Met Val His Arg Gln Trp Phe Phe Asp Leu Pro Leu
485 490 495
Pro Trp Thr Ser Gly Ala Ser Ala Glu Thr Pro Thr Trp Asn Arg Lys
500 505 510
Glu Leu Leu Val Thr Phe Lys Asn Ala His Ala Lys Lys Gln Glu Val
515 520 525
Val Val Leu Gly Ser Gln Glu Gly Ala Met His Thr Ala Leu Thr Gly
530 535 540
Ala Thr Glu Ile Gln Thr Ser Gly Gly Thr Ser Ile Phe Ala Gly His
545 550 555 560
Leu Lys Cys Arg Leu Lys Met Asp Lys Leu Glu Leu Lys Gly Met Ser
565 570 575
Tyr Ala Met Cys Leu Ser Ser Phe Val Leu Lys Lys Glu Val Ser Glu
580 585 590
Thr Gln His Gly Thr Ile Leu Ile Lys Val Glu Tyr Lys Gly Glu Asp
595 600 605
Ala Pro Cys Lys Ile Pro Phe Ser Thr Glu Asp Gly Gln Gly Lys Ala
610 615 620
Leu Asn Gly Arg Leu Ile Thr Ala Asn Pro Val Val Thr Lys Lys Glu
625 630 635 640
Glu Pro Val Asn Ile Glu Ala Glu Pro Pro Phe Gly Glu Ser Asn Ile
645 650 655
Val Ile Gly Ile Gly Asp Lys Ala Leu Lys Ile Asn Trp Tyr Lys Lys
660 665 670
Gly Ser Ser Ile Gly Lys Met Phe Glu Ala Thr Ala Arg Gly Ala Arg
675 680 685
Arg Met Ala Ile Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser Val Gly
690 695 700
Gly Val Leu Asn Ser Leu Gly Lys Met Val His Gln Ile Phe Gly Ser
705 710 715 720
Ala Tyr Thr Ala Leu Phe Gly Gly Val Ser Trp Met Met Lys Ile Gly
725 730 735
Ile Gly Val Leu Leu Thr Trp Ile Gly Leu Asn Ser Lys Asn Thr Ser
740 745 750
Met Ser Phe Ser Cys Ile Ala Ala Gly Ile Val Thr Leu Tyr Leu Gly
755 760 765
Val Met Val Gln Ala Asp Ser Gly Cys Val Val Ser Trp Lys Asn Lys
770 775 780
Glu Leu Lys Cys Gly Ser Gly Ile Phe Ile Thr Asp Asn Val His Thr
785 790 795 800
Trp Thr Glu Gln Tyr Lys Phe Gln Pro Glu Ser Pro Ser Lys Leu Ala
805 810 815
Ser Ala Ile Gln Lys Ala His Glu Glu Asp Ile Cys Gly Ile Arg Ser
820 825 830
Val Thr Arg Leu Glu Asn Leu Met Trp Lys Gln Ile Thr Pro Glu Leu
835 840 845
Asn His Ile Leu Ser Glu Asn Glu Val Lys Leu Thr Ile Met Thr Gly
850 855 860
Asp Ile Lys Gly Ile Met Gln Ala Gly Lys Arg Ser Leu Arg Pro Gln
865 870 875 880
Pro Thr Glu Leu Lys Tyr Ser Trp Lys Thr Trp Gly Lys Ala Lys Met
885 890 895
Leu Ser Thr Glu Ser His Asn Gln Thr Phe Leu Ile Asp Gly Pro Glu
900 905 910
Thr Ala Glu Cys Pro Asn Thr Asn Arg Ala Trp Asn Ser Leu Glu Val
915 920 925
Glu Asp Tyr Gly Phe Gly Val Phe Thr Thr Asn Ile Trp Leu Lys Leu
930 935 940
Lys Glu Lys Gln Asp Val Phe Cys Asp Ser Lys Leu Met Ser Ala Ala
945 950 955 960
Ile Lys Asp Asn Arg Ala Val His Ala Asp Met Gly Tyr Trp Ile Glu
965 970 975
Ser Ala Leu Asn Asp Thr Trp Lys Ile Glu Lys Ala Ser Phe Ile Glu
980 985 990
Val Lys Asn Cys His Trp Pro Lys Ser His Thr Leu Trp Ser Asn Gly
995 1000 1005
Val Leu Glu Ser Glu Met Ile Ile Pro Lys Asn Leu Ala Gly Pro
1010 1015 1020
Val Ser Gln His Asn Tyr Arg Pro Gly Tyr His Thr Gln Ile Thr
1025 1030 1035
Gly Pro Trp His Leu Gly Lys Leu Glu Met Asp Phe Asp Phe Cys
1040 1045 1050
Asp Gly Thr Thr Val Val Val Thr Glu Asp Cys Gly Asn Arg Gly
1055 1060 1065
Pro Ser Leu Arg Thr Thr Thr Ala Ser Gly Lys Leu Ile Thr Glu
1070 1075 1080
Trp Cys Cys Arg Ser Cys Thr Leu Pro Pro Leu Arg Tyr Arg Gly
1085 1090 1095
Glu Asp Gly Cys Trp Tyr Gly Met Glu Ile Arg Pro Leu Lys Glu
1100 1105 1110
Lys Glu Glu Asn Leu Val Asn Ser Leu Val Thr Ala Gly His Gly
1115 1120 1125
Gln Val Asp Asn Phe Ser Leu Gly Val Leu Gly Met Ala Leu Phe
1130 1135 1140
Leu Glu Glu Met Leu Arg Thr Arg Val Gly Thr Lys His Ala Ile
1145 1150 1155
Leu Leu Val Ala Val Ser Phe Val Thr Leu Ile Thr Gly Asn Met
1160 1165 1170
Ser Phe Arg Asp Leu Gly Arg Val Met Val Met Val Gly Ala Thr
1175 1180 1185
Met Thr Asp Asp Ile Gly Met Gly Val Thr Tyr Leu Ala Leu Leu
1190 1195 1200
Ala Ala Phe Lys Val Arg Pro Thr Phe Ala Ala Gly Leu Leu Leu
1205 1210 1215
Arg Lys Leu Thr Ser Lys Glu Leu Met Met Thr Thr Ile Gly Ile
1220 1225 1230
Val Leu Leu Ser Gln Ser Thr Ile Pro Glu Thr Ile Leu Glu Leu
1235 1240 1245
Thr Asp Ala Leu Ala Leu Gly Met Met Val Leu Lys Met Val Arg
1250 1255 1260
Asn Met Glu Lys Tyr Gln Leu Ala Val Thr Ile Met Ala Ile Leu
1265 1270 1275
Cys Val Pro Asn Ala Val Ile Leu Gln Asn Ala Trp Lys Val Ser
1280 1285 1290
Cys Thr Ile Leu Ala Val Val Ser Val Ser Pro Leu Phe Leu Thr
1295 1300 1305
Ser Ser Gln Gln Lys Thr Asp Trp Ile Pro Leu Ala Leu Thr Ile
1310 1315 1320
Lys Gly Leu Asn Pro Thr Ala Ile Phe Leu Thr Thr Leu Ser Arg
1325 1330 1335
Thr Ser Lys Lys Arg Ser Trp Pro Leu Asn Glu Ala Ile Met Ala
1340 1345 1350
Val Gly Met Val Ser Ile Leu Ala Ser Ser Leu Leu Lys Asn Asp
1355 1360 1365
Ile Pro Met Thr Gly Pro Leu Val Ala Gly Gly Leu Leu Thr Val
1370 1375 1380
Cys Tyr Val Leu Thr Gly Arg Ser Ala Asp Leu Glu Leu Glu Arg
1385 1390 1395
Ala Ala Asp Val Lys Trp Glu Asp Gln Ala Glu Ile Ser Gly Ser
1400 1405 1410
Ser Pro Ile Leu Ser Ile Thr Ile Ser Glu Asp Gly Ser Met Ser
1415 1420 1425
Ile Lys Asn Glu Glu Glu Glu Gln Thr Leu Thr Ile Leu Ile Arg
1430 1435 1440
Thr Gly Leu Leu Val Ile Ser Gly Leu Phe Pro Val Ser Ile Pro
1445 1450 1455
Ile Thr Ala Ala Ala Trp Tyr Leu Trp Glu Val Lys Lys Gln Arg
1460 1465 1470
Ala Gly Val Leu Trp Asp Val Pro Ser Pro Pro Pro Met Gly Lys
1475 1480 1485
Ala Glu Leu Glu Asp Gly Ala Tyr Arg Ile Lys Gln Lys Gly Ile
1490 1495 1500
Leu Gly Tyr Ser Gln Ile Gly Ala Gly Val Tyr Lys Glu Gly Thr
1505 1510 1515
Phe His Thr Met Trp His Val Thr Arg Gly Ala Val Leu Met His
1520 1525 1530
Lys Gly Lys Arg Ile Glu Pro Ser Trp Ala Asp Val Lys Lys Asp
1535 1540 1545
Leu Ile Ser Tyr Gly Gly Gly Trp Lys Leu Glu Gly Glu Trp Lys
1550 1555 1560
Glu Gly Glu Glu Val Gln Val Leu Ala Leu Glu Pro Gly Lys Asn
1565 1570 1575
Pro Arg Ala Val Gln Thr Lys Pro Gly Leu Phe Lys Thr Asn Ala
1580 1585 1590
Gly Thr Ile Gly Ala Val Ser Leu Asp Phe Ser Pro Gly Thr Ser
1595 1600 1605
Gly Ser Pro Ile Ile Asp Lys Lys Gly Lys Val Val Gly Leu Tyr
1610 1615 1620
Gly Asn Gly Val Val Thr Arg Ser Gly Ala Tyr Val Ser Ala Ile
1625 1630 1635
Ala Gln Thr Glu Lys Ser Ile Glu Asp Asn Pro Glu Ile Glu Asp
1640 1645 1650
Asp Ile Phe Arg Lys Arg Arg Leu Thr Ile Met Asp Leu His Pro
1655 1660 1665
Gly Ala Gly Lys Thr Lys Arg Tyr Leu Pro Ala Ile Val Arg Glu
1670 1675 1680
Ala Ile Lys Arg Gly Leu Arg Thr Leu Ile Leu Ala Pro Thr Arg
1685 1690 1695
Val Val Ala Ala Glu Met Glu Glu Ala Leu Arg Gly Leu Pro Ile
1700 1705 1710
Arg Tyr Gln Thr Pro Ala Ile Arg Ala Val His Thr Gly Arg Glu
1715 1720 1725
Ile Val Asp Leu Met Cys His Ala Thr Phe Thr Met Arg Leu Leu
1730 1735 1740
Ser Pro Val Arg Val Pro Asn Tyr Asn Leu Ile Ile Met Asp Glu
1745 1750 1755
Ala His Phe Thr Asp Pro Ala Ser Ile Ala Ala Arg Gly Tyr Ile
1760 1765 1770
Ser Thr Arg Val Glu Met Gly Glu Ala Ala Gly Ile Phe Met Thr
1775 1780 1785
Ala Thr Pro Pro Gly Ser Arg Asp Pro Phe Pro Gln Ser Asn Ala
1790 1795 1800
Pro Ile Ile Asp Glu Glu Arg Glu Ile Pro Glu Arg Ser Trp Asn
1805 1810 1815
Ser Gly His Glu Trp Val Thr Asp Phe Lys Gly Lys Thr Val Trp
1820 1825 1830
Phe Val Pro Ser Ile Lys Ala Gly Asn Asp Ile Ala Ala Cys Leu
1835 1840 1845
Arg Lys Asn Gly Lys Lys Val Ile Gln Leu Ser Arg Lys Thr Phe
1850 1855 1860
Asp Ser Glu Tyr Val Lys Thr Arg Thr Asn Asp Trp Asp Phe Val
1865 1870 1875
Val Thr Thr Asp Ile Ser Glu Met Gly Ala Asn Phe Lys Ala Glu
1880 1885 1890
Arg Val Ile Asp Pro Arg Arg Cys Met Lys Pro Val Ile Leu Thr
1895 1900 1905
Asp Gly Glu Glu Arg Val Ile Leu Ala Gly Pro Met Pro Val Thr
1910 1915 1920
His Ser Ser Ala Ala Gln Arg Arg Gly Arg Ile Gly Arg Asn Pro
1925 1930 1935
Lys Asn Glu Asn Asp Gln Tyr Ile Tyr Met Gly Glu Pro Leu Glu
1940 1945 1950
Asn Asp Glu Asp Cys Ala His Trp Lys Glu Ala Lys Met Leu Leu
1955 1960 1965
Asp Asn Ile Asn Thr Pro Glu Gly Ile Ile Pro Ser Met Phe Glu
1970 1975 1980
Pro Glu Arg Glu Lys Val Asp Ala Ile Asp Gly Glu Tyr Arg Leu
1985 1990 1995
Arg Gly Glu Ala Arg Lys Thr Phe Val Asp Leu Met Arg Arg Gly
2000 2005 2010
Asp Leu Pro Val Trp Leu Ala Tyr Arg Val Ala Ala Glu Gly Ile
2015 2020 2025
Asn Tyr Ala Asp Arg Arg Trp Cys Phe Asp Gly Val Lys Asn Asn
2030 2035 2040
Gln Ile Leu Glu Glu Asn Val Glu Val Glu Ile Trp Thr Lys Glu
2045 2050 2055
Gly Glu Arg Lys Lys Leu Lys Pro Arg Trp Leu Asp Ala Arg Ile
2060 2065 2070
Tyr Ser Asp Pro Leu Ala Leu Lys Glu Phe Lys Glu Phe Ala Ala
2075 2080 2085
Gly Arg Lys Ser Leu Thr Leu Asn Leu Ile Thr Glu Met Gly Arg
2090 2095 2100
Leu Pro Thr Phe Met Thr Gln Lys Ala Arg Asn Ala Leu Asp Asn
2105 2110 2115
Leu Ala Val Leu His Thr Ala Glu Ala Gly Gly Arg Ala Tyr Asn
2120 2125 2130
His Ala Leu Ser Glu Leu Pro Glu Thr Leu Glu Thr Leu Leu Leu
2135 2140 2145
Leu Thr Leu Leu Ala Thr Val Thr Gly Gly Ile Phe Leu Phe Leu
2150 2155 2160
Met Ser Ala Arg Gly Ile Gly Lys Met Thr Leu Gly Met Cys Cys
2165 2170 2175
Ile Ile Thr Ala Ser Ile Leu Leu Trp Tyr Ala Gln Ile Gln Pro
2180 2185 2190
His Trp Ile Ala Ala Ser Ile Ile Leu Glu Phe Phe Leu Ile Val
2195 2200 2205
Leu Leu Ile Pro Glu Pro Glu Lys Gln Arg Thr Pro Gln Asp Asn
2210 2215 2220
Gln Leu Thr Tyr Val Val Ile Ala Ile Leu Thr Val Val Ala Ala
2225 2230 2235
Thr Met Ala Asn Glu Met Gly Phe Leu Glu Lys Thr Lys Lys Asp
2240 2245 2250
Leu Gly Leu Gly Ser Ile Ala Thr Gln Gln Pro Glu Ser Asn Ile
2255 2260 2265
Leu Asp Ile Asp Leu Arg Pro Ala Ser Ala Trp Thr Leu Tyr Ala
2270 2275 2280
Val Ala Thr Thr Phe Val Thr Pro Met Leu Arg His Ser Ile Glu
2285 2290 2295
Asn Ser Ser Val Asn Val Ser Leu Thr Ala Ile Ala Asn Gln Ala
2300 2305 2310
Thr Val Leu Met Gly Leu Gly Lys Gly Trp Pro Leu Ser Lys Met
2315 2320 2325
Asp Ile Gly Val Pro Leu Leu Ala Ile Gly Cys Tyr Ser Gln Val
2330 2335 2340
Asn Pro Ile Thr Leu Thr Ala Ala Leu Phe Leu Leu Val Ala His
2345 2350 2355
Tyr Ala Ile Ile Gly Pro Gly Leu Gln Ala Lys Ala Thr Arg Glu
2360 2365 2370
Ala Gln Lys Arg Ala Ala Ala Gly Ile Met Lys Asn Pro Thr Val
2375 2380 2385
Asp Gly Ile Thr Val Ile Asp Leu Asp Pro Ile Pro Tyr Asp Pro
2390 2395 2400
Lys Phe Glu Lys Gln Leu Gly Gln Val Met Leu Leu Val Leu Cys
2405 2410 2415
Val Thr Gln Val Leu Met Met Arg Thr Thr Trp Ala Leu Cys Glu
2420 2425 2430
Ala Leu Thr Leu Ala Thr Gly Pro Ile Ser Thr Leu Trp Glu Gly
2435 2440 2445
Asn Pro Gly Arg Phe Trp Asn Thr Thr Ile Ala Val Ser Met Ala
2450 2455 2460
Asn Ile Phe Arg Gly Ser Tyr Leu Ala Gly Ala Gly Leu Leu Phe
2465 2470 2475
Ser Ile Met Lys Asn Thr Thr Asn Thr Arg Arg Gly Thr Gly Asn
2480 2485 2490
Ile Gly Glu Thr Leu Gly Glu Lys Trp Lys Ser Arg Leu Asn Ala
2495 2500 2505
Leu Gly Lys Ser Glu Phe Gln Ile Tyr Lys Lys Ser Gly Ile Gln
2510 2515 2520
Glu Val Asp Arg Thr Leu Ala Lys Glu Gly Ile Lys Arg Gly Glu
2525 2530 2535
Thr Asp His His Ala Val Ser Arg Gly Ser Ala Lys Leu Arg Trp
2540 2545 2550
Phe Val Glu Arg Asn Met Val Thr Pro Glu Gly Lys Val Val Asp
2555 2560 2565
Leu Gly Cys Gly Arg Gly Gly Trp Ser Tyr Tyr Cys Gly Gly Leu
2570 2575 2580
Lys Asn Val Arg Glu Val Lys Gly Leu Thr Lys Gly Gly Pro Gly
2585 2590 2595
His Glu Glu Pro Ile Pro Met Ser Thr Tyr Gly Trp Asn Leu Val
2600 2605 2610
Arg Leu Gln Ser Gly Val Asp Val Phe Phe Ile Pro Pro Glu Lys
2615 2620 2625
Cys Asp Thr Leu Leu Cys Asp Ile Gly Glu Ser Ser Pro Asn Pro
2630 2635 2640
Thr Val Glu Ala Gly Arg Thr Leu Arg Val Leu Asn Leu Val Glu
2645 2650 2655
Asn Trp Leu Asn Asn Asn Thr Gln Phe Cys Ile Lys Val Leu Asn
2660 2665 2670
Pro Tyr Met Pro Ser Val Ile Glu Lys Met Glu Ala Leu Gln Arg
2675 2680 2685
Lys Tyr Gly Gly Ala Leu Val Arg Asn Pro Leu Ser Arg Asn Ser
2690 2695 2700
Thr His Glu Met Tyr Trp Val Ser Asn Ala Ser Gly Asn Ile Val
2705 2710 2715
Ser Ser Val Asn Met Ile Ser Arg Met Leu Ile Asn Arg Phe Thr
2720 2725 2730
Met Arg Tyr Lys Lys Ala Thr Tyr Glu Pro Asp Val Asp Leu Gly
2735 2740 2745
Ser Gly Thr Arg Asn Ile Gly Ile Glu Ser Glu Ile Pro Asn Leu
2750 2755 2760
Asp Ile Ile Gly Lys Arg Ile Glu Lys Ile Lys Gln Glu His Glu
2765 2770 2775
Thr Ser Trp His Tyr Asp Gln Asp His Pro Tyr Lys Thr Trp Ala
2780 2785 2790
Tyr His Gly Ser Tyr Glu Thr Lys Gln Thr Gly Ser Ala Ser Ser
2795 2800 2805
Met Val Asn Gly Val Val Arg Leu Leu Thr Lys Pro Trp Asp Val
2810 2815 2820
Val Pro Met Val Thr Gln Met Ala Met Thr Asp Thr Thr Pro Phe
2825 2830 2835
Gly Gln Gln Arg Val Phe Lys Glu Lys Val Asp Thr Arg Thr Gln
2840 2845 2850
Glu Pro Lys Glu Gly Thr Lys Lys Leu Met Lys Ile Thr Ala Glu
2855 2860 2865
Trp Leu Trp Lys Glu Leu Gly Lys Lys Lys Thr Pro Arg Met Cys
2870 2875 2880
Thr Arg Glu Glu Phe Thr Arg Lys Val Arg Ser Asn Ala Ala Leu
2885 2890 2895
Gly Ala Ile Phe Thr Asp Glu Asn Lys Trp Lys Ser Ala Arg Glu
2900 2905 2910
Ala Val Glu Asp Ser Arg Phe Trp Glu Leu Val Asp Lys Glu Arg
2915 2920 2925
Asn Leu His Leu Glu Gly Lys Cys Glu Thr Cys Val Tyr Asn Met
2930 2935 2940
Met Gly Lys Arg Glu Lys Lys Leu Gly Glu Phe Gly Lys Ala Lys
2945 2950 2955
Gly Ser Arg Ala Ile Trp Tyr Met Trp Leu Gly Ala Arg Phe Leu
2960 2965 2970
Glu Phe Glu Ala Leu Gly Phe Leu Asn Glu Asp His Trp Phe Ser
2975 2980 2985
Arg Glu Asn Ser Leu Ser Gly Val Glu Gly Glu Gly Leu His Lys
2990 2995 3000
Leu Gly Tyr Ile Leu Arg Asp Val Ser Lys Lys Glu Gly Gly Ala
3005 3010 3015
Met Tyr Ala Asp Asp Thr Ala Gly Trp Asp Thr Arg Ile Thr Leu
3020 3025 3030
Glu Asp Leu Lys Asn Glu Glu Met Val Thr Asn His Met Glu Gly
3035 3040 3045
Glu His Lys Lys Leu Ala Glu Ala Ile Phe Lys Leu Thr Tyr Gln
3050 3055 3060
Asn Lys Val Val Arg Val Gln Arg Pro Thr Pro Arg Gly Thr Val
3065 3070 3075
Met Asp Ile Ile Ser Arg Arg Asp Gln Arg Gly Ser Gly Gln Val
3080 3085 3090
Gly Thr Tyr Gly Leu Asn Thr Phe Thr Asn Met Glu Ala Gln Leu
3095 3100 3105
Ile Arg Gln Met Glu Gly Glu Gly Val Phe Lys Ser Ile Gln His
3110 3115 3120
Leu Thr Ile Thr Glu Glu Ile Ala Val Gln Asn Trp Leu Ala Arg
3125 3130 3135
Val Gly Arg Glu Arg Leu Ser Arg Met Ala Ile Ser Gly Asp Asp
3140 3145 3150
Cys Val Val Lys Pro Leu Asp Asp Arg Phe Ala Ser Ala Leu Thr
3155 3160 3165
Ala Leu Asn Asp Met Gly Lys Ile Arg Lys Asp Ile Gln Gln Trp
3170 3175 3180
Glu Pro Ser Arg Gly Trp Asn Asp Trp Thr Gln Val Pro Phe Cys
3185 3190 3195
Ser His His Phe His Glu Leu Ile Met Lys Asp Gly Arg Val Leu
3200 3205 3210
Val Val Pro Cys Arg Asn Gln Asp Glu Leu Ile Gly Arg Ala Arg
3215 3220 3225
Ile Ser Gln Gly Ala Gly Trp Ser Leu Arg Glu Thr Ala Cys Leu
3230 3235 3240
Gly Lys Ser Tyr Ala Gln Met Trp Ser Leu Met Tyr Phe His Arg
3245 3250 3255
Arg Asp Leu Arg Leu Ala Ala Asn Ala Ile Cys Ser Ala Val Pro
3260 3265 3270
Ser His Trp Val Pro Thr Ser Arg Thr Thr Trp Ser Ile His Ala
3275 3280 3285
Lys His Glu Trp Met Thr Thr Glu Asp Met Leu Thr Val Trp Asn
3290 3295 3300
Arg Val Trp Ile Gln Glu Asn Pro Trp Met Glu Asp Lys Thr Pro
3305 3310 3315
Val Glu Ser Trp Glu Glu Ile Pro Tyr Leu Gly Lys Arg Glu Asp
3320 3325 3330
Gln Trp Cys Gly Ser Leu Ile Gly Leu Thr Ser Arg Ala Thr Trp
3335 3340 3345
Ala Lys Asn Ile Gln Ala Ala Ile Asn Gln Val Arg Ser Leu Ile
3350 3355 3360
Gly Asn Glu Glu Tyr Thr Asp Tyr Met Pro Ser Met Lys Arg Phe
3365 3370 3375
Arg Arg Glu Glu Glu Glu Ala Gly Val Leu Trp
3380 3385
<210> 9
<211> 3389
<212> PRT
<213> 人工序列
<220>
<223> 登革热病毒血清型3, MVS
<400> 9
Met Asn Asn Gln Arg Lys Lys Ala Lys Asn Thr Pro Phe Asn Met Leu
1 5 10 15
Lys Arg Glu Arg Asn Arg Val Ser Thr Val Gln Gln Leu Thr Lys Arg
20 25 30
Phe Ser Leu Gly Met Leu Gln Gly Arg Gly Pro Leu Lys Leu Phe Met
35 40 45
Ala Leu Val Ala Phe Leu Arg Phe Leu Thr Ile Pro Pro Thr Ala Gly
50 55 60
Ile Leu Lys Arg Trp Gly Thr Ile Lys Lys Ser Lys Ala Ile Asn Val
65 70 75 80
Leu Arg Gly Phe Arg Lys Glu Ile Gly Arg Met Leu Asn Ile Leu Asn
85 90 95
Arg Arg Arg Arg Ser Ala Gly Met Ile Ile Met Leu Ile Pro Thr Val
100 105 110
Met Ala Phe His Leu Thr Thr Arg Asp Gly Glu Pro Arg Met Ile Val
115 120 125
Gly Lys Asn Glu Arg Gly Lys Ser Leu Leu Phe Lys Thr Ala Ser Gly
130 135 140
Ile Asn Met Cys Thr Leu Ile Ala Met Asp Leu Gly Glu Met Cys Asp
145 150 155 160
Asp Thr Val Thr Tyr Lys Cys Pro His Ile Thr Glu Val Glu Pro Glu
165 170 175
Asp Ile Asp Cys Trp Cys Asn Leu Thr Ser Thr Trp Val Thr Tyr Gly
180 185 190
Thr Cys Asn Gln Ala Gly Glu His Arg Arg Asp Lys Arg Ser Val Ala
195 200 205
Leu Ala Pro His Val Gly Met Gly Leu Asp Thr Arg Thr Gln Thr Trp
210 215 220
Met Ser Ala Glu Gly Ala Trp Arg Gln Val Glu Lys Val Glu Thr Trp
225 230 235 240
Ala Leu Arg His Pro Gly Phe Thr Ile Leu Ala Leu Phe Leu Ala His
245 250 255
Tyr Ile Gly Thr Ser Leu Thr Gln Lys Val Val Ile Phe Ile Leu Leu
260 265 270
Met Leu Val Thr Pro Ser Met Thr Met Arg Cys Val Gly Val Gly Asn
275 280 285
Arg Asp Phe Val Glu Gly Leu Ser Gly Ala Thr Trp Val Asp Val Val
290 295 300
Leu Glu His Gly Gly Cys Val Thr Thr Met Ala Lys Asn Lys Pro Thr
305 310 315 320
Leu Asp Ile Glu Leu Gln Lys Thr Glu Ala Thr Gln Leu Ala Thr Leu
325 330 335
Arg Lys Leu Cys Ile Glu Gly Lys Ile Thr Asn Ile Thr Thr Asp Ser
340 345 350
Arg Cys Pro Thr Gln Gly Glu Ala Ile Leu Pro Glu Glu Gln Asp Gln
355 360 365
Asn Tyr Val Cys Lys His Thr Tyr Val Asp Arg Gly Trp Gly Asn Gly
370 375 380
Cys Gly Leu Phe Gly Lys Gly Ser Leu Val Thr Cys Ala Lys Phe Gln
385 390 395 400
Cys Leu Glu Ser Ile Glu Gly Lys Val Val Gln His Glu Asn Leu Lys
405 410 415
