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CN109851678A - 一种改良的亚稳定态牛呼吸道合胞病毒融合前体f蛋白质及编码的dna分子和其应用 - Google Patents

一种改良的亚稳定态牛呼吸道合胞病毒融合前体f蛋白质及编码的dna分子和其应用 Download PDF

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CN109851678A
CN109851678A CN201910171850.0A CN201910171850A CN109851678A CN 109851678 A CN109851678 A CN 109851678A CN 201910171850 A CN201910171850 A CN 201910171850A CN 109851678 A CN109851678 A CN 109851678A
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周建国
戴敏
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Abstract

本发明属于生物技术领域，公开了一种经过基因改良的亚稳定态的牛呼吸道合胞病毒融合前体F蛋白质、编码的DNA分子及其应用。本发明将结构生物学的方法应用到疫苗设计和改良中，通过对牛呼吸道合胞病毒F蛋白质三维结构的观察，发现其发生构象变化的生物学机制。在F蛋白质三维结构的基础上进行抗原设计和基因工程改造，获得保持在亚稳定态F蛋白质融合前体的牛呼吸道合胞病毒F蛋白质疫苗。在此蛋白质疫苗基础上进行基因工程改造，构建可以在细胞表面表达牛呼吸道胞合体病毒F蛋白融合前体蛋白质的质粒做为DNA疫苗，使接受免疫动物获得良好的保护。通过分子生物学，生物学，细胞生物学，免疫学的方法确定了疫苗的稳定性，有效性和安全性。

Description

一种改良的亚稳定态牛呼吸道合胞病毒融合前体F蛋白质及 编码的DNA分子和其应用
技术领域
本发明属于生物技术领域，具体涉及一种牛呼吸道合胞病毒融合前体F蛋白质及所述蛋白质编码的DNA分子和其应用。
背景技术
牛呼吸道合胞病毒(Bovine Respiratory Syncytial Virus,BRSV)主要引起2～6月龄犊牛的细支气管炎和间质性肺炎。即使存在中等水平的母牛抗体，出生后第一年犊牛的患病率仍可高达50％以上。尽管市场上已有多种获得许可的BRSV灭活，弱毒疫苗。但由于这些疫苗的稳定性和对动物的保护率低等原因，目前尚没有理想的疫苗可供使用。虽然BRSV致死率不高，但因其并发症导致的无法逆转的肺部和呼吸道伤害及治疗费用、饲养成本的增加会给养牛业造成严重的经济损失。仅2015年美国当年损失接近10亿美元。国内相关的数据未见报道，但是2017年国内牛的存栏数近一亿头，略高于美国的数量。
RSV属于副粘病毒科、肺炎病毒属中的成员，其基因组为非节段的单负链RNA，由15225个核苷酸组成。RSV基因组编码主要有11种蛋白质，基因组从3'至5'端编码的蛋白依次为NSl、NS2、N、P、M、SH、G、F、M2(M2-l、M2-2)和L等，其中表面蛋白糖蛋白G和融合蛋白F在病毒囊膜表面形成刺突。RSV感染宿主细胞时，需要病毒膜与寄主细胞膜发生融合。基于当前对副黏液病毒发生融合的研究，RSV F蛋白质最初形成时折叠成一种"融合前"构象(Pro-fusion)。在两者的膜发生融合时，该融合前体的构象进行再折叠和构象变化成为"融合后"构象(Post-fusion)。因此，该RSV F融合前体蛋白的是一种亚稳定态的蛋白质，它通过最初折叠成一种亚稳形式(融合前构象)，该亚稳形式随后进行不连续，逐步和不可逆的构象变化成一个较低能量稳定态的构象(融合后构象)。构象变化的生物学机制在于病毒通过F蛋白的构象变化介导病毒和宿主细胞膜发生融合，致使病毒的遗传物质侵入宿主细胞内，借助宿主细胞系统完成病毒的复制。所以F蛋白融合前体是最佳的疫苗候选物，是主要中和抗原成分。机体感染RSV后，病毒编码的F和G抗原蛋白能刺激机体免疫应答，产生血清IgG中和抗体和呼吸道黏膜的分泌型IgA抗体，在抗RSV感染具有重要作用。但是问题在于天然的F蛋白融合前体很不稳定。这也是使用传统的灭活和减毒的方法无法获得长期有效和稳定的疫苗的原因。需要对其进行构象稳定化的改良。
使用电子显微镜技术对RSV F蛋白进行观察的结果显示融合前与融合后三聚体之间存在巨大的结构差异。这些结构的变化也通过蛋白质结晶学方法得到了确认McLellanJ.S.et al.Science.136(2013)和McLellan J.S.et al.158(2013)。动物实验的结果显示融合前和融合后RSV F蛋白质在抗原性上是独特的Calder L.J.et al.Virology 271(2000)。
人类呼吸道合胞病毒(Human Respiratory Syncytial Virus,HRSV)与牛呼吸道合胞病毒具有遗传相似性，可高达85％。人类RSV是儿童出生后第一年毛细支气管炎和肺炎的最常见原因。RSV还可引起反复感染，包括严重的下呼吸道疾病，这可能发生在任何年龄，特别是老年人或心脏，肺部或免疫系统受损的人。每年有4 300万人幼儿和老年人的因严重呼吸道疾病导致住院治疗。相关的HRSV疫苗已经在小鼠，棉鼠和非人灵长类动物(NHP)动物模型中进行了评估。但是上个世纪七十年代Merck公司研发的灭活hRSV疫苗临床实验造成了受试婴儿死亡的重大事故。2013年美国国立卫生研究院疫苗研究中心将结构生物学的方法应用疫苗研发的领域。他们的基于蛋白质原子结构设计的亚稳态融合前(F)糖蛋白质(Pre-fusion F)疫苗候选物在临床前非人灵长类动物实验中取得了良好的结果。目前这种人类RSV疫苗已经进入了临床I期阶段。(参见例如WO20101149745、WO2010/1149743、WO2009/1079796、WO 2012/158613)。将这些疫苗设计转化为牛背景的疫苗候选物在理化学，生物学及犊牛动物实验中也显示了非常好的前景。然而，他们改良的疫苗在产量和抗原活性上仍有可改良的空间。
目前国外市场上有几种已经被认可使用的bRSV疫苗。这些传统疫苗的研制和生产方式主要是通过改变培养条件，或在不同培养细胞上传代使致病微生物毒性减弱的减毒疫苗，如(ZOETIS,Bovi-Shield Gold 5,SKU:540492)。它们的缺点在于有效期短，保护率低。这类疫苗的局限性还表现在：(1)动物和人类的病毒需要在动物细胞中培养，这使得疫苗生产的成本很高；(2)疫苗中的致病物质在疫苗生产过程中有可能没有充分减毒，这会导致疫苗中含有强毒性致病物质，进而使得疾病在更大的范围内传播；(3)减毒菌株有可能会发生突变，引起病害。
发明内容
有鉴于此，本发明的目的在于针对现有疫苗存在的问题，提供一种构象稳定的牛呼吸道合胞病毒F蛋白的融合前体蛋白、编码所述蛋白的DNA及其应用。
为实现本发明的目的，本发明采用如下技术方案：
一种牛呼吸道合胞病毒F蛋白的融合前体蛋白，包含至少一处选自下组的对F蛋白野生型的改造：
A、增加F蛋白三聚体每个单体内部以及单体之间二硫键的连接数量；
B、突变F蛋白三聚体内部至少一个氨基酸，将侧链较小的氨基酸突变成为侧链较大的氨基酸或增加内部的疏水结合；
C、突变剔除至少一个蛋白酶的酶切位点；
D、切除F蛋白三聚体至少一个动态较大氨基酸，代之以较短的连接肽；
E、在F-蛋白质的C-终端延长螺旋构造。
作为优选，所述对F蛋白野生型的改造包含第143位甘氨酸、第404位丝氨酸、第103位丝氨酸、第262位丝氨酸突变为半胱氨酸，第288位异亮氨酸突变为苯丙氨酸，第187位缬氨酸突变为亮氨酸。更优选地，所述牛呼吸道合胞病毒F蛋白的融合前体蛋白在下述三种设计改造下抗原的产量、稳定性及与特异抗体结合的亲和力有较好的表现：
1)M1设计：第159位组氨酸、第291位缬氨酸突变为半胱氨酸，剪除从109至137氨基酸序列，加入连接肽丝氨酸－谷氨酸－丝氨酸－丝氨酸－谷氨酸－丝氨酸－丝氨酸－谷氨酸；简写为H159C，V291C；G143C，S404C；S103C，S262C；I288F；V187L；Δ109-137(SGSSGSSG)；
2)M2设计：第158位亮氨酸、第290位丝氨酸突变为半胱氨酸，剪除从114至132氨基酸序列，加入连接肽丝氨酸－谷氨酸－丝氨酸－丝氨酸－谷氨酸；简写为L158C，S290C；G143C，S404C；S103C，S262C；I288F；V187L；Δ114-132(SGSSG)；
3)M3设计：第158位亮氨酸、第290位丝氨酸突变为半胱氨酸，剪除从114至132氨基酸序列，加入连接肽丝氨酸－谷氨酸－丝氨酸－丝氨酸－谷氨酸－丝氨酸－丝氨酸－谷氨酸；简写为L158C，S290C；G143C，S404C；S103C，S262C；I288F；V187L；Δ109-137(SGSSGSSG)。
不同病毒株的牛呼吸道胞合体病毒F蛋白质的基因序列不同，改造后的牛呼吸道合胞病毒F蛋白的融合前体蛋白的序列也就不同。进一步的，牛呼吸道合胞病毒F蛋白的融合前体蛋白，选自下述序列中的一种：
(1)牛呼吸道合胞病毒NP_048055.1(ATue51908)的M1、M2、M3三种设计改造的F蛋白的融合前体蛋白的氨基酸序列依次如SEQ ID NO:1、2、3所示；
(2)牛呼吸道合胞病毒YP_009505455(ATCC 51908)的M1、M2、M3三种设计改造的F蛋白的融合前体蛋白的氨基酸序列依次如SEQ ID NO:4、5、6所示；
(3)牛呼吸道合胞病毒ANN02895(USII/S1)的M1、M2、M3三种设计改造的F蛋白的融合前体蛋白的氨基酸序列依次如SEQ ID NO:7、8、9所示；
(4)牛呼吸道合胞病毒AAL49410(A51908)的M1、M2、M3三种设计改造的F蛋白的融合前体蛋白的氨基酸序列依次如SEQ ID NO:10、11、12所示；
(5)牛呼吸道合胞病毒BAA00798.1(RB94)的M1、M2、M3三种设计改造的F蛋白的融合前体蛋白的氨基酸序列依次如SEQ ID NO:13、14、15所示；
(6)牛呼吸道合胞病毒P22167.1(Copenhagen)的M1、M2、M3三种设计改造的F蛋白的融合前体蛋白的氨基酸序列依次如SEQ ID NO:16、17、18所示；
(7)牛呼吸道合胞病毒AAB22601(391-2)的M1、M2、M3三种设计改造的F蛋白的融合前体蛋白的氨基酸序列依次如SEQ ID NO:19、20、21所示。
本发明还提供了编码上述的F蛋白的融合前体蛋白的DNA分子。
在一些实施方案中，所述上述SEQ ID NO:1-21所示F蛋白的融合前体蛋的核苷酸序列依次如SEQ ID NO:22-42所示。
实验表明本发明所述的牛呼吸道合胞病毒F蛋白的融合前体蛋白构象稳定可作为疫苗候选物用于预防牛呼吸道合胞病毒。因此本发明还提供了本发明所述的牛呼吸道合胞病毒F蛋白的融合前体蛋白和编码所述本发明所述的牛呼吸道合胞病毒F蛋白的融合前体蛋白的DNA分子在制备预防牛呼吸道合胞病毒的产品中的应用。
本发明还提供了一种蛋白疫苗，由上述的牛呼吸道合胞病毒F蛋白的融合前体蛋白中的至少一种制成。进一步的，所述蛋白疫苗，由氨基酸序列如SEQ ID NO.1-21中任意一种所示的牛呼吸道合胞病毒F蛋白的融合前体蛋白制成。
本发明还提供了一种DNA疫苗，包含编码上述的牛呼吸道合胞病毒F蛋白的融合前体蛋白的DNA分子中至少一种和质粒载体。
进一步的，所述DNA疫苗，由核苷酸序列如SEQ ID NO.22-42中任意一种所示的牛呼吸道合胞病毒F蛋白的融合前体蛋白的DNA分子和质粒载体。
作为优选，所述的DNA疫苗中所述质粒载体为pVAC1-mcs质粒。
由上述技术方案可知，本发明提供了一种牛呼吸道合胞病毒F蛋白的融合前体蛋白、编码所述蛋白的DNA分子及其应用。本发明将结构生物学的方法应用到疫苗设计和改良中，通过对F-蛋白质结构的观察，发现F-蛋白发生构象变化的生物学机制。在牛呼吸道合胞病毒F蛋白的三维结构的基础上进行抗原设计和基因工程改造，获得保持在亚稳定态F-蛋白融合前体的牛呼吸道合胞病毒F蛋白的融合前体蛋白。在此蛋白质疫苗基础上进一步进行基因工程改造，构建可以在宿主细胞表面表达牛呼吸道胞合体病毒F蛋白融合前体蛋白的质粒做为DNA疫苗，使其在哺乳细胞表达分泌出的蛋白质大部分能够保持在融合前体的亚稳定状态。通过分子生物学，生物学，细胞生物学，免疫学的方法确定疫苗的稳定性，有效性和安全性。
附图说明
图1示M1，M2和M3三种设计的F蛋白在蛋白质三维结构(5TGD)上的空间位置示意图；
图2示病毒株391-2F蛋白突变体M1的凝胶过滤纯化色谱图；
图3示病毒株391-2F蛋白突变体M1SDS-PAGE凝胶电泳图；
图4示pVAC1-mcs质粒图谱；
图5示病毒株391-2和ATue51908的M1，M2和M3三种突变体设计的DNA疫苗和蛋白疫苗在小鼠动物实验中第五周血清的抗病毒的活性结果图；
图6示病毒株ATue51908的DNA及其蛋白质的突变体M1免疫原在犊牛免疫实验结果图；
图7示受免疫小牛的BRSV攻毒实验的实验结果图。
具体实施方式
本发明提供了一种构象稳定的牛呼吸道合胞病毒F蛋白的融合前体蛋白、编码所述蛋白的DNA及其应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本文中所用的细胞培养、分子遗传学、核酸化学、生物化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
抗原(antigen,Ag)，是指能够刺激机体产生(特异性)免疫应答，并能与免疫应答产物抗体和致敏淋巴细胞在体外结合，发生免疫效应(特异性反应)的物质。抗原为能够在呈递至宿主动物后诱导免疫反应的一段DNA或DNA片段；多肽、表位、半抗原或其任何组合。
“多肽”和“蛋白质”在本文中可互换使用，意指连续氨基酸残基的聚合物。术语“核酸”、“核苷酸”可互换使用，是指RNA、DNA、cDNA(互补DNA)或cRNA (互补RNA)及其衍生物，例如包含经修饰主链的形式。
“融合”是指将两种或更多种蛋白融合表达以形成融合蛋白的技术。通常，通过使用重组DNA技术将编码两种或更多种蛋白的DNA片段符合读框地连接在一起，并进行蛋白质表达来获得融合蛋白。融合使用是指将MS与sPD-1的序列融合在一起，制得疫苗的方法。
“预防”是指预防疾病或与之相关的病况或症状。
“载体(vector)”是指可将多聚核苷酸插入其中的一种核酸运载工具。一种载体可以含有多种控制表达的元件，包括但不限于启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。
“宿主细胞”是指可用于导入载体的细胞，其包括但不限于如大肠杆菌或枯草菌等的原核细胞，如酵母细胞或曲霉菌等的真菌细胞，如S2果蝇细胞或Sf9等的昆虫细胞，或者如BHK细胞、HEK293细胞或人细胞等的动物细胞。
“佐剂”是指非特异性免疫增强剂，当其与抗原一起或预先递送入机体时，其可增强机体对抗原的免疫应答或改变免疫应答类型。佐剂有很多种，包括但不限于铝佐剂(例如氢氧化铝)、弗氏佐剂(例如完全弗氏佐剂和不完全弗氏佐剂)、短小棒状杆菌、脂多糖、细胞因子等。弗氏佐剂是目前动物试验中最常用的佐剂。氢氧化铝佐剂则在临床实验中使用较多。
疫苗(vaccine)是指为了预防、控制传染病的发生、流行，用于人体预防接种的疫苗类预防性生物制品。“DNA载体疫苗”是指基于DNA或RNA(例如质粒，如表达质粒)的疫苗，其任选地还包含佐剂。
本发明采用的试材、试剂、仪器皆为普通市售品，皆可于市场购得。
下面结合实施例，进一步阐述本发明：
实施例1：构象稳定的牛呼吸道合胞病毒F蛋白的融合前体蛋白的设计与筛选
(1)根据结构生物学的原理，观察三个蛋白质的三维结构PDB编码为5TGD，5TDL和3RRR，确定RSV病毒F蛋白融合前体和融合后体的构象变化，在F蛋白的三维结构的基础上对七种野生的BRSV病毒株进行抗原设计和基因工程改造，以使表达的F蛋白尽可能够保持在融合前体的亚稳定状态。
其中所述七种牛呼吸道胞合体病毒F蛋白的野生病毒株的基因序列的基因库登录号(毒株名)分别为：(1)NP_048055.1(ATue51908)；(2)YP_009505455(ATCC 51908)；(3)ANN02895(USII/S1)；(4)AAL49410(A51908)；(5)AM746678.1(RB94)；(6)P22167.1(Copenhagen)；(7)AAB22601(391-2)。
在F蛋白的三维结构的基础上进行抗原设计和基因工程改造的目标是：
1)增加F蛋白三聚体每个单体内部以及单体之间二硫键的连接数量，阻碍其发生构象变化，将其锁定在融合前体的亚稳定状态。
2)突变F蛋白三聚体内部一些氨基酸，主要是将一些侧链较小的氨基酸突变成为侧链较大的氨基酸填充内部当空隙，增加内部的疏水结合，减少构象变化所需的移动空间，限制其向融合后体发生构象变化。
3)突变剔除某些蛋白酶的酶切位点，保护融合肽，阻止因蛋白酶酶切后引发的构象变化。
4)切除F蛋白三聚体内部一些动态较大氨基酸，代之以一些较短的连接肽，提高蛋白质的稳定性。
5)在F-蛋白质的C-终端延长螺旋构造，增强三聚体形成的机率和及其稳定度。
按照上述目标，申请人在F蛋白的三维结构的基础上进行第一轮抗原设计对病毒株ATu51908的F蛋白的原始序列基因工程改造的获得了56种突变体抗原，其中各抗原的设计和改良理念、F蛋白的形态、突变的序列位点、与一些特异性抗体结合的亲和力和蛋白质表达水平，如表1所示。蛋白质疫苗是通过使用Expi293F细胞Thermo Fisher Scientific，MA和293Fectin(Thermo Fisher Scientific)表达RSV F蛋白的突变体。使用的质粒是pcDNA3.0.瞬时转染5天后收获细胞培养物上清液。以10,000×g离心以除去细胞碎片。上清液通过无菌过滤，使用镍(Ni)(Roche)和Strep-Tactin(iba)亲和层析对RSV F突变体蛋白进行分离纯化。然后通过分子筛柱子进一步分离纯化，获得了RSV F的蛋白质疫苗。
表1第一轮抗原设计和基因工程改造的获得的56种抗原
结合第一轮的优良设计，主要是抗原的表达水平及与特异抗体结合的亲和力，在病毒株ATu51908原始序列的基础上进行了第二轮F蛋白的设计和改良，经过筛选获得了20种抗原在一些极端环境下，如高低温度(50-70℃)、冷冻解冻五次、酸碱(pH3-10)和盐浓度(10-3000mM)及长时间在室温放置等条件的理化学性质及与一些特异性抗体结合的亲和力及其蛋白质表达水平。
表2第二轮F蛋白抗原设计的获得的20种抗原在特殊物理和化学条件下的稳定性质
表3第二轮F蛋白抗原设计的获得的20种抗原和抗体相互作用的亲和力
结果发现，三种突变体抗原的设计在产量、稳定性及与特异抗体结合的亲和力方面有较好的表现，分别为：
M1：H159C、V291C，G143C、S404C，S103C、N262C，I288F，V187L，Δ109-137(SGSSGSSG)；即牛呼吸道胞合体病毒F蛋白氨基酸序列第159位组氨酸、第291位缬氨酸、第143位甘氨酸、第404位丝氨酸、第103位丝氨酸、第262位丝氨酸突变为半胱氨酸，第288位异亮氨酸突变为苯丙氨酸，第187位缬氨酸突变为亮氨酸，剪除从109至137氨基酸序列，并在剪除位点相同位置加入连接肽SGSSGSSG(丝氨酸－谷氨酸－丝氨酸－丝氨酸－谷氨酸－丝氨酸－丝氨酸－谷氨酸)。
M2：L158C、S290C，G143C、S404C，S103C、N262C，I288F，V187L，Δ114-132(SGSSG)；即牛呼吸道胞合体病毒F蛋白氨基酸序列第158位亮氨酸、第290位丝氨酸、第143位甘氨酸、第404位丝氨酸、第103位丝氨酸、第262位丝氨酸突变为半胱氨酸，第288位异亮氨酸突变为苯丙氨酸，第187位缬氨酸变为亮氨酸，剪除从114至132氨基酸序列，加入连接肽SGSSG(丝氨酸－谷氨酸－丝氨酸－丝氨酸－谷氨酸)；
M3：L158C、S290C，G143C、S404C，S103C、N262C，I288F，V187L，Δ109-137(SGSSGSSG)；即牛呼吸道胞合体病毒F蛋白氨基酸序列在M2的基础上延长了连接肽的长度，突变成为SGSSGSSG(丝氨酸－谷氨酸－丝氨酸－丝氨酸－谷氨酸－丝氨酸－丝氨酸－谷氨酸)。
M1，M2和M3三种设计在蛋白三维结构(5TGD)上的空间位置如图1所示。
七种牛呼吸道胞合体病毒F蛋白的融合前体蛋白的氨基酸序列及相应的DNA序列如表4所示
表4 BRSV F蛋白的融合前体蛋白的氨基酸序列及相应的DNA序列
以病毒株391-2为例，对391-2M1设计的F蛋白的稳定性进行检测，结果如图2和图3。
由图2的凝胶过滤色谱图可知391-2M1设计的F蛋白大部分保留在融合前体的三聚体的构象中，表现为较单一的吸收峰。而图3所示的凝胶图像显示391-2M1设计的F蛋白保持在较稳定的三聚体,在还原剂存在的情况下只有一个带。但是没有进行改良的391-2毒株的F蛋白则处在不稳定、易被蛋白酶切断的状态，表现为具有多个带。
391-2M2和M3设计的F蛋白稳定性结果与391-2M1相似。
实施例2：DNA疫苗的制备
将经过验证的筛选获得的抗原蛋白序列进行相应的DNA合成、提纯和检测，使用来源于InvivoGen，USA的pVAC1-mcs质粒(图4)做为DNA疫苗载体。克隆在此质粒的抗原蛋白可以表达并靶向的锚定在细胞表面，通过肌肉注射刺激体液免疫反应。肌肉细胞表面产生的抗原被认为是通过抗原表达细胞(APCs)吸收的，并通过主要的组织相容性复合体(MHC)II类途径进行处理。
实施例3：小鼠免疫实验
将抗原蛋白产量高及抗原抗体亲和力较好的391-2M1、391-2M2、391-2M3和ATue51908M1、ATue51908M2、ATue51908M3候选物进行相应的DNA合成，序列如表3所示。然后分别用BamHI和EcoRI的酶切克隆至加入pVAC1-mcs质粒中用于小鼠的DNA疫苗免疫实验，同时设置同源的蛋白质疫苗免疫实验，比较小鼠接受免疫后血清的抗病毒活性结果如图5所示。
在Bovine 391-2和A51908的F蛋白的基础上经M1，M2和M3三种设计改良的DNA疫苗和同源的蛋白质疫苗在小鼠在零周免疫，两周后加强，五周后同源的DNA疫苗和蛋白质疫苗诱导的血清的抗病毒效价非常接近。比没有改良的处于融合后体状态的F蛋白对照平均高数百倍。显示接受改良疫苗免疫较没有改良的疫苗的小鼠的血清有很强的抗病毒活性。每个动物的中性滴度显示为单个点,以黑色水平线表示几何均。
实施例4：犊牛免疫实验
为了进一步观察所设计的DNA和蛋白质疫苗在犊牛的表现，进行了以下临床前的动物实验。选择了高度稳定的ATue51908DNA及其蛋白质的M1免疫原，它们在小鼠实验中获得最高中和滴度，分别为EC50 26,716.8和19,605.8。作为对照，选择了融合后体的ATue51908DNA及蛋白质抗原,并使用磷酸盐缓冲生理盐水(PBS)对安慰剂组进行免疫。10个犊牛一组，对1-3周大的雄性小牛进行了免疫接种。在第0周和第4周两次,每周2周后收集血清免疫。每次注射由DNA抗原或者蛋白质疫苗并加入疫苗佐剂Montanide TM ISA71VG(Seppic,France)。观察EC50中和滴度，小牛在第2周、第4周呈指数级增长。第6周注射融合前体DNA疫苗和蛋白质抗原的小牛血清抗病毒效价分别是同比的没有进行改良的融合后体疫苗的894倍和642倍。第6周达到高峰后持平衡(图6)。
实施例5：受免疫小牛的BRSV攻毒实验
为了证明经过改良的疫苗可以诱导小牛的免疫系统产生较高的抗病毒活性，进行BRSV攻毒实验方法。实施例4的小牛都受到鼻内和气管内的喷毒，每天对小牛进行疾病临床症状监测和病毒滴度，在鼻咽喷毒挑战6天后小牛被安乐死。采样分析支气管肺泡从肺的三个区域灌洗(bal)和肺活检获得,以确定病毒滴度、中性粒细胞浸润、微观和宏观病变的程度。观察相关组织的BRSV的残余情况，结果如图7。观察的器官组织包括右心脏(rc)，左心(lc)，气管上皮(trsc)、肺清洗细胞(lwc)样本。
结果显示，本发明改良的的DNA疫苗和蛋白质疫苗对犊牛起到了很好保护效果，在被融合前体免疫的个体所观察的组织器官内无病毒的残余。但是没有改良的融合后体和缓冲溶液的对照组发现很多病毒的侵染残余。
以上仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
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<110> 苏州宇之波生物科技有限公司
<120> 一种改良的亚稳定态牛呼吸道合胞病毒融合前体F蛋白质及编码的DNA分子和其应用
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Gln Val Asn Ala Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Arg Ser
485 490 495
Asp Glu Leu Leu His
500
<210> 3
<211> 504
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Met Ala Thr Thr Ala Met Arg Met Ile Ile Ser Ile Ile Phe Ile Ser
1 5 10 15
Thr Tyr Val Thr His Ile Thr Leu Cys Gln Asn Ile Thr Glu Glu Phe
20 25 30
Tyr Gln Ser Thr Cys Ser Ala Val Ser Arg Gly Tyr Leu Ser Ala Leu
35 40 45
Arg Thr Gly Trp Tyr Thr Ser Val Val Thr Ile Glu Leu Ser Lys Ile
50 55 60
Gln Lys Asn Val Cys Lys Ser Thr Asp Ser Lys Val Lys Leu Ile Lys
65 70 75 80
Gln Glu Leu Glu Arg Tyr Asn Asn Ala Val Val Glu Leu Gln Ser Leu
85 90 95
Met Gln Asn Glu Pro Ala Cys Phe Ser Arg Ala Lys Arg Gly Ile Pro
100 105 110
Glu Ser Gly Ser Ser Gly Ser Ser Gly Lys Arg Lys Arg Arg Phe Leu
115 120 125
Gly Phe Leu Leu Cys Ile Gly Ser Ala Val Ala Ser Gly Val Ala Val
130 135 140
Ser Lys Val Cys His Leu Glu Gly Glu Val Asn Lys Ile Lys Asn Ala
145 150 155 160
Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val Ser
165 170 175
Leu Leu Thr Ser Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys Glu
180 185 190
Leu Leu Pro Gln Val Asn Asn His Asp Cys Arg Ile Ser Asn Ile Glu
195 200 205
Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile Ala
210 215 220
Arg Glu Phe Ser Val Asn Ala Gly Ile Thr Thr Pro Leu Ser Thr Tyr
225 230 235 240
Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Cys Asp Met Pro Ile
245 250 255
Thr Asn Asp Gln Lys Lys Leu Met Ser Ser Asn Val Gln Ile Val Arg
260 265 270
Gln Gln Ser Tyr Ser Phe Met Cys Val Val Lys Glu Glu Val Ile Ala
