CN111918666A - 一种新型疫苗佐剂 - Google Patents

一种新型疫苗佐剂 Download PDF

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CN111918666A
CN111918666A CN201980023133.1A CN201980023133A CN111918666A CN 111918666 A CN111918666 A CN 111918666A CN 201980023133 A CN201980023133 A CN 201980023133A CN 111918666 A CN111918666 A CN 111918666A
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徐龙福
崔永祐
沈湘禧
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Sl Wasi Gene
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Abstract

本发明提供了一种新的疫苗佐剂，更具体而言，一种用于刺激T淋巴细胞特异性免疫反应的疫苗佐剂，所述疫苗佐剂包括作为活性成分的IL‑12蛋白和IL‑21蛋白，或包括作为活性成分的编码IL‑12蛋白的多核苷酸和编码IL‑21蛋白的多核苷酸。

Description

一种新型疫苗佐剂
相关申请
本申请要求2018年2月2日提交的韩国专利申请10-2018-0013329的优先权。上述申请的说明书通过引用全文合并于此。
技术领域
本发明涉及疫苗佐剂，更具体地，涉及通过选择性增强T细胞特异性免疫应答能使抗癌疫苗等的治疗效果最大化的新型疫苗佐剂。
背景技术
疫苗是用于产生针对抗原的免疫应答以防御病原体感染的药物，并且最近开发的疫苗主要使用重组蛋白作为抗原。与灭活疫苗或减毒活疫苗相比，重组蛋白安全且毒性较低，但重组蛋白的免疫原性较低。因此，其与疫苗佐剂一起使用以提供足够的免疫力来抵抗感染。
疫苗佐剂分为三种类型，即抗原的载体，免疫刺激剂和一种根据作用机理在刺激免疫应答的同时充当抗原基质的载体。有效使用疫苗佐剂可以带来多种效果，例如：(1)提高重组抗原的免疫原性，(2)降低抗原剂量或减少免疫接种的次数，(3)改善免疫力较弱的婴儿和老年人的免疫原性。但是，尽管这些疫苗佐剂有用，仍然很少有许可用于人类疫苗的疫苗佐剂，这是因为要选择疫苗佐剂需要考虑许多因素。疫苗佐剂的理想条件是它们应安全，耐药性优良，制造简单且生产成本低。另外，疫苗佐剂在体内应具有长的半衰期并且是可生物降解的，并且不应诱导针对免疫佐剂本身的任何免疫应答。特别地，其中疫苗佐剂的安全性问题最为重要。
目前在欧洲和美国通过批准用于疫苗的疫苗佐剂包括铝盐、MF59、AS03、AS04等。铝盐主要以Al(OH)3或AlPO4的形式使用，其被认为是通过吸附并缓慢释放蛋白质抗原而表现出免疫刺激作用。然而，近来已表明铝盐能激活树突状细胞并促进细胞因子(例如，IL-1β和IL-18)的分泌。铝盐广泛用于几种疫苗中，被认为是非常安全的，但据推测铝盐可能引起过敏反应并具有神经毒性。另外，铝盐强烈地诱导抗体介导的体液免疫反应，但是铝盐的缺点是它们几乎不诱导细胞介导的免疫反应并且不能进行冷冻保存。
Novartis(诺华)的MF59(Ott et al.,Pharm Biotechnol.6:277-296,1995)和GSK的AS03(Li et al.,J.Virol.80(3):1414-1426,2006)都是以角鲨烯为基本成分的水包油(o/w)乳液形式的免疫佐剂。o/w乳剂免疫佐剂激活抗原呈递细胞从而刺激抗原摄取和细胞因子分泌，增加趋化因子受体的表达，从而增加抗原呈递细胞的迁移。MF59和AS03用于增强对抗2009年新型流感病毒大爆发的疫苗中抗原的免疫原性并减少抗原用量，通过临床试验证实MF59和AS03具有提高抗体产生率和交叉防御的作用。除了角鲨烯以外，各种乳剂免疫佐剂已经开发出来，其使用的是自然界中制备的可用于人类的乳剂，但是由于过度的免疫反应和毒性，这些乳剂免疫佐剂的商业化是困难的。诸如脂质体，免疫刺激复合物和病毒样颗粒之类的颗粒载体也正在被开发为免疫佐剂，并且它们通常通过封装抗原而被开发为疫苗载体和佐剂。特别地，脂质体已被积极研究。脂质体是构成脂质层的合成球体，脂质层可以封装抗原并同时发挥疫苗输送和免疫佐剂的作用，脂质体的活性随脂质层的数量，电荷，组成和制备方法而变化。脂质体可增强针对蛋白质和多糖抗原的体液免疫和细胞免疫。然而，由于制备、稳定性等方面的困难，脂质体的使用受到限制，并且必须添加免疫刺激成分达到强烈的免疫活性，尤其因为相比佐剂增强免疫反应，免疫刺激成分的作用更接近抗原载体。目前，GSK已开发出一种免疫佐剂AS01B，其中向脂质体中添加了单磷酰脂质A(MPL)，并且GSK目前正尝试发展该免疫佐剂作为结核病和疟疾患者的疫苗佐剂(WO96/33739A)。
然而，尽管这些疫苗佐剂增强了细胞介导的免疫反应，但是这些疫苗佐剂大多已经开发用于增强疫苗组合物的免疫应答，以预防主要需要体液免疫的传染病(例如病毒等)，因此尚未确认疫苗在预防和治疗需要细胞介导的免疫反应的疾病(例如癌症等)中的作用。
发明概述
如上所述，仍然迫切需要开发通过增强T细胞特异性免疫反应的疫苗佐剂，该佐剂可有效预防和治疗需要增强细胞介导免疫反应的疾病(如癌症)，以及由病毒感染引起的常规传染病的预防和治疗等，就这一方面而言为了解决包括上述问题在内的各种问题，本发明的发明人提供了一种新型疫苗佐剂，其可以通过增强T细胞免疫应答特异性免疫来增强疫苗的作用。但本发明不受该目的的限制。
一方面，本发明提供了一种用于刺激T淋巴细胞特异性免疫反应的疫苗佐剂，其中所述疫苗佐剂包括编码IL-12蛋白的多核苷酸和编码IL-21蛋白的多核苷酸作为活性成分。
另一方面，本发明提供一种用于刺激T淋巴细胞特异性免疫反应的疫苗佐剂，其中所述疫苗佐剂包括作为活性成分的IL-12蛋白和IL-21蛋白。
根据本发明的另一方面，提供了一种用于刺激T淋巴细胞特异性免疫反应的疫苗组合物，其中所述疫苗组合物包括作为活性成分的疫苗佐剂和免疫抗原。
根据本发明的另一个方面，提供了一种通过疫苗组合物刺激T淋巴细胞特异性免疫应答的方法，其中所述方法包括在向个体施用疫苗组合物同时、或之前或之后施用疫苗佐剂。
发明效果
根据本发明一个实施方案中的疫苗佐剂通过选择性增加对抗原特异性反应的T细胞而显著增强T细胞特异性免疫应答，并且不影响抗体产生。因此，该疫苗佐剂可有效地用作治疗宫颈癌等癌症的免疫治疗剂，以及通过增强细胞免疫来治疗各种感染性病毒感染的传染病。
附图说明
结合附图，可以从以下描述中更详细地理解示例性实施方案，其中：
图1a为显示BD-121的基因构建体的示意图，根据示例性实施方案，BD-121是一种疫苗佐剂；图1b为显示BD-121A的基因构建体的示意图，根据示例性实施方案，BD-121A是一种疫苗佐剂；
图2为显示BD-14的基因构建体的示意图，BD-14是根据示例性实施方案制备的14价HPV DNA疫苗；
图3为显示根据示例性实施方案制备的CMV DNA疫苗的基因构建体的示意图；
图4a为显示BD-02B的基因构建体的示意图，BD-02B包含在根据示例性实施案制备的HSV-2DNA疫苗中；图4b为显示BD-02C的基因构建体的示意图，所述BD-02C包含在根据示例性实施方案制备的HSV-2DNA疫苗中；
图5为显示根据示例性实施方案制备的SFTS DNA疫苗的基因构建体的示意图；
图6显示根据示例性实施方案，当传统的2价HPV DNA疫苗单独或与疫苗佐剂BD-121组合施用时，抗原特异性T细胞免疫应答的ELISPOT分析结果；
图7a显示转染hBD-121构建体的COS-7细胞培养上清液中IL-12和IL-21浓度的ELISA分析结果，根据示例性实施方案，hBD-121构建体是疫苗佐剂；
图7b是示出用mBD-121构建体转染COS-7细胞培养上清液中IL-12和IL-21浓度的ELISA分析结果的图，根据示例性实施方案，mBD-121构体是疫苗佐剂；
图7c显示分别转染hBD-121构建体和mBD-121构建体的COS-7细胞培养上清中IL-12和IL-21表达水平的蛋白印迹分析结果，根据示例性实施方案，二者为细胞疫苗佐剂；
图8a显示根据示例性实施方案制备的CMV DNA疫苗和BD-121的给药时间表，BD-121是根据示例性实施方案制备的疫苗佐剂；
图8b显示当根据示例性实施方案制备的CMV DNA疫苗单独或与IL-12、IL-21或BD-121联合施用时，以CMV抗原特异性方式响应的脾细胞数量的计数结果示图，其为根据示范性实施例制备的疫苗佐剂；
图8c显示当根据示例性实施方案制备的CMV DNA疫苗单独或与IL-12、IL-21或BD-121(根据示例性实施方案为制备的疫苗佐剂)组合施用时抗原特异性抗体反应的分析结果示图；
图9a显示当根据示例性实施方案制备的HBV DNA疫苗单独或与BD-121组合施用时，以HBV抗原(HBsAg、HBcAg和PreS1/S2)特异性方式响应的脾细胞数量的示图，BD-121是根据示例性实施方案制备的疫苗佐剂；
图9b显示当根据示例性实施方案制备的HBV DNA疫苗单独或与BD-121(根据示例性实施方案制备的疫苗佐剂)组合施用时，HBsAg特异性抗体反应的分析结果示图；
图10a显示根据示例性实施方案制备的SFTS DNA疫苗和BD-121的给药时间表，BD-121是根据示例性实施方案制备的疫苗佐剂；
图10b显示当根据示例性实施方案制备的SFTS DNA疫苗单独或与BD-121组合施用时，以SFTS抗原(GnGc、NP和NS)特异性方式响应的脾细胞数量的计数结果，BD-121是根据示例性实施方案制备的疫苗佐剂(初始免疫)；
图10c显示当根据示例性实施方案制备的SFTS DNA疫苗单独或与BD-121组合施用两次时，以SFTS抗原(GnGc、NP和NS)特异性方式响应的脾细胞数量的计数结果的示图，BD-121为根据示例性实施方案制备的疫苗佐剂(初始免疫和加强免疫)；
图11a显示当根据示例性实施方案制备的SFTS DNA疫苗单独施用或与BD-121组合施用(改变混合比)一次时，以SFTS抗原(GnGc、NP和NS)特异性方式响应的脾细胞数量示意图，BD-121为根据示例性实施方案制备的疫苗佐剂(初始免疫)；
图11b显示当根据示例性实施方案制备的SFTS DNA疫苗单独或两次与BD-121结合施用(改变混合比)时，以SFTS抗原(GnGc、NP和NS)特异方式响应的脾细胞数量的计数结果示意图，BD-121为根据示例性实施方案制备的疫苗佐剂(初始免疫和加强免疫)；
图12a显示根据示例性实施方案制备的HSV-2DNA疫苗(BD02)和BD-121的给药时间表，BD-121是根据示例性实施方案制备的疫苗佐剂；
图12b显示在单独(BD-02B+BD-02C)施用根据示例性实施方案制备的HSV-2DNA疫苗(BD02)的组和与BD-121组合施用的组中HSV-2的病理指数随时间变化的示意图，BD-121为根据示例性实施方案制备之疫苗佐剂(BD02 DP)；
图12c显示在单独(BD-02B+BD-02C)施用根据示例性实施方案制备的HSV-2DNA疫苗(BD02)的组中和与BD-121联合施用的组中实验动物存活率随时间的变化的曲线图，BD-121为根据示例性实施方案制备的疫苗佐剂(BD02 DP)；
图13a为显示实验动物给药时间表的示意图，其中通过调整剂量施用根据示例性实施方案制备的HSV-2DNA疫苗(BD02)和BD-121(根据示例性实施方案制备的疫苗佐剂)；
图13b显示根据实验动物中疫苗佐剂的剂量，以HSV-2抗原(gD、UL39和ICP4)特异性方式响应的脾细胞数量的曲线图，其中实验动物经过剂量调整进行的初次接种(初次免疫)根据示例性实施方案制备的HSV-2DNA疫苗(BD02)和BD-121，BD-121是根据示例性实施方案制备的疫苗佐剂(BD02-DP)；
图14显示BD-121改善T细胞特异性免疫应答效果的分析结果，BD-121是根据示例性实施方案的疫苗佐剂，对于常规2价HPV DNA疫苗是通过计算以抗原特异性方式响应的脾细胞的数量而获得的；
图15a显示根据示例性实施方案制备的14价HPV DNA疫苗和BD-121或BD-121A的给药时间表，BD-121或BD-121A是根据示例性实施方案制备的疫苗佐剂；
图15b显示根据示例性实施方案制备的免疫佐剂BD-121和BD-121A对改善T细胞特异性免疫应答的效果分析结果图，对于14价HPV DNA疫苗，通过计数以抗原特异性方式反应的脾细胞数量获得；
图16a显示临床前实验中用于分析根据示例性实施方案的HSV-2DNA疫苗(BD-02)和BD-121A的效果的给药时间表，根据示例性实施方案，BD-121A是用于食蟹猴(是实验动物)的疫苗佐剂；以及
图16b显示根据示例性实施方案，在没有疫苗佐剂的情况下单独施用BD-02的组(左)和BD-02与BD-121A联合施用的组(右)中，以HSV-2抗原(gD2和UL39)特异性方式响应的脾细胞数目的计数结果，给药前(VS)、一次给药(VT1)和两次给药(VT2)。
发明详述
在本发明的一个方面，促进T淋巴细胞特异性免疫反应的疫苗佐剂包括编码IL-12蛋白的多核苷酸和编码IL-21蛋白的多核苷酸作为活性成分。
特别地，所述疫苗佐剂可包含选自由以下组成的组中的至少一种：
一至三个载体，每个载体包括：分别编码组成所述IL-12蛋白的p35链(IL-12p35)和p40链(IL-12p40)的多核苷酸；和
mRNA分子，每个分子都编码所述IL-12p35、IL-12p40和IL-21蛋白。
此外，可能在上述疫苗佐剂中所述IL-21p35、IL-12p40和IL-21的一部分可以作为蛋白被包括，而其余部分可以用作异质分子的混合物，例如表达载体和/或mRNA分子。
具体而言，所述一至三个载体可包括一个基因构建体，其中多核苷酸可操作地连接到控制序列(例如，启动子)，从而可以表达所述IL-21p35、IL-12p40和IL-21。疫苗佐剂可以由一至三个载体构建，以使分别编码IL-21p35、IL-12p40和IL-21的多核苷酸插入单个表达载体(三重载体系统)，或插入一个或两个表达载体(单载体或双载体系统)。这种单载体系统到三载体系统的具体示例性实施方案如下：
i)第4表达载体，其包括第1基因构建体至第3基因构建体，其中分别编码所述IL-21p35、IL-12p40和IL-21蛋白的多核苷酸分别可操作地连接到启子；
ii)第2表达载体至第4表达载体，其分别包括第1基因构建体至第3基因构建体；
iii)第5表达载体和第6表达载体，每一表达载体分别包括第1基因构建体至第3基因构建体中的两个和剩余的一个基因构建体；
iv)一个融合蛋白，其中IL-21连接到所述IL-12p35和所述IL-12p40中任何一个；以及在所述IL-12p35和所述IL-12p40之间未包含在融合蛋白中的肽；
v)第7表达载体，其包括第4基因构建体，其中编码iv)的所述融合蛋白的多核苷酸可操作地连接到启动子，以及第5基因构建体，其中编码iv)的所述肽的多核苷酸可操作地连接到启动子；
vi)第8表达载体和第9表达载体，分别包括第4基因构建体和第5基因构建体；
vii)第10表达载体，其包括：第6基因构建体，其中至少两个分别编码所述IL-12p35、IL-12p40和IL-21蛋白的多核苷酸连接到内部核糖体进入位点(IRES)；以及可选的第7基因构建体，其中在三个多核苷酸中，未包含在所述第6基因构建体的剩余多核苷酸可操作地连接到启动子；以及
viii)第11表达载体，包括第9基因构建体；以及第12表达载体，其可选地包括所述第7基因构建体。
所述疫苗佐剂可促进T淋巴细胞特异性免疫应答而不诱导B淋巴细胞特异性免疫应答。
在所述疫苗佐剂中，所述IL-12p35蛋白可由与人IL-12p35具有至少90％且优选地至少95％的序列同源性的氨基酸序列组成，所述人IL-12p35由如序列ID NO:1所示的氨基酸序列组成，并且还可以使用有高度同源性的、不会在人体内引起任何免疫反应的来自非人类(例如，灵长类或猿猴)的IL-12p35。所述IL-12p40蛋白可由与人IL-12p40具有至少90％且较佳地至少95％的序列同源性的氨基酸序列组成，所述人IL-12p40氨包含如SEQ IDNO:2所示的氨基酸序列，且也可使用衍生自具有高度同源性的非人类(例如，灵长类或猿类)的IL-12p40在人体内不会引起任何免疫反应。也可以使用如韩国专利NO.0399728中描述的IL-12p35和IL-12p40序列。所述IL-21蛋白可由与人IL-21具有至少90％且优选地至少95％的序列同源性的氨基酸序列组成，所述人IL-21由如序列ID NO:3所示的氨基酸序列组成，并且还可以使用具有高度同源性的来自非人类的IL-21(例如，(灵长类或猿类)，不会在人体内引起任何免疫反应。
疫苗佐剂可进一步包括由以下组成的组中选择的至少一种：
一种MIP-1α基因构建体，其中编码MIP-1α蛋白的多核苷酸可操作地连接到启动子；
一种复合基因构建体，其中编码MIP-1α蛋白的多核苷酸通过编码IRES或连接肽的多核苷酸与编码所述IL-12p35、IL-12p40和IL-21蛋白中每一种的多核苷酸中的至少一种可操作地连接；以及
编码MIP-1α蛋白的mRNA分子。
在所述疫苗佐剂中，编码所述MIP-1α蛋白的多核苷酸可通过编码肽连接体的多核苷酸或IRES与编码IL-12p35、IL-12p40和IL-21蛋白质中的每一个的多核苷酸可操作地连接，或可以单独的基因构建体形式提供。也就是说，所述MIP-1α基因构建体可以包括在单独的表达载体中，或者所述MIP-1α基因构建体可以包括在所述一至三个载体中的任何一个或多个载体中，其中每个载体包括：分别编码组成所述IL-12蛋白的p35链(IL-12p35)和p40链(IL-12p40)；以及一个编码所述IL-21蛋白的多核苷酸。也就是说，所述MIP-1α基因构建体可以包括在关于疫苗佐剂的示例性实施方案中描述的从由第1个表达载体到第12个表达载体组成的组中选择的至少一个表达载体中。
在所述疫苗佐剂中，所述MIP-1α蛋白可由与MIP-1α蛋白具有至少90％且优选地至少95％的序列同源性的氨基酸序列组成，所述MIP-1α蛋白质由如序列ID NO:10所示的氨基酸序列组成，并且还可以使用具有高度同源性的来自非人类的MIP-1α蛋白(例如，灵长类或猿类)，不会在人体内引起任何免疫反应。
在本发明的另一方面，提供了一种疫苗佐剂，包括作为活性成分的IL-12蛋白和IL-21蛋白。
在所述疫苗佐剂中，所述IL-12蛋白可由p35链(IL-12p35)和p40链(IL-12p40)组成。
所述疫苗佐剂可进一步包括MIP-1α蛋白。
IL-12蛋白、IL-21蛋白和MIP-1α如上文所述。
如本文所用，术语“可操作地连接到”意指目的核酸序列(例如，在体外转录/翻译系统或在宿主细胞中)连接到控制序列，以便可以表达异源核酸序列。
术语“控制序列”是包括启动子、增强子和其他控制序列(例如，聚腺苷酸化信号)的术语。所述控制序列包括：一个指导所述异源核酸在多个宿主细胞中基本表达的序列，一个指导所述控制序列仅在特定组织的细胞中表达(例如，组织特异性控制序列)，和一个指导所述表达由特定信号(例如，可诱导的控制序列)诱导。本领域普通技术人员将能够理解，表达载体的设计可以取决于诸如要转化的宿主细胞的选择、期望的蛋白表达水平等因素。本发明的表达载体可以引入宿主细胞来表达融合蛋白。本领域技术人员熟知允许在真核细胞和原核细胞中表达的控制序列。如上所述，这些控制序列包括通常负责转录起始的序列和负责转录的选择性终止及其稳定的poly-A信号。除转录控制因子外，额外的控制序列可包括翻译增强因子和/或天然结合或异源启动子区域。例如，能够在哺乳动物宿主细胞中表达的可能的控制序列可包括CMV-HSV胸苷激酶启动子、SV40、RSV启动子(鲁斯式肉瘤病毒)、人类延伸因子1α-启动子、糖皮质激素诱导的MMTV启动子(莫洛尼小鼠肿瘤病毒)、金属硫蛋白诱导的或四环素诱导的启动子，或放大剂，例如CMV放大剂或SV40放大剂。神经丝启动子、PGDF启动子、NSE启动子、PrP启动子或thy-1启动子可用于神经元的表达。启动子在技术领域是众所周知的，并且在文献中有描述(Charron,J.Biol.Chem.270:25739-25745)。为了在原核细胞中表达，公开了许多启动子，包括lac启动子、tac启动子和trp启动子。除了这些可以启动转录的因子外，这些控制序列还可以包括根据本公开的示例性实施方案的多核苷酸下游的转录终止信号(例如，SV40-poly-A区域或TK-poly-A区域)。在本公开中，合适的表达载体是本领域公知的，例如，冈山博格(Okayama-Berg)cDNA表达载体pcDV1(Parmacia)、pRc/CMV、pcDNA1、pcDNA3(In-vitrogene)、pSPORT1(GIBCO BRL)、pGX27(韩国专利No.1442254)、pX(Pagano(1992)Science 255,1144-1147)、酵母双杂交载体，例如，pEG202和dpJG4-5((Gyuris(1995)Cell 75,791-803)或真核表达载体，例如lambda gt11或pGEX(Amersham Pharmacia)。除了本公开的核酸分子外，载体还可以包括编码分泌信号肽的多核苷酸。本领域技术人员知晓所述的分泌信号肽。另外，根据本发明公开的示例性实施方案，根据所使用的表达系统，将能够使融合蛋白定向到细胞室的先导序列与多核苷酸的编码序列相结合，且优选地为可直接将所翻译的蛋白或其蛋白分泌到细胞质或胞外介质中的先导序列。
此外，本发明的载体可以通过，例如标准重组DNA技术制备。标准重组DNA技术的实施例包括平末端和粘性末端的连接、限制性酶处理以提供适当的末端、通过碱性磷酸酶处理去除磷酸基以防止不适当的结合、通过T4 DNA连接酶进行酶连接等。通过重组由化学合成或遗传重组技术获得的信号肽的DNA，或重组编码本发明的IL-12和IL-21蛋白到包含适当控制序列的载体上的DNA可以制备本发明的载体。含有控制序列的载体可以购买或商业制造，并且在本发明的一个示例性实施方案中，公开了pGX27，其为用于制备DNA疫苗的载体。
根据本发明示例性实施方案的表达载体可以是能够表达所述融合蛋白的表达载体，并且所述表达载体可以表示但不限于任何形式的质粒载体、病毒载体、共生体载体、噬菌体载体、人工人类染色体等。
如本文所用，术语“融合蛋白”是指一种重组蛋白，其中在该蛋白中负责固有功能的两个或多个蛋白或结构域被连接起来以便每个蛋白或结构域都可以负责固有功能。通常，具有柔性结构的连接肽可插入所述两个或多个蛋白质或结构域之间。所述连接肽可以包括(G4S)n(单位：SEQ ID NO:50，n是1到10的整数)、(GS)n(n是1到10的整数)、(GSSGGS)n(单位：SEQ ID NO:51，n是1到10的整数)、KESGSVSSEQLAQFRSLD(SEQ ID NO:52)、EGKSSGSGSESKST(SEQ ID NO:53),GSAGSAAGSGEF(SEQ ID NO:54)、(EAAAK)n(单位：SEQ IDNO:55，n是1到10的整数)、CRRRRRREAEAC(SEQ ID NO:56)、A(EAAAK)4ALEA(EAAAK)4A(SEQID NO:57)、GGGGGGGG(SEQ ID NO:58)、GGGGGG(SEQ ID NO:59)、AEAAAKEAAAAKA(SEQ IDNO:60)、PAPAP(SEQ ID NO:61)、(Ala Pro)n(n为1至10的整数整数)、VSQTSKLTRAETVFPDV(SEQ ID NO:62)、PLGLWA(SEQ ID NO:63)、TRHRQPRGWE(SEQ ID NO:64)、AGNRVRRSVG(SEQID NO:65)、RRRRRRRR(SEQ ID NO:66)、GFLG(SEQ ID NO:67)、GSSGGSGSSGGSGGGDEADGSRGSQKAGVDE(SEQ ID NO:68)等。
所述表达载体可进一步包括编码一个或两个或多个用于增强免疫的肽的多核苷酸，所述增强免疫的肽可以是CD28、诱导共刺激物(ICOS)、细胞毒性T淋巴细胞相关蛋白4(CTLA4)、程序性细胞死亡蛋白1(PD1)、B和T淋巴细胞相关蛋白(BTLA)、死亡受体3(DR3)、4-1BB、CD2、CD40、CD30、CD27、信号性淋巴细胞活化分子(SLAM)、2B4(CD244)、自然杀伤分子2、成员D(NKG2D)/DNAX激活蛋白12(DAP12)、T细胞免疫球蛋白和含粘蛋白结构域的蛋白1(TIM1)、TIM2、TIM3、TIGIT、CD226、CD160、淋巴细胞活化基因3(LAG3)、B7-1，B7-H1、糖皮质激素诱导的TNFR家族相关蛋白(GITR)、fms样酪氨酸激酶3配体(Flt3配体)、鞭毛蛋白、疱疹病毒进入介体(HVEM)或OX40L的细胞质域[CD134(OX40)和CD252的配体]或其中至少两个的连接体。
所述表达载体可以进一步包括编码分泌信号序列的多核苷酸，所述分泌信号序列诱导细胞表达的重组蛋白分泌到细胞外，所述表达载体可以是组织纤溶酶原激活物(tPA)信号序列、单纯疱疹病毒糖蛋白(HSV gDs)信号序列，或者生长激素信号序列。
在本发明的另一方面，提供了一种用于预防和治疗病毒感染的疫苗组合物，所述疫苗组合物包括作为活性成分的疫苗佐剂和来自感染性病毒的抗原，其中编码所述抗原的多核苷酸可操作地连接到启动子，或包含基因构建体的表达载体。
在所述疫苗组合物中，来自传染性病毒的抗原可以是来自非洲淋巴瘤病毒(EBV)、甲型肝炎病毒(HAV)、乙型肝炎病毒(HBV)、丙型肝炎病毒(HCV)、丁型肝炎病毒(HDV)、戊型肝炎病毒(HEV)、汉坦病毒、巨细胞病毒(CMV)、人类免疫缺陷病毒(HIV)，流感病毒、人乳头瘤病毒(HPV)、脊髓灰质炎病毒、埃博拉病毒、轮状病毒、登革热病毒、西尼罗河病毒、黄热病病毒、腺病毒、日本脑炎病毒、BK病毒、天花病毒、寨卡病毒、高热伴血小板减少综合征(SFTS)病毒或单纯疱疹病毒(HSV)的抗原。
根据本发明的另一方面，提供了一种用于癌症治疗的疫苗组合物，包括作为活性成分的疫苗佐剂和癌症抗原、编码癌症抗原的多核苷酸、其中所述多核苷酸可操作地连接到启动子的抗原基因构建体，或包含基因构建体的表达载体。
在用于癌症治疗的疫苗组合物中，癌症抗原可以是人乳头瘤病毒(HPV)衍生抗原、癌胚抗原、前列腺特异性膜抗原(PSMA)、Her2/neu、MUC-1、BCR/ABL、α-胎蛋白(AFP)、非洲淋巴瘤病毒(EBV)衍生抗原、人类乙型肝炎病毒(HBV)衍生抗原、人丙型肝炎病毒(HCV)衍生抗原、癌抗原125(CA-125)、癌抗原72-4(CA-72-4)、癌抗原15-3(CA-15-3)或癌抗原19-9(CA-19-9)。
除所述载体外，所述疫苗组合物可进一步包括药学上可接受的佐剂、赋形剂或稀释剂。
如本文所用，术语“药学上可接受”系指对人类施用时生理上可接受且通常不会引起过敏反应(例如，胃肠道紊乱、头晕等)或其类似反应的组合物。载体、赋形剂和稀释剂的实施例可包括乳糖、葡萄糖、蔗糖、山梨醇、甘露醇、木糖醇、赤藓糖醇、麦芽糖醇、淀粉、阿拉伯胶、海藻酸钠、明胶、磷酸钙、硅酸钙、纤维素、甲基纤维素、聚乙烯吡咯烷酮、水、甲基羟基苯甲酸丙酯、滑石粉、硬脂酸镁，矿物油。此外，还可进一步包括填料、抗凝固剂、润滑剂、保湿剂、香精、乳化剂、防腐剂等。
除上述疫苗佐剂外，所述疫苗组合物可进一步包括常规使用的疫苗佐剂。作为常规使用的疫苗佐剂，可以使用氢氧化铝、磷酸铝、明矾(硫酸铝钾)、MF59、病毒体、AS04[氢氧化铝和单磷酸脂A(MPL)的混合物]、AS03(DL-α-生育酚、角鲨烯和聚山梨酯80(乳化剂)的混合物)、CpG、鞭毛蛋白、聚I:C、AS01、AS02、ISCOMs，ISCOMMATRIX等。
如本文所用，术语“佐剂”或“疫苗佐剂”是指为了改善疫苗的免疫应答而施用的药物或免疫制剂。
此外，根据本发明的示例性实施方案的疫苗佐剂或含有疫苗佐剂的疫苗组合物可使用本领域已知的方法来配制，以便在将活性成分投给哺乳动物时能够快速释放或持续或延迟释放。这些制剂可包括粉末、颗粒、片剂、乳剂、糖浆、气溶胶、软或硬明胶胶囊、无菌注射溶液和无菌粉末。
根据本发明的示例性实施方案，疫苗佐剂或含有疫苗佐剂的疫苗组合物可通过各种途径施用，例如口服、肠外(例如栓剂、经皮、静脉、腹腔内、肌肉内、病变内、鼻、椎管，或可使用植入式装置进行持续、持续或反复释放。给药次数可在所需范围内每天给药一次或多次，给药周期不受特别限制。
根据本发明的示例性实施例，疫苗佐剂或含有疫苗佐剂的疫苗组合物可通过常规全身或局部施用(例如，肌肉注射或静脉注射)，如果作为DNA疫苗组合物提供，则所述疫苗组合物可最好用电穿孔器注射。对于电穿孔器，可使用用于将市场上买得到的DNA药物注射到体内的电穿孔器(例如，意大利IGEA的GlinporatorTM、韩国JCBIO的CUY21EDIT、瑞士Supertech的SP-4a、韩国SLVAXiGEN的Orbiector等)。
只要疫苗组合物能够到达靶组织，根据本发明示例性实施方案的疫苗佐剂或含有疫苗佐剂的疫苗组合物的给药途径可通过任何常规途径施用。给药途径可包括但不限于肠外给药(例如，腹腔内、静脉内、肌肉内、皮下和鞘内给药)。
根据本发明示例性实施方案的疫苗佐剂或含有疫苗佐剂的疫苗组合物的给药途径可与常用的药学上可接受载体一起以合适的形式配制。药学上可接受的可包括例如水、合适的油、生理盐水、肠外载体(例如含水葡萄糖、二醇等)，并且可进一步包含稳定剂和防腐剂。合适的稳定剂可包括抗氧化剂(例如，亚硫酸氢钠、亚硫酸钠或抗坏血酸)。合适的防腐剂可包括苯扎氯铵、对羟基苯甲酸甲酯或丙酯和氯丁醇。另外，如有必要，根据给药方法和制剂，根据本发明的组合物可适当地包括悬浮剂、溶解佐剂、稳定剂、等渗剂、防腐剂、吸附抑制剂、表面活性剂、稀释剂、赋形剂、pH调节剂、镇痛剂、缓冲剂、抗氧化剂等。文献[Remington's Pharmaceutical Sciences，New Edition]中详细描述了适合本发明的合适的药学上可接受的载体和制剂的合适实例，包括上述的举例说明。
针对患者的疫苗组合物的给药剂量可能因许多因素而变化，包括患者的身高、体表面积、年龄、所给药的特定化合物、性别、给药时间和途径、总体健康状况以及同时施用的其他药物。药学上的活性DNA可以100ng/体重(kg)至10mg/体重(kg)的量给药，更优选从1μg/kg至500μg/kg(体重)，且最优选5μg/kg至50μg/kg(体重)，且给药剂量可就因素而论进行调整。
此外，本发明的疫苗组合物可以药学上的有效量给药。
如本文所用，术语“药学上的有效量”是指足以以适用于医疗的合理效益/风险比率治疗疾病的量，并且有效剂量的水平可由以下因素决定，包括：受试者的种类、疾病的严重程度、年龄、性别，药物活性、药物敏感性、给药时间、给药途径和溶解速率、治疗时间、包括联合用药在内的因素，以及其他医学领域众所周知的因素。本发明的疫苗组合物可以0.1mg/kg至1g/kg，且更优选1mg/kg至500mg/kg的剂量给药。同时，给药剂量可根据患者的年龄、性别和健康状况进行适当调整。
此外，根据本公开的另一个方面，提供了一种通过疫苗组合物刺激T淋巴细胞特异性免疫应答的方法，包括向个体施用疫苗佐剂和疫苗组合物，或在注射疫苗成分之前或之后施用。
在所述方法中，疫苗佐剂可通过体内电穿孔给药。
所述方法的特征在于，所述方法仅选择性地诱导T淋巴细胞特异性免疫应答，而不诱导个体的B细胞特异性免疫应答。
具体实施方式
在下文中，将通过实施例和实验实施例对本发明进行更详细的描述。但是，本发明不限于以下描述的实施例和实验实施例中，而是可以各种其他形式实现。提供以下实施例和实验实施例是为了使本发明的公开更加充分，并将本发明的范围完全传达给本发明所属领域的技术人员。
实施例1：人IL-12和IL-21表达载体的制备
1-1:单载体系统
本发明的发明者设计了单载体系统，使IL-12和IL-21通过单载体表达。
为此，特别地，本发明的发明者制备了一种基因构建体，通过连接编码人IL-12蛋白两个亚单位(即，如SEQ ID NO:1所示的氨基酸序列组成的hIL-12p35多肽和如SEQ IDNO:2所示的氨基酸序列组成的hIL-12p40多肽)多核苷酸(如SEQ ID NOS:4和5所示)到具有如SEQ ID NO:6所示的核酸序列的EMCV衍生的内部核糖体进入位点(IRES)；并将如SEQ IDNO:7所示的RSV启动子(pRSV)和编码由如SEQ ID NO:3所示的氨基酸序列组成的人IL-21蛋白(hIL-21)的多核苷酸(如SEQ ID NO:8所示)顺序连接到编码hIL-12p40多肽的多核苷酸的3′端，然后将所述基因构建体插入pGX-27的多克隆位点载体(韩国专利No.1442254)，由此制备根据本发明的示例性实施方案的载体。这样制备的载体命名为“hBD-121”(图1a)。
1-2:双载体系统
本发明者设计了一种双载体系统，使IL-12和IL-21插入到单独的载体中进行表达。
双载体系统制备如下：
将编码hIL-12p35多肽的多核苷酸(如SEQ ID NO:4所示)和编码hIL-12p40多肽的多核苷酸(如SEQ ID NO:5所示)连接到具有如SEQ ID NO:6所示的核酸序列的EMCV-IRES；由此制得的产物插入到pGX-27载体的多克隆位点；同样，编码人IL-21蛋白(hIL-21)的多核苷酸(如SEQ ID NO:8所示)也插入到pGX-27载体的多克隆位点，从而制备了双载体系统。
1-3:三重载体系统(Triple vector system)
由于IL-12是由hIL-12p35多肽和hIL-12p40多肽组成的二聚体蛋白质，因此可以从独立载体表达hIL-12p35多肽和hIL-12p40多肽。因此，根据本发明的一个示例性实施方案，hIL-12p35多肽、hIL-12p40多肽和IL-21可通过三个独立构成的载体中的每一个来表达，并且为了方便起见，将其命名为“三重载体系统”。
三重载体系统的制备方法如下：
将编码hIL-12p35多肽、hIL-12p40多肽和hIL-21中的每一个的多核苷酸(如SEQID NOS:4、5和8所示)插入pGX-27载体的多克隆位点，从而制备三重载体系统。
实施例2：人IL-12、IL-21和MIP-1α表达载体的制备
制备一种基因构建体，通过将编码如SEQ ID NO:1所示的氨基酸序列组成的hIL-12p35多肽和如SEQ ID NO:2所示的氨基酸序列组成的hIL-12p40多肽编码的多核苷酸(如SEQ ID NOS:4和5所示)连接到EMCV衍生的具有如SEQ ID NO:6所示的核酸序列的内部核糖体进入位点(IRES)；并将又如SEQ ID NO:9所示的核酸序列组成的人EF-1α启动子(pEF-1α)和编码由如SEQ ID NO:10所示的氨基酸序列组成的人MIP-1α蛋白质(hMIP-1α)的多核苷酸(如SEQ ID NO:11所示)顺序连接到如SEQ ID NO:8所示的多核苷酸，该核苷酸上顺序连接有如SEQ ID NO:7所示的RSV启动子(pRSV)和编码由如SEQ ID NO:3所示的氨基酸序列构成的人IL-21蛋白(hIL-21)的多核苷酸；并将基因构建体插入pGX-27载体的多克隆位点；这样制备的载体命名为“hBD-121A”(图1b)。
实施例3：小鼠IL-12和IL-21表达载体的制备
制备一种基因构建体，通过连接编码小鼠IL-12蛋白的两个亚基中每一个(即，由如SEQ ID NO:12的氨基酸序列组成的mIL-12p35多肽和由如SEQ ID NO:13的氨基酸序列组成的mIL-12p40多肽)的核苷酸序列(SEQ ID NOS:14和15)到具有如SEQ ID NO:6的核酸序列的EMCV衍生的内部核糖体进入位点(IRES)；并且将如SEQ ID NO:7所示的RSV启动子(pRSV)和编码小鼠IL-21蛋白(mIL-21)的多核苷酸(如SEQ-ID NO:17所示)顺序连接到编码mIL-12p40多肽的多核苷酸的3′端；以及将所述基因构建体插入pGX-27载体的多克隆位点中，从而制备根据本发明的示例性实施方案的载体。这样制备的载体命名为“mBD-121”(图1a)。
实施例4：小鼠IL-12、IL-21和MIP-1α表达载体的制备
构建一种基因构建体，通过将编码由如SEQ ID NO:12所示的氨基酸序列组成的mIL-12p35多肽的和如SEQ ID NO:13所示的氨基酸序列组成的mIL-12p40多肽的多核苷酸(如SEQ ID NOS:14和15所示)连接到具有如SEQ ID NO:6所示的核酸序列的EMCV衍生的内部核糖体进入位点(IRES)；并将由如SEQ ID NO:9所示的核酸序列组成的人EF-1α启动子(pEF-1α)，以及包含如SEQ ID NO:18所示的氨基酸序列的编码小鼠MIP-1α蛋白(mMIP-1α)的多核苷酸(如SEQ ID NO:19所示)顺序连接到编码mIL-12p40多肽(如SEQ ID NO:8所示)的多核苷酸的3’端，该多核苷酸上顺序连接有如SEQ ID NO:7所示的RSV启动子(pRSV)和编码由如SEQ ID NO:16所示的氨基酸序列组成的小鼠IL-21蛋白(mIL-21)的多核苷酸(如SEQID NO:17)；将所述基因构建体插入所述pGX-27载体的多克隆位点中，由此制备的载体命名为“mBD-121A”(图1b)。
实施例5:HPV DNA疫苗构建体的制备
为了制备14价HPV DNA疫苗，本发明者通过PCR获得了编码E6和E7抗原中每种类型的N端片段和C端片段的多核苷酸，以便以重组融合蛋白的形式表达E6和E7抗原，其中，HPV类型6、11、16、18、31、33、35、39、45、51、52、56、58和59型的早期表达蛋白E6和E7属于高危组，然后按照图2和表1所示的顺序连接多核苷酸，从而制备三个基因构建体。将三个基因构建体分别插入pGX-27载体中，从而制备HPV-DNA构建体。这三个基因构建体分别命名为BD-14A、BD-14B和BD-14C，并且含有这三种载体的组合命名为BD-14。多价HPV-DNA疫苗构建体是一种三重载体系统，其原因是当待插入的基因构建体过大时，pGX-27载体的容量不够。
如图2和表1所示，每种HPV类型的E6和E7抗原是在这样的结构中构建的，其中这些抗原分别分为部分序列重叠的N端片段和C端片段，然后是融合多肽，其中E7的C端片段与E6的N端片段末端和融合多肽(其中E6的C端片段连接到E7的N端片段的末端)通过(GS)5连接肽再次连接，使得每种类型的4到5个抗原单元连接到(GS)5连接肽以包含在单载体中。表1示例性地显示了要插入到一个表达载体中的每一个亚型型的种类，但它们仅用于说明目的，并且可以以任何其他顺序制备。
表1：本公开14价HPV DNA疫苗的构建
Figure BDA0002706264950000151
Figure BDA0002706264950000161
实施例6:CMV DNA疫苗构建体的制备
为了制备CMV DNA疫苗构建体，本发明者制备了编码包含CMV糖蛋白B(gB)和pp65蛋白的蛋白的多核苷酸，然后将所述多核苷酸插入pGX-27载体的多克隆位点，从而制备出CMV DNA疫苗构建体(图5)。
实施例7:HBV DNA疫苗构建体的制备
为了制备HBV DNA疫苗构建体，本发明者制备了表达前S1、前S2、S抗原、表面抗原和核抗原的HBV DNA疫苗构建体，并将其插入到pGX-27载体中。
实施例8:HSV-2DNA疫苗构建体的制备
为了制备HSV-2DNA疫苗构建体，本发明者将HSV-2的UL39抗原分成5个片段(即UL3921-154(Δ78-104)、C2:UL391117-1142、N2:UL39165-227、N4-C1:UL39398-1116以及N3:UL39208-398)，然后将HSV-2的重组UL-39抗原和编码重组UL-39的基因构建体插入pGX-27载体的多克隆位点，制备HSV-2DNA疫苗构建体。
具体而言，通过将包含多核苷酸(如SEQ ID NO:38所示)的基因构建体插入pGX-27质粒载体来制备重组-UL39质粒DNA，所述多核苷酸对按照UL39-N1、UL39-C2、UL39-N2、UL39-N4-C1和UL39-N3的顺序连接的形式进行编码，基于所述形式分为UL39-N1(如SEQ IDNO:32所示)、UL39-C2(如SEQ ID NO:33所示)、UL39-N2(如SEQ ID NO:34所示)、UL39-N4-C1(如SEQ ID NO:35所示)和UL39-N3(如SEQ ID NO:36所示)(图4a)，并且重组UL39质粒DNA命名为“BD-02B”。
此外，本发明者通过将基因建构体插入pGX-27载体来制备tPA-Flt3L-gD-ICP0-ICP4质粒DNA，其中该基因建构体包含编码tPA-Flt3L-gD-ICP0-ICP4融合蛋白(如SEQ IDNO:42所示)的多核苷酸(如SEQ ID NO:43所示)，其中含HSV-2抗原而非重组UL39抗原，即为，连接信号序列(gD1-25)和跨膜结构域(gD350-363)被移除的糖蛋白D(gD26-349)(如SEQ IDNO:39所示)、核定位序列(NLS,ICP0510-516)被移除的受感染的细胞多肽0((ICP0Δ510-516,如SEQ ID NO:40所示)、和RS1.3部分(ICP767-1318)被移除的受感染细胞多肽4(ICP4Δ767-1318,如SEQ ID NO:41所示)(图4b)。上述两个质粒DNA都设计为以融合蛋白形式表达的质粒DNA，其中在N末端添加了一个tPA分泌信号序列(如SEQ ID NO:22所示)，该序列是为高效表达而优化的密码子，而且也添加了一个类似FMS的酪氨酸激酶3配体(Flt3L，如SEQ ID NO:24所示)(即免疫活性蛋白)。由此制备的DNA疫苗质粒DNA命名为“BD-02C”，由“BD-02B”和“BD-02C”组成的DNA疫苗组合物命名为“BD-02”。
实施例9:SFTS DNA疫苗构建体的制备
为了表达糖蛋白N和C(GnGc，如SEQ ID NO:44所示)、核衣壳(NP，如SEQ ID NO:45所示)和非结构(NS)蛋白(如SEQ ID NO:46所示)抗原，除了SFTS的抗原RNA依赖性RNA聚合酶(RdRP)，本发明者利用SFTS病毒基因组作为模板，通过PCR扩增编码每种蛋白质(如SEQID NOS:47至49所示)的多核苷酸。每个扩增的多核苷酸可操作地连接到CMV启动子(pCMV)、RSV启动子(pRSV)和EF-1a启动子(pEF-1α)，从而制备重组质粒DNA(SFTS质粒DNA)，其为SFTS DNA疫苗(图5)。
实验实施例1:确认IL-12和IL-21作为疫苗佐剂的潜力
本发明者进行了初步实验，以确认IL-12和IL-21以蛋白质和DNA形式作为疫苗佐剂的潜力。为此，具体而言，通过单独给个体接种HPV 2价DNA疫苗，并结合IL-12和/或IL-21检测hpv16和18型的免疫应答。
具体而言，通过体内电穿孔，使用
Figure BDA0002706264950000171