Tyr Thr Val Ile Ile Thr Val His Thr Gly Asp Gln His Gln Val Gly
420 425 430
Asn Glu Thr Gln Gly Val Thr Ala Glu Ile Thr Pro Gln Ala Ser Thr
435 440 445
Ala Glu Ala Ile Leu Pro Glu Tyr Gly Thr Leu Gly Leu Glu Cys Ser
450 455 460
Pro Arg Thr Gly Leu Asp Phe Asn Glu Met Ile Ser Leu Thr Met Lys
465 470 475 480
Asn Lys Ala Trp Met Val His Arg Gln Trp Phe Phe Asp Leu Pro Leu
485 490 495
Pro Trp Thr Ser Gly Ala Ser Ala Glu Thr Pro Thr Trp Asn Arg Lys
500 505 510
Glu Leu Leu Val Thr Phe Lys Asn Ala His Ala Lys Lys Gln Glu Val
515 520 525
Val Val Leu Gly Ser Gln Glu Gly Ala Met His Thr Ala Leu Thr Gly
530 535 540
Ala Thr Glu Ile Gln Thr Ser Gly Gly Thr Ser Ile Phe Ala Gly His
545 550 555 560
Leu Lys Cys Arg Leu Lys Met Asp Lys Leu Glu Leu Lys Gly Met Ser
565 570 575
Tyr Ala Met Cys Leu Ser Ser Phe Val Leu Lys Lys Glu Val Ser Glu
580 585 590
Thr Gln His Gly Thr Ile Leu Ile Lys Val Glu Tyr Lys Gly Glu Asp
595 600 605
Ala Pro Cys Lys Ile Pro Phe Ser Thr Glu Asp Gly Gln Gly Lys Ala
610 615 620
Leu Asn Gly Arg Leu Ile Thr Ala Asn Pro Val Val Thr Lys Lys Glu
625 630 635 640
Glu Pro Val Asn Ile Glu Ala Glu Pro Pro Phe Gly Glu Ser Asn Ile
645 650 655
Val Ile Gly Ile Gly Asp Lys Ala Leu Lys Ile Asn Trp Tyr Lys Lys
660 665 670
Gly Ser Ser Ile Gly Lys Met Phe Glu Ala Thr Ala Arg Gly Ala Arg
675 680 685
Arg Met Ala Ile Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser Val Gly
690 695 700
Gly Val Leu Asn Ser Leu Gly Lys Met Val His Gln Ile Phe Gly Ser
705 710 715 720
Ala Tyr Thr Ala Leu Phe Gly Gly Val Ser Trp Met Met Lys Ile Gly
725 730 735
Ile Gly Val Leu Leu Thr Trp Ile Gly Leu Asn Ser Lys Asn Thr Ser
740 745 750
Met Ser Phe Ser Cys Ile Ala Ala Gly Ile Val Thr Leu Tyr Leu Gly
755 760 765
Val Met Val Gln Ala Asp Ser Gly Cys Val Val Ser Trp Lys Asn Lys
770 775 780
Glu Leu Lys Cys Gly Ser Gly Ile Phe Ile Thr Asp Asn Val His Thr
785 790 795 800
Trp Thr Glu Gln Tyr Lys Phe Gln Pro Glu Ser Pro Ser Lys Leu Ala
805 810 815
Ser Ala Ile Gln Lys Ala His Glu Glu Asp Ile Cys Gly Ile Arg Ser
820 825 830
Val Thr Arg Leu Glu Asn Leu Met Trp Lys Gln Ile Thr Pro Glu Leu
835 840 845
Asn His Ile Leu Ser Glu Asn Glu Val Lys Leu Thr Ile Met Thr Gly
850 855 860
Asp Ile Lys Gly Ile Met Gln Ala Gly Lys Arg Ser Leu Arg Pro Gln
865 870 875 880
Pro Thr Glu Leu Lys Tyr Ser Trp Lys Thr Trp Gly Lys Ala Lys Met
885 890 895
Leu Ser Thr Glu Ser His Asn Gln Thr Phe Leu Ile Asp Gly Pro Glu
900 905 910
Thr Ala Glu Cys Pro Asn Thr Asn Arg Ala Trp Asn Ser Leu Glu Val
915 920 925
Glu Asp Tyr Gly Phe Gly Val Phe Thr Thr Asn Ile Trp Leu Lys Leu
930 935 940
Lys Glu Lys Gln Asp Val Phe Cys Asp Ser Lys Leu Met Ser Ala Ala
945 950 955 960
Ile Lys Asp Asn Arg Ala Val His Ala Asp Met Gly Tyr Trp Ile Glu
965 970 975
Ser Ala Leu Asn Asp Thr Trp Lys Ile Glu Lys Ala Ser Phe Ile Glu
980 985 990
Val Lys Asn Cys His Trp Pro Lys Ser His Thr Leu Trp Ser Asn Gly
995 1000 1005
Val Leu Glu Ser Glu Met Ile Ile Pro Lys Asn Leu Ala Gly Pro
1010 1015 1020
Val Ser Gln His Asn Tyr Arg Pro Gly Tyr His Thr Gln Ile Thr
1025 1030 1035
Gly Pro Trp His Leu Gly Lys Leu Glu Met Asp Phe Asp Phe Cys
1040 1045 1050
Asp Gly Thr Thr Val Val Val Thr Glu Asp Cys Gly Asn Arg Gly
1055 1060 1065
Pro Ser Leu Arg Thr Thr Thr Ala Ser Gly Lys Leu Ile Thr Glu
1070 1075 1080
Trp Cys Cys Arg Ser Cys Thr Leu Pro Pro Leu Arg Tyr Arg Gly
1085 1090 1095
Glu Asp Gly Cys Trp Tyr Gly Met Glu Ile Arg Pro Leu Lys Glu
1100 1105 1110
Lys Glu Glu Asn Leu Val Asn Ser Leu Val Thr Ala Gly His Gly
1115 1120 1125
Gln Val Asp Asn Phe Ser Leu Gly Val Leu Gly Met Ala Leu Phe
1130 1135 1140
Leu Glu Glu Met Leu Arg Thr Arg Val Gly Thr Lys His Ala Ile
1145 1150 1155
Leu Leu Val Ala Val Ser Phe Val Thr Leu Ile Thr Gly Asn Met
1160 1165 1170
Ser Phe Arg Asp Leu Gly Arg Val Met Val Met Val Gly Ala Thr
1175 1180 1185
Met Thr Asp Asp Ile Gly Met Gly Val Thr Tyr Leu Ala Leu Leu
1190 1195 1200
Ala Ala Phe Lys Val Arg Pro Thr Phe Ala Ala Gly Leu Leu Leu
1205 1210 1215
Arg Lys Leu Thr Ser Lys Glu Leu Met Met Thr Thr Ile Gly Ile
1220 1225 1230
Val Leu Leu Ser Gln Ser Thr Ile Pro Glu Thr Ile Leu Glu Leu
1235 1240 1245
Thr Asp Ala Leu Ala Leu Gly Met Met Val Leu Lys Met Val Arg
1250 1255 1260
Asn Met Glu Lys Tyr Gln Leu Ala Val Thr Ile Met Ala Ile Leu
1265 1270 1275
Cys Val Pro Asn Ala Val Ile Leu Gln Asn Ala Trp Lys Val Ser
1280 1285 1290
Cys Thr Ile Leu Ala Val Val Ser Val Ser Pro Leu Phe Leu Thr
1295 1300 1305
Ser Ser Gln Gln Lys Thr Asp Trp Ile Pro Leu Ala Leu Thr Ile
1310 1315 1320
Lys Gly Leu Asn Pro Thr Ala Ile Phe Leu Thr Thr Leu Ser Arg
1325 1330 1335
Thr Ser Lys Lys Arg Ser Trp Pro Leu Asn Glu Ala Ile Met Ala
1340 1345 1350
Val Gly Met Val Ser Ile Leu Ala Ser Ser Leu Leu Lys Asn Asp
1355 1360 1365
Ile Pro Met Thr Gly Pro Leu Val Ala Gly Gly Leu Leu Thr Val
1370 1375 1380
Cys Tyr Val Leu Thr Gly Arg Ser Ala Asp Leu Glu Leu Glu Arg
1385 1390 1395
Ala Ala Asp Val Lys Trp Glu Asp Gln Ala Glu Ile Ser Gly Ser
1400 1405 1410
Ser Pro Ile Leu Ser Ile Thr Ile Ser Glu Asp Gly Ser Met Ser
1415 1420 1425
Ile Lys Asn Glu Glu Glu Glu Gln Thr Leu Thr Ile Leu Ile Arg
1430 1435 1440
Thr Gly Leu Leu Val Ile Ser Gly Leu Phe Pro Val Ser Ile Pro
1445 1450 1455
Ile Thr Ala Ala Ala Trp Tyr Leu Trp Glu Val Lys Lys Gln Arg
1460 1465 1470
Ala Gly Val Leu Trp Asp Val Pro Ser Pro Pro Pro Met Gly Lys
1475 1480 1485
Ala Glu Leu Glu Asp Gly Ala Tyr Arg Ile Lys Gln Lys Gly Ile
1490 1495 1500
Leu Gly Tyr Ser Gln Ile Gly Ala Gly Val Tyr Lys Glu Gly Thr
1505 1510 1515
Phe His Thr Met Trp His Val Thr Arg Gly Ala Val Leu Met His
1520 1525 1530
Lys Gly Lys Arg Ile Glu Pro Ser Trp Ala Asp Val Lys Lys Asp
1535 1540 1545
Leu Ile Ser Tyr Gly Gly Gly Trp Lys Leu Glu Gly Glu Trp Lys
1550 1555 1560
Glu Gly Glu Glu Val Gln Val Leu Ala Leu Glu Pro Gly Lys Asn
1565 1570 1575
Pro Arg Ala Val Gln Thr Lys Pro Gly Leu Phe Lys Thr Asn Ala
1580 1585 1590
Gly Thr Ile Gly Ala Val Ser Leu Asp Phe Ser Pro Gly Thr Ser
1595 1600 1605
Gly Ser Pro Ile Ile Asp Lys Lys Gly Lys Val Val Gly Leu Tyr
1610 1615 1620
Gly Asn Gly Val Val Thr Arg Ser Gly Ala Tyr Val Ser Ala Ile
1625 1630 1635
Ala Gln Thr Glu Lys Ser Ile Glu Asp Asn Pro Glu Ile Glu Asp
1640 1645 1650
Asp Ile Phe Arg Lys Arg Arg Leu Thr Ile Met Asp Leu His Pro
1655 1660 1665
Gly Ala Gly Lys Thr Lys Arg Tyr Leu Pro Ala Ile Val Arg Glu
1670 1675 1680
Ala Ile Lys Arg Gly Leu Arg Thr Leu Ile Leu Ala Pro Thr Arg
1685 1690 1695
Val Val Ala Ala Glu Met Glu Glu Ala Leu Arg Gly Leu Pro Ile
1700 1705 1710
Arg Tyr Gln Thr Pro Ala Ile Arg Ala Val His Thr Gly Arg Glu
1715 1720 1725
Ile Val Asp Leu Met Cys His Ala Thr Phe Thr Met Arg Leu Leu
1730 1735 1740
Ser Pro Val Arg Val Pro Asn Tyr Asn Leu Ile Ile Met Asp Glu
1745 1750 1755
Ala His Phe Thr Asp Pro Ala Ser Ile Ala Ala Arg Gly Tyr Ile
1760 1765 1770
Ser Thr Arg Val Glu Met Gly Glu Ala Ala Gly Ile Phe Met Thr
1775 1780 1785
Ala Thr Pro Pro Gly Ser Arg Asp Pro Phe Pro Gln Ser Asn Ala
1790 1795 1800
Pro Ile Ile Asp Glu Glu Arg Glu Ile Pro Glu Arg Ser Trp Asn
1805 1810 1815
Ser Gly His Glu Trp Val Thr Asp Phe Lys Gly Lys Thr Val Trp
1820 1825 1830
Phe Val Pro Ser Ile Lys Ala Gly Asn Asp Ile Ala Ala Cys Leu
1835 1840 1845
Arg Lys Asn Gly Lys Lys Val Ile Gln Leu Ser Arg Lys Thr Phe
1850 1855 1860
Asp Ser Glu Tyr Val Lys Thr Arg Thr Asn Asp Trp Asp Phe Val
1865 1870 1875
Val Thr Thr Asp Ile Ser Glu Met Gly Ala Asn Phe Lys Ala Glu
1880 1885 1890
Arg Val Ile Asp Pro Arg Arg Cys Met Lys Pro Val Ile Leu Thr
1895 1900 1905
Asp Gly Glu Glu Arg Val Ile Leu Ala Gly Pro Met Pro Val Thr
1910 1915 1920
His Ser Ser Ala Ala Gln Arg Arg Gly Arg Ile Gly Arg Asn Pro
1925 1930 1935
Lys Asn Glu Asn Asp Gln Tyr Ile Tyr Met Gly Glu Pro Leu Glu
1940 1945 1950
Asn Asp Glu Asp Cys Ala His Trp Lys Glu Ala Lys Met Leu Leu
1955 1960 1965
Asp Asn Ile Asn Thr Pro Glu Gly Ile Ile Pro Ser Met Phe Glu
1970 1975 1980
Pro Glu Arg Glu Lys Val Asp Ala Ile Asp Gly Glu Tyr Arg Leu
1985 1990 1995
Arg Gly Glu Ala Arg Lys Thr Phe Val Asp Leu Met Arg Arg Gly
2000 2005 2010
Asp Leu Pro Val Trp Leu Ala Tyr Arg Val Ala Ala Glu Gly Ile
2015 2020 2025
Asn Tyr Ala Asp Arg Arg Trp Cys Phe Asp Gly Val Lys Asn Asn
2030 2035 2040
Gln Ile Leu Glu Glu Asn Val Glu Val Glu Ile Trp Thr Lys Glu
2045 2050 2055
Gly Glu Arg Lys Lys Leu Lys Pro Arg Trp Leu Asp Ala Arg Ile
2060 2065 2070
Tyr Ser Asp Pro Leu Ala Leu Lys Glu Phe Lys Glu Phe Ala Ala
2075 2080 2085
Gly Arg Lys Ser Leu Thr Leu Asn Leu Ile Thr Glu Met Gly Arg
2090 2095 2100
Leu Pro Thr Phe Met Thr Gln Lys Ala Arg Asp Ala Leu Asp Asn
2105 2110 2115
Leu Ala Val Leu His Thr Ala Glu Ala Gly Gly Arg Ala Tyr Asn
2120 2125 2130
His Ala Leu Ser Glu Leu Pro Glu Thr Leu Glu Thr Leu Leu Leu
2135 2140 2145
Leu Thr Leu Leu Ala Thr Val Thr Gly Gly Ile Phe Leu Phe Leu
2150 2155 2160
Met Ser Ala Arg Gly Ile Gly Lys Met Thr Leu Gly Met Cys Cys
2165 2170 2175
Ile Ile Thr Ala Ser Ile Leu Leu Trp Tyr Ala Gln Ile Gln Pro
2180 2185 2190
His Trp Ile Ala Ala Ser Ile Ile Leu Glu Phe Phe Leu Ile Val
2195 2200 2205
Leu Leu Ile Pro Glu Pro Glu Lys Gln Arg Thr Pro Gln Asp Asn
2210 2215 2220
Gln Leu Thr Tyr Val Val Ile Ala Ile Leu Thr Val Val Ala Ala
2225 2230 2235
Thr Met Ala Asn Glu Met Gly Phe Leu Glu Lys Thr Lys Lys Asp
2240 2245 2250
Leu Gly Leu Gly Ser Ile Ala Thr Gln Gln Pro Glu Ser Asn Ile
2255 2260 2265
Leu Asp Ile Asp Leu Arg Pro Ala Ser Ala Trp Thr Leu Tyr Ala
2270 2275 2280
Val Ala Thr Thr Phe Val Thr Pro Met Leu Arg His Ser Ile Glu
2285 2290 2295
Asn Ser Ser Val Asn Val Ser Leu Thr Ala Ile Ala Asn Gln Ala
2300 2305 2310
Thr Val Leu Met Gly Leu Gly Lys Gly Trp Pro Leu Ser Lys Met
2315 2320 2325
Asp Ile Gly Val Pro Leu Leu Ala Ile Gly Cys Tyr Ser Gln Val
2330 2335 2340
Asn Pro Ile Thr Leu Thr Ala Ala Leu Phe Leu Leu Val Ala His
2345 2350 2355
Tyr Ala Ile Ile Gly Pro Gly Leu Gln Ala Lys Ala Thr Arg Glu
2360 2365 2370
Ala Gln Lys Arg Ala Ala Ala Gly Ile Met Lys Asn Pro Thr Val
2375 2380 2385
Asp Gly Ile Thr Val Ile Asp Leu Asp Pro Ile Pro Tyr Asp Pro
2390 2395 2400
Lys Phe Glu Lys Gln Leu Gly Gln Val Met Leu Leu Val Leu Cys
2405 2410 2415
Val Thr Gln Val Leu Met Met Arg Thr Thr Trp Ala Leu Cys Glu
2420 2425 2430
Ala Leu Thr Leu Ala Thr Gly Pro Ile Ser Thr Leu Trp Glu Gly
2435 2440 2445
Asn Pro Gly Arg Phe Trp Asn Thr Thr Ile Ala Val Ser Met Ala
2450 2455 2460
Asn Ile Phe Arg Gly Ser Tyr Leu Ala Gly Ala Gly Leu Leu Phe
2465 2470 2475
Ser Ile Met Lys Asn Thr Thr Asn Thr Arg Arg Gly Thr Gly Asn
2480 2485 2490
Ile Gly Glu Thr Leu Gly Glu Lys Trp Lys Ser Arg Leu Asn Ala
2495 2500 2505
Leu Gly Lys Ser Glu Phe Gln Ile Tyr Lys Lys Ser Gly Ile Gln
2510 2515 2520
Glu Val Asp Arg Thr Leu Ala Lys Glu Gly Ile Lys Arg Gly Glu
2525 2530 2535
Thr Asp His His Ala Val Ser Arg Gly Ser Ala Lys Leu Arg Trp
2540 2545 2550
Phe Val Glu Arg Asn Met Val Thr Pro Glu Gly Lys Val Val Asp
2555 2560 2565
Leu Gly Cys Gly Arg Gly Gly Trp Ser Tyr Tyr Cys Gly Gly Leu
2570 2575 2580
Lys Asn Val Arg Glu Val Lys Gly Leu Thr Lys Gly Gly Pro Gly
2585 2590 2595
His Glu Glu Pro Ile Pro Met Ser Thr Tyr Gly Trp Asn Leu Val
2600 2605 2610
Arg Leu Gln Ser Gly Val Asp Val Phe Phe Ile Pro Pro Glu Lys
2615 2620 2625
Cys Asp Thr Leu Leu Cys Asp Ile Gly Glu Ser Ser Pro Asn Pro
2630 2635 2640
Thr Val Glu Ala Gly Arg Thr Leu Arg Val Leu Asn Leu Val Glu
2645 2650 2655
Asn Trp Leu Asn Asn Asn Thr Gln Phe Cys Ile Lys Val Leu Asn
2660 2665 2670
Pro Tyr Met Pro Ser Val Ile Glu Lys Met Glu Ala Leu Gln Arg
2675 2680 2685
Lys Tyr Gly Gly Ala Leu Val Arg Asn Pro Leu Ser Arg Asn Ser
2690 2695 2700
Thr His Glu Met Tyr Trp Val Ser Asn Ala Ser Gly Asn Ile Val
2705 2710 2715
Ser Ser Val Asn Met Ile Ser Arg Met Leu Ile Asn Arg Phe Thr
2720 2725 2730
Met Arg Tyr Lys Lys Ala Thr Tyr Glu Pro Asp Val Asp Leu Gly
2735 2740 2745
Ser Gly Thr Arg Asn Ile Gly Ile Glu Ser Glu Ile Pro Asn Leu
2750 2755 2760
Asp Ile Ile Gly Lys Arg Ile Glu Lys Ile Lys Gln Glu His Glu
2765 2770 2775
Thr Ser Trp His Tyr Asp Gln Asp His Pro Tyr Lys Thr Trp Ala
2780 2785 2790
Tyr His Gly Ser Tyr Glu Thr Lys Gln Thr Gly Ser Ala Ser Ser
2795 2800 2805
Met Val Asn Gly Val Val Arg Leu Leu Thr Lys Pro Trp Asp Val
2810 2815 2820
Val Pro Met Val Thr Gln Met Ala Met Thr Asp Thr Thr Pro Phe
2825 2830 2835
Gly Gln Gln Arg Val Phe Lys Glu Lys Val Asp Thr Arg Thr Gln
2840 2845 2850
Glu Pro Lys Glu Gly Thr Lys Lys Leu Met Lys Ile Thr Ala Glu
2855 2860 2865
Trp Leu Trp Lys Glu Leu Gly Lys Lys Lys Thr Pro Arg Met Cys
2870 2875 2880
Thr Arg Glu Glu Phe Thr Arg Lys Val Arg Ser Asn Ala Ala Leu
2885 2890 2895
Gly Ala Ile Phe Thr Asp Glu Asn Lys Trp Lys Ser Ala Arg Glu
2900 2905 2910
Ala Val Glu Asp Ser Arg Phe Trp Glu Leu Val Asp Lys Glu Arg
2915 2920 2925
Asn Leu His Leu Glu Gly Lys Cys Glu Thr Cys Val Tyr Asn Met
2930 2935 2940
Met Gly Lys Arg Glu Lys Lys Leu Gly Glu Phe Gly Lys Ala Lys
2945 2950 2955
Gly Ser Arg Ala Ile Trp Tyr Met Trp Leu Gly Ala Arg Phe Leu
2960 2965 2970
Glu Phe Glu Ala Leu Gly Phe Leu Asn Glu Asp His Trp Phe Ser
2975 2980 2985
Arg Glu Asn Ser Leu Ser Gly Val Glu Gly Glu Gly Leu His Lys
2990 2995 3000
Leu Gly Tyr Ile Leu Arg Asp Val Ser Lys Lys Glu Gly Gly Ala
3005 3010 3015
Met Tyr Ala Asp Asp Thr Ala Gly Trp Asp Thr Arg Ile Thr Leu
3020 3025 3030
Glu Asp Leu Lys Asn Glu Glu Met Val Thr Asn His Met Glu Gly
3035 3040 3045
Glu His Lys Lys Leu Ala Glu Ala Ile Phe Lys Leu Thr Tyr Gln
3050 3055 3060
Asn Lys Val Val Arg Val Gln Arg Pro Thr Pro Arg Gly Thr Val
3065 3070 3075
Met Asp Ile Ile Ser Arg Arg Asp Gln Arg Gly Ser Gly Gln Val
3080 3085 3090
Gly Thr Tyr Gly Leu Asn Thr Phe Thr Asn Met Glu Ala Gln Leu
3095 3100 3105
Ile Arg Gln Met Glu Gly Glu Gly Val Phe Lys Ser Ile Gln His
3110 3115 3120
Leu Thr Ile Thr Glu Glu Ile Ala Val Gln Asn Trp Leu Ala Arg
3125 3130 3135
Val Gly Arg Glu Arg Leu Ser Arg Met Ala Ile Ser Gly Asp Asp
3140 3145 3150
Cys Val Val Lys Pro Leu Asp Asp Arg Phe Ala Ser Ala Leu Thr
3155 3160 3165
Ala Leu Asn Asp Met Gly Lys Ile Arg Lys Asp Ile Gln Gln Trp
3170 3175 3180
Glu Pro Ser Arg Gly Trp Asn Asp Trp Thr Gln Val Pro Phe Cys
3185 3190 3195
Ser His His Phe His Glu Leu Ile Met Lys Asp Gly Arg Val Leu
3200 3205 3210
Val Val Pro Cys Arg Asn Gln Asp Glu Leu Ile Gly Arg Ala Arg
3215 3220 3225
Ile Ser Gln Gly Ala Gly Trp Ser Leu Arg Glu Thr Ala Cys Leu
3230 3235 3240
Gly Lys Ser Tyr Ala Gln Met Trp Ser Leu Met Tyr Phe His Arg
3245 3250 3255
Arg Asp Leu Arg Leu Ala Ala Asn Ala Ile Cys Ser Ala Val Pro
3260 3265 3270
Ser His Trp Val Pro Thr Ser Arg Thr Thr Trp Ser Ile His Ala
3275 3280 3285
Lys His Glu Trp Met Thr Thr Glu Asp Met Leu Thr Val Trp Asn
3290 3295 3300
Arg Val Trp Ile Gln Glu Asn Pro Trp Met Glu Asp Lys Thr Pro
3305 3310 3315
Val Glu Ser Trp Glu Glu Ile Pro Tyr Leu Gly Lys Arg Glu Asp
3320 3325 3330
Gln Trp Cys Gly Ser Leu Ile Gly Leu Thr Ser Arg Ala Thr Trp
3335 3340 3345
Ala Lys Asn Ile Gln Ala Ala Ile Asn Gln Val Arg Ser Leu Ile
3350 3355 3360
Gly Asn Glu Glu Tyr Thr Asp Tyr Met Pro Ser Met Lys Arg Phe
3365 3370 3375
Arg Arg Glu Glu Glu Glu Ala Gly Val Leu Trp
3380 3385
<210> 10
<211> 10723
<212> DNA
<213> 人工序列
<220>
<223> 登革热病毒血清型4, BVS
<400> 10
agttgttagt ctacgtggac cgacaaagac agattctttg agggagctaa gctcaatgta 60
gttctaacag ttttttaatt agagagcaga tctctgatga ataaccaacg gaaaaaggcg 120
aaaaacacgc ctttcaatat gctgaaacgc gagagaaacc gcgtgtcgac tgtgcaacag 180
ctgacaaaga gattctcact tggaatgctg cagggacgag gacctttaaa actgttcatg 240
gccctggtgg cgttccttcg tttcctaaca atcccaccaa cagcagggat attgaagaga 300
tggggaacaa ttaaaaaatc aaaagctatt aatgttttga gagggttcag gaaagagatt 360
ggaaggatgc tgaacatctt gaataggaga cgcagctctg caggcatgat cattatgctg 420
attccaacag tgatggcgtt ccatttaacc acgcgtgatg gcgaacccct catgatagtg 480
gcaaaacatg aaagggggag acctctcttg tttaagacaa cagaggggat caacaaatgc 540
actctcattg ccatggactt gggtgaaatg tgtgaggaca ctgtcacgta taaatgcccc 600
ttactggtca ataccgaacc tgaagacatt gattgctggt gcaatctcac gtctacctgg 660
gtcatgtatg ggacatgcac ccagagcgga gaacggagac gagagaagcg ctcagtagct 720
ttaacaccac attcaggaat gggattggaa acaagagctg agacatggat gtcatcggaa 780
ggggcttgga agcatgctca gagagtagag agctggatac tcagaaaccc aggattcgcg 840
ctcttggcag gatttatggc ttatatgatt gggcaaacag gaatccagcg aactgtcttc 900
tttgtcctaa tgatgctggt cgccccatcc tacggaatgc gatgcgtagg agtaggaaac 960
agagactttg tggaaggagt ctcaggtgga gcatgggtcg atctggtgct agaacatgga 1020
ggatgcgtca caaccatggc ccagggaaaa ccaaccttgg attttgaact gactaagaca 1080
acagccaagg aagtggctct gttaagaacc tattgcattg aagcctcaat atcaaacata 1140
accacggcaa caagatgtcc aacgcaagga gagccttatc taaaagagga acaagaccaa 1200
cagtacattt gccggagaga tgtggtagac agagggtggg gcaatggctg tggcttgttt 1260
ggaaaaggag gagttgtgac atgtgcgaag ttttcatgtt cggggaagat aacaggcaat 1320
ttggtccaaa ttgagaacct tgaatacaca gtggttgtaa cagtccacaa tggagacacc 1380