275 280 285
Tyr Val Val Gln Leu Pro Ile Tyr Gly Val Ile Asp Thr Pro Cys Trp
290 295 300
Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asp Asn Lys Glu Gly Ser
305 310 315 320
Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala
325 330 335
Gly Ser Val Ser Phe Phe Pro Gln Thr Glu Thr Cys Lys Val Gln Ser
340 345 350
Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Thr Asp
355 360 365
Val Asn Leu Cys Asn Thr Asp Ile Phe Asn Thr Lys Tyr Asp Cys Lys
370 375 380
Ile Met Thr Ser Lys Thr Asp Ile Ser Cys Ser Val Ile Thr Ser Ile
385 390 395 400
Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn
405 410 415
Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val
420 425 430
Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr
435 440 445
Val Asn Lys Leu Glu Gly Lys Ala Leu Tyr Ile Lys Gly Glu Pro Ile
450 455 460
Ile Asn Tyr Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp Ala
465 470 475 480
Ser Ile Ala Gln Val Asn Ala Lys Ile Asn Gln Ser Leu Ala Phe Ile
485 490 495
Arg Arg Ser Asp Glu Leu Leu His
500
<210> 4
<211> 494
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Met Ala Thr Thr Ala Met Arg Met Ile Ile Ser Ile Ile Phe Ile Ser
1 5 10 15
Thr Tyr Val Thr His Ile Thr Leu Cys Gln Asn Ile Thr Glu Glu Phe
20 25 30
Tyr Gln Ser Thr Cys Ser Ala Val Ser Arg Gly Tyr Leu Ser Ala Leu
35 40 45
Arg Thr Gly Trp Tyr Thr Ser Val Val Thr Ile Glu Leu Ser Lys Ile
50 55 60
Gln Lys Asn Val Cys Asn Ser Thr Asp Ser Lys Val Lys Leu Ile Lys
65 70 75 80
Gln Glu Leu Glu Arg Tyr Asn Asn Ala Val Val Glu Leu Gln Ser Leu
85 90 95
Met Gln Asn Glu Pro Ala Cys Phe Ser Arg Ala Lys Ser Gly Ser Ser
100 105 110
Gly Ser Ser Gly Leu Gly Phe Leu Leu Cys Ile Gly Ser Ala Ile Ala
115 120 125
Ser Gly Val Ala Val Ser Lys Val Leu Cys Leu Glu Gly Glu Val Asn
130 135 140
Lys Ile Lys Asn Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Ala
145 150 155 160
Gly Ile Thr Thr Pro Leu Ser Leu Ser Asn Gly Val Ser Leu Leu Thr
165 170 175
Ser Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys Glu Leu Leu Pro
180 185 190
Lys Val Asn Asn His Asp Cys Arg Ile Ser Lys Ile Glu Thr Val Ile
195 200 205
Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile Ala Arg Glu Phe
210 215 220
Ser Val Asn Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile
225 230 235 240
Cys Asp Met Pro Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Ser Asn
245 250 255
Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Phe Met Ser Cys Val Lys
260 265 270
Glu Glu Val Ile Ala Tyr Val Val Gln Leu Pro Ile Tyr Gly Val Ile
275 280 285
Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asp
290 295 300
Asn Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp
305 310 315 320
Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Thr Glu Thr
325 330 335
Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu
340 345 350
Thr Leu Pro Thr Asp Val Asn Leu Cys Asn Thr Asp Ile Phe Asn Thr
355 360 365
Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr Asp Ile Ser Cys Ser
370 375 380
Val Ile Thr Ser Ile Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys
385 390 395 400
Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn
405 410 415
Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly
420 425 430
Asn Thr Leu Tyr Tyr Val Asn Lys Leu Glu Gly Lys Ala Leu Tyr Ile
435 440 445
Lys Gly Glu Pro Ile Ile Asn Tyr Tyr Asp Pro Leu Val Phe Pro Ser
450 455 460
Asp Glu Phe Asp Ala Ser Ile Ala Gln Val Asn Ala Lys Ile Asn Gln
465 470 475 480
Ser Leu Ala Phe Ile Arg Arg Ser Asp Glu Leu Leu His Ser
485 490
<210> 5
<211> 501
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Met Ala Thr Thr Ala Met Arg Met Ile Ile Ser Ile Ile Phe Ile Ser
1 5 10 15
Thr Tyr Val Thr His Ile Thr Leu Cys Gln Asn Ile Thr Glu Glu Phe
20 25 30
Tyr Gln Ser Thr Cys Ser Ala Val Ser Arg Gly Tyr Leu Ser Ala Leu
35 40 45
Arg Thr Gly Trp Tyr Thr Ser Val Val Thr Ile Glu Leu Ser Lys Ile
50 55 60
Gln Lys Asn Val Cys Asn Ser Thr Asp Ser Lys Val Lys Leu Ile Lys
65 70 75 80
Gln Glu Leu Glu Arg Tyr Asn Asn Ala Val Val Glu Leu Gln Ser Leu
85 90 95
Met Gln Asn Glu Pro Ala Cys Phe Ser Arg Ala Lys Arg Gly Ile Pro
100 105 110
Glu Ser Gly Ser Ser Gly Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu
115 120 125
Cys Ile Gly Ser Ala Ile Ala Ser Gly Val Ala Val Ser Lys Val Cys
130 135 140
His Leu Glu Gly Glu Val Asn Lys Ile Lys Asn Ala Leu Leu Ser Thr
145 150 155 160
Asn Lys Ala Val Val Ser Ala Gly Ile Thr Thr Pro Leu Ser Leu Ser
165 170 175
Asn Gly Val Ser Leu Leu Thr Ser Lys Val Leu Asp Leu Lys Asn Tyr
180 185 190
Ile Asp Lys Glu Leu Leu Pro Lys Val Asn Asn His Asp Cys Arg Ile
195 200 205
Ser Lys Ile Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu
210 215 220
Leu Glu Ile Ala Arg Glu Phe Ser Val Asn Thr Tyr Met Leu Thr Asn
225 230 235 240
Ser Glu Leu Leu Ser Leu Ile Cys Asp Met Pro Ile Thr Asn Asp Gln
245 250 255
Lys Lys Leu Met Ser Ser Asn Val Gln Ile Val Arg Gln Gln Ser Tyr
260 265 270
Ser Phe Met Cys Val Val Lys Glu Glu Val Ile Ala Tyr Val Val Gln
275 280 285
Leu Pro Ile Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr
290 295 300
Ser Pro Leu Cys Thr Thr Asp Asn Lys Glu Gly Ser Asn Ile Cys Leu
305 310 315 320
Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser
325 330 335
Phe Phe Pro Gln Thr Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe
340 345 350
Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Thr Asp Val Asn Leu Cys
355 360 365
Asn Thr Asp Ile Phe Asn Thr Lys Tyr Asp Cys Lys Ile Met Thr Ser
370 375 380
Lys Thr Asp Ile Ser Cys Ser Val Ile Thr Ser Ile Gly Ala Ile Val
385 390 395 400
Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly
405 410 415
Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly
420 425 430
Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Leu
435 440 445
Glu Gly Lys Ala Leu Tyr Ile Lys Gly Glu Pro Ile Ile Asn Tyr Tyr
450 455 460
Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ala Gln
465 470 475 480
Val Asn Ala Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Arg Ser Asp
485 490 495
Glu Leu Leu His Ser
500
<210> 6
<211> 494
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Met Ala Thr Thr Ala Met Arg Met Ile Ile Ser Ile Ile Phe Ile Ser
1 5 10 15
Thr Tyr Val Thr His Ile Thr Leu Cys Gln Asn Ile Thr Glu Glu Phe
20 25 30
Tyr Gln Ser Thr Cys Ser Ala Val Ser Arg Gly Tyr Leu Ser Ala Leu
35 40 45
Arg Thr Gly Trp Tyr Thr Ser Val Val Thr Ile Glu Leu Ser Lys Ile
50 55 60
Gln Lys Asn Val Cys Asn Ser Thr Asp Ser Lys Val Lys Leu Ile Lys
65 70 75 80
Gln Glu Leu Glu Arg Tyr Asn Asn Ala Val Val Glu Leu Gln Ser Leu
85 90 95
Met Gln Asn Glu Pro Ala Cys Phe Ser Arg Ala Lys Ser Gly Ser Ser
100 105 110
Gly Ser Ser Gly Leu Gly Phe Leu Leu Cys Ile Gly Ser Ala Ile Ala
115 120 125
Ser Gly Val Ala Val Ser Lys Val Cys His Leu Glu Gly Glu Val Asn
130 135 140
Lys Ile Lys Asn Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Ala
145 150 155 160
Gly Ile Thr Thr Pro Leu Ser Leu Ser Asn Gly Val Ser Leu Leu Thr
165 170 175
Ser Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys Glu Leu Leu Pro
180 185 190
Lys Val Asn Asn His Asp Cys Arg Ile Ser Lys Ile Glu Thr Val Ile
195 200 205
Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile Ala Arg Glu Phe
210 215 220
Ser Val Asn Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile
225 230 235 240
Cys Asp Met Pro Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Ser Asn
245 250 255
Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Phe Met Cys Val Val Lys
260 265 270
Glu Glu Val Ile Ala Tyr Val Val Gln Leu Pro Ile Tyr Gly Val Ile
275 280 285
Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asp
290 295 300
Asn Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp
305 310 315 320
Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Thr Glu Thr
325 330 335
Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu
340 345 350
Thr Leu Pro Thr Asp Val Asn Leu Cys Asn Thr Asp Ile Phe Asn Thr
355 360 365
Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr Asp Ile Ser Cys Ser
370 375 380
Val Ile Thr Ser Ile Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys
385 390 395 400
Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn
405 410 415
Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly
420 425 430
Asn Thr Leu Tyr Tyr Val Asn Lys Leu Glu Gly Lys Ala Leu Tyr Ile
435 440 445
Lys Gly Glu Pro Ile Ile Asn Tyr Tyr Asp Pro Leu Val Phe Pro Ser
450 455 460
Asp Glu Phe Asp Ala Ser Ile Ala Gln Val Asn Ala Lys Ile Asn Gln
465 470 475 480
Ser Leu Ala Phe Ile Arg Arg Ser Asp Glu Leu Leu His Ser
485 490
<210> 7
<211> 494
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Met Ala Ala Met Ala Met Arg Met Ile Ile Ser Ile Ile Phe Ile Ser
1 5 10 15
Thr Tyr Met Thr His Ile Thr Leu Cys Gln Asn Ile Thr Glu Glu Phe
20 25 30
Tyr Gln Ser Thr Cys Ser Ala Val Ser Arg Gly Tyr Leu Ser Ala Leu
35 40 45
Arg Thr Gly Trp Tyr Thr Ser Val Val Thr Ile Glu Leu Ser Lys Ile
50 55 60
Gln Lys Asp Val Cys Lys Ser Thr Asp Ser Lys Val Lys Leu Ile Lys
65 70 75 80
Gln Glu Leu Glu Arg Tyr Asn Asn Ala Val Thr Glu Leu Gln Ser Leu
85 90 95
Met Gln Asn Val Pro Ala Cys Phe Asn Arg Ala Lys Ser Gly Ser Ser
100 105 110
Gly Ser Ser Gly Leu Gly Phe Leu Leu Cys Ile Gly Ser Ala Ile Ala
115 120 125
Ser Gly Val Ala Val Ser Lys Val Leu Cys Leu Glu Gly Glu Val Asn
130 135 140
Lys Ile Lys Asn Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu
145 150 155 160
Ser Asn Gly Val Ser Leu Leu Thr Ser Lys Val Leu Asp Leu Lys Asn
165 170 175
Tyr Ile Asp Lys Glu Leu Leu Pro Lys Val Asn Asn His Asp Cys Arg
180 185 190
Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg
195 200 205
Leu Leu Glu Ile Ala Arg Glu Phe Ser Val Asn Ala Gly Ile Thr Thr
210 215 220
Pro Leu Ser Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile
225 230 235 240
Cys Asp Met Pro Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Ser Asn
245 250 255
Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Phe Met Ser Cys Val Lys
260 265 270
Glu Glu Val Ile Ala Tyr Val Val Gln Leu Pro Ile Tyr Gly Val Ile
275 280 285
Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asp
290 295 300
Asn Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp
305 310 315 320
Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr
325 330 335
Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu
340 345 350
Thr Leu Pro Thr Asp Val Asn Leu Cys Asn Thr Asp Ile Phe Asn Thr
355 360 365
Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr Asp Ile Ser Cys Ser
370 375 380
Val Ile Thr Ser Ile Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys
385 390 395 400
Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn
405 410 415
Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly
420 425 430
Asn Thr Leu Tyr Tyr Val Asn Lys Leu Glu Gly Lys Ala Leu Tyr Ile
435 440 445
Lys Gly Glu Pro Ile Ile Asn Tyr Tyr Asp Pro Leu Val Phe Pro Ser
450 455 460
Asp Glu Phe Asp Ala Ser Ile Ala Gln Val Asn Ala Lys Ile Asn Gln
465 470 475 480
Ser Leu Ala Phe Ile Arg Arg Ser Asp Glu Leu Leu His Ser
485 490
<210> 8
<211> 501
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Met Ala Ala Met Ala Met Arg Met Ile Ile Ser Ile Ile Phe Ile Ser
1 5 10 15
Thr Tyr Met Thr His Ile Thr Leu Cys Gln Asn Ile Thr Glu Glu Phe
20 25 30
Tyr Gln Ser Thr Cys Ser Ala Val Ser Arg Gly Tyr Leu Ser Ala Leu
35 40 45
Arg Thr Gly Trp Tyr Thr Ser Val Val Thr Ile Glu Leu Ser Lys Ile
50 55 60
Gln Lys Asp Val Cys Lys Ser Thr Asp Ser Lys Val Lys Leu Ile Lys
65 70 75 80
Gln Glu Leu Glu Arg Tyr Asn Asn Ala Val Thr Glu Leu Gln Ser Leu
85 90 95
Met Gln Asn Val Pro Ala Cys Phe Asn Arg Ala Lys Arg Gly Ile Pro
100 105 110
Glu Ser Gly Ser Ser Gly Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu
115 120 125
Cys Ile Gly Ser Ala Ile Ala Ser Gly Val Ala Val Ser Lys Val Cys
130 135 140
His Leu Glu Gly Glu Val Asn Lys Ile Lys Asn Ala Leu Leu Ser Thr
145 150 155 160
Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val Ser Leu Leu Thr Ser
165 170 175
Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys Glu Leu Leu Pro Lys
180 185 190
Val Asn Asn His Asp Cys Arg Ile Ser Asn Ile Glu Thr Val Ile Glu
195 200 205
Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile Ala Arg Glu Phe Ser
210 215 220
Val Asn Ala Gly Ile Thr Thr Pro Leu Ser Thr Tyr Met Leu Thr Asn
225 230 235 240
Ser Glu Leu Leu Ser Leu Ile Cys Asp Met Pro Ile Thr Asn Asp Gln
245 250 255