(韩国SLVAXiGEN)将各2μg的韩国专利公开出版物No.10-2017-0045254中所述的16/18型HPV 2价DNA疫苗构建体，每隔两周给C57BL/6小鼠的股肌注射两次，并与各2μg的mIL-12构建体和/或mIL-21构建体组合使用。然后，在第2次给药后两周，提取脾脏，计数对每种hpv16和HPV18有脾免疫反应细胞的数量(图6)。
结果，如图6所示，与单独使用IL-12或IL-21治疗相比，同时接受IL-12和IL-21治疗的小鼠中显示免疫应答的细胞显著增加，尤其是已证实在18型HPV中效果显著。
这些结果表明，IL-12和IL-21是可以协同提高疫苗的免疫应答的细胞因子。
实验实验例2:BD-121表达分析
2-1:酶联免疫吸附试验
本发明者根据示例性实施方案将实施例2和实施例4中制备的hBD-121构建体和mBD-121构建体分别转化至细胞，并检查IL-12和IL-21是否在转化细胞中正常表达。具体而言，利用脂质体2000将COS-7细胞系接种于每个100mm培养皿上并培养16小时，用空载体(对照(mock)质粒DNA)、实施例2-1中制备的hBD-121质粒DNA和实施例4中制备的mBD-121质粒DNA中的每一个进行转化。每个转化子在培养箱(37℃，CO2)中培养3天，回收每种条件下的COS-7细胞培养上清液并作为样本。每个样本中存在的IL-12和IL-21蛋白分别利用特异性识别IL-12和IL-21的抗体(IL-12:R&D Systems,Cat#D1200,IL-21:BioLegend,Cat#433808)通过ELISA分析进行定量(图7a和7b)。
结果如图7a显示，当以4μg DNA的量引入hIL-12时，引入hBD-121质粒DNA的样品中蛋白的表达水平大于4000pg/mL，从而证实这些蛋白正常表达，然而，当以4μg DNA的量引入hIL-21时，样品中蛋白的表达水平高达接近200ng/mL，因此证实这些蛋白以高水平表达。此外，如图7b所示，小鼠构建体显示出与人类构建体相似的结果。同时，在引入空载体的对照组中，这两种蛋白均未表达，从而证实本发明的疫苗佐剂表达系统工作正常。
1-2:蛋白免疫印迹分析
本发明者使用在实验实施例1-1中获得的细胞裂解物进行SDS-PAGE电泳，转移到尼龙膜上，并使用抗IL-12A抗体(Abcam,Cat#ab131039)、抗IL-12B抗体(Abcam,Cat#ab133752)和抗IL-21抗体(Abcam,Cat#ab5978)(图7c)进行蛋白免疫印迹分析。
结果如图7c所示，根据本发明的示例性实施方案，证实IL-12和IL-21通常通过转染BD-121质粒DNA来表达。
实验实施例3:BD-121对多种病毒增强免疫应答作用的分析
随后，根据实验实施例2的结果，本发明者检查根据本发明的示例性实施方案的BD-121构建体是否实际上能够改善针对各种病毒的免疫应答。
3-1:CMV的T细胞特异性免疫应答和抗体应答
首先，本发明者使用
Figure BDA0002706264950000182
(韩国SLVAXiGEN)通过体内电穿孔，将实施例6中制备的CMV DNA疫苗构建体(0.5μg)单独接种或与BD-121(0.5μg)组合接种到C57BL/6小鼠的股肌。特别地，作为对照组，分别表达IL-12和IL-21(即，构成BD121的组分)的表达载体单独或联合(IL-12+IL-21)与CMV DNA疫苗构建体(0.5μg)相结合(表2)。接种疫苗两周后，处死实验动物，提取脾脏，用CMVpp65(即CMV DNA疫苗中包含的CMV抗原)和CMVgB多肽库(图8a)分析抗CMV的T细胞特异性免疫应答。利用获得的血浆，通过CMV特异性抗体反应ELISA试验方法分析CMVgB特异性抗体反应(图8a至8c)。
[表2]本发明的CMV疫苗给药组合物
Figure BDA0002706264950000181
Figure BDA0002706264950000191
结果如图8b所示，与单独施用CMV DNA疫苗时相比，根据本发明示例性实施方案的BD-121疫苗佐剂产生显著更高的T细胞特异性免疫应答。具体而言，当CMV DNA疫苗单独施用时，CMVP65和CMVgB特异性免疫应答分别为300个斑点形成细胞(SFCs)和700个SFCs，而当CMV DNA疫苗与BD-121联合施用时，CMVP65和CMVgB特异性免疫应答分别为2000个SFC和2100个SFC。因此，表明抗原特异性T细胞反应分别增加约3至7倍。同时，如图8c所示，通过对CMV特异性抗体产生水平的分析，当单独使用CMV DNA疫苗治疗时，本发明的BD-121诱导了相当低的抗体反应。这些结果表明，根据本发明示例性实施方案，BD-121通过选择性地增强T细胞特异性免疫应答(即，细胞介导的免疫应答)来诱导免疫增强，而不是通过非选择性地增加免疫应答的方法。
3-2:HBV的T细胞特异性免疫应答和抗体应答
本发明者使用
Figure BDA0002706264950000192
(韩国SLVAXiGEN)通过体内电穿孔将在实施例7中制备的HBV DNA疫苗构建体(2μg)单独或与BD-121(2μg)组合接种到C57BL/6小鼠的股肌，两周间隔两次(初始免疫和加强免疫)。在第二个接种日，即从增强后两周起，处死实验动物，提取脾脏，使用HBV DNA疫苗构建体中包含的HBV抗原(HBsAg、HBcAg和PreS1/S2)多肽库，通过体外ELISPOT分析来分析抗HBV的T细胞特异性免疫应答(图8a)。此外，对于HBsAg特异性抗体反应，利用获得的血浆，用ELISA检测方法分析HBV特异性抗体反应(图9a和9b)。
结果如图9a所示，与单独施用HBV DNA疫苗构建体相比，根据本发明示例性实施方案的BD-121疫苗佐剂导致显著更高的T细胞特异性免疫应答。尤其是对HBsAg和HBcAg抗原有反应的脾脏免疫细胞数量急剧增加。然而，如图9b所示，通过对HBV特异性抗体产生水平的分析，证实与预期相反，本发明的BD-121与使用HBV DNA疫苗构建体单独处理相比，没有增加抗体滴度。这些结果证实，本发明的BD-121选择性增强T细胞特异性免疫应答并不局限于特定的病毒抗原，而是出现在大多数感染性病毒的类似特征中的现象。
3-3:SFTS的T细胞特异性免疫应答和抗体应答
将pGX-27(G1)作为空载体给药，或将SFTS DNA疫苗(12μg)单独给药(G2)或与BD-121(12μg)(G3)联合，使用
Figure BDA0002706264950000202
(韩国SLVAXiGEN)通过体内电穿孔(促进)对C57BL/6小鼠的股骨肌肉进行两次为期两周的注射。然后，在第一次接种(初始免疫)后处死小鼠提取脾脏，然后使用SFTS DNA疫苗中包含的SFTS抗原GnGc、NP和NS进行ELISPOT分析，并将剩下的实验动物(n＝5)处死两周在第2天接种(加强免疫)后提取脾脏，通过ELISPOT分析对T细胞特异性免疫应答进行如上所述的分析(图10a至10c)。
结果如图10b所示，与单独用SFTS DNA疫苗处理时相比，根据本发明示例性实施方案的BD-121疫苗佐剂即使仅基于初始免疫也诱导显著的T细胞特异性免疫应答，如图10c所示，在加强免疫时T细胞特异性免疫反应进一步增强。同时，单独施用SFTS DNA疫苗时，未观察到有因加强免疫而增强免疫反应的现象。因此，证实根据本发明示例性实施方案的BD-121在通过第二次接种加强以及通过第一次接种的初始免疫表现出更有效的疫苗佐剂效果。
然后，根据本发明的一个示例性实施方案，本发明者通过改变SFTS DNA疫苗和BD-121疫苗佐剂的接种率来比较初始免疫和加强免疫的效果。
具体来说，通过
Figure BDA0002706264950000203
(韩国SLVAXiGEN)采用体内电穿孔法将一种空载体(对照DNA，G1)、一种SFTS DNA疫苗(G2)、SFTS DNA疫苗BD-121的量为1:0.3(G3)的疫苗、SFTSDNA疫苗BD-121的量为1：1(G4)的疫苗和SFTS DNA疫苗BD-121的量为1：3(G5)的疫苗施用于C57BL/6小鼠股肌，每隔两周施用两次(加强免疫)。然后，在第一次接种(初始免疫)后处死部分小鼠，提取脾脏，然后利用SFTS DNA疫苗中SFTS的抗原GnGc、NP和NS进行ELISPOT分析，分析针对SFTS的T细胞特异性免疫反应，其余实验动物(n＝5)在第二次接种(加强免疫)后2周处死取脾脏，取血浆进行ELISPOT分析SFTS特异性抗体反应(表3，图11a和11b)。
[表3]BD-121对SFTS DNA疫苗剂量变化实验概要
Figure BDA0002706264950000201
结果如图11a所示，在单独施用SFTS DNA疫苗的组和接种疫苗的组之间(其中，根据本发明的示例性实施方案的SFTS DNA疫苗和BD-121以1:0.3的比例混合)没有因初始反应引起显著变化。然而，当剂量比增加到1:1时，实验组的T细胞特异性免疫反应的效果明显增强。同时，在加强免疫的情况下，如图11b所示，证实了与单独给药SFTS组相比，即使剂量比为1:0.3，T细胞特异性免疫应答也显著增加。这些佐剂均能以剂量依赖性的方式改善疫苗的免疫应答。
3-4:HSV-2的T细胞特异性免疫应答
当施用实施例8中制备的HSV-2DNA疫苗构建体时，本发明者测试了根据本发明的示例性实施方案制备的BD-121A是否能够增强T细胞特异性免疫应答。
具体而言，将实验动物分为使用空载体(pGX-27)(n＝10)施用的组；施用实施例8中制备的BD-02B(0.49μg)和BD-02C(0.49μg)的组(n＝11)；施用实施例8制备的BD-02B(1.4μg)和BD-02C(1.4μg)的组(n＝9)；使用实施例8制备的BD-02B(0.49μg)、BD-02C(0.49μg)和BD121A(0.49μg)(n＝11)施用的组；以及施用实施例8中制备的BD-02B(1.4μg)、BD-02C(1.4μg)和BD121A(1.4μg)的组(n＝10)，并使用
Figure BDA0002706264950000212
(韩国SLVAXiGEN)通过体内电穿孔(初始免疫和加强免疫)对C57BL/6小鼠的股骨肌肉施用两次，间隔两周。然后，在加强免疫一周后，施用Depo-provera(2mg)来调节雌性小鼠之间的性激素周期，并在加强免疫后两周感染HSV-2，然后分析实验动物随时间的病理评分和存活率(图12a至12c)。
结果如图12b所示，联合施用根据本发明示例性实施方案的BD121A的实验组的病理评分低于单独施用BD-02的组中的病理评分，并且这种现象被证明是剂量依赖性的。此外，生存率分析的结果，如图12c所示，与对照组相比，单独施用BD-02的组显示生存率以剂量依赖的方式增加，而根据本发明示例性实施方案的BD-121A的组(BD02-DP)则是共同给药后，实验动物的存活率显著提高。特别是，在分别施用BD-02和BD-121A(BD02-DP)1.4μg的剂量的组中，即使在感染18天后也没有观察到动物死亡。
然后，为了证实BD-02和BD-121A之间的最佳混合比例，本发明者以不同的比率(BD-02B:BD-02C:BD121A＝2:2:0.2、2:2:0.6、2:2:2和n＝5+5每组)调整BD-02和BD-121A，并使用
Figure BDA0002706264950000213
(韩国SLVAXiGEN)通过体内电穿孔(初始免疫和加强免疫)每隔两周接种两次将BD-02和BD-121A接种到C57BL/6小鼠的股肌中。初始免疫两周后每组处死5只小鼠，加强免疫两周后处死每组剩下的5只。之后从中提取脾脏，用ELISPOT分析法计算对BD-02中包含的各种HSV-2抗原反应的脾免疫细胞的数量(表4和图13a和13b)。
[表4]对HSV-2DNA疫苗的BD-121剂量变化实验的概要
Figure BDA0002706264950000211
Figure BDA0002706264950000221
结果如图13b所示，在单独给药BD-02的实验组(BD-02B+BD-02C)中，在初始免疫后两周内未观察到T细胞特异性免疫反应。同时，在联合施用根据本发明示例性实施方案的BD121A的组(BD02 DP)中，以BD121A浓度依赖方式的T细胞特异性免疫应答确认为1:1:0.6比例。然而，在较高剂量下，未观察到T细胞特异性免疫反应的进一步增强。因此，在根据本发明的示例性实施方案中的BD-02和BD121A疫苗组合物，证实即使在混合比例(BD-02B:BD-02C:BD121A＝2:2:0.6)下也显示出显著的免疫刺激效果。
3-5:HPV的T细胞特异性免疫反应分析
本发明者使用韩国专利公开出版物No.10-2017-0045254中描述的2价HPV类型16和18的DNA疫苗构建体来检测BD-121是否能够增强T细胞特异性免疫应答。
具体而言，将C57BL/6小鼠(实验动物)分为以下组：一组施用空载体(施用pGX-27，n＝10)，一组单独施用2价HPV DNA疫苗构建体(n＝10)，一组施用2价HPV DNA疫苗构建体(8μg)+BD-121(1μg)(n＝10)，以及一组施用2价HPV DNA疫苗构建物(8μg)+BD-121(3μg)(n＝10)。然后，在第一次接种疫苗后两周(每个实验组，n＝5)处死一半实验动物，提取脾脏用于根据初始免疫情况分析T细胞特异性免疫反应，而剩下的一半(每个实验组，n＝5)初始免疫后两周进行第二次接种(加强免疫)，同样，其余一半在加强免疫后两周处死，提取脾脏，通过ELISPOT分析计算对抗原特异性反应的脾免疫细胞数量的方法分析T细胞特异性免疫反应(图14)。
结果如图14所示，当单独施用2价HPV DNA疫苗构建体时，T细胞特异性免疫应答可忽略不计，而当根据本发明的示例性实施方案施用BD-121时，浓度依赖性的T细胞特异性免疫应答增强。
然后，为了确认作为疫苗佐剂的BD-121和BD-121A对于根据本发明的示例性实施方案制备的HPV14 DNA疫苗(BD-14A)的性能，本发明者施用在实施例5中制备的BD-14s(BD-14A、BD-14B和BD-14C)，单独或与BD-121A结合施用，之后对结果进行分析(表5)。
[表5]不同疫苗佐剂对14价HPV DNA疫苗的效果分析实验概况
Figure BDA0002706264950000231
具体而言，将C57BL/6小鼠(即实验动物)分为空载体(n＝10)给药组、BD-14(2μg)(n＝10)给药组、BD-14(2μg)+MIP-1α(2μg)(n＝10)给药组、BD-14(2μg)+BD-121(2μg)(n＝10)给药组和BD-14(2μg)+BD-121(2μg)(n＝10)给药组和BD-14(2μg)+BD-121(2μg)+Mip-1α(2μg)(n＝10)给药组。然后，在第一次接种疫苗(初始免疫)后两周处死一半实验动物(每个实验组，n＝5)，提取脾脏用于根据初始免疫情况分析T细胞特异性免疫反应，而剩下的一半(每个实验组，n＝5)给药后2周进行第二次接种(加强免疫)，同样，在加强免疫2周后处死这一半实验动物，提取脾脏，通过ELISPOT分析计算对抗原特异性反应的脾免疫细胞数量的方法分析T细胞特异性免疫反应(图15a和15b)。
结果如图15b所示，与单独施用BD-10A组和联合施用BD-10A与MIP-1α组相比，BD-121和BD-121A都显著增强了T细胞特异性免疫应答。
实验实施例4：临床前分析
根据实施例3的结果，本发明者证实，根据本发明的示例性实施方案的BD-121能够增强针对各种病毒抗原的T细胞特异性免疫应答，而不会对抗体产生太大影响。在这方面，本发明者检查了与单独使用DNA疫苗相比，BD-121是否能够减轻由病毒感染引起的病理症状，即使在使用真实动物模型的临床前研究中也是如此。
具体而言，作为实验动物，每个实验组使用三只食蟹猴，给药组分为BD-02单独给药组和与BD-121A联合给药组，每组每隔3周给药2次，每次经肌肉电穿孔给药1.8mg DNA，并且在给药前(VS，第14天和第5天)、第一次给药后的时间点(VT1，第21天)和第二次给药后的时间点(VT2，第35天)对BD-02特异性免疫应答进行评估(图16a)。在每个时间点，分离的外周血单个核细胞(PBMCs)被BD-02中的HSV-2gD和UL39抗原的多肽库刺激，并通过体外ELIPOST分析法评估表现出特异性反应的细胞，将其标记为斑点形成细胞(SFCs)。
结果如图16b所示，证实在给药前的时间点未观察到BD-02特异性免疫应答，但BD-02-特异性免疫应答在给药后以剂量-频率依赖性方式增加。特别地，证实与单独给药组相比，BD-121A联合给药可显著提高BD-02特异性免疫应答，且随着给药频率的增加/减少，差异变得更大。
如上所述，经证实，根据本发明的示例性实施方案的BD-121和BD-121A能够显著增强针对来自各种病毒感染的各种病毒抗原的T细胞特异性免疫应答，而不会对抗体产生太大影响。因此，由于根据本发明示例性实施方案的BD-121和BD-121A可显著增强T细胞特异性免疫应答，而该免疫应答选择性地增加对抗原特异性反应的T细胞而不影响抗体的产生，根据本发明的示例性实施方案的BD-121和BD-121A通过癌症细胞介导的对癌症(例如，宫颈癌)的免疫增强和对各种感染性病毒的预防和治疗来用作免疫治疗剂。例如，在抗肿瘤免疫治疗应答的情况下，已知抗体应答对肿瘤细胞或肿瘤相关抗原的治疗作用很小，而有效提高肿瘤细胞或肿瘤相关抗原特异性的T细胞应答非常重要。即使在对HSV-2或HPV等病毒感染的治疗性免疫应答中，有效地提高T细胞应答也至关重要，因为T细胞应答起着至关重要的作用，而非抗体应答。基于这些原因，BD-121和BD-121A的应用具有一个优点，即可以导致开发用于上述各种疾病的新型治疗剂，其仅能选择性地改善对抗原或疫苗特异的T细胞反应。
虽然已参照具体实施例描述了新型疫苗佐剂，但它们仅用于说明目的。因此，本领域技术人员将理解，在不脱离由所附权利要求限定的本发明的精神和范围的情况下，可以对其进行各种修改和改变。因此，本发明的实际保护范围应当由所附权利要求的技术范围确定。
工业适用性
通过对癌症(例如宫颈癌)的细胞免疫增强和对各种感染性病毒的感染的预防和治疗，根据本发明实施方案的免疫佐剂可有效地用作免疫治疗剂。
独立文本的序列表
SEQ ID NO:1是人IL-12p35多肽的氨基酸序列。
SEQ ID NO:2是人IL-12p40多肽的氨基酸序列。
SEQ ID NO:3是人IL-21多肽的氨基酸序列。
SEQ ID NO:4是编码人IL-12p35多肽的多核苷酸的核酸序列。
SEQ ID NO:5是编码人IL-12p40多肽的多核苷酸的核酸序列。
SEQ ID NO:6是EMCV衍生的内部核糖体进入位点的核酸序列。
SEQ ID NO:7是RSV启动子的核酸序列。
SEQ ID NO:8是编码人IL-12多肽的多核苷酸的核酸序列。
SEQ ID NO:9是人EF-1α启动子的核酸序列。
SEQ ID NO:10是人MIP-1α多肽的氨基酸序列。
SEQ ID NO:11是编码人MIP-1α多肽的多核苷酸的核酸序列。
SEQ ID NO:12是小鼠IL-12p35多肽的氨基酸序列。
SEQ ID NO:13是小鼠IL-12p40多肽的氨基酸序列。
SEQ ID NO:14是编码小鼠IL-12p35多肽的多核苷酸的核酸序列。
SEQ ID NO:15是编码小鼠IL-12p40多肽的多核苷酸的核酸序列。
SEQ ID NO:16是小鼠IL-21多肽的氨基酸序列。
SEQ ID NO:17是编码小鼠IL-21多肽的多核苷酸的核酸序列。
SEQ ID NO:18是小鼠MIP-1α多肽的氨基酸序列。
SEQ ID NO:19是编码小鼠MIP-1α多肽的多核苷酸的核酸序列。
SEQ ID NO:20是(GS)5连接子多肽的氨基酸序列。
SEQ ID NO:21是编码(GS)5连接子多肽的多核苷酸的核酸序列。
SEQ ID NO:22是tPA先导肽的氨基酸序列。
SEQ ID NO:23是编码tPA先导肽的多核苷酸的核酸序列。
SEQ ID NO:24是信号序列被截断的Flt3L多肽的氨基酸序列。
SEQ ID NO:25是编码Flt3L多肽的多核苷酸的核酸序列。
SEQ ID NOs:26、28和30分别是根据本发明实施方案的DNA疫苗构建物中包含的重组抗原融合蛋白的氨基酸序列。
SEQ ID NOs:27、29和31分别为编码重组抗原复合物的多核苷酸核酸序列。
SEQ ID NO:32是UL39-N1多肽的氨基酸序列。
SEQ ID NO:33是UL39-C2多肽的氨基酸序列。
SEQ ID NO:34是UL39-N2多肽的氨基酸序列。
SEQ ID NO:35是UL39-N4-C1多肽的氨基酸序列。
SEQ ID NO:36是UL39-N3多肽的氨基酸序列。
SEQ ID NO:37是UL39-N1、UL39-C2、UL39-N2、UL39-N4-C1和UL39-N3顺序连接的重组UL39抗原蛋白的氨基酸序列。
SEQ ID NO:39是HSV-2糖蛋白D(gD)的氨基酸序列，已移除信号序列和跨膜结构域。
SEQ ID NO:40是已移除核定位序列的ICP0蛋白的氨基酸序列。
SEQ ID NO:41是已移除RS1.3部分的ICP4蛋白质的氨基酸序列。
SEQ ID NO:42是tPA-Flt3L-gD-ICP0-ICP4融合蛋白的氨基酸序列。
SEQ ID NO:43是编码如SEQ ID NO:42所示的融合蛋白的多核苷酸的核酸序列。
SEQ ID NO:44是SFTS的抗原GnGc蛋白的氨基酸序列。
SEQ ID NO:45是SFTS的NP蛋白的氨基酸序列。
SEQ ID NO:46是SFTS的NS蛋白的氨基酸序列。
SEQ ID NOs:47至49分别为编码GnGc、NP和NS蛋白的多核苷酸的核酸序列。
SEQ ID NOs:50至68是可用于本发明的各种连接子多肽的氨基酸序列。
SEQUENCE LISTING
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<120> 一种新型疫苗佐剂
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aaagagccca agaacaaaac cttcctgaga tgcgaagcca agaattatag cgggaggttt 420
acctgttggt ggctgaccac aatctctact gacctgacct ttagtgtgaa gtctagtagg 480
gggtcaagcg atcctcaggg agtgacctgc ggagcagcta cactgagcgc agagcgggtc 540
agaggagaca acaaggagta cgaatattcc gtggagtgcc aggaagattc tgcatgtccc 600
gcagccgagg aatccctgcc tatcgaagtg atggtggacg ccgtgcacaa gctgaaatac 660
gaaaactaca catcctcttt ctttatccgg gacatcatta agccagatcc ccctaaaaac 720
ctgcagctga agcccctgaa aaattcacga caggtggagg tcagctggga ataccctgat 780
acatggagca ctccacattc ttatttcagt ctgacttttt gcgtgcaggt ccagggcaag 840
agtaaacgag agaagaaaga ccgggtcttc accgataaga catccgctac tgtgatctgt 900
cggaaaaacg ccagtatttc agtgagggct caggaccgct actatagttc aagctggtca 960
gagtgggcaa gcgtgccctg ttcctag 987
<210> 6
<211> 579
<212> DNA
<213> Artificial Sequence
<220>
<223> ECMV IRES
<400> 6
cccctctccc tccccccccc ctaacgttac tggccgaagc cgcttggaat aaggccggtg 60
tgcgtttgtc tatatgttat tttccaccat attgccgtct tttggcaatg tgagggcccg 120
gaaacctggc cctgtcttct tgacgagcat tcctaggggt ctttcccctc tcgccaaagg 180
aatgcaaggt ctgttgaatg tcgtgaagga agcagttcct ctggaagctt cttgaagaca 240
aacaacgtct gtagcgaccc tttgcaggca gcggaacccc ccacctggcg acaggtgcct 300
ctgcggccaa aagccacgtg tataagatac acctgcaaag gcggcacaac cccagtgcca 360
cgttgtgagt tggatagttg tggaaagagt caaatggctc tcctcaagcg tattcaacaa 420
ggggctgaag gatgcccaga aggtacccca ttgtatggga tctgatctgg ggcctcggtg 480
cacatgcttt acatgtgttt agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg 540
ggacgtggtt ttcctttgaa aaacacgatg ataatatgg 579
<210> 7
<211> 527
<212> DNA
<213> Artificial Sequence
<220>
<223> RSV promoter
<400> 7
ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca 60
acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg 120
ctgcttcgcg atgtacgggc cagatatacg cgtatctgag gggactaggg tgtgtttagg 180
cgaaaagcgg ggcttcggtt gtacgcggtt aggagtcccc tcaggatata gtagtttcgc 240
ttttgcatag ggagggggaa atgtagtctt atgcaatact cttgtagtct tgcaacatgg 300
taacgatgag ttagcaacat gccttacaag gagagaaaaa gcaccgtgca tgccgattgg 360
tggaagtaag gtggtacgat cgtgccttat taggaaggca acagacgggt ctgacatgga 420
ttggacgaac cactgaattc cgcattgcag agatattgta tttaagtgcc tagctcgata 480
caataaacgc catttgacca ttcaccacat tggtgtgcac ctccaag 527
<210> 8
<211> 468
<212> DNA
<213> Homo sapiens
<400> 8
atggaacgga ttgtcatttg cctgatggtc atttttctgg gaaccctggt ccacaagtca 60
agcagtcagg gccaggatag gcacatgatt aggatgcgcc agctgatcga cattgtggat 120
cagctgaaga actacgtgaa tgacctggtc cctgagtttc tgcctgcacc agaggatgtc 180
gaaacaaact gcgaatggag cgccttctcc tgttttcaga aggcccagct gaaatccgct 240
aacaccggca acaatgagcg aatcatcaac gtgagcatca agaagctgaa gcggaaaccc 300
cctagcacta atgctgggcg gagacagaaa catagactga cctgcccctc ttgtgacagt 360
tatgaaaaga aaccacccaa ggagttcctg gaacgcttta aaagtctgct gcagaaaatg 420
attcaccagc acctgtcctc cagaactcac gggtccgaag attcctaa 468
<210> 9
<211> 1189
<212> DNA
<213> Artificial Sequence
<220>
<223> hEF-1alpha promoter
<400> 9
cgtgaggctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt 60
tggggggagg ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg 120
aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa 180
gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggtaa 240
gtgccgtgtg tggttcccgc gggcctggcc tctttacggg ttatggccct tgcgtgcctt 300
gaattacttc cacgcccctg gctgcagtac gtgattcttg atcccgagct tcgggttgga 360
agtgggtggg agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt 420
gaggcctggc ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt 480
ctcgctgctt tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt 540
tttttctggc aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt 600
tttggggccg cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg 660
ggcctgcgag cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct 720
ctggtgcctg gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg 780
tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca 840
aaatggagga cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg 900
gcctttccgt cctcagccgt cgcttcatgt gactccacgg agtaccgggc gccgtccagg 960
cacctcgatt agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt 1020
tatgcgatgg agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac 1080
ttgatgtaat tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag 1140
cctcagacag tggttcaaag tttttttctt ccatttcagg tgtcgtgaa 1189
<210> 10
<211> 92
<212> PRT
<213> Homo sapiens
<400> 10
Met Gln Val Ser Thr Ala Ala Leu Ala Val Leu Leu Cys Thr Met Ala
1 5 10 15
Leu Cys Asn Gln Phe Ser Ala Ser Leu Ala Ala Asp Thr Pro Thr Ala
20 25 30
Cys Cys Phe Ser Tyr Thr Ser Arg Gln Ile Pro Gln Asn Phe Ile Ala
35 40 45
Asp Tyr Phe Glu Thr Ser Ser Gln Cys Ser Lys Pro Gly Val Ile Phe
50 55 60
Leu Thr Lys Arg Ser Arg Gln Val Cys Ala Asp Pro Ser Glu Glu Trp
65 70 75 80
Val Gln Lys Tyr Val Ser Asp Leu Glu Leu Ser Ala
85 90
<210> 11
<211> 279
<212> DNA
<213> Homo sapiens
<400> 11
atgcaggtgt caaccgccgc cctggctgtc ctgctgtgca ctatggctct gtgcaatcag 60
ttttccgcaa gtctggccgc tgatactccc accgcctgct gtttctctta cacaagtagg 120
cagatccctc agaacttcat tgctgactat tttgagacta gctcccagtg cagcaagccc 180
ggcgtgatct ttctgaccaa gcggagccgg caggtctgtg ccgatccctc cgaagaatgg 240
gtgcagaagt atgtctccga cctggaactg tcagcataa 279
<210> 12
<211> 215
<212> PRT
<213> Mus musculus
<400> 12
Met Cys Gln Ser Arg Tyr Leu Leu Phe Leu Ala Thr Leu Ala Leu Leu
1 5 10 15
Asn His Leu Ser Leu Ala Arg Val Ile Pro Val Ser Gly Pro Ala Arg
20 25 30
Cys Leu Ser Gln Ser Arg Asn Leu Leu Lys Thr Thr Asp Asp Met Val
35 40 45
Lys Thr Ala Arg Glu Lys Leu Lys His Tyr Ser Cys Thr Ala Glu Asp
50 55 60
Ile Asp His Glu Asp Ile Thr Arg Asp Gln Thr Ser Thr Leu Lys Thr
65 70 75 80
Cys Leu Pro Leu Glu Leu His Lys Asn Glu Ser Cys Leu Ala Thr Arg
85 90 95
Glu Thr Ser Ser Thr Thr Arg Gly Ser Cys Leu Pro Pro Gln Lys Thr
100 105 110
Ser Leu Met Met Thr Leu Cys Leu Gly Ser Ile Tyr Glu Asp Leu Lys
115 120 125
Met Tyr Gln Thr Glu Phe Gln Ala Ile Asn Ala Ala Leu Gln Asn His
130 135 140
Asn His Gln Gln Ile Ile Leu Asp Lys Gly Met Leu Val Ala Ile Asp
145 150 155 160
Glu Leu Met Gln Ser Leu Asn His Asn Gly Glu Thr Leu Arg Gln Lys
165 170 175
Pro Pro Val Gly Glu Ala Asp Pro Tyr Arg Val Lys Met Lys Leu Cys
180 185 190
Ile Leu Leu His Ala Phe Ser Thr Arg Val Val Thr Ile Asn Arg Val
195 200 205
Met Gly Tyr Leu Ser Ser Ala
210 215
<210> 13
<211> 335
<212> PRT
<213> Mus musculus
<400> 13
Met Cys Pro Gln Lys Leu Thr Ile Ser Trp Phe Ala Ile Val Leu Leu
1 5 10 15
Val Ser Pro Leu Met Ala Met Trp Glu Leu Glu Lys Asp Val Tyr Val
20 25 30
Val Glu Val Asp Trp Thr Pro Asp Ala Pro Gly Glu Thr Val Asn Leu
35 40 45
Thr Cys Asp Thr Pro Glu Glu Asp Asp Ile Thr Trp Thr Ser Asp Gln
50 55 60
Arg His Gly Val Ile Gly Ser Gly Lys Thr Leu Thr Ile Thr Val Lys
65 70 75 80
Glu Phe Leu Asp Ala Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Thr
85 90 95
Leu Ser His Ser His Leu Leu Leu His Lys Lys Glu Asn Gly Ile Trp
100 105 110
Ser Thr Glu Ile Leu Lys Asn Phe Lys Asn Lys Thr Phe Leu Lys Cys
115 120 125
Glu Ala Pro Asn Tyr Ser Gly Arg Phe Thr Cys Ser Trp Leu Val Gln
130 135 140
Arg Asn Met Asp Leu Lys Phe Asn Ile Lys Ser Ser Ser Ser Ser Pro
145 150 155 160
Asp Ser Arg Ala Val Thr Cys Gly Met Ala Ser Leu Ser Ala Glu Lys
165 170 175
Val Thr Leu Asp Gln Arg Asp Tyr Glu Lys Tyr Ser Val Ser Cys Gln
180 185 190
Glu Asp Val Thr Cys Pro Thr Ala Glu Glu Thr Leu Pro Ile Glu Leu
195 200 205
Ala Leu Glu Ala Arg Gln Gln Asn Lys Tyr Glu Asn Tyr Ser Thr Ser
210 215 220
Phe Phe Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln
225 230 235 240
Met Lys Pro Leu Lys Asn Ser Gln Val Glu Val Ser Trp Glu Tyr Pro
245 250 255
Asp Ser Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Lys Phe Phe Val
260 265 270
Arg Ile Gln Arg Lys Lys Glu Lys Met Lys Glu Thr Glu Glu Gly Cys
275 280 285
Asn Gln Lys Gly Ala Phe Leu Val Glu Lys Thr Ser Thr Glu Val Gln
290 295 300
Cys Lys Gly Gly Asn Val Cys Val Gln Ala Gln Asp Arg Tyr Tyr Asn
305 310 315 320
Ser Ser Cys Ser Lys Trp Ala Cys Val Pro Cys Arg Val Arg Ser
325 330 335
<210> 14
<211> 648
<212> DNA
<213> Mus musculus
<400> 14
atgtgccaga gcagatacct gctgttcctg gccaccctgg ccctgctgaa ccacctgagc 60
ctggccagag tgatccccgt gagcggcccc gccagatgcc tgagccagag cagaaacctg 120
ctgaagacaa ccgacgacat ggtgaagacc gccagagaga agctgaagca ctacagctgc 180
accgccgagg acatcgacca cgaggacatc accagagacc agaccagcac cctgaagacc 240
tgcctgcccc tggagctgca caagaacgag agctgcctgg ccacaagaga gaccagcagc 300
accacaagag gcagctgcct gcctccccag aagaccagcc tgatgatgac cctgtgcctg 360
ggcagcatct acgaggacct gaagatgtac cagaccgagt tccaggccat caacgctgcc 420
ctgcagaacc acaatcacca gcagatcatc ctggacaagg gcatgctggt ggccatcgac 480
gagctgatgc agagcctgaa ccacaacggc gagaccctga gacagaagcc ccctgtgggc 540
gaggccgatc cctacagagt gaagatgaag ctgtgcatcc tgctgcacgc cttcagcacc 600
agagtggtga ccatcaacag agtgatgggc tacctgagca gcgcctga 648
<210> 15
<211> 1008
<212> DNA
<213> Mus musculus
<400> 15
atgtgccccc agaagctgac catcagctgg ttcgccatcg tgctgctggt gagccccctg 60
atggccatgt gggagctgga gaaggacgtg tacgtggtgg aggtggactg gacccccgac 120
gcccccggcg agaccgtgaa cctgacctgc gacacccccg aggaggacga catcacctgg 180
accagcgacc agaggcacgg cgtgatcggc agcggcaaga ccctgaccat caccgtgaag 240
gagttcctgg acgccggcca gtacacctgc cacaagggcg gcgagaccct gagccacagc 300
cacctgctgc tgcacaagaa ggagaacggc atctggagca ccgagatcct gaagaacttc 360