catgcagtag gaaatgacac gtccaatcat ggagttacag ccacgataac tcccaggtca 1440
ccatcggtgg aagtcaaatt gccggactat ggagaactaa cactcgattg tgaacccagg 1500
tctggaattg actttaatga gatgattctg atgaaaatga aaaagaaaac atggcttgtg 1560
cataagcaat ggtttttgga tctacctcta ccatggacag caggagcaga cacatcagag 1620
gttcactgga attacaaaga gagaatggtg acatttaagg ttcctcatgc caagagacag 1680
gatgtgacag tgctgggatc tcaggaagga gccatgcatt ctgccctcgc tggagccaca 1740
gaagtggact ccggtgatgg aaatcacatg tttgcaggac atctcaagtg caaagtccgt 1800
atggagaaat tgagaatcaa gggaatgtca tacacgatgt gttcaggaaa gttctcaatt 1860
gacaaagaga tggcagaaac acagcatggg acaacagtgg tgaaagtcaa gtatgaaggt 1920
gctggagctc cgtgtaaagt ccccatagag ataagagatg tgaacaagga aaaagtggtt 1980
gggcgtatca tctcatccac ccctttggct gagaatacca acagtgtaac caacatagag 2040
ttagaacccc cctttgggga cagctacata gtgataggtg ttggaaacag tgcattaaca 2100
ctccattggt tcaggaaagg gagttccatt ggcaagatgt ttgagtccac atacagaggt 2160
gcaaaacgaa tggccattct aggtgaaaca gcttgggatt ttggttccgt tggtggactg 2220
ttcacatcat tgggaaaggc tgtgcaccag gtttttggaa gtgtgtatac aaccctgttt 2280
ggaggagtct catggatgat tagaatccta attgggttcc tagtgttgtg gattggcacg 2340
aactcaagga acacttcaat ggctatgacg tgcatagctg ccggcattgt gacactgtat 2400
ttgggagtca tggtgcaggc cgatagtggt tgcgttgtga gctggaaaaa caaagaactg 2460
aaatgtggca gtgggatttt catcacagac aacgtgcaca catggacaga acaatacaag 2520
ttccaaccag aatccccttc aaaactagct tcagctatcc agaaagccca tgaagaggac 2580
atttgtggaa tccgctcagt aacaagactg gagaatctga tgtggaaaca aataacacca 2640
gaattgaatc acattctatc agaaaatgag gtgaagttaa ctattatgac aggagacatc 2700
aaaggaatca tgcaggcagg aaaacgatct ctgcggcctc agcccactga gctgaagtat 2760
tcatggaaaa catggggcaa agcaaaaatg ctctctacag agtctcataa ccagaccttt 2820
ctcattgatg gccccgaaac agcagaatgc cccaacacaa atagagcttg gaattcgttg 2880
gaagttgaag actatggctt tggagtattc accaccaata tatggctaaa attgaaagaa 2940
aaacaggatg tattctgcga ctcaaaactc atgtcagcgg ccataaaaga caacagagcc 3000
gtccatgccg atatgggtta ttggatagaa agtgcactca atgacacatg gaagatagag 3060
aaagcctctt tcattgaagt taaaaactgc cactggccaa aatcacacac cctctggagc 3120
aatggagtgc tagaaagtga gatgataatt ccaaagaatc tcgctggacc agtgtctcaa 3180
cacaactata gaccaggcta ccatacacaa ataacaggac catggcatct aggtaagctt 3240
gagatggact ttgatttctg tgatggaaca acagtggtag tgactgagga ctgcggaaat 3300
agaggaccct ctttgagaac aaccactgcc tctggaaaac tcataacaga atggtgctgc 3360
cgatcttgca cattaccacc gctaagatac agaggtgagg atgggtgctg gtacgggatg 3420
gaaatcagac cattgaagga gaaagaagag aatttggtca actccttggt cacagctgga 3480
catgggcagg tcgacaactt ttcactagga gtcttgggaa tggcattgtt cctggaggaa 3540
atgcttagga cccgagtagg aacgaaacat gcaatactac tagttgcagt ttcttttgtg 3600
acattgatca cagggaacat gtcctttaga gacctgggaa gagtgatggt tatggtaggc 3660
gccactatga cgggtgacat aggtatgggc gtgacttatc ttgccctact agcagccttc 3720
aaagtcagac caacttttgc agctggacta ctcttgagaa agctgacctc cagggaattg 3780
atgatgacta ctataggaat tgtactcctc tcccagagca ccataccaga gaccattctt 3840
gagttgactg atgcgttagc cttaggcatg atggtcctca aaatggtgag aaatatggaa 3900
aagtatcaat tggcagtgac tatcatggct atcttgtgcg tcccaaacgc agtgatatta 3960
caaaacgcat ggaaagtgag ttgcacaata ttggcagtgg tgtccgtttc cccactgttc 4020
ttaacatcct cacagcaaaa aacagattgg ataccattag cattgacgat caaaggtctc 4080
aatccaacag ctatttttct aacaaccctc tcaagaacca gcaagaaaag gagctggcca 4140
ttaaatgagg ctatcatggc agtcgggatg gtgagcattt tagccagttc tctcctaaaa 4200
aatgatattc ccatgacagg accattagtg gctggagggc tcctcactgt gtgctacgtg 4260
ctcactggac gatcggccga tttggaactg gagagagcag ccgatgtcaa atgggaagac 4320
caggcagaga tatcaggaag cagtccaatc ctgtcaataa caatatcaga agatggtagc 4380
atgtcgataa aaaatgaaga ggaagaacaa acactgacca tactcattag aacaggattg 4440
ctggtgatct caggactttt tcctgtatca ataccaatca cggcagcagc atggtacctg 4500
tgggaagtga agaaacaacg ggccggagta ttgtgggatg ttccttcacc cccacccatg 4560
ggaaaggctg aactggaaga tggagcctat agaattaagc aaaaagggat tcttggatat 4620
tcccagatcg gagccggagt ttacaaagaa ggaacattcc atacaatgtg gcatgtcaca 4680
cgtggcgctg ttctaatgca taaaggaaag aggattgaac catcatgggc ggacgtcaag 4740
aaagacctaa tatcatatgg aggaggctgg aagttagaag gagaatggaa ggaaggagaa 4800
gaagtccagg tattggcact ggagcctgga aaaaatccaa gagccgtcca aacgaaacct 4860
ggtcttttca aaaccaacgc cggaacaata ggtgctgtat ctctggactt ttctcctgga 4920
acgtcaggat ctccaattat cgacaaaaaa ggaaaagttg tgggtcttta tggtaatggt 4980
gttgttacaa ggagtggagc atatgtgagt gctatagccc agactgaaaa aagcattgaa 5040
gacaacccag agatcgaaga tgacattttc cgaaagagaa gactgaccat catggacctc 5100
cacccaggag cgggaaagac gaagagatac cttccggcca tagtcagaga agctataaaa 5160
cggggtttga gaacattaat cttggccccc actagagttg tggcagctga aatggaggaa 5220
gcccttagag gacttccaat aagataccag accccagcca tcagagctgt gcacaccggg 5280
cgggagattg tggacctaat gtgtcatgcc acatttacca tgaggctgct atcaccagtt 5340
agagtgccaa actacaacct gattatcatg gacgaagccc atttcacaga tccagcaagt 5400
atagcagcta gaggatacat ctcaactcga gtggagatgg gtgaggcagc tgggattttt 5460
atgacagcca ctcccccggg aagcagagac ccatttcctc agagcaatgc accaatcata 5520
gatgaagaaa gagaaatccc tgaacgctcg tggaattccg gacatgaatg ggtcacggat 5580
tttaaaggga agactgtttg gttcgttcca agtataaaag caggaaatga tatagcagct 5640
tgcctgagga aaaatggaaa gaaagtgata caactcagta ggaagacctt tgattctgag 5700
tatgtcaaga ctagaaccaa tgattgggac ttcgtggtta caactgacat ttcagaaatg 5760
ggtgccaatt tcaaggctga gagggttata gaccccagac gctgcatgaa accagtcata 5820
ctaacagatg gtgaagagcg ggtgattctg gcaggaccta tgccagtgac ccactctagt 5880
gcagcacaaa gaagagggag aataggaaga aatccaaaaa atgagaatga ccagtacata 5940
tacatggggg aacctctgga aaatgatgaa gactgtgcac actggaaaga agctaaaatg 6000
ctcctagata acatcaacac gccagaagga atcattccta gcatgttcga accagagcgt 6060
gaaaaggtgg atgccattga tggcgaatac cgcttgagag gagaagcaag gaaaaccttt 6120
gtagacttaa tgagaagagg agacctacca gtctggttgg cctacagagt ggcagctgaa 6180
ggcatcaact acgcagacag aaggtggtgt tttgatggag tcaagaacaa ccaaatccta 6240
gaagaaaacg tggaagttga aatctggaca aaagaagggg aaaggaagaa attgaaaccc 6300
agatggttgg atgctaggat ctattctgac ccactggcgc taaaagaatt taaggaattt 6360
gcagccggaa gaaagtctct gaccctgaac ctaatcacag aaatgggtag gctcccaacc 6420
ttcatgactc agaaggtaag agacgcactg gacaacttag cagtgctgca cacggctgag 6480
gcaggtggaa gggcgtacaa ccatgctctc agtgaactgc cggagaccct ggagacattg 6540
cttttactga cacttctggc tacagtcacg ggagggatct ttttattctt gatgagcgca 6600
aggggcatag ggaagatgac cctgggaatg tgctgcataa tcacggctag catcctccta 6660
tggtacgcac aaatacagcc acactggata gcagcttcaa taatactgga gttttttctc 6720
atagttttgc ttattccaga acctgaaaaa cagagaacac cccaagacaa ccaactgacc 6780
tacgttgtca tagccatcct cacagtggtg gccgcaacca tggcaaacga gatgggtttc 6840
ctagaaaaaa cgaagaaaga tctcggattg ggaagcattg caacccagca acccgagagc 6900
aacatcctgg acatagatct acgtcctgca tcagcatgga cgctgtatgc cgtggccaca 6960
acatttgtta caccaatgtt gagacatagc attgaaaatt cctcagtgaa tgtgtcccta 7020
acagccatag ccaaccaagc cacagtgtta atgggtctcg ggaaaggatg gccattgtca 7080
aagatggaca tcggagttcc ccttctcgcc attggatgct actcacaagt caaccccata 7140
actctcacag cagctctttt cttattggta gcacattatg ccatcatagg gccaggactc 7200
caagcaaaag caaccagaga agctcagaaa agagcagcgg cgggcatcat gaaaaaccca 7260
actgtcgatg gaataacagt gattgaccta gatccaatac cttatgatcc aaagtttgaa 7320
aagcagttgg gacaagtaat gctcctagtc ctctgcgtga ctcaagtatt gatgatgagg 7380
actacatggg ctctgtgtga ggctttaacc ttagctaccg ggcccatctc cacattgtgg 7440
gaaggaaatc cagggaggtt ttggaacact accattgcgg tgtcaatggc taacattttt 7500
agagggagtt acttggccgg agctggactt ctctttttta ttatgaagaa cacaaccaac 7560
acaagaaggg gaactggcaa cataggagag acgcttggag agaaatggaa aagccgattg 7620
aacgcattgg gaaaaagtga attccagatc tacaagaaaa gtggaatcca ggaagtggat 7680
agaaccttag caaaagaagg cattaaaaga ggagaaacgg accatcacgc tgtgtcgcga 7740
ggctcagcaa aactgagatg gttcgttgag agaaacatgg tcacaccaga agggaaagta 7800
gtggacctcg gttgtggcag aggaggctgg tcatactatt gtggaggact aaagaatgta 7860
agagaagtca aaggcctaac aaaaggagga ccaggacacg aagaacccat ccccatgtca 7920
acatatgggt ggaatctagt gcgtcttcaa agtggagttg acgttttctt catcccgcca 7980
gaaaagtgtg acacattatt gtgtgacata ggggagtcat caccaaatcc cacagtggaa 8040
gcaggacgaa cactcagagt ccttaactta gtagaaaatt ggttgaacaa caacactcaa 8100
ttttgcataa aggttctcaa cccatatatg ccctcagtca tagaaaaaat ggaagcacta 8160
caaaggaaat atggaggagc cttagtgagg aatccactct cacgaaactc cacacatgag 8220
atgtactggg tatccaatgc ttccgggaac atagtgtcat cagtgaacat gatttcaagg 8280
atgttgatca acagatttac aatgagatac aagaaagcca cttacgagcc ggatgttgac 8340
ctcggaagcg gaacccgtaa catcgggatt gaaagtgaga taccaaacct agatataatt 8400
gggaaaagaa tagaaaaaat aaagcaagag catgaaacat catggcacta tgaccaagac 8460
cacccataca aaacgtgggc ataccatggt agctatgaaa caaaacagac tggatcagca 8520
tcatccatgg tcaacggagt ggtcaggctg ctgacaaaac cttgggacgt cgtccccatg 8580
gtgacacaga tggcaatgac agacacgact ccatttggac aacagcgcgt ttttaaagag 8640
aaagtggaca cgagaaccca agaaccgaaa gaaggcacga agaaactaat gaaaataaca 8700
gcagagtggc tttggaaaga attagggaag aaaaagacac ccaggatgtg caccagagaa 8760
gaattcacaa gaaaggtgag aagcaatgca gccttggggg ccatattcac tgatgagaac 8820
aagtggaagt cggcacgtga ggctgttgaa gatagtaggt tttgggagct ggttgacaag 8880
gaaaggaatc tccatcttga aggaaagtgt gaaacatgtg tgtacaacat gatgggaaaa 8940
agagagaaga agctagggga attcggcaag gcaaaaggca gcagagccat atggtacatg 9000
tggcttggag cacgcttctt agagtttgaa gccctaggat tcttaaatga agatcactgg 9060
ttctccagag agaactccct gagtggagtg gaaggagaag ggctgcacaa gctaggttac 9120
attctaagag acgtgagcaa gaaagaggga ggagcaatgt atgccgatga caccgcagga 9180
tgggatacaa gaatcacact agaagaccta aaaaatgaag aaatggtaac aaaccacatg 9240
gaaggagaac acaagaaact agccgaggcc attttcaaac taacgtacca aaacaaggtg 9300
gtgcgtgtgc aaagaccaac accaagaggc acagtaatgg acatcatatc gagaagagac 9360
caaagaggta gtggacaagt tggcacctat ggactcaata ctttcaccaa tatggaagcc 9420
caactaatca gacagatgga gggagaagga gtctttaaaa gcattcagca cctaacaatc 9480
acagaagaaa tcgctgtgca aaactggtta gcaagagtgg ggcgcgaaag gttatcaaga 9540
atggccatca gtggagatga ttgtgttgtg aaacctttag atgacaggtt cgcaagcgct 9600
ttaacagctc taaatgacat gggaaagatt aggaaagaca tacaacaatg ggaaccttca 9660
agaggatgga atgattggac acaagtgccc ttctgttcac accatttcca tgagttaatc 9720
atgaaagacg gtcgcgtact cgttgttccc tgtagaaacc aagatgaact gattggcaga 9780
gcccgaatct cccaaggagc agggtggtct ttgcgggaga cggcctgttt ggggaagtct 9840
tacgcccaaa tgtggagctt gatgtacttc cacagacgcg acctcaggct ggcggcaaat 9900
gctatttgct cggcagtacc atcacattgg gttccaacaa gtcgaacaac ctggtccata 9960
catgctaaac atgaatggat gacaacggaa gacatgctga cagtctggaa cagggtgtgg 10020
attcaagaaa acccatggat ggaagacaaa actccagtgg aatcatggga ggaaatccca 10080
tacttgggga aaagagaaga ccaatggtgc ggctcattga ttgggttaac aagcagggcc 10140
acctgggcaa agaacatcca agcagcaata aatcaagtta gatcccttat aggcaatgaa 10200
gaatacacag attacatgcc atccatgaaa agattcagaa gagaagagga agaagcagga 10260
gttctgtggt agaaagcaaa actaacatga aacaaggcta gaagtcaggt cggattaagc 10320
catagtacgg aaaaaactat gctacctgtg agccccgtcc aaggacgtta aaagaagtca 10380
ggccatcata aatgccatag cttgagtaaa ctatgcagcc tgtagctcca cctgagaagg 10440
tgtaaaaaat ccgggaggcc acaaaccatg gaagctgtac gcatggcgta gtggactagc 10500
ggttagagga gacccctccc ttacaaatcg cagcaacaat gggggcccaa ggcgagatga 10560
agctgtagtc tcgctggaag gactagaggt tagaggagac ccccccgaaa caaaaaacag 10620
catattgacg ctgggaaaga ccagagatcc tgctgtctcc tcagcatcat tccaggcaca 10680
gaacgccaga aaatggaatg gtgctgttga atcaacaggt tct 10723
<210> 11
<211> 3391
<212> PRT
<213> 人工序列
<220>
<223> 登革热病毒血清型4, BVS
<400> 11
Met Asn Asn Gln Arg Lys Lys Ala Lys Asn Thr Pro Phe Asn Met Leu
1 5 10 15
Lys Arg Glu Arg Asn Arg Val Ser Thr Val Gln Gln Leu Thr Lys Arg
20 25 30
Phe Ser Leu Gly Met Leu Gln Gly Arg Gly Pro Leu Lys Leu Phe Met
35 40 45
Ala Leu Val Ala Phe Leu Arg Phe Leu Thr Ile Pro Pro Thr Ala Gly
50 55 60
Ile Leu Lys Arg Trp Gly Thr Ile Lys Lys Ser Lys Ala Ile Asn Val
65 70 75 80
Leu Arg Gly Phe Arg Lys Glu Ile Gly Arg Met Leu Asn Ile Leu Asn
85 90 95
Arg Arg Arg Ser Ser Ala Gly Met Ile Ile Met Leu Ile Pro Thr Val
100 105 110
Met Ala Phe His Leu Thr Thr Arg Asp Gly Glu Pro Leu Met Ile Val
115 120 125
Ala Lys His Glu Arg Gly Arg Pro Leu Leu Phe Lys Thr Thr Glu Gly
130 135 140
Ile Asn Lys Cys Thr Leu Ile Ala Met Asp Leu Gly Glu Met Cys Glu
145 150 155 160
Asp Thr Val Thr Tyr Lys Cys Pro Leu Leu Val Asn Thr Glu Pro Glu
165 170 175
Asp Ile Asp Cys Trp Cys Asn Leu Thr Ser Thr Trp Val Met Tyr Gly
180 185 190
Thr Cys Thr Gln Ser Gly Glu Arg Arg Arg Glu Lys Arg Ser Val Ala
195 200 205
Leu Thr Pro His Ser Gly Met Gly Leu Glu Thr Arg Ala Glu Thr Trp
210 215 220
Met Ser Ser Glu Gly Ala Trp Lys His Ala Gln Arg Val Glu Ser Trp
225 230 235 240
Ile Leu Arg Asn Pro Gly Phe Ala Leu Leu Ala Gly Phe Met Ala Tyr
245 250 255
Met Ile Gly Gln Thr Gly Ile Gln Arg Thr Val Phe Phe Val Leu Met
260 265 270
Met Leu Val Ala Pro Ser Tyr Gly Met Arg Cys Val Gly Val Gly Asn
275 280 285
Arg Asp Phe Val Glu Gly Val Ser Gly Gly Ala Trp Val Asp Leu Val
290 295 300
Leu Glu His Gly Gly Cys Val Thr Thr Met Ala Gln Gly Lys Pro Thr
305 310 315 320
Leu Asp Phe Glu Leu Thr Lys Thr Thr Ala Lys Glu Val Ala Leu Leu
325 330 335
Arg Thr Tyr Cys Ile Glu Ala Ser Ile Ser Asn Ile Thr Thr Ala Thr
340 345 350
Arg Cys Pro Thr Gln Gly Glu Pro Tyr Leu Lys Glu Glu Gln Asp Gln
355 360 365
Gln Tyr Ile Cys Arg Arg Asp Val Val Asp Arg Gly Trp Gly Asn Gly
370 375 380
Cys Gly Leu Phe Gly Lys Gly Gly Val Val Thr Cys Ala Lys Phe Ser
385 390 395 400
Cys Ser Gly Lys Ile Thr Gly Asn Leu Val Gln Ile Glu Asn Leu Glu
405 410 415
Tyr Thr Val Val Val Thr Val His Asn Gly Asp Thr His Ala Val Gly
420 425 430
Asn Asp Thr Ser Asn His Gly Val Thr Ala Thr Ile Thr Pro Arg Ser
435 440 445
Pro Ser Val Glu Val Lys Leu Pro Asp Tyr Gly Glu Leu Thr Leu Asp
450 455 460
Cys Glu Pro Arg Ser Gly Ile Asp Phe Asn Glu Met Ile Leu Met Lys
465 470 475 480
Met Lys Lys Lys Thr Trp Leu Val His Lys Gln Trp Phe Leu Asp Leu
485 490 495
Pro Leu Pro Trp Thr Ala Gly Ala Asp Thr Ser Glu Val His Trp Asn
500 505 510
Tyr Lys Glu Arg Met Val Thr Phe Lys Val Pro His Ala Lys Arg Gln
515 520 525
Asp Val Thr Val Leu Gly Ser Gln Glu Gly Ala Met His Ser Ala Leu
530 535 540
Ala Gly Ala Thr Glu Val Asp Ser Gly Asp Gly Asn His Met Phe Ala
545 550 555 560
Gly His Leu Lys Cys Lys Val Arg Met Glu Lys Leu Arg Ile Lys Gly
565 570 575
Met Ser Tyr Thr Met Cys Ser Gly Lys Phe Ser Ile Asp Lys Glu Met
580 585 590
Ala Glu Thr Gln His Gly Thr Thr Val Val Lys Val Lys Tyr Glu Gly
595 600 605
Ala Gly Ala Pro Cys Lys Val Pro Ile Glu Ile Arg Asp Val Asn Lys
610 615 620
Glu Lys Val Val Gly Arg Ile Ile Ser Ser Thr Pro Leu Ala Glu Asn
625 630 635 640
Thr Asn Ser Val Thr Asn Ile Glu Leu Glu Pro Pro Phe Gly Asp Ser
645 650 655
Tyr Ile Val Ile Gly Val Gly Asn Ser Ala Leu Thr Leu His Trp Phe
660 665 670
Arg Lys Gly Ser Ser Ile Gly Lys Met Phe Glu Ser Thr Tyr Arg Gly
675 680 685
Ala Lys Arg Met Ala Ile Leu Gly Glu Thr Ala Trp Asp Phe Gly Ser
690 695 700
Val Gly Gly Leu Phe Thr Ser Leu Gly Lys Ala Val His Gln Val Phe
705 710 715 720
Gly Ser Val Tyr Thr Thr Leu Phe Gly Gly Val Ser Trp Met Ile Arg
725 730 735
Ile Leu Ile Gly Phe Leu Val Leu Trp Ile Gly Thr Asn Ser Arg Asn
740 745 750
Thr Ser Met Ala Met Thr Cys Ile Ala Ala Gly Ile Val Thr Leu Tyr
755 760 765
Leu Gly Val Met Val Gln Ala Asp Ser Gly Cys Val Val Ser Trp Lys
770 775 780
Asn Lys Glu Leu Lys Cys Gly Ser Gly Ile Phe Ile Thr Asp Asn Val
785 790 795 800
His Thr Trp Thr Glu Gln Tyr Lys Phe Gln Pro Glu Ser Pro Ser Lys
805 810 815
Leu Ala Ser Ala Ile Gln Lys Ala His Glu Glu Asp Ile Cys Gly Ile
820 825 830
Arg Ser Val Thr Arg Leu Glu Asn Leu Met Trp Lys Gln Ile Thr Pro
835 840 845
Glu Leu Asn His Ile Leu Ser Glu Asn Glu Val Lys Leu Thr Ile Met
850 855 860
Thr Gly Asp Ile Lys Gly Ile Met Gln Ala Gly Lys Arg Ser Leu Arg
865 870 875 880
Pro Gln Pro Thr Glu Leu Lys Tyr Ser Trp Lys Thr Trp Gly Lys Ala
885 890 895
Lys Met Leu Ser Thr Glu Ser His Asn Gln Thr Phe Leu Ile Asp Gly
900 905 910
Pro Glu Thr Ala Glu Cys Pro Asn Thr Asn Arg Ala Trp Asn Ser Leu
915 920 925
Glu Val Glu Asp Tyr Gly Phe Gly Val Phe Thr Thr Asn Ile Trp Leu
930 935 940
Lys Leu Lys Glu Lys Gln Asp Val Phe Cys Asp Ser Lys Leu Met Ser
945 950 955 960
Ala Ala Ile Lys Asp Asn Arg Ala Val His Ala Asp Met Gly Tyr Trp
965 970 975
Ile Glu Ser Ala Leu Asn Asp Thr Trp Lys Ile Glu Lys Ala Ser Phe
980 985 990
Ile Glu Val Lys Asn Cys His Trp Pro Lys Ser His Thr Leu Trp Ser
995 1000 1005
Asn Gly Val Leu Glu Ser Glu Met Ile Ile Pro Lys Asn Leu Ala
1010 1015 1020
Gly Pro Val Ser Gln His Asn Tyr Arg Pro Gly Tyr His Thr Gln
1025 1030 1035
Ile Thr Gly Pro Trp His Leu Gly Lys Leu Glu Met Asp Phe Asp
1040 1045 1050
Phe Cys Asp Gly Thr Thr Val Val Val Thr Glu Asp Cys Gly Asn
1055 1060 1065
Arg Gly Pro Ser Leu Arg Thr Thr Thr Ala Ser Gly Lys Leu Ile
1070 1075 1080
Thr Glu Trp Cys Cys Arg Ser Cys Thr Leu Pro Pro Leu Arg Tyr
1085 1090 1095
Arg Gly Glu Asp Gly Cys Trp Tyr Gly Met Glu Ile Arg Pro Leu
1100 1105 1110