Lys Lys Leu Met Ser Ser Asn Val Gln Ile Val Arg Gln Gln Ser Tyr
260 265 270
Ser Phe Met Cys Val Val Lys Glu Glu Val Ile Ala Tyr Val Val Gln
275 280 285
Leu Pro Ile Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr
290 295 300
Ser Pro Leu Cys Thr Thr Asp Asn Lys Glu Gly Ser Asn Ile Cys Leu
305 310 315 320
Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser
325 330 335
Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe
340 345 350
Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Thr Asp Val Asn Leu Cys
355 360 365
Asn Thr Asp Ile Phe Asn Thr Lys Tyr Asp Cys Lys Ile Met Thr Ser
370 375 380
Lys Thr Asp Ile Ser Cys Ser Val Ile Thr Ser Ile Gly Ala Ile Val
385 390 395 400
Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly
405 410 415
Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly
420 425 430
Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Leu
435 440 445
Glu Gly Lys Ala Leu Tyr Ile Lys Gly Glu Pro Ile Ile Asn Tyr Tyr
450 455 460
Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ala Gln
465 470 475 480
Val Asn Ala Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Arg Ser Asp
485 490 495
Glu Leu Leu His Ser
500
<210> 9
<211> 494
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Met Ala Ala Met Ala Met Arg Met Ile Ile Ser Ile Ile Phe Ile Ser
1 5 10 15
Thr Tyr Met Thr His Ile Thr Leu Cys Gln Asn Ile Thr Glu Glu Phe
20 25 30
Tyr Gln Ser Thr Cys Ser Ala Val Ser Arg Gly Tyr Leu Ser Ala Leu
35 40 45
Arg Thr Gly Trp Tyr Thr Ser Val Val Thr Ile Glu Leu Ser Lys Ile
50 55 60
Gln Lys Asp Val Cys Lys Ser Thr Asp Ser Lys Val Lys Leu Ile Lys
65 70 75 80
Gln Glu Leu Glu Arg Tyr Asn Asn Ala Val Thr Glu Leu Gln Ser Leu
85 90 95
Met Gln Asn Val Pro Ala Cys Phe Asn Arg Ala Lys Ser Gly Ser Ser
100 105 110
Gly Ser Ser Gly Leu Gly Phe Leu Leu Cys Ile Gly Ser Ala Ile Ala
115 120 125
Ser Gly Val Ala Val Ser Lys Val Cys His Leu Glu Gly Glu Val Asn
130 135 140
Lys Ile Lys Asn Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu
145 150 155 160
Ser Asn Gly Val Ser Leu Leu Thr Ser Lys Val Leu Asp Leu Lys Asn
165 170 175
Tyr Ile Asp Lys Glu Leu Leu Pro Lys Val Asn Asn His Asp Cys Arg
180 185 190
Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg
195 200 205
Leu Leu Glu Ile Ala Arg Glu Phe Ser Val Asn Ala Gly Ile Thr Thr
210 215 220
Pro Leu Ser Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile
225 230 235 240
Cys Asp Met Pro Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Ser Asn
245 250 255
Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Phe Met Cys Leu Val Lys
260 265 270
Glu Glu Val Ile Ala Tyr Val Val Gln Leu Pro Ile Tyr Gly Val Ile
275 280 285
Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asp
290 295 300
Asn Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp
305 310 315 320
Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr
325 330 335
Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu
340 345 350
Thr Leu Pro Thr Asp Val Asn Leu Cys Asn Thr Asp Ile Phe Asn Thr
355 360 365
Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr Asp Ile Ser Cys Ser
370 375 380
Val Ile Thr Ser Ile Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys
385 390 395 400
Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn
405 410 415
Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly
420 425 430
Asn Thr Leu Tyr Tyr Val Asn Lys Leu Glu Gly Lys Ala Leu Tyr Ile
435 440 445
Lys Gly Glu Pro Ile Ile Asn Tyr Tyr Asp Pro Leu Val Phe Pro Ser
450 455 460
Asp Glu Phe Asp Ala Ser Ile Ala Gln Val Asn Ala Lys Ile Asn Gln
465 470 475 480
Ser Leu Ala Phe Ile Arg Arg Ser Asp Glu Leu Leu His Ser
485 490
<210> 10
<211> 489
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Met Arg Met Ile Ile Ser Ile Ile Leu Ile Ser Thr Tyr Val Pro His
1 5 10 15
Ile Thr Leu Cys Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys
20 25 30
Ser Ala Val Ser Arg Gly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr
35 40 45
Thr Ser Val Val Thr Ile Glu Leu Ser Lys Ile Gln Lys Asn Val Cys
50 55 60
Asn Gly Thr Asp Ser Lys Val Lys Leu Ile Lys Gln Glu Leu Glu Arg
65 70 75 80
Tyr Asn Asn Ala Val Val Glu Leu Gln Ser Leu Met Gln Asn Glu Pro
85 90 95
Thr Cys Ser Ser Arg Ala Lys Ser Gly Ser Ser Gly Ser Ser Gly Leu
100 105 110
Gly Phe Leu Leu Cys Ile Gly Ser Ala Ile Ala Ser Gly Val Ala Val
115 120 125
Ser Lys Val Leu Cys Leu Glu Gly Glu Val Asn Lys Ile Lys Asn Ala
130 135 140
Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val Ser
145 150 155 160
Leu Leu Thr Ser Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys Glu
165 170 175
Leu Leu Pro Lys Val Asn Asn His Asp Cys Arg Ile Ser Asn Ile Ala
180 185 190
Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile Ala
195 200 205
Arg Glu Phe Ser Val Asn Ala Gly Ile Thr Thr Pro Leu Ser Thr Tyr
210 215 220
Met Leu Thr Asn Ser Glu Leu Leu Ser Ile Ile Cys Asp Met Pro Ile
225 230 235 240
Thr Asn Asp Gln Lys Lys Leu Met Ser Ser Asn Val Gln Ile Val Arg
245 250 255
Gln Gln Ser Tyr Ser Phe Met Ser Cys Val Lys Glu Glu Val Ile Ala
260 265 270
Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp
275 280 285
Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asp Asn Glu Glu Gly Ser
290 295 300
Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala
305 310 315 320
Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln Ser
325 330 335
Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Thr Asp
340 345 350
Val Asn Leu Cys Asn Thr Asp Ile Phe Asn Ala Lys Tyr Asp Cys Lys
355 360 365
Ile Met Thr Ser Lys Thr Asp Ile Ser Cys Ser Val Ile Thr Ser Ile
370 375 380
Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn
385 390 395 400
Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val
405 410 415
Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr
420 425 430
Val Asn Lys Leu Glu Gly Lys Ala Leu Tyr Ile Lys Gly Glu Pro Ile
435 440 445
Ile Asn Tyr Tyr Asn Pro Leu Val Phe Pro Ser Asp Glu Phe Asp Ala
450 455 460
Ser Ile Ala Gln Val Asn Ala Lys Ile Asn Gln Ser Leu Ala Phe Ile
465 470 475 480
Arg Arg Ser Asp Glu Leu Leu His Ser
485
<210> 11
<211> 496
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Met Arg Met Ile Ile Ser Ile Ile Leu Ile Ser Thr Tyr Val Pro His
1 5 10 15
Ile Thr Leu Cys Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys
20 25 30
Ser Ala Val Ser Arg Gly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr
35 40 45
Thr Ser Val Val Thr Ile Glu Leu Ser Lys Ile Gln Lys Asn Val Cys
50 55 60
Asn Gly Thr Asp Ser Lys Val Lys Leu Ile Lys Gln Glu Leu Glu Arg
65 70 75 80
Tyr Asn Asn Ala Val Val Glu Leu Gln Ser Leu Met Gln Asn Glu Pro
85 90 95
Thr Cys Ser Ser Arg Ala Lys Arg Gly Ile Pro Glu Ser Gly Ser Ser
100 105 110
Gly Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Cys Ile Gly Ser Ala
115 120 125
Ile Ala Ser Gly Val Ala Val Ser Lys Val Cys His Leu Glu Gly Glu
130 135 140
Val Asn Lys Ile Lys Asn Ala Leu Leu Ser Thr Asn Lys Ala Val Val
145 150 155 160
Ser Leu Ser Asn Gly Val Ser Leu Leu Thr Ser Lys Val Leu Asp Leu
165 170 175
Lys Asn Tyr Ile Asp Lys Glu Leu Leu Pro Lys Val Asn Asn His Asp
180 185 190
Cys Arg Ile Ser Asn Ile Ala Thr Val Ile Glu Phe Gln Gln Lys Asn
195 200 205
Asn Arg Leu Leu Glu Ile Ala Arg Glu Phe Ser Val Asn Ala Gly Ile
210 215 220
Thr Thr Pro Leu Ser Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser
225 230 235 240
Ile Ile Cys Asp Met Pro Ile Thr Asn Asp Gln Lys Lys Leu Met Ser
245 250 255
Ser Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Phe Met Cys Val
260 265 270
Val Lys Glu Glu Val Ile Ala Tyr Val Val Gln Leu Pro Leu Tyr Gly
275 280 285
Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr
290 295 300
Thr Asp Asn Glu Glu Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg
305 310 315 320
Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Ala
325 330 335
Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn
340 345 350
Ser Leu Thr Leu Pro Thr Asp Val Asn Leu Cys Asn Thr Asp Ile Phe
355 360 365
Asn Ala Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr Asp Ile Ser
370 375 380
Cys Ser Val Ile Thr Ser Ile Gly Ala Ile Val Ser Cys Tyr Gly Lys
385 390 395 400
Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe
405 410 415
Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp Thr Val Ser
420 425 430
Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Leu Glu Gly Lys Ala Leu
435 440 445
Tyr Ile Lys Gly Glu Pro Ile Ile Asn Tyr Tyr Asn Pro Leu Val Phe
450 455 460
Pro Ser Asp Glu Phe Asp Ala Ser Ile Ala Gln Val Asn Ala Lys Ile
465 470 475 480
Asn Gln Ser Leu Ala Phe Ile Arg Arg Ser Asp Glu Leu Leu His Ser
485 490 495
<210> 12
<211> 489
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Met Arg Met Ile Ile Ser Ile Ile Leu Ile Ser Thr Tyr Val Pro His
1 5 10 15
Ile Thr Leu Cys Gln Asn Ile Thr Glu Glu Phe Tyr Gln Ser Thr Cys
20 25 30
Ser Ala Val Ser Arg Gly Tyr Leu Ser Ala Leu Arg Thr Gly Trp Tyr
35 40 45
Thr Ser Val Val Thr Ile Glu Leu Ser Lys Ile Gln Lys Asn Val Cys
50 55 60
Asn Gly Thr Asp Ser Lys Val Lys Leu Ile Lys Gln Glu Leu Glu Arg
65 70 75 80
Tyr Asn Asn Ala Val Val Glu Leu Gln Ser Leu Met Gln Asn Glu Pro
85 90 95
Thr Cys Ser Ser Arg Ala Lys Ser Gly Ser Ser Gly Ser Ser Gly Leu
100 105 110
Gly Phe Leu Leu Cys Ile Gly Ser Ala Ile Ala Ser Gly Val Ala Val
115 120 125
Ser Lys Val Cys His Leu Glu Gly Glu Val Asn Lys Ile Lys Asn Ala
130 135 140
Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val Ser
145 150 155 160
Leu Leu Thr Ser Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys Glu
165 170 175
Leu Leu Pro Lys Val Asn Asn His Asp Cys Arg Ile Ser Asn Ile Ala
180 185 190
Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile Ala
195 200 205
Arg Glu Phe Ser Val Asn Ala Gly Ile Thr Thr Pro Leu Ser Thr Tyr
210 215 220
Met Leu Thr Asn Ser Glu Leu Leu Ser Ile Ile Cys Asp Met Pro Ile
225 230 235 240
Thr Asn Asp Gln Lys Lys Leu Met Ser Ser Asn Val Gln Ile Val Arg
245 250 255
Gln Gln Ser Tyr Ser Phe Met Cys Val Val Lys Glu Glu Val Ile Ala
260 265 270
Tyr Val Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp
275 280 285
Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asp Asn Glu Glu Gly Ser
290 295 300
Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala
305 310 315 320
Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln Ser
325 330 335
Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Thr Asp
340 345 350
Val Asn Leu Cys Asn Thr Asp Ile Phe Asn Ala Lys Tyr Asp Cys Lys
355 360 365
Ile Met Thr Ser Lys Thr Asp Ile Ser Cys Ser Val Ile Thr Ser Ile
370 375 380
Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn
385 390 395 400
Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val
405 410 415
Ser Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr
420 425 430
Val Asn Lys Leu Glu Gly Lys Ala Leu Tyr Ile Lys Gly Glu Pro Ile
435 440 445
Ile Asn Tyr Tyr Asn Pro Leu Val Phe Pro Ser Asp Glu Phe Asp Ala
450 455 460
Ser Ile Ala Gln Val Asn Ala Lys Ile Asn Gln Ser Leu Ala Phe Ile
465 470 475 480
Arg Arg Ser Asp Glu Leu Leu His Ser
485
<210> 13
<211> 494
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Met Ala Thr Thr Ala Met Arg Met Ile Ile Ser Ile Ile Phe Ile Ser
1 5 10 15
Thr Tyr Val Thr His Ile Thr Leu Cys Gln Asn Ile Thr Glu Glu Phe
20 25 30
Tyr Gln Ser Thr Cys Ser Ala Val Ser Arg Gly Tyr Leu Ser Ala Leu
35 40 45
Arg Thr Gly Trp Tyr Thr Ser Val Val Thr Ile Glu Leu Ser Lys Ile
50 55 60
Gln Lys Asn Val Cys Asn Ser Thr Asp Ser Asn Val Lys Leu Ile Lys
65 70 75 80
Gln Glu Leu Glu Arg Tyr Asn Asn Ala Val Val Glu Leu Gln Ser Leu
85 90 95
Met Gln Asn Glu Pro Ala Cys Ser Ser Arg Ala Lys Ser Gly Ser Ser
100 105 110
Gly Ser Ser Gly Leu Gly Phe Leu Leu Cys Ile Gly Ser Ala Ile Ala
115 120 125
Ser Gly Val Ala Val Ser Lys Val Leu Cys Leu Glu Gly Glu Val Asn
130 135 140
Lys Ile Lys Asn Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu
145 150 155 160
Ser Asn Gly Val Ser Leu Leu Thr Ser Lys Val Leu Asp Leu Lys Asn
165 170 175
Tyr Ile Asp Lys Glu Leu Leu Pro Lys Val Asn Asn His Asp Cys Lys
180 185 190
Ile Ser Asn Ile Ala Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg
195 200 205
Leu Leu Glu Ile Ala Arg Glu Phe Ser Val Asn Ala Gly Ile Thr Thr
210 215 220
Pro Leu Ser Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile
225 230 235 240
Cys Asp Met Pro Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Ser Asn
245 250 255
Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Phe Met Ser Cys Val Lys
260 265 270
