具体实施方式
当在权利要求和/或说明书中与术语“包含”联用时,词语“一(a)”或“一(an)”可以指“一个”,但也可以指“一个或多个”、“至少一个”以及“一个或多于一个”。
如在权利要求和说明书中所使用的,词语“包含”、“具有”、“包括”或“含有”是指包括在内的或开放式的,并不排除额外的、未引述的元件或方法步骤。
在整个申请文件中,术语“约”表示:一个值包括测定该值所使用的装置或方法的误差的标准偏差。
虽然所公开的内容支持术语“或”的定义仅为替代物以及“和/或”,但除非明确表示仅为替代物或替代物之间相互排斥外,权利要求中的术语“或”是指“和/或”。
如本公开所使用的,术语“多肽”、“肽”和“蛋白质”在本文中互换地使用并且为任意长度的氨基酸聚合物。该聚合物可以是线形或分支的,它可以包含修饰的氨基酸,并且它可以由非氨基酸隔断。该术语也包括已经被修饰(例如,二硫键形成、糖基化、脂质化、乙酰化、磷酸化或任何其他操作,如以标记组分缀合)的氨基酸聚合物。
如本公开所使用的,术语“环状RNA”是一种呈封闭环形RNA分子,主要由外显子、IRES元件、蛋白编码区和间隔区。在一些优选的实施方案中,环状RNA具有如下结构:“第二外显子E2-间隔区-IRES元件-编码区-间隔区-第一外显子E1”。本公开所使用的环状RNA具有蛋白翻译活性,又可称为“环状mRNA”。
如本公开所使用的,术语“线状RNA”是指能够环化形成环状RNA的RNA前体,其一般由线状的DNA分子转录形成。
如本公开所使用的,术语“线性RNA”是指包括5’帽子结构(5’Cap),3’多聚腺苷尾巴(PolyA tail),5’非翻译序列(5’untranslational region,5’UTR),3’非翻译序列(3’untranslational region,3’UTR)以及开放阅读框(Open reading frame,ORF)等结构的具有翻译功能的RNA。
如本公开所使用的,术语“IRES”(Internal ribosome entry site,IRES)又称内部核糖体进入位点,“内部核糖体进入位点”(IRES)属于翻译控制序列,通常位于所关注基因的5’端,并使得以帽非依赖性方式翻译RNA。经转录的IRES可直接结合核糖体亚单位,以使得mRNA起始密码子在核糖体中适当地取向以进行翻译。IRES序列通常位于mRNA的5’UTR中(起始密码子的正上游)。IRES在功能上取代对各种与真核生物翻译机制相互作用的蛋白因子的需求。在一些具体的实施方案中,本公开的IRES元件来源于EV病毒或柯萨奇病毒(CVB病毒)。在一些优选的实施方案中,本公开的IRES元件选自EV24 IRES,EV29 IRES,EV33IRES,CVB3 IRES,或者CVB3v IRES与EV24 IRES、EV29 IRES、EV33 IRES中任意1种的嵌合体序列。本公开中的“CVB3v”是指CVB3 IRES的v结构域,本公开中嵌合体序列包括:以CVB3IRES的v结构域替换EV24 IRES的v结构域所得的EV24+CVB3v嵌合体,以CVB3 IRES的v结构域替换EV29 IRES的v结构域所得的EV29+CVB3v嵌合体,以CVB3 IRES的v结构域替换EV33IRES的v结构域所得的EV33+CVB3v嵌合体。
如本公开所使用的,术语“编码区”(Coding Region)是指能够转录信使RNA,并最终翻译为目标多肽、蛋白的基因序列。
如本公开所使用的,术语“上游”或“下游”是指沿编码区的蛋白翻译方向的上游及下游。
在一些实施方案中,本公开的编码区编码选自抗原、抗体、抗原结合受体、配体、融合蛋白和重组蛋白中的一种或多种的目标多肽。
在一些实施方案中,本公开的抗原选自病毒来源的抗体或肿瘤特异性抗原。
在一些实施方案中,本公开的抗体选自Fab、Fab’、F(ab’)2、Fv、scFv、sdAb、双抗体、骆驼科抗体或单克隆抗体。
在一些实施方案中,本公开的抗原结合受体选自嵌合抗原受体或T细胞受体。
在一些实施方案中,本公开的目标多肽选自抗原、抗体、抗原结合受体、配体、融合蛋白和重组蛋白中的一种或多种。
如本公开所使用的,术语“取代、重复、缺失或添加一个或多个氨基酸”其中,取代是指用不同氨基酸置换占用一个位置的核苷酸或氨基酸。缺失是指去除占据某一位置的氨基酸。***是指在邻接并且紧随占据位置的氨基酸之后添加氨基酸。示例性的,本公开中的“突变”包括“保守突变”。