Lys Glu Lys Glu Glu Asn Leu Val Asn Ser Leu Val Thr Ala Gly
1115 1120 1125
His Gly Gln Val Asp Asn Phe Ser Leu Gly Val Leu Gly Met Ala
1130 1135 1140
Leu Phe Leu Glu Glu Met Leu Arg Thr Arg Val Gly Thr Lys His
1145 1150 1155
Ala Ile Leu Leu Val Ala Val Ser Phe Val Thr Leu Ile Thr Gly
1160 1165 1170
Asn Met Ser Phe Arg Asp Leu Gly Arg Val Met Val Met Val Gly
1175 1180 1185
Ala Thr Met Thr Gly Asp Ile Gly Met Gly Val Thr Tyr Leu Ala
1190 1195 1200
Leu Leu Ala Ala Phe Lys Val Arg Pro Thr Phe Ala Ala Gly Leu
1205 1210 1215
Leu Leu Arg Lys Leu Thr Ser Arg Glu Leu Met Met Thr Thr Ile
1220 1225 1230
Gly Ile Val Leu Leu Ser Gln Ser Thr Ile Pro Glu Thr Ile Leu
1235 1240 1245
Glu Leu Thr Asp Ala Leu Ala Leu Gly Met Met Val Leu Lys Met
1250 1255 1260
Val Arg Asn Met Glu Lys Tyr Gln Leu Ala Val Thr Ile Met Ala
1265 1270 1275
Ile Leu Cys Val Pro Asn Ala Val Ile Leu Gln Asn Ala Trp Lys
1280 1285 1290
Val Ser Cys Thr Ile Leu Ala Val Val Ser Val Ser Pro Leu Phe
1295 1300 1305
Leu Thr Ser Ser Gln Gln Lys Thr Asp Trp Ile Pro Leu Ala Leu
1310 1315 1320
Thr Ile Lys Gly Leu Asn Pro Thr Ala Ile Phe Leu Thr Thr Leu
1325 1330 1335
Ser Arg Thr Ser Lys Lys Arg Ser Trp Pro Leu Asn Glu Ala Ile
1340 1345 1350
Met Ala Val Gly Met Val Ser Ile Leu Ala Ser Ser Leu Leu Lys
1355 1360 1365
Asn Asp Ile Pro Met Thr Gly Pro Leu Val Ala Gly Gly Leu Leu
1370 1375 1380
Thr Val Cys Tyr Val Leu Thr Gly Arg Ser Ala Asp Leu Glu Leu
1385 1390 1395
Glu Arg Ala Ala Asp Val Lys Trp Glu Asp Gln Ala Glu Ile Ser
1400 1405 1410
Gly Ser Ser Pro Ile Leu Ser Ile Thr Ile Ser Glu Asp Gly Ser
1415 1420 1425
Met Ser Ile Lys Asn Glu Glu Glu Glu Gln Thr Leu Thr Ile Leu
1430 1435 1440
Ile Arg Thr Gly Leu Leu Val Ile Ser Gly Leu Phe Pro Val Ser
1445 1450 1455
Ile Pro Ile Thr Ala Ala Ala Trp Tyr Leu Trp Glu Val Lys Lys
1460 1465 1470
Gln Arg Ala Gly Val Leu Trp Asp Val Pro Ser Pro Pro Pro Met
1475 1480 1485
Gly Lys Ala Glu Leu Glu Asp Gly Ala Tyr Arg Ile Lys Gln Lys
1490 1495 1500
Gly Ile Leu Gly Tyr Ser Gln Ile Gly Ala Gly Val Tyr Lys Glu
1505 1510 1515
Gly Thr Phe His Thr Met Trp His Val Thr Arg Gly Ala Val Leu
1520 1525 1530
Met His Lys Gly Lys Arg Ile Glu Pro Ser Trp Ala Asp Val Lys
1535 1540 1545
Lys Asp Leu Ile Ser Tyr Gly Gly Gly Trp Lys Leu Glu Gly Glu
1550 1555 1560
Trp Lys Glu Gly Glu Glu Val Gln Val Leu Ala Leu Glu Pro Gly
1565 1570 1575
Lys Asn Pro Arg Ala Val Gln Thr Lys Pro Gly Leu Phe Lys Thr
1580 1585 1590
Asn Ala Gly Thr Ile Gly Ala Val Ser Leu Asp Phe Ser Pro Gly
1595 1600 1605
Thr Ser Gly Ser Pro Ile Ile Asp Lys Lys Gly Lys Val Val Gly
1610 1615 1620
Leu Tyr Gly Asn Gly Val Val Thr Arg Ser Gly Ala Tyr Val Ser
1625 1630 1635
Ala Ile Ala Gln Thr Glu Lys Ser Ile Glu Asp Asn Pro Glu Ile
1640 1645 1650
Glu Asp Asp Ile Phe Arg Lys Arg Arg Leu Thr Ile Met Asp Leu
1655 1660 1665
His Pro Gly Ala Gly Lys Thr Lys Arg Tyr Leu Pro Ala Ile Val
1670 1675 1680
Arg Glu Ala Ile Lys Arg Gly Leu Arg Thr Leu Ile Leu Ala Pro
1685 1690 1695
Thr Arg Val Val Ala Ala Glu Met Glu Glu Ala Leu Arg Gly Leu
1700 1705 1710
Pro Ile Arg Tyr Gln Thr Pro Ala Ile Arg Ala Val His Thr Gly
1715 1720 1725
Arg Glu Ile Val Asp Leu Met Cys His Ala Thr Phe Thr Met Arg
1730 1735 1740
Leu Leu Ser Pro Val Arg Val Pro Asn Tyr Asn Leu Ile Ile Met
1745 1750 1755
Asp Glu Ala His Phe Thr Asp Pro Ala Ser Ile Ala Ala Arg Gly
1760 1765 1770
Tyr Ile Ser Thr Arg Val Glu Met Gly Glu Ala Ala Gly Ile Phe
1775 1780 1785
Met Thr Ala Thr Pro Pro Gly Ser Arg Asp Pro Phe Pro Gln Ser
1790 1795 1800
Asn Ala Pro Ile Ile Asp Glu Glu Arg Glu Ile Pro Glu Arg Ser
1805 1810 1815
Trp Asn Ser Gly His Glu Trp Val Thr Asp Phe Lys Gly Lys Thr
1820 1825 1830
Val Trp Phe Val Pro Ser Ile Lys Ala Gly Asn Asp Ile Ala Ala
1835 1840 1845
Cys Leu Arg Lys Asn Gly Lys Lys Val Ile Gln Leu Ser Arg Lys
1850 1855 1860
Thr Phe Asp Ser Glu Tyr Val Lys Thr Arg Thr Asn Asp Trp Asp
1865 1870 1875
Phe Val Val Thr Thr Asp Ile Ser Glu Met Gly Ala Asn Phe Lys
1880 1885 1890
Ala Glu Arg Val Ile Asp Pro Arg Arg Cys Met Lys Pro Val Ile
1895 1900 1905
Leu Thr Asp Gly Glu Glu Arg Val Ile Leu Ala Gly Pro Met Pro
1910 1915 1920
Val Thr His Ser Ser Ala Ala Gln Arg Arg Gly Arg Ile Gly Arg
1925 1930 1935
Asn Pro Lys Asn Glu Asn Asp Gln Tyr Ile Tyr Met Gly Glu Pro
1940 1945 1950
Leu Glu Asn Asp Glu Asp Cys Ala His Trp Lys Glu Ala Lys Met
1955 1960 1965
Leu Leu Asp Asn Ile Asn Thr Pro Glu Gly Ile Ile Pro Ser Met
1970 1975 1980
Phe Glu Pro Glu Arg Glu Lys Val Asp Ala Ile Asp Gly Glu Tyr
1985 1990 1995
Arg Leu Arg Gly Glu Ala Arg Lys Thr Phe Val Asp Leu Met Arg
2000 2005 2010
Arg Gly Asp Leu Pro Val Trp Leu Ala Tyr Arg Val Ala Ala Glu
2015 2020 2025
Gly Ile Asn Tyr Ala Asp Arg Arg Trp Cys Phe Asp Gly Val Lys
2030 2035 2040
Asn Asn Gln Ile Leu Glu Glu Asn Val Glu Val Glu Ile Trp Thr
2045 2050 2055
Lys Glu Gly Glu Arg Lys Lys Leu Lys Pro Arg Trp Leu Asp Ala
2060 2065 2070
Arg Ile Tyr Ser Asp Pro Leu Ala Leu Lys Glu Phe Lys Glu Phe
2075 2080 2085
Ala Ala Gly Arg Lys Ser Leu Thr Leu Asn Leu Ile Thr Glu Met
2090 2095 2100
Gly Arg Leu Pro Thr Phe Met Thr Gln Lys Val Arg Asp Ala Leu
2105 2110 2115
Asp Asn Leu Ala Val Leu His Thr Ala Glu Ala Gly Gly Arg Ala
2120 2125 2130
Tyr Asn His Ala Leu Ser Glu Leu Pro Glu Thr Leu Glu Thr Leu
2135 2140 2145
Leu Leu Leu Thr Leu Leu Ala Thr Val Thr Gly Gly Ile Phe Leu
2150 2155 2160
Phe Leu Met Ser Ala Arg Gly Ile Gly Lys Met Thr Leu Gly Met
2165 2170 2175
Cys Cys Ile Ile Thr Ala Ser Ile Leu Leu Trp Tyr Ala Gln Ile
2180 2185 2190
Gln Pro His Trp Ile Ala Ala Ser Ile Ile Leu Glu Phe Phe Leu
2195 2200 2205
Ile Val Leu Leu Ile Pro Glu Pro Glu Lys Gln Arg Thr Pro Gln
2210 2215 2220
Asp Asn Gln Leu Thr Tyr Val Val Ile Ala Ile Leu Thr Val Val
2225 2230 2235
Ala Ala Thr Met Ala Asn Glu Met Gly Phe Leu Glu Lys Thr Lys
2240 2245 2250
Lys Asp Leu Gly Leu Gly Ser Ile Ala Thr Gln Gln Pro Glu Ser
2255 2260 2265
Asn Ile Leu Asp Ile Asp Leu Arg Pro Ala Ser Ala Trp Thr Leu
2270 2275 2280
Tyr Ala Val Ala Thr Thr Phe Val Thr Pro Met Leu Arg His Ser
2285 2290 2295
Ile Glu Asn Ser Ser Val Asn Val Ser Leu Thr Ala Ile Ala Asn
2300 2305 2310
Gln Ala Thr Val Leu Met Gly Leu Gly Lys Gly Trp Pro Leu Ser
2315 2320 2325
Lys Met Asp Ile Gly Val Pro Leu Leu Ala Ile Gly Cys Tyr Ser
2330 2335 2340
Gln Val Asn Pro Ile Thr Leu Thr Ala Ala Leu Phe Leu Leu Val
2345 2350 2355
Ala His Tyr Ala Ile Ile Gly Pro Gly Leu Gln Ala Lys Ala Thr
2360 2365 2370
Arg Glu Ala Gln Lys Arg Ala Ala Ala Gly Ile Met Lys Asn Pro
2375 2380 2385
Thr Val Asp Gly Ile Thr Val Ile Asp Leu Asp Pro Ile Pro Tyr
2390 2395 2400
Asp Pro Lys Phe Glu Lys Gln Leu Gly Gln Val Met Leu Leu Val
2405 2410 2415
Leu Cys Val Thr Gln Val Leu Met Met Arg Thr Thr Trp Ala Leu
2420 2425 2430
Cys Glu Ala Leu Thr Leu Ala Thr Gly Pro Ile Ser Thr Leu Trp
2435 2440 2445
Glu Gly Asn Pro Gly Arg Phe Trp Asn Thr Thr Ile Ala Val Ser
2450 2455 2460
Met Ala Asn Ile Phe Arg Gly Ser Tyr Leu Ala Gly Ala Gly Leu
2465 2470 2475
Leu Phe Phe Ile Met Lys Asn Thr Thr Asn Thr Arg Arg Gly Thr
2480 2485 2490
Gly Asn Ile Gly Glu Thr Leu Gly Glu Lys Trp Lys Ser Arg Leu
2495 2500 2505
Asn Ala Leu Gly Lys Ser Glu Phe Gln Ile Tyr Lys Lys Ser Gly
2510 2515 2520
Ile Gln Glu Val Asp Arg Thr Leu Ala Lys Glu Gly Ile Lys Arg
2525 2530 2535
Gly Glu Thr Asp His His Ala Val Ser Arg Gly Ser Ala Lys Leu
2540 2545 2550
Arg Trp Phe Val Glu Arg Asn Met Val Thr Pro Glu Gly Lys Val
2555 2560 2565
Val Asp Leu Gly Cys Gly Arg Gly Gly Trp Ser Tyr Tyr Cys Gly
2570 2575 2580
Gly Leu Lys Asn Val Arg Glu Val Lys Gly Leu Thr Lys Gly Gly
2585 2590 2595
Pro Gly His Glu Glu Pro Ile Pro Met Ser Thr Tyr Gly Trp Asn
2600 2605 2610
Leu Val Arg Leu Gln Ser Gly Val Asp Val Phe Phe Ile Pro Pro
2615 2620 2625
Glu Lys Cys Asp Thr Leu Leu Cys Asp Ile Gly Glu Ser Ser Pro
2630 2635 2640
Asn Pro Thr Val Glu Ala Gly Arg Thr Leu Arg Val Leu Asn Leu
2645 2650 2655
Val Glu Asn Trp Leu Asn Asn Asn Thr Gln Phe Cys Ile Lys Val
2660 2665 2670
Leu Asn Pro Tyr Met Pro Ser Val Ile Glu Lys Met Glu Ala Leu
2675 2680 2685
Gln Arg Lys Tyr Gly Gly Ala Leu Val Arg Asn Pro Leu Ser Arg
2690 2695 2700
Asn Ser Thr His Glu Met Tyr Trp Val Ser Asn Ala Ser Gly Asn
2705 2710 2715
Ile Val Ser Ser Val Asn Met Ile Ser Arg Met Leu Ile Asn Arg
2720 2725 2730
Phe Thr Met Arg Tyr Lys Lys Ala Thr Tyr Glu Pro Asp Val Asp
2735 2740 2745
Leu Gly Ser Gly Thr Arg Asn Ile Gly Ile Glu Ser Glu Ile Pro
2750 2755 2760
Asn Leu Asp Ile Ile Gly Lys Arg Ile Glu Lys Ile Lys Gln Glu
2765 2770 2775
His Glu Thr Ser Trp His Tyr Asp Gln Asp His Pro Tyr Lys Thr
2780 2785 2790
Trp Ala Tyr His Gly Ser Tyr Glu Thr Lys Gln Thr Gly Ser Ala
2795 2800 2805
Ser Ser Met Val Asn Gly Val Val Arg Leu Leu Thr Lys Pro Trp
2810 2815 2820
Asp Val Val Pro Met Val Thr Gln Met Ala Met Thr Asp Thr Thr
2825 2830 2835
Pro Phe Gly Gln Gln Arg Val Phe Lys Glu Lys Val Asp Thr Arg
2840 2845 2850
Thr Gln Glu Pro Lys Glu Gly Thr Lys Lys Leu Met Lys Ile Thr
2855 2860 2865
Ala Glu Trp Leu Trp Lys Glu Leu Gly Lys Lys Lys Thr Pro Arg
2870 2875 2880
Met Cys Thr Arg Glu Glu Phe Thr Arg Lys Val Arg Ser Asn Ala
2885 2890 2895
Ala Leu Gly Ala Ile Phe Thr Asp Glu Asn Lys Trp Lys Ser Ala
2900 2905 2910
Arg Glu Ala Val Glu Asp Ser Arg Phe Trp Glu Leu Val Asp Lys
2915 2920 2925
Glu Arg Asn Leu His Leu Glu Gly Lys Cys Glu Thr Cys Val Tyr
2930 2935 2940
Asn Met Met Gly Lys Arg Glu Lys Lys Leu Gly Glu Phe Gly Lys
2945 2950 2955
Ala Lys Gly Ser Arg Ala Ile Trp Tyr Met Trp Leu Gly Ala Arg
2960 2965 2970
Phe Leu Glu Phe Glu Ala Leu Gly Phe Leu Asn Glu Asp His Trp
2975 2980 2985
Phe Ser Arg Glu Asn Ser Leu Ser Gly Val Glu Gly Glu Gly Leu
2990 2995 3000
His Lys Leu Gly Tyr Ile Leu Arg Asp Val Ser Lys Lys Glu Gly
3005 3010 3015
Gly Ala Met Tyr Ala Asp Asp Thr Ala Gly Trp Asp Thr Arg Ile
3020 3025 3030
Thr Leu Glu Asp Leu Lys Asn Glu Glu Met Val Thr Asn His Met
3035 3040 3045
Glu Gly Glu His Lys Lys Leu Ala Glu Ala Ile Phe Lys Leu Thr
3050 3055 3060
Tyr Gln Asn Lys Val Val Arg Val Gln Arg Pro Thr Pro Arg Gly
3065 3070 3075
Thr Val Met Asp Ile Ile Ser Arg Arg Asp Gln Arg Gly Ser Gly
3080 3085 3090
Gln Val Gly Thr Tyr Gly Leu Asn Thr Phe Thr Asn Met Glu Ala
3095 3100 3105
Gln Leu Ile Arg Gln Met Glu Gly Glu Gly Val Phe Lys Ser Ile
3110 3115 3120
Gln His Leu Thr Ile Thr Glu Glu Ile Ala Val Gln Asn Trp Leu
3125 3130 3135
Ala Arg Val Gly Arg Glu Arg Leu Ser Arg Met Ala Ile Ser Gly
3140 3145 3150
Asp Asp Cys Val Val Lys Pro Leu Asp Asp Arg Phe Ala Ser Ala
3155 3160 3165
Leu Thr Ala Leu Asn Asp Met Gly Lys Ile Arg Lys Asp Ile Gln
3170 3175 3180
Gln Trp Glu Pro Ser Arg Gly Trp Asn Asp Trp Thr Gln Val Pro
3185 3190 3195
Phe Cys Ser His His Phe His Glu Leu Ile Met Lys Asp Gly Arg
3200 3205 3210
Val Leu Val Val Pro Cys Arg Asn Gln Asp Glu Leu Ile Gly Arg
3215 3220 3225
Ala Arg Ile Ser Gln Gly Ala Gly Trp Ser Leu Arg Glu Thr Ala
3230 3235 3240
Cys Leu Gly Lys Ser Tyr Ala Gln Met Trp Ser Leu Met Tyr Phe
3245 3250 3255
His Arg Arg Asp Leu Arg Leu Ala Ala Asn Ala Ile Cys Ser Ala
3260 3265 3270
Val Pro Ser His Trp Val Pro Thr Ser Arg Thr Thr Trp Ser Ile
3275 3280 3285
His Ala Lys His Glu Trp Met Thr Thr Glu Asp Met Leu Thr Val
3290 3295 3300
Trp Asn Arg Val Trp Ile Gln Glu Asn Pro Trp Met Glu Asp Lys
3305 3310 3315
Thr Pro Val Glu Ser Trp Glu Glu Ile Pro Tyr Leu Gly Lys Arg
3320 3325 3330
Glu Asp Gln Trp Cys Gly Ser Leu Ile Gly Leu Thr Ser Arg Ala
3335 3340 3345
Thr Trp Ala Lys Asn Ile Gln Ala Ala Ile Asn Gln Val Arg Ser
3350 3355 3360
Leu Ile Gly Asn Glu Glu Tyr Thr Asp Tyr Met Pro Ser Met Lys
3365 3370 3375
Arg Phe Arg Arg Glu Glu Glu Glu Ala Gly Val Leu Trp
3380 3385 3390
<210> 12
<211> 3391
<212> PRT
<213> 人工序列
<220>
<223> 登革热病毒血清型4, MVS
<400> 12
Met Asn Asn Gln Arg Lys Lys Ala Lys Asn Thr Pro Phe Asn Met Leu
1 5 10 15
Lys Arg Glu Arg Asn Arg Val Ser Thr Val Gln Gln Leu Thr Lys Arg
20 25 30
Phe Ser Leu Gly Met Leu Gln Gly Arg Gly Pro Leu Lys Leu Phe Met
35 40 45
Ala Leu Val Ala Phe Leu Arg Phe Leu Thr Ile Pro Pro Thr Ala Gly
50 55 60
Ile Leu Lys Arg Trp Gly Thr Ile Lys Lys Ser Lys Ala Ile Asn Val
65 70 75 80
Leu Arg Gly Phe Arg Lys Glu Ile Gly Arg Met Leu Asn Ile Leu Asn
85 90 95
Arg Arg Arg Ser Ser Ala Gly Met Ile Ile Met Leu Ile Pro Thr Val
100 105 110
Met Ala Phe His Leu Thr Thr Arg Asp Gly Glu Pro Leu Met Ile Val
115 120 125
Ala Lys His Glu Arg Gly Arg Pro Leu Leu Phe Lys Thr Thr Glu Gly
130 135 140
Ile Asn Lys Cys Thr Leu Ile Ala Met Asp Leu Gly Glu Met Cys Glu
145 150 155 160
Asp Thr Val Thr Tyr Lys Cys Pro Leu Leu Val Asn Thr Glu Pro Glu
165 170 175
Asp Ile Asp Cys Trp Cys Asn Leu Thr Ser Thr Trp Val Met Tyr Gly
180 185 190
Thr Cys Thr Gln Ser Gly Glu Arg Arg Arg Glu Lys Arg Ser Val Ala
195 200 205
Leu Thr Pro His Ser Gly Met Gly Leu Glu Thr Arg Ala Glu Thr Trp
210 215 220
Met Ser Ser Glu Gly Ala Trp Lys His Ala Gln Arg Val Glu Ser Trp
225 230 235 240
Ile Leu Arg Asn Pro Gly Phe Ala Leu Leu Ala Gly Phe Met Ala Tyr
245 250 255
Met Ile Gly Gln Thr Gly Ile Gln Arg Thr Val Phe Phe Val Leu Met
260 265 270
Met Leu Val Ala Pro Ser Tyr Gly Met Arg Cys Val Gly Val Gly Asn
275 280 285
Arg Asp Phe Val Glu Gly Val Ser Gly Gly Ala Trp Val Asp Leu Val
290 295 300
Leu Glu His Gly Gly Cys Val Thr Thr Met Ala Gln Gly Lys Pro Thr
305 310 315 320
Leu Asp Phe Glu Leu Thr Lys Thr Thr Ala Lys Glu Val Ala Leu Leu
325 330 335
Arg Thr Tyr Cys Ile Glu Ala Ser Ile Ser Asn Ile Thr Thr Ala Thr
340 345 350
Arg Cys Pro Thr Gln Gly Glu Pro Tyr Leu Lys Glu Glu Gln Asp Gln
355 360 365
Gln Tyr Ile Cys Arg Arg Asp Val Val Asp Arg Gly Trp Gly Asn Gly
370 375 380
Cys Gly Leu Phe Gly Lys Gly Gly Val Val Thr Cys Ala Lys Phe Ser
385 390 395 400
Cys Ser Gly Lys Ile Thr Gly Asn Leu Val Gln Ile Glu Asn Leu Glu
405 410 415
Tyr Thr Val Val Val Thr Val His Asn Gly Asp Thr His Ala Val Gly
420 425 430
Asn Asp Thr Ser Asn His Gly Val Thr Ala Thr Ile Thr Pro Arg Ser
435 440 445
Pro Ser Val Glu Val Lys Leu Pro Asp Tyr Gly Glu Leu Thr Leu Asp
450 455 460
Cys Glu Pro Arg Ser Gly Ile Asp Phe Asn Glu Met Ile Leu Met Lys
465 470 475 480
Met Lys Lys Lys Thr Trp Leu Val His Lys Gln Trp Phe Leu Asp Leu
485 490 495
Pro Leu Pro Trp Thr Ala Gly Ala Asp Thr Ser Glu Val His Trp Asn
500 505 510
Tyr Lys Glu Arg Met Val Thr Phe Lys Val Pro His Ala Lys Arg Gln
515 520 525
Asp Val Thr Val Leu Gly Ser Gln Glu Gly Ala Met His Ser Ala Leu
530 535 540
Ala Gly Ala Thr Glu Val Asp Ser Gly Asp Gly Asn His Met Phe Ala
545 550 555 560
Gly His Leu Lys Cys Lys Val Arg Met Glu Lys Leu Arg Ile Lys Gly
565 570 575
Met Ser Tyr Thr Met Cys Ser Gly Lys Phe Ser Ile Asp Lys Glu Met
580 585 590
Ala Glu Thr Gln His Gly Thr Thr Val Val Lys Val Lys Tyr Glu Gly
595 600 605
Ala Gly Ala Pro Cys Lys Val Pro Ile Glu Ile Arg Asp Val Asn Lys
610 615 620
Glu Lys Val Val Gly Arg Ile Ile Ser Ser Thr Pro Leu Ala Glu Asn
625 630 635 640
Thr Asn Ser Val Thr Asn Ile Glu Leu Glu Pro Pro Phe Gly Asp Ser
645 650 655
Tyr Ile Val Ile Gly Val Gly Asn Ser Ala Leu Thr Leu His Trp Phe
660 665 670
Arg Lys Gly Ser Ser Ile Gly Lys Met Phe Glu Ser Thr Tyr Arg Gly
675 680 685
Ala Lys Arg Met Ala Ile Leu Gly Glu Thr Ala Trp Asp Phe Gly Ser
690 695 700
Val Gly Gly Leu Phe Thr Ser Leu Gly Lys Ala Val His Gln Val Phe
705 710 715 720
Gly Ser Val Tyr Thr Thr Leu Phe Gly Gly Val Ser Trp Met Ile Arg
725 730 735
Ile Leu Ile Gly Phe Leu Val Leu Trp Ile Gly Thr Asn Ser Arg Asn
740 745 750
Thr Ser Met Ala Met Thr Cys Ile Ala Ala Gly Ile Val Thr Leu Tyr
755 760 765
Leu Gly Val Met Val Gln Ala Asp Ser Gly Cys Val Val Ser Trp Lys
770 775 780
Asn Lys Glu Leu Lys Cys Gly Ser Gly Ile Phe Ile Thr Asp Asn Val
785 790 795 800
His Thr Trp Thr Glu Gln Tyr Lys Phe Gln Pro Glu Ser Pro Ser Lys
805 810 815
Leu Ala Ser Ala Ile Gln Lys Ala His Glu Glu Asp Ile Cys Gly Ile
820 825 830
Arg Ser Val Thr Arg Leu Glu Asn Leu Met Trp Lys Gln Ile Thr Pro
835 840 845
Glu Leu Asn His Ile Leu Ser Glu Asn Glu Val Lys Leu Thr Ile Met
850 855 860
Thr Gly Asp Ile Lys Gly Ile Met Gln Ala Gly Lys Arg Ser Leu Arg
865 870 875 880
Pro Gln Pro Thr Glu Leu Lys Tyr Ser Trp Lys Thr Trp Gly Lys Ala
885 890 895
Lys Met Leu Ser Thr Glu Ser His Asn Gln Thr Phe Leu Ile Asp Gly
900 905 910
Pro Glu Thr Ala Glu Cys Pro Asn Thr Asn Arg Ala Trp Asn Ser Leu
915 920 925
Glu Val Glu Asp Tyr Gly Phe Gly Val Phe Thr Thr Asn Ile Trp Leu
930 935 940
Lys Leu Lys Glu Lys Gln Asp Val Phe Cys Asp Ser Lys Leu Met Ser
945 950 955 960
Ala Ala Ile Lys Asp Asn Arg Ala Val His Ala Asp Met Gly Tyr Trp
965 970 975
Ile Glu Ser Ala Leu Asn Asp Thr Trp Lys Ile Glu Lys Ala Ser Phe
980 985 990
Ile Glu Val Lys Asn Cys His Trp Pro Lys Ser His Thr Leu Trp Ser
995 1000 1005
Asn Gly Val Leu Glu Ser Glu Met Ile Ile Pro Lys Asn Leu Ala
1010 1015 1020
Gly Pro Val Ser Gln His Asn Tyr Arg Pro Gly Tyr His Thr Gln
1025 1030 1035
Ile Thr Gly Pro Trp His Leu Gly Lys Leu Glu Met Asp Phe Asp
1040 1045 1050