Glu Glu Val Met Ala Tyr Val Val Gln Leu Pro Ile Tyr Gly Val Ile
275 280 285
Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asp
290 295 300
Asn Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp
305 310 315 320
Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr
325 330 335
Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu
340 345 350
Thr Leu Pro Thr Asp Val Asn Leu Cys Asn Thr Asp Ile Phe Asn Ala
355 360 365
Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr Asp Ile Ser Cys Ser
370 375 380
Val Ile Thr Ser Ile Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys
385 390 395 400
Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn
405 410 415
Gly Cys Asp Tyr Val Ser Asn Arg Gly Val Asp Thr Val Ser Val Gly
420 425 430
Asn Thr Leu Tyr Tyr Val Asn Lys Leu Glu Gly Lys Ala Leu Tyr Ile
435 440 445
Lys Gly Glu Pro Ile Ile Asn Tyr Tyr Asp Pro Leu Val Phe Pro Ser
450 455 460
Asp Glu Phe Asp Ala Ser Ile Ala Gln Val Asn Ala Lys Ile Asn Gln
465 470 475 480
Ser Leu Ala Phe Ile Arg Arg Ser Asp Glu Leu Leu His Ser
485 490
<210> 14
<211> 501
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 14
Met Ala Thr Thr Ala Met Arg Met Ile Ile Ser Ile Ile Phe Ile Ser
1 5 10 15
Thr Tyr Val Thr His Ile Thr Leu Cys Gln Asn Ile Thr Glu Glu Phe
20 25 30
Tyr Gln Ser Thr Cys Ser Ala Val Ser Arg Gly Tyr Leu Ser Ala Leu
35 40 45
Arg Thr Gly Trp Tyr Thr Ser Val Val Thr Ile Glu Leu Ser Lys Ile
50 55 60
Gln Lys Asn Val Cys Asn Ser Thr Asp Ser Asn Val Lys Leu Ile Lys
65 70 75 80
Gln Glu Leu Glu Arg Tyr Asn Asn Ala Val Val Glu Leu Gln Ser Leu
85 90 95
Met Gln Asn Glu Pro Ala Cys Ser Ser Arg Ala Lys Arg Gly Ile Pro
100 105 110
Glu Ser Gly Ser Ser Gly Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu
115 120 125
Cys Ile Gly Ser Ala Ile Ala Ser Gly Val Ala Val Ser Lys Val Cys
130 135 140
His Leu Glu Gly Glu Val Asn Lys Ile Lys Asn Ala Leu Leu Ser Thr
145 150 155 160
Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val Ser Leu Leu Thr Ser
165 170 175
Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys Glu Leu Leu Pro Lys
180 185 190
Val Asn Asn His Asp Cys Lys Ile Ser Asn Ile Ala Thr Val Ile Glu
195 200 205
Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile Ala Arg Glu Phe Ser
210 215 220
Val Asn Ala Gly Ile Thr Thr Pro Leu Ser Thr Tyr Met Leu Thr Asn
225 230 235 240
Ser Glu Leu Leu Ser Leu Ile Cys Asp Met Pro Ile Thr Asn Asp Gln
245 250 255
Lys Lys Leu Met Ser Ser Asn Val Gln Ile Val Arg Gln Gln Ser Tyr
260 265 270
Ser Phe Met Cys Val Val Lys Glu Glu Val Met Ala Tyr Val Val Gln
275 280 285
Leu Pro Ile Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr
290 295 300
Ser Pro Leu Cys Thr Thr Asp Asn Lys Glu Gly Ser Asn Ile Cys Leu
305 310 315 320
Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser
325 330 335
Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn Arg Val Phe
340 345 350
Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Thr Asp Val Asn Leu Cys
355 360 365
Asn Thr Asp Ile Phe Asn Ala Lys Tyr Asp Cys Lys Ile Met Thr Ser
370 375 380
Lys Thr Asp Ile Ser Cys Ser Val Ile Thr Ser Ile Gly Ala Ile Val
385 390 395 400
Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly
405 410 415
Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Arg Gly
420 425 430
Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Leu
435 440 445
Glu Gly Lys Ala Leu Tyr Ile Lys Gly Glu Pro Ile Ile Asn Tyr Tyr
450 455 460
Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ala Gln
465 470 475 480
Val Asn Ala Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Arg Ser Asp
485 490 495
Glu Leu Leu His Ser
500
<210> 15
<211> 494
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 15
Met Ala Thr Thr Ala Met Arg Met Ile Ile Ser Ile Ile Phe Ile Ser
1 5 10 15
Thr Tyr Val Thr His Ile Thr Leu Cys Gln Asn Ile Thr Glu Glu Phe
20 25 30
Tyr Gln Ser Thr Cys Ser Ala Val Ser Arg Gly Tyr Leu Ser Ala Leu
35 40 45
Arg Thr Gly Trp Tyr Thr Ser Val Val Thr Ile Glu Leu Ser Lys Ile
50 55 60
Gln Lys Asn Val Cys Asn Ser Thr Asp Ser Asn Val Lys Leu Ile Lys
65 70 75 80
Gln Glu Leu Glu Arg Tyr Asn Asn Ala Val Val Glu Leu Gln Ser Leu
85 90 95
Met Gln Asn Glu Pro Ala Cys Ser Ser Arg Ala Lys Ser Gly Ser Ser
100 105 110
Gly Ser Ser Gly Leu Gly Phe Leu Leu Cys Ile Gly Ser Ala Ile Ala
115 120 125
Ser Gly Val Ala Val Ser Lys Val Cys His Leu Glu Gly Glu Val Asn
130 135 140
Lys Ile Lys Asn Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser Leu
145 150 155 160
Ser Asn Gly Val Ser Leu Leu Thr Ser Lys Val Leu Asp Leu Lys Asn
165 170 175
Tyr Ile Asp Lys Glu Leu Leu Pro Lys Val Asn Asn His Asp Cys Lys
180 185 190
Ile Ser Asn Ile Ala Thr Val Ile Glu Phe Gln Gln Lys Asn Asn Arg
195 200 205
Leu Leu Glu Ile Ala Arg Glu Phe Ser Val Asn Ala Gly Ile Thr Thr
210 215 220
Pro Leu Ser Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile
225 230 235 240
Cys Asp Met Pro Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Ser Asn
245 250 255
Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Phe Met Cys Val Val Lys
260 265 270
Glu Glu Val Met Ala Tyr Val Val Gln Leu Pro Ile Tyr Gly Val Ile
275 280 285
Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr Asp
290 295 300
Asn Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp
305 310 315 320
Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu Thr
325 330 335
Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser Leu
340 345 350
Thr Leu Pro Thr Asp Val Asn Leu Cys Asn Thr Asp Ile Phe Asn Ala
355 360 365
Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr Asp Ile Ser Cys Ser
370 375 380
Val Ile Thr Ser Ile Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys
385 390 395 400
Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn
405 410 415
Gly Cys Asp Tyr Val Ser Asn Arg Gly Val Asp Thr Val Ser Val Gly
420 425 430
Asn Thr Leu Tyr Tyr Val Asn Lys Leu Glu Gly Lys Ala Leu Tyr Ile
435 440 445
Lys Gly Glu Pro Ile Ile Asn Tyr Tyr Asp Pro Leu Val Phe Pro Ser
450 455 460
Asp Glu Phe Asp Ala Ser Ile Ala Gln Val Asn Ala Lys Ile Asn Gln
465 470 475 480
Ser Leu Ala Phe Ile Arg Arg Ser Asp Glu Leu Leu His Ser
485 490
<210> 16
<211> 504
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 16
Met Ala Ala Thr Ala Met Arg Met Ile Ile Ser Ile Ile Phe Ile Ser
1 5 10 15
Thr Tyr Met Thr His Ile Thr Leu Cys Gln Asn Ile Thr Glu Glu Phe
20 25 30
Tyr Gln Ser Thr Cys Ser Ala Val Ser Arg Gly Tyr Leu Ser Ala Leu
35 40 45
Arg Thr Gly Trp Tyr Thr Ser Val Val Thr Ile Glu Leu Ser Lys Ile
50 55 60
Gln Lys Asn Val Cys Lys Ser Thr Asp Ser Lys Val Lys Leu Ile Lys
65 70 75 80
Gln Glu Leu Glu Arg Tyr Asn Asn Ala Val Ile Glu Leu Gln Ser Leu
85 90 95
Met Gln Asn Glu Pro Ala Cys Phe Ser Arg Ala Lys Arg Gly Ile Pro
100 105 110
Glu Ser Gly Ser Ser Gly Ser Ser Gly Arg Lys Arg Arg Phe Leu Gly
115 120 125
Phe Leu Leu Cys Ile Gly Ser Ala Ile Ala Ser Gly Val Ala Val Ser
130 135 140
Lys Val Leu Cys Leu Glu Gly Glu Val Asn Lys Ile Lys Asn Ala Leu
145 150 155 160
Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val Ser Leu
165 170 175
Leu Thr Ser Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys Glu Leu
180 185 190
Leu Pro Lys Val Asn Asn His Asp Cys Arg Ile Ser Asn Ile Glu Thr
195 200 205
Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile Ala Arg
210 215 220
Glu Phe Ser Val Asn Ala Gly Ile Thr Thr Pro Leu Ser Thr Tyr Met
225 230 235 240
Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Cys Asp Met Pro Ile Thr
245 250 255
Asn Asp Gln Lys Lys Leu Met Ser Ser Asn Val Gln Ile Val Arg Gln
260 265 270
Gln Ser Tyr Ser Phe Met Leu Cys Val Lys Glu Glu Val Ile Ala Tyr
275 280 285
Val Val Gln Leu Pro Ile Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys
290 295 300
Leu His Thr Ser Pro Leu Cys Thr Thr Asp Asn Lys Glu Gly Ser Asn
305 310 315 320
Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly
325 330 335
Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn
340 345 350
Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Thr Asp Val
355 360 365
Asn Leu Cys Asn Thr Asp Ile Phe Asn Thr Lys Tyr Asp Cys Lys Ile
370 375 380
Met Thr Ser Lys Thr Asp Ile Ser Cys Ser Val Ile Thr Ser Ile Gly
385 390 395 400
Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys
405 410 415
Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser
420 425 430
Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val
435 440 445
Asn Lys Leu Glu Gly Lys Ala Leu Tyr Ile Lys Gly Glu Pro Ile Ile
450 455 460
Asn Tyr Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser
465 470 475 480
Ile Ala Gln Val Asn Ala Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg
485 490 495
Arg Ser Asp Glu Leu Leu His Ser
500
<210> 17
<211> 511
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 17
Met Ala Ala Thr Ala Met Arg Met Ile Ile Ser Ile Ile Phe Ile Ser
1 5 10 15
Thr Tyr Met Thr His Ile Thr Leu Cys Gln Asn Ile Thr Glu Glu Phe
20 25 30
Tyr Gln Ser Thr Cys Ser Ala Val Ser Arg Gly Tyr Leu Ser Ala Leu
35 40 45
Arg Thr Gly Trp Tyr Thr Ser Val Val Thr Ile Glu Leu Ser Lys Ile
50 55 60
Gln Lys Asn Val Cys Lys Ser Thr Asp Ser Lys Val Lys Leu Ile Lys
65 70 75 80
Gln Glu Leu Glu Arg Tyr Asn Asn Ala Val Ile Glu Leu Gln Ser Leu
85 90 95
Met Gln Asn Glu Pro Ala Cys Phe Ser Arg Ala Lys Arg Gly Ile Pro
100 105 110
Glu Arg Gly Ile Pro Glu Ser Gly Ser Ser Gly Arg Lys Arg Arg Phe
115 120 125
Arg Lys Arg Arg Phe Leu Gly Phe Leu Leu Cys Ile Gly Ser Ala Ile
130 135 140
Ala Ser Gly Val Ala Val Ser Lys Val Cys His Leu Glu Gly Glu Val
145 150 155 160
Asn Lys Ile Lys Asn Ala Leu Leu Ser Thr Asn Lys Ala Val Val Ser
165 170 175
Leu Ser Asn Gly Val Ser Leu Leu Thr Ser Lys Val Leu Asp Leu Lys
180 185 190
Asn Tyr Ile Asp Lys Glu Leu Leu Pro Lys Val Asn Asn His Asp Cys
195 200 205
Arg Ile Ser Asn Ile Glu Thr Val Ile Glu Phe Gln Gln Lys Asn Asn
210 215 220
Arg Leu Leu Glu Ile Ala Arg Glu Phe Ser Val Asn Ala Gly Ile Thr
225 230 235 240
Thr Pro Leu Ser Thr Tyr Met Leu Thr Asn Ser Glu Leu Leu Ser Leu
245 250 255
Ile Cys Asp Met Pro Ile Thr Asn Asp Gln Lys Lys Leu Met Ser Ser
260 265 270
Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Phe Met Cys Val Val
275 280 285
Lys Glu Glu Val Ile Ala Tyr Val Val Gln Leu Pro Ile Tyr Gly Val
290 295 300
Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser Pro Leu Cys Thr Thr
305 310 315 320
Asp Asn Lys Glu Gly Ser Asn Ile Cys Leu Thr Arg Thr Asp Arg Gly
325 330 335
Trp Tyr Cys Asp Asn Ala Gly Ser Val Ser Phe Phe Pro Gln Ala Glu
340 345 350
Thr Cys Lys Val Gln Ser Asn Arg Val Phe Cys Asp Thr Met Asn Ser
355 360 365
Leu Thr Leu Pro Thr Asp Val Asn Leu Cys Asn Thr Asp Ile Phe Asn
370 375 380
Thr Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr Asp Ile Ser Cys
385 390 395 400
Ser Val Ile Thr Ser Ile Gly Ala Ile Val Ser Cys Tyr Gly Lys Thr
405 410 415
Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile Lys Thr Phe Ser
420 425 430
Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp Thr Val Ser Val
435 440 445
Gly Asn Thr Leu Tyr Tyr Val Asn Lys Leu Glu Gly Lys Ala Leu Tyr
450 455 460
Ile Lys Gly Glu Pro Ile Ile Asn Tyr Tyr Asp Pro Leu Val Phe Pro
465 470 475 480
Ser Asp Glu Phe Asp Ala Ser Ile Ala Gln Val Asn Ala Lys Ile Asn
485 490 495
Gln Ser Leu Ala Phe Ile Arg Arg Ser Asp Glu Leu Leu His Ser
500 505 510
<210> 18
<211> 504
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 18
Met Ala Ala Thr Ala Met Arg Met Ile Ile Ser Ile Ile Phe Ile Ser
1 5 10 15
Thr Tyr Met Thr His Ile Thr Leu Cys Gln Asn Ile Thr Glu Glu Phe
20 25 30
Tyr Gln Ser Thr Cys Ser Ala Val Ser Arg Gly Tyr Leu Ser Ala Leu
35 40 45
Arg Thr Gly Trp Tyr Thr Ser Val Val Thr Ile Glu Leu Ser Lys Ile
50 55 60
Gln Lys Asn Val Cys Lys Ser Thr Asp Ser Lys Val Lys Leu Ile Lys
65 70 75 80
Gln Glu Leu Glu Arg Tyr Asn Asn Ala Val Ile Glu Leu Gln Ser Leu
85 90 95
Met Gln Asn Glu Pro Ala Cys Phe Ser Arg Ala Lys Arg Gly Ile Pro
100 105 110
Glu Ser Gly Ser Ser Gly Ser Ser Gly Arg Lys Arg Arg Phe Leu Gly
115 120 125
Phe Leu Leu Cys Ile Gly Ser Ala Ile Ala Ser Gly Val Ala Val Ser
130 135 140
Lys Val Cys His Leu Glu Gly Glu Val Asn Lys Ile Lys Asn Ala Leu
145 150 155 160
Leu Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val Ser Leu
165 170 175
Leu Thr Ser Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys Glu Leu
180 185 190
Leu Pro Lys Val Asn Asn His Asp Cys Arg Ile Ser Asn Ile Glu Thr
195 200 205
Val Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile Ala Arg