aagaacaaga ccttcctgaa gtgcgaggcc cccaactaca gcggcaggtt cacctgcagc 420
tggctggtgc agaggaacat ggacctgaag ttcaacatca agagcagcag cagcagcccc 480
gacagcaggg ccgtgacctg cggcatggcc agcctgagcg ccgagaaggt gaccctggac 540
cagagggact acgagaagta cagcgtgagc tgccaggagg acgtgacctg ccccaccgcc 600
gaggagaccc tgcccatcga gctggccctg gaggccaggc agcagaacaa gtacgagaac 660
tacagcacca gcttcttcat cagggacatc atcaagcccg acccccccaa gaacctgcag 720
atgaagcccc tgaagaacag ccaggtggag gtgagctggg agtaccccga cagctggagc 780
accccccaca gctacttcag cctgaagttc ttcgtgagaa tccagaggaa gaaggagaag 840
atgaaggaga ccgaggaggg ctgcaaccag aagggcgcct tcctggtgga gaagaccagc 900
accgaggtgc agtgcaaggg cggcaacgtg tgcgtgcagg cccaggacag gtactacaac 960
agcagctgca gcaagtgggc ctgcgtgccc tgcagggtga ggagctaa 1008
<210> 16
<211> 146
<212> PRT
<213> Mus musculus
<400> 16
Met Glu Arg Thr Leu Val Cys Leu Val Val Ile Phe Leu Gly Thr Val
1 5 10 15
Ala His Lys Ser Ser Pro Gln Gly Pro Asp Arg Leu Leu Ile Arg Leu
20 25 30
Arg His Leu Ile Asp Ile Val Glu Gln Leu Lys Ile Tyr Glu Asn Asp
35 40 45
Leu Asp Pro Glu Leu Leu Ser Ala Pro Gln Asp Val Lys Gly His Cys
50 55 60
Glu His Ala Ala Phe Ala Cys Phe Gln Lys Ala Lys Leu Lys Pro Ser
65 70 75 80
Asn Pro Gly Asn Asn Lys Thr Phe Ile Ile Asp Leu Val Ala Gln Leu
85 90 95
Arg Arg Arg Leu Pro Ala Arg Arg Gly Gly Lys Lys Gln Lys His Ile
100 105 110
Ala Lys Cys Pro Ser Cys Asp Ser Tyr Glu Lys Arg Thr Pro Lys Glu
115 120 125
Phe Leu Glu Arg Leu Lys Trp Leu Leu Gln Lys Met Ile His Gln His
130 135 140
Leu Ser
145
<210> 17
<211> 441
<212> DNA
<213> Mus musculus
<400> 17
atggagagaa cactggtctg cctcgtggtc atcttcctgg gtactgtggc tcataaatcc 60
tcacctcagg gtcccgatag actgctgatc aggctgcggc acctcatcga cattgtggag 120
cagctcaaaa tctacgaaaa cgacctggac cccgagctgc tctctgcccc ccaggatgtc 180
aaggggcact gcgaacatgc cgctttcgca tgttttcaga aggccaaact gaagcccagc 240
aatcctggca acaataagac cttcatcatt gacctggtgg ctcagctcag gagacggctg 300
ccagcacgac gaggaggaaa gaaacagaaa catatcgcta agtgccctag ctgtgattcc 360
tatgagaaaa gaacaccaaa ggagttcctc gaaaggctca aatggctcct ccagaagatg 420
attcaccagc acctctccta a 441
<210> 18
<211> 92
<212> PRT
<213> Mus musculus
<400> 18
Met Lys Val Ser Thr Thr Ala Leu Ala Val Leu Leu Cys Thr Met Thr
1 5 10 15
Leu Cys Asn Gln Val Phe Ser Ala Pro Tyr Gly Ala Asp Thr Pro Thr
20 25 30
Ala Cys Cys Phe Ser Tyr Ser Arg Lys Ile Pro Arg Gln Phe Ile Val
35 40 45
Asp Tyr Phe Glu Thr Ser Ser Leu Cys Ser Gln Pro Gly Val Ile Phe
50 55 60
Leu Thr Lys Arg Asn Arg Gln Ile Cys Ala Asp Ser Lys Glu Thr Trp
65 70 75 80
Val Gln Glu Tyr Ile Thr Asp Leu Glu Leu Asn Ala
85 90
<210> 19
<211> 279
<212> DNA
<213> Mus musculus
<400> 19
atgaaggtga gcaccaccgc tctggctgtg ctgctctgca ccatgaccct ctgcaaccag 60
gtgttctcag ctccctacgg cgctgatacc cccaccgcct gctgcttcag ctacagccgg 120
aagatccccc ggcagttcat cgtggactac ttcgaaacca gcagcctgtg cagccagccc 180
ggcgtgatct tcctgaccaa acggaaccgg cagatctgcg ctgacagcaa agagacctgg 240
gtgcaggaat acatcaccga cctggaactg aacgcctaa 279
<210> 20
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> (GS)5 linker
<400> 20
Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser
1 5 10
<210> 21
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> polynucleotide encoding (GS)5 linker
<400> 21
ggatcaggca gtggttcagg atcaggtagt 30
<210> 22
<211> 25
<212> PRT
<213> Artificial Sequence
<220>
<223> tPA signal sequence
<400> 22
Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly
1 5 10 15
Ala Val Phe Val Ser Pro Ser His Ala
20 25
<210> 23
<211> 75
<212> DNA
<213> Artificial Sequence
<220>
<223> polynucleotide encoding tPA signal sequence
<400> 23
atggacgcca tgaagagagg cctgtgctgc gtgctgctgc tgtgcggcgc cgtgttcgtg 60
agccccagcc acgcc 75
<210> 24
<211> 156
<212> PRT
<213> Artificial Sequence
<220>
<223> Flt3L_27-182
<400> 24
Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala
1 5 10 15
Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val
20 25 30
Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp
35 40 45
Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala
50 55 60
Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His
65 70 75 80
Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe
85 90 95
Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu
100 105 110
Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu
115 120 125
Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Pro Pro Pro Trp Ser
130 135 140
Pro Arg Pro Leu Glu Ala Thr Ala Pro Thr Ala Pro
145 150 155
<210> 25
<211> 468
<212> DNA
<213> Artificial Sequence
<220>
<223> polynucleotide encoding Flt3L_27-182
<400> 25
acccaggact gcagcttcca gcacagcccc atcagcagcg acttcgccgt gaagatcaga 60
gagctgagcg actacctgct gcaggactac cccgtgaccg tggccagcaa cctgcaggac 120
gaggagctgt gcggcggcct gtggagactg gtgctggccc agagatggat ggagagactg 180
aagaccgtgg ccggcagcaa gatgcagggc ctgctggaga gagtgaacac cgagatccac 240
ttcgtgacca agtgcgcctt ccagcctccc cccagctgcc tgaggttcgt gcagaccaac 300
atcagcagac tgctgcagga gaccagcgag cagctggtgg ccctgaagcc ctggatcacc 360
agacagaact tcagcagatg cctggagctg cagtgccagc ccgacagcag caccctgccc 420
cctccctgga gccccagacc cctggaggcc accgctccca cagcccct 468
<210> 26
<211> 1957
<212> PRT
<213> Artificial Sequence
<220>
<223> BD-14A polypeptide
<400> 26
Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly Ala
1 5 10 15
Val Phe Val Ser Pro Ser His Ala Thr Gln Asp Cys Ser Phe Gln His
20 25 30
Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu Ser Asp
35 40 45
Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu Gln Asp
50 55 60
Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln Arg Trp
65 70 75 80
Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly Leu Leu
85 90 95
Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala Phe Gln
100 105 110
Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu
115 120 125
Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp Ile Thr
130 135 140
Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro Asp Ser
145 150 155 160
Ser Thr Leu Pro Pro Pro Trp Ser Pro Arg Pro Leu Glu Ala Thr Ala
165 170 175
Pro Thr Ala Pro Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Met Asp
180 185 190
Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly Ala Val
195 200 205
Phe Val Ser Pro Ser His Ala Thr Gln Asp Cys Ser Phe Gln His Ser
210 215 220
Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr
225 230 235 240
Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu Gln Asp Glu
245 250 255
Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln Arg Trp Met
260 265 270
Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly Leu Leu Glu
275 280 285
Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala Phe Gln Pro
290 295 300
Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu Leu
305 310 315 320
Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp Ile Thr Arg
325 330 335
Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro Asp Ser Ser
340 345 350
Thr Leu Pro Pro Pro Trp Ser Pro Arg Pro Leu Glu Ala Thr Ala Pro
355 360 365
Thr Ala Pro Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Met His Gln
370 375 380
Lys Arg Thr Ala Met Phe Gln Asp Pro Gln Glu Arg Pro Arg Lys Leu
385 390 395 400
Pro Gln Leu Cys Thr Glu Leu Gln Thr Thr Ile His Asp Ile Ile Leu
405 410 415
Glu Cys Val Tyr Cys Lys Gln Gln Leu Leu Arg Arg Glu Val Tyr Asp
420 425 430
Phe Ala Phe Arg Asp Leu Cys Ile Val Tyr Arg Asp Gly Asn Pro Tyr
435 440 445
Ala Val Cys Asp Lys Cys Leu Lys Phe Tyr Ser Lys Ile Ser Glu Tyr
450 455 460
Arg His Pro Ala Gly Gln Ala Glu Pro Asp Arg Ala His Tyr Asn Ile
465 470 475 480
Val Thr Phe Cys Cys Lys Cys Asp Ser Thr Leu Arg Leu Cys Val Gln
485 490 495
Ser Thr His Val Asp Ile Arg Thr Leu Glu Asp Leu Leu Met Gly Thr
500 505 510
Leu Gly Ile Val Cys Pro Ile Cys Ser Gln Lys Pro Gly Ser Gly Ser
515 520 525
Gly Ser Gly Ser Gly Ser Met His Gly Asp Thr Pro Thr Leu His Glu
530 535 540
Tyr Met Leu Asp Leu Gln Pro Glu Thr Thr Asp Leu Tyr Cys Tyr Glu
545 550 555 560
Gln Leu Asn Asp Ser Ser Glu Glu Glu Asp Glu Ile Asp Gly Pro Ala
565 570 575
Gly Gln Ala Glu Pro Asp Arg Ala His Tyr Asn Ile Val Thr Phe Cys
580 585 590
Cys Lys Pro Tyr Ala Val Cys Asp Lys Cys Leu Lys Phe Tyr Ser Lys
595 600 605
Ile Ser Glu Tyr Arg His Tyr Cys Tyr Ser Val Tyr Gly Thr Thr Leu
610 615 620
Glu Gln Gln Tyr Asn Lys Pro Leu Cys Asp Leu Leu Ile Arg Cys Ile
625 630 635 640
Asn Cys Gln Lys Pro Leu Cys Pro Glu Glu Lys Gln Arg His Leu Asp
645 650 655
Lys Lys Gln Arg Phe His Asn Ile Arg Gly Arg Trp Thr Gly Arg Cys
660 665 670
Met Ser Cys Cys Arg Ser Ser Arg Thr Arg Arg Glu Thr Gln Leu Gly
675 680 685
Ser Gly Ser Gly Ser Gly Ser Gly Ser Met Ala Arg Phe Glu Asp Pro
690 695 700
Thr Arg Arg Pro Tyr Lys Leu Pro Asp Leu Cys Thr Glu Leu Asn Thr
705 710 715 720
Ser Leu Gln Asp Ile Glu Ile Thr Cys Val Tyr Cys Lys Thr Val Leu
725 730 735
Glu Leu Thr Glu Val Phe Glu Phe Ala Phe Lys Asp Leu Phe Val Val
740 745 750
Tyr Arg Asp Ser Ile Pro His Ala Ala Cys His Lys Cys Ile Asp Phe
755 760 765
Tyr Ser Arg Ile Arg Glu Leu Arg His Tyr Ser Asp Ser Val Ile Asp
770 775 780
Gly Val Asn His Gln His Leu Pro Ala Arg Arg Ala Glu Pro Gln Arg
785 790 795 800
His Thr Met Leu Cys Met Cys Cys Lys Cys Glu Ala Arg Ile Glu Leu
805 810 815
Val Val Glu Ser Ser Ala Asp Asp Leu Arg Ala Phe Gln Gln Leu Phe
820 825 830
Leu Ser Thr Leu Ser Phe Val Cys Pro Trp Cys Ala Ser Gln Gln Gly
835 840 845
Ser Gly Ser Gly Ser Gly Ser Gly Ser Met Tyr Gly Pro Lys Ala Thr
850 855 860
Leu Gln Asp Ile Val Leu His Leu Glu Pro Gln Asn Glu Ile Pro Val
865 870 875 880
Asp Leu Leu Cys His Glu Gln Leu Ser Asp Ser Glu Glu Glu Asn Asp
885 890 895
Glu Ile Asp Gly Val Asn His Gln His Leu Pro Ala Arg Arg Ala Glu
900 905 910
Pro Gln Arg His Thr His Lys Cys Ile Asp Phe Tyr Ser Arg Ile Arg
915 920 925
Glu Leu Arg His Tyr Ser Asp Ser Val Tyr Gly Asp Thr Leu Glu Lys
930 935 940
Leu Thr Asn Thr Gly Leu Tyr Asn Leu Leu Ile Arg Cys Leu Arg Cys
945 950 955 960
Gln Lys Pro Leu Asn Pro Ala Glu Lys Leu Arg His Leu Asn Glu Lys
965 970 975
Arg Arg Phe His Asn Ile Ala Gly His Tyr Arg Gly Gln Cys His Ser
980 985 990
Cys Cys Asn Arg Ala Arg Gln Glu Arg Leu Gln Arg Arg Arg Glu Thr
995 1000 1005
Gln Val Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Leu Glu Met
1010 1015 1020
Phe Gln Asp Pro Ala Glu Arg Pro Tyr Lys Leu His Asp Leu Cys
1025 1030 1035
Asn Glu Val Glu Glu Ser Ile His Glu Ile Cys Leu Asn Cys Val
1040 1045 1050
Tyr Cys Lys Gln Glu Leu Gln Arg Ser Glu Val Tyr Asp Phe Ala
1055 1060 1065
Cys Tyr Asp Leu Cys Ile Val Tyr Arg Glu Gly Gln Pro Tyr Gly
1070 1075 1080
Val Cys Met Lys Cys Leu Lys Phe Tyr Ser Lys Ile Ser Glu Tyr
1085 1090 1095
Arg Trp Pro Ala Gly Gln Ala Lys Pro Asp Thr Ser Asn Tyr Asn
1100 1105 1110
Ile Val Thr Ser Cys Cys Lys Cys Glu Ala Thr Leu Arg Leu Cys
1115 1120 1125
Val Gln Ser Thr His Ile Asp Ile Arg Lys Leu Glu Asp Leu Leu
1130 1135 1140
Met Gly Thr Phe Gly Ile Val Cys Pro Gly Cys Ser Gln Arg Ala
1145 1150 1155
Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Met His Gly Glu Ile
1160 1165 1170
Thr Thr Leu Gln Asp Tyr Val Leu Asp Leu Glu Pro Glu Ala Thr
1175 1180 1185
Asp Leu Tyr Cys Tyr Glu Gln Leu Cys Asp Ser Ser Glu Glu Glu
1190 1195 1200
Glu Asp Thr Ile Asp Gly Pro Ala Gly Gln Ala Lys Pro Asp Thr
1205 1210 1215
Ser Asn Tyr Asn Ile Val Thr Ser Cys Cys Lys Pro Tyr Gly Val
1220 1225 1230
Cys Met Lys Cys Leu Lys Phe Tyr Ser Lys Ile Ser Glu Tyr Arg
1235 1240 1245
Trp Tyr Arg Tyr Ser Val Tyr Gly Glu Thr Leu Glu Lys Gln Cys
1250 1255 1260
Asn Lys Gln Leu Cys His Leu Leu Ile Arg Cys Ile Thr Cys Gln
1265 1270 1275
Lys Pro Leu Cys Pro Val Glu Lys Gln Arg His Leu Glu Glu Lys
1280 1285 1290
Lys Arg Phe His Asn Ile Gly Gly Arg Trp Thr Gly Arg Cys Met
1295 1300 1305
Ser Cys Trp Lys Pro Thr Arg Arg Glu Thr Glu Val Gly Ser Gly
1310 1315 1320
Ser Gly Ser Gly Ser Gly Ser Val Glu Gly Ser Met Ala Arg Phe
1325 1330 1335
Asp Asp Pro Lys Gln Arg Pro Tyr Lys Leu Pro Asp Leu Cys Thr
1340 1345 1350
Glu Leu Asn Thr Ser Leu Gln Asp Val Ser Ile Ala Cys Val Tyr
1355 1360 1365
Cys Lys Ala Thr Leu Glu Arg Thr Glu Val Tyr Gln Phe Ala Phe
1370 1375 1380
Lys Asp Leu Cys Ile Val Tyr Arg Asp Cys Ile Ala Tyr Ala Ala
1385 1390 1395
Cys His Lys Cys Ile Asp Phe Tyr Ser Arg Ile Arg Glu Leu Arg
1400 1405 1410
Tyr Tyr Ser Asn Ser Val Glu Ala Asp Gly Val Ser His Ala Gln
1415 1420 1425
Leu Pro Ala Arg Arg Ala Glu Pro Gln Arg His Lys Ile Leu Cys
1430 1435 1440
Val Cys Cys Lys Cys Asp Gly Arg Ile Asp Leu Thr Val Glu Ser
1445 1450 1455
Ser Ala Asp Asp Leu Arg Thr Leu Gln Gln Leu Phe Leu Ser Thr
1460 1465 1470
Leu Ser Phe Val Cys Pro Trp Cys Ala Thr Asn Gln Gly Ser Gly
1475 1480 1485
Ser Gly Ser Gly Ser Gly Ser Met His Gly Pro Arg Ala Thr Leu
1490 1495 1500
Gln Glu Ile Val Leu His Leu Glu Pro Gln Asn Glu Leu Asp Pro
1505 1510 1515
Val Asp Leu Leu Cys Tyr Glu Gln Leu Ser Glu Ser Glu Glu Glu
1520 1525 1530
Asn Asp Glu Ala Asp Gly Val Ser His Ala Gln Leu Pro Ala Arg
1535 1540 1545
Arg Ala Glu Pro Gln Arg His His Lys Cys Ile Asp Phe Tyr Ser
1550 1555 1560
Arg Ile Arg Glu Leu Arg Tyr Tyr Ser Asn Ser Val Tyr Gly Glu
1565 1570 1575
Thr Leu Glu Lys Ile Thr Asn Thr Glu Leu Tyr Asn Leu Leu Ile
1580 1585 1590
Arg Cys Leu Arg Cys Gln Lys Pro Leu Asn Pro Ala Glu Lys Arg
1595 1600 1605
Arg His Leu Lys Asp Lys Arg Arg Phe His Ser Ile Ala Gly Gln
1610 1615 1620
Tyr Arg Gly Gln Cys Asn Thr Cys Cys Asp Gln Ala Arg Gln Glu
1625 1630 1635
Arg Leu Arg Arg Arg Arg Glu Thr Gln Val Gly Ser Gly Ser Gly
1640 1645 1650
Ser Gly Ser Gly Ser Met Phe Gln Asp Ala Glu Glu Lys Pro Arg
1655 1660 1665
Thr Leu His Asp Leu Cys Gln Ala Leu Glu Thr Ser Val His Glu
1670 1675 1680
Ile Glu Leu Lys Cys Val Glu Cys Lys Lys Thr Leu Gln Arg Ser
1685 1690 1695
Glu Val Tyr Asp Phe Val Phe Ala Asp Leu Arg Ile Val Tyr Arg
1700 1705 1710
Asp Gly Asn Pro Phe Ala Val Cys Lys Val Cys Leu Arg Leu Leu
1715 1720 1725
Ser Lys Ile Ser Glu Tyr Arg His Tyr Asn Tyr Ser Leu Tyr Gly
1730 1735 1740
Arg Pro Asp Gly Gln Ala Gln Pro Ala Thr Ala Asn Tyr Tyr Ile
1745 1750 1755
Val Thr Cys Cys Tyr Thr Cys Asp Thr Thr Val Arg Leu Cys Ile
1760 1765 1770
Asn Ser Thr Thr Thr Asp Val Arg Thr Leu Gln Gln Leu Leu Met
1775 1780 1785
Gly Thr Cys Thr Ile Val Cys Pro Ser Cys Ala Gln Gln Gly Ser
1790 1795 1800
Gly Ser Gly Ser Gly Ser Gly Ser Met Arg Gly Asn Asn Pro Thr
1805 1810 1815
Leu Arg Glu Tyr Ile Leu Asp Leu His Pro Glu Pro Thr Asp Leu
1820 1825 1830
Phe Cys Tyr Glu Gln Leu Cys Asp Ser Ser Asp Glu Asp Glu Ile
1835 1840 1845
Gly Leu Asp Arg Pro Asp Gly Gln Ala Gln Pro Ala Thr Ala Asn
1850 1855 1860
Tyr Tyr Ile Val Thr Cys Cys Tyr Cys Leu Arg Leu Leu Ser Lys
1865 1870 1875
Ile Ser Glu Tyr Arg His Tyr Asn Tyr Ser Leu Tyr Gly Asp Thr
1880 1885 1890
Leu Glu Gln Thr Leu Lys Lys Cys Leu Asn Glu Ile Leu Ile Arg
1895 1900 1905
Cys Ile Ile Cys Gln Arg Pro Leu Cys Pro Gln Glu Lys Lys Arg
1910 1915 1920
His Val Asp Leu Asn Lys Arg Phe His Asn Ile Ser Gly Arg Trp
1925 1930 1935
Thr Gly Arg Cys Ala Val Cys Trp Arg Pro Arg Arg Arg Gln Thr
1940 1945 1950
Gln Val Gly Ser
1955
<210> 27
<211> 5304
<212> DNA
<213> Artificial Sequence
<220>
<223> polynucleotide encoding BD-14A polypeptide
<400> 27
atggacgcca tgaagagagg cctgtgctgc gtgctgctgc tgtgcggcgc cgtgttcgtg 60
agccccagcc acgccaccca ggactgcagc ttccagcaca gccccatcag cagcgacttc 120
gccgtgaaga tcagagagct gagcgactac ctgctgcagg actaccccgt gaccgtggcc 180
agcaacctgc aggacgagga gctgtgcggc ggcctgtgga gactggtgct ggcccagaga 240
tggatggaga gactgaagac cgtggccggc agcaagatgc agggcctgct ggagagagtg 300
aacaccgaga tccacttcgt gaccaagtgc gccttccagc ctccccccag ctgcctgagg 360
ttcgtgcaga ccaacatcag cagactgctg caggagacca gcgagcagct ggtggccctg 420
aagccctgga tcaccagaca gaacttcagc agatgcctgg agctgcagtg ccagcccgac 480
agcagcaccc tgccccctcc ctggagcccc agacccctgg aggccaccgc tcccacagcc 540
cctggcagcg ggtccggaag tgggtctgga tctatgcacc agaagagaac cgccatgttc 600
caggaccccc aggagagacc cagaaagctg ccccagctgt gcaccgagct gcagaccaca 660
atccacgaca tcatcctgga gtgcgtgtac tgcaagcagc agctgctgag aagagaggtg 720
tacgacttcg ccttcagaga cctgtgcatc gtgtacagag atggcaaccc ttatgctgtc 780
tgtgataaat gtctcaaatt ttattccaaa attagtgaat ataggcatcc agcaggacag 840
gctgaaccag atagggctca ttataatatt gtcacatttt gttgtaaatg cgacagcacc 900
ctgagactgt gcgtgcagag cacccacgtg gacatcagaa ccctggagga cctgctgatg 960
ggcaccctgg gcatcgtgtg ccccatctgc agccagaagc ctggcagcgg ctctggctcc 1020
ggcagtggct caatgcacgg cgacacaccc accctgcacg agtacatgct ggacctgcag 1080
cccgagacta ccgacctgta ctgctacgag cagctgaacg acagcagcga ggaagaggac 1140
gagatcgacg gccctgctgg ccaggccgag cccgacagag cccactacaa catcgtgacc 1200
ttctgctgca agccctacgc cgtgtgcgac aagtgcctga agttctacag caagatcagc 1260
gagtacagac actactgcta cagcgtgtac ggcaccaccc tggagcagca gtacaacaag 1320
cccctgtgcg acctgctgat cagatgcatc aactgccaga agcccctgtg ccccgaggag 1380
aagcagagac acctggacaa gaagcagaga ttccacaaca tcagaggcag atggaccggc 1440
agatgcatga gctgctgcag aagcagcaga accagaagag agacccagct gggatctggc 1500
agtggatctg gaagcggctc tatggccaga ttcgaagatc ccaccagaag accctacaag 1560
ctgcccgacc tgtgcaccga gctgaacacc agcctgcagg acatcgagat cacctgcgtg 1620
tactgcaaga ccgtgctgga gctgaccgag gtgttcgagt tcgccttcaa ggacctgttc 1680
gtggtgtaca gagacagcat cccccacgct gcctgccata aatgtattga tttttattcc 1740
aggattaggg aactcaggca ttatagtgat tctgtcattg atggtgtcaa tcatcagcat 1800
ctcccagcta ggagggctga acctcagagg cataccatgc tgtgcatgtg ctgcaagtgc 1860
gaggccagaa tcgagctggt ggtggagagc agcgccgacg acctgagagc cttccagcag 1920
ctgttcctga gcaccctgag cttcgtgtgc ccctggtgcg ccagccagca gggctcagga 1980
tctggcagcg gaagtggatc tatgtacggc cccaaggcta ccctgcagga catcgtgctg 2040
cacctggagc cccagaacga gatccccgtg gacctgctgt gccacgagca gctgagcgac 2100
agcgaggaag aaaacgacga gatcgacggc gtgaaccacc agcacctgcc tgccagaaga 2160
gccgagcccc agagacacac ccacaagtgc atcgacttct acagcagaat cagagagctg 2220
agacactaca gcgacagcgt gtacggcgac accctggaga agctgaccaa caccggcctg 2280
tacaacctgc tgatcagatg cctgagatgc cagaagcccc tgaaccctgc cgagaagctg 2340
agacacctga acgagaagag aagattccac aacatcgccg gccactacag aggccagtgc 2400
cacagctgct gcaacagagc cagacaggag agactgcaga gaagaagaga gacccaggtg 2460
ggatctggca gcggctctgg ctccggctca ctcgagatgt tccaggaccc tgccgaaaga 2520
ccctacaagc tgcatgatct gtgcaatgaa gtcgaagaga gtatccatga aatctgtctg 2580
aattgcgtgt actgtaagca ggagctgcag cgcagtgaag tctacgactt cgcctgctat 2640
gacctgtgca tcgtgtaccg agagggacag ccatatggcg tctgcatgaa gtgtctgaag 2700
ttctactcta agatcagtga atataggtgg ccagccggcc aggctaaacc cgacacttcc 2760
aactataata ttgtgacctc ttgctgtaaa tgcgaggcta ccctgagact gtgcgtgcag 2820
agcacacaca tcgacattag gaagctggag gacctgctga tggggacctt cggaatcgtg 2880
tgcccaggat gttcccagcg agctggatct ggcagtgggt caggaagcgg ctccatgcat 2940
ggagagatta ccacactgca ggactacgtc ctggatctgg agcctgaagc aactgacctg 3000
tactgctatg aacagctgtg cgatagctcc gaggaagagg aagacaccat cgatggccct 3060
gcagggcagg ccaagccaga tacaagtaac tacaacatcg tgacttcatg ctgtaaaccc 3120
tacggcgtct gcatgaaatg tctgaaattc tactcaaaga tcagcgagta tcggtggtac 3180