Phe Cys Asp Gly Thr Thr Val Val Val Thr Glu Asp Cys Gly Asn
1055 1060 1065
Arg Gly Pro Ser Leu Arg Thr Thr Thr Ala Ser Gly Lys Leu Ile
1070 1075 1080
Thr Glu Trp Cys Cys Arg Ser Cys Thr Leu Pro Pro Leu Arg Tyr
1085 1090 1095
Arg Gly Glu Asp Gly Cys Trp Tyr Gly Met Glu Ile Arg Pro Leu
1100 1105 1110
Lys Glu Lys Glu Glu Asn Leu Val Asn Ser Leu Val Thr Ala Gly
1115 1120 1125
His Gly Gln Val Asp Asn Phe Ser Leu Gly Val Leu Gly Met Ala
1130 1135 1140
Leu Phe Leu Glu Glu Met Leu Arg Thr Arg Val Gly Thr Lys His
1145 1150 1155
Ala Ile Leu Leu Val Ala Val Ser Phe Val Thr Leu Ile Thr Gly
1160 1165 1170
Asn Met Ser Phe Arg Asp Leu Gly Arg Val Met Val Met Val Gly
1175 1180 1185
Ala Thr Met Thr Gly Asp Ile Gly Met Gly Val Thr Tyr Leu Ala
1190 1195 1200
Leu Leu Ala Ala Phe Lys Val Arg Pro Thr Phe Ala Ala Gly Leu
1205 1210 1215
Leu Leu Arg Lys Leu Thr Ser Arg Glu Leu Met Met Thr Thr Ile
1220 1225 1230
Gly Ile Val Leu Leu Ser Gln Ser Thr Ile Pro Glu Thr Ile Leu
1235 1240 1245
Glu Leu Thr Asp Ala Leu Ala Leu Gly Met Met Val Leu Lys Met
1250 1255 1260
Val Arg Asn Met Glu Lys Tyr Gln Leu Ala Val Thr Ile Met Ala
1265 1270 1275
Ile Leu Cys Val Pro Asn Ala Val Ile Leu Gln Asn Ala Trp Lys
1280 1285 1290
Val Ser Cys Thr Ile Leu Ala Val Val Ser Val Ser Pro Leu Phe
1295 1300 1305
Leu Thr Ser Ser Gln Gln Lys Thr Asp Trp Ile Pro Leu Ala Leu
1310 1315 1320
Thr Ile Lys Gly Leu Asn Pro Thr Ala Ile Phe Leu Thr Thr Leu
1325 1330 1335
Ser Arg Thr Ser Lys Lys Arg Ser Trp Pro Leu Asn Glu Ala Ile
1340 1345 1350
Met Ala Val Gly Met Val Ser Ile Leu Ala Ser Ser Leu Leu Lys
1355 1360 1365
Asn Asp Ile Pro Met Thr Gly Pro Leu Val Ala Gly Gly Leu Leu
1370 1375 1380
Thr Val Cys Tyr Val Leu Thr Gly Arg Ser Ala Asp Leu Glu Leu
1385 1390 1395
Glu Arg Ala Ala Asp Val Lys Trp Glu Asp Gln Ala Glu Ile Ser
1400 1405 1410
Gly Ser Ser Pro Ile Leu Ser Ile Thr Ile Ser Glu Asp Gly Ser
1415 1420 1425
Met Ser Ile Lys Asn Glu Glu Glu Glu Gln Thr Leu Thr Ile Leu
1430 1435 1440
Ile Arg Thr Gly Leu Leu Val Ile Ser Gly Leu Phe Pro Val Ser
1445 1450 1455
Ile Pro Ile Thr Ala Ala Ala Trp Tyr Leu Trp Glu Val Lys Lys
1460 1465 1470
Gln Arg Ala Gly Val Leu Trp Asp Val Pro Ser Pro Pro Pro Met
1475 1480 1485
Gly Lys Ala Glu Leu Glu Asp Gly Ala Tyr Arg Ile Lys Gln Lys
1490 1495 1500
Gly Ile Leu Gly Tyr Ser Gln Ile Gly Ala Gly Val Tyr Lys Glu
1505 1510 1515
Gly Thr Phe His Thr Met Trp His Val Thr Arg Gly Ala Val Leu
1520 1525 1530
Met His Lys Gly Lys Arg Ile Glu Pro Ser Trp Ala Asp Val Lys
1535 1540 1545
Lys Asp Leu Ile Ser Tyr Gly Gly Gly Trp Lys Leu Glu Gly Glu
1550 1555 1560
Trp Lys Glu Gly Glu Glu Val Gln Val Leu Ala Leu Glu Pro Gly
1565 1570 1575
Lys Asn Pro Arg Ala Val Gln Thr Lys Pro Gly Leu Phe Lys Thr
1580 1585 1590
Asn Ala Gly Thr Ile Gly Ala Val Ser Leu Asp Phe Ser Pro Gly
1595 1600 1605
Thr Ser Gly Ser Pro Ile Ile Asp Lys Lys Gly Lys Val Val Gly
1610 1615 1620
Leu Tyr Gly Asn Gly Val Val Thr Arg Ser Gly Ala Tyr Val Ser
1625 1630 1635
Ala Ile Ala Gln Thr Glu Lys Ser Ile Glu Asp Asn Pro Glu Ile
1640 1645 1650
Glu Asp Asp Ile Phe Arg Lys Arg Arg Leu Thr Ile Met Asp Leu
1655 1660 1665
His Pro Gly Ala Gly Lys Thr Lys Arg Tyr Leu Pro Ala Ile Val
1670 1675 1680
Arg Glu Ala Ile Lys Arg Gly Leu Arg Thr Leu Ile Leu Ala Pro
1685 1690 1695
Thr Arg Val Val Ala Ala Glu Met Glu Glu Ala Leu Arg Gly Leu
1700 1705 1710
Pro Ile Arg Tyr Gln Thr Pro Ala Ile Arg Ala Val His Thr Gly
1715 1720 1725
Arg Glu Ile Val Asp Leu Met Cys His Ala Thr Phe Thr Met Arg
1730 1735 1740
Leu Leu Ser Pro Val Arg Val Pro Asn Tyr Asn Leu Ile Ile Met
1745 1750 1755
Asp Glu Ala His Phe Thr Asp Pro Ala Ser Ile Ala Ala Arg Gly
1760 1765 1770
Tyr Ile Ser Thr Arg Val Glu Met Gly Glu Ala Ala Gly Ile Phe
1775 1780 1785
Met Thr Ala Thr Pro Pro Gly Ser Arg Asp Pro Phe Pro Gln Ser
1790 1795 1800
Asn Ala Pro Ile Ile Asp Glu Glu Arg Glu Ile Pro Glu Arg Ser
1805 1810 1815
Trp Asn Ser Gly His Glu Trp Val Thr Asp Phe Lys Gly Lys Thr
1820 1825 1830
Val Trp Phe Val Pro Ser Ile Lys Ala Gly Asn Asp Ile Ala Ala
1835 1840 1845
Cys Leu Arg Lys Asn Gly Lys Lys Val Ile Gln Leu Ser Arg Lys
1850 1855 1860
Thr Phe Asp Ser Glu Tyr Val Lys Thr Arg Thr Asn Asp Trp Asp
1865 1870 1875
Phe Val Val Thr Thr Asp Ile Ser Glu Met Gly Ala Asn Phe Lys
1880 1885 1890
Ala Glu Arg Val Ile Asp Pro Arg Arg Cys Met Lys Pro Val Ile
1895 1900 1905
Leu Thr Asp Gly Glu Glu Arg Val Ile Leu Ala Gly Pro Met Pro
1910 1915 1920
Val Thr His Ser Ser Ala Ala Gln Arg Arg Gly Arg Ile Gly Arg
1925 1930 1935
Asn Pro Lys Asn Glu Asn Asp Gln Tyr Ile Tyr Met Gly Glu Pro
1940 1945 1950
Leu Glu Asn Asp Glu Asp Cys Ala His Trp Lys Glu Ala Lys Met
1955 1960 1965
Leu Leu Asp Asn Ile Asn Thr Pro Glu Gly Ile Ile Pro Ser Met
1970 1975 1980
Phe Glu Pro Glu Arg Glu Lys Val Asp Ala Ile Asp Gly Glu Tyr
1985 1990 1995
Arg Leu Arg Gly Glu Ala Arg Lys Thr Phe Val Asp Leu Met Arg
2000 2005 2010
Arg Gly Asp Leu Pro Val Trp Leu Ala Tyr Arg Val Ala Ala Glu
2015 2020 2025
Gly Ile Asn Tyr Ala Asp Arg Arg Trp Cys Phe Asp Gly Val Lys
2030 2035 2040
Asn Asn Gln Ile Leu Glu Glu Asn Val Glu Val Glu Ile Trp Thr
2045 2050 2055
Lys Glu Gly Glu Arg Lys Lys Leu Lys Pro Arg Trp Leu Asp Ala
2060 2065 2070
Arg Ile Tyr Ser Asp Pro Leu Ala Leu Lys Glu Phe Lys Glu Phe
2075 2080 2085
Ala Ala Gly Arg Lys Ser Leu Thr Leu Asn Leu Ile Thr Glu Met
2090 2095 2100
Gly Arg Leu Pro Thr Phe Met Thr Gln Lys Val Arg Asp Ala Leu
2105 2110 2115
Asp Asn Leu Ala Val Leu His Thr Ala Glu Ala Gly Gly Arg Ala
2120 2125 2130
Tyr Asn His Ala Leu Ser Glu Leu Pro Glu Thr Leu Glu Thr Leu
2135 2140 2145
Leu Leu Leu Thr Leu Leu Ala Thr Val Thr Gly Gly Ile Phe Leu
2150 2155 2160
Phe Leu Met Ser Ala Arg Gly Ile Gly Lys Met Thr Leu Gly Met
2165 2170 2175
Cys Cys Ile Ile Thr Ala Ser Ile Leu Leu Trp Tyr Ala Gln Ile
2180 2185 2190
Gln Pro His Trp Ile Ala Ala Ser Ile Ile Leu Glu Phe Phe Leu
2195 2200 2205
Ile Val Leu Leu Ile Pro Glu Pro Glu Lys Gln Arg Thr Pro Gln
2210 2215 2220
Asp Asn Gln Leu Thr Tyr Val Val Ile Ala Ile Leu Thr Val Val
2225 2230 2235
Ala Ala Thr Met Ala Asn Glu Met Gly Phe Leu Glu Lys Thr Lys
2240 2245 2250
Lys Asp Leu Gly Leu Gly Ser Ile Ala Thr Gln Gln Pro Glu Ser
2255 2260 2265
Asn Ile Leu Asp Ile Asp Leu Arg Pro Ala Ser Ala Trp Thr Leu
2270 2275 2280
Tyr Ala Val Ala Thr Thr Phe Val Thr Pro Met Leu Arg His Ser
2285 2290 2295
Ile Glu Asn Ser Ser Val Asn Val Ser Leu Thr Ala Ile Ala Asn
2300 2305 2310
Gln Ala Thr Val Leu Met Gly Leu Gly Lys Gly Trp Pro Leu Ser
2315 2320 2325
Lys Met Asp Ile Gly Val Pro Leu Leu Ala Ile Gly Cys Tyr Ser
2330 2335 2340
Gln Val Asn Pro Ile Thr Leu Thr Ala Ala Leu Phe Leu Leu Val
2345 2350 2355
Ala His Tyr Ala Ile Ile Gly Pro Gly Leu Gln Ala Lys Ala Thr
2360 2365 2370
Arg Glu Ala Gln Lys Arg Ala Ala Ala Gly Ile Met Lys Asn Pro
2375 2380 2385
Thr Val Asp Gly Ile Thr Val Ile Asp Leu Asp Pro Ile Pro Tyr
2390 2395 2400
Asp Pro Lys Phe Glu Lys Gln Leu Gly Gln Val Met Leu Leu Val
2405 2410 2415
Leu Cys Val Thr Gln Val Leu Met Met Arg Thr Thr Trp Ala Leu
2420 2425 2430
Cys Glu Ala Leu Thr Leu Ala Thr Gly Pro Ile Ser Thr Leu Trp
2435 2440 2445
Glu Gly Asn Pro Gly Arg Phe Trp Asn Thr Thr Ile Ala Val Ser
2450 2455 2460
Met Ala Asn Ile Phe Arg Gly Ser Tyr Leu Ala Gly Ala Gly Leu
2465 2470 2475
Leu Phe Ser Ile Met Lys Asn Thr Thr Asn Thr Arg Arg Gly Thr
2480 2485 2490
Gly Asn Ile Gly Glu Thr Leu Gly Glu Lys Trp Lys Ser Arg Leu
2495 2500 2505
Asn Ala Leu Gly Lys Ser Glu Phe Gln Ile Tyr Lys Lys Ser Gly
2510 2515 2520
Ile Gln Glu Val Asp Arg Thr Leu Ala Lys Glu Gly Ile Lys Arg
2525 2530 2535
Gly Glu Thr Asp His His Ala Val Ser Arg Gly Ser Ala Lys Leu
2540 2545 2550
Arg Trp Phe Val Glu Arg Asn Met Val Thr Pro Glu Gly Lys Val
2555 2560 2565
Val Asp Leu Gly Cys Gly Arg Gly Gly Trp Ser Tyr Tyr Cys Gly
2570 2575 2580
Gly Leu Lys Asn Val Arg Glu Val Lys Gly Leu Thr Lys Gly Gly
2585 2590 2595
Pro Gly His Glu Glu Pro Ile Pro Met Ser Thr Tyr Gly Trp Asn
2600 2605 2610
Leu Val Arg Leu Gln Ser Gly Val Asp Val Phe Phe Ile Pro Pro
2615 2620 2625
Glu Lys Cys Asp Thr Leu Leu Cys Asp Ile Gly Glu Ser Ser Pro
2630 2635 2640
Asn Pro Thr Val Glu Ala Gly Arg Thr Leu Arg Val Leu Asn Leu
2645 2650 2655
Val Glu Asn Trp Leu Asn Asn Asn Thr Gln Phe Cys Ile Lys Val
2660 2665 2670
Leu Asn Pro Tyr Met Pro Ser Val Ile Glu Lys Met Glu Ala Leu
2675 2680 2685
Gln Arg Lys Tyr Gly Gly Ala Leu Val Arg Asn Pro Leu Ser Arg
2690 2695 2700
Asn Ser Thr His Glu Met Tyr Trp Val Ser Asn Ala Ser Gly Asn
2705 2710 2715
Ile Val Ser Ser Val Asn Met Ile Ser Arg Met Leu Ile Asn Arg
2720 2725 2730
Phe Thr Met Arg Tyr Lys Lys Ala Thr Tyr Glu Pro Asp Val Asp
2735 2740 2745
Leu Gly Ser Gly Thr Arg Asn Ile Gly Ile Glu Ser Glu Ile Pro
2750 2755 2760
Asn Leu Asp Ile Ile Gly Lys Arg Ile Glu Lys Ile Lys Gln Glu
2765 2770 2775
His Glu Thr Ser Trp His Tyr Asp Gln Asp His Pro Tyr Lys Thr
2780 2785 2790
Trp Ala Tyr His Gly Ser Tyr Glu Thr Lys Gln Thr Gly Ser Ala
2795 2800 2805
Ser Ser Met Val Asn Gly Val Val Arg Leu Leu Thr Lys Pro Trp
2810 2815 2820
Asp Val Val Pro Met Val Thr Gln Met Ala Met Thr Asp Thr Thr
2825 2830 2835
Pro Phe Gly Gln Gln Arg Val Phe Lys Glu Lys Val Asp Thr Arg
2840 2845 2850
Thr Gln Glu Pro Lys Glu Gly Thr Lys Lys Leu Met Lys Ile Thr
2855 2860 2865
Ala Glu Trp Leu Trp Lys Glu Leu Gly Lys Lys Lys Thr Pro Arg
2870 2875 2880
Met Cys Thr Arg Glu Glu Phe Thr Arg Lys Val Arg Ser Asn Ala
2885 2890 2895
Ala Leu Gly Ala Ile Phe Thr Asp Glu Asn Lys Trp Lys Ser Ala
2900 2905 2910
Arg Glu Ala Val Glu Asp Ser Arg Phe Trp Glu Leu Val Asp Lys
2915 2920 2925
Glu Arg Asn Leu His Leu Glu Gly Lys Cys Glu Thr Cys Val Tyr
2930 2935 2940
Asn Met Met Gly Lys Arg Glu Lys Lys Leu Gly Glu Phe Gly Lys
2945 2950 2955
Ala Lys Gly Ser Arg Ala Ile Trp Tyr Met Trp Leu Gly Ala Arg
2960 2965 2970
Phe Leu Glu Phe Glu Ala Leu Gly Phe Leu Asn Glu Asp His Trp
2975 2980 2985
Phe Ser Arg Glu Asn Ser Leu Ser Gly Val Glu Gly Glu Gly Leu
2990 2995 3000
His Lys Leu Gly Tyr Ile Leu Arg Asp Val Ser Lys Lys Glu Gly
3005 3010 3015
Gly Ala Met Tyr Ala Asp Asp Thr Ala Gly Trp Asp Thr Arg Ile
3020 3025 3030
Thr Leu Glu Asp Leu Lys Asn Glu Glu Met Val Thr Asn His Met
3035 3040 3045
Glu Gly Glu His Lys Lys Leu Ala Glu Ala Ile Phe Lys Leu Thr
3050 3055 3060
Tyr Gln Asn Lys Val Val Arg Val Gln Arg Pro Thr Pro Arg Gly
3065 3070 3075
Thr Val Met Asp Ile Ile Ser Arg Arg Asp Gln Arg Gly Ser Gly
3080 3085 3090
Gln Val Gly Thr Tyr Gly Leu Asn Thr Phe Thr Asn Met Glu Ala
3095 3100 3105
Gln Leu Ile Arg Gln Met Glu Gly Glu Gly Val Phe Lys Ser Ile
3110 3115 3120
Gln His Leu Thr Ile Thr Glu Glu Ile Ala Val Gln Asn Trp Leu
3125 3130 3135
Ala Arg Val Gly Arg Glu Arg Leu Ser Arg Met Ala Ile Ser Gly
3140 3145 3150
Asp Asp Cys Val Val Lys Pro Leu Asp Asp Arg Phe Ala Ser Ala
3155 3160 3165
Leu Thr Ala Leu Asn Asp Met Gly Lys Ile Arg Lys Asp Ile Gln
3170 3175 3180
Gln Trp Glu Pro Ser Arg Gly Trp Asn Asp Trp Thr Gln Val Pro
3185 3190 3195
Phe Cys Ser His His Phe His Glu Leu Ile Met Lys Asp Gly Arg
3200 3205 3210
Val Leu Val Val Pro Cys Arg Asn Gln Asp Glu Leu Ile Gly Arg
3215 3220 3225
Ala Arg Ile Ser Gln Gly Ala Gly Trp Ser Leu Arg Glu Thr Ala
3230 3235 3240
Cys Leu Gly Lys Ser Tyr Ala Gln Met Trp Ser Leu Met Tyr Phe
3245 3250 3255
His Arg Arg Asp Leu Arg Leu Ala Ala Asn Ala Ile Cys Ser Ala
3260 3265 3270
Val Pro Ser His Trp Val Pro Thr Ser Arg Thr Thr Trp Ser Ile
3275 3280 3285
His Ala Lys His Glu Trp Met Thr Thr Glu Asp Met Leu Thr Val
3290 3295 3300
Trp Asn Arg Val Trp Ile Gln Glu Asn Pro Trp Met Glu Asp Lys
3305 3310 3315
Thr Pro Val Glu Ser Trp Glu Glu Ile Pro Tyr Leu Gly Lys Arg
3320 3325 3330
Glu Asp Gln Trp Cys Gly Ser Leu Ile Gly Leu Thr Ser Arg Ala
3335 3340 3345
Thr Trp Ala Lys Asn Ile Gln Ala Ala Ile Asn Gln Val Arg Ser
3350 3355 3360
Leu Ile Gly Asn Glu Glu Tyr Thr Asp Tyr Met Pro Ser Met Lys
3365 3370 3375
Arg Phe Arg Arg Glu Glu Glu Glu Ala Gly Val Leu Trp
3380 3385 3390
<210> 13
<211> 10723
<212> DNA
<213> 人工序列
<220>
<223> 登革热病毒血清型1, MVS
<400> 13
agttgttagt ctacgtggac cgacaaagac agattctttg agggagctaa gctcaatgta 60
gttctaacag ttttttaatt agagagcaga tctctgatga ataaccaacg gaaaaaggcg 120
aaaaacacgc ctttcaatat gctgaaacgc gagagaaacc gcgtgtcgac tgtgcaacag 180
ctgacaaaga gattctcact tggaatgctg cagggacgag gaccattaaa actgttcatg 240
gccctggtgg cgttccttcg tttcctaaca atcccaccaa cagcagggat attgaagaga 300
tggggaacaa ttaaaaaatc aaaagctatt aatgttttga gagggttcag gaaagagatt 360
ggaaggatgc tgaacatctt gaataggaga cgcagatctg caggcatgat cattatgctg 420
attccaacag tgatggcgtt ccatttaacc acgcgtgggg gagagccgca tatgatagtt 480
agcaagcagg aaagaggaaa gtcacttttg ttcaagacct ctgcaggtgt caacatgtgc 540
accctcattg cgatggattt gggagagttg tgtgaggaca cgatgaccta caaatgcccc 600
cggatcactg aggcggaacc agatgacgtt gactgttggt gcaatgccac ggacacatgg 660
gtgacctatg gaacgtgctc tcaaactggc gaacaccgac gagacaaacg ttccgtcgca 720
ttggccccac acgtggggct tggcctagaa acaagagccg aaacgtggat gtcctctgaa 780
ggtgcttgga aacagataca aaaagtagag acttgggctc tgagacatcc aggattcacg 840
gtgatagccc tttttctagc acatgccata ggaacatcca tcacccagaa agggatcatt 900
ttcattttgc tgatgctggt aacaccatct atggccatgc gatgcgtggg aataggcaac 960
agagacttcg tggaaggact gtcaggagca acatgggtgg atgtggtact ggagcatgga 1020
agttgcgtca ccaccatggc aaaaaacaaa ccaacactgg acattgaact cttgaagacg 1080
gaggtcacaa accctgcagt tctgcgtaaa ttgtgcattg aagctaaaat atcaaacacc 1140
accaccgatt cgagatgtcc aacacaagga gaagccacac tggtggaaga acaagacgcg 1200
aactttgtgt gccgacgaac gttcgtggac agaggctggg gcaatggctg tgggctattc 1260
ggaaaaggta gtctaataac gtgtgccaag tttaagtgtg tgacaaaact agaaggaaag 1320
atagttcaat atgaaaacct aaaatattca gtgatagtca ccgtccacac tggagatcag 1380
caccaggtgg gaaatgagac tacagaacat ggaacaactg caaccataac acctcaagct 1440
cctacgtcgg aaatacagct gaccgactac ggaaccctta cattagattg ttcacctagg 1500
acagggctag attttaacga gatggtgttg ctgacaatga aagaaagatc atggcttgtc 1560
cacaaacaat ggttcctaga cttaccactg ccttggacct ctggggcttc aacatcccaa 1620
gagacttgga acagacaaga tttactggtc acatttaaga cagctcatgc aaagaagcag 1680
gaagtagtcg tactaggatc acaagaagga gcaatgcaca ctgcgctgac tggagcgaca 1740
gaaatccaaa cgtcaggaac gacaacaatt ttcgcaggac acctaaaatg cagactaaaa 1800
atggacaaac taactttaaa agggatgtca tatgtgatgt gcacaggctc attcaagtta 1860
gagaaagaag tggctgagac ccagcatgga actgttctgg tgcaggttaa atatgaagga 1920
acagacgcac catgcaagat tcccttttcg acccaagatg agaaaggagc aacccagaat 1980
gggagattaa taacagccaa ccccatagtc actgacaaag aaaaaccagt caatattgag 2040
gcagaaccac cctttggtga gagctacatc gtggtaggag caggtgaaaa agctttgaaa 2100
ctaagctggt tcaagaaagg aagcagcata gggaaaatgt ttgaagcaac tgcccgagga 2160
gcacgaagga tggccattct gggagacacc gcatgggact tcggttctat aggaggagtg 2220
ttcacgtcta tgggaaaact ggtacaccag gtttttggaa ctgcatatgg agttttgttt 2280
agcggagttt cttggaccat gaaaatagga atagggattc tgctgacatg gctaggatta 2340
aattcaagga acacgtccct ttcgatgatg tgcatcgcag ccggcattgt gacactgtat 2400
ttgggagtca tggtgcaggc cgatagtggt tgcgttgtga gctggaaaaa caaagaactg 2460
aaatgtggca gtgggatttt catcacagac aacgtgcaca catggacaga acaatacaag 2520
ttccaaccag aatccccttc aaaactagct tcagctatcc agaaagccca tgaagaggac 2580
atttgtggaa tccgctcagt aacaagactg gagaatctga tgtggaaaca aataacacca 2640
gaattgaatc acattctatc agaaaatgag gtgaagttaa ctattatgac aggagacatc 2700
aaaggaatca tgcaggcagg aaaacgatct ctgcggcctc agcccactga gctgaagtat 2760
tcatggaaaa catggggcaa agcaaaaatg ctctctacag agtctcataa ccagaccttt 2820
ctcattgatg gccccgaaac agcagaatgc cccaacacaa atagagcttg gaattcgttg 2880
gaagttgaag actatggctt tggagtattc accaccaata tatggctaaa attgaaagaa 2940
aaacaggatg tattctgcga ctcaaaactc atgtcagcgg ccataaaaga caacagagcc 3000
gtccatgccg atatgggtta ttggatagaa agtgcactca atgacacatg gaagatagag 3060
aaagcctctt tcattgaagt taaaaactgc cactggccaa aatcacacac cctctggagc 3120
aatggagtgc tagaaagtga gatgataatt ccaaagaatc tcgctggacc agtgtctcaa 3180
cacaactata gaccaggcta ccatacacaa ataacaggac catggcatct aggtaagctt 3240
gagatggact ttgatttctg tgatggaaca acagtggtag tgactgagga ctgcggaaat 3300
agaggaccct ctttgagaac aaccactgcc tctggaaaac tcataacaga atggtgctgc 3360
cgatcttgca cattaccacc gctaagatac agaggtgagg atgggtgctg gtacgggatg 3420
gaaatcagac cattgaagga gaaagaagag aatttggtca actccttggt cacagctgga 3480
catgggcagg tcgacaactt ttcactagga gtcttgggaa tggcattgtt cctggaggaa 3540
atgcttagga cccgagtagg aacgaaacat gcaatactac tagttgcagt ttcttttgtg 3600
acattgatca cagggaacat gtcctttaga gacctgggaa gagtgatggt tatggtaggc 3660
gccactatga cggatgacat aggtatgggc gtgacttatc ttgccctact agcagccttc 3720
aaagtcagac caacttttgc agctggacta ctcttgagaa agctgacctc caaggaattg 3780
atgatgacta ctataggaat tgtactcctc tcccagagca ccctaccaga gaccattctt 3840
gagttgactg atgcgttagc cttaggcatg atggtcctca aaatggtgag aaatatggaa 3900
aagtatcaat tggcagtgac tatcatggct atcttgtgcg tcccaaacgc agtgatatta 3960
caaaacgcat ggaaagtgag ttgcacaata ttggcagtgg tgtccgtttc cccactgttc 4020