210 215 220
Glu Phe Ser Val Asn Ala Gly Ile Thr Thr Pro Leu Ser Thr Tyr Met
225 230 235 240
Leu Thr Asn Ser Glu Leu Leu Ser Leu Ile Cys Asp Met Pro Ile Thr
245 250 255
Asn Asp Gln Lys Lys Leu Met Ser Ser Asn Val Gln Ile Val Arg Gln
260 265 270
Gln Ser Tyr Ser Phe Met Cys Val Val Lys Glu Glu Val Ile Ala Tyr
275 280 285
Val Val Gln Leu Pro Ile Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys
290 295 300
Leu His Thr Ser Pro Leu Cys Thr Thr Asp Asn Lys Glu Gly Ser Asn
305 310 315 320
Ile Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly
325 330 335
Ser Val Ser Phe Phe Pro Gln Ala Glu Thr Cys Lys Val Gln Ser Asn
340 345 350
Arg Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Thr Asp Val
355 360 365
Asn Leu Cys Asn Thr Asp Ile Phe Asn Thr Lys Tyr Asp Cys Lys Ile
370 375 380
Met Thr Ser Lys Thr Asp Ile Ser Cys Ser Val Ile Thr Ser Ile Gly
385 390 395 400
Ala Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys
405 410 415
Asn Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser
420 425 430
Asn Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val
435 440 445
Asn Lys Leu Glu Gly Lys Ala Leu Tyr Ile Lys Gly Glu Pro Ile Ile
450 455 460
Asn Tyr Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser
465 470 475 480
Ile Ala Gln Val Asn Ala Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg
485 490 495
Arg Ser Asp Glu Leu Leu His Ser
500
<210> 19
<211> 517
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 19
Met Ala Leu Ser Lys Val Lys Leu Asn Asp Thr Phe Asn Lys Asp Gln
1 5 10 15
Leu Leu Ser Thr Ser Lys Tyr Thr Ile Gln Arg Ser Thr Gly Asp Asn
20 25 30
Ile Asp Ile Pro Asn Tyr Asp Val Gln Lys His Leu Asn Lys Leu Cys
35 40 45
Gly Met Leu Leu Ile Thr Glu Asp Ala Asn His Lys Phe Thr Gly Leu
50 55 60
Ile Gly Met Leu Tyr Ala Met Ser Arg Leu Gly Arg Glu Asp Thr Leu
65 70 75 80
Lys Ile Leu Lys Asp Ala Gly Tyr Gln Val Arg Ala Asn Gly Val Asp
85 90 95
Val Ile Thr His Arg Gln Cys Val Asn Gly Lys Ser Gly Ser Ser Gly
100 105 110
Ser Ser Gly Gln Gly Asn Ile Glu Cys Glu Ser Arg Lys Ser Tyr Lys
115 120 125
Lys Met Leu Lys Glu Met Gly Glu Val Ala Cys Glu Tyr Arg His Asp
130 135 140
Phe Pro Asp Cys Gly Met Ile Val Leu Cys Val Ala Ala Leu Val Ile
145 150 155 160
Thr Lys Leu Leu Ala Gly Asp Arg Ser Gly Leu Thr Ala Val Ile Arg
165 170 175
Arg Ala Asn Asn Val Leu Arg Asn Glu Met Lys Arg Tyr Lys Gly Leu
180 185 190
Ile Pro Lys Asp Ile Ala Asn Ser Phe Tyr Glu Val Phe Glu Lys Tyr
195 200 205
Pro His Tyr Ile Asp Val Phe Val His Phe Gly Ile Ala Gln Ser Ser
210 215 220
Thr Arg Gly Gly Ser Arg Val Glu Gly Ile Phe Ala Cys Leu Phe Met
225 230 235 240
Asn Ala Tyr Gly Ala Gly Gln Val Met Leu Arg Trp Gly Val Leu Ala
245 250 255
Lys Ser Val Lys Asn Phe Met Leu Cys His Ala Ser Val Gln Ala Glu
260 265 270
Met Glu Gln Val Val Glu Val Tyr Glu Tyr Ala Gln Lys Leu Gly Gly
275 280 285
Glu Ala Gly Phe Tyr His Ile Leu Asn Asn Pro Lys Ala Ser Leu Leu
290 295 300
Ser Leu Thr Gln Phe Pro Asn Phe Ser Ser Val Val Leu Gly Asn Ala
305 310 315 320
Ala Gly Leu Gly Ile Met Gly Glu Tyr Arg Gly Thr Pro Arg Asn Gln
325 330 335
Asp Leu Tyr Asp Ala Ala Lys Ala Tyr Ala Glu Gln Leu Lys Glu Asn
340 345 350
Gly Val Ile Asn Tyr Ser Val Leu Asp Leu Thr Thr Glu Glu Leu Glu
355 360 365
Ala Ile Lys Asn Gln Leu Asn Pro Lys Asp Asn Asp Val Glu Leu Cys
370 375 380
Asn Thr Asp Ile Phe Asn Thr Lys Tyr Asp Cys Lys Ile Met Thr Ser
385 390 395 400
Lys Thr Asp Ile Ser Cys Ser Val Ile Thr Ser Ile Gly Ala Ile Val
405 410 415
Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly
420 425 430
Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly
435 440 445
Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Leu
450 455 460
Glu Gly Lys Ala Leu Tyr Ile Lys Gly Glu Pro Ile Ile Asn Tyr Tyr
465 470 475 480
Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ala Gln
485 490 495
Val Asn Ala Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Arg Ser Asp
500 505 510
Glu Leu Leu His Ser
515
<210> 20
<211> 514
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 20
Met Ala Leu Ser Lys Val Lys Leu Asn Asp Thr Phe Asn Lys Asp Gln
1 5 10 15
Leu Leu Ser Thr Ser Lys Tyr Thr Ile Gln Arg Ser Thr Gly Asp Asn
20 25 30
Ile Asp Ile Pro Asn Tyr Asp Val Gln Lys His Leu Asn Lys Leu Cys
35 40 45
Gly Met Leu Leu Ile Thr Glu Asp Ala Asn His Lys Phe Thr Gly Leu
50 55 60
Ile Gly Met Leu Tyr Ala Met Ser Arg Leu Gly Arg Glu Asp Thr Leu
65 70 75 80
Lys Ile Leu Lys Asp Ala Gly Tyr Gln Val Arg Ala Asn Gly Val Asp
85 90 95
Val Ile Thr His Arg Gln Cys Val Asn Gly Lys Ser Gly Ser Ser Gly
100 105 110
Gln Gly Asn Ile Glu Cys Glu Ser Arg Lys Ser Tyr Lys Lys Met Leu
115 120 125
Lys Glu Met Gly Glu Val Ala Cys Glu Tyr Arg His Asp Phe Pro Asp
130 135 140
Cys Gly Met Ile Val Leu Cys Val Ala Ala Leu Val Ile Thr Lys Leu
145 150 155 160
Leu Ala Gly Asp Arg Ser Gly Leu Thr Ala Val Ile Arg Arg Ala Asn
165 170 175
Asn Val Leu Arg Asn Glu Met Lys Arg Tyr Lys Gly Leu Ile Pro Lys
180 185 190
Asp Ile Ala Asn Ser Phe Tyr Glu Val Phe Glu Lys Tyr Pro His Tyr
195 200 205
Ile Asp Val Phe Val His Phe Gly Ile Ala Gln Ser Ser Thr Arg Gly
210 215 220
Gly Ser Arg Val Glu Gly Ile Phe Ala Cys Leu Phe Met Asn Ala Tyr
225 230 235 240
Gly Ala Gly Gln Val Met Leu Arg Trp Gly Val Leu Ala Lys Ser Val
245 250 255
Lys Asn Phe Met Leu Cys His Ala Ser Val Gln Ala Glu Met Glu Gln
260 265 270
Val Val Glu Val Tyr Glu Tyr Ala Gln Lys Leu Gly Gly Glu Ala Gly
275 280 285
Phe Tyr His Ile Leu Asn Asn Pro Lys Ala Ser Leu Leu Ser Leu Thr
290 295 300
Gln Phe Pro Asn Phe Ser Ser Val Val Leu Gly Asn Ala Ala Gly Leu
305 310 315 320
Gly Ile Met Gly Glu Tyr Arg Gly Thr Pro Arg Asn Gln Asp Leu Tyr
325 330 335
Asp Ala Ala Lys Ala Tyr Ala Glu Gln Leu Lys Glu Asn Gly Val Ile
340 345 350
Asn Tyr Ser Val Leu Asp Leu Thr Thr Glu Glu Leu Glu Ala Ile Lys
355 360 365
Asn Gln Leu Asn Pro Lys Asp Asn Asp Val Glu Leu Cys Asn Thr Asp
370 375 380
Ile Phe Asn Thr Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr Asp
385 390 395 400
Ile Ser Cys Ser Val Ile Thr Ser Ile Gly Ala Ile Val Ser Cys Tyr
405 410 415
Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile Lys
420 425 430
Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly Val Asp Thr
435 440 445
Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Leu Glu Gly Lys
450 455 460
Ala Leu Tyr Ile Lys Gly Glu Pro Ile Ile Asn Tyr Tyr Asp Pro Leu
465 470 475 480
Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ala Gln Val Asn Ala
485 490 495
Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Arg Ser Asp Glu Leu Leu
500 505 510
His Ser
<210> 21
<211> 517
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 21
Met Ala Leu Ser Lys Val Lys Leu Asn Asp Thr Phe Asn Lys Asp Gln
1 5 10 15
Leu Leu Ser Thr Ser Lys Tyr Thr Ile Gln Arg Ser Thr Gly Asp Asn
20 25 30
Ile Asp Ile Pro Asn Tyr Asp Val Gln Lys His Leu Asn Lys Leu Cys
35 40 45
Gly Met Leu Leu Ile Thr Glu Asp Ala Asn His Lys Phe Thr Gly Leu
50 55 60
Ile Gly Met Leu Tyr Ala Met Ser Arg Leu Gly Arg Glu Asp Thr Leu
65 70 75 80
Lys Ile Leu Lys Asp Ala Gly Tyr Gln Val Arg Ala Asn Gly Val Asp
85 90 95
Val Ile Thr His Arg Gln Cys Val Asn Gly Lys Ser Gly Ser Ser Gly
100 105 110
Ser Ser Gly Gln Gly Asn Ile Glu Cys Glu Ser Arg Lys Ser Tyr Lys
115 120 125
Lys Met Leu Lys Glu Met Gly Glu Val Ala Cys Glu Tyr Arg His Asp
130 135 140
Phe Pro Asp Cys Gly Met Ile Val Leu Cys Val Ala Ala Leu Val Ile
145 150 155 160
Thr Lys Leu Leu Ala Gly Asp Arg Ser Gly Leu Thr Ala Val Ile Arg
165 170 175
Arg Ala Asn Asn Val Leu Arg Asn Glu Met Lys Arg Tyr Lys Gly Leu
180 185 190
Ile Pro Lys Asp Ile Ala Asn Ser Phe Tyr Glu Val Phe Glu Lys Tyr
195 200 205
Pro His Tyr Ile Asp Val Phe Val His Phe Gly Ile Ala Gln Ser Ser
210 215 220
Thr Arg Gly Gly Ser Arg Val Glu Gly Ile Phe Ala Cys Leu Phe Met
225 230 235 240
Asn Ala Tyr Gly Ala Gly Gln Val Met Leu Arg Trp Gly Val Leu Ala
245 250 255
Lys Ser Val Lys Asn Phe Met Leu Cys His Ala Ser Val Gln Ala Glu
260 265 270
Met Glu Gln Val Val Glu Val Tyr Glu Tyr Ala Gln Lys Leu Gly Gly
275 280 285
Glu Ala Gly Phe Tyr His Ile Leu Asn Asn Pro Lys Ala Ser Leu Leu
290 295 300
Ser Leu Thr Gln Phe Pro Asn Phe Ser Ser Val Val Leu Gly Asn Ala
305 310 315 320
Ala Gly Leu Gly Ile Met Gly Glu Tyr Arg Gly Thr Pro Arg Asn Gln
325 330 335
Asp Leu Tyr Asp Ala Ala Lys Ala Tyr Ala Glu Gln Leu Lys Glu Asn
340 345 350
Gly Val Ile Asn Tyr Ser Val Leu Asp Leu Thr Thr Glu Glu Leu Glu
355 360 365
Ala Ile Lys Asn Gln Leu Asn Pro Lys Asp Asn Asp Val Glu Leu Cys
370 375 380
Asn Thr Asp Ile Phe Asn Thr Lys Tyr Asp Cys Lys Ile Met Thr Ser
385 390 395 400
Lys Thr Asp Ile Ser Cys Ser Val Ile Thr Ser Ile Gly Ala Ile Val
405 410 415
Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly
420 425 430
Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly
435 440 445
Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn Lys Leu
450 455 460
Glu Gly Lys Ala Leu Tyr Ile Lys Gly Glu Pro Ile Ile Asn Tyr Tyr
465 470 475 480
Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile Ala Gln
485 490 495
Val Asn Ala Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Arg Ser Asp
500 505 510
Glu Leu Leu His Ser
515
<210> 22
<211> 1482
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
atggccacca ccgccatgag gatgatcatc agcatcatct tcatcagcac ctacgtgacc 60
cacatcaccc tgtgccagaa catcaccgag gagttctacc agagcacctg cagcgccgtg 120
agcaggggct acctgagcgc cctgaggacc ggctggtaca ccagcgtggt gaccatcgag 180
ctgagcaaga tccagaagaa cgtgtgcaag agcaccgaca gcaaggtgaa gctgatcaag 240
caggagctgg agaggtacaa caacgccgtg gtggagctgc agagcctgat gcagaacgag 300
cccgcctgct tcagcagggc caagagcggc agcagcggca gcagcggcct gggcttcctg 360
ctgtgcatcg gcagcgccgt ggccagcggc gtggccgtga gcaaggtgct gtgcctggag 420
ggcgaggtga acaagatcaa gaacgccctg ctgagcacca acaaggccgt ggtgagcctg 480
agcaacggcg tgagcctgct gaccagcaag gtgctggacc tgaagaacta catcgacaag 540
gagctgctgc cccaggtgaa caaccacgac tgcaggatca gcaacatcga gaccgtgatc 600
gagttccagc agaagaacaa caggctgctg gagatcgcca gggagttcag cgtgaacgcc 660
ggcatcacca cccccctgag cacctacatg ctgaccaaca gcgagctgct gagcctgatc 720
tgcgacatgc ccatcaccaa cgaccagaag aagctgatga gcagcaacgt gcagatcgtg 780
aggcagcaga gctacagctt catgagctgc gtgaaggagg aggtgatcgc ctacgtggtg 840
cagctgccca tctacggcgt gatcgacacc ccctgctgga agctgcacac cagccccctg 900
tgcaccaccg acaacaagga gggcagcaac atctgcctga ccaggaccga caggggctgg 960
tactgcgaca acgccggcag cgtgagcttc ttcccccaga ccgagacctg caaggtgcag 1020
agcaacaggg tgttctgcga caccatgaac agcctgaccc tgcccaccga cgtgaacctg 1080
tgcaacaccg acatcttcaa caccaagtac gactgcaaga tcatgaccag caagaccgac 1140
atcagctgca gcgtgatcac cagcatcggc gccatcgtga gctgctacgg caagaccaag 1200
tgcaccgcca gcaacaagaa caggggcatc atcaagacct tcagcaacgg ctgcgactac 1260
gtgagcaaca agggcgtgga caccgtgagc gtgggcaaca ccctgtacta cgtgaacaag 1320
ctggagggca aggccctgta catcaagggc gagcccatca tcaactacta cgaccccctg 1380
gtgttcccca gcgacgagtt cgacgccagc atcgcccagg tgaacgccaa gatcaaccag 1440
agcctggcct tcatcaggag gagcgacgag ctgctgcaca gc 1482
<210> 23
<211> 1503
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
atggccacca ccgccatgag gatgatcatc agcatcatct tcatcagcac ctacgtgacc 60
cacatcaccc tgtgccagaa catcaccgag gagttctacc agagcacctg cagcgccgtg 120
agcaggggct acctgagcgc cctgaggacc ggctggtaca ccagcgtggt gaccatcgag 180
ctgagcaaga tccagaagaa cgtgtgcaag agcaccgaca gcaaggtgaa gctgatcaag 240
caggagctgg agaggtacaa caacgccgtg gtggagctgc agagcctgat gcagaacgag 300
cccgcctgct tcagcagggc caagaggggc atccccgaga gcggcagcag cggcaagagg 360
aagaggaggt tcctgggctt cctgctgtgc atcggcagcg ccgtggccag cggcgtggcc 420
gtgagcaagg tgtgccacct ggagggcgag gtgaacaaga tcaagaacgc cctgctgagc 480
accaacaagg ccgtggtgag cctgagcaac ggcgtgagcc tgctgaccag caaggtgctg 540
gacctgaaga actacatcga caaggagctg ctgccccagg tgaacaacca cgactgcagg 600
atcagcaaca tcgagaccgt gatcgagttc cagcagaaga acaacaggct gctggagatc 660
gccagggagt tcagcgtgaa cgccggcatc accacccccc tgagcaccta catgctgacc 720
aacagcgagc tgctgagcct gatctgcgac atgcccatca ccaacgacca gaagaagctg 780
atgagcagca acgtgcagat cgtgaggcag cagagctaca gcttcatgtg cgtggtgaag 840
gaggaggtga tcgcctacgt ggtgcagctg cccatctacg gcgtgatcga caccccctgc 900
tggaagctgc acaccagccc cctgtgcacc accgacaaca aggagggcag caacatctgc 960
ctgaccagga ccgacagggg ctggtactgc gacaacgccg gcagcgtgag cttcttcccc 1020
cagaccgaga cctgcaaggt gcagagcaac agggtgttct gcgacaccat gaacagcctg 1080
accctgccca ccgacgtgaa cctgtgcaac accgacatct tcaacaccaa gtacgactgc 1140