agatatagcg tgtacgggga gacactggaa aagcagtgca acaaacagct gtgccacctg 3240
ctgatccggt gcattacttg tcagaagccc ctgtgccctg tggagaaaca gcgacacctg 3300
gaggaaaaga aacggtttca taatattggc gggaggtgga caggccgctg catgagctgt 3360
tggaagccta ccagacggga gaccgaagtg ggcagcggca gtgggagcgg aagcgggagt 3420
gtcgagggat ctatggccag attcgacgac cccaagcaga gaccctacaa gctgcccgac 3480
ctgtgcaccg agctgaacac cagcctgcag gacgtgagca tcgcctgcgt gtactgcaag 3540
gccaccctgg agagaaccga ggtgtaccag ttcgccttca aggacctgtg catcgtgtac 3600
agagactgca tcgcctacgc cgcctgccat aaatgtattg atttttattc caggattcgg 3660
gagctccgct attattctaa tagtgtcgaa gctgatggag tcagtcatgc tcagctccct 3720
gctcggaggg cagaacctca gaggcataag atcctgtgcg tgtgctgcaa gtgcgacggc 3780
agaatcgacc tgaccgtgga gagcagcgcc gacgacctga gaaccctgca gcagctgttc 3840
ctgagcaccc tgagcttcgt gtgcccctgg tgcgccacca accagggcag cggaagcgga 3900
agcggcagcg gcagcatgca cggccccaga gccaccctgc aggagatcgt gctgcacctg 3960
gagccccaga acgagctgga ccccgtggac ctgttgtgct acgagcagct gagcgaaagc 4020
gaggaagaga acgacgaggc cgacggcgtg agccacgccc agctgcccgc cagaagagcc 4080
gagccccaga gacaccacaa gtgcatcgac ttctacagca gaatcagaga gctgagatac 4140
tacagcaaca gcgtgtacgg cgagaccctg gagaagatca ccaacaccga gctgtacaac 4200
ctgttgatca gatgcctgag atgccagaag cccctgaacc ccgccgagaa gagaagacac 4260
ctgaaggaca agagaagatt ccacagcatc gccggccagt acagaggcca gtgcaacacc 4320
tgctgcgacc aggccagaca ggagagactg agaaggagga gagagaccca ggtgggatca 4380
ggaagtggat ctgggtccgg cagcatgttc caggacgccg aggagaagcc cagaaccctg 4440
cacgacctgt gccaggccct ggagaccagc gtgcacgaga tcgagctgaa gtgcgtggag 4500
tgcaagaaga ccctgcagag aagcgaggtg tatgacttcg tgttcgccga cctgagaatc 4560
gtgtatagag acggcaaccc cttcgccgtg tgcaaggtgt gtttgaggct cctctccaaa 4620
atttctgaat atcggcatta taactattcc ctctatggaa ggcctgatgg acaggctcag 4680
ccagctacag caaattatta tattgtcaca tgttgctata cctgcgacac caccgtgaga 4740
ctgtgcatca acagcaccac aaccgacgtg agaaccctgc agcagctgct gatgggcacc 4800
tgcaccatcg tgtgccccag ctgcgcccag cagggctcag gcagcggctc cggcagcgga 4860
tctatgagag gcaacaaccc caccctgaga gagtacatcc tggacctgca ccccgagccc 4920
accgacctgt tctgctacga gcagctgtgc gacagcagcg acgaggacga gatcggcctg 4980
gacagacccg acggccaggc ccagcccgcc accgccaact actacatcgt gacctgctgc 5040
tactgcctga gactgctgag caagatcagc gagtacagac actacaacta cagcctgtac 5100
ggcgacaccc tggagcagac cctgaagaag tgcctgaacg agatcctgat cagatgcatc 5160
atctgccaga gacccctgtg cccccaggag aagaagagac acgtggacct gaacaagaga 5220
ttccacaaca tcagcggcag atggaccggc agatgcgccg tgtgctggag acccagaagg 5280
agacagaccc aggtgggatc ctaa 5304
<210> 28
<211> 1909
<212> PRT
<213> Artificial Sequence
<220>
<223> BD-14B polypeptide
<400> 28
Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly Ala
1 5 10 15
Val Phe Val Ser Pro Ser His Ala Thr Gln Asp Cys Ser Phe Gln His
20 25 30
Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu Ser Asp
35 40 45
Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu Gln Asp
50 55 60
Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln Arg Trp
65 70 75 80
Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly Leu Leu
85 90 95
Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala Phe Gln
100 105 110
Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu
115 120 125
Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp Ile Thr
130 135 140
Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro Asp Ser
145 150 155 160
Ser Thr Leu Pro Pro Pro Trp Ser Pro Arg Pro Leu Glu Ala Thr Ala
165 170 175
Pro Thr Ala Pro Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Met Asp
180 185 190
Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly Ala Val
195 200 205
Phe Val Ser Pro Ser His Ala Thr Gln Asp Cys Ser Phe Gln His Ser
210 215 220
Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr
225 230 235 240
Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu Gln Asp Glu
245 250 255
Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln Arg Trp Met
260 265 270
Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly Leu Leu Glu
275 280 285
Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala Phe Gln Pro
290 295 300
Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu Leu
305 310 315 320
Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp Ile Thr Arg
325 330 335
Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro Asp Ser Ser
340 345 350
Thr Leu Pro Pro Pro Trp Ser Pro Arg Pro Leu Glu Ala Thr Ala Pro
355 360 365
Thr Ala Pro Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Met Phe Lys
370 375 380
Asn Pro Ala Glu Arg Pro Arg Lys Leu His Glu Leu Ser Ser Ala Leu
385 390 395 400
Glu Ile Pro Tyr Asp Glu Leu Arg Leu Asn Cys Val Tyr Cys Lys Gly
405 410 415
Gln Leu Thr Glu Thr Glu Val Leu Asp Phe Ala Phe Thr Asp Leu Thr
420 425 430
Ile Val Tyr Arg Asp Asp Thr Pro Tyr Gly Val Cys Thr Lys Cys Leu
435 440 445
Arg Phe Tyr Ser Lys Val Ser Glu Phe Arg Trp Tyr Arg Tyr Ser Val
450 455 460
Tyr Gly Pro Ala Gly Gln Ala Lys Pro Asp Thr Ser Asn Tyr Asn Ile
465 470 475 480
Val Thr Phe Cys Cys Gln Cys Glu Ser Thr Leu Arg Leu Cys Val Gln
485 490 495
Ser Thr Gln Val Asp Ile Arg Ile Leu Gln Glu Leu Leu Met Gly Ser
500 505 510
Phe Gly Ile Val Cys Pro Asn Cys Ser Thr Arg Leu Gly Ser Gly Ser
515 520 525
Gly Ser Gly Ser Gly Ser Met Arg Gly Glu Thr Pro Thr Leu Gln Asp
530 535 540
Tyr Val Leu Asp Leu Gln Pro Glu Ala Thr Asp Leu His Cys Tyr Glu
545 550 555 560
Gln Leu Pro Asp Ser Ser Asp Glu Glu Asp Val Ile Asp Ser Pro Ala
565 570 575
Gly Gln Ala Lys Pro Asp Thr Ser Asn Tyr Asn Ile Val Thr Phe Cys
580 585 590
Cys Gln Cys Leu Arg Phe Tyr Ser Lys Val Ser Glu Phe Arg Trp Tyr
595 600 605
Arg Tyr Ser Val Tyr Gly Thr Thr Leu Glu Lys Leu Thr Asn Lys Gly
610 615 620
Ile Cys Asp Leu Leu Ile Arg Cys Ile Thr Cys Gln Arg Pro Leu Cys
625 630 635 640
Pro Glu Glu Lys Gln Arg His Leu Asp Lys Lys Lys Arg Phe His Asn
645 650 655
Ile Gly Gly Arg Trp Thr Gly Arg Cys Ile Val Cys Trp Arg Arg Pro
660 665 670
Arg Thr Glu Thr Gln Val Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser
675 680 685
Met Phe Gln Asp Thr Glu Glu Lys Pro Arg Thr Leu His Asp Leu Cys
690 695 700
Gln Ala Leu Glu Thr Thr Ile His Asn Ile Glu Leu Gln Cys Val Glu
705 710 715 720
Cys Lys Asn Pro Leu Gln Arg Ser Glu Val Tyr Asp Phe Ala Phe Ala
725 730 735
Asp Leu Thr Val Val Tyr Arg Glu Gly Asn Pro Phe Gly Ile Cys Lys
740 745 750
Leu Cys Leu Arg Phe Leu Ser Lys Ile Ser Glu Tyr Arg His Tyr Asn
755 760 765
Tyr Ser Val Tyr Gly Pro Asp Gly Gln Ala Gln Pro Ala Thr Ala Asp
770 775 780
Tyr Tyr Ile Val Thr Cys Cys His Thr Cys Asn Thr Thr Val Arg Leu
785 790 795 800
Cys Val Asn Ser Thr Ala Ser Asp Leu Arg Thr Ile Gln Gln Leu Leu
805 810 815
Met Gly Thr Val Asn Ile Val Cys Pro Thr Cys Ala Gln Leu Gly Ser
820 825 830
Gly Ser Gly Ser Gly Ser Gly Ser Met Arg Gly His Lys Pro Thr Leu
835 840 845
Lys Glu Tyr Val Leu Asp Leu Tyr Pro Glu Pro Thr Asp Leu Tyr Cys
850 855 860
Tyr Glu Gln Leu Ser Asp Ser Ser Asp Glu Asp Glu Gly Leu Asp Arg
865 870 875 880
Pro Asp Gly Gln Ala Gln Pro Ala Thr Ala Asp Tyr Tyr Ile Val Thr
885 890 895
Cys Cys His Thr Cys Leu Arg Phe Leu Ser Lys Ile Ser Glu Tyr Arg
900 905 910
His Tyr Asn Tyr Ser Val Tyr Gly His Thr Leu Glu Gln Thr Val Lys
915 920 925
Lys Pro Leu Asn Glu Ile Leu Ile Arg Cys Ile Ile Cys Gln Arg Pro
930 935 940
Leu Cys Pro Gln Glu Lys Lys Arg His Val Asp Leu Asn Lys Arg Phe
945 950 955 960
His Asn Ile Ser Gly Arg Trp Ala Gly Arg Cys Ala Ala Cys Trp Arg
965 970 975
Ser Arg Arg Arg Glu Thr Ala Leu Gly Ser Gly Ser Gly Ser Gly Ser
980 985 990
Gly Ser Met Glu Ser Ala Asn Ala Ser Thr Ser Ala Thr Thr Ile Asp
995 1000 1005
Gln Leu Cys Lys Thr Phe Asn Leu Ser Met His Thr Leu Gln Ile
1010 1015 1020
Asn Cys Val Phe Cys Lys Asn Ala Leu Thr Thr Ala Glu Ile Tyr
1025 1030 1035
Ser Tyr Ala Tyr Lys His Leu Lys Val Leu Phe Arg Gly Gly Tyr
1040 1045 1050
Pro Tyr Ala Ala Cys Ala Cys Cys Leu Glu Phe His Gly Lys Ile
1055 1060 1065
Asn Gln Tyr Arg His Phe Asp Tyr Ala Gly Tyr Asp Gly Gln Asp
1070 1075 1080
Ser Gln Pro Leu Lys Gln His Tyr Gln Ile Val Thr Cys Cys Cys
1085 1090 1095
Gly Cys Asp Ser Asn Val Arg Leu Val Val Gln Cys Thr Glu Thr
1100 1105 1110
Asp Ile Arg Glu Val Gln Gln Leu Leu Leu Gly Thr Leu Asn Ile
1115 1120 1125
Val Cys Pro Ile Cys Ala Pro Lys Thr Gly Ser Gly Ser Gly Ser
1130 1135 1140
Gly Ser Gly Ser Met His Gly Arg His Val Thr Leu Lys Asp Ile
1145 1150 1155
Val Leu Asp Leu Gln Pro Pro Asp Pro Val Gly Leu His Cys Tyr
1160 1165 1170
Glu Gln Leu Val Asp Ser Ser Glu Asp Glu Val Asp Glu Val Asp
1175 1180 1185
Gly Gln Asp Ser Gln Pro Leu Lys Gln His Tyr Gln Ile Val Thr
1190 1195 1200
Cys Cys Cys Gly Cys Cys Leu Glu Phe His Gly Lys Ile Asn Gln
1205 1210 1215
Tyr Arg His Phe Asp Tyr Ala Gly Tyr Ala Thr Thr Val Glu Glu
1220 1225 1230
Glu Thr Lys Gln Asp Ile Leu Asp Val Leu Ile Arg Cys Tyr Leu
1235 1240 1245
Cys His Lys Pro Leu Cys Glu Val Glu Lys Val Lys His Ile Leu
1250 1255 1260
Thr Lys Ala Arg Phe Ile Lys Leu Asn Cys Thr Trp Lys Gly Arg
1265 1270 1275
Cys Leu His Cys Trp Thr Thr Cys Met Glu Asp Met Leu Pro Gly
1280 1285 1290
Ser Gly Ser Gly Ser Gly Ser Gly Ser Met Glu Ser Lys Asp Ala
1295 1300 1305
Ser Thr Ser Ala Thr Ser Ile Asp Gln Leu Cys Lys Thr Phe Asn
1310 1315 1320
Leu Ser Leu His Thr Leu Gln Ile Gln Cys Val Phe Cys Arg Asn
1325 1330 1335
Ala Leu Thr Thr Ala Glu Ile Tyr Ala Tyr Ala Tyr Lys Asn Leu
1340 1345 1350
Lys Val Val Trp Arg Asp Asn Phe Pro Phe Ala Ala Cys Ala Cys
1355 1360 1365
Cys Leu Glu Leu Gln Gly Lys Ile Asn Gln Tyr Arg His Phe Asn
1370 1375 1380
Tyr Ala Ala Tyr Asp Lys Gln Asp Ser Gln Pro Leu Thr Gln His
1385 1390 1395
Tyr Gln Ile Leu Thr Cys Cys Cys Gly Cys Asp Ser Asn Val Arg
1400 1405 1410
Leu Val Val Glu Cys Thr Asp Gly Asp Ile Arg Gln Leu Gln Asp
1415 1420 1425
Leu Leu Leu Gly Thr Leu Asn Ile Val Cys Pro Ile Cys Ala Pro
1430 1435 1440
Lys Pro Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Met His Gly
1445 1450 1455
Arg Leu Val Thr Leu Lys Asp Ile Val Leu Asp Leu Gln Pro Pro
1460 1465 1470
Asp Pro Val Gly Leu His Cys Tyr Glu Gln Leu Glu Asp Ser Ser
1475 1480 1485
Glu Asp Glu Val Asp Lys Val Asp Lys Gln Asp Ser Gln Pro Leu
1490 1495 1500
Thr Gln His Tyr Gln Ile Leu Thr Cys Cys Cys Gly Cys Cys Leu
1505 1510 1515
Glu Leu Gln Gly Lys Ile Asn Gln Tyr Arg His Phe Asn Tyr Ala
1520 1525 1530
Ala Tyr Ala Pro Thr Val Glu Glu Glu Thr Asn Glu Asp Ile Leu
1535 1540 1545
Lys Val Leu Ile Arg Cys Tyr Leu Cys His Lys Pro Leu Cys Glu
1550 1555 1560
Ile Glu Lys Leu Lys His Ile Leu Gly Lys Ala Arg Phe Ile Lys
1565 1570 1575
Leu Asn Asn Gln Trp Lys Gly Arg Cys Leu His Cys Trp Thr Thr
1580 1585 1590
Cys Met Glu Asp Leu Leu Pro Gly Ser Gly Ser Gly Ser Gly Ser
1595 1600 1605
Gly Ser Met Phe Glu Asp Pro Ala Thr Arg Pro Arg Thr Leu His
1610 1615 1620
Glu Leu Cys Glu Val Leu Glu Glu Ser Val His Glu Ile Arg Leu
1625 1630 1635
Gln Cys Val Gln Cys Lys Lys Glu Leu Gln Arg Arg Glu Val Tyr
1640 1645 1650
Lys Phe Leu Phe Thr Asp Leu Arg Ile Val Tyr Arg Asp Asn Asn
1655 1660 1665
Pro Tyr Gly Val Cys Ile Met Cys Leu Arg Phe Leu Ser Lys Ile
1670 1675 1680
Ser Glu Tyr Arg His Tyr Gln Tyr Ser Leu Tyr Gly Asp Arg Pro
1685 1690 1695
Asp Gly Gln Ala Glu Gln Ala Thr Ser Asn Tyr Tyr Ile Val Thr
1700 1705 1710
Tyr Cys His Ser Cys Asp Ser Thr Leu Arg Leu Cys Ile His Ser
1715 1720 1725
Thr Ala Thr Asp Leu Arg Thr Leu Gln Gln Met Leu Leu Gly Thr
1730 1735 1740
Leu Gln Val Val Cys Pro Gly Cys Ala Arg Leu Gly Ser Gly Ser
1745 1750 1755
Gly Ser Gly Ser Gly Ser Met Arg Gly Asp Lys Ala Thr Ile Lys
1760 1765 1770
Asp Tyr Ile Leu Asp Leu Gln Pro Glu Thr Thr Asp Leu His Cys
1775 1780 1785
Tyr Glu Gln Leu Gly Asp Ser Ser Asp Glu Glu Asp Thr Asp Gly
1790 1795 1800
Val Asp Arg Pro Asp Gly Gln Ala Glu Gln Ala Thr Ser Asn Tyr
1805 1810 1815
Tyr Ile Val Thr Tyr Cys Cys Leu Arg Phe Leu Ser Lys Ile Ser
1820 1825 1830
Glu Tyr Arg His Tyr Gln Tyr Ser Leu Tyr Gly Lys Thr Leu Glu
1835 1840 1845
Glu Arg Val Lys Lys Pro Leu Ser Glu Ile Thr Ile Arg Cys Ile
1850 1855 1860
Ile Cys Gln Thr Pro Leu Cys Pro Glu Glu Lys Glu Arg His Val
1865 1870 1875
Asn Ala Asn Lys Arg Phe His Asn Ile Met Gly Arg Trp Thr Gly
1880 1885 1890
Arg Cys Ser Glu Cys Trp Arg Pro Arg Pro Val Thr Gln Val Gly
1895 1900 1905
Ser
<210> 29
<211> 5160
<212> DNA
<213> Artificial Sequence
<220>
<223> polynucleotide encoding BD-14B polypeptide
<400> 29
atggacgcca tgaagagagg cctgtgctgc gtgctgctgc tgtgcggcgc cgtgttcgtg 60
agccccagcc acgccaccca ggactgcagc ttccagcaca gccccatcag cagcgacttc 120
gccgtgaaga tcagagagct gagcgactac ctgctgcagg actaccccgt gaccgtggcc 180
agcaacctgc aggacgagga gctgtgcggc ggcctgtgga gactggtgct ggcccagaga 240
tggatggaga gactgaagac cgtggccggc agcaagatgc agggcctgct ggagagagtg 300
aacaccgaga tccacttcgt gaccaagtgc gccttccagc ctccccccag ctgcctgagg 360
ttcgtgcaga ccaacatcag cagactgctg caggagacca gcgagcagct ggtggccctg 420
aagccctgga tcaccagaca gaacttcagc agatgcctgg agctgcagtg ccagcccgac 480
agcagcaccc tgccccctcc ctggagcccc agacccctgg aggccaccgc tcccacagcc 540
cctggcagcg ggtccggaag tgggtctgga tctatgttca agaaccccgc cgagagaccc 600
agaaagctgc acgagctgag cagcgccctg gagatcccct acgacgagct gagactgaac 660
tgcgtgtact gcaagggcca gctgaccgag accgaggtgc tggacttcgc cttcaccgac 720
ctgaccatcg tgtacagaga cgacaccccc tacggcgtgt gcaccaagtg tctcaggttt 780
tatagtaaag tctctgaatt taggtggtat aggtattccg tctatggtcc tgcaggacag 840
gctaaacctg atacaagtaa ttataatatt gtcacatttt gttgtcagtg tgagagcacc 900
ctgagactgt gcgtgcagag cacccaggtg gacatcagaa tcctgcagga gctgctgatg 960
ggcagcttcg gcatcgtgtg ccccaactgc agcaccagac tgggcagtgg aagcggctca 1020
ggaagcggca gcatgagagg cgagaccccc accctgcagg actacgtgct ggacctgcag 1080
cccgaggcca ccgacctgca ctgctacgag cagctgcccg acagcagcga cgaggaggat 1140
gtgatcgaca gccccgccgg ccaggccaag cccgacacca gcaactacaa catcgtgacc 1200
ttctgctgcc agtgcctgag attctacagc aaggtgagcg agttcagatg gtacagatac 1260
agcgtgtacg gcaccaccct ggagaagctg accaacaagg gcatctgcga cctgctgatc 1320
agatgcatca cctgccagag acccctgtgc cccgaggaga agcagagaca cctggacaag 1380
aagaaaagat tccacaacat cggcggcaga tggaccggca gatgcatcgt gtgctggaga 1440
agacccagaa ccgagaccca ggtgggcagc ggctccggat caggcagcgg aagtatgttc 1500
caggacaccg aggagaagcc cagaaccctg cacgacctgt gccaggccct ggagaccacc 1560
atccacaaca tcgagctgca gtgcgtggag tgcaagaacc ccctgcagag aagcgaggtg 1620
tacgacttcg ccttcgccga cctgaccgtg gtgtacagag agggcaaccc cttcggcatc 1680
tgcaagctgt gtctcaggtt tctcagtaaa atttctgaat ataggcatta taattattcc 1740
gtctatggac ctgatggaca ggctcagcct gctacagcag attattatat tgtcacatgt 1800
tgtcatacat gtaacaccac cgtgagactg tgcgtgaaca gcaccgccag cgatctgaga 1860
accatccagc agctgctgat gggcaccgtg aacatcgtgt gccccacctg cgcccagctg 1920
ggctcaggaa gtggaagcgg ctctggatct atgagaggcc acaagcccac cctgaaggag 1980
tacgtgctgg acctgtaccc cgagcccacc gacctgtact gctacgagca gctgagcgat 2040
agcagcgacg aggacgaggg cctggacaga cccgatggcc aggcccagcc cgccaccgcc 2100
gactactaca tcgtgacctg ctgccacacc tgcctgagat tcctgagcaa gatcagcgag 2160
tacagacact acaactacag cgtgtacggc cacaccctgg agcagaccgt gaagaagccc 2220
ctgaacgaga tcctgatcag atgcatcatc tgccagagac ccctgtgccc ccaggagaag 2280
aagagacacg tggacctgaa caagagattc cacaacatca gcggcagatg ggccggcaga 2340
tgcgccgcct gctggagaag cagaagaaga gagaccgccc tgggcagcgg ctctggctcc 2400
ggctcaggat ctatggagtc tgctaacgct tccacatccg ctacaactat cgaccagctg 2460
tgcaagactt tcaacctcag catgcacacc ttgcagatca actgtgtgtt ttgcaaaaac 2520
gccctgacca cagcagaaat ttacagttac gcctacaaac atctgaaggt gctctttcgg 2580
gggggctatc cctacgccgc atgcgcttgt tgcttggaat ttcatggaaa aatcaaccag 2640
tatcggcatt tcgattatgc cggatacgat gggcaggata gtcagcctct gaaacagcac 2700
tatcagattg tgacctgttg ctgtggatgt gacagcaacg tgaggctggt cgtgcagtgt 2760
acagaaacag acatcagaga ggtgcagcag cttcttctgg gcactctcaa catcgtgtgt 2820
cccatctgcg ctccaaaaac cgggtccggc agcggatctg gaagcggctc catgcacggg 2880
cggcacgtga cacttaaaga catcgtcctg gaccttcagc cccctgatcc tgtcggcttg 2940
cactgttacg agcagctggt ggactcatct gaggatgagg tggacgaagt ggacggacag 3000
gattcacagc ctctgaaaca gcattaccag attgtgacct gctgctgcgg ctgttgtctt 3060
gagttccatg gaaaaatcaa ccagtacaga catttcgatt atgccggata cgcaacaacc 3120
gtcgaagagg agactaaaca ggacatcctc gacgtcctga ttcgctgcta cctgtgtcac 3180
aaaccactgt gtgaggtcga aaaggtgaaa cacattctta ccaaggcaag attcatcaaa 3240
ctcaattgta cctggaaggg acggtgcctg cactgttgga ctacatgcat ggaagatatg 3300
cttccaggaa gtgggagcgg ctcaggaagc gggagcatgg aaagtaaaga cgcttccaca 3360
agtgccactt caatcgacca gctctgtaag acattcaact tgagtctgca caccctgcag 3420
atccagtgcg tgttttgcag aaacgcactc acaaccgctg agatttacgc ctatgcttac 3480
aagaacctca aggtcgtgtg gagggataat ttccccttcg ctgcctgcgc ctgttgcctg 3540
gaactgcagg ggaaaatcaa tcagtatcgg catttcaact atgctgctta cgacaaacag 3600
gattctcagc ctctgaccca gcactaccag attctcacct gctgctgcgg ctgcgatagt 3660
aatgtgaggc tcgtggtcga gtgtaccgac ggcgacatta ggcagctcca ggatcttctc 3720
cttggcacac tgaatatcgt gtgtcctatt tgtgccccaa aacccgggtc tggaagtggc 3780
tccggatctg ggagtatgca tggacgcctc gtgacactga aggatattgt gctcgatctg 3840
cagccacctg atcccgtggg cctccactgt tatgagcagc tggaggattc ctcagaagat 3900
gaggtggata aagtggacaa acaggactcc cagcctctta cccagcatta tcagatcctg 3960
acctgctgct gcggatgttg tctggaattg cagggcaaaa tcaaccagta tagacatttc 4020
aattatgctg catacgcccc tacagtcgag gaggaaacca atgaagacat cctcaaggtg 4080
ctgatcagat gttacctctg tcacaagcct ctttgcgaaa tcgagaaact gaagcatatc 4140
ctgggaaagg ctcgctttat caagcttaac aatcagtgga aaggcaggtg cctgcactgc 4200
tggaccacct gtatggaaga cctgctgccc gggtccggct caggaagcgg ctccggctct 4260
atgtttgaag acccagccac caggccaaga acattgcacg agctttgcga agtcctcgaa 4320
gagagtgtgc atgagattag gctccagtgt gtgcagtgca agaaggaact tcagcgcaga 4380
gaggtctaca agttcttgtt tacagacctg cggatcgtgt acagggataa taatccctat 4440
ggcgtctgca ttatgtgtct taggttcctg tccaagattt cagagtacag acattaccag 4500
tattcactgt atggggacag gccagatggc caggccgagc aggctactag taactactac 4560
attgtgacct actgtcactc ctgcgactca accctccggc tgtgcattca cagcaccgcc 4620
accgaccttc gcactctgca gcagatgctg ctcggcacct tgcaggtggt gtgtcccgga 4680
tgcgccaggt tgggcagcgg gagtgggtcc ggaagcggca gtatgagagg cgataaggca 4740
accatcaagg actacatcct ggacctgcag cctgagacca ctgatttgca ttgctacgaa 4800
cagctgggag actcaagcga tgaagaagac actgatggcg tggacaggcc cgacggacag 4860
gccgaacagg ccaccagtaa ctattatatc gtcacctatt gctgcctgag gtttctcagt 4920
aaaatttctg agtacagaca ctatcagtac tcactttacg gcaagacatt ggaggagagg 4980
gtgaagaagc ctctgtccga gatcactatt aggtgcatca tctgtcagac tcccctgtgt 5040
cctgaggaaa aggagcggca tgtcaatgct aacaagagat tccacaacat catgggacgg 5100
tggacaggcc gctgctctga atgctggcgc cccaggccag tgactcaggt gggatcctaa 5160
<210> 30
<211> 1467
<212> PRT
<213> Artificial Sequence
<220>
<223> BD-14C polypeptide
<400> 30
Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly Ala
1 5 10 15
Val Phe Val Ser Pro Ser His Ala Thr Gln Asp Cys Ser Phe Gln His
20 25 30
Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu Ser Asp
35 40 45
Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu Gln Asp
50 55 60
Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln Arg Trp
65 70 75 80
Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly Leu Leu
85 90 95
Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala Phe Gln
100 105 110
Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu
115 120 125
Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp Ile Thr
130 135 140
Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro Asp Ser
145 150 155 160
Ser Thr Leu Pro Pro Pro Trp Ser Pro Arg Pro Leu Glu Ala Thr Ala
165 170 175
Pro Thr Ala Pro Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Met Ala
180 185 190
Arg Phe His Asn Pro Ala Glu Arg Pro Tyr Lys Leu Pro Asp Leu Cys
195 200 205
Thr Thr Leu Asp Thr Thr Leu Gln Asp Ile Thr Ile Ala Cys Val Tyr
210 215 220
Cys Arg Arg Pro Leu Gln Gln Thr Glu Val Tyr Glu Phe Ala Phe Ser
225 230 235 240
Asp Leu Tyr Val Val Tyr Arg Asp Gly Glu Pro Leu Ala Ala Cys Gln
245 250 255
Ser Cys Ile Lys Phe Tyr Ala Lys Ile Arg Glu Leu Arg Tyr Tyr Ser
260 265 270
Asp Ser Val Gln Leu Leu Ala Arg Arg Asp Glu Pro Gln Arg His Thr
275 280 285
Ile Gln Cys Ser Cys Cys Lys Cys Asn Asn Thr Leu Gln Leu Val Val
290 295 300
Glu Ala Ser Arg Asp Thr Leu Arg Gln Leu Gln Gln Leu Phe Met Asp
305 310 315 320
Ser Leu Gly Phe Val Cys Pro Trp Cys Ala Thr Ala Asn Gln Gly Ser
325 330 335
Gly Ser Gly Ser Gly Ser Gly Ser Met Arg Gly Pro Lys Pro Thr Leu
340 345 350
Gln Glu Ile Val Leu Asp Leu Cys Pro Tyr Asn Glu Ile Gln Pro Val
355 360 365
Asp Leu Val Cys His Glu Gln Leu Gly Glu Ser Glu Asp Glu Ile Asp
370 375 380
Glu Pro Asp His Ala Val Asn His Gln His Gln Leu Leu Ala Arg Arg
385 390 395 400
Asp Glu Pro Gln Arg His Thr Ile Gln Cys Ser Cys Cys Lys Gln Ser
405 410 415
Cys Ile Lys Phe Tyr Ala Lys Ile Arg Glu Leu Arg Tyr Tyr Ser Asp
420 425 430
Ser Val Tyr Ala Thr Thr Leu Glu Asn Ile Thr Asn Thr Lys Leu Tyr
435 440 445
Asn Leu Leu Ile Arg Cys Met Cys Cys Leu Lys Pro Leu Cys Pro Ala
450 455 460
Glu Lys Leu Arg His Leu Asn Ser Lys Arg Arg Phe His Lys Ile Ala
465 470 475 480
Gly Ser Tyr Thr Gly Gln Cys Arg Arg Cys Trp Thr Thr Lys Arg Glu
485 490 495
Asp Arg Arg Leu Thr Arg Arg Glu Thr Gln Val Gly Ser Gly Ser Gly
500 505 510
Ser Gly Ser Gly Ser Met Phe Glu Asp Lys Arg Glu Arg Pro Arg Thr
515 520 525
Leu His Glu Leu Cys Glu Ala Leu Asn Val Ser Met His Asn Ile Gln
530 535 540
Val Val Cys Val Tyr Cys Lys Lys Glu Leu Cys Arg Ala Asp Val Tyr
545 550 555 560
Asn Val Ala Phe Thr Glu Ile Lys Ile Val Tyr Arg Asp Asn Asn Pro
565 570 575
Tyr Ala Val Cys Lys Gln Cys Leu Leu Phe Tyr Ser Lys Ile Arg Glu
580 585 590
Tyr Arg Arg Tyr Ser Arg Ser Val Leu Pro Glu Arg Arg Ala Gly Gln
595 600 605
Ala Thr Cys Tyr Arg Ile Glu Ala Pro Cys Cys Arg Cys Ser Ser Val
610 615 620
Val Gln Leu Ala Val Glu Ser Ser Gly Asp Thr Leu Arg Val Val Gln
625 630 635 640
Gln Met Leu Met Gly Glu Leu Ser Leu Val Cys Pro Cys Cys Ala Asn
645 650 655
Asn Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Met Arg Gly Asn Val
660 665 670
Pro Gln Leu Lys Asp Val Val Leu His Leu Thr Pro Gln Thr Glu Ile
675 680 685
Asp Leu Gln Cys Tyr Glu Gln Phe Asp Ser Ser Glu Glu Glu Asp Glu
690 695 700
Val Asp Asn Met Arg Asp Gln Leu Pro Glu Arg Arg Ala Gly Gln Ala
705 710 715 720
Thr Cys Tyr Arg Ile Glu Ala Pro Cys Cys Arg Lys Gln Cys Leu Leu
725 730 735
Phe Tyr Ser Lys Ile Arg Glu Tyr Arg Arg Tyr Ser Arg Ser Val Tyr
740 745 750
Gly Thr Thr Leu Glu Ala Ile Thr Lys Lys Ser Leu Tyr Asp Leu Ser
755 760 765
Ile Arg Cys His Arg Cys Gln Arg Pro Leu Gly Pro Glu Glu Lys Gln
770 775 780
Lys Leu Val Asp Glu Lys Lys Arg Phe His Glu Ile Ala Gly Arg Trp
785 790 795 800
Thr Gly Gln Cys Ala Asn Cys Trp Gln Arg Thr Arg Gln Arg Asn Glu
805 810 815
Thr Gln Val Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Met Glu Pro
820 825 830
Gln Phe Asn Asn Pro Gln Glu Arg Pro Arg Ser Leu His His Leu Ser
835 840 845
Glu Val Leu Glu Ile Pro Leu Ile Asp Leu Arg Leu Ser Cys Val Tyr
850 855 860
Cys Lys Lys Glu Leu Thr Arg Ala Glu Val Tyr Asn Phe Ala Cys Thr
865 870 875 880
Glu Leu Lys Leu Val Tyr Arg Asp Asp Phe Pro Tyr Ala Val Cys Arg
885 890 895
Val Cys Leu Leu Phe Tyr Ser Lys Val Arg Lys Tyr Arg Tyr Tyr Asp
900 905 910
Tyr Ser Val Gln Ala Arg Gln Ala Lys Gln His Thr Cys Tyr Leu Ile
915 920 925
His Val Pro Cys Cys Glu Cys Lys Phe Val Val Gln Leu Asp Ile Gln
930 935 940
Ser Thr Lys Glu Asp Leu Arg Val Val Gln Gln Leu Leu Met Gly Ala
945 950 955 960
Leu Thr Val Thr Cys Pro Leu Cys Ala Ser Ser Asn Gly Ser Gly Ser
965 970 975
Gly Ser Gly Ser Gly Ser Met His Gly Lys Val Pro Thr Leu Gln Asp
980 985 990
Val Val Leu Glu Leu Thr Pro Gln Thr Glu Ile Asp Leu Gln Cys Asn
995 1000 1005
Glu Gln Leu Asp Ser Ser Glu Asp Glu Asp Glu Asp Glu Val Asp
1010 1015 1020
His Leu Gln Glu Arg Pro Gln Gln Ala Arg Gln Ala Lys Gln His
1025 1030 1035
Thr Cys Tyr Leu Ile His Val Pro Cys Cys Glu Arg Val Cys Leu
1040 1045 1050
Leu Phe Tyr Ser Lys Val Arg Lys Tyr Arg Tyr Tyr Asp Tyr Ser
1055 1060 1065
Val Tyr Gly Ala Thr Leu Glu Ser Ile Thr Lys Lys Gln Leu Cys
1070 1075 1080
Asp Leu Leu Ile Arg Cys Tyr Arg Cys Gln Ser Pro Leu Thr Pro
1085 1090 1095
Glu Glu Lys Gln Leu His Cys Asp Arg Lys Arg Arg Phe His Leu
1100 1105 1110
Ile Ala His Gly Trp Thr Gly Ser Cys Leu Gly Cys Trp Arg Gln
1115 1120 1125
Thr Ser Arg Glu Pro Arg Glu Ser Thr Val Gly Ser Gly Ser Gly
1130 1135 1140
Ser Gly Ser Gly Ser Met Ala Arg Phe Glu Asp Pro Thr Gln Arg
1145 1150 1155
Pro Tyr Lys Leu Pro Asp Leu Ser Thr Thr Leu Asn Ile Pro Leu
1160 1165 1170
His Asp Ile Arg Ile Asn Cys Val Phe Cys Lys Gly Glu Leu Gln
1175 1180 1185
Glu Arg Glu Val Phe Glu Phe Ala Phe Asn Asp Leu Phe Ile Val
1190 1195 1200
Tyr Arg Asp Cys Thr Pro Tyr Ala Ala Cys Leu Lys Cys Ile Ser
1205 1210 1215
Phe Tyr Ala Arg Val Arg Glu Leu Arg Tyr Tyr Arg Asp Ser Val
1220 1225 1230
Leu Leu Leu Ala Arg Arg Ala Glu Pro Gln Arg His Asn Ile Val
1235 1240 1245
Cys Val Cys Cys Lys Cys Asn Asn Gln Leu Gln Leu Val Val Glu
1250 1255 1260
Thr Ser Gln Asp Gly Leu Arg Ala Leu Gln Gln Leu Phe Met Asp
1265 1270 1275
Thr Leu Ser Phe Val Cys Pro Leu Cys Ala Ala Asn Gln Gly Ser
1280 1285 1290
Gly Ser Gly Ser Gly Ser Gly Ser Met His Gly Pro Lys Ala Thr
1295 1300 1305
Leu Cys Asp Ile Val Leu Asp Leu Glu Pro Gln Asn Tyr Glu Glu
1310 1315 1320
Val Asp Leu Val Cys Tyr Glu Gln Leu Pro Asp Ser Asp Ser Glu
1325 1330 1335
Asn Glu Lys Asp Glu Pro Asp Gly Val Asn His Pro Leu Leu Leu
1340 1345 1350
Ala Arg Arg Ala Glu Pro Gln Arg His Asn Ile Val Cys Val Cys
1355 1360 1365
Cys Lys Leu Lys Cys Ile Ser Phe Tyr Ala Arg Val Arg Glu Leu
1370 1375 1380
Arg Tyr Tyr Arg Asp Ser Val Tyr Gly Glu Thr Leu Glu Ala Glu
1385 1390 1395
Thr Lys Thr Pro Leu His Glu Leu Leu Ile Arg Cys Tyr Arg Cys
1400 1405 1410
Leu Lys Pro Leu Cys Pro Thr Asp Lys Leu Lys His Ile Thr Glu
1415 1420 1425
Lys Arg Arg Phe His Asn Ile Ala Gly Ile Tyr Thr Gly Gln Cys
1430 1435 1440
Arg Gly Cys Arg Thr Arg Ala Arg His Leu Arg Gln Gln Arg Gln
1445 1450 1455
Ala Arg Ser Glu Thr Leu Val Gly Ser
1460 1465
<210> 31
<211> 4407
<212> DNA
<213> Artificial Sequence
<220>
<223> polynucleotide encoding BD-14C polypeptide
<400> 31
atggacgcca tgaagagagg cctgtgctgc gtgctgctgc tgtgcggcgc cgtgttcgtg 60
agccccagcc acgccaccca ggactgcagc ttccagcaca gccccatcag cagcgacttc 120
gccgtgaaga tcagagagct gagcgactac ctgctgcagg actaccccgt gaccgtggcc 180
agcaacctgc aggacgagga gctgtgcggc ggcctgtgga gactggtgct ggcccagaga 240
tggatggaga gactgaagac cgtggccggc agcaagatgc agggcctgct ggagagagtg 300
aacaccgaga tccacttcgt gaccaagtgc gccttccagc ctccccccag ctgcctgagg 360
ttcgtgcaga ccaacatcag cagactgctg caggagacca gcgagcagct ggtggccctg 420
aagccctgga tcaccagaca gaacttcagc agatgcctgg agctgcagtg ccagcccgac 480
agcagcaccc tgccccctcc ctggagcccc agacccctgg aggccaccgc tcccacagcc 540
cctggcagcg ggtccggaag tgggtctgga tctatggcta gatttcataa ccccgccgag 600
cgcccttaca aactgcccga cctgtgcacc actctggata ccactctgca ggacatcact 660
atcgcatgcg tgtactgtcg gagaccactg cagcagaccg aggtctatga gttcgccttt 720
tccgacctgt acgtggtcta tagagatggc gagcccctgg ccgcttgcca gtcttgtatc 780
aagttttacg ctaagatcag ggagctgcgc tactatagcg actccgtgca gctgctggca 840
aggcgcgatg aaccccagag gcacaccatc cagtgctcct gctgtaagtg taacaataca 900
ctgcagctgg tggtcgaggc ctcacgcgac actctgcgac agctgcagca gctgtttatg 960
gatagcctgg ggttcgtgtg cccttggtgt gccactgcta accagggctc tgggagtgga 1020
tcaggcagcg ggtccatgcg aggaccaaag cctaccctgc aggagatcgt gctggacctg 1080
tgcccctaca acgaaattca gcctgtggat ctggtctgtc acgagcagct gggcgaaagc 1140
gaggatgaaa tcgacgagcc agatcatgca gtgaatcacc agcatcagct gctggcccga 1200
cgggacgaac cacagcggca cacaattcag tgcagctgct gtaagcagtc ctgtatcaag 1260
ttctacgcaa aaattcgaga gctgcggtac tattctgata gcgtgtacgc caccacactg 1320
gaaaacatca ctaataccaa actgtataac ctgctgatta gatgcatgtg ctgtctgaag 1380
ccactgtgcc ccgccgagaa actgaggcac ctgaatagca agagaaggtt tcataaaatc 1440
gctgggtcct acaccggaca gtgccgccga tgttggacta ccaagagaga ggaccggaga 1500
ctgaccaggc gcgaaacaca agtgggatca ggcagcgggt ccggatctgg cagtatgttc 1560
gaggataaac gggaaagacc aaggacactg cacgagctgt gcgaagccct gaacgtgtcc 1620
atgcataata ttcaggtggt ctgcgtctac tgtaagaaag aactgtgccg cgcagacgtg 1680
tataatgtcg cctttactga gatcaagatc gtgtaccggg ataacaatcc ctatgccgtc 1740
tgcaagcagt gtctgctgtt ctactctaaa atccgcgaat accgacggta ttcacggagc 1800
gtgctgcctg agagaagggc aggccaggcc acttgctata gaattgaggc cccatgctgt 1860
aggtgtagct ccgtggtcca gctggctgtg gaatctagtg gagacaccct gagagtggtc 1920
cagcagatgc tgatgggaga gctgagcctg gtgtgcccat gctgtgccaa caatgggtcc 1980
ggatctggca gtgggtcagg aagcatgagg ggcaacgtgc cacagctgaa ggacgtggtc 2040
ctgcacctga ctccacagac cgagatcgac ctgcagtgct acgaacagtt tgattcaagc 2100
gaggaagagg acgaagtgga taatatgcga gatcagctgc cagagcgccg agctggacag 2160
gcaacctgct accgcatcga ggcaccttgc tgtcggaaac agtgcctgct gttctattcc 2220
aaaattagag agtaccggcg gtacagccgg agcgtgtacg gcacaactct ggaagctatc 2280
acaaagaaat ctctgtatga cctgagtatt agatgccaca ggtgtcagcg ccctctggga 2340
ccagaagaga agcagaaact ggtggatgag aagaaacgct ttcatgaaat cgcaggccgg 2400
tggaccggac agtgcgctaa ctgttggcag cgcacacgac agcggaatga gactcaagtg 2460
ggcagtgggt caggaagcgg ctccgggtct atggagcccc agttcaacaa tcctcaggaa 2520
agaccaaggt cactgcacca tctgagcgag gtgctggaaa tccctctgat tgacctgaga 2580
ctgagctgcg tgtactgtaa gaaagagctg acaagggctg aagtctataa ctttgcatgc 2640
actgagctga agctggtgta ccgcgacgat tttccctatg ccgtgtgccg ggtctgtctg 2700
ctgttctact ccaaggtgcg aaaataccgg tactatgatt atagtgtcca ggcccgccag 2760
gctaaacagc acacatgcta tctgatccat gtgccatgct gtgagtgtaa gttcgtggtc 2820
cagctggaca ttcagagcac taaagaggac ctgcgggtgg tccagcagct gctgatggga 2880
gctctgacag tgacttgccc cctgtgcgca tcctctaacg gaagtggctc agggagcggc 2940
agcggctcta tgcacggcaa ggtgcccaca ctgcaggacg tggtcctgga gctgacacct 3000
cagactgaaa tcgacctgca gtgcaatgag cagctggata gttcagagga cgaagatgag 3060
gacgaagtgg atcatctgca ggaaagacct cagcaggcaa ggcaggccaa gcagcacacc 3120
tgctacctga ttcacgtccc atgctgtgag cgcgtctgtc tgctgtttta cagcaaggtg 3180
agaaaatata ggtactatga ctacagtgtc tatggcgcca ctctggagtc aatcaccaag 3240
aaacagctgt gcgatctgct gattcgatgc taccggtgcc agagcccact gacccctgaa 3300
gagaagcagc tgcactgcga cagaaaaagg cgcttccacc tgatcgccca tggatggaca 3360
ggcagctgcc tgggctgttg gaggcagact tcccgggagc ctagagaatc taccgtgggg 3420
agtggatcag gcagcgggtc cggatctatg gctagatttg aggaccccac acagaggcct 3480
tacaagctgc ccgacctgag caccaccctg aacattccac tgcatgacat ccgcattaat 3540
tgcgtcttct gtaaaggcga gctgcaggag cgggaagtgt tcgaatttgc cttcaacgac 3600
ctgtttatcg tgtacaggga ttgcaccccc tatgcagcct gcctgaagtg tatttccttc 3660
tacgctcgcg tgcgagagct gaggtactat cgcgattctg tcctgctgct ggctcgacgg 3720
gcagaacctc agcgccacaa tatcgtgtgc gtctgctgta aatgtaacaa tcagctgcag 3780
ctggtcgtgg agaccagcca ggacggactg cgggccctgc aacaactgtt tatggataca 3840
ctgagcttcg tgtgccctct gtgcgctgca aaccaaggca gtgggtcagg aagcggctcc 3900
gggtctatgc atggaccaaa ggccaccctg tgcgacatcg tgctggatct ggaaccccag 3960
aattacgaag aggtggacct ggtctgttat gagcagctgc ctgatagtga ctcagagaac 4020
gaaaaagacg aaccagatgg cgtgaatcac ccactgctgc tggccagaag ggctgagcca 4080
cagagacata acatcgtgtg cgtctgctgc aagctgaaat gtattagttt ttacgctcgg 4140
gtgagagaac tgcgatacta tcgggactct gtctatgggg agactctgga ggcagaaacc 4200
aagacacccc tgcacgagct gctgatcaga tgctacaggt gtctgaaacc tctgtgcccc 4260
accgataagc tgaaacacat tacagagaaa cgccgattcc ataatatcgc cggaatctac 4320
accggccagt gcagggggtg tagaacacga gcaaggcatc tgaggcagca gcggcaggca 4380
aggtccgaga ctctggtggg atcctaa 4407
<210> 32
<211> 115
<212> PRT
<213> Artificial Sequence
<220>
<223> HSV-2 UL39-N1 peptide
<400> 32
Met Arg Ser Pro Ser Glu Arg Gln Glu Pro Arg Glu Pro Glu Val Ala
1 5 10 15
Pro Pro Gly Gly Asp His Val Phe Cys Arg Lys Val Ser Gly Val Met
20 25 30
Val Leu Ser Ser Asp Pro Pro Gly Pro Ala Ala Tyr Arg Ile Ser Asp
35 40 45
Ser Ser Phe Val Gln Cys Gly Ser Asn Cys Ser Met Ile Ile Asp Gly
50 55 60
Asp Gly Asp Gly Arg Thr Ala Val Val Ala Leu Gly Gly Thr Ser Gly
65 70 75 80
Pro Ser Ala Thr Thr Ser Val Gly Thr Gln Thr Ser Gly Glu Phe Leu
85 90 95
His Gly Asn Pro Arg Thr Pro Glu Pro Gln Gly Pro Gln Ala Val Pro
100 105 110
Pro Pro Pro
115
<210> 33
<211> 26
<212> PRT
<213> Artificial Sequence
<220>
<223> HSV-2 UL39-C2 peptide
<400> 33
Tyr Cys Lys Val Arg Lys Ala Thr Asn Ser Gly Val Phe Ala Gly Asp
1 5 10 15
Asp Asn Ile Val Cys Thr Ser Cys Ala Leu
20 25
<210> 34
<211> 73
<212> PRT
<213> Artificial Sequence
<220>
<223> HSV-2 UL39-N2 peptide
<400> 34
Pro Pro Pro Phe Pro Trp Gly His Glu Cys Cys Ala Arg Arg Asp Ala
1 5 10 15
Arg Gly Gly Ala Glu Lys Asp Val Gly Ala Ala Glu Ser Trp Ser Asp
20 25 30
Gly Pro Ser Ser Asp Ser Glu Thr Glu Asp Ser Asp Ser Ser Asp Glu
35 40 45
Asp Thr Gly Ser Glu Thr Leu Ser Arg Ser Ser Ser Ile Trp Ala Ala
50 55 60
Gly Ala Thr Asp Asp Asp Asp Ser Asp
65 70
<210> 35
<211> 718
<212> PRT
<213> Artificial Sequence
<220>
<223> HSV-2 UL39-N4-C1 peptide
<400> 35
Pro Asn Ala Tyr Thr Pro Tyr His Leu Arg Glu Tyr Ala Thr Arg Leu
1 5 10 15
Val Asn Gly Phe Lys Pro Leu Val Arg Arg Ser Ala Arg Leu Tyr Arg
20 25 30
Ile Leu Gly Val Leu Val His Leu Arg Ile Arg Thr Arg Glu Ala Ser
35 40 45
Phe Glu Glu Trp Met Arg Ser Lys Glu Val Asp Leu Asp Phe Gly Leu
50 55 60
Thr Glu Arg Leu Arg Glu His Glu Ala Gln Leu Met Ile Leu Ala Gln
65 70 75 80
Ala Leu Asn Pro Tyr Asp Cys Leu Ile His Ser Thr Pro Asn Thr Leu
85 90 95
Val Glu Arg Gly Leu Gln Ser Ala Leu Lys Tyr Glu Glu Phe Tyr Leu
100 105 110
Lys Arg Phe Gly Gly His Tyr Met Glu Ser Val Phe Gln Met Tyr Thr
115 120 125
Arg Ile Ala Gly Phe Leu Ala Cys Arg Ala Thr Arg Gly Met Arg His
130 135 140
Ile Ala Leu Gly Arg Gln Gly Ser Trp Trp Glu Met Phe Lys Phe Phe
145 150 155 160
Phe His Arg Leu Tyr Asp His Gln Ile Val Pro Ser Thr Pro Ala Met
165 170 175
Leu Asn Leu Gly Thr Arg Asn Tyr Tyr Thr Ser Ser Cys Tyr Leu Val
180 185 190
Asn Pro Gln Ala Thr Thr Asn Gln Ala Thr Leu Arg Ala Ile Thr Gly
195 200 205
Asn Val Ser Ala Ile Leu Ala Arg Asn Gly Gly Ile Gly Leu Cys Met
210 215 220
Gln Ala Phe Asn Asp Ala Ser Pro Gly Thr Ala Ser Ile Met Pro Ala
225 230 235 240
Leu Lys Val Leu Asp Ser Leu Val Ala Ala His Asn Lys Gln Ser Thr
245 250 255
Arg Pro Thr Gly Ala Cys Val Tyr Leu Glu Pro Trp His Ser Asp Val
260 265 270
Arg Ala Val Leu Arg Met Lys Gly Val Leu Ala Gly Glu Glu Ala Gln
275 280 285
Arg Cys Asp Asn Ile Phe Ser Ala Leu Trp Met Pro Asp Leu Phe Phe
290 295 300
Lys Arg Leu Ile Arg His Leu Asp Gly Glu Lys Asn Val Thr Trp Ser
305 310 315 320
Leu Phe Asp Arg Asp Thr Ser Met Ser Leu Ala Asp Phe His Gly Glu
325 330 335
Glu Phe Glu Lys Leu Tyr Glu His Leu Glu Ala Met Gly Phe Gly Glu
340 345 350
Thr Ile Pro Ile Gln Asp Leu Ala Tyr Ala Ile Val Arg Ser Ala Ala
355 360 365
Thr Thr Gly Ser Pro Phe Ile Met Phe Lys Asp Ala Val Asn Arg His
370 375 380
Tyr Ile Tyr Asp Thr Gln Gly Ala Ala Ile Ala Gly Ser Asn Leu Cys
385 390 395 400
Thr Glu Ile Val His Pro Ala Ser Lys Arg Ser Ser Gly Val Cys Asn
405 410 415
Leu Gly Ser Val Asn Leu Ala Arg Cys Val Ser Arg Gln Thr Phe Asp
420 425 430
Phe Gly Arg Leu Arg Asp Ala Val Gln Ala Cys Val Leu Met Val Asn
435 440 445
Ile Met Ile Asp Ser Thr Leu Gln Pro Thr Pro Gln Cys Thr Arg Gly
450 455 460
Asn Asp Asn Leu Arg Ser Met Gly Ile Gly Met Gln Gly Leu His Thr
465 470 475 480
Ala Cys Leu Lys Met Gly Leu Asp Leu Glu Ser Ala Glu Phe Arg Asp
485 490 495
Leu Asn Thr His Ile Ala Glu Val Met Leu Leu Ala Ala Met Lys Thr
500 505 510
Ser Asn Ala Leu Cys Val Arg Gly Ala Arg Pro Phe Ser His Phe Lys
515 520 525
Arg Ser Met Tyr Arg Ala Gly Arg Phe His Trp Glu Arg Phe Ser Asn
530 535 540
Ala Ser Pro Arg Tyr Glu Gly Glu Trp Glu Met Leu Arg Gln Ser Met
545 550 555 560
Met Lys His Gly Leu Arg Asn Ser Gln Phe Ile Ala Leu Met Pro Thr
565 570 575
Ala Ala Ser Ala Gln Ile Ser Asp Val Ser Glu Gly Phe Ala Pro Leu
580 585 590
Phe Thr Asn Leu Phe Ser Lys Val Thr Arg Asp Gly Glu Thr Leu Arg
595 600 605
Pro Asn Thr Leu Leu Leu Lys Glu Leu Glu Arg Thr Phe Gly Gly Lys
610 615 620
Arg Leu Leu Asp Ala Met Asp Gly Leu Glu Ala Lys Gln Trp Ser Val
625 630 635 640
Ala Gln Ala Leu Pro Cys Leu Asp Pro Ala His Pro Leu Arg Arg Phe
645 650 655
Lys Thr Ala Phe Asp Tyr Asp Gln Glu Leu Leu Ile Asp Leu Cys Ala
660 665 670
Asp Arg Ala Pro Tyr Val Asp His Ser Gln Ser Met Thr Leu Tyr Val
675 680 685
Thr Glu Lys Ala Asp Gly Thr Leu Pro Ala Ser Thr Leu Val Arg Leu
690 695 700
Leu Val His Ala Tyr Lys Arg Gly Leu Lys Thr Gly Met Tyr
705 710 715
<210> 36
<211> 191
<212> PRT
<213> Artificial Sequence
<220>
<223> HSV-2 UL39-N3 peptide
<400> 36
Thr Leu Ser Arg Ser Ser Ser Ile Trp Ala Ala Gly Ala Thr Asp Asp
1 5 10 15
Asp Asp Ser Asp Ser Asp Ser Arg Ser Asp Asp Ser Val Gln Pro Asp
20 25 30
Val Val Val Arg Arg Arg Trp Ser Asp Gly Pro Ala Pro Val Ala Phe
35 40 45
Pro Lys Pro Arg Arg Pro Gly Asp Ser Pro Gly Asn Pro Gly Leu Gly
50 55 60
Ala Gly Thr Gly Pro Gly Ser Ala Thr Asp Pro Arg Ala Ser Ala Asp
65 70 75 80
Ser Asp Ser Ala Ala His Ala Ala Ala Pro Gln Ala Asp Val Ala Pro
85 90 95
Val Leu Asp Ser Gln Pro Thr Val Gly Thr Asp Pro Gly Tyr Pro Val
100 105 110
Pro Leu Glu Leu Thr Pro Glu Asn Ala Glu Ala Val Ala Arg Phe Leu
115 120 125
Gly Asp Ala Val Asp Arg Glu Pro Ala Leu Met Leu Glu Tyr Phe Cys
130 135 140
Arg Cys Ala Arg Glu Glu Ser Lys Arg Val Pro Pro Arg Thr Phe Gly
145 150 155 160
Ser Ala Pro Arg Leu Thr Glu Asp Asp Phe Gly Leu Leu Asn Tyr Ala
165 170 175
Leu Ala Glu Met Arg Arg Leu Cys Leu Asp Leu Pro Pro Val Pro
180 185 190
<210> 37
<211> 1123
<212> PRT
<213> Artificial Sequence
<220>
<223> shuffled UL39 polypeptide
<400> 37
Met Arg Ser Pro Ser Glu Arg Gln Glu Pro Arg Glu Pro Glu Val Ala
1 5 10 15
Pro Pro Gly Gly Asp His Val Phe Cys Arg Lys Val Ser Gly Val Met
20 25 30
Val Leu Ser Ser Asp Pro Pro Gly Pro Ala Ala Tyr Arg Ile Ser Asp
35 40 45
Ser Ser Phe Val Gln Cys Gly Ser Asn Cys Ser Met Ile Ile Asp Gly
50 55 60
Asp Gly Asp Gly Arg Thr Ala Val Val Ala Leu Gly Gly Thr Ser Gly
65 70 75 80
Pro Ser Ala Thr Thr Ser Val Gly Thr Gln Thr Ser Gly Glu Phe Leu
85 90 95
His Gly Asn Pro Arg Thr Pro Glu Pro Gln Gly Pro Gln Ala Val Pro
100 105 110
Pro Pro Pro Tyr Cys Lys Val Arg Lys Ala Thr Asn Ser Gly Val Phe