ttaacatcct cacagcaaaa aacagattgg ataccattag cattgacgat caaaggtctc 4080
aatccaacag ctatttttct aacaaccctc tcaagaacca gcaagaaaag gagctggcca 4140
ttaaatgagg ctatcatggc agtcgggatg gtgagcattt tagccagttc tctcctaaaa 4200
aatgatattc ccatgacagg accattagtg gctggagggc tcctcactgt gtgctacgtg 4260
ctcactggac gatcggccga tttggaactg gagagagcag ccgatgtcaa atgggaagac 4320
caggcagaga tatcaggaag cagtccaatc ctgtcaataa caatatcaga agatggtagc 4380
atgtcgataa aaaatgaaga ggaagatcaa acactgacca tactcattag aacaggattg 4440
ctggtgatct caggactttt tcctgtatca ataccaatca cggcagcagc atggtacctg 4500
tgggaagtga agaaacaacg ggccggagta ttgtgggatg ttccttcacc cccacccatg 4560
ggaaaggctg aactggaaga tggagcctat agaattaagc aaaaagggat tcttggatat 4620
tcccagatcg gagccggagt ttacaaagaa ggaacattcc atacaatgtg gcatgtcaca 4680
cgtggcgctg ttctaatgca taaaggaaag aggattgaac catcatgggc ggacgtcaag 4740
aaagacctaa tatcatatgg aggaggctgg aagttagaag gagaatggaa ggaaggagaa 4800
gaagtccagg tattggcact ggagcctgga aaaaatccaa gagccgtcca aacgaaacct 4860
ggtcttttca aaaccaacgc cggaacaata ggtgctgtat ctctggactt ttctcctgga 4920
acgtcaggat ctccaattat cgacaaaaaa ggaaaagttg tgggtcttta tggtaatggt 4980
gttgttacaa ggagtggagc atatgtgagt gctatagccc agactgaaaa aagcattgaa 5040
gacaacccag agatcgaaga tgacattttc cgaaagagaa gactgaccat catggacctc 5100
cacccaggag cgggaaagac gaagagatac cttccggcca tagtcagaga agctataaaa 5160
cggggtttga gaacattaat cttggccccc actagagttg tggcagctga aatggaggaa 5220
gcccttagag gacttccaat aagataccag accccagcca tcagagctgt gcacaccggg 5280
cgggagattg tggacctaat gtgtcatgcc acatttacca tgaggctgct atcaccagtt 5340
agagtgccaa actacaacct gattatcatg gacgaagccc atttcacaga cccagcaagt 5400
atagcagcta gaggatacat ctcaactcga gtggagatgg gtgaggcagc tgggattttt 5460
atgacagcca ctcccccggg aagcagagac ccatttcctc agagcaatgc accaatcata 5520
gatgaagaaa gagaaatccc tgaacgctcg tggaattccg gacatgaatg ggtcacggat 5580
tttaaaggga agactgtttg gttcgttcca agtataaaag caggaaatga tatagcagct 5640
tgcctgagga aaaatggaaa gaaagtgata caactcagta ggaagacctt tgattctgag 5700
tatgtcaaga ctagaaccaa tgattgggac ttcgtggtta caactgacat ttcagaaatg 5760
ggtgccaatt tcaaggctga gagggttata gaccccagac gctgcatgaa accagtcata 5820
ctaacagatg gtgaagagcg ggtgattctg gcaggaccta tgccagtgac ccactctagt 5880
gcagcacaaa gaagagggag aataggaaga aatccaaaaa atgagaatga ccagtacata 5940
tacatggggg aacctctgga aaatgatgaa gactgtgcac actggaaaga agctaaaatg 6000
ctcctagata acatcaacac gccagaagga atcattccta gcatgttcga accagagcgt 6060
gaaaaggtgg atgccattga tggcgaatac cgcttgagag gagaagcaag gaaaaccttt 6120
gtagacttaa tgagaagagg agacctacca gtctggttgg cctacagagt ggcagctgaa 6180
ggcatcaact acgcagacag aaggtggtgt tttgatggag tcaagaacaa ccaaatccta 6240
gaagaaaacg tggaagttga aatctggaca aaagaagggg aaaggaagaa attgaaaccc 6300
agatggttgg atgctaggat ctattctgac ccactggcgc taaaagaatt taaggaattt 6360
gcagccggaa gaaagtctct gaccctgaac ctaatcacag aaatgggtag gctcccaacc 6420
ttcatgactc agaaggcaag agacgcactg gacaacttag cagtgctgca cacggctgag 6480
gcaggtggaa gggcgtacaa ccatgctctc agtgaactgc cggagaccct ggagacattg 6540
cttttactga cacttctggc tacagtcacg ggagggatct ttttattctt gatgagcgca 6600
aggggcatag ggaagatgac cctgggaatg tgctgcataa tcacggctag catcctccta 6660
tggtacgcac aaatacagcc acactggata gcagcttcaa taatactgga gttttttctc 6720
atagttttgc ttattccaga acctgaaaaa cagagaacac cccaagacaa ccaactgacc 6780
tacgttgtca tagccatcct cacagtggtg gccgcaacca tggcaaacga gatgggtttc 6840
ctagaaaaaa cgaagaaaga tctcggattg ggaagcattg caacccagca acccgagagc 6900
aacatcctgg acatagatct acgtcctgca tcagcatgga cgctgtatgc cgtggccaca 6960
acatttgtta caccaatgtt gagacatagc attgaaaatt cctcagtgaa tgtgtcccta 7020
acagctatag ccaaccaagc cacagtgtta atgggtctcg ggaaaggatg gccattgtca 7080
aagatggaca tcggagttcc ccttctcgcc attggatgct actcacaagt caaccccata 7140
actctcacag cagctctttt cttattggta gcacattatg ccatcatagg gccaggactc 7200
caagcaaaag caaccagaga agctcagaaa agagcagcgg cgggcatcat gaaaaaccca 7260
actgtcgatg gaataacagt gattgaccta gatccaatac cttatgatcc gaagtttgaa 7320
aagcagttgg gacaagtaat gctcctagtc ctctgcgtga ctcaagtatt gatgatgagg 7380
actacatggg ctctgtgtga ggctttaacc ttagctaccg ggcccatctc cacattgtgg 7440
gaaggaaatc cagggaggtt ttggaacact accattgcgg tgtcaatggc taacattttt 7500
agagggagtt acttggccgg agctggactt ctcttttcta ttatgaagaa cacaaccaac 7560
acaagaaggg gaactggcaa cataggagag acgcttggag agaaatggaa aagccgattg 7620
aacgcattgg gaaaaagtga attccagatc tacaagaaaa gtggaatcca ggaagtggat 7680
agaaccttag caaaagaagg cattaaaaga ggagaaacgg accatcacgc tgtgtcgcga 7740
ggctcagcaa aactgagatg gttcgttgag agaaacatgg tcacaccaga agggaaagta 7800
gtggacctcg gttgtggcag aggaggctgg tcatactatt gtggaggact aaagaatgta 7860
agagaagtca aaggcctaac aaaaggagga ccaggacacg aagaacccat ccccatgtca 7920
acatatgggt ggaatctagt gcgtcttcaa agtggagttg acgttttctt catcccgcca 7980
gaaaagtgtg acacattatt gtgtgacata ggggagtcat caccaaatcc cacagtggaa 8040
gcaggacgaa cactcagagt ccttaactta gtagaaaatt ggttgaacaa caacactcaa 8100
ttttgcataa aggttctcaa cccatatatg ccctcagtca tagaaaaaat ggaagcacta 8160
caaaggaaat atggaggagc cttagtgagg aatccactct cacgaaactc cacacatgag 8220
atgtactggg tatccaatgc ttccgggaac atagtgtcat cagtgaacat gatttcaagg 8280
atgttgatca acagatttac aatgagatac aagaaagcca cttacgagcc ggatgttgac 8340
ctcggaagcg gaacccgtaa catcgggatt gaaagtgaga taccaaacct agatataatt 8400
gggaaaagaa tagaaaaaat aaagcaagag catgaaacat catggcacta tgaccaagac 8460
cacccataca aaacgtgggc ataccatggt agctatgaaa caaaacagac tggatcagca 8520
tcatccatgg tcaacggagt ggtcaggctg ctgacaaaac cttgggacgt cgtccccatg 8580
gtgacacaga tggcaatgac agacacgact ccatttggac aacagcgcgt ttttaaagag 8640
aaagtggaca cgagaaccca agaaccgaaa gaaggcacga agaaactaat gaaaataaca 8700
gcagagtggc tttggaaaga attagggaag aaaaagacac ccaggatgtg caccagagaa 8760
gaattcacaa gaaaggtgag aagcaatgca gccttggggg ccatattcac tgatgagaac 8820
aagtggaagt cggcacgtga ggctgttgaa gatagtaggt tttgggagct ggttgacaag 8880
gaaaggaatc tccatcttga aggaaagtgt gaaacatgtg tgtacaacat gatgggaaaa 8940
agagagaaga agctagggga attcggcaag gcaaaaggca gcagagccat atggtacatg 9000
tggcttggag cacgcttctt agagtttgaa gccctaggat tcttaaatga agatcactgg 9060
ttctccagag agaactccct gagtggagtg gaaggagaag ggctgcacaa gctaggttac 9120
attctaagag acgtgagcaa gaaagaggga ggagcaatgt atgccgatga caccgcagga 9180
tgggatacaa gaatcacact agaagaccta aaaaatgaag aaatggtaac aaaccacatg 9240
gaaggagaac acaagaaact agccgaggcc attttcaaac taacgtacca aaacaaggtg 9300
gtgcgtgtgc aaagaccaac accaagaggc acagtaatgg acatcatatc gagaagagac 9360
caaagaggta gtggacaagt tggcacctat ggactcaata ctttcaccaa tatggaagcc 9420
caactaatca gacagatgga gggagaagga gtctttaaaa gcattcagca cctaacaatc 9480
acagaagaaa tcgctgtgca aaactggtta gcaagagtgg ggcgcgaaag gttatcaaga 9540
atggccatca gtggagatga ttgtgttgtg aaacctttag atgacaggtt cgcaagcgct 9600
ttaacagctc taaatgacat gggaaagatt aggaaagaca tacaacaatg ggaaccttca 9660
agaggatgga atgattggac acaagtgccc ttctgttcac accatttcca tgagttaatc 9720
atgaaagacg gtcgcgtact cgttgttcca tgtagaaacc aagatgaact gattggcaga 9780
gcccgaatct cccaaggagc agggtggtct ttgcgggaga cggcctgttt ggggaagtct 9840
tacgcccaaa tgtggagctt gatgtacttc cacagacgcg acctcaggct ggcggcaaat 9900
gctatttgct cggcagtacc atcacattgg gttccaacaa gtcgaacaac ctggtccata 9960
catgctaaac atgaatggat gacaacggaa gacatgctga cagtctggaa cagggtgtgg 10020
attcaagaaa acccatggat ggaagacaaa actccagtgg aatcatggga ggaaatccca 10080
tacttgggga aaagagaaga ccaatggtgc ggctcattga ttgggttaac aagcagggcc 10140
acctgggcaa agaacatcca agcagcaata aatcaagtta gatcccttat aggcaatgaa 10200
gaatacacag attacatgcc atccatgaaa agattcagaa gagaagagga agaagcagga 10260
gttctgtggt agaaagcaaa actaacatga aacaaggcta gaagtcaggt cggattaagc 10320
catagtacgg aaaaaactat gctacctgtg agccccgtcc aaggacgtta aaagaagtca 10380
ggccatcata aatgccatag cttgagtaaa ctatgcagcc tgtagctcca cctgagaagg 10440
tgtaaaaaat ccgggaggcc acaaaccatg gaagctgtac gcatggcgta gtggactagc 10500
ggttagagga gacccctccc ttacaaatcg cagcaacaat gggggcccaa ggcgagatga 10560
agctgtagtc tcgctggaag gactagaggt tagaggagac ccccccgaaa caaaaaacag 10620
catattgacg ctgggaaaga ccagagatcc tgctgtctcc tcagcatcat tccaggcaca 10680
gaacgccaga aaatggaatg gtgctgttga atcaacaggt tct 10723
<210> 14
<211> 10723
<212> DNA
<213> 人工序列
<220>
<223> 登革热病毒血清型2, MVS
<400> 14
agttgttagt ctacgtggac cgacaaagac agattctttg agggagctaa gctcaatgta 60
gttctaacag ttttttaatt agagagcaga tctctgatga ataaccaacg gaaaaaggcg 120
aaaaacacgc ctttcaatat gctgaaacgc gagagaaacc gcgtgtcgac tgtgcaacag 180
ctgacaaaga gattctcact tggaatgctg cagggacgag gaccattaaa actgttcatg 240
gccctggtgg cgttccttcg tttcctaaca atcccaccaa cagcagggat attgaagaga 300
tggggaacaa ttaaaaaatc aaaagctatt aatgttttga gagggttcag gaaagagatt 360
ggaaggatgc tgaacatctt gaataggaga cgcagatctg caggcatgat cattatgctg 420
attccaacag tgatggcgtt ccatttaacc acacgtaacg gagaaccaca catgatcgtc 480
agcagacaag agaaagggaa aagtcttctg tttaaaacag aggttggcgt gaacatgtgt 540
accctcatgg ccatggacct tggtgaattg tgtgaagaca caatcacgta cgagtgtccc 600
cttctcaggc agaatgagcc agaagacata gactgttggt gcaactctac gtccacgtgg 660
gtaacttatg ggacgtgtac caccatggga gaacatagaa gagaaaaaag atcagtggca 720
ctcgttccac atgtgggaat gggactggag acacgaactg aaacatggat gtcatcagaa 780
ggggcctgga aacatgtcca gagaattgaa acttggatct tgagacatcc aggcttcacc 840
atgatggcag caatcctggc atacaccata ggaacgacac atttccaaag agccctgatc 900
ttcatcttac tgacagctgt cactccttca atgacaatgc gttgcatagg aatgtcaaat 960
agagactttg tggaaggggt ttcaggagga agctgggttg acatagtctt agaacatgga 1020
agctgtgtga cgacgatggc aaaaaacaaa ccaacattgg attttgaact gataaaaaca 1080
gaagccaaac agcctgccac cctaaggaag tactgtatag aggcaaagct aaccaacaca 1140
acaacagaat ctcgctgccc aacacaaggg gaacccagcc taaatgaaga gcaggacaaa 1200
aggttcgtct gcaaacactc catggtagac agaggatggg gaaatggatg tggactattt 1260
ggaaagggag gcattgtgac ctgtgctatg ttcagatgca aaaagaacat ggaaggaaaa 1320
gttgtgcaac cagaaaactt ggaatacacc attgtgataa cacctcactc aggggaagag 1380
catgcagtcg gaaatgacac aggaaaacat ggcaaggaaa tcaaaataac accacagagt 1440
tccatcacag aagcagaatt gacaggttat ggcactgtca caatggagtg ctctccaaga 1500
acgggcctcg acttcaatga gatggtgttg ctgcagatgg aaaataaagc ttggctggtg 1560
cacaggcaat ggttcctaga cctgccgtta ccatggttgc ccggagcgga cacacaaggg 1620
tcaaattgga tacagaaaga gacattggtc actttcaaaa atccccatgc gaagaaacag 1680
gatgttgttg ttttaggatc ccaagaaggg gccatgcaca cagcacttac aggggccaca 1740
gaaatccaaa tgtcatcagg aaacttactc ttcacaggac atctcaagtg caggctgaga 1800
atggacaagc tacagctcaa aggaatgtca tactctatgt gcacaggaaa gtttaaagtt 1860
gtgaaggaaa tagcagaaac acaacatgga acaatagtta tcagagtgca atatgaaggg 1920
gacggctctc catgcaagat cccttttgag ataatggatt tggaaaaaag acatgtctta 1980
ggtcgcctga ttacagtcaa cccaattgtg acagaaaaag atagcccagt caacatagaa 2040
gcagaacctc catttggaga cagctacatc atcataggag tagagccggg acaactgaag 2100
ctcaactggt ttaagaaagg aagttctatc ggccaaatgt ttgagacaac aatgaggggg 2160
gcgaagagaa tggccatttt aggtgacaca gcctgggatt ttggatcctt gggaggagtg 2220
tttacatcta taggaaaggc tctccaccaa gtctttggag caatctatgg agctgccttc 2280
agtggggttt catggactat gaaaatcctc ataggagtca ttatcacatg gataggaatg 2340
aattcacgca gcacctcact gtctgtgaca ctagtattgg tgggaattgt gacactgtat 2400
ttgggagtca tggtgcaggc cgatagtggt tgcgttgtga gctggaaaaa caaagaactg 2460
aaatgtggca gtgggatttt catcacagac aacgtgcaca catggacaga acaatacaag 2520
ttccaaccag aatccccttc aaaactagct tcagctatcc agaaagccca tgaagaggac 2580
atttgtggaa tccgctcagt aacaagactg gagaatctga tgtggaaaca aataacacca 2640
gaattgaatc acattctatc agaaaatgag gtgaagttaa ctattatgac aggagacatc 2700
aaaggaatca tgcaggcagg aaaacgatct ctgcggcctc agcccactga gctgaagtat 2760
tcatggaaaa catggggcaa agcaaaaatg ctctctacag agtctcataa ccagaccttt 2820
ctcattgatg gccccgaaac agcagaatgc cccaacacaa atagagcttg gaattcgttg 2880
gaagttgaag actatggctt tggagtattc accaccaata tatggctaaa attgaaagaa 2940
aaacaggatg tattctgcga ctcaaaactc atgtcagcgg ccataaaaga caacagagcc 3000
gtccatgccg atatgggtta ttggatagaa agtgcactca atgacacatg gaagatagag 3060
aaagcctctt tcattgaagt taaaaactgc cactggccaa aatcacacac cctctggagc 3120
aatggagtgc tagaaagtga gatgataatt ccaaagaatc tcgctggacc agtgtctcaa 3180
cacaactata gaccaggcta ccatacacaa ataacaggac catggcatct aggtaagctt 3240
gagatggact ttgatttctg tgatggaaca acagtggtag tgactgagga ctgcggaaat 3300
agaggaccct ctttgagaac aaccactgcc tctggaaaac tcataacaga atggtgctgc 3360
cgatcttgca cattaccacc gctaagatac agaggtgagg atgggtgctg gtacgggatg 3420
gaaatcagac cattgaagga gaaagaagag aatttggtca actccttggt cacagctgga 3480
catgggcagg tcgacaactt ttcactagga gtcttgggaa tggcattgtt cctggaggaa 3540
atgcttagga cccgagtagg aacgaaacat gcaatactac tagttgcagt ttcttttgtg 3600
acattgatca cagggaacat gtcctttaga gacctgggaa gagtgatggt tatggtaggc 3660
gccactatga cggatgacat aggtatgggc gtgacttatc ttgccctact agcagccttc 3720
aaagtcagac caacttttgc agctggacta ctcttgagaa agctgacctc caaggaattg 3780
atgatgacta ctataggaat tgtactcctc tcccagagca ccataccaga gaccattctt 3840
gagttgactg atgcgttagc cttaggcatg atggtcctca aaatggtgag aaatatggaa 3900
aagtatcaat tggcagtgac tatcatggct atcttgtgcg tcccaaacgc agtgatatta 3960
caaaacgcat ggaaagtgag ttgcacaata ttggcagtgg tgtccgtttc cccactgttc 4020
ttaacatcct cacagcaaaa aacagattgg ataccattag cattgacgat caaaggtctc 4080
aatccaacag ctatttttct aacaaccctc tcaagaacca gcaagaaaag gagctggcca 4140
ttaaatgagg ctatcatggc agtcgggatg gtgagcattt tagccagttc tctcctaaaa 4200
aatgatattc ccatgacagg accattagtg gctggagggc tcctcactgt gtgctacgtg 4260
ctcactggac gatcggccga tttggaactg gagagagcag ccgatgtcaa atgggaagac 4320
caggcagaga tatcaggaag cagtccaatc ctgtcaataa caatatcaga agatggtagc 4380
atgtcgataa aaaatgaaga ggaagaacaa acactgacca tactcattag aacaggattg 4440
ctggtgatct caggactttt tcctgtatca ataccaatca cggcagcagc atggtacctg 4500
tgggaagtga agaaacaacg ggccggagta ttgtgggatg ttccttcacc cccacccatg 4560
ggaaaggctg aactggaaga tggagcctat agaattaagc aaaaagggat tcttggatat 4620
tcccagatcg gagccggagt ttacaaagaa ggaacattcc atacaatgtg gcatgtcaca 4680
cgtggcgctg ttctaatgca taaaggaaag aggattgaac catcatgggc ggacgtcaag 4740
aaagacctaa tatcatatgg aggaggctgg aagttagaag gagaatggaa ggaaggagaa 4800
gaagtccagg tattggcact ggagcctgga aaaaatccaa gagccgtcca aacgaaacct 4860
ggtcttttca aaaccaacgc cggaacaata ggtgctgtat ctctggactt ttctcctgga 4920
acgtcaggat ctccaattat cgacaaaaaa ggaaaagttg tgggtcttta tggtaatggt 4980
gttgttacaa ggagtggagc atatgtgagt gctatagccc agactgaaaa aagcattgaa 5040
gacaacccag agatcgaaga tgacattttc cgaaagagaa gactgaccat catggacctc 5100
cacccaggag cgggaaagac gaagagatac cttccggcca tagtcagaga agctataaaa 5160
cggggtttga gaacattaat cttggccccc actagagttg tggcagctga aatggaggaa 5220
gcccttagag gacttccaat aagataccag accccagcca tcagagctgt gcacaccggg 5280
cgggagattg tggacctaat gtgtcatgcc acatttacca tgaggctgct atcaccagtt 5340
agagtgccaa actacaacct gattatcatg gacgaagccc atttcacaga cccagcaagt 5400
atagcagcta gaggatacat ctcaactcga gtggagatgg gtgaggcagc tgggattttt 5460
atgacagcca ctcccccggg aagcagagac ccatttcctc agagcaatgc accaatcata 5520
gatgaagaaa gagaaatccc tgaacgctcg tggaattccg gacatgaatg ggtcacggat 5580
tttaaaggga agactgtttg gttcgttcca agtataaaag caggaaatga tatagcagct 5640
tgcctgagga aaaatggaaa gaaagtgata caactcagta ggaagacctt tgattctgag 5700
tatgtcaaga ctagaaccaa tgattgggac ttcgtggtta caactgacat ttcagaaatg 5760
ggtgccaatt tcaaggctga gagggttata gaccccagac gctgcatgaa accagtcata 5820
ctaacagatg gtgaagagcg ggtgattctg gcaggaccta tgccagtgac ccactctagt 5880
gcagcacaaa gaagagggag aataggaaga aatccaaaaa atgagaatga ccagtacata 5940
tacatggggg aacctctgga aaatgatgaa gactgtgcac actggaaaga agctaaaatg 6000
ctcctagata acatcaacac gccagaagga atcattccta gcatgttcga accagagcgt 6060
gaaaaggtgg atgccattga tggcgaatac cgcttgagag gagaagcaag gaaaaccttt 6120
gtagacttaa tgagaagagg agacctacca gtctggttgg cctacagagt ggcagctgaa 6180
ggcatcaact acgcagacag aaggtggtgt tttgatggag tcaagaacaa ccaaatccta 6240
gaagaaaacg tggaagttga aatctggaca aaagaagggg aaaggaagaa attgaaaccc 6300
agatggttgg atgctaggat ctattctgac ccactggcgc taaaagaatt taaggaattt 6360
gcagccggaa gaaagtctct gaccctgaac ctaatcacag aaatgggtag gctcccaacc 6420
ttcatgactc agaaggcaag agacgcactg gacaacttag cagtgctgca cacggctgag 6480
gcaggtggaa gggcgtacaa ccatgctctc agtgaactgc cggagaccct ggagacattg 6540
cttttactga cacttctggc tacagtcacg ggagggatct ttttattctt gatgagcgca 6600
aggggcatag ggaagatgac cctgggaatg tgctgcataa tcacggctag catcctccta 6660
tggtacgcac aaatacagcc acactggata gcagcttcaa taatactgga gttttttctc 6720
atagttttgc ttattccaga acctgaaaaa cagagaacac cccaagacaa ccaactgacc 6780
tacgttgtca tagccatcct cacagtggtg gccgcaacca tggcaaacga gatgggtttc 6840
ctagaaaaaa cgaagaaaga tctcggattg ggaagcattg caacccagca acccgagagc 6900
aacatcctgg acatagatct acgtcctgca tcagcatgga cgctgtatgc cgtggccaca 6960
acatttgtta caccaatgtt gagacatagc attgaaaatt cctcagtgaa tgtgtcccta 7020
acagctatag ccaaccaagc cacagtgtta atgggtctcg ggaaaggatg gccattgtca 7080
aagatggaca tcggagttcc ccttctcgcc attggatgct actcacaagt caaccccata 7140