aagatcatga ccagcaagac cgacatcagc tgcagcgtga tcaccagcat cggcgccatc 1200
gtgagctgct acggcaagac caagtgcacc gccagcaaca agaacagggg catcatcaag 1260
accttcagca acggctgcga ctacgtgagc aacaagggcg tggacaccgt gagcgtgggc 1320
aacaccctgt actacgtgaa caagctggag ggcaaggccc tgtacatcaa gggcgagccc 1380
atcatcaact actacgaccc cctggtgttc cccagcgacg agttcgacgc cagcatcgcc 1440
caggtgaacg ccaagatcaa ccagagcctg gccttcatca ggaggagcga cgagctgctg 1500
cac 1503
<210> 24
<211> 1512
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
atggccacca ccgccatgag gatgatcatc agcatcatct tcatcagcac ctacgtgacc 60
cacatcaccc tgtgccagaa catcaccgag gagttctacc agagcacctg cagcgccgtg 120
agcaggggct acctgagcgc cctgaggacc ggctggtaca ccagcgtggt gaccatcgag 180
ctgagcaaga tccagaagaa cgtgtgcaag agcaccgaca gcaaggtgaa gctgatcaag 240
caggagctgg agaggtacaa caacgccgtg gtggagctgc agagcctgat gcagaacgag 300
cccgcctgct tcagcagggc caagaggggc atccccgaga gcggcagcag cggcagcagc 360
ggcaagagga agaggaggtt cctgggcttc ctgctgtgca tcggcagcgc cgtggccagc 420
ggcgtggccg tgagcaaggt gtgccacctg gagggcgagg tgaacaagat caagaacgcc 480
ctgctgagca ccaacaaggc cgtggtgagc ctgagcaacg gcgtgagcct gctgaccagc 540
aaggtgctgg acctgaagaa ctacatcgac aaggagctgc tgccccaggt gaacaaccac 600
gactgcagga tcagcaacat cgagaccgtg atcgagttcc agcagaagaa caacaggctg 660
ctggagatcg ccagggagtt cagcgtgaac gccggcatca ccacccccct gagcacctac 720
atgctgacca acagcgagct gctgagcctg atctgcgaca tgcccatcac caacgaccag 780
aagaagctga tgagcagcaa cgtgcagatc gtgaggcagc agagctacag cttcatgtgc 840
gtggtgaagg aggaggtgat cgcctacgtg gtgcagctgc ccatctacgg cgtgatcgac 900
accccctgct ggaagctgca caccagcccc ctgtgcacca ccgacaacaa ggagggcagc 960
aacatctgcc tgaccaggac cgacaggggc tggtactgcg acaacgccgg cagcgtgagc 1020
ttcttccccc agaccgagac ctgcaaggtg cagagcaaca gggtgttctg cgacaccatg 1080
aacagcctga ccctgcccac cgacgtgaac ctgtgcaaca ccgacatctt caacaccaag 1140
tacgactgca agatcatgac cagcaagacc gacatcagct gcagcgtgat caccagcatc 1200
ggcgccatcg tgagctgcta cggcaagacc aagtgcaccg ccagcaacaa gaacaggggc 1260
atcatcaaga ccttcagcaa cggctgcgac tacgtgagca acaagggcgt ggacaccgtg 1320
agcgtgggca acaccctgta ctacgtgaac aagctggagg gcaaggccct gtacatcaag 1380
ggcgagccca tcatcaacta ctacgacccc ctggtgttcc ccagcgacga gttcgacgcc 1440
agcatcgccc aggtgaacgc caagatcaac cagagcctgg ccttcatcag gaggagcgac 1500
gagctgctgc ac 1512
<210> 25
<211> 1482
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
atggccacca ccgccatgag gatgatcatc agcatcatct tcatcagcac ctacgtgacc 60
cacatcaccc tgtgccagaa catcaccgag gagttctacc agagcacctg cagcgccgtg 120
agcaggggct acctgagcgc cctgaggacc ggctggtaca ccagcgtggt gaccatcgag 180
ctgagcaaga tccagaagaa cgtgtgcaac agcaccgaca gcaaggtgaa gctgatcaag 240
caggagctgg agaggtacaa caacgccgtg gtggagctgc agagcctgat gcagaacgag 300
cccgcctgct tcagcagggc caagagcggc agcagcggca gcagcggcct gggcttcctg 360
ctgtgcatcg gcagcgccat cgccagcggc gtggccgtga gcaaggtgct gtgcctggag 420
ggcgaggtga acaagatcaa gaacgccctg ctgagcacca acaaggccgt ggtgagcgcc 480
ggcatcacca cccccctgag cctgagcaac ggcgtgagcc tgctgaccag caaggtgctg 540
gacctgaaga actacatcga caaggagctg ctgcccaagg tgaacaacca cgactgcagg 600
atcagcaaga tcgagaccgt gatcgagttc cagcagaaga acaacaggct gctggagatc 660
gccagggagt tcagcgtgaa cacctacatg ctgaccaaca gcgagctgct gagcctgatc 720
tgcgacatgc ccatcaccaa cgaccagaag aagctgatga gcagcaacgt gcagatcgtg 780
aggcagcaga gctacagctt catgagctgc gtgaaggagg aggtgatcgc ctacgtggtg 840
cagctgccca tctacggcgt gatcgacacc ccctgctgga agctgcacac cagccccctg 900
tgcaccaccg acaacaagga gggcagcaac atctgcctga ccaggaccga caggggctgg 960
tactgcgaca acgccggcag cgtgagcttc ttcccccaga ccgagacctg caaggtgcag 1020
agcaacaggg tgttctgcga caccatgaac agcctgaccc tgcccaccga cgtgaacctg 1080
tgcaacaccg acatcttcaa caccaagtac gactgcaaga tcatgaccag caagaccgac 1140
atcagctgca gcgtgatcac cagcatcggc gccatcgtga gctgctacgg caagaccaag 1200
tgcaccgcca gcaacaagaa caggggcatc atcaagacct tcagcaacgg ctgcgactac 1260
gtgagcaaca agggcgtgga caccgtgagc gtgggcaaca ccctgtacta cgtgaacaag 1320
ctggagggca aggccctgta catcaagggc gagcccatca tcaactacta cgaccccctg 1380
gtgttcccca gcgacgagtt cgacgccagc atcgcccagg tgaacgccaa gatcaaccag 1440
agcctggcct tcatcaggag gagcgacgag ctgctgcaca gc 1482
<210> 26
<211> 1503
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
atggccacca ccgccatgag gatgatcatc agcatcatct tcatcagcac ctacgtgacc 60
cacatcaccc tgtgccagaa catcaccgag gagttctacc agagcacctg cagcgccgtg 120
agcaggggct acctgagcgc cctgaggacc ggctggtaca ccagcgtggt gaccatcgag 180
ctgagcaaga tccagaagaa cgtgtgcaac agcaccgaca gcaaggtgaa gctgatcaag 240
caggagctgg agaggtacaa caacgccgtg gtggagctgc agagcctgat gcagaacgag 300
cccgcctgct tcagcagggc caagaggggc atccccgaga gcggcagcag cggcaggaag 360
aggaggttcc tgggcttcct gctgtgcatc ggcagcgcca tcgccagcgg cgtggccgtg 420
agcaaggtgt gccacctgga gggcgaggtg aacaagatca agaacgccct gctgagcacc 480
aacaaggccg tggtgagcgc cggcatcacc acccccctga gcctgagcaa cggcgtgagc 540
ctgctgacca gcaaggtgct ggacctgaag aactacatcg acaaggagct gctgcccaag 600
gtgaacaacc acgactgcag gatcagcaag atcgagaccg tgatcgagtt ccagcagaag 660
aacaacaggc tgctggagat cgccagggag ttcagcgtga acacctacat gctgaccaac 720
agcgagctgc tgagcctgat ctgcgacatg cccatcacca acgaccagaa gaagctgatg 780
agcagcaacg tgcagatcgt gaggcagcag agctacagct tcatgtgcgt ggtgaaggag 840
gaggtgatcg cctacgtggt gcagctgccc atctacggcg tgatcgacac cccctgctgg 900
aagctgcaca ccagccccct gtgcaccacc gacaacaagg agggcagcaa catctgcctg 960
accaggaccg acaggggctg gtactgcgac aacgccggca gcgtgagctt cttcccccag 1020
accgagacct gcaaggtgca gagcaacagg gtgttctgcg acaccatgaa cagcctgacc 1080
ctgcccaccg acgtgaacct gtgcaacacc gacatcttca acaccaagta cgactgcaag 1140
atcatgacca gcaagaccga catcagctgc agcgtgatca ccagcatcgg cgccatcgtg 1200
agctgctacg gcaagaccaa gtgcaccgcc agcaacaaga acaggggcat catcaagacc 1260
ttcagcaacg gctgcgacta cgtgagcaac aagggcgtgg acaccgtgag cgtgggcaac 1320
accctgtact acgtgaacaa gctggagggc aaggccctgt acatcaaggg cgagcccatc 1380
atcaactact acgaccccct ggtgttcccc agcgacgagt tcgacgccag catcgcccag 1440
gtgaacgcca agatcaacca gagcctggcc ttcatcagga ggagcgacga gctgctgcac 1500
agc 1503
<210> 27
<211> 1482
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
atggccacca ccgccatgag gatgatcatc agcatcatct tcatcagcac ctacgtgacc 60
cacatcaccc tgtgccagaa catcaccgag gagttctacc agagcacctg cagcgccgtg 120
agcaggggct acctgagcgc cctgaggacc ggctggtaca ccagcgtggt gaccatcgag 180
ctgagcaaga tccagaagaa cgtgtgcaac agcaccgaca gcaaggtgaa gctgatcaag 240
caggagctgg agaggtacaa caacgccgtg gtggagctgc agagcctgat gcagaacgag 300
cccgcctgct tcagcagggc caagagcggc agcagcggca gcagcggcct gggcttcctg 360
ctgtgcatcg gcagcgccat cgccagcggc gtggccgtga gcaaggtgtg ccacctggag 420
ggcgaggtga acaagatcaa gaacgccctg ctgagcacca acaaggccgt ggtgagcgcc 480
ggcatcacca cccccctgag cctgagcaac ggcgtgagcc tgctgaccag caaggtgctg 540
gacctgaaga actacatcga caaggagctg ctgcccaagg tgaacaacca cgactgcagg 600
atcagcaaga tcgagaccgt gatcgagttc cagcagaaga acaacaggct gctggagatc 660
gccagggagt tcagcgtgaa cacctacatg ctgaccaaca gcgagctgct gagcctgatc 720
tgcgacatgc ccatcaccaa cgaccagaag aagctgatga gcagcaacgt gcagatcgtg 780
aggcagcaga gctacagctt catgtgcgtg gtgaaggagg aggtgatcgc ctacgtggtg 840
cagctgccca tctacggcgt gatcgacacc ccctgctgga agctgcacac cagccccctg 900
tgcaccaccg acaacaagga gggcagcaac atctgcctga ccaggaccga caggggctgg 960
tactgcgaca acgccggcag cgtgagcttc ttcccccaga ccgagacctg caaggtgcag 1020
agcaacaggg tgttctgcga caccatgaac agcctgaccc tgcccaccga cgtgaacctg 1080
tgcaacaccg acatcttcaa caccaagtac gactgcaaga tcatgaccag caagaccgac 1140
atcagctgca gcgtgatcac cagcatcggc gccatcgtga gctgctacgg caagaccaag 1200
tgcaccgcca gcaacaagaa caggggcatc atcaagacct tcagcaacgg ctgcgactac 1260
gtgagcaaca agggcgtgga caccgtgagc gtgggcaaca ccctgtacta cgtgaacaag 1320
ctggagggca aggccctgta catcaagggc gagcccatca tcaactacta cgaccccctg 1380
gtgttcccca gcgacgagtt cgacgccagc atcgcccagg tgaacgccaa gatcaaccag 1440
agcctggcct tcatcaggag gagcgacgag ctgctgcaca gc 1482
<210> 28
<211> 1482
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
atggccgcca tggccatgag gatgatcatc agcatcatct tcatcagcac ctacatgacc 60
cacatcaccc tgtgccagaa catcaccgag gagttctacc agagcacctg cagcgccgtg 120
agcaggggct acctgagcgc cctgaggacc ggctggtaca ccagcgtggt gaccatcgag 180
ctgagcaaga tccagaagga cgtgtgcaag agcaccgaca gcaaggtgaa gctgatcaag 240
caggagctgg agaggtacaa caacgccgtg accgagctgc agagcctgat gcagaacgtg 300
cccgcctgct tcaacagggc caagagcggc agcagcggca gcagcggcct gggcttcctg 360
ctgtgcatcg gcagcgccat cgccagcggc gtggccgtga gcaaggtgct gtgcctggag 420
ggcgaggtga acaagatcaa gaacgccctg ctgagcacca acaaggccgt ggtgagcctg 480
agcaacggcg tgagcctgct gaccagcaag gtgctggacc tgaagaacta catcgacaag 540
gagctgctgc ccaaggtgaa caaccacgac tgcaggatca gcaacatcga gaccgtgatc 600
gagttccagc agaagaacaa caggctgctg gagatcgcca gggagttcag cgtgaacgcc 660
ggcatcacca cccccctgag cacctacatg ctgaccaaca gcgagctgct gagcctgatc 720
tgcgacatgc ccatcaccaa cgaccagaag aagctgatga gcagcaacgt gcagatcgtg 780
aggcagcaga gctacagctt catgagctgc gtgaaggagg aggtgatcgc ctacgtggtg 840
cagctgccca tctacggcgt gatcgacacc ccctgctgga agctgcacac cagccccctg 900
tgcaccaccg acaacaagga gggcagcaac atctgcctga ccaggaccga caggggctgg 960
tactgcgaca acgccggcag cgtgagcttc ttcccccagg ccgagacctg caaggtgcag 1020
agcaacaggg tgttctgcga caccatgaac agcctgaccc tgcccaccga cgtgaacctg 1080
tgcaacaccg acatcttcaa caccaagtac gactgcaaga tcatgaccag caagaccgac 1140
atcagctgca gcgtgatcac cagcatcggc gccatcgtga gctgctacgg caagaccaag 1200
tgcaccgcca gcaacaagaa caggggcatc atcaagacct tcagcaacgg ctgcgactac 1260
gtgagcaaca agggcgtgga caccgtgagc gtgggcaaca ccctgtacta cgtgaacaag 1320
ctggagggca aggccctgta catcaagggc gagcccatca tcaactacta cgaccccctg 1380
gtgttcccca gcgacgagtt cgacgccagc atcgcccagg tgaacgccaa gatcaaccag 1440
agcctggcct tcatcaggag gagcgacgag ctgctgcaca gc 1482
<210> 29
<211> 1503
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
atggccgcca tggccatgag gatgatcatc agcatcatct tcatcagcac ctacatgacc 60
cacatcaccc tgtgccagaa catcaccgag gagttctacc agagcacctg cagcgccgtg 120
agcaggggct acctgagcgc cctgaggacc ggctggtaca ccagcgtggt gaccatcgag 180
ctgagcaaga tccagaagga cgtgtgcaag agcaccgaca gcaaggtgaa gctgatcaag 240
caggagctgg agaggtacaa caacgccgtg accgagctgc agagcctgat gcagaacgtg 300
cccgcctgct tcaacagggc caagaggggc atccccgaga gcggcagcag cggcaggaag 360
aggaggttcc tgggcttcct gctgtgcatc ggcagcgcca tcgccagcgg cgtggccgtg 420
agcaaggtgt gccacctgga gggcgaggtg aacaagatca agaacgccct gctgagcacc 480
aacaaggccg tggtgagcct gagcaacggc gtgagcctgc tgaccagcaa ggtgctggac 540
ctgaagaact acatcgacaa ggagctgctg cccaaggtga acaaccacga ctgcaggatc 600
agcaacatcg agaccgtgat cgagttccag cagaagaaca acaggctgct ggagatcgcc 660
agggagttca gcgtgaacgc cggcatcacc acccccctga gcacctacat gctgaccaac 720
agcgagctgc tgagcctgat ctgcgacatg cccatcacca acgaccagaa gaagctgatg 780
agcagcaacg tgcagatcgt gaggcagcag agctacagct tcatgtgcgt ggtgaaggag 840
gaggtgatcg cctacgtggt gcagctgccc atctacggcg tgatcgacac cccctgctgg 900
aagctgcaca ccagccccct gtgcaccacc gacaacaagg agggcagcaa catctgcctg 960
accaggaccg acaggggctg gtactgcgac aacgccggca gcgtgagctt cttcccccag 1020
gccgagacct gcaaggtgca gagcaacagg gtgttctgcg acaccatgaa cagcctgacc 1080
ctgcccaccg acgtgaacct gtgcaacacc gacatcttca acaccaagta cgactgcaag 1140
atcatgacca gcaagaccga catcagctgc agcgtgatca ccagcatcgg cgccatcgtg 1200
agctgctacg gcaagaccaa gtgcaccgcc agcaacaaga acaggggcat catcaagacc 1260
ttcagcaacg gctgcgacta cgtgagcaac aagggcgtgg acaccgtgag cgtgggcaac 1320
accctgtact acgtgaacaa gctggagggc aaggccctgt acatcaaggg cgagcccatc 1380
atcaactact acgaccccct ggtgttcccc agcgacgagt tcgacgccag catcgcccag 1440
gtgaacgcca agatcaacca gagcctggcc ttcatcagga ggagcgacga gctgctgcac 1500
agc 1503
<210> 30
<211> 1482
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
atggccgcca tggccatgag gatgatcatc agcatcatct tcatcagcac ctacatgacc 60
cacatcaccc tgtgccagaa catcaccgag gagttctacc agagcacctg cagcgccgtg 120
agcaggggct acctgagcgc cctgaggacc ggctggtaca ccagcgtggt gaccatcgag 180
ctgagcaaga tccagaagga cgtgtgcaag agcaccgaca gcaaggtgaa gctgatcaag 240
caggagctgg agaggtacaa caacgccgtg accgagctgc agagcctgat gcagaacgtg 300
cccgcctgct tcaacagggc caagagcggc agcagcggca gcagcggcct gggcttcctg 360
ctgtgcatcg gcagcgccat cgccagcggc gtggccgtga gcaaggtgtg ccacctggag 420
ggcgaggtga acaagatcaa gaacgccctg ctgagcacca acaaggccgt ggtgagcctg 480
agcaacggcg tgagcctgct gaccagcaag gtgctggacc tgaagaacta catcgacaag 540
gagctgctgc ccaaggtgaa caaccacgac tgcaggatca gcaacatcga gaccgtgatc 600
gagttccagc agaagaacaa caggctgctg gagatcgcca gggagttcag cgtgaacgcc 660