115 120 125
Ala Gly Asp Asp Asn Ile Val Cys Thr Ser Cys Ala Leu Pro Pro Pro
130 135 140
Phe Pro Trp Gly His Glu Cys Cys Ala Arg Arg Asp Ala Arg Gly Gly
145 150 155 160
Ala Glu Lys Asp Val Gly Ala Ala Glu Ser Trp Ser Asp Gly Pro Ser
165 170 175
Ser Asp Ser Glu Thr Glu Asp Ser Asp Ser Ser Asp Glu Asp Thr Gly
180 185 190
Ser Glu Thr Leu Ser Arg Ser Ser Ser Ile Trp Ala Ala Gly Ala Thr
195 200 205
Asp Asp Asp Asp Ser Asp Pro Asn Ala Tyr Thr Pro Tyr His Leu Arg
210 215 220
Glu Tyr Ala Thr Arg Leu Val Asn Gly Phe Lys Pro Leu Val Arg Arg
225 230 235 240
Ser Ala Arg Leu Tyr Arg Ile Leu Gly Val Leu Val His Leu Arg Ile
245 250 255
Arg Thr Arg Glu Ala Ser Phe Glu Glu Trp Met Arg Ser Lys Glu Val
260 265 270
Asp Leu Asp Phe Gly Leu Thr Glu Arg Leu Arg Glu His Glu Ala Gln
275 280 285
Leu Met Ile Leu Ala Gln Ala Leu Asn Pro Tyr Asp Cys Leu Ile His
290 295 300
Ser Thr Pro Asn Thr Leu Val Glu Arg Gly Leu Gln Ser Ala Leu Lys
305 310 315 320
Tyr Glu Glu Phe Tyr Leu Lys Arg Phe Gly Gly His Tyr Met Glu Ser
325 330 335
Val Phe Gln Met Tyr Thr Arg Ile Ala Gly Phe Leu Ala Cys Arg Ala
340 345 350
Thr Arg Gly Met Arg His Ile Ala Leu Gly Arg Gln Gly Ser Trp Trp
355 360 365
Glu Met Phe Lys Phe Phe Phe His Arg Leu Tyr Asp His Gln Ile Val
370 375 380
Pro Ser Thr Pro Ala Met Leu Asn Leu Gly Thr Arg Asn Tyr Tyr Thr
385 390 395 400
Ser Ser Cys Tyr Leu Val Asn Pro Gln Ala Thr Thr Asn Gln Ala Thr
405 410 415
Leu Arg Ala Ile Thr Gly Asn Val Ser Ala Ile Leu Ala Arg Asn Gly
420 425 430
Gly Ile Gly Leu Cys Met Gln Ala Phe Asn Asp Ala Ser Pro Gly Thr
435 440 445
Ala Ser Ile Met Pro Ala Leu Lys Val Leu Asp Ser Leu Val Ala Ala
450 455 460
His Asn Lys Gln Ser Thr Arg Pro Thr Gly Ala Cys Val Tyr Leu Glu
465 470 475 480
Pro Trp His Ser Asp Val Arg Ala Val Leu Arg Met Lys Gly Val Leu
485 490 495
Ala Gly Glu Glu Ala Gln Arg Cys Asp Asn Ile Phe Ser Ala Leu Trp
500 505 510
Met Pro Asp Leu Phe Phe Lys Arg Leu Ile Arg His Leu Asp Gly Glu
515 520 525
Lys Asn Val Thr Trp Ser Leu Phe Asp Arg Asp Thr Ser Met Ser Leu
530 535 540
Ala Asp Phe His Gly Glu Glu Phe Glu Lys Leu Tyr Glu His Leu Glu
545 550 555 560
Ala Met Gly Phe Gly Glu Thr Ile Pro Ile Gln Asp Leu Ala Tyr Ala
565 570 575
Ile Val Arg Ser Ala Ala Thr Thr Gly Ser Pro Phe Ile Met Phe Lys
580 585 590
Asp Ala Val Asn Arg His Tyr Ile Tyr Asp Thr Gln Gly Ala Ala Ile
595 600 605
Ala Gly Ser Asn Leu Cys Thr Glu Ile Val His Pro Ala Ser Lys Arg
610 615 620
Ser Ser Gly Val Cys Asn Leu Gly Ser Val Asn Leu Ala Arg Cys Val
625 630 635 640
Ser Arg Gln Thr Phe Asp Phe Gly Arg Leu Arg Asp Ala Val Gln Ala
645 650 655
Cys Val Leu Met Val Asn Ile Met Ile Asp Ser Thr Leu Gln Pro Thr
660 665 670
Pro Gln Cys Thr Arg Gly Asn Asp Asn Leu Arg Ser Met Gly Ile Gly
675 680 685
Met Gln Gly Leu His Thr Ala Cys Leu Lys Met Gly Leu Asp Leu Glu
690 695 700
Ser Ala Glu Phe Arg Asp Leu Asn Thr His Ile Ala Glu Val Met Leu
705 710 715 720
Leu Ala Ala Met Lys Thr Ser Asn Ala Leu Cys Val Arg Gly Ala Arg
725 730 735
Pro Phe Ser His Phe Lys Arg Ser Met Tyr Arg Ala Gly Arg Phe His
740 745 750
Trp Glu Arg Phe Ser Asn Ala Ser Pro Arg Tyr Glu Gly Glu Trp Glu
755 760 765
Met Leu Arg Gln Ser Met Met Lys His Gly Leu Arg Asn Ser Gln Phe
770 775 780
Ile Ala Leu Met Pro Thr Ala Ala Ser Ala Gln Ile Ser Asp Val Ser
785 790 795 800
Glu Gly Phe Ala Pro Leu Phe Thr Asn Leu Phe Ser Lys Val Thr Arg
805 810 815
Asp Gly Glu Thr Leu Arg Pro Asn Thr Leu Leu Leu Lys Glu Leu Glu
820 825 830
Arg Thr Phe Gly Gly Lys Arg Leu Leu Asp Ala Met Asp Gly Leu Glu
835 840 845
Ala Lys Gln Trp Ser Val Ala Gln Ala Leu Pro Cys Leu Asp Pro Ala
850 855 860
His Pro Leu Arg Arg Phe Lys Thr Ala Phe Asp Tyr Asp Gln Glu Leu
865 870 875 880
Leu Ile Asp Leu Cys Ala Asp Arg Ala Pro Tyr Val Asp His Ser Gln
885 890 895
Ser Met Thr Leu Tyr Val Thr Glu Lys Ala Asp Gly Thr Leu Pro Ala
900 905 910
Ser Thr Leu Val Arg Leu Leu Val His Ala Tyr Lys Arg Gly Leu Lys
915 920 925
Thr Gly Met Tyr Thr Leu Ser Arg Ser Ser Ser Ile Trp Ala Ala Gly
930 935 940
Ala Thr Asp Asp Asp Asp Ser Asp Ser Asp Ser Arg Ser Asp Asp Ser
945 950 955 960
Val Gln Pro Asp Val Val Val Arg Arg Arg Trp Ser Asp Gly Pro Ala
965 970 975
Pro Val Ala Phe Pro Lys Pro Arg Arg Pro Gly Asp Ser Pro Gly Asn
980 985 990
Pro Gly Leu Gly Ala Gly Thr Gly Pro Gly Ser Ala Thr Asp Pro Arg
995 1000 1005
Ala Ser Ala Asp Ser Asp Ser Ala Ala His Ala Ala Ala Pro Gln
1010 1015 1020
Ala Asp Val Ala Pro Val Leu Asp Ser Gln Pro Thr Val Gly Thr
1025 1030 1035
Asp Pro Gly Tyr Pro Val Pro Leu Glu Leu Thr Pro Glu Asn Ala
1040 1045 1050
Glu Ala Val Ala Arg Phe Leu Gly Asp Ala Val Asp Arg Glu Pro
1055 1060 1065
Ala Leu Met Leu Glu Tyr Phe Cys Arg Cys Ala Arg Glu Glu Ser
1070 1075 1080
Lys Arg Val Pro Pro Arg Thr Phe Gly Ser Ala Pro Arg Leu Thr
1085 1090 1095
Glu Asp Asp Phe Gly Leu Leu Asn Tyr Ala Leu Ala Glu Met Arg
1100 1105 1110
Arg Leu Cys Leu Asp Leu Pro Pro Val Pro
1115 1120
<210> 38
<211> 3372
<212> DNA
<213> Artificial Sequence
<220>
<223> polynucleotide encoding shuffled UL39 polypeptide
<400> 38
atgagaagtc caagcgaaag gcaggaacct agagagccag aagtggcacc acctggaggg 60
gaccacgtct tctgcaggaa ggtgtctggg gtcatggtgc tgagctccga tccaccagga 120
cctgcagctt accggatcag tgactctagt tttgtgcagt gcgggagcaa ctgttccatg 180
atcattgacg gcgatgggga cggaagaaca gctgtggtcg cactgggagg cacttctgga 240
cccagtgcca ccacatccgt gggcacccag acatctgggg aatttctgca cggaaatccc 300
cgaacccctg agccacaggg acctcaggcc gtgcctccac ccccttattg taaggtcaga 360
aaagctacca acagtggagt gttcgcaggc gacgataata ttgtctgcac atcatgtgcc 420
ctgccacccc cttttccatg gggccatgag tgctgtgccc ggagagatgc taggggggga 480
gcagaaaaag acgtgggggc agccgagagt tggtcagatg gaccctcaag cgacagcgag 540
accgaagatt ccgactcctc tgatgaagac actggcagtg agaccctgtc acgcagttca 600
agcatctggg ctgcaggggc cacagacgat gacgattccg acccaaacgc ttacacaccc 660
tatcacctga gggaatacgc aactcgcctg gtgaatgggt tcaagcctct ggtcaggcgc 720
agcgcccgcc tgtatagaat cctgggagtc ctggtgcatc tgagaatcag gacacgcgag 780
gctagcttcg aggaatggat gaggtccaaa gaagtggatc tggactttgg cctgactgag 840
cgactgcggg agcacgaagc ccagctgatg attctggcac aggccctgaa cccatacgat 900
tgcctgatcc attcaactcc caataccctg gtggaacgag gactgcagag cgccctgaag 960
tacgaggagt tctacctgaa acggtttggc gggcactaca tggagagcgt gttccagatg 1020
tataccagaa ttgccggctt tctggcctgt cgggctacaa gagggatgag gcatatcgct 1080
ctgggccgcc aggggagctg gtgggagatg tttaagttct ttttccaccg gctgtacgac 1140
catcagatcg tgccatccac ccccgccatg ctgaacctgg gcactcggaa ttactatacc 1200
tcctcttgct atctggtgaa cccccaggcc actaccaatc aggccacact gcgcgctatc 1260
actgggaacg tgagcgcaat tctggcccga aatggaggca tcggactgtg catgcaggca 1320
ttcaacgatg cctctcctgg caccgccagt atcatgccag ctctgaaggt cctggactcc 1380
ctggtggccg ctcacaacaa gcagtctaca aggcctactg gcgcctgcgt gtacctggag 1440
ccatggcatt ctgatgtcag agctgtgctg aggatgaagg gggtgctggc tggagaggaa 1500
gcacagcggt gcgataacat tttcagcgcc ctgtggatgc ctgacctgtt tttcaagcgc 1560
ctgatccgac acctggatgg agagaaaaat gtgacctgga gcctgtttga tcgggacaca 1620
agcatgtccc tggccgactt ccacggcgag gaatttgaaa aactgtacga acatctggag 1680
gctatgggct tcggggagac catcccaatt caggacctgg cttatgcaat tgtgaggagt 1740
gcagccacaa ctggctcacc ctttatcatg ttcaaggatg ccgtcaaccg ccactacatc 1800
tacgacactc agggcgctgc aatcgctggg tctaatctgt gcaccgagat cgtgcatcca 1860
gcaagtaaaa gaagttcagg agtctgcaac ctgggctcag tgaatctggc acgatgcgtg 1920
agccggcaga ccttcgactt cggaagactg agggacgctg tgcaggcatg cgtcctgatg 1980
gtgaacatca tgattgatag cacactgcag cccactcctc agtgtacccg aggcaacgac 2040
aatctgcggt ccatgggaat tggcatgcag ggactgcaca cagcctgcct gaagatgggc 2100
ctggatctgg aatctgccga gttccgggac ctgaacacac atatcgctga agtgatgctg 2160
ctggccgcta tgaagactag caatgcactg tgcgtgcggg gagccagacc tttttcacac 2220
ttcaaaagaa gcatgtacag ggcaggccgc ttccattggg agcggttttc aaacgccagc 2280
ccaagatatg agggggaatg ggagatgctg agacagagta tgatgaagca cggactgcgg 2340
aacagccagt ttattgcact gatgcccact gcagcctctg cccagatcag cgacgtgagc 2400
gaaggctttg cccctctgtt caccaacctg tttagcaaag tcaccagaga cggggagaca 2460
ctgaggccta atactctgct gctgaaggaa ctggagcgca ctttcggggg aaaacgactg 2520
ctggatgcta tggacggcct ggaggcaaag cagtggtccg tggcacaggc tctgccatgc 2580
ctggaccctg ctcatccact gcgacggttc aaaacagcat ttgattacga ccaggaactg 2640
ctgatcgacc tgtgcgccga cagagctccc tacgtggatc actctcagag tatgacactg 2700
tatgtcactg agaaggccga cggcaccctg cctgctagca cactggtcag gctgctggtg 2760
catgcctaca agcgcggcct gaaaaccggg atgtatacac tgtccaggag ctcctctatc 2820
tgggccgccg gggccaccga cgatgacgat tcagatagcg actcccgctc tgacgattcc 2880
gtgcagccag atgtggtcgt gagaaggcgc tggtctgacg gaccagcacc agtggcattc 2940
cctaaaccac gacggcccgg agatagccca ggcaaccctg gactgggagc aggcactggg 3000
cctggatctg ctaccgaccc aagggcaagt gccgatagtg actcagccgc tcacgcagcc 3060
gctccacagg ctgatgtcgc acctgtgctg gactcccagc caaccgtggg gacagacccc 3120
ggataccccg tccctctgga gctgacccct gaaaatgctg aggcagtggc ccgattcctg 3180
ggcgatgcag tggaccggga accagccctg atgctggagt atttttgccg atgtgcccgg 3240
gaggaaagca agcgggtgcc accaagaact ttcggctccg ctccacgact gaccgaggac 3300
gattttgggc tgctgaacta tgccctggcc gaaatgcgga gactgtgcct ggacctgccc 3360
cccgtgccct aa 3372
<210> 39
<211> 344
<212> PRT
<213> Artificial Sequence
<220>
<223> HSV-2 gD_26-349 polypeptide
<400> 39
Lys Tyr Ala Leu Ala Asp Pro Ser Leu Lys Met Ala Asp Pro Asn Arg
1 5 10 15
Phe Arg Gly Lys Asn Leu Pro Val Leu Asp Gln Leu Thr Asp Pro Pro
20 25 30
Gly Val Lys Arg Val Tyr His Ile Gln Pro Ser Leu Glu Asp Pro Phe
35 40 45
Gln Pro Pro Ser Ile Pro Ile Thr Val Tyr Tyr Ala Val Leu Glu Arg
50 55 60
Ala Cys Arg Ser Val Leu Leu His Ala Pro Ser Glu Ala Pro Gln Ile
65 70 75 80
Val Arg Gly Ala Ser Asp Glu Ala Arg Lys His Thr Tyr Asn Leu Thr
85 90 95
Ile Ala Trp Tyr Arg Met Gly Asp Asn Cys Ala Ile Pro Ile Thr Val
100 105 110
Met Glu Tyr Thr Glu Cys Pro Tyr Asn Lys Ser Leu Gly Val Cys Pro
115 120 125
Ile Arg Thr Gln Pro Arg Trp Ser Tyr Tyr Asp Ser Phe Ser Ala Val
130 135 140
Ser Glu Asp Asn Leu Gly Phe Leu Met His Ala Pro Ala Phe Glu Thr
145 150 155 160
Ala Gly Thr Tyr Leu Arg Leu Val Lys Ile Asn Asp Trp Thr Glu Ile
165 170 175
Thr Gln Phe Ile Leu Glu His Arg Ala Arg Ala Ser Cys Lys Tyr Ala
180 185 190
Leu Pro Leu Arg Ile Pro Pro Ala Ala Cys Leu Thr Ser Lys Ala Tyr
195 200 205
Gln Gln Gly Val Thr Val Asp Ser Ile Gly Met Leu Pro Arg Phe Ile
210 215 220
Pro Glu Asn Gln Arg Thr Val Ala Leu Tyr Ser Leu Lys Ile Ala Gly
225 230 235 240
Trp His Gly Pro Lys Pro Pro Tyr Thr Ser Thr Leu Leu Pro Pro Glu
245 250 255
Leu Ser Asp Thr Thr Asn Ala Thr Gln Pro Glu Leu Val Pro Glu Asp
260 265 270
Pro Glu Asp Ser Ala Leu Leu Glu Asp Pro Ala Gly Thr Val Ser Ser
275 280 285
Gln Ile Pro Pro Asn Trp His Ile Pro Ser Ile Gln Asp Val Ala Pro
290 295 300
His His Ala Pro Ala Ala Pro Ser Asn Pro Arg Arg Arg Ala Gln Met
305 310 315 320
Ala Pro Lys Arg Leu Arg Leu Pro His Ile Arg Asp Asp Asp Ala Pro
325 330 335
Pro Ser His Gln Pro Leu Phe Tyr
340
<210> 40
<211> 818
<212> PRT
<213> Artificial Sequence
<220>
<223> HSV-2 ICP0 polypeptide w/o NLS sequence (a.a. 510-516)
<400> 40
Met Glu Pro Arg Pro Gly Thr Ser Ser Arg Ala Asp Pro Gly Pro Glu
1 5 10 15
Arg Pro Pro Arg Gln Thr Pro Gly Thr Gln Pro Ala Ala Pro His Ala
20 25 30
Trp Gly Met Leu Asn Asp Met Gln Trp Leu Ala Ser Ser Asp Ser Glu
35 40 45
Glu Glu Thr Glu Val Gly Ile Ser Asp Asp Asp Leu His Arg Asp Ser
50 55 60
Thr Ser Glu Ala Gly Ser Thr Asp Thr Glu Met Phe Glu Ala Gly Leu
65 70 75 80
Met Asp Ala Ala Thr Pro Pro Ala Arg Pro Pro Ala Glu Arg Gln Gly
85 90 95
Ser Pro Thr Pro Ala Asp Ala Gln Gly Ser Cys Gly Gly Gly Pro Val
100 105 110
Gly Glu Glu Glu Ala Glu Ala Gly Gly Gly Gly Asp Val Cys Ala Val
115 120 125
Cys Thr Asp Glu Ile Ala Pro Pro Leu Arg Cys Gln Ser Phe Pro Cys
130 135 140
Leu His Pro Phe Cys Ile Pro Cys Met Lys Thr Trp Ile Pro Leu Arg
145 150 155 160
Asn Thr Cys Pro Leu Cys Asn Thr Pro Val Ala Tyr Leu Ile Val Gly
165 170 175
Val Thr Ala Ser Gly Ser Phe Ser Thr Ile Pro Ile Val Asn Asp Pro
180 185 190
Arg Thr Arg Val Glu Ala Glu Ala Ala Val Arg Ala Gly Thr Ala Val
195 200 205
Asp Phe Ile Trp Thr Gly Asn Pro Arg Thr Ala Pro Arg Ser Leu Ser
210 215 220
Leu Gly Gly His Thr Val Arg Ala Leu Ser Pro Thr Pro Pro Trp Pro
225 230 235 240
Gly Thr Asp Asp Glu Asp Asp Asp Leu Ala Asp Val Asp Tyr Val Pro
245 250 255
Pro Ala Pro Arg Arg Ala Pro Arg Arg Gly Gly Gly Gly Ala Gly Ala
260 265 270
Thr Arg Gly Thr Ser Gln Pro Ala Ala Thr Arg Pro Ala Pro Pro Gly
275 280 285
Ala Pro Arg Ser Ser Ser Ser Gly Gly Ala Pro Leu Arg Ala Gly Val
290 295 300
Gly Ser Gly Ser Gly Gly Gly Pro Ala Val Ala Ala Val Val Pro Arg
305 310 315 320
Val Ala Ser Leu Pro Pro Ala Ala Gly Gly Gly Arg Ala Gln Ala Arg
325 330 335
Arg Val Gly Glu Asp Ala Ala Ala Ala Glu Gly Arg Thr Pro Pro Ala
340 345 350
Arg Gln Pro Arg Ala Ala Gln Glu Pro Pro Ile Val Ile Ser Asp Ser
355 360 365
Pro Pro Pro Ser Pro Arg Arg Pro Ala Gly Pro Gly Pro Leu Ser Phe
370 375 380
Val Ser Ser Ser Ser Ala Gln Val Ser Ser Gly Pro Gly Gly Gly Gly
385 390 395 400
Leu Pro Gln Ser Ser Gly Arg Ala Ala Arg Pro Arg Ala Ala Val Ala
405 410 415
Pro Arg Val Arg Ser Pro Pro Arg Ala Ala Ala Ala Pro Val Val Ser
420 425 430
Ala Ser Ala Asp Ala Ala Gly Pro Ala Pro Pro Ala Val Pro Val Asp
435 440 445
Ala His Arg Ala Pro Arg Ser Arg Met Thr Gln Ala Gln Thr Asp Thr
450 455 460
Gln Ala Gln Ser Leu Gly Arg Ala Gly Ala Thr Asp Ala Arg Gly Ser
465 470 475 480
Gly Gly Pro Gly Ala Glu Gly Gly Pro Gly Val Pro Arg Gly Thr Asn
485 490 495
Thr Pro Gly Ala Ala Pro His Ala Ala Glu Gly Ala Ala Gly Ser Asp
500 505 510
Ser Gly Pro Ala Ala Ser Ser Ser Ala Ser Ser Ser Ala Ala Pro Arg
515 520 525
Ser Pro Leu Ala Pro Gln Gly Val Gly Ala Lys Arg Ala Ala Pro Arg
530 535 540
Arg Ala Pro Asp Ser Asp Ser Gly Asp Arg Gly His Gly Pro Leu Ala
545 550 555 560
Pro Ala Ser Ala Gly Ala Ala Pro Pro Ser Ala Ser Pro Ser Ser Gln
565 570 575
Ala Ala Val Ala Ala Ala Ser Ser Ser Ser Ala Ser Ser Ser Ser Ala
580 585 590
Ser Ser Ser Ser Ala Ser Ser Ser Ser Ala Ser Ser Ser Ser Ala Ser
595 600 605
Ser Ser Ser Ala Ser Ser Ser Ser Ala Ser Ser Ser Ala Gly Gly Ala
610 615 620
Gly Gly Ser Val Ala Ser Ala Ser Gly Ala Gly Glu Arg Arg Glu Thr
625 630 635 640
Ser Leu Gly Pro Arg Ala Ala Ala Pro Arg Gly Pro Arg Lys Cys Ala
645 650 655
Arg Lys Thr Arg His Ala Glu Gly Gly Pro Glu Pro Gly Ala Arg Asp
660 665 670
Pro Ala Pro Gly Leu Thr Arg Tyr Leu Pro Ile Ala Gly Val Ser Ser
675 680 685
Val Val Ala Leu Ala Pro Tyr Val Asn Lys Thr Val Thr Gly Asp Cys
690 695 700
Leu Pro Val Leu Asp Met Glu Thr Gly His Ile Gly Ala Tyr Val Val
705 710 715 720
Leu Val Asp Gln Thr Gly Asn Val Ala Asp Leu Leu Arg Ala Ala Ala
725 730 735
Pro Ala Trp Ser Arg Arg Thr Leu Leu Pro Glu His Ala Arg Asn Cys
740 745 750
Val Arg Pro Pro Asp Tyr Pro Thr Pro Pro Ala Ser Glu Trp Asn Ser
755 760 765
Leu Trp Met Thr Pro Val Gly Asn Met Leu Phe Asp Gln Gly Thr Leu
770 775 780
Val Gly Ala Leu Asp Phe His Gly Leu Arg Ser Arg His Pro Trp Ser
785 790 795 800
Arg Glu Gln Gly Ala Pro Ala Pro Ala Gly Asp Ala Pro Ala Gly His
805 810 815
Gly Glu
<210> 41
<211> 766
<212> PRT
<213> Artificial Sequence
<220>
<223> HSV-2 ICP4 polypeptide w/o RS1.3 region (a.a. 767-1318)
<400> 41
Met Ser Ala Glu Gln Arg Lys Lys Lys Lys Thr Thr Thr Thr Thr Gln
1 5 10 15
Gly Arg Gly Ala Glu Val Ala Met Ala Asp Glu Asp Gly Gly Arg Leu
20 25 30
Arg Ala Ala Ala Glu Thr Thr Gly Gly Pro Gly Ser Pro Asp Pro Ala
35 40 45
Asp Gly Pro Pro Pro Thr Pro Asn Pro Asp Arg Arg Pro Ala Ala Arg
50 55 60
Pro Gly Phe Gly Trp His Gly Gly Pro Glu Glu Asn Glu Asp Glu Ala
65 70 75 80
Asp Asp Ala Ala Ala Asp Ala Asp Ala Asp Glu Ala Ala Pro Ala Ser
85 90 95
Gly Glu Ala Val Asp Glu Pro Ala Ala Asp Gly Val Val Ser Pro Arg
100 105 110
Gln Leu Ala Leu Leu Ala Ser Met Val Asp Glu Ala Val Arg Thr Ile
115 120 125
Pro Ser Pro Pro Pro Glu Arg Asp Gly Ala Gln Glu Glu Ala Ala Arg
130 135 140
Ser Pro Ser Pro Pro Arg Thr Pro Ser Met Arg Ala Asp Tyr Gly Glu
145 150 155 160
Glu Asn Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp Arg Asp Ala
165 170 175
Gly Arg Trp Val Arg Gly Pro Glu Thr Thr Ser Ala Val Arg Gly Ala
180 185 190
Tyr Pro Asp Pro Met Ala Ser Leu Ser Pro Arg Pro Pro Ala Pro Arg
195 200 205
Arg His His His His His His His Arg Arg Arg Arg Ala Pro Arg Arg
210 215 220
Arg Ser Ala Ala Ser Asp Ser Ser Lys Ser Gly Ser Ser Ser Ser Ala
225 230 235 240
Ser Ser Ala Ser Ser Ser Ala Ser Ser Ser Ser Ser Ala Ser Ala Ser
245 250 255
Ser Ser Asp Asp Asp Asp Asp Asp Asp Ala Ala Arg Ala Pro Ala Ser
260 265 270
Ala Ala Asp His Ala Ala Gly Gly Thr Leu Gly Ala Asp Asp Glu Glu
275 280 285
Ala Gly Val Pro Ala Arg Ala Pro Gly Ala Ala Pro Arg Pro Ser Pro
290 295 300
Pro Arg Ala Glu Pro Ala Pro Ala Arg Thr Pro Ala Ala Thr Ala Gly
305 310 315 320
Arg Leu Glu Arg Arg Arg Ala Arg Ala Ala Val Ala Gly Arg Asp Ala
325 330 335
Thr Gly Arg Phe Thr Ala Gly Arg Pro Arg Arg Val Glu Leu Asp Ala
340 345 350
Asp Ala Ala Ser Gly Ala Phe Tyr Ala Arg Tyr Arg Asp Gly Tyr Val
355 360 365
Ser Gly Glu Pro Trp Pro Gly Ala Gly Pro Pro Pro Pro Gly Arg Val
370 375 380
Leu Tyr Gly Gly Leu Gly Asp Ser Arg Pro Gly Leu Trp Gly Ala Pro
385 390 395 400
Glu Ala Glu Glu Ala Arg Ala Arg Phe Glu Ala Ser Gly Ala Pro Ala
405 410 415
Pro Val Trp Ala Pro Glu Leu Gly Asp Ala Ala Gln Gln Tyr Ala Leu
420 425 430
Ile Thr Arg Leu Leu Tyr Thr Pro Asp Ala Glu Ala Met Gly Trp Leu
435 440 445
Gln Asn Pro Arg Val Ala Pro Gly Asp Val Ala Leu Asp Gln Ala Cys
450 455 460
Phe Arg Ile Ser Gly Ala Ala Arg Asn Ser Ser Ser Phe Ile Ser Gly
465 470 475 480
Ser Val Ala Arg Ala Val Pro His Leu Gly Tyr Ala Met Ala Ala Gly
485 490 495
Arg Phe Gly Trp Gly Leu Ala His Val Ala Ala Ala Val Ala Met Ser
500 505 510
Arg Arg Tyr Asp Arg Ala Gln Lys Gly Phe Leu Leu Thr Ser Leu Arg
515 520 525
Arg Ala Tyr Ala Pro Leu Leu Ala Arg Glu Asn Ala Ala Leu Thr Gly
530 535 540
Ala Arg Thr Pro Asp Asp Gly Gly Asp Ala Asn Arg His Asp Gly Asp
545 550 555 560
Asp Ala Arg Gly Lys Pro Ala Ala Ala Ala Ala Pro Leu Pro Ser Ala
565 570 575
Ala Ala Ser Pro Ala Asp Glu Arg Ala Val Pro Ala Gly Tyr Gly Ala
580 585 590
Ala Gly Val Leu Ala Ala Leu Gly Arg Leu Ser Ala Ala Pro Ala Ser
595 600 605
Ala Pro Ala Gly Ala Asp Asp Asp Asp Asp Asp Asp Gly Ala Gly Gly
610 615 620
Gly Gly Gly Gly Arg Arg Ala Glu Ala Gly Arg Val Ala Val Glu Cys
625 630 635 640
Leu Ala Ala Cys Arg Gly Ile Leu Glu Ala Leu Ala Glu Gly Phe Asp
645 650 655
Gly Asp Leu Ala Ala Val Pro Gly Leu Ala Gly Ala Arg Pro Ala Ala
660 665 670
Pro Pro Arg Pro Gly Pro Ala Gly Ala Ala Ala Pro Pro His Ala Asp
675 680 685
Ala Pro Arg Leu Arg Ala Trp Leu Arg Glu Leu Arg Phe Val Arg Asp
690 695 700
Ala Leu Val Leu Met Arg Leu Arg Gly Asp Leu Arg Val Ala Gly Gly
705 710 715 720
Ser Glu Ala Ala Val Ala Ala Val Arg Ala Val Ser Leu Val Ala Gly
725 730 735
Ala Leu Gly Pro Ala Leu Pro Arg Ser Pro Arg Leu Leu Ser Ser Ala
740 745 750
Ala Ala Ala Ala Ala Asp Leu Leu Phe Gln Asn Gln Ser Leu
755 760 765
<210> 42
<211> 2172
<212> PRT
<213> Artificial Sequence
<220>
<223> tPA-Flt3L-gD-IPC0-ICP4 fusion protein