actctcacag cagctctttt cttattggta gcacattatg ccatcatagg gccaggactc 7200
caagcaaaag caaccagaga agctcagaaa agagcagcgg cgggcatcat gaaaaaccca 7260
actgtcgatg gaataacagt gattgaccta gatccaatac cttatgatcc aaagtttgaa 7320
aagcagttgg gacaagtaat gctcctagtc ctctgcgtga ctcaagtatt gatgatgagg 7380
actacatggg ctctgtgtga ggctttaacc ttagctaccg ggcccatctc cacattgtgg 7440
gaaggaaatc cagggaggtt ttggaacact accattgcgg tgtcaatggc taacattttt 7500
agagggagtt acttggccgg agctggactt ctcttttcta ttatgaagaa cacaaccaac 7560
acaagaaggg gaactggcaa cataggagag acgcttggag agaaatggaa aagccgattg 7620
aacgcattgg gaaaaagtga attccagatc tacaagaaaa gtggaatcca ggaagtggat 7680
agaaccttag caaaagaagg cattaaaaga ggagaaacgg accatcacgc tgtgtcgcga 7740
ggctcagcaa aactgagatg gttcgttgag agaaacatgg tcacaccaga agggaaagta 7800
gtggacctcg gttgtggcag aggaggctgg tcatactatt gtggaggact aaagaatgta 7860
agagaagtca aaggcctaac aaaaggagga ccaggacacg aagaacccat ccccatgtca 7920
acatatgggt ggaatctagt gcgtcttcaa agtggagttg acgttttctt catcccgcca 7980
gaaaagtgtg acacattatt gtgtgacata ggggagtcat caccaaatcc cacagtggaa 8040
gcaggacgaa cactcagagt ccttaactta gtagaaaatt ggttgaacaa caacactcaa 8100
ttttgcataa aggttctcaa cccatatatg ccctcagtca tagaaaaaat ggaagcacta 8160
caaaggaaat atggaggagc cttagtgagg aatccactct cacgaaactc cacacatgag 8220
atgtactggg tatccaatgc ttccgggaac atagtgtcat cagtgaacat gatttcaagg 8280
atgttgatca acagatttac aatgagatac aagaaagcca cttacgagcc ggatgttgac 8340
ctcggaagcg gaacccgtaa catcgggatt gaaagtgaga taccaaacct agatataatt 8400
gggaaaagaa tagaaaaaat aaagcaagag catgaaacat catggcacta tgaccaagac 8460
cacccataca aaacgtgggc ataccatggt agctatgaaa caaaacagac tggatcagca 8520
tcatccatgg tcaacggagt ggtcaggctg ctgacaaaac cttgggacgt cgtccccatg 8580
gtgacacaga tggcaatgac agacacgact ccatttggac aacagcgcgt ttttaaagag 8640
aaagtggaca cgagaaccca agaaccgaaa gaaggcacga agaaactaat gaaaataaca 8700
gcagagtggc tttggaaaga attagggaag aaaaagacac ccaggatgtg caccagagaa 8760
gaattcacaa gaaaggtgag aagcaatgca gccttggggg ccgtattcac tgatgagaac 8820
aagtggaagt cggcacgtga ggctgttgaa gatagtaggt tttgggagct ggttgacaag 8880
gaaaggaatc tccatcttga aggaaagtgt gaaacatgtg tgtacaacat gatgggaaaa 8940
agagagaaga agctagggga attcggcaag gcaaaaggca gcagagccat atggtacatg 9000
tggcttggag cacgcttctt agagtttgaa gccctaggat tcttaaatga agatcactgg 9060
ttctccagag agaactccct gagtggagtg gaaggagaag ggctgcacaa gctaggttac 9120
attctaagag acgtgagcaa gaaagaggga ggagcaatgt atgccgatga caccgcagga 9180
tgggatacaa gaatcacact agaagaccta aaaaatgaag aaatggtaac aaaccacatg 9240
gaaggagaac acaagaaact agccgaggcc attttcaaac taacgtacca aaacaaggtg 9300
gtgcgtgtgc aaagaccaac accaagaggc acagtaatgg acatcatatc gagaagagac 9360
caaagaggta gtggacaagt tggcacctat ggactcaata ctttcaccaa tatggaagcc 9420
caactaatca gacagatgga gggagaagga gtctttaaaa gcattcagca cctaacaatc 9480
acagaagaaa tcgctgtgca aaactggtta gcaagagtgg ggcgcgaaag gttatcaaga 9540
atggccatca gtggagatga ttgtgttgtg aaacctttag atgacaggtt cgcaagcgct 9600
ttaacagctc taaatgacat gggaaagatt aggaaagaca tacaacaatg ggaaccttca 9660
agaggatgga atgattggac acaagtgccc ttctgttcac accatttcca tgagttaatc 9720
atgaaagacg gtcgcgtact cgttgttcca tgtagaaacc aagatgaact gattggcaga 9780
gcccgaatct cccaaggagc agggtggtct ttgcgggaga cggcctgttt ggggaagtct 9840
tacgcccaaa tgtggagctt gatgtacttc cacagacgcg acctcaggct ggcggcaaat 9900
gctatttgct cggcagtacc atcacattgg gttccaacaa gtcgaacaac ctggtccata 9960
catgctaaac atgaatggat gacaacggaa gacatgctga cagtctggaa cagggtgtgg 10020
attcaagaaa acccatggat ggaagacaaa actccagtgg aatcatggga ggaaatccca 10080
tacttgggga aaagagaaga ccaatggtgc ggctcattga ttgggttaac aagcagggcc 10140
acctgggcaa agaacatcca agcagcaata aatcaagtta gatcccttat aggcaatgaa 10200
gaatacacag attacatgcc atccatgaaa agattcagaa gagaagagga agaagcagga 10260
gttctgtggt agaaagcaaa actaacatga aacaaggcta gaagtcaggt cggattaagc 10320
catagtacgg aaaaaactat gctacctgtg agccccgtcc aaggacgtta aaagaagtca 10380
ggccatcata aatgccatag cttgagtaaa ctatgcagcc tgtagctcca cctgagaagg 10440
tgtaaaaaat ccgggaggcc acaaaccatg gaagctgtac gcatggcgta gtggactagc 10500
ggttagagga gacccctccc ttacaaatcg cagcaacaat gggggcccaa ggcgagatga 10560
agctgtagtc tcgctggaag gactagaggt tagaggagac ccccccgaaa caaaaaacag 10620
catattgacg ctgggaaaga ccagagatcc tgctgtctcc tcagcatcat tccaggcaca 10680
gaacgccaga aaatggaatg gtgctgttga atcaacaggt tct 10723
<210> 15
<211> 10717
<212> DNA
<213> 人工序列
<220>
<223> 登革热病毒血清型3, MVS
<400> 15
agttgttagt ctacgtggac cgacaaagac agattctttg agggagctaa gctcaatgta 60
gttctaacag ttttttaatt agagagcaga tctctgatga ataaccaacg gaaaaaggcg 120
aaaaacacgc ctttcaatat gctgaaacgc gagagaaacc gcgtgtcgac tgtgcaacag 180
ctgacaaaga gattctcact tggaatgctg cagggacgag gaccattaaa actgttcatg 240
gccctggtgg cgttccttcg tttcctaaca atcccaccaa cagcagggat attgaagaga 300
tggggaacaa ttaaaaaatc aaaagctatt aatgttttga gagggttcag gaaagagatt 360
ggaaggatgc tgaacatctt gaataggaga cgcagatctg caggcatgat cattatgctg 420
attccaacag tgatggcgtt ccatttaacc acgcgtgatg gagagccgcg catgattgtg 480
gggaagaatg aaagaggaaa atccctactt ttcaagacag cctctggaat caacatgtgc 540
acactcatag ctatggatct gggagagatg tgtgatgaca cggtcactta caaatgcccc 600
cacattaccg aagtggagcc tgaagacatt gactgctggt gcaaccttac atcgacatgg 660
gtgacttatg gaacatgcaa tcaagctgga gagcatagac gcgataagag atcagtggcg 720
ttagctcccc atgttggcat gggactggac acacgcactc aaacctggat gtcggctgaa 780
ggagcttgga gacaagtcga gaaggtagag acatgggccc ttaggcaccc agggtttacc 840
atactagccc tatttcttgc ccattacata ggcacttcct tgacccagaa agtggttatt 900
tttatactat taatgctggt taccccatcc atgacaatga gatgtgtagg agtaggaaac 960
agagattttg tggaaggcct atcgggagct acgtgggttg acgtggtgct cgagcacggt 1020
gggtgtgtga ctaccatggc taagaacaag cccacgctgg acatagagct tcagaagacc 1080
gaggccaccc aactggcgac cctaaggaag ctatgcattg agggaaaaat taccaacata 1140
acaaccgact caagatgtcc cacccaaggg gaagcgattt tacctgagga gcaggaccag 1200
aactacgtgt gtaagcatac atacgtggac agaggctggg gaaacggttg tggtttgttt 1260
ggcaagggaa gcttggtgac atgcgcgaaa tttcaatgtt tagaatcaat agagggaaaa 1320
gtggtgcaac atgagaacct caaatacacc gtcatcatca cagtgcacac aggagaccaa 1380
caccaggtgg gaaatgaaac gcagggagtc acggctgaga taacacccca ggcatcaacc 1440
gctgaagcca ttttacctga atatggaacc ctcgggctag aatgctcacc acggacaggt 1500
ttggatttca atgaaatgat ctcattgaca atgaagaaca aagcatggat ggtacataga 1560
caatggttct ttgacttacc cctaccatgg acatcaggag cttcagcaga aacaccaact 1620
tggaacagga aagagcttct tgtgacattt aaaaatgcac atgcaaaaaa gcaagaagta 1680
gttgttcttg gatcacaaga gggagcaatg catacagcac tgacaggagc tacagagatc 1740
caaacctcag gaggcacaag tatctttgcg gggcacttaa aatgtagact caagatggac 1800
aaattggaac tcaaggggat gagctatgca atgtgcttga gtagctttgt gttgaagaaa 1860
gaagtctccg aaacgcagca tgggacaata ctcattaagg ttgagtacaa aggggaagat 1920
gcaccctgca agattccttt ctccacggag gatggacaag gaaaagctct caatggcaga 1980
ctgatcacag ccaatccagt ggtgaccaag aaggaggagc ctgtcaacat tgaggctgaa 2040
cctccttttg gagaaagtaa catagtaatt ggaattggag acaaagccct gaaaatcaac 2100
tggtacaaga agggaagctc gattgggaag atgttcgagg ccactgccag aggtgcaagg 2160
cgcatggcca tcttgggaga cacagcctgg gactttggat cagtgggtgg tgttttgaat 2220
tcattaggga aaatggtcca ccaaatattt gggagtgctt acacagccct atttggtgga 2280
gtctcctgga tgatgaaaat tggaataggt gtcctcttaa cctggatagg gttgaactca 2340
aaaaatactt ctatgtcatt ttcatgcatc gcggccggca ttgtgacact gtatttggga 2400
gtcatggtgc aggccgatag tggttgcgtt gtgagctgga aaaacaaaga actgaaatgt 2460
ggcagtggga ttttcatcac agacaacgtg cacacatgga cagaacaata caagttccaa 2520
ccagaatccc cttcaaaact agcttcagct atccagaaag cccatgaaga ggacatttgt 2580
ggaatccgct cagtaacaag actggagaat ctgatgtgga aacaaataac accagaattg 2640
aatcacattc tatcagaaaa tgaggtgaag ttaactatta tgacaggaga catcaaagga 2700
atcatgcagg caggaaaacg atctctgcgg cctcagccca ctgagctgaa gtattcatgg 2760
aaaacatggg gcaaagcaaa aatgctctct acagagtctc ataaccagac ctttctcatt 2820
gatggccccg aaacagcaga atgccccaac acaaatagag cttggaattc gttggaagtt 2880
gaagactatg gctttggagt attcaccacc aatatatggc taaaattgaa agaaaaacag 2940
gatgtattct gcgactcaaa actcatgtca gcggccataa aagacaacag agccgtccat 3000
gccgatatgg gttattggat agaaagtgca ctcaatgaca catggaagat agagaaagcc 3060
tctttcattg aagttaaaaa ctgccactgg ccaaaatcac acaccctctg gagcaatgga 3120
gtgctagaaa gtgagatgat aattccaaag aatctcgctg gaccagtgtc tcaacacaac 3180
tatagaccag gctaccatac acaaataaca ggaccatggc atctaggtaa gcttgagatg 3240
gactttgatt tctgtgatgg aacaacagtg gtagtgactg aggactgcgg aaatagagga 3300
ccctctttga gaacaaccac tgcctctgga aaactcataa cagaatggtg ctgccgatct 3360
tgcacattac caccgctaag atacagaggt gaggatgggt gctggtacgg gatggaaatc 3420
agaccattga aggagaaaga agagaatttg gtcaactcct tggtcacagc tggacatggg 3480
caggtcgaca acttttcact aggagtcttg ggaatggcat tgttcctgga ggaaatgctt 3540
aggacccgag taggaacgaa acatgcaata ctactagttg cagtttcttt tgtgacattg 3600
atcacaggga acatgtcctt tagagacctg ggaagagtga tggttatggt aggcgccact 3660
atgacggatg acataggtat gggcgtgact tatcttgccc tactagcagc cttcaaagtc 3720
agaccaactt ttgcagctgg actactcttg agaaagctga cctccaagga attgatgatg 3780
actactatag gaattgtact cctctcccag agcaccatac cagagaccat tcttgagttg 3840
actgatgcgt tagccttagg catgatggtc ctcaaaatgg tgagaaatat ggaaaagtat 3900
caattggcag tgactatcat ggctatcttg tgcgtcccaa acgcagtgat attacaaaac 3960
gcatggaaag tgagttgcac aatattggca gtggtgtccg tttccccact gttcttaaca 4020
tcctcacagc aaaaaacaga ttggatacca ttagcattga cgatcaaagg tctcaatcca 4080
acagctattt ttctaacaac cctctcaaga accagcaaga aaaggagctg gccattaaat 4140
gaggctatca tggcagtcgg gatggtgagc attttagcca gttctctcct aaaaaatgat 4200
attcccatga caggaccatt agtggctgga gggctcctca ctgtgtgcta cgtgctcact 4260
ggacgatcgg ccgatttgga actggagaga gcagccgatg tcaaatggga agaccaggca 4320
gagatatcag gaagcagtcc aatcctgtca ataacaatat cagaagatgg tagcatgtcg 4380
ataaaaaatg aagaggaaga acaaacactg accatactca ttagaacagg attgctggtg 4440
atctcaggac tttttcctgt atcaatacca atcacggcag cagcatggta cctgtgggaa 4500
gtgaagaaac aacgggccgg agtattgtgg gatgttcctt cacccccacc catgggaaag 4560
gctgaactgg aagatggagc ctatagaatt aagcaaaaag ggattcttgg atattcccag 4620
atcggagccg gagtttacaa agaaggaaca ttccatacaa tgtggcatgt cacacgtggc 4680
gctgttctaa tgcataaagg aaagaggatt gaaccatcat gggcggacgt caagaaagac 4740
ctaatatcat atggaggagg ctggaagtta gaaggagaat ggaaggaagg agaagaagtc 4800
caggtattgg cactggagcc tggaaaaaat ccaagagccg tccaaacgaa acctggtctt 4860
ttcaaaacca acgccggaac aataggtgct gtatctctgg acttttctcc tggaacgtca 4920
ggatctccaa ttatcgacaa aaaaggaaaa gttgtgggtc tttatggtaa tggtgttgtt 4980
acaaggagtg gagcatatgt gagtgctata gcccagactg aaaaaagcat tgaagacaac 5040
ccagagatcg aagatgacat tttccgaaag agaagactga ccatcatgga cctccaccca 5100
ggagcgggaa agacgaagag ataccttccg gccatagtca gagaagctat aaaacggggt 5160
ttgagaacat taatcttggc ccccactaga gttgtggcag ctgaaatgga ggaagccctt 5220
agaggacttc caataagata ccagacccca gccatcagag ctgtgcacac cgggcgggag 5280
attgtggacc taatgtgtca tgccacattt accatgaggc tgctatcacc agttagagtg 5340
ccaaactaca acctgattat catggacgaa gcccatttca cagacccagc aagtatagca 5400
gctagaggat acatctcaac tcgagtggag atgggtgagg cagctgggat ttttatgaca 5460
gccactcccc cgggaagcag agacccattt cctcagagca atgcaccaat catagatgaa 5520
gaaagagaaa tccctgaacg ctcgtggaat tccggacatg aatgggtcac ggattttaaa 5580
gggaagactg tttggttcgt tccaagtata aaagcaggaa atgatatagc agcttgcctg 5640
aggaaaaatg gaaagaaagt gatacaactc agtaggaaga cctttgattc tgagtatgtc 5700
aagactagaa ccaatgattg ggacttcgtg gttacaactg acatttcaga aatgggtgcc 5760
aatttcaagg ctgagagggt tatagacccc agacgctgca tgaaaccagt catactaaca 5820
gatggtgaag agcgggtgat tctggcagga cctatgccag tgacccactc tagtgcagca 5880
caaagaagag ggagaatagg aagaaatcca aaaaatgaga atgaccagta catatacatg 5940
ggggaacctc tggaaaatga tgaagactgt gcacactgga aagaagctaa aatgctccta 6000
gataacatca acacgccaga aggaatcatt cctagcatgt tcgaaccaga gcgtgaaaag 6060
gtggatgcca ttgatggcga ataccgcttg agaggagaag caaggaaaac ctttgtagac 6120
ttaatgagaa gaggagacct accagtctgg ttggcctaca gagtggcagc tgaaggcatc 6180
aactacgcag acagaaggtg gtgttttgat ggagtcaaga acaaccaaat cctagaagaa 6240
aacgtggaag ttgaaatctg gacaaaagaa ggggaaagga agaaattgaa acccagatgg 6300
ttggatgcta ggatctattc tgacccactg gcgctaaaag aatttaagga atttgcagcc 6360
ggaagaaagt ctctgaccct gaacctaatc acagaaatgg gtaggctccc aaccttcatg 6420
actcagaagg caagagacgc actggacaac ttagcagtgc tgcacacggc tgaggcaggt 6480
ggaagggcgt acaaccatgc tctcagtgaa ctgccggaga ccctggagac attgctttta 6540
ctgacacttc tggctacagt cacgggaggg atctttttat tcttgatgag cgcaaggggc 6600
atagggaaga tgaccctggg aatgtgctgc ataatcacgg ctagcatcct cctatggtac 6660
gcacaaatac agccacactg gatagcagct tcaataatac tggagttttt tctcatagtt 6720
ttgcttattc cagaacctga aaaacagaga acaccccaag acaaccaact gacctacgtt 6780
gtcatagcca tcctcacagt ggtggccgca accatggcaa acgagatggg tttcctagaa 6840
aaaacgaaga aagatctcgg attgggaagc attgcaaccc agcaacccga gagcaacatc 6900
ctggacatag atctacgtcc tgcatcagca tggacgctgt atgccgtggc cacaacattt 6960
gttacaccaa tgttgagaca tagcattgaa aattcctcag tgaatgtgtc cctaacagct 7020
atagccaacc aagccacagt gttaatgggt ctcgggaaag gatggccatt gtcaaagatg 7080
gacatcggag ttccccttct cgccattgga tgctactcac aagtcaaccc cataactctc 7140
acagcagctc ttttcttatt ggtagcacat tatgccatca tagggccagg actccaagca 7200
aaagcaacca gagaagctca gaaaagagca gcggcgggca tcatgaaaaa cccaactgtc 7260
gatggaataa cagtgattga cctagatcca ataccttatg atccaaagtt tgaaaagcag 7320
ttgggacaag taatgctcct agtcctctgc gtgactcaag tattgatgat gaggactaca 7380
tgggctctgt gtgaggcttt aaccttagct accgggccca tctccacatt gtgggaagga 7440
aatccaggga ggttttggaa cactaccatt gcggtgtcaa tggctaacat ttttagaggg 7500
agttacttgg ccggagctgg acttctcttt tctattatga agaacacaac caacacaaga 7560
aggggaactg gcaacatagg agagacgctt ggagagaaat ggaaaagccg attgaacgcg 7620
ttgggaaaaa gtgaattcca gatctacaag aaaagtggaa tccaggaagt ggatagaacc 7680
ttagcaaaag aaggcattaa aagaggagaa acggaccatc acgctgtgtc gcgaggctca 7740
gcaaaactga gatggttcgt tgagagaaac atggtcacac cagaagggaa agtagtggac 7800
ctcggttgtg gcagaggagg ctggtcatac tattgtggag gactaaagaa tgtaagagaa 7860
gtcaaaggcc taacaaaagg aggaccagga cacgaagaac ccatccccat gtcaacatat 7920
gggtggaatc tagtgcgtct tcaaagtgga gttgacgttt tcttcatccc gccagaaaag 7980
tgtgacacat tattgtgtga cataggggag tcatcaccaa atcccacagt ggaagcagga 8040
cgaacactca gagtccttaa cttagtagaa aattggttga acaacaacac tcaattttgc 8100
ataaaggttc tcaacccata tatgccctca gtcatagaaa aaatggaagc actacaaagg 8160
aaatatggag gagccttagt gaggaatcca ctctcacgaa actccacaca tgagatgtac 8220
tgggtatcca atgcttccgg gaacatagtg tcatcagtga acatgatttc aaggatgttg 8280
atcaacagat ttacaatgag atacaagaaa gccacttacg agccggatgt tgacctcgga 8340
agcggaaccc gtaacatcgg gattgaaagt gagataccaa acctagatat aattgggaaa 8400
agaatagaaa aaataaagca agagcatgaa acatcatggc actatgacca agaccaccca 8460
tacaaaacgt gggcatacca tggtagctat gaaacaaaac agactggatc agcatcatcc 8520
atggtcaacg gagtggtcag gctgctgaca aaaccttggg acgtcgtccc catggtgaca 8580
cagatggcaa tgacagacac gactccattt ggacaacagc gcgtttttaa agagaaagtg 8640
gacacgagaa cccaagaacc gaaagaaggc acgaagaaac taatgaaaat aacagcagag 8700
tggctttgga aagaattagg gaagaaaaag acacccagga tgtgcaccag agaagaattc 8760
acaagaaagg tgagaagcaa tgcagccttg ggggccatat tcactgatga gaacaagtgg 8820
aagtcggcac gtgaggctgt tgaagatagt aggttttggg agctggttga caaggaaagg 8880
aatctccatc ttgaaggaaa gtgtgaaaca tgtgtgtaca acatgatggg aaaaagagag 8940
aagaagctag gggaattcgg caaggcaaaa ggcagcagag ccatatggta catgtggctt 9000
ggagcacgct tcttagagtt tgaagcccta ggattcttaa atgaagatca ctggttctcc 9060
agagagaact ccctgagtgg agtggaagga gaagggctgc acaagctagg ttacattcta 9120
agagacgtga gcaagaaaga gggaggagca atgtatgccg atgacaccgc aggatgggat 9180
acaagaatca cactagaaga cctaaaaaat gaagaaatgg taacaaacca catggaagga 9240
gaacacaaga aactagccga ggccattttc aaactaacgt accaaaacaa ggtggtgcgt 9300
gtgcaaagac caacaccaag aggcacagta atggacatca tatcgagaag agaccaaaga 9360
ggtagtggac aagttggcac ctatggactc aatactttca ccaatatgga agcccaacta 9420
atcagacaga tggagggaga aggagtcttt aaaagcattc agcacctaac aatcacagaa 9480
gaaatcgctg tgcaaaactg gttagcaaga gtggggcgcg aaaggttatc aagaatggcc 9540
atcagtggag atgattgtgt tgtgaaacct ttagatgaca ggttcgcaag cgctttaaca 9600
gctctaaatg acatgggaaa gattaggaaa gacatacaac aatgggaacc ttcaagagga 9660
tggaatgatt ggacacaagt gcccttctgt tcacaccatt tccatgagtt aatcatgaaa 9720
gacggtcgcg tactcgttgt tccatgtaga aaccaagatg aactgattgg cagagcccga 9780
atctcccaag gagcagggtg gtctttgcgg gagacggcct gtttggggaa gtcttacgcc 9840
caaatgtgga gcttgatgta cttccacaga cgcgacctca ggctggcggc aaatgctatt 9900
tgctcggcag taccatcaca ttgggttcca acaagtcgaa caacctggtc catacatgct 9960
aaacatgaat ggatgacaac ggaagacatg ctgacagtct ggaacagggt gtggattcaa 10020
gaaaacccat ggatggaaga caaaactcca gtggaatcat gggaggaaat cccatacttg 10080
gggaaaagag aagaccaatg gtgcggctca ttgattgggt taacaagcag ggccacctgg 10140
gcaaagaaca tccaagcagc aataaatcaa gttagatccc ttataggcaa tgaagaatac 10200
acagattaca tgccatccat gaaaagattc agaagagaag aggaagaagc aggagttctg 10260