ggcatcacca cccccctgag cacctacatg ctgaccaaca gcgagctgct gagcctgatc 720
tgcgacatgc ccatcaccaa cgaccagaag aagctgatga gcagcaacgt gcagatcgtg 780
aggcagcaga gctacagctt catgtgcctg gtgaaggagg aggtgatcgc ctacgtggtg 840
cagctgccca tctacggcgt gatcgacacc ccctgctgga agctgcacac cagccccctg 900
tgcaccaccg acaacaagga gggcagcaac atctgcctga ccaggaccga caggggctgg 960
tactgcgaca acgccggcag cgtgagcttc ttcccccagg ccgagacctg caaggtgcag 1020
agcaacaggg tgttctgcga caccatgaac agcctgaccc tgcccaccga cgtgaacctg 1080
tgcaacaccg acatcttcaa caccaagtac gactgcaaga tcatgaccag caagaccgac 1140
atcagctgca gcgtgatcac cagcatcggc gccatcgtga gctgctacgg caagaccaag 1200
tgcaccgcca gcaacaagaa caggggcatc atcaagacct tcagcaacgg ctgcgactac 1260
gtgagcaaca agggcgtgga caccgtgagc gtgggcaaca ccctgtacta cgtgaacaag 1320
ctggagggca aggccctgta catcaagggc gagcccatca tcaactacta cgaccccctg 1380
gtgttcccca gcgacgagtt cgacgccagc atcgcccagg tgaacgccaa gatcaaccag 1440
agcctggcct tcatcaggag gagcgacgag ctgctgcaca gc 1482
<210> 31
<211> 1467
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
atgaggatga tcatcagcat catcctgatc agcacctacg tgccccacat caccctgtgc 60
cagaacatca ccgaggagtt ctaccagagc acctgcagcg ccgtgagcag gggctacctg 120
agcgccctga ggaccggctg gtacaccagc gtggtgacca tcgagctgag caagatccag 180
aagaacgtgt gcaacggcac cgacagcaag gtgaagctga tcaagcagga gctggagagg 240
tacaacaacg ccgtggtgga gctgcagagc ctgatgcaga acgagcccac ctgcagcagc 300
agggccaaga gcggcagcag cggcagcagc ggcctgggct tcctgctgtg catcggcagc 360
gccatcgcca gcggcgtggc cgtgagcaag gtgctgtgcc tggagggcga ggtgaacaag 420
atcaagaacg ccctgctgag caccaacaag gccgtggtga gcctgagcaa cggcgtgagc 480
ctgctgacca gcaaggtgct ggacctgaag aactacatcg acaaggagct gctgcccaag 540
gtgaacaacc acgactgcag gatcagcaac atcgccaccg tgatcgagtt ccagcagaag 600
aacaacaggc tgctggagat cgccagggag ttcagcgtga acgccggcat caccaccccc 660
ctgagcacct acatgctgac caacagcgag ctgctgagca tcatctgcga catgcccatc 720
accaacgacc agaagaagct gatgagcagc aacgtgcaga tcgtgaggca gcagagctac 780
agcttcatga gctgcgtgaa ggaggaggtg atcgcctacg tggtgcagct gcccctgtac 840
ggcgtgatcg acaccccctg ctggaagctg cacaccagcc ccctgtgcac caccgacaac 900
gaggagggca gcaacatctg cctgaccagg accgacaggg gctggtactg cgacaacgcc 960
ggcagcgtga gcttcttccc ccaggccgag acctgcaagg tgcagagcaa cagggtgttc 1020
tgcgacacca tgaacagcct gaccctgccc accgacgtga acctgtgcaa caccgacatc 1080
ttcaacgcca agtacgactg caagatcatg accagcaaga ccgacatcag ctgcagcgtg 1140
atcaccagca tcggcgccat cgtgagctgc tacggcaaga ccaagtgcac cgccagcaac 1200
aagaacaggg gcatcatcaa gaccttcagc aacggctgcg actacgtgag caacaagggc 1260
gtggacaccg tgagcgtggg caacaccctg tactacgtga acaagctgga gggcaaggcc 1320
ctgtacatca agggcgagcc catcatcaac tactacaacc ccctggtgtt ccccagcgac 1380
gagttcgacg ccagcatcgc ccaggtgaac gccaagatca accagagcct ggccttcatc 1440
aggaggagcg acgagctgct gcacagc 1467
<210> 32
<211> 1488
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
atgaggatga tcatcagcat catcctgatc agcacctacg tgccccacat caccctgtgc 60
cagaacatca ccgaggagtt ctaccagagc acctgcagcg ccgtgagcag gggctacctg 120
agcgccctga ggaccggctg gtacaccagc gtggtgacca tcgagctgag caagatccag 180
aagaacgtgt gcaacggcac cgacagcaag gtgaagctga tcaagcagga gctggagagg 240
tacaacaacg ccgtggtgga gctgcagagc ctgatgcaga acgagcccac ctgcagcagc 300
agggccaaga ggggcatccc cgagagcggc agcagcggca ggaagaggag gttcctgggc 360
ttcctgctgt gcatcggcag cgccatcgcc agcggcgtgg ccgtgagcaa ggtgtgccac 420
ctggagggcg aggtgaacaa gatcaagaac gccctgctga gcaccaacaa ggccgtggtg 480
agcctgagca acggcgtgag cctgctgacc agcaaggtgc tggacctgaa gaactacatc 540
gacaaggagc tgctgcccaa ggtgaacaac cacgactgca ggatcagcaa catcgccacc 600
gtgatcgagt tccagcagaa gaacaacagg ctgctggaga tcgccaggga gttcagcgtg 660
aacgccggca tcaccacccc cctgagcacc tacatgctga ccaacagcga gctgctgagc 720
atcatctgcg acatgcccat caccaacgac cagaagaagc tgatgagcag caacgtgcag 780
atcgtgaggc agcagagcta cagcttcatg tgcgtggtga aggaggaggt gatcgcctac 840
gtggtgcagc tgcccctgta cggcgtgatc gacaccccct gctggaagct gcacaccagc 900
cccctgtgca ccaccgacaa cgaggagggc agcaacatct gcctgaccag gaccgacagg 960
ggctggtact gcgacaacgc cggcagcgtg agcttcttcc cccaggccga gacctgcaag 1020
gtgcagagca acagggtgtt ctgcgacacc atgaacagcc tgaccctgcc caccgacgtg 1080
aacctgtgca acaccgacat cttcaacgcc aagtacgact gcaagatcat gaccagcaag 1140
accgacatca gctgcagcgt gatcaccagc atcggcgcca tcgtgagctg ctacggcaag 1200
accaagtgca ccgccagcaa caagaacagg ggcatcatca agaccttcag caacggctgc 1260
gactacgtga gcaacaaggg cgtggacacc gtgagcgtgg gcaacaccct gtactacgtg 1320
aacaagctgg agggcaaggc cctgtacatc aagggcgagc ccatcatcaa ctactacaac 1380
cccctggtgt tccccagcga cgagttcgac gccagcatcg cccaggtgaa cgccaagatc 1440
aaccagagcc tggccttcat caggaggagc gacgagctgc tgcacagc 1488
<210> 33
<211> 1467
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
atgaggatga tcatcagcat catcctgatc agcacctacg tgccccacat caccctgtgc 60
cagaacatca ccgaggagtt ctaccagagc acctgcagcg ccgtgagcag gggctacctg 120
agcgccctga ggaccggctg gtacaccagc gtggtgacca tcgagctgag caagatccag 180
aagaacgtgt gcaacggcac cgacagcaag gtgaagctga tcaagcagga gctggagagg 240
tacaacaacg ccgtggtgga gctgcagagc ctgatgcaga acgagcccac ctgcagcagc 300
agggccaaga gcggcagcag cggcagcagc ggcctgggct tcctgctgtg catcggcagc 360
gccatcgcca gcggcgtggc cgtgagcaag gtgtgccacc tggagggcga ggtgaacaag 420
atcaagaacg ccctgctgag caccaacaag gccgtggtga gcctgagcaa cggcgtgagc 480
ctgctgacca gcaaggtgct ggacctgaag aactacatcg acaaggagct gctgcccaag 540
gtgaacaacc acgactgcag gatcagcaac atcgccaccg tgatcgagtt ccagcagaag 600
aacaacaggc tgctggagat cgccagggag ttcagcgtga acgccggcat caccaccccc 660
ctgagcacct acatgctgac caacagcgag ctgctgagca tcatctgcga catgcccatc 720
accaacgacc agaagaagct gatgagcagc aacgtgcaga tcgtgaggca gcagagctac 780
agcttcatgt gcgtggtgaa ggaggaggtg atcgcctacg tggtgcagct gcccctgtac 840
ggcgtgatcg acaccccctg ctggaagctg cacaccagcc ccctgtgcac caccgacaac 900
gaggagggca gcaacatctg cctgaccagg accgacaggg gctggtactg cgacaacgcc 960
ggcagcgtga gcttcttccc ccaggccgag acctgcaagg tgcagagcaa cagggtgttc 1020
tgcgacacca tgaacagcct gaccctgccc accgacgtga acctgtgcaa caccgacatc 1080
ttcaacgcca agtacgactg caagatcatg accagcaaga ccgacatcag ctgcagcgtg 1140
atcaccagca tcggcgccat cgtgagctgc tacggcaaga ccaagtgcac cgccagcaac 1200
aagaacaggg gcatcatcaa gaccttcagc aacggctgcg actacgtgag caacaagggc 1260
gtggacaccg tgagcgtggg caacaccctg tactacgtga acaagctgga gggcaaggcc 1320
ctgtacatca agggcgagcc catcatcaac tactacaacc ccctggtgtt ccccagcgac 1380
gagttcgacg ccagcatcgc ccaggtgaac gccaagatca accagagcct ggccttcatc 1440
aggaggagcg acgagctgct gcacagc 1467
<210> 34
<211> 1482
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 34
atggccacca ccgccatgag gatgatcatc agcatcatct tcatcagcac ctacgtgacc 60
cacatcaccc tgtgccagaa catcaccgag gagttctacc agagcacctg cagcgccgtg 120
agcaggggct acctgagcgc cctgaggacc ggctggtaca ccagcgtggt gaccatcgag 180
ctgagcaaga tccagaagaa cgtgtgcaac agcaccgaca gcaacgtgaa gctgatcaag 240
caggagctgg agaggtacaa caacgccgtg gtggagctgc agagcctgat gcagaacgag 300
cccgcctgca gcagcagggc caagagcggc agcagcggca gcagcggcct gggcttcctg 360
ctgtgcatcg gcagcgccat cgccagcggc gtggccgtga gcaaggtgct gtgcctggag 420
ggcgaggtga acaagatcaa gaacgccctg ctgagcacca acaaggccgt ggtgagcctg 480
agcaacggcg tgagcctgct gaccagcaag gtgctggacc tgaagaacta catcgacaag 540
gagctgctgc ccaaggtgaa caaccacgac tgcaagatca gcaacatcgc caccgtgatc 600
gagttccagc agaagaacaa caggctgctg gagatcgcca gggagttcag cgtgaacgcc 660
ggcatcacca cccccctgag cacctacatg ctgaccaaca gcgagctgct gagcctgatc 720
tgcgacatgc ccatcaccaa cgaccagaag aagctgatga gcagcaacgt gcagatcgtg 780
aggcagcaga gctacagctt catgagctgc gtgaaggagg aggtgatggc ctacgtggtg 840
cagctgccca tctacggcgt gatcgacacc ccctgctgga agctgcacac cagccccctg 900
tgcaccaccg acaacaagga gggcagcaac atctgcctga ccaggaccga caggggctgg 960
tactgcgaca acgccggcag cgtgagcttc ttcccccagg ccgagacctg caaggtgcag 1020
agcaacaggg tgttctgcga caccatgaac agcctgaccc tgcccaccga cgtgaacctg 1080
tgcaacaccg acatcttcaa cgccaagtac gactgcaaga tcatgaccag caagaccgac 1140
atcagctgca gcgtgatcac cagcatcggc gccatcgtga gctgctacgg caagaccaag 1200
tgcaccgcca gcaacaagaa caggggcatc atcaagacct tcagcaacgg ctgcgactac 1260
gtgagcaaca ggggcgtgga caccgtgagc gtgggcaaca ccctgtacta cgtgaacaag 1320
ctggagggca aggccctgta catcaagggc gagcccatca tcaactacta cgaccccctg 1380
gtgttcccca gcgacgagtt cgacgccagc atcgcccagg tgaacgccaa gatcaaccag 1440
agcctggcct tcatcaggag gagcgacgag ctgctgcaca gc 1482
<210> 35
<211> 1503
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 35
atggccacca ccgccatgag gatgatcatc agcatcatct tcatcagcac ctacgtgacc 60
cacatcaccc tgtgccagaa catcaccgag gagttctacc agagcacctg cagcgccgtg 120
agcaggggct acctgagcgc cctgaggacc ggctggtaca ccagcgtggt gaccatcgag 180
ctgagcaaga tccagaagaa cgtgtgcaac agcaccgaca gcaacgtgaa gctgatcaag 240
caggagctgg agaggtacaa caacgccgtg gtggagctgc agagcctgat gcagaacgag 300
cccgcctgca gcagcagggc caagaggggc atccccgaga gcggcagcag cggcaggaag 360
aggaggttcc tgggcttcct gctgtgcatc ggcagcgcca tcgccagcgg cgtggccgtg 420
agcaaggtgt gccacctgga gggcgaggtg aacaagatca agaacgccct gctgagcacc 480
aacaaggccg tggtgagcct gagcaacggc gtgagcctgc tgaccagcaa ggtgctggac 540
ctgaagaact acatcgacaa ggagctgctg cccaaggtga acaaccacga ctgcaagatc 600
agcaacatcg ccaccgtgat cgagttccag cagaagaaca acaggctgct ggagatcgcc 660
agggagttca gcgtgaacgc cggcatcacc acccccctga gcacctacat gctgaccaac 720
agcgagctgc tgagcctgat ctgcgacatg cccatcacca acgaccagaa gaagctgatg 780
agcagcaacg tgcagatcgt gaggcagcag agctacagct tcatgtgcgt ggtgaaggag 840
gaggtgatgg cctacgtggt gcagctgccc atctacggcg tgatcgacac cccctgctgg 900
aagctgcaca ccagccccct gtgcaccacc gacaacaagg agggcagcaa catctgcctg 960
accaggaccg acaggggctg gtactgcgac aacgccggca gcgtgagctt cttcccccag 1020
gccgagacct gcaaggtgca gagcaacagg gtgttctgcg acaccatgaa cagcctgacc 1080
ctgcccaccg acgtgaacct gtgcaacacc gacatcttca acgccaagta cgactgcaag 1140
atcatgacca gcaagaccga catcagctgc agcgtgatca ccagcatcgg cgccatcgtg 1200
agctgctacg gcaagaccaa gtgcaccgcc agcaacaaga acaggggcat catcaagacc 1260
ttcagcaacg gctgcgacta cgtgagcaac aggggcgtgg acaccgtgag cgtgggcaac 1320
accctgtact acgtgaacaa gctggagggc aaggccctgt acatcaaggg cgagcccatc 1380
atcaactact acgaccccct ggtgttcccc agcgacgagt tcgacgccag catcgcccag 1440
gtgaacgcca agatcaacca gagcctggcc ttcatcagga ggagcgacga gctgctgcac 1500
agc 1503
<210> 36
<211> 1482
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 36
atggccacca ccgccatgag gatgatcatc agcatcatct tcatcagcac ctacgtgacc 60
cacatcaccc tgtgccagaa catcaccgag gagttctacc agagcacctg cagcgccgtg 120
agcaggggct acctgagcgc cctgaggacc ggctggtaca ccagcgtggt gaccatcgag 180
ctgagcaaga tccagaagaa cgtgtgcaac agcaccgaca gcaacgtgaa gctgatcaag 240
caggagctgg agaggtacaa caacgccgtg gtggagctgc agagcctgat gcagaacgag 300
cccgcctgca gcagcagggc caagagcggc agcagcggca gcagcggcct gggcttcctg 360
ctgtgcatcg gcagcgccat cgccagcggc gtggccgtga gcaaggtgtg ccacctggag 420
ggcgaggtga acaagatcaa gaacgccctg ctgagcacca acaaggccgt ggtgagcctg 480
agcaacggcg tgagcctgct gaccagcaag gtgctggacc tgaagaacta catcgacaag 540
gagctgctgc ccaaggtgaa caaccacgac tgcaagatca gcaacatcgc caccgtgatc 600
gagttccagc agaagaacaa caggctgctg gagatcgcca gggagttcag cgtgaacgcc 660
ggcatcacca cccccctgag cacctacatg ctgaccaaca gcgagctgct gagcctgatc 720
tgcgacatgc ccatcaccaa cgaccagaag aagctgatga gcagcaacgt gcagatcgtg 780
aggcagcaga gctacagctt catgtgcgtg gtgaaggagg aggtgatggc ctacgtggtg 840
cagctgccca tctacggcgt gatcgacacc ccctgctgga agctgcacac cagccccctg 900
tgcaccaccg acaacaagga gggcagcaac atctgcctga ccaggaccga caggggctgg 960
tactgcgaca acgccggcag cgtgagcttc ttcccccagg ccgagacctg caaggtgcag 1020
agcaacaggg tgttctgcga caccatgaac agcctgaccc tgcccaccga cgtgaacctg 1080
tgcaacaccg acatcttcaa cgccaagtac gactgcaaga tcatgaccag caagaccgac 1140
atcagctgca gcgtgatcac cagcatcggc gccatcgtga gctgctacgg caagaccaag 1200
tgcaccgcca gcaacaagaa caggggcatc atcaagacct tcagcaacgg ctgcgactac 1260
gtgagcaaca ggggcgtgga caccgtgagc gtgggcaaca ccctgtacta cgtgaacaag 1320
ctggagggca aggccctgta catcaagggc gagcccatca tcaactacta cgaccccctg 1380
gtgttcccca gcgacgagtt cgacgccagc atcgcccagg tgaacgccaa gatcaaccag 1440
agcctggcct tcatcaggag gagcgacgag ctgctgcaca gc 1482
<210> 37
<211> 1512
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 37
atggccgcca ccgccatgag gatgatcatc agcatcatct tcatcagcac ctacatgacc 60
cacatcaccc tgtgccagaa catcaccgag gagttctacc agagcacctg cagcgccgtg 120
agcaggggct acctgagcgc cctgaggacc ggctggtaca ccagcgtggt gaccatcgag 180
ctgagcaaga tccagaagaa cgtgtgcaag agcaccgaca gcaaggtgaa gctgatcaag 240