<400> 42
Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly
1 5 10 15
Ala Val Phe Val Ser Pro Ser His Ala Thr Gln Asp Cys Ser Phe Gln
20 25 30
His Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu Ser
35 40 45
Asp Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu Gln
50 55 60
Asp Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln Arg
65 70 75 80
Trp Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly Leu
85 90 95
Leu Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala Phe
100 105 110
Gln Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser Arg
115 120 125
Leu Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp Ile
130 135 140
Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro Asp
145 150 155 160
Ser Ser Thr Leu Pro Pro Pro Trp Ser Pro Arg Pro Leu Glu Ala Thr
165 170 175
Ala Pro Thr Ala Pro Gly Ser Gly Ser Gly Ser Gly Ser Gly Ser Gly
180 185 190
Arg Ala Lys Tyr Ala Leu Ala Asp Pro Ser Leu Lys Met Ala Asp Pro
195 200 205
Asn Arg Phe Arg Gly Lys Asn Leu Pro Val Leu Asp Gln Leu Thr Asp
210 215 220
Pro Pro Gly Val Lys Arg Val Tyr His Ile Gln Pro Ser Leu Glu Asp
225 230 235 240
Pro Phe Gln Pro Pro Ser Ile Pro Ile Thr Val Tyr Tyr Ala Val Leu
245 250 255
Glu Arg Ala Cys Arg Ser Val Leu Leu His Ala Pro Ser Glu Ala Pro
260 265 270
Gln Ile Val Arg Gly Ala Ser Asp Glu Ala Arg Lys His Thr Tyr Asn
275 280 285
Leu Thr Ile Ala Trp Tyr Arg Met Gly Asp Asn Cys Ala Ile Pro Ile
290 295 300
Thr Val Met Glu Tyr Thr Glu Cys Pro Tyr Asn Lys Ser Leu Gly Val
305 310 315 320
Cys Pro Ile Arg Thr Gln Pro Arg Trp Ser Tyr Tyr Asp Ser Phe Ser
325 330 335
Ala Val Ser Glu Asp Asn Leu Gly Phe Leu Met His Ala Pro Ala Phe
340 345 350
Glu Thr Ala Gly Thr Tyr Leu Arg Leu Val Lys Ile Asn Asp Trp Thr
355 360 365
Glu Ile Thr Gln Phe Ile Leu Glu His Arg Ala Arg Ala Ser Cys Lys
370 375 380
Tyr Ala Leu Pro Leu Arg Ile Pro Pro Ala Ala Cys Leu Thr Ser Lys
385 390 395 400
Ala Tyr Gln Gln Gly Val Thr Val Asp Ser Ile Gly Met Leu Pro Arg
405 410 415
Phe Ile Pro Glu Asn Gln Arg Thr Val Ala Leu Tyr Ser Leu Lys Ile
420 425 430
Ala Gly Trp His Gly Pro Lys Pro Pro Tyr Thr Ser Thr Leu Leu Pro
435 440 445
Pro Glu Leu Ser Asp Thr Thr Asn Ala Thr Gln Pro Glu Leu Val Pro
450 455 460
Glu Asp Pro Glu Asp Ser Ala Leu Leu Glu Asp Pro Ala Gly Thr Val
465 470 475 480
Ser Ser Gln Ile Pro Pro Asn Trp His Ile Pro Ser Ile Gln Asp Val
485 490 495
Ala Pro His His Ala Pro Ala Ala Pro Ser Asn Pro Arg Arg Arg Ala
500 505 510
Gln Met Ala Pro Lys Arg Leu Arg Leu Pro His Ile Arg Asp Asp Asp
515 520 525
Ala Pro Pro Ser His Gln Pro Leu Phe Tyr Gly Gly Gly Gly Ser Gly
530 535 540
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
545 550 555 560
Gly Gly Ser Met Glu Pro Arg Pro Gly Thr Ser Ser Arg Ala Asp Pro
565 570 575
Gly Pro Glu Arg Pro Pro Arg Gln Thr Pro Gly Thr Gln Pro Ala Ala
580 585 590
Pro His Ala Trp Gly Met Leu Asn Asp Met Gln Trp Leu Ala Ser Ser
595 600 605
Asp Ser Glu Glu Glu Thr Glu Val Gly Ile Ser Asp Asp Asp Leu His
610 615 620
Arg Asp Ser Thr Ser Glu Ala Gly Ser Thr Asp Thr Glu Met Phe Glu
625 630 635 640
Ala Gly Leu Met Asp Ala Ala Thr Pro Pro Ala Arg Pro Pro Ala Glu
645 650 655
Arg Gln Gly Ser Pro Thr Pro Ala Asp Ala Gln Gly Ser Cys Gly Gly
660 665 670
Gly Pro Val Gly Glu Glu Glu Ala Glu Ala Gly Gly Gly Gly Asp Val
675 680 685
Cys Ala Val Cys Thr Asp Glu Ile Ala Pro Pro Leu Arg Cys Gln Ser
690 695 700
Phe Pro Cys Leu His Pro Phe Cys Ile Pro Cys Met Lys Thr Trp Ile
705 710 715 720
Pro Leu Arg Asn Thr Cys Pro Leu Cys Asn Thr Pro Val Ala Tyr Leu
725 730 735
Ile Val Gly Val Thr Ala Ser Gly Ser Phe Ser Thr Ile Pro Ile Val
740 745 750
Asn Asp Pro Arg Thr Arg Val Glu Ala Glu Ala Ala Val Arg Ala Gly
755 760 765
Thr Ala Val Asp Phe Ile Trp Thr Gly Asn Pro Arg Thr Ala Pro Arg
770 775 780
Ser Leu Ser Leu Gly Gly His Thr Val Arg Ala Leu Ser Pro Thr Pro
785 790 795 800
Pro Trp Pro Gly Thr Asp Asp Glu Asp Asp Asp Leu Ala Asp Val Asp
805 810 815
Tyr Val Pro Pro Ala Pro Arg Arg Ala Pro Arg Arg Gly Gly Gly Gly
820 825 830
Ala Gly Ala Thr Arg Gly Thr Ser Gln Pro Ala Ala Thr Arg Pro Ala
835 840 845
Pro Pro Gly Ala Pro Arg Ser Ser Ser Ser Gly Gly Ala Pro Leu Arg
850 855 860
Ala Gly Val Gly Ser Gly Ser Gly Gly Gly Pro Ala Val Ala Ala Val
865 870 875 880
Val Pro Arg Val Ala Ser Leu Pro Pro Ala Ala Gly Gly Gly Arg Ala
885 890 895
Gln Ala Arg Arg Val Gly Glu Asp Ala Ala Ala Ala Glu Gly Arg Thr
900 905 910
Pro Pro Ala Arg Gln Pro Arg Ala Ala Gln Glu Pro Pro Ile Val Ile
915 920 925
Ser Asp Ser Pro Pro Pro Ser Pro Arg Arg Pro Ala Gly Pro Gly Pro
930 935 940
Leu Ser Phe Val Ser Ser Ser Ser Ala Gln Val Ser Ser Gly Pro Gly
945 950 955 960
Gly Gly Gly Leu Pro Gln Ser Ser Gly Arg Ala Ala Arg Pro Arg Ala
965 970 975
Ala Val Ala Pro Arg Val Arg Ser Pro Pro Arg Ala Ala Ala Ala Pro
980 985 990
Val Val Ser Ala Ser Ala Asp Ala Ala Gly Pro Ala Pro Pro Ala Val
995 1000 1005
Pro Val Asp Ala His Arg Ala Pro Arg Ser Arg Met Thr Gln Ala
1010 1015 1020
Gln Thr Asp Thr Gln Ala Gln Ser Leu Gly Arg Ala Gly Ala Thr
1025 1030 1035
Asp Ala Arg Gly Ser Gly Gly Pro Gly Ala Glu Gly Gly Pro Gly
1040 1045 1050
Val Pro Arg Gly Thr Asn Thr Pro Gly Ala Ala Pro His Ala Ala
1055 1060 1065
Glu Gly Ala Ala Gly Ser Asp Ser Gly Pro Ala Ala Ser Ser Ser
1070 1075 1080
Ala Ser Ser Ser Ala Ala Pro Arg Ser Pro Leu Ala Pro Gln Gly
1085 1090 1095
Val Gly Ala Lys Arg Ala Ala Pro Arg Arg Ala Pro Asp Ser Asp
1100 1105 1110
Ser Gly Asp Arg Gly His Gly Pro Leu Ala Pro Ala Ser Ala Gly
1115 1120 1125
Ala Ala Pro Pro Ser Ala Ser Pro Ser Ser Gln Ala Ala Val Ala
1130 1135 1140
Ala Ala Ser Ser Ser Ser Ala Ser Ser Ser Ser Ala Ser Ser Ser
1145 1150 1155
Ser Ala Ser Ser Ser Ser Ala Ser Ser Ser Ser Ala Ser Ser Ser
1160 1165 1170
Ser Ala Ser Ser Ser Ser Ala Ser Ser Ser Ala Gly Gly Ala Gly
1175 1180 1185
Gly Ser Val Ala Ser Ala Ser Gly Ala Gly Glu Arg Arg Glu Thr
1190 1195 1200
Ser Leu Gly Pro Arg Ala Ala Ala Pro Arg Gly Pro Arg Lys Cys
1205 1210 1215
Ala Arg Lys Thr Arg His Ala Glu Gly Gly Pro Glu Pro Gly Ala
1220 1225 1230
Arg Asp Pro Ala Pro Gly Leu Thr Arg Tyr Leu Pro Ile Ala Gly
1235 1240 1245
Val Ser Ser Val Val Ala Leu Ala Pro Tyr Val Asn Lys Thr Val
1250 1255 1260
Thr Gly Asp Cys Leu Pro Val Leu Asp Met Glu Thr Gly His Ile
1265 1270 1275
Gly Ala Tyr Val Val Leu Val Asp Gln Thr Gly Asn Val Ala Asp
1280 1285 1290
Leu Leu Arg Ala Ala Ala Pro Ala Trp Ser Arg Arg Thr Leu Leu
1295 1300 1305
Pro Glu His Ala Arg Asn Cys Val Arg Pro Pro Asp Tyr Pro Thr
1310 1315 1320
Pro Pro Ala Ser Glu Trp Asn Ser Leu Trp Met Thr Pro Val Gly
1325 1330 1335
Asn Met Leu Phe Asp Gln Gly Thr Leu Val Gly Ala Leu Asp Phe
1340 1345 1350
His Gly Leu Arg Ser Arg His Pro Trp Ser Arg Glu Gln Gly Ala
1355 1360 1365
Pro Ala Pro Ala Gly Asp Ala Pro Ala Gly His Gly Glu Gly Gly
1370 1375 1380
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
1385 1390 1395
Gly Gly Ser Gly Gly Gly Gly Ser Met Ser Ala Glu Gln Arg Lys
1400 1405 1410
Lys Lys Lys Thr Thr Thr Thr Thr Gln Gly Arg Gly Ala Glu Val
1415 1420 1425
Ala Met Ala Asp Glu Asp Gly Gly Arg Leu Arg Ala Ala Ala Glu
1430 1435 1440
Thr Thr Gly Gly Pro Gly Ser Pro Asp Pro Ala Asp Gly Pro Pro
1445 1450 1455
Pro Thr Pro Asn Pro Asp Arg Arg Pro Ala Ala Arg Pro Gly Phe
1460 1465 1470
Gly Trp His Gly Gly Pro Glu Glu Asn Glu Asp Glu Ala Asp Asp
1475 1480 1485
Ala Ala Ala Asp Ala Asp Ala Asp Glu Ala Ala Pro Ala Ser Gly
1490 1495 1500
Glu Ala Val Asp Glu Pro Ala Ala Asp Gly Val Val Ser Pro Arg
1505 1510 1515
Gln Leu Ala Leu Leu Ala Ser Met Val Asp Glu Ala Val Arg Thr
1520 1525 1530
Ile Pro Ser Pro Pro Pro Glu Arg Asp Gly Ala Gln Glu Glu Ala
1535 1540 1545
Ala Arg Ser Pro Ser Pro Pro Arg Thr Pro Ser Met Arg Ala Asp
1550 1555 1560
Tyr Gly Glu Glu Asn Asp Asp Asp Asp Asp Asp Asp Asp Asp Asp
1565 1570 1575
Asp Arg Asp Ala Gly Arg Trp Val Arg Gly Pro Glu Thr Thr Ser
1580 1585 1590
Ala Val Arg Gly Ala Tyr Pro Asp Pro Met Ala Ser Leu Ser Pro
1595 1600 1605
Arg Pro Pro Ala Pro Arg Arg His His His His His His His Arg
1610 1615 1620
Arg Arg Arg Ala Pro Arg Arg Arg Ser Ala Ala Ser Asp Ser Ser
1625 1630 1635
Lys Ser Gly Ser Ser Ser Ser Ala Ser Ser Ala Ser Ser Ser Ala
1640 1645 1650
Ser Ser Ser Ser Ser Ala Ser Ala Ser Ser Ser Asp Asp Asp Asp
1655 1660 1665
Asp Asp Asp Ala Ala Arg Ala Pro Ala Ser Ala Ala Asp His Ala
1670 1675 1680
Ala Gly Gly Thr Leu Gly Ala Asp Asp Glu Glu Ala Gly Val Pro
1685 1690 1695
Ala Arg Ala Pro Gly Ala Ala Pro Arg Pro Ser Pro Pro Arg Ala
1700 1705 1710
Glu Pro Ala Pro Ala Arg Thr Pro Ala Ala Thr Ala Gly Arg Leu
1715 1720 1725
Glu Arg Arg Arg Ala Arg Ala Ala Val Ala Gly Arg Asp Ala Thr
1730 1735 1740
Gly Arg Phe Thr Ala Gly Arg Pro Arg Arg Val Glu Leu Asp Ala
1745 1750 1755
Asp Ala Ala Ser Gly Ala Phe Tyr Ala Arg Tyr Arg Asp Gly Tyr
1760 1765 1770
Val Ser Gly Glu Pro Trp Pro Gly Ala Gly Pro Pro Pro Pro Gly
1775 1780 1785
Arg Val Leu Tyr Gly Gly Leu Gly Asp Ser Arg Pro Gly Leu Trp
1790 1795 1800
Gly Ala Pro Glu Ala Glu Glu Ala Arg Ala Arg Phe Glu Ala Ser
1805 1810 1815
Gly Ala Pro Ala Pro Val Trp Ala Pro Glu Leu Gly Asp Ala Ala
1820 1825 1830
Gln Gln Tyr Ala Leu Ile Thr Arg Leu Leu Tyr Thr Pro Asp Ala
1835 1840 1845
Glu Ala Met Gly Trp Leu Gln Asn Pro Arg Val Ala Pro Gly Asp
1850 1855 1860
Val Ala Leu Asp Gln Ala Cys Phe Arg Ile Ser Gly Ala Ala Arg
1865 1870 1875
Asn Ser Ser Ser Phe Ile Ser Gly Ser Val Ala Arg Ala Val Pro
1880 1885 1890
His Leu Gly Tyr Ala Met Ala Ala Gly Arg Phe Gly Trp Gly Leu
1895 1900 1905
Ala His Val Ala Ala Ala Val Ala Met Ser Arg Arg Tyr Asp Arg
1910 1915 1920
Ala Gln Lys Gly Phe Leu Leu Thr Ser Leu Arg Arg Ala Tyr Ala
1925 1930 1935
Pro Leu Leu Ala Arg Glu Asn Ala Ala Leu Thr Gly Ala Arg Thr
1940 1945 1950
Pro Asp Asp Gly Gly Asp Ala Asn Arg His Asp Gly Asp Asp Ala
1955 1960 1965
Arg Gly Lys Pro Ala Ala Ala Ala Ala Pro Leu Pro Ser Ala Ala
1970 1975 1980
Ala Ser Pro Ala Asp Glu Arg Ala Val Pro Ala Gly Tyr Gly Ala
1985 1990 1995
Ala Gly Val Leu Ala Ala Leu Gly Arg Leu Ser Ala Ala Pro Ala
2000 2005 2010
Ser Ala Pro Ala Gly Ala Asp Asp Asp Asp Asp Asp Asp Gly Ala
2015 2020 2025
Gly Gly Gly Gly Gly Gly Arg Arg Ala Glu Ala Gly Arg Val Ala
2030 2035 2040
Val Glu Cys Leu Ala Ala Cys Arg Gly Ile Leu Glu Ala Leu Ala
2045 2050 2055
Glu Gly Phe Asp Gly Asp Leu Ala Ala Val Pro Gly Leu Ala Gly
2060 2065 2070
Ala Arg Pro Ala Ala Pro Pro Arg Pro Gly Pro Ala Gly Ala Ala
2075 2080 2085
Ala Pro Pro His Ala Asp Ala Pro Arg Leu Arg Ala Trp Leu Arg
2090 2095 2100
Glu Leu Arg Phe Val Arg Asp Ala Leu Val Leu Met Arg Leu Arg
2105 2110 2115
Gly Asp Leu Arg Val Ala Gly Gly Ser Glu Ala Ala Val Ala Ala
2120 2125 2130
Val Arg Ala Val Ser Leu Val Ala Gly Ala Leu Gly Pro Ala Leu
2135 2140 2145
Pro Arg Ser Pro Arg Leu Leu Ser Ser Ala Ala Ala Ala Ala Ala
2150 2155 2160
Asp Leu Leu Phe Gln Asn Gln Ser Leu
2165 2170
<210> 43
<211> 6519
<212> DNA
<213> Artificial Sequence
<220>
<223> polynucleotide encoding tPA-Flt3L-gD-IPC0-ICP4 fusion protein
<400> 43
atggacgcca tgaagagagg cctgtgctgc gtgctgctgc tgtgcggcgc cgtgttcgtg 60
agccccagcc acgccaccca ggactgcagc ttccagcaca gccccatcag cagcgacttc 120
gccgtgaaga tcagagagct gagcgactac ctgctgcagg actaccccgt gaccgtggcc 180
agcaacctgc aggacgagga gctgtgcggc ggcctgtgga gactggtgct ggcccagaga 240
tggatggaga gactgaagac cgtggccggc agcaagatgc agggcctgct ggagagagtg 300
aacaccgaga tccacttcgt gaccaagtgc gccttccagc ctccccccag ctgcctgagg 360
ttcgtgcaga ccaacatcag cagactgctg caggagacca gcgagcagct ggtggccctg 420
aagccctgga tcaccagaca gaacttcagc agatgcctgg agctgcagtg ccagcccgac 480
agcagcaccc tgccccctcc ctggagcccc agacccctgg aggccaccgc tcccacagcc 540
cctggcagcg ggtccggaag tgggtctgga tccgggcgcg ccaagtatgc tctggccgac 600
ccaagcctga agatggccga ccctaataga ttccggggca agaacctgcc tgtcctggac 660
cagctgaccg atccccctgg cgtgaagcgc gtctaccaca tccagccttc actggaggac 720
ccattccagc cacccagcat ccctattacc gtgtactatg ctgtcctgga acgcgcatgc 780
cgaagtgtgc tgctgcacgc tccctcagag gcacctcaga tcgtcagagg agcctccgat 840
gaagctagga agcatactta caacctgacc attgcctggt atcggatggg cgacaattgt 900
gctatcccca ttacagtgat ggagtacact gaatgccctt ataacaaatc tctgggcgtc 960
tgtcccattc gaacacagcc tcggtggtcc tactatgatt cattcagcgc cgtgtctgag 1020
gacaacctgg gcttcctgat gcacgctcca gcatttgaaa cagccgggac ttatctgaga 1080
ctggtgaaaa tcaatgattg gaccgagatc acacagttta ttctggaaca tagggcccgc 1140
gctagctgca agtacgcact gccactgagg attcctccag cagcttgtct gaccagtaaa 1200
gcttatcagc agggagtgac agtggactca atcggcatgc tgccacgctt cattcccgag 1260
aaccagcgaa ccgtggcact gtacagcctg aagatcgctg ggtggcacgg acccaaaccc 1320
ccttatacta gcaccctgct gccacccgag ctgtccgata ccacaaatgc cacacagccc 1380
gaactggtgc ctgaggaccc agaagacagc gcactgctgg aggaccctgc cggcactgtg 1440
agctcccaga tccctccaaa ctggcatatc cctagcattc aggacgtggc accacaccat 1500
gcaccagcag caccttccaa tccacggaga agggctcaga tggcaccaaa gcgactgcgg 1560
ctgccccaca ttagagacga tgacgccccc ccttcccatc agcctctgtt ttacggagga 1620
ggaggctccg gaggaggagg atctggaggc gggggaagtg gcgggggagg ctcaggggga 1680
ggcggatcta tggaacccag accaggaact tcaagcagag ccgacccagg accagaaaga 1740
cctccccgac agacaccagg cactcagcca gccgcaccac acgcctgggg catgctgaat 1800
gatatgcagt ggctggccag ctccgactct gaggaggaga cagaggtggg catctccgat 1860
gacgatctgc acagggactc cacaagcgag gccggctcca ccgataccga gatgtttgag 1920
gccggcctga tggacgccgc cacccctcca gcccggcccc cagccgagag gcagggctct 1980
ccaacacccg ccgatgccca gggctcctgc ggcggcggcc ctgtgggcga ggaggaggcc 2040
gaggccggcg gcggcggcga cgtgtgcgcc gtgtgcacag acgagatcgc cccacctctg 2100
agatgccaga gctttccatg tctgcaccca ttctgcatcc catgcatgaa gacctggata 2160
cctctgagga atacctgtcc tctgtgcaat accccagtgg cctacctgat cgtgggcgtg 2220
accgcctctg gctcctttag cacaatcccc atcgtgaacg atcctagaac ccgggtggag 2280
gccgaggccg ccgtgcgggc cggcaccgcc gtggatttca tctggaccgg caacccacgg 2340
acagccccta ggtctctgag cctgggcggc cacaccgtga gagccctgag ccccacaccc 2400
ccttggccag gcacagacga tgaggatgac gacctggccg acgtggatta tgtgccccct 2460
gcccccagaa gagccccccg gagaggcggc ggcggcgccg gcgccacaag aggcacatcc 2520
cagcctgccg ccacaaggcc tgccccacct ggcgccccac ggtcctcctc cagcggcggc 2580
gccccactgc gggccggcgt gggctccggc tccggcggcg gcccagccgt ggccgccgtg 2640
gtgcccagag tggccagcct gcctccagcc gccggcggcg gcagagccca ggccagacgg 2700
gtgggcgagg atgccgccgc cgccgagggc cggacacccc ctgccagaca gccaagagcc 2760
gcccaggagc cccctatcgt gatcagcgac tctccacctc cctccccaag aaggccagcc 2820
ggcccaggcc ctctgtcctt tgtgagctct tcttccgccc aggtgtcttc cggcccaggc 2880
ggcggcggcc tgcctcagtc ctccggcaga gccgccagac cacgggccgc cgtggcccct 2940
agagtgagga gccccccaag ggccgccgcc gccccagtgg tgtccgcctc cgccgatgcc 3000
gccggcccag ccccacccgc cgtgccagtg gacgcccaca gggccccacg gagcagaatg 3060
acccaggccc agaccgatac ccaggcccag agcctgggca gggccggcgc caccgatgcc 3120
agaggctccg gcggcccagg cgccgagggc ggccccggcg tgcccagggg cacaaataca 3180
ccaggcgccg ccccccacgc cgccgagggc gccgccggca gcgattccgg ccctgccgcc 3240
tcctcttctg cctccagcag cgccgcccca cgctctcccc tggccccaca gggcgtgggc 3300
gccaagcggg ccgccccaag acgggcccct gactccgatt ccggcgatcg gggccacggc 3360
ccactggccc ctgcctctgc cggcgccgcc cctcctagcg cctccccaag ctctcaggcc 3420
gccgtggccg ccgccagctc cagctccgcc agctcttcct ctgccagctc ctcctctgcc 3480
tcctcctcct ctgcctcttc cagctctgcc agctctagca gcgcctcctc tagctccgcc 3540
tcttctagcg ccggcggcgc cggcggctcc gtggcctctg cctccggcgc cggcgagaga 3600
agagagacct ctctgggccc aagagccgcc gcccccagag gccctagaaa gtgtgccagg 3660
aagaccaggc acgccgaggg cggcccagag ccaggcgccc gcgaccctgc cccaggcctg 3720
acacggtatc tgccaatcgc cggcgtgagc tctgtggtgg ccctggcccc atatgtgaac 3780
aagacagtga caggcgactg tctgccagtg ctggacatgg agacaggcca catcggcgcc 3840
tatgtggtgc tggtggacca gacaggcaat gtggccgatc tgctgagagc cgccgcccct 3900
gcctggtcca gaaggacact gctgccagag cacgccagaa actgcgtgag accacctgac 3960
tatcccacac cacccgcctc tgagtggaat agcctgtgga tgacaccagt gggcaacatg 4020
ctgtttgatc agggcacact ggtgggcgcc ctggatttcc acggcctgag atcccgccac 4080
ccttggtcca gagagcaggg cgcccccgcc ccagccggcg atgccccagc cggccacggc 4140
gagggcggcg gcggctcagg gggggggggc agcggcggag gtggctcagg aggtggaggt 4200
agcggaggtg gcggatctat gtcagcagaa cagaggaaga agaagaaaac aacaacaact 4260
actcagggaa ggggagcaga agtggcaatg gcagacgagg atggaggcag actgagggcc 4320
gccgccgaga caaccggcgg cccaggctct cctgacccag ccgatggccc accaccaaca 4380
cccaacccag accgcagacc agccgccaga cctggctttg gctggcacgg cggcccagag 4440
gagaacgagg atgaggccga cgatgccgcc gccgatgccg acgccgatga ggccgccccc 4500
gcctccggcg aggccgtgga cgagccagcc gccgacggcg tggtgtctcc tagacagctg 4560
gccctgctgg cctccatggt ggacgaggcc gtgaggacca tcccatctcc tcctccagag 4620
agagatggcg cccaggagga ggccgccagg agcccatccc ccccaagaac ccctagcatg 4680
agagccgact atggcgagga gaacgatgat gacgacgacg atgacgatga tgatgacagg 4740
gacgccggcc gctgggtgcg gggcccagag accacatctg ccgtgagagg cgcctatcct 4800
gacccaatgg cctctctgag cccaagacct cccgccccaa gacggcacca ccaccaccac 4860
caccacagga gacggagggc cccaagaagg cggagcgccg ccagcgactc ctctaagtct 4920
ggctccagca gctctgcctc cagcgccagc tcctctgcca gctcctcctc tagcgccagc 4980
gcctcttcct ccgacgatga tgacgatgat gacgccgcca gggcccctgc ctccgccgcc 5040
gatcacgccg ccggcggcac cctgggcgcc gatgacgagg aggccggcgt gccagccaga 5100
gccccaggcg ccgcccccag gccttctcca ccaagagccg agcccgcccc agccaggacc 5160
ccagccgcca cagccggcag actggagcgg agacgcgcca gagccgccgt ggccggcaga 5220
gatgccacag gcaggtttac cgccggcagg cccagaaggg tggagctgga tgccgatgcc 5280
gcctccggcg ccttttacgc cagatacagg gacggctacg tgagcggcga gccctggcca 5340
ggcgccggcc ctcccccacc tggcagggtg ctgtatggcg gcctgggcga tagcagacct 5400
ggcctgtggg gcgcccctga ggccgaggag gccagagcca gattcgaggc ctctggcgcc 5460
ccagcccccg tgtgggcccc agagctgggc gacgccgccc agcagtacgc cctgatcacc 5520
agactgctgt ataccccaga cgccgaggcc atgggctggc tgcagaaccc tagagtggcc 5580
ccaggcgacg tggccctgga tcaggcctgc ttccggatct ctggcgccgc ccggaacagc 5640
tctagcttta tctccggctc tgtggccaga gccgtgccac acctgggcta cgccatggcc 5700
gccggcaggt tcggctgggg cctggcccac gtagccgccg ccgtggccat gtccagacgg 5760
tatgacagag cccagaaggg ctttctgctg acatctctga ggcgggccta tgcccctctg 5820
ctggccaggg agaatgccgc cctgacaggc gccagaaccc ctgacgacgg cggcgatgcc 5880
aacagacacg atggcgacga tgccagaggc aagccagccg ccgccgccgc ccccctgcca 5940
tccgccgccg cctcccccgc cgatgagaga gccgtgcctg ccggctatgg cgccgccggc 6000
gtgctggccg ccctgggccg gctgtctgcc gccccagcct ccgcccccgc cggcgccgac 6060
gatgatgacg acgatgatgg cgccggcggc ggcggcggcg gcagacgggc cgaggccggc 6120
agggtggccg tggagtgtct ggccgcctgc cgcggcatcc tggaggccct ggccgagggc 6180
tttgatggcg acctggccgc cgtgcccggc ctggccggcg ccagacctgc cgccccacct 6240
agacctggcc ccgccggcgc cgccgccccc cctcacgccg acgccccacg gctgagagcc 6300
tggctgagag agctgagatt cgtgcgggac gccctggtgc tgatgcggct gagaggcgat 6360
ctgcgggtgg ccggcggctc tgaggccgcc gtggccgccg tgagagccgt gtccctggtg 6420
gccggcgccc tgggcccagc cctgcctaga tccccaagac tgctgtcctc cgccgctgcc 6480
gctgccgccg acctgctgtt tcagaaccag agcctgtaa 6519
<210> 44
<211> 1073
<212> PRT
<213> Artificial Sequence
<220>
<223> SFTS GnGc protein
<400> 44
Met Met Lys Val Ile Trp Phe Ser Ser Leu Ile Cys Leu Val Ile Gln