tggtagaaag caaaactaac atgaaacaag gctagaagtc aggtcggatt aagccatagt 10320
acggaaaaaa ctatgctacc tgtgagcccc gtccaaggac gttaaaagaa gtcaggccat 10380
cataaatgcc atagcttgag taaactatgc agcctgtagc tccacctgag aaggtgtaaa 10440
aaatccggga ggccacaaac catggaagct gtacgcatgg cgtagtggac tagcggttag 10500
aggagacccc tcccttacaa atcgcagcaa caatgggggc ccaaggcgag atgaagctgt 10560
agtctcgctg gaaggactag aggttagagg agaccccccc gaaacaaaaa acagcatatt 10620
gacgctggga aagaccagag atcctgctgt ctcctcagca tcattccagg cacagaacgc 10680
cagaaaatgg aatggtgctg ttgaatcaac aggttct 10717
<210> 16
<211> 10723
<212> DNA
<213> 人工序列
<220>
<223> 登革热病毒血清型4, MVS
<400> 16
agttgttagt ctacgtggac cgacaaagac agattctttg agggagctaa gctcaatgta 60
gttctaacag ttttttaatt agagagcaga tctctgatga ataaccaacg gaaaaaggcg 120
aaaaacacgc ctttcaatat gctgaaacgc gagagaaacc gcgtgtcgac tgtgcaacag 180
ctgacaaaga gattctcact tggaatgctg cagggacgag gacctttaaa actgttcatg 240
gccctggtgg cgttccttcg tttcctaaca atcccaccaa cagcagggat attgaagaga 300
tggggaacaa ttaaaaaatc aaaagctatt aatgttttga gagggttcag gaaagagatt 360
ggaaggatgc tgaacatctt gaataggaga cgcagctctg caggcatgat cattatgctg 420
attccaacag tgatggcgtt ccatttaacc acgcgtgatg gcgaacccct catgatagtg 480
gcaaaacatg aaagggggag acctctcttg tttaagacaa cagaggggat caacaaatgc 540
actctcattg ccatggactt gggtgaaatg tgtgaggaca ctgtcacgta taaatgcccc 600
ttactggtca ataccgaacc tgaagacatt gattgctggt gcaatctcac gtctacctgg 660
gtcatgtatg ggacatgcac ccagagcgga gaacggagac gagagaagcg ctcagtagct 720
ttaacaccac attcaggaat gggattggaa acaagagctg agacatggat gtcatcggaa 780
ggggcttgga agcatgctca gagagtagag agctggatac tcagaaaccc aggattcgcg 840
ctcttggcag gatttatggc ttatatgatt gggcaaacag gaatccagcg aactgtcttc 900
tttgtcctaa tgatgctggt cgccccatcc tacggaatgc gatgcgtagg agtaggaaac 960
agagactttg tggaaggagt ctcaggtgga gcatgggtcg atctggtgct agaacatgga 1020
ggatgcgtca caaccatggc ccagggaaaa ccaaccttgg attttgaact gactaagaca 1080
acagccaagg aagtggctct gttaagaacc tattgcattg aagcctcaat atcaaacata 1140
accacggcaa caagatgtcc aacgcaagga gagccttatc taaaagagga acaagaccaa 1200
cagtacattt gccggagaga tgtggtagac agagggtggg gcaatggctg tggcttgttt 1260
ggaaaaggag gagttgtgac atgtgcgaag ttttcatgtt cggggaagat aacaggcaat 1320
ttggtccaaa ttgagaacct tgaatacaca gtggttgtaa cagtccacaa tggagacacc 1380
catgcagtag gaaatgacac gtccaatcat ggagttacag ccacgataac tcccaggtca 1440
ccatcggtgg aagtcaaatt gccggactat ggagaactaa cactcgattg tgaacccagg 1500
tctggaattg actttaatga gatgattctg atgaaaatga aaaagaaaac atggcttgtg 1560
cataagcaat ggtttttgga tctacctcta ccatggacag caggagcaga cacatcagag 1620
gttcactgga attacaaaga gagaatggtg acatttaagg ttcctcatgc caagagacag 1680
gatgtgacag tgctgggatc tcaggaagga gccatgcatt ctgccctcgc tggagccaca 1740
gaagtggact ccggtgatgg aaatcacatg tttgcaggac atctcaagtg caaagtccgt 1800
atggagaaat tgagaatcaa gggaatgtca tacacgatgt gttcaggaaa gttctcaatt 1860
gacaaagaga tggcagaaac acagcatggg acaacagtgg tgaaagtcaa gtatgaaggt 1920
gctggagctc cgtgtaaagt ccccatagag ataagagatg tgaacaagga aaaagtggtt 1980
gggcgtatca tctcatccac ccctttggct gagaatacca acagtgtaac caacatagag 2040
ttagaacccc cctttgggga cagctacata gtgataggtg ttggaaacag tgcattaaca 2100
ctccattggt tcaggaaagg gagttccatt ggcaagatgt ttgagtccac atacagaggt 2160
gcaaaacgaa tggccattct aggtgaaaca gcttgggatt ttggttccgt tggtggactg 2220
ttcacatcat tgggaaaggc tgtgcaccag gtttttggaa gtgtgtatac aaccctgttt 2280
ggaggagtct catggatgat tagaatccta attgggttcc tagtgttgtg gattggcacg 2340
aactcaagga acacttcaat ggctatgacg tgcatagctg ccggcattgt gacactgtat 2400
ttgggagtca tggtgcaggc cgatagtggt tgcgttgtga gctggaaaaa caaagaactg 2460
aaatgtggca gtgggatttt catcacagac aacgtgcaca catggacaga acaatacaag 2520
ttccaaccag aatccccttc aaaactagct tcagctatcc agaaagccca tgaagaggac 2580
atttgtggaa tccgctcagt aacaagactg gagaatctga tgtggaaaca aataacacca 2640
gaattgaatc acattctatc agaaaatgag gtgaagttaa ctattatgac aggagacatc 2700
aaaggaatca tgcaggcagg aaaacgatct ctgcggcctc agcccactga gctgaagtat 2760
tcatggaaaa catggggcaa agcaaaaatg ctctctacag agtctcataa ccagaccttt 2820
ctcattgatg gccccgaaac agcagaatgc cccaacacaa atagagcttg gaattcgttg 2880
gaagttgaag actatggctt tggagtattc accaccaata tatggctaaa attgaaagaa 2940
aaacaggatg tattctgcga ctcaaaactc atgtcagcgg ccataaaaga caacagagcc 3000
gtccatgccg atatgggtta ttggatagaa agtgcactca atgacacatg gaagatagag 3060
aaagcctctt tcattgaagt taaaaactgc cactggccaa aatcacacac cctctggagc 3120
aatggagtgc tagaaagtga gatgataatt ccaaagaatc tcgctggacc agtgtctcaa 3180
cacaactata gaccaggcta ccatacacaa ataacaggac catggcatct aggtaagctt 3240
gagatggact ttgatttctg tgatggaaca acagtggtag tgactgagga ctgcggaaat 3300
agaggaccct ctttgagaac aaccactgcc tctggaaaac tcataacaga atggtgctgc 3360
cgatcttgca cattaccacc gctaagatac agaggtgagg atgggtgctg gtacgggatg 3420
gaaatcagac cattgaagga gaaagaagag aatttggtca actccttggt cacagctgga 3480
catgggcagg tcgacaactt ttcactagga gtcttgggaa tggcattgtt cctggaggaa 3540
atgcttagga cccgagtagg aacgaaacat gcaatactac tagttgcagt ttcttttgtg 3600
acattgatca cagggaacat gtcctttaga gacctgggaa gagtgatggt tatggtaggc 3660
gccactatga cgggtgacat aggtatgggc gtgacttatc ttgccctact agcagccttc 3720
aaagtcagac caacttttgc agctggacta ctcttgagaa agctgacctc cagggaattg 3780
atgatgacta ctataggaat tgtactcctc tcccagagca ccataccaga gaccattctt 3840
gagttgactg atgcgttagc cttaggcatg atggtcctca aaatggtgag aaatatggaa 3900
aagtatcaat tggcagtgac tatcatggct atcttgtgcg tcccaaacgc agtgatatta 3960
caaaacgcat ggaaagtgag ttgcacaata ttggcagtgg tgtccgtttc cccactgttc 4020
ttaacatcct cacagcaaaa aacagattgg ataccattag cattgacgat caaaggtctc 4080
aatccaacag ctatttttct aacaaccctc tcaagaacca gcaagaaaag gagctggcca 4140
ttaaatgagg ctatcatggc agtcgggatg gtgagcattt tagccagttc tctcctaaaa 4200
aatgatattc ccatgacagg accattagtg gctggagggc tcctcactgt gtgctacgtg 4260
ctcactggac gatcggccga tttggaactg gagagagcag ccgatgtcaa atgggaagac 4320
caggcagaga tatcaggaag cagtccaatc ctgtcaataa caatatcaga agatggtagc 4380
atgtcgataa aaaatgaaga ggaagaacaa acactgacca tactcattag aacaggattg 4440
ctggtgatct caggactttt tcctgtatca ataccaatca cggcagcagc atggtacctg 4500
tgggaagtga agaaacaacg ggccggagta ttgtgggatg ttccttcacc cccacccatg 4560
ggaaaggctg aactggaaga tggagcctat agaattaagc aaaaagggat tcttggatat 4620
tcccagatcg gagccggagt ttacaaagaa ggaacattcc atacaatgtg gcatgtcaca 4680
cgtggcgctg ttctaatgca taaaggaaag aggattgaac catcatgggc ggacgtcaag 4740
aaagacctaa tatcatatgg aggaggctgg aagttagaag gagaatggaa ggaaggagaa 4800
gaagtccagg tattggcact ggagcctgga aaaaatccaa gagccgtcca aacgaaacct 4860
ggtcttttca aaaccaacgc cggaacaata ggtgctgtat ctctggactt ttctcctgga 4920
acgtcaggat ctccaattat cgacaaaaaa ggaaaagttg tgggtcttta tggtaatggt 4980
gttgttacaa ggagtggagc atatgtgagt gctatagccc agactgaaaa aagcattgaa 5040
gacaacccag agatcgaaga tgacattttc cgaaagagaa gactgaccat catggacctc 5100
cacccaggag cgggaaagac gaagagatac cttccggcca tagtcagaga agctataaaa 5160
cggggtttga gaacattaat cttggccccc actagagttg tggcagctga aatggaggaa 5220
gcccttagag gacttccaat aagataccag accccagcca tcagagctgt gcacaccggg 5280
cgggagattg tggacctaat gtgtcatgcc acatttacca tgaggctgct atcaccagtt 5340
agagtgccaa actacaacct gattatcatg gacgaagccc atttcacaga tccagcaagt 5400
atagcagcta gaggatacat ctcaactcga gtggagatgg gtgaggcagc tgggattttt 5460
atgacagcca ctcccccggg aagcagagac ccatttcctc agagcaatgc accaatcata 5520
gatgaagaaa gagaaatccc tgaacgctcg tggaattccg gacatgaatg ggtcacggat 5580
tttaaaggga agactgtttg gttcgttcca agtataaaag caggaaatga tatagcagct 5640
tgcctgagga aaaatggaaa gaaagtgata caactcagta ggaagacctt tgattctgag 5700
tatgtcaaga ctagaaccaa tgattgggac ttcgtggtta caactgacat ttcagaaatg 5760
ggtgccaatt tcaaggctga gagggttata gaccccagac gctgcatgaa accagtcata 5820
ctaacagatg gtgaagagcg ggtgattctg gcaggaccta tgccagtgac ccactctagt 5880
gcagcacaaa gaagagggag aataggaaga aatccaaaaa atgagaatga ccagtacata 5940
tacatggggg aacctctgga aaatgatgaa gactgtgcac actggaaaga agctaaaatg 6000
ctcctagata acatcaacac gccagaagga atcattccta gcatgttcga accagagcgt 6060
gaaaaggtgg atgccattga tggcgaatac cgcttgagag gagaagcaag gaaaaccttt 6120
gtagacttaa tgagaagagg agacctacca gtctggttgg cctacagagt ggcagctgaa 6180
ggcatcaact acgcagacag aaggtggtgt tttgatggag tcaagaacaa ccaaatccta 6240
gaagaaaacg tggaagttga aatctggaca aaagaagggg aaaggaagaa attgaaaccc 6300
agatggttgg atgctaggat ctattctgac ccactggcgc taaaagaatt taaggaattt 6360
gcagccggaa gaaagtctct gaccctgaac ctaatcacag aaatgggtag gctcccaacc 6420
ttcatgactc agaaggtaag agacgcactg gacaacttag cagtgctgca cacggctgag 6480
gcaggtggaa gggcgtacaa ccatgctctc agtgaactgc cggagaccct ggagacattg 6540
cttttactga cacttctggc tacagtcacg ggagggatct ttttattctt gatgagcgca 6600
aggggcatag ggaagatgac cctgggaatg tgctgcataa tcacggctag catcctccta 6660
tggtacgcac aaatacagcc acactggata gcagcttcaa taatactgga gttttttctc 6720
atagttttgc ttattccaga acctgaaaaa cagagaacac cccaagacaa ccaactgacc 6780
tacgttgtca tagccatcct cacagtggtg gccgcaacca tggcaaacga gatgggtttc 6840
ctagaaaaaa cgaagaaaga tctcggattg ggaagcattg caacccagca acccgagagc 6900
aacatcctgg acatagatct acgtcctgca tcagcatgga cgctgtatgc cgtggccaca 6960
acatttgtta caccaatgtt gagacatagc attgaaaatt cctcagtgaa tgtgtcccta 7020
acagccatag ccaaccaagc cacagtgtta atgggtctcg ggaaaggatg gccattgtca 7080
aagatggaca tcggagttcc ccttctcgcc attggatgct actcacaagt caaccccata 7140
actctcacag cagctctttt cttattggta gcacattatg ccatcatagg gccaggactc 7200
caagcaaaag caaccagaga agctcagaaa agagcagcgg cgggcatcat gaaaaaccca 7260
actgtcgatg gaataacagt gattgaccta gatccaatac cttatgatcc aaagtttgaa 7320
aagcagttgg gacaagtaat gctcctagtc ctctgcgtga ctcaagtatt gatgatgagg 7380
actacatggg ctctgtgtga ggctttaacc ttagctaccg ggcccatctc cacattgtgg 7440
gaaggaaatc cagggaggtt ttggaacact accattgcgg tgtcaatggc taacattttt 7500
agagggagtt acttggccgg agctggactt ctcttttcta ttatgaagaa cacaaccaac 7560
acaagaaggg gaactggcaa cataggagag acgcttggag agaaatggaa aagccgattg 7620
aacgcattgg gaaaaagtga attccagatc tacaagaaaa gtggaatcca ggaagtggat 7680
agaaccttag caaaagaagg cattaaaaga ggagaaacgg accatcacgc tgtgtcgcga 7740
ggctcagcaa aactgagatg gttcgttgag agaaacatgg tcacaccaga agggaaagta 7800
gtggacctcg gttgtggcag aggaggctgg tcatactatt gtggaggact aaagaatgta 7860
agagaagtca aaggcctaac aaaaggagga ccaggacacg aagaacccat ccccatgtca 7920
acatatgggt ggaatctagt gcgtcttcaa agtggagttg acgttttctt catcccgcca 7980
gaaaagtgtg acacattatt gtgtgacata ggggagtcat caccaaatcc cacagtggaa 8040
gcaggacgaa cactcagagt ccttaactta gtagaaaatt ggttgaacaa caacactcaa 8100
ttttgcataa aggttctcaa cccatatatg ccctcagtca tagaaaaaat ggaagcacta 8160
caaaggaaat atggaggagc cttagtgagg aatccactct cacgaaactc cacacatgag 8220
atgtactggg tatccaatgc ttccgggaac atagtgtcat cagtgaacat gatttcaagg 8280
atgttgatca acagatttac aatgagatac aagaaagcca cttacgagcc ggatgttgac 8340
ctcggaagcg gaacccgtaa catcgggatt gaaagtgaga taccaaacct agatataatt 8400
gggaaaagaa tagaaaaaat aaagcaagag catgaaacat catggcacta tgaccaagac 8460
cacccataca aaacgtgggc ataccatggt agctatgaaa caaaacagac tggatcagca 8520
tcatccatgg tcaacggagt ggtcaggctg ctgacaaaac cttgggacgt cgtccccatg 8580
gtgacacaga tggcaatgac agacacgact ccatttggac aacagcgcgt ttttaaagag 8640
aaagtggaca cgagaaccca agaaccgaaa gaaggcacga agaaactaat gaaaataaca 8700
gcagagtggc tttggaaaga attagggaag aaaaagacac ccaggatgtg caccagagaa 8760
gaattcacaa gaaaggtgag aagcaatgca gccttggggg ccatattcac tgatgagaac 8820
aagtggaagt cggcacgtga ggctgttgaa gatagtaggt tttgggagct ggttgacaag 8880
gaaaggaatc tccatcttga aggaaagtgt gaaacatgtg tgtacaacat gatgggaaaa 8940
agagagaaga agctagggga attcggcaag gcaaaaggca gcagagccat atggtacatg 9000
tggcttggag cacgcttctt agagtttgaa gccctaggat tcttaaatga agatcactgg 9060
ttctccagag agaactccct gagtggagtg gaaggagaag ggctgcacaa gctaggttac 9120
attctaagag acgtgagcaa gaaagaggga ggagcaatgt atgccgatga caccgcagga 9180
tgggatacaa gaatcacact agaagaccta aaaaatgaag aaatggtaac aaaccacatg 9240
gaaggagaac acaagaaact agccgaggcc attttcaaac taacgtacca aaacaaggtg 9300
gtgcgtgtgc aaagaccaac accaagaggc acagtaatgg acatcatatc gagaagagac 9360
caaagaggta gtggacaagt tggcacctat ggactcaata ctttcaccaa tatggaagcc 9420
caactaatca gacagatgga gggagaagga gtctttaaaa gcattcagca cctaacaatc 9480
acagaagaaa tcgctgtgca aaactggtta gcaagagtgg ggcgcgaaag gttatcaaga 9540
atggccatca gtggagatga ttgtgttgtg aaacctttag atgacaggtt cgcaagcgct 9600
ttaacagctc taaatgacat gggaaagatt aggaaagaca tacaacaatg ggaaccttca 9660
agaggatgga atgattggac acaagtgccc ttctgttcac accatttcca tgagttaatc 9720
atgaaagacg gtcgcgtact cgttgttccc tgtagaaacc aagatgaact gattggcaga 9780
gcccgaatct cccaaggagc agggtggtct ttgcgggaga cggcctgttt ggggaagtct 9840
tacgcccaaa tgtggagctt gatgtacttc cacagacgcg acctcaggct ggcggcaaat 9900
gctatttgct cggcagtacc atcacattgg gttccaacaa gtcgaacaac ctggtccata 9960
catgctaaac atgaatggat gacaacggaa gacatgctga cagtctggaa cagggtgtgg 10020
attcaagaaa acccatggat ggaagacaaa actccagtgg aatcatggga ggaaatccca 10080
tacttgggga aaagagaaga ccaatggtgc ggctcattga ttgggttaac aagcagggcc 10140
acctgggcaa agaacatcca agcagcaata aatcaagtta gatcccttat aggcaatgaa 10200
gaatacacag attacatgcc atccatgaaa agattcagaa gagaagagga agaagcagga 10260
gttctgtggt agaaagcaaa actaacatga aacaaggcta gaagtcaggt cggattaagc 10320
catagtacgg aaaaaactat gctacctgtg agccccgtcc aaggacgtta aaagaagtca 10380
ggccatcata aatgccatag cttgagtaaa ctatgcagcc tgtagctcca cctgagaagg 10440
tgtaaaaaat ccgggaggcc acaaaccatg gaagctgtac gcatggcgta gtggactagc 10500
ggttagagga gacccctccc ttacaaatcg cagcaacaat gggggcccaa ggcgagatga 10560
agctgtagtc tcgctggaag gactagaggt tagaggagac ccccccgaaa caaaaaacag 10620
catattgacg ctgggaaaga ccagagatcc tgctgtctcc tcagcatcat tccaggcaca 10680
gaacgccaga aaatggaatg gtgctgttga atcaacaggt tct 10723

Claims (31)

1.编码修饰的活减毒登革热-2病毒株PDK-53多肽分子的多核苷酸分子，其中所述多核苷酸包含至少一种突变，其中所述至少一种突变包括：
在位置592处由鸟嘌呤取代腺嘌呤的突变，其编码的谷氨酸替代多肽分子的对应于prM-52处的氨基酸位置166的赖氨酸；以及
在位置8803处由鸟嘌呤取代腺嘌呤的突变，其编码的缬氨酸替代多肽分子中对应于NS5-412处的氨基酸位置2903的异亮氨酸。
2.如权利要求1所述的多核苷酸分子，其还包含以下的至少一个额外突变：
在核酸位置6481处由胞嘧啶取代鸟嘌呤的突变，其编码的脯氨酸替代多肽分子中对应于NS4A-36处的氨基酸位置2129的丙氨酸；和
在位置7156处由胸腺嘧啶取代胞嘧啶的突变，其编码的苯丙氨酸替代多肽分子中对应于NS4B-111处的氨基酸位置2354的亮氨酸。
3.根据权利要求1所述的多核苷酸分子，其包括SEQ ID NO:14所示的多核苷酸。
4.根据权利要求1或3所述的多核苷酸分子，其编码修饰的活减毒登革热-2病毒株PDK-53，其中所述多肽由SEQ ID NO:6所示。
5.由根据权利要求1-4任一项所述的多核苷酸分子编码的多肽分子。
6.修饰的活减毒登革热-2病毒株PDK-53，其包含一种或多种根据权利要求5所述的多肽分子。
7.药物组合物，其包含根据权利要求1-4任一项所述的多核苷酸分子、根据权利要求5所述的多肽分子或根据权利要求6所述的登革热-2病毒以及药学上可接受的赋形剂。
8.药学上可接受量的权利要求7所述的组合物在制备用于在个体中诱导免疫应答的药物中的用途，其中所述组合物在个体中针对登革热病毒诱导免疫应答。
9.载体，其编码权利要求1-4任一项所述的多核苷酸分子。
10.根据权利要求9所述的载体，其中所述载体是质粒载体。
11.根据权利要求9所述的载体，其中所述载体是cDNA传染性克隆。
12.细胞，其包含根据权利要求1-4任一项所述的多核苷酸分子或者根据权利要求9所述的载体。
13.免疫原性组合物，其包含至少一种根据权利要求1-4中任一项所述的多核苷酸分子以及药学上可接受的载剂。
14.权利要求13所述的免疫原性组合物，其进一步包含编码登革热-1/登革热-2多肽嵌合体的多核苷酸分子，所述多核苷酸包含编码来自修饰的活减毒登革热-2病毒株PDK-53的非结构蛋白质的第一核苷酸序列以及编码至少一种来自登革热-1的结构蛋白质的第二核苷酸序列，其中所述多核苷酸包含至少一个突变，其中所述至少一个突变包括以下的一个或多个：
在位置3823处由胞嘧啶取代腺嘌呤的突变，其编码的亮氨酸替代所述嵌合体中对应于NS2A 116处的氨基酸位置1243的异亮氨酸；
在位置4407处由胸腺嘧啶取代腺嘌呤的突变，其编码的天冬氨酸替代所述嵌合体中对应于NS2B-92处的氨基酸位置1437的谷氨酸；和
在位置7311处由鸟嘌呤取代腺嘌呤的突变。
15.权利要求14所述的免疫原性组合物，其中编码登革热-1/登革热-2多肽嵌合体的所述多核苷酸分子进一步包含以下的至少一个额外的突变：
在位置7148处由胸腺嘧啶取代胞嘧啶的突变，其编码的异亮氨酸替代所述嵌合体中对应于NS4B-108处的氨基酸位置2351的苏氨酸；和
在位置2384处由胞嘧啶取代鸟嘌呤的突变，其编码的丙氨酸替代所述嵌合体中对应于E-483处的氨基酸位置763的甘氨酸。
16.权利要求14所述的免疫原性组合物，其中编码登革热-1/登革热-2多肽嵌合体的所述多核苷酸分子包含由SEQ ID NO:13所示的多核苷酸。
17.权利要求14或16所述的免疫原性组合物，其中所述多核苷酸分子编码由SEQ IDNO:3所示的登革热-1/登革热-2多肽嵌合体。
18.权利要求13所述的免疫原性组合物，其进一步包含编码登革热-3/登革热-2多肽嵌合体的多核苷酸分子，所述多核苷酸包含编码来自修饰的活减毒登革热-2病毒株PDK-53的非结构蛋白质的第一核苷酸序列以及编码至少一种来自登革热-3的结构蛋白质的第二核苷酸序列，其中所述多核苷酸包含至少一个突变，其中所述至少一个突变包括：
在位置1603处由胸腺嘧啶取代腺嘌呤的突变，其编码的丝氨酸替代所述嵌合体中对应于E-223处的氨基酸位置503的苏氨酸；和
在位置7620处由鸟嘌呤取代腺嘌呤的突变。
19.权利要求18所述的免疫原性组合物，其中编码登革热-3/登革热-2多肽嵌合体的所述多核苷酸分子进一步包含：
在位置6436处由胸腺嘧啶取代腺嘌呤的突变，其编码的天冬酰胺替代所述嵌合体中对应于N42A-23处的氨基酸位置1243的天冬氨酸。
20.权利要求18所述的免疫原性组合物，其中编码登革热-3/登革热-2多肽嵌合体的所述多核苷酸分子包含由SEQ ID NO:15所示的多核苷酸。
21.权利要求18或20所述的免疫原性组合物，其中所述多核苷酸分子编码由SEQ IDNO:9所示的登革热-3/登革热-2多肽嵌合体。
22.权利要求13所述的免疫原性组合物，其进一步包含编码登革热-4/登革热-2多肽嵌合体的多核苷酸分子，所述多核苷酸包含编码来自修饰的活减毒登革热-2病毒株PDK-53的非结构蛋白质的第一核苷酸序列以及编码至少一种来自登革热-4的结构蛋白质的第二核苷酸序列，其中所述多核苷酸包含至少一个突变，其中所述至少一个突变包括：
在位置225处由胸腺嘧啶取代腺嘌呤的突变；
在位置3674处由鸟嘌呤取代腺嘌呤的突变，其编码的甘氨酸替代所述嵌合体中对应于NS2A-66处的氨基酸位置1193的天冬氨酸；
在位置5391处由胸腺嘧啶取代胞嘧啶的突变；
在位置6437处由胸腺嘧啶取代胞嘧啶的突变，其编码的缬氨酸替代所述嵌合体中对应于NS4A-21处的氨基酸位置2114的丙氨酸；
在位置7026处由胞嘧啶取代胸腺嘧啶的突变；和
在位置9750处由胞嘧啶取代腺嘌呤的突变。
23.权利要求22所述的免疫原性组合物，其中编码登革热-4/登革热-2多肽嵌合体的所述多核苷酸分子进一步包含：
在位置3773处由鸟嘌呤取代腺嘌呤的突变，其编码的精氨酸替代所述嵌合体中对应于NS2A-99处的氨基酸位置1226的赖氨酸。
24.权利要求22或23所述的免疫原性组合物，其中编码登革热-4/登革热-2多肽嵌合体的所述多核苷酸分子进一步包含：
在位置7538处由胸腺嘧啶取代胞嘧啶的突变，其编码的苯丙氨酸替代所述嵌合体中对应于NS4B-238处的氨基酸位置2481的丝氨酸。
25.权利要求22或23所述的免疫原性组合物，其中编码登革热-4/登革热-2多肽嵌合体的所述多核苷酸分子包含由SEQ ID NO:16所示的多核苷酸。
26.权利要求22或23所述的免疫原性组合物，其中所述多核苷酸分子编码由SEQ IDNO:12所示的登革热-4/登革热-2多肽嵌合体。
27.权利要求22或23所述的免疫原性组合物，其中所述组合物包含全部四种登革热病毒血清型，其为四价组合物。
28.权利要求22或23所述的免疫原性组合物，其还包含针对选自以下的黄病毒的免疫原性组合物：黄热病毒、蜱媒脑炎病毒、日本脑炎病毒、西尼罗病毒、丙型肝炎病毒、及其中两种或更多种的组合。
29.试剂盒，其包含至少一种权利要求1-4中任一项所述的多核苷酸分子或至少一种权利要求7和13-28中任一项所述的组合物以及容器。
30.组合物，其包含一种或多种权利要求6所述的活减毒病毒和药学上可接受的载剂。
31.权利要求30所述的组合物，其中所述组合物包含能够在个体内诱导对抗全部四种登革热病毒血清型的免疫应答的四价登革热病毒组合物。
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