caggagctgg agaggtacaa caacgccgtg atcgagctgc agagcctgat gcagaacgag 300
cccgcctgct tcagcagggc caagaggggc atccccgaga gcggcagcag cggcagcagc 360
ggcaggaaga ggaggttcct gggcttcctg ctgtgcatcg gcagcgccat cgccagcggc 420
gtggccgtga gcaaggtgct gtgcctggag ggcgaggtga acaagatcaa gaacgccctg 480
ctgagcacca acaaggccgt ggtgagcctg agcaacggcg tgagcctgct gaccagcaag 540
gtgctggacc tgaagaacta catcgacaag gagctgctgc ccaaggtgaa caaccacgac 600
tgcaggatca gcaacatcga gaccgtgatc gagttccagc agaagaacaa caggctgctg 660
gagatcgcca gggagttcag cgtgaacgcc ggcatcacca cccccctgag cacctacatg 720
ctgaccaaca gcgagctgct gagcctgatc tgcgacatgc ccatcaccaa cgaccagaag 780
aagctgatga gcagcaacgt gcagatcgtg aggcagcaga gctacagctt catgctgtgc 840
gtgaaggagg aggtgatcgc ctacgtggtg cagctgccca tctacggcgt gatcgacacc 900
ccctgctgga agctgcacac cagccccctg tgcaccaccg acaacaagga gggcagcaac 960
atctgcctga ccaggaccga caggggctgg tactgcgaca acgccggcag cgtgagcttc 1020
ttcccccagg ccgagacctg caaggtgcag agcaacaggg tgttctgcga caccatgaac 1080
agcctgaccc tgcccaccga cgtgaacctg tgcaacaccg acatcttcaa caccaagtac 1140
gactgcaaga tcatgaccag caagaccgac atcagctgca gcgtgatcac cagcatcggc 1200
gccatcgtga gctgctacgg caagaccaag tgcaccgcca gcaacaagaa caggggcatc 1260
atcaagacct tcagcaacgg ctgcgactac gtgagcaaca agggcgtgga caccgtgagc 1320
gtgggcaaca ccctgtacta cgtgaacaag ctggagggca aggccctgta catcaagggc 1380
gagcccatca tcaactacta cgaccccctg gtgttcccca gcgacgagtt cgacgccagc 1440
atcgcccagg tgaacgccaa gatcaaccag agcctggcct tcatcaggag gagcgacgag 1500
ctgctgcaca gc 1512
<210> 38
<211> 1533
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 38
atggccgcca ccgccatgag gatgatcatc agcatcatct tcatcagcac ctacatgacc 60
cacatcaccc tgtgccagaa catcaccgag gagttctacc agagcacctg cagcgccgtg 120
agcaggggct acctgagcgc cctgaggacc ggctggtaca ccagcgtggt gaccatcgag 180
ctgagcaaga tccagaagaa cgtgtgcaag agcaccgaca gcaaggtgaa gctgatcaag 240
caggagctgg agaggtacaa caacgccgtg atcgagctgc agagcctgat gcagaacgag 300
cccgcctgct tcagcagggc caagaggggc atccccgaga ggggcatccc cgagagcggc 360
agcagcggca ggaagaggag gttcaggaag aggaggttcc tgggcttcct gctgtgcatc 420
ggcagcgcca tcgccagcgg cgtggccgtg agcaaggtgt gccacctgga gggcgaggtg 480
aacaagatca agaacgccct gctgagcacc aacaaggccg tggtgagcct gagcaacggc 540
gtgagcctgc tgaccagcaa ggtgctggac ctgaagaact acatcgacaa ggagctgctg 600
cccaaggtga acaaccacga ctgcaggatc agcaacatcg agaccgtgat cgagttccag 660
cagaagaaca acaggctgct ggagatcgcc agggagttca gcgtgaacgc cggcatcacc 720
acccccctga gcacctacat gctgaccaac agcgagctgc tgagcctgat ctgcgacatg 780
cccatcacca acgaccagaa gaagctgatg agcagcaacg tgcagatcgt gaggcagcag 840
agctacagct tcatgtgcgt ggtgaaggag gaggtgatcg cctacgtggt gcagctgccc 900
atctacggcg tgatcgacac cccctgctgg aagctgcaca ccagccccct gtgcaccacc 960
gacaacaagg agggcagcaa catctgcctg accaggaccg acaggggctg gtactgcgac 1020
aacgccggca gcgtgagctt cttcccccag gccgagacct gcaaggtgca gagcaacagg 1080
gtgttctgcg acaccatgaa cagcctgacc ctgcccaccg acgtgaacct gtgcaacacc 1140
gacatcttca acaccaagta cgactgcaag atcatgacca gcaagaccga catcagctgc 1200
agcgtgatca ccagcatcgg cgccatcgtg agctgctacg gcaagaccaa gtgcaccgcc 1260
agcaacaaga acaggggcat catcaagacc ttcagcaacg gctgcgacta cgtgagcaac 1320
aagggcgtgg acaccgtgag cgtgggcaac accctgtact acgtgaacaa gctggagggc 1380
aaggccctgt acatcaaggg cgagcccatc atcaactact acgaccccct ggtgttcccc 1440
agcgacgagt tcgacgccag catcgcccag gtgaacgcca agatcaacca gagcctggcc 1500
ttcatcagga ggagcgacga gctgctgcac agc 1533
<210> 39
<211> 1512
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 39
atggccgcca ccgccatgag gatgatcatc agcatcatct tcatcagcac ctacatgacc 60
cacatcaccc tgtgccagaa catcaccgag gagttctacc agagcacctg cagcgccgtg 120
agcaggggct acctgagcgc cctgaggacc ggctggtaca ccagcgtggt gaccatcgag 180
ctgagcaaga tccagaagaa cgtgtgcaag agcaccgaca gcaaggtgaa gctgatcaag 240
caggagctgg agaggtacaa caacgccgtg atcgagctgc agagcctgat gcagaacgag 300
cccgcctgct tcagcagggc caagaggggc atccccgaga gcggcagcag cggcagcagc 360
ggcaggaaga ggaggttcct gggcttcctg ctgtgcatcg gcagcgccat cgccagcggc 420
gtggccgtga gcaaggtgtg ccacctggag ggcgaggtga acaagatcaa gaacgccctg 480
ctgagcacca acaaggccgt ggtgagcctg agcaacggcg tgagcctgct gaccagcaag 540
gtgctggacc tgaagaacta catcgacaag gagctgctgc ccaaggtgaa caaccacgac 600
tgcaggatca gcaacatcga gaccgtgatc gagttccagc agaagaacaa caggctgctg 660
gagatcgcca gggagttcag cgtgaacgcc ggcatcacca cccccctgag cacctacatg 720
ctgaccaaca gcgagctgct gagcctgatc tgcgacatgc ccatcaccaa cgaccagaag 780
aagctgatga gcagcaacgt gcagatcgtg aggcagcaga gctacagctt catgtgcgtg 840
gtgaaggagg aggtgatcgc ctacgtggtg cagctgccca tctacggcgt gatcgacacc 900
ccctgctgga agctgcacac cagccccctg tgcaccaccg acaacaagga gggcagcaac 960
atctgcctga ccaggaccga caggggctgg tactgcgaca acgccggcag cgtgagcttc 1020
ttcccccagg ccgagacctg caaggtgcag agcaacaggg tgttctgcga caccatgaac 1080
agcctgaccc tgcccaccga cgtgaacctg tgcaacaccg acatcttcaa caccaagtac 1140
gactgcaaga tcatgaccag caagaccgac atcagctgca gcgtgatcac cagcatcggc 1200
gccatcgtga gctgctacgg caagaccaag tgcaccgcca gcaacaagaa caggggcatc 1260
atcaagacct tcagcaacgg ctgcgactac gtgagcaaca agggcgtgga caccgtgagc 1320
gtgggcaaca ccctgtacta cgtgaacaag ctggagggca aggccctgta catcaagggc 1380
gagcccatca tcaactacta cgaccccctg gtgttcccca gcgacgagtt cgacgccagc 1440
atcgcccagg tgaacgccaa gatcaaccag agcctggcct tcatcaggag gagcgacgag 1500
ctgctgcaca gc 1512
<210> 40
<211> 1551
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 40
atggccctga gcaaggtgaa gctgaacgac accttcaaca aggaccagct gctgagcacc 60
agcaagtaca ccatccagag gagcaccggc gacaacatcg acatccccaa ctacgacgtg 120
cagaagcacc tgaacaagct gtgcggcatg ctgctgatca ccgaggacgc caaccacaag 180
ttcaccggcc tgatcggcat gctgtacgcc atgagcaggc tgggcaggga ggacaccctg 240
aagatcctga aggacgccgg ctaccaggtg agggccaacg gcgtggacgt gatcacccac 300
aggcagtgcg tgaacggcaa gagcggcagc agcggcagca gcggccaggg caacatcgag 360
tgcgagagca ggaagagcta caagaagatg ctgaaggaga tgggcgaggt ggcctgcgag 420
tacaggcacg acttccccga ctgcggcatg atcgtgctgt gcgtggccgc cctggtgatc 480
accaagctgc tggccggcga caggagcggc ctgaccgccg tgatcaggag ggccaacaac 540
gtgctgagga acgagatgaa gaggtacaag ggcctgatcc ccaaggacat cgccaacagc 600
ttctacgagg tgttcgagaa gtacccccac tacatcgacg tgttcgtgca cttcggcatc 660
gcccagagca gcaccagggg cggcagcagg gtggagggca tcttcgcctg cctgttcatg 720
aacgcctacg gcgccggcca ggtgatgctg aggtggggcg tgctggccaa gagcgtgaag 780
aacttcatgc tgtgccacgc cagcgtgcag gccgagatgg agcaggtggt ggaggtgtac 840
gagtacgccc agaagctggg cggcgaggcc ggcttctacc acatcctgaa caaccccaag 900
gccagcctgc tgagcctgac ccagttcccc aacttcagca gcgtggtgct gggcaacgcc 960
gccggcctgg gcatcatggg cgagtacagg ggcaccccca ggaaccagga cctgtacgac 1020
gccgccaagg cctacgccga gcagctgaag gagaacggcg tgatcaacta cagcgtgctg 1080
gacctgacca ccgaggagct ggaggccatc aagaaccagc tgaaccccaa ggacaacgac 1140
gtggagctgt gcaacaccga catcttcaac accaagtacg actgcaagat catgaccagc 1200
aagaccgaca tcagctgcag cgtgatcacc agcatcggcg ccatcgtgag ctgctacggc 1260
aagaccaagt gcaccgccag caacaagaac aggggcatca tcaagacctt cagcaacggc 1320
tgcgactacg tgagcaacaa gggcgtggac accgtgagcg tgggcaacac cctgtactac 1380
gtgaacaagc tggagggcaa ggccctgtac atcaagggcg agcccatcat caactactac 1440
gaccccctgg tgttccccag cgacgagttc gacgccagca tcgcccaggt gaacgccaag 1500
atcaaccaga gcctggcctt catcaggagg agcgacgagc tgctgcacag c 1551
<210> 41
<211> 1542
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 41
atggccctga gcaaggtgaa gctgaacgac accttcaaca aggaccagct gctgagcacc 60
agcaagtaca ccatccagag gagcaccggc gacaacatcg acatccccaa ctacgacgtg 120
cagaagcacc tgaacaagct gtgcggcatg ctgctgatca ccgaggacgc caaccacaag 180
ttcaccggcc tgatcggcat gctgtacgcc atgagcaggc tgggcaggga ggacaccctg 240
aagatcctga aggacgccgg ctaccaggtg agggccaacg gcgtggacgt gatcacccac 300
aggcagtgcg tgaacggcaa gagcggcagc agcggccagg gcaacatcga gtgcgagagc 360
aggaagagct acaagaagat gctgaaggag atgggcgagg tggcctgcga gtacaggcac 420
gacttccccg actgcggcat gatcgtgctg tgcgtggccg ccctggtgat caccaagctg 480
ctggccggcg acaggagcgg cctgaccgcc gtgatcagga gggccaacaa cgtgctgagg 540
aacgagatga agaggtacaa gggcctgatc cccaaggaca tcgccaacag cttctacgag 600
gtgttcgaga agtaccccca ctacatcgac gtgttcgtgc acttcggcat cgcccagagc 660
agcaccaggg gcggcagcag ggtggagggc atcttcgcct gcctgttcat gaacgcctac 720
ggcgccggcc aggtgatgct gaggtggggc gtgctggcca agagcgtgaa gaacttcatg 780
ctgtgccacg ccagcgtgca ggccgagatg gagcaggtgg tggaggtgta cgagtacgcc 840
cagaagctgg gcggcgaggc cggcttctac cacatcctga acaaccccaa ggccagcctg 900
ctgagcctga cccagttccc caacttcagc agcgtggtgc tgggcaacgc cgccggcctg 960
ggcatcatgg gcgagtacag gggcaccccc aggaaccagg acctgtacga cgccgccaag 1020
gcctacgccg agcagctgaa ggagaacggc gtgatcaact acagcgtgct ggacctgacc 1080
accgaggagc tggaggccat caagaaccag ctgaacccca aggacaacga cgtggagctg 1140
tgcaacaccg acatcttcaa caccaagtac gactgcaaga tcatgaccag caagaccgac 1200
atcagctgca gcgtgatcac cagcatcggc gccatcgtga gctgctacgg caagaccaag 1260
tgcaccgcca gcaacaagaa caggggcatc atcaagacct tcagcaacgg ctgcgactac 1320
gtgagcaaca agggcgtgga caccgtgagc gtgggcaaca ccctgtacta cgtgaacaag 1380
ctggagggca aggccctgta catcaagggc gagcccatca tcaactacta cgaccccctg 1440
gtgttcccca gcgacgagtt cgacgccagc atcgcccagg tgaacgccaa gatcaaccag 1500
agcctggcct tcatcaggag gagcgacgag ctgctgcaca gc 1542
<210> 42
<211> 1551
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 42
atggccctga gcaaggtgaa gctgaacgac accttcaaca aggaccagct gctgagcacc 60
agcaagtaca ccatccagag gagcaccggc gacaacatcg acatccccaa ctacgacgtg 120
cagaagcacc tgaacaagct gtgcggcatg ctgctgatca ccgaggacgc caaccacaag 180
ttcaccggcc tgatcggcat gctgtacgcc atgagcaggc tgggcaggga ggacaccctg 240
aagatcctga aggacgccgg ctaccaggtg agggccaacg gcgtggacgt gatcacccac 300
aggcagtgcg tgaacggcaa gagcggcagc agcggcagca gcggccaggg caacatcgag 360
tgcgagagca ggaagagcta caagaagatg ctgaaggaga tgggcgaggt ggcctgcgag 420
tacaggcacg acttccccga ctgcggcatg atcgtgctgt gcgtggccgc cctggtgatc 480
accaagctgc tggccggcga caggagcggc ctgaccgccg tgatcaggag ggccaacaac 540
gtgctgagga acgagatgaa gaggtacaag ggcctgatcc ccaaggacat cgccaacagc 600
ttctacgagg tgttcgagaa gtacccccac tacatcgacg tgttcgtgca cttcggcatc 660
gcccagagca gcaccagggg cggcagcagg gtggagggca tcttcgcctg cctgttcatg 720
aacgcctacg gcgccggcca ggtgatgctg aggtggggcg tgctggccaa gagcgtgaag 780
aacttcatgc tgtgccacgc cagcgtgcag gccgagatgg agcaggtggt ggaggtgtac 840
gagtacgccc agaagctggg cggcgaggcc ggcttctacc acatcctgaa caaccccaag 900
gccagcctgc tgagcctgac ccagttcccc aacttcagca gcgtggtgct gggcaacgcc 960
gccggcctgg gcatcatggg cgagtacagg ggcaccccca ggaaccagga cctgtacgac 1020
gccgccaagg cctacgccga gcagctgaag gagaacggcg tgatcaacta cagcgtgctg 1080
gacctgacca ccgaggagct ggaggccatc aagaaccagc tgaaccccaa ggacaacgac 1140
gtggagctgt gcaacaccga catcttcaac accaagtacg actgcaagat catgaccagc 1200
aagaccgaca tcagctgcag cgtgatcacc agcatcggcg ccatcgtgag ctgctacggc 1260
aagaccaagt gcaccgccag caacaagaac aggggcatca tcaagacctt cagcaacggc 1320
tgcgactacg tgagcaacaa gggcgtggac accgtgagcg tgggcaacac cctgtactac 1380
gtgaacaagc tggagggcaa ggccctgtac atcaagggcg agcccatcat caactactac 1440
gaccccctgg tgttccccag cgacgagttc gacgccagca tcgcccaggt gaacgccaag 1500
atcaaccaga gcctggcctt catcaggagg agcgacgagc tgctgcacag c 1551

Claims (10)

1.一种牛呼吸道合胞病毒F蛋白的融合前体蛋白，包含至少一处选自下组的对野生型F蛋白的改造：
A、增加F蛋白三聚体每个单体内部以及单体之间二硫键的连接数量；
B、突变F蛋白三聚体内部至少一个氨基酸，将侧链较小的氨基酸突变成为侧链较大的氨基酸或增加内部的疏水结合；
C、突变剔除至少一个蛋白酶的酶切位点；
D、切除F蛋白三聚体至少一个动态较大氨基酸，代之以较短的连接肽；
E、延长F-蛋白质的C-终端阿尔法(α)螺旋构造。
2.根据权利要求1所述的融合前体蛋白，所述改造包含第143位甘氨酸、第404位丝氨酸、第103位丝氨酸、第262位丝氨酸突变为半胱氨酸，第288位异亮氨酸突变为苯丙氨酸，第187位缬氨酸突变为亮氨酸。
3.根据权利要求2所述的融合前体蛋白，还包含下述改造中的一种：
1)第159位组氨酸、第291位缬氨酸突变为半胱氨酸，剪除从109至137氨基酸序列，加入连接肽丝氨酸－谷氨酸－丝氨酸－丝氨酸－谷氨酸－丝氨酸－丝氨酸－谷氨酸；
2)第158位亮氨酸、第290位丝氨酸突变为半胱氨酸，剪除从114至132氨基酸序列，加入连接肽丝氨酸－谷氨酸－丝氨酸－丝氨酸－谷氨酸；
3)第158位亮氨酸、第290位丝氨酸突变为半胱氨酸，剪除从114至132氨基酸序列，加入连接肽丝氨酸－谷氨酸－丝氨酸－丝氨酸－谷氨酸－丝氨酸－丝氨酸－谷氨酸。
4.根据权利要求3所述的融合前体蛋白，选自下述序列中的一种：
(1)其氨基酸序列如SEQ ID NO:1、2或3所示；
(2)其氨基酸序列如SEQ ID NO:4、5或6所示；
(3)其氨基酸序列如SEQ ID NO:7、8或9所示；
(4)其氨基酸序列如SEQ ID NO:10、11或12所示；
(5)其氨基酸序列如SEQ ID NO:13、14或15所示；
(6)其氨基酸序列如SEQ ID NO:16、17或18所示；
(7)其氨基酸序列如SEQ ID NO:19、20或21所示。
5.编码权利要求1-4任意一项所述的融合前体蛋白的DNA分子。
6.根据权利要求5所述的DNA分子，其核苷酸序列选自SEQ ID NO:22-42所示序列中的一种。
7.权利要求1-4任意一项所述的牛呼吸道合胞病毒F蛋白的融合前体蛋白或权利要求5-6任意一项所述DNA分子在制备预防牛呼吸道合胞病毒的产品中的应用。
8.一种蛋白疫苗，由权利要求1-4任意一项所述的牛呼吸道合胞病毒F蛋白的融合前体蛋白中的至少一种制成。
9.一种DNA疫苗，包含权利要求5-6任意一项所述DNA分子中至少一种和质粒载体。
10.根据权利要求4所述的DNA疫苗，所述质粒载体为pVAC1-mcs质粒。
CN201910171850.0A 2019-03-07 2019-03-07 一种改良的亚稳定态牛呼吸道合胞病毒融合前体f蛋白质及编码的dna分子和其应用 Pending CN109851678A (zh)

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