1 5 10 15
Cys Ser Gly Asp Ser Gly Pro Ile Ile Cys Ala Gly Pro Ile His Ser
20 25 30
Asn Lys Ser Ala Ser Ile Pro His Leu Leu Gly Tyr Ser Glu Lys Ile
35 40 45
Cys Gln Ile Asp Arg Leu Ile His Val Ser Ser Trp Leu Arg Asn His
50 55 60
Ser Gln Phe Gln Gly Tyr Val Gly Gln Arg Gly Gly Arg Ser Gln Val
65 70 75 80
Ser Tyr Tyr Pro Ala Glu Asn Ser Tyr Ser Arg Trp Ser Gly Leu Leu
85 90 95
Ser Pro Cys Asp Ala Asp Trp Leu Gly Met Leu Val Val Lys Lys Ala
100 105 110
Lys Gly Ser Asp Met Ile Val Pro Gly Pro Ser Tyr Lys Gly Lys Val
115 120 125
Phe Phe Glu Arg Pro Thr Phe Asp Gly Tyr Val Gly Trp Gly Cys Gly
130 135 140
Ser Gly Lys Ser Arg Thr Glu Ser Gly Glu Leu Cys Ser Ser Asp Ser
145 150 155 160
Gly Thr Ser Ser Gly Leu Leu Pro Ser Asp Arg Val Leu Trp Ile Gly
165 170 175
Asp Val Ala Cys Gln Pro Met Thr Pro Ile Pro Glu Glu Thr Phe Leu
180 185 190
Glu Leu Lys Ser Phe Ser Gln Ser Glu Phe Pro Asp Ile Cys Lys Ile
195 200 205
Asp Gly Ile Val Phe Asn Gln Cys Glu Gly Glu Ser Leu Pro Gln Pro
210 215 220
Phe Asp Val Ala Trp Met Asp Val Gly His Ser His Lys Ile Ile Met
225 230 235 240
Arg Glu His Lys Thr Lys Trp Val Gln Glu Ser Ser Ser Lys Asp Phe
245 250 255
Val Cys Tyr Lys Glu Gly Thr Gly Pro Cys Ser Glu Ser Glu Glu Lys
260 265 270
Ala Cys Lys Thr Ser Gly Ser Cys Arg Gly Asp Met Gln Phe Cys Lys
275 280 285
Val Ala Gly Cys Glu His Gly Glu Glu Ala Ser Asp Ala Lys Cys Arg
290 295 300
Cys Ser Leu Val His Lys Pro Gly Glu Val Val Val Ser Tyr Gly Gly
305 310 315 320
Met Arg Val Arg Pro Lys Cys Tyr Gly Phe Ser Arg Met Met Ala Thr
325 330 335
Leu Glu Val Asn Pro Pro Glu Gln Arg Ile Gly Gln Cys Thr Gly Cys
340 345 350
His Leu Glu Cys Ile Asn Gly Gly Val Arg Leu Ile Thr Leu Thr Ser
355 360 365
Glu Leu Lys Ser Ala Thr Val Cys Ala Ser His Phe Cys Ser Ser Ala
370 375 380
Thr Ser Gly Lys Lys Ser Thr Glu Ile Gln Phe His Ser Gly Ser Leu
385 390 395 400
Val Gly Lys Thr Ala Ile His Val Lys Gly Ala Leu Val Asp Gly Thr
405 410 415
Glu Phe Thr Phe Glu Gly Ser Cys Met Phe Pro Asp Gly Cys Asp Ala
420 425 430
Val Asp Cys Thr Phe Cys Arg Glu Phe Leu Lys Asn Pro Gln Cys Tyr
435 440 445
Pro Ala Lys Lys Trp Leu Phe Ile Leu Ile Val Ile Leu Leu Gly Tyr
450 455 460
Ala Gly Leu Met Leu Ile Thr Asn Val Leu Lys Ala Ile Gly Val Trp
465 470 475 480
Gly Ser Trp Val Ile Ala Pro Val Lys Leu Met Phe Ala Ile Ile Lys
485 490 495
Lys Leu Met Arg Thr Val Ser Cys Leu Met Gly Lys Leu Met Asp Arg
500 505 510
Gly Arg Gln Val Ile His Glu Glu Ile Gly Glu Asn Gly Glu Gly Asn
515 520 525
Gln Asp Asp Val Arg Ile Glu Met Ala Arg Pro Arg Arg Val Arg His
530 535 540
Trp Met Tyr Ser Pro Val Ile Leu Thr Ile Leu Ala Ile Gly Leu Ala
545 550 555 560
Glu Gly Cys Asp Glu Met Val His Ala Asp Ser Lys Leu Val Ser Cys
565 570 575
Arg Gln Gly Ser Gly Asn Met Lys Glu Cys Val Thr Thr Gly Arg Ala
580 585 590
Leu Leu Pro Ala Val Asn Pro Gly Gln Glu Ala Cys Leu His Phe Thr
595 600 605
Ala Pro Gly Ser Pro Asp Ser Lys Cys Leu Lys Ile Lys Val Lys Arg
610 615 620
Ile Asn Leu Lys Cys Lys Lys Ser Ser Ser Tyr Phe Val Pro Asp Ala
625 630 635 640
Arg Ser Arg Cys Thr Ser Val Arg Arg Cys Arg Trp Ala Gly Asp Cys
645 650 655
Gln Ser Gly Cys Pro Pro His Phe Thr Ser Asn Ser Phe Ser Asp Asp
660 665 670
Trp Ala Gly Lys Met Asp Arg Ala Gly Leu Gly Phe Ser Gly Cys Ser
675 680 685
Asp Gly Cys Gly Gly Ala Ala Cys Gly Cys Phe Asn Ala Ala Pro Ser
690 695 700
Cys Ile Phe Trp Arg Lys Trp Val Glu Asn Pro His Gly Ile Ile Trp
705 710 715 720
Lys Val Ser Pro Cys Ala Ala Trp Val Pro Ser Ala Val Ile Glu Leu
725 730 735
Thr Met Pro Ser Gly Glu Val Arg Thr Phe His Pro Met Ser Gly Ile
740 745 750
Pro Thr Gln Val Phe Lys Gly Val Ser Val Thr Tyr Leu Gly Ser Asp
755 760 765
Met Glu Val Ser Gly Leu Thr Asp Leu Cys Glu Ile Glu Glu Leu Lys
770 775 780
Ser Lys Lys Leu Ala Leu Ala Pro Cys Asn Gln Ala Gly Met Gly Val
785 790 795 800
Val Gly Lys Val Gly Glu Ile Gln Cys Ser Ser Glu Glu Ser Ala Arg
805 810 815
Thr Ile Lys Lys Asp Gly Cys Ile Trp Asn Ala Asp Leu Val Gly Ile
820 825 830
Glu Leu Arg Val Asp Asp Ala Val Cys Tyr Ser Lys Ile Thr Ser Val
835 840 845
Glu Ala Val Ala Asn Tyr Ser Ala Ile Pro Thr Thr Ile Gly Gly Leu
850 855 860
Arg Phe Glu Arg Ser His Asp Ser Gln Gly Lys Ile Ser Gly Ser Pro
865 870 875 880
Leu Asp Ile Thr Ala Ile Arg Gly Ser Phe Ser Val Asn Tyr Arg Gly
885 890 895
Leu Arg Leu Ser Leu Ser Glu Ile Thr Ala Thr Cys Thr Gly Glu Val
900 905 910
Thr Asn Val Ser Gly Cys Tyr Ser Cys Met Thr Gly Ala Lys Val Ser
915 920 925
Ile Lys Leu His Ser Ser Lys Asn Ser Thr Ala His Val Arg Cys Lys
930 935 940
Gly Asp Glu Thr Ala Phe Ser Val Leu Glu Gly Val His Ser Tyr Thr
945 950 955 960
Val Ser Leu Ser Phe Asp His Ala Val Val Asp Glu Gln Cys Gln Leu
965 970 975
Asn Cys Gly Gly His Glu Ser Gln Val Thr Leu Lys Gly Asn Leu Ile
980 985 990
Phe Leu Asp Val Pro Lys Phe Val Asp Gly Ser Tyr Met Gln Thr Tyr
995 1000 1005
His Ser Thr Val Pro Thr Gly Ala Asn Ile Pro Ser Pro Thr Asp
1010 1015 1020
Trp Leu Asn Ala Leu Phe Gly Asn Gly Leu Ser Arg Trp Ile Leu
1025 1030 1035
Gly Val Ile Gly Val Leu Leu Gly Gly Leu Ala Leu Phe Phe Leu
1040 1045 1050
Ile Met Ser Leu Phe Lys Leu Gly Thr Lys Gln Val Phe Arg Ser
1055 1060 1065
Arg Thr Lys Leu Ala
1070
<210> 45
<211> 245
<212> PRT
<213> Artificial Sequence
<220>
<223> SFTS NP protein
<400> 45
Met Ser Glu Trp Ser Arg Ile Ala Val Glu Phe Gly Glu Gln Gln Leu
1 5 10 15
Asn Leu Ser Glu Leu Glu Asp Phe Ala Arg Glu Leu Ala Tyr Glu Gly
20 25 30
Leu Asp Pro Ala Leu Ile Ile Lys Lys Leu Lys Glu Thr Gly Gly Asp
35 40 45
Asp Trp Val Lys Asp Thr Lys Phe Ile Ile Val Phe Ala Leu Thr Arg
50 55 60
Gly Asn Lys Ile Val Lys Ala Ser Gly Lys Met Ser Asn Ser Gly Ser
65 70 75 80
Lys Arg Leu Met Ala Leu Gln Glu Lys Tyr Gly Leu Val Glu Arg Ala
85 90 95
Glu Thr Arg Leu Ser Ile Thr Pro Val Arg Val Ala Gln Ser Leu Pro
100 105 110
Thr Trp Thr Cys Ala Ala Ala Ala Ala Leu Lys Glu Tyr Leu Pro Val
115 120 125
Gly Pro Ala Val Met Asn Leu Lys Val Glu Asn Tyr Pro Pro Glu Met
130 135 140
Met Cys Met Ala Phe Gly Ser Leu Ile Pro Thr Ala Gly Val Ser Glu
145 150 155 160
Ala Thr Thr Lys Thr Leu Met Glu Ala Tyr Ser Leu Trp Gln Asp Ala
165 170 175
Phe Thr Lys Thr Ile Asn Val Lys Met Arg Gly Ala Ser Lys Thr Glu
180 185 190
Val Tyr Asn Ser Phe Arg Asp Pro Leu His Ala Ala Val Asn Ser Val
195 200 205
Phe Phe Pro Asn Asp Val Arg Val Lys Trp Leu Lys Ala Lys Gly Ile
210 215 220
Leu Gly Pro Asp Gly Val Pro Ser Arg Ala Ala Glu Val Ala Ala Ala
225 230 235 240
Ala Tyr Arg Asn Leu
245
<210> 46
<211> 293
<212> PRT
<213> Artificial Sequence
<220>
<223> SFTS NSs protein
<400> 46
Met Ser Leu Ser Lys Ser Ser Asn Val Asp Leu Lys Ser Val Ala Met
1 5 10 15
Asn Ala Asn Thr Val Arg Leu Glu Pro Ser Leu Gly Glu Tyr Pro Thr
20 25 30
Leu Arg Arg Asp Leu Val Glu Cys Ser Cys Ser Val Leu Thr Leu Ser
35 40 45
Met Val Lys Arg Met Gly Lys Met Thr Asn Thr Val Trp Leu Phe Gly
50 55 60
Asn Pro Lys Asn Pro Leu His Gln Leu Glu Pro Gly Leu Glu Gln Leu
65 70 75 80
Leu Asp Met Tyr Tyr Lys Asp Met Arg Cys Tyr Ser Gln Arg Glu Leu
85 90 95
Ser Ala Leu Arg Trp Pro Ser Gly Lys Pro Ser Val Trp Phe Leu Gln
100 105 110
Ala Ala His Met Phe Phe Ser Ile Lys Asn Ser Trp Ala Met Glu Thr
115 120 125
Gly Lys Glu Asn Trp Arg Gly Leu Phe His Arg Ile Thr Lys Gly Gln
130 135 140
Lys Tyr Leu Phe Glu Gly Asp Met Ile Leu Asp Ser Leu Glu Ala Ile
145 150 155 160
Glu Lys Arg Arg Leu Arg Leu Gly Leu Pro Glu Ile Leu Ile Thr Gly
165 170 175
Leu Ser Pro Ile Leu Asp Val Ala Leu Leu Gln Ile Glu Ser Leu Ala
180 185 190
Arg Leu Arg Gly Met Ser Leu Asn His His Leu Phe Thr Ser Ser Ser
195 200 205
Leu Arg Lys Pro Leu Leu Asp Cys Trp Asp Phe Phe Ile Pro Val Arg
210 215 220
Lys Lys Lys Thr Asp Gly Ser Tyr Ser Val Leu Asp Glu Asp Asp Glu
225 230 235 240
Pro Gly Ala Leu Gln Gly Tyr Pro His Leu Met Ala His Tyr Leu Asn
245 250 255
Arg Cys Pro Phe His Asn Leu Ile Arg Phe Asp Glu Glu Leu Arg Thr
260 265 270
Ala Ala Leu Asn Thr Ile Trp Gly Arg Asp Trp Pro Ala Ile Gly Asp
275 280 285
Leu Pro Lys Glu Val
290
<210> 47
<211> 3222
<212> DNA
<213> Artificial Sequence
<220>
<223> polynucleotide encoding SFTS GnGc protein
<400> 47
atgatgaaag tgatttggtt ctcctctctg atttgtctgg tcattcagtg tagcggggat 60
tctggaccta ttatctgtgc tgggccaatc cacagcaaca agagcgcctc catcccccac 120
ctgctgggct actccgagaa gatctgccag atcgaccgcc tgatccacgt gagctcctgg 180
ctgcggaacc acagccagtt ccagggatac gtgggacaga ggggaggccg cagccaggtg 240
tcctactatc cagccgagaa ttcttatagc agatggtccg gcctgctgtc tccatgtgac 300
gcagattggc tgggcatgct ggtggtgaag aaggccaagg gctctgatat gatcgtgcct 360
ggcccaagct acaagggcaa ggtgttcttt gagcggccca ccttcgacgg atatgtggga 420
tggggatgcg gatctggcaa gagcaggaca gagtccggcg agctgtgcag cagcgattct 480
ggcacctcct ctggcctgct gcctagcgat cgcgtgctgt ggatcggcga cgtggcatgc 540
cagccaatga cacccatccc tgaggagaca ttcctggagc tgaagtcctt ctctcagagc 600
gagtttcctg atatctgcaa gatcgacggc atcgtgttca atcagtgtga gggcgagagc 660
ctgccacagc cctttgatgt ggcctggatg gacgtgggcc actcccacaa gatcatcatg 720
cgggagcaca agaccaagtg ggtgcaggag agctcctcta aggacttcgt gtgctacaag 780
gagggcacag gcccatgttc cgagtctgag gagaaggcct gcaagaccag cggctcctgt 840
agaggcgata tgcagttttg caaggtggca ggatgtgagc acggagagga ggcctctgac 900
gccaagtgca ggtgtagcct ggtgcacaag ccaggagagg tggtggtgtc ttacggagga 960
atgcgggtgc ggcccaagtg ctatggcttc agcagaatga tggccacact ggaggtgaac 1020
ccccctgagc agaggatcgg ccagtgcacc ggctgtcacc tggagtgtat caatggcggc 1080
gtgaggctga tcaccctgac aagcgagctg aagtccgcca cagtgtgcgc cagccacttc 1140
tgtagctccg ccacatctgg caagaagagc accgagatcc agtttcactc tggcagcctg 1200
gtgggcaaga ccgcaatcca cgtgaagggc gccctggtgg atggcacaga gttcaccttt 1260
gagggctcct gcatgttccc agacggctgt gatgccgtgg actgcacctt ctgtagagag 1320
tttctgaaga acccacagtg ctaccccgcc aagaagtggc tgtttatcct gatcgtgatc 1380
ctgctgggct atgccggcct gatgctgatc acaaatgtgc tgaaggccat cggcgtgtgg 1440
ggatcttggg tcatcgcacc cgtgaagctg atgtttgcca tcatcaagaa gctgatgagg 1500
accgtgtcct gcctgatggg caagctgatg gacaggggcc ggcaggtcat ccacgaggag 1560
atcggcgaga acggcgaggg caatcaggac gatgtgcgga tcgagatggc cagacctcgg 1620
agagtgaggc actggatgta cagcccagtg atcctgacaa tcctggcaat cggcctggca 1680
gagggatgcg atgagatggt gcacgccgac tccaagctgg tgtcttgccg ccagggctcc 1740
ggcaacatga aggagtgcgt gaccacaggc cgggccctgc tgcctgcagt gaatccaggc 1800
caggaggcat gtctgcactt caccgcacca ggctcccctg actctaagtg cctgaagatc 1860
aaggtgaagc gcatcaacct gaagtgtaag aagtctagct cctattttgt gcccgatgcc 1920
cggagcagat gcacatccgt gaggcgctgt agatgggcag gcgactgcca gagcggctgt 1980
ccaccccact tcacctccaa ttcttttagc gacgattggg ccggcaagat ggatagagca 2040
ggcctgggct tcagcggatg ctccgacgga tgtggaggag cagcatgcgg atgtttcaac 2100
gcagccccct cctgcatctt ttggaggaag tgggtggaga atcctcacgg catcatctgg 2160
aaggtgagcc catgtgcagc atgggtgccc tccgccgtga tcgagctgac aatgccatcc 2220
ggagaggtgc gcaccttcca ccccatgtct ggcatcccta cacaggtgtt taagggcgtg 2280
agcgtgacct acctgggcag cgatatggag gtgtccggcc tgaccgacct gtgcgagatc 2340
gaggagctga agtccaagaa gctggccctg gcaccttgta accaggcagg aatgggagtg 2400
gtgggcaagg tcggcgagat ccagtgctct agcgaggaga gcgcccgcac aatcaagaag 2460
gatggctgta tctggaacgc agacctggtg ggaatcgagc tgagggtgga cgatgccgtg 2520
tgctactcta agatcaccag cgtggaggcc gtggccaatt attccgccat ccctaccaca 2580
atcggcggcc tgcggttcga gagatctcac gatagccagg gcaagatctc cggctctcca 2640
ctggacatca ccgccatccg cggcagcttt tccgtgaact acaggggcct gcgcctgtct 2700
ctgagcgaga tcaccgccac atgcaccggc gaggtgacaa acgtgagcgg ctgctattct 2760
tgtatgaccg gcgccaaggt gtctatcaag ctgcactcct ctaagaacag cacagcccac 2820
gtgcggtgta agggcgatga gacagccttc tccgtgctgg agggcgtgca ctcttataca 2880
gtgtccctgt cttttgatca cgccgtggtg gacgagcagt gccagctgaa ctgtggcggc 2940
cacgagagcc aggtgaccct gaagggcaat ctgatcttcc tggatgtgcc aaagtttgtg 3000
gacggcagct acatgcagac atatcactcc acagtgccca ccggcgccaa tatcccttcc 3060
ccaaccgact ggctgaacgc cctgttcggc aatggcctgt ctaggtggat cctgggcgtg 3120
atcggcgtgc tgctgggagg cctggccctg ttctttctga tcatgtctct gttcaaactg 3180
ggcactaagc aggtctttcg gtcccgcaca aaactggctt aa 3222
<210> 48
<211> 735
<212> DNA
<213> Artificial Sequence
<220>
<223> polynucleotide encoding SFTS NP protein
<400> 48
atgtcggagt ggtccaggat tgcagtggag tttggtgagc agcagctcaa tttgtctgag 60
cttgaggatt tcgcgagaga actggcctat gaaggccttg accctgcttt gatcatcaag 120
aagctgaagg agacaggtgg agatgattgg gtgaaggata caaagttcat cattgtcttt 180
gccctgactc gaggcaacaa gatcgtcaag gcatcaggga aaatgtcaaa ctcagggtcc 240
aagaggttga tggcactcca agagaaatat ggactggttg agagggcaga gaccaggctc 300
tcaatcactc ctgtgagggt tgcgcagagc cttcccactt ggacatgtgc tgcagcagca 360
gccttgaagg agtatctccc tgtggggcca gccgtcatga acctgaaggt cgagaattat 420
ccccctgaga tgatgtgcat ggcctttgga tccctgattc caactgcggg ggtatctgaa 480
gccacaacca agaccctgat ggaggcctac tctctgtggc aagatgcctt caccaagact 540
atcaatgtga agatgcgcgg agccagcaag acagaagttt acaactcctt cagggaccct 600
cttcatgctg ctgtgaactc tgtcttcttt cccaatgatg ttcgggtaaa gtggctgaag 660
gccaagggga tccttggccc agatggggtc cccagcagag ctgctgaggt tgctgctgct 720
gcttacagaa acctg 735
<210> 49
<211> 882
<212> DNA
<213> Artificial Sequence
<220>
<223> polynucleotide encoding SFTS NSs protein
<400> 49
atgagcctga gtaagagcag caatgtggat ctgaaaagcg tcgcaatgaa tgccaacacc 60
gtccgcctgg aaccatcact gggggaatac cctaccctga ggcgggacct ggtggagtgc 120
agctgttccg tgctgacact gtctatggtg aagaggatgg gcaagatgac caacacagtg 180
tggctgttcg gcaaccccaa gaatcctctg caccagctgg agccaggcct ggagcagctg 240
ctggacatgt actataagga tatgaggtgc tactctcaga gggagctgag cgccctgagg 300
tggccctccg gcaagcccag cgtgtggttt ctccaggccg cccacatgtt ctttagcatc 360
aagaactcct gggctatgga gacaggcaag gagaattggc ggggcctgtt ccacagaatc 420
acaaagggcc agaagtatct gtttgagggc gacatgatcc tggatagcct ggaggcaatc 480
gagaagaggc gcctgaggct gggcctgcca gagatcctga tcaccggcct gtcccctatc 540
ctggacgtgg ccctgctcca gatcgagtcc ctggcccggc tgagaggcat gtctctgaat 600
caccacctgt tcaccagctc ctctctgcgg aagccactgc tggactgctg ggatttcttt 660
atccccgtgc ggaagaagaa gaccgacggc tcttatagcg tgctggatga ggacgatgag 720
cccggcgccc tccagggcta cccacacctg atggcccact atctgaacag gtgtcccttc 780
cacaatctga tcaggtttga tgaggagctg cgcacagccg ccctgaatac catctggggg 840
cgggactggc ctgctatcgg cgacctgcct aaggaagtgt aa 882
<210> 50
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> (GGGGS)n linker peptide unit
<400> 50
Gly Gly Gly Gly Ser
1 5
<210> 51
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> (GSSGGS)n linker peptide unit
<400> 51
Gly Ser Ser Gly Gly Ser
1 5
<210> 52
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> linker peptide
<400> 52
Lys Glu Ser Gly Ser Val Ser Ser Glu Gln Leu Ala Gln Phe Arg Ser
1 5 10 15
Leu Asp
<210> 53
<211> 14
<212> PRT
<213> Artificial Sequence
<220>
<223> linker peptide
<400> 53
Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr
1 5 10
<210> 54
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> linker peptide
<400> 54
Gly Ser Ala Gly Ser Ala Ala Gly Ser Gly Glu Phe
1 5 10
<210> 55
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> (EAAAK)n linker peptide unit
<400> 55
Glu Ala Ala Ala Lys
1 5
<210> 56
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> linker peptide
<400> 56
Cys Arg Arg Arg Arg Arg Arg Glu Ala Glu Ala Cys
1 5 10
<210> 57
<211> 46
<212> PRT
<213> Artificial Sequence
<220>
<223> linker peptide
<400> 57
Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys
1 5 10 15
Glu Ala Ala Ala Lys Ala Leu Glu Ala Glu Ala Ala Ala Lys Glu Ala
20 25 30
Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Ala
35 40 45
<210> 58
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> linker peptide
<400> 58
Gly Gly Gly Gly Gly Gly Gly Gly
1 5
<210> 59
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> linker peptide
<400> 59
Gly Gly Gly Gly Gly Gly
1 5
<210> 60
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> linker peptide
<400> 60
Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Ala Lys Ala
1 5 10
<210> 61
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> linker peptide
<400> 61
Pro Ala Pro Ala Pro
1 5
<210> 62
<211> 17
<212> PRT
<213> Artificial Sequence
<220>
<223> linker peptide
<400> 62
Val Ser Gln Thr Ser Lys Leu Thr Arg Ala Glu Thr Val Phe Pro Asp
1 5 10 15
Val
<210> 63
<211> 6
<212> PRT
<213> Artificial Sequence
<220>
<223> linker peptide
<400> 63
Pro Leu Gly Leu Trp Ala
1 5
<210> 64
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> linker peptide
<400> 64
Thr Arg His Arg Gln Pro Arg Gly Trp Glu
1 5 10
<210> 65
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> linker peptide
<400> 65
Ala Gly Asn Arg Val Arg Arg Ser Val Gly
1 5 10
<210> 66
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> linker peptide
<400> 66
Arg Arg Arg Arg Arg Arg Arg Arg
1 5
<210> 67
<211> 4
<212> PRT
<213> Artificial Sequence
<220>
<223> linker peptide
<400> 67
Gly Phe Leu Gly
1
<210> 68
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> linker peptide
<400> 68
Gly Ser Ser Gly Gly Ser Gly Ser Ser Gly Gly Ser Gly Gly Gly Asp
1 5 10 15
Glu Ala Asp Gly Ser Arg Gly Ser Gln Lys Ala Gly Val Asp Glu
20 25 30

Claims (16)

1.一种用于刺激T淋巴细胞特异性免疫应答的疫苗佐剂，其包括作为活性成分的编码IL-12蛋白的多核苷酸和编码IL-21蛋白的多核苷酸。
2.如权利要求1所述的疫苗佐剂，其特征在于，所述疫苗佐剂包含选自由以下组成的组中的至少一种：
一至三个载体，每个载体包含：分别编码组成所述IL-12蛋白的p35链(IL-12p35)和p40链(IL-12p40)的多核苷酸；和编码所述IL-21蛋白质的多核苷酸；以及
mRNA分子，每个分子都编码所述IL-12p35、所述IL-12p40和所述IL-21蛋白。
3.如权利要求1所述的疫苗佐剂，其特征在于，所述疫苗佐剂促进T淋巴细胞特异性免疫反应而不诱导B淋巴细胞特异性免疫反应。
4.如权利要求1所述的疫苗佐剂，其特征在于，所述IL-12p35蛋白包含如SEQ ID NO:1所示的氨基酸序列。
5.如权利要求1所述的疫苗佐剂，其特征在于，所述IL-12p40蛋白为人IL-12p40，所述IL-12p40由如SEQ ID NO:2所示的氨基酸序列组成。
6.如权利要求1所述的疫苗佐剂，其特征在于，所述IL-21蛋白为人IL-21，所述IL-21由如SEQ ID NO:3所示的氨基酸序列组成。
7.如权利要求1所述的疫苗佐剂，其特征在于，所述疫苗佐剂进一步包含以下中的至少一种：
一种MIP-1α基因构建体，其中编码MIP-1α蛋白的多核苷酸可操作地连接到启动子；
一种复合基因构建体，其中编码MIP-1α蛋白的多核苷酸通过编码内部核糖体进入位点(IRES)或连接肽的多核苷酸可操作地连接到所述IL-12p35、所述IL-12p40m和所述IL-21蛋白中的至少一种；和
一种编码MIP-1α蛋白的mRNA分子。
8.如权利要求7所述的疫苗佐剂，其特征在于，所述MIP-1α蛋白由如SEQ ID NO:10所示的氨基酸序列组成。
9.如权利要求7所述的疫苗佐剂，其特征在于，所述MIP-1α基因构建体包含在一种单独表达载体中或包含在所述一至三种载体中的任何一个或多个载体里，每个所述载体包括：分别编码组成所述IL-12蛋白的p35链(IL-12p35)和p40链(IL-12p40)的多核苷酸；编码所述IL-21蛋白的多核苷酸。
10.一种预防和治疗病毒感染的疫苗组合物，包括作为活性成分的：
如权利要求1至9中任一项所述的疫苗佐剂；和
源自传染性病毒的抗原；
编码所述抗原的多核苷酸；
抗原基因构建体，其中所述多核苷酸可操作地连接到启动子；或
包含所述抗原基因构建体的表达载体。
11.如权利要求10所述的疫苗组合物，其特征在于，来自传染性病毒的所述抗原是源自爱泼斯坦-巴尔病毒(EBV)、甲肝病毒(HAV)、乙型肝炎病毒(HBV)、丙型肝炎病毒(HCV)、丁型肝炎病毒(HDV)、戊型肝炎病毒(HEV)、汉坦病毒、巨细胞病毒(CMV)、人类免疫缺陷病毒(HIV)的抗原，流感病毒、人乳头瘤病毒(HPV)、脊髓灰质炎病毒、埃博拉病毒、轮状病毒、登革热病毒、西尼罗河病毒、黄热病病毒、腺病毒、日本脑炎病毒、BK病毒、天花病毒、寨卡病毒、高热伴血小板减少综合征(SFTS)病毒或单纯疱疹病毒(HSV)的抗原。
12.一种治疗癌症的疫苗组合物，其包括作为活性成分的：
如权利要求1至9中任一项所述的疫苗佐剂；和
癌症抗原，
编码所述癌症抗原的多核苷酸，
基因构建体，其中所述多核苷酸可操作地连接到启动子；或
包含所述基因构建体的表达载体。
13.如权利要求12所述的疫苗组合物，其特征在于，所述癌症抗原是人类乳头状瘤病毒(HPV)衍生抗原、癌胚抗原、前列腺特异性膜抗原(PSMA)、Her2/neu、MUC-1、BCR/ABL、甲胎蛋白(AFP)、爱泼斯坦-巴尔病毒(EBV)衍生抗原、人类乙型肝炎病毒(HBV)衍生抗原、人丙型肝炎病毒(HCV)衍生抗原、癌抗原125(CA-125)、癌抗原72-4(CA-72-4)、癌抗原15-3(CA-15-3)或癌抗原19-9(CA-19-9)。
14.一种通过疫苗组合物刺激T淋巴细胞特异性免疫反应的方法，其包括向个体施用疫苗组合物的同时、或之前或之后施用如权利要求1至9中任一项所述疫苗佐剂所述。
15.如权利要求14所述的方法，其特征在于，通过体内电穿孔将所述疫苗佐剂施用于个体。
16.如权利要求14所述的方法，其特征在于，所述方法仅选择性地诱导T淋巴细胞特异性免疫应答，而不诱导个体的B细胞特异性免疫应答。
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