本公开中的术语“保守突变”是指可正常维持蛋白质的功能的保守突变。保守突变的代表性例子为保守置换。保守置换是指,例如,在置换部位为芳香族氨基酸的情况下,在Phe、Trp、Tyr间相互置换的突变;在置换部位为疏水性氨基酸的情况下,在Leu、Ile、Val间相互置换的突变;在为极性氨基酸的情况下,在Gln、Asn间相互置换的突变;在为碱性氨基酸的情况下,在Lys、Arg、His间相互置换的突变;在为酸性氨基酸的情况下,在Asp、Glu间相互置换的突变;在为具有羟基的氨基酸的情况下,在Ser、Thr间相互置换的突变。作为被视作保守置换的置换,具体而言,可以举出Ala向Ser或Thr的置换、Arg向Gln、His或Lys的置换、Asn向Glu、Gln、Lys、His或Asp的置换、Asp向Asn、Glu或Gln的置换、Cys向Ser或Ala的置换、Gln向Asn、Glu、Lys、His、Asp或Arg的置换、Glu向Gly、Asn、Gln、Lys或Asp的置换、Gly向Pro的置换、His向Asn、Lys、Gln、Arg或Tyr的置换、Ile向Leu、Met、Val或Phe的置换、Leu向Ile、Met、Val或Phe的置换、Lys向Asn、Glu、Gln、His或Arg的置换、Met向Ile、Leu、Val或Phe的置换、Phe向Trp、Tyr、Met、Ile或Leu的置换、Ser向Thr或Ala的置换、Thr向Ser或Ala的置换、Trp向Phe或Tyr的置换、Tyr向His、Phe或Trp的置换、及Val向Met、Ile或Leu的置换。此外,保守突变还包括起因于基因所来源的个体差异、株、种的差异等天然产生的突变。
本公开中的“序列同一性”和“同一性百分比”指两个或更多个多核苷酸或多肽之间相同(即同一)的核苷酸或氨基酸的百分比。两个或更多个多核苷酸或多肽之间的序列同一性可通过以下方法测定:将多核苷酸或多肽的核苷酸或氨基酸序列对准且对经对准的多核苷酸或多肽中含有相同核苷酸或氨基酸残基的位置数目进行评分,且将其与经对准的多核苷酸或多肽中含有不同核苷酸或氨基酸残基的位置数目进行比较。多核苷酸可例如通过含有不同核苷酸(即取代或突变)或缺失核苷酸(即一个或两个多核苷酸中的核苷酸***或核苷酸缺失)而在一个位置处不同。多肽可例如通过含有不同氨基酸(即取代或突变)或缺失氨基酸(即一个或两个多肽中的氨基酸***或氨基酸缺失)而在一个位置处不同。序列同一性可通过用含有相同核苷酸或氨基酸残基的位置数目除以多核苷酸或多肽中氨基酸残基的总数来计算。举例而言,可通过用含有相同核苷酸或氨基酸残基的位置数目除以多核苷酸或多肽中核苷酸或氨基酸残基的总数且乘以100来计算同一性百分比。
示例性的,在本公开中,当使用序列比较算法或通过目视检查测量以最大的对应性进行比较和比对时,两个或多个序列或子序列具有至少40%、50%、60%、70%、80%、85%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%核苷酸或氨基酸残基的“序列同一性”或“同一性百分比”。“序列同一性”或“同一性百分比”的判断/计算可以基于序列任何合适的区域上。例如,长度至少约50个残基的区域、至少约100个残基的区域,至少约200个残基的区域,至少约400个残基的区域,或至少约500个残基的区域。在某些实施方案中,所述序列在任一或两个相比较的生物聚合物(就是核酸或多肽)的整个长度上基本相同。
如本公开所使用的,术语“反向互补序列”(Reverse Complementary Sequence)的含义为:和原始多核苷酸的序列的方向相反,并且与原始多核苷酸的序列也互补的序列。示例性的,如果原始多核苷酸序列为ACTGAAC,则其反向互补序列为GTTCAGT。
如本公开所使用的,术语“多核苷酸”指由核苷酸组成的聚合物。多核苷酸可以是单独片段的形式,也可以是更大的核苷酸序列结构的一个组成部分,其是从至少在数量或浓度上分离一次的核苷酸序列衍生而来的,能够通过标准分子生物学方法(例如,使用克隆载体)识别、操纵以及恢复序列及其组分核苷酸序列。当一个核苷酸序列通过一个DNA序列(即A、T、G、C)表示时,这也包括一个RNA序列(即A、U、G、C),其中“U”取代“T”。换句话说,“多核苷酸”指从其他核苷酸(单独的片段或整个片段)中去除的核苷酸聚合物,或者可以是一个较大核苷酸结构的组成部分或成分,如表达载体或多顺反子序列。多核苷酸包括DNA、RNA和cDNA序列。“重组多核苷酸”、“重组核酸分子”属于“多核苷酸”中的一种。
如本公开所使用的,术语“重组核酸分子”指具有在自然界中不连接在一起的序列的多核苷酸。重组多核苷酸可包括在合适的载体中,且该载体可用于转化至合适的宿主细胞。然后多核苷酸在重组宿主细胞中表达以产生例如“重组多肽”“重组蛋白”“融合蛋白”等。在本公开中,重组核酸分子包含编码目标多肽的编码区,和连接于所述编码区上游的IRES元件。在一些具体的实施方案中,本公开的重组核酸分子包含如下的序列结构:
5’同源臂-3’内含子-第二外显子E2-5’间隔区-IRES元件-编码区-3’间隔区-第一外显子E1-5’内含子-3’同源臂。利用内含子的核酶特性,在GTP的引发下,5’内含子与第一外显子的连接处发生断裂;第一外显子的核酶裂口进一步攻击3’内含子与第二外显子的连接处,使该处发生断裂,3’内含子解离,第一外显子和第二外显子连接形成环状RNA。
如本公开所使用的,术语“载体”指的是DNA构建体,其含有与合适的控制序列可操作地连接的DNA序列,从而在合适的宿主中表达目的基因。
如本公开所使用的,术语“重组表达载体”指用于表达例如编码所需多肽的多核苷酸的DNA结构。重组表达载体可包括,例如包含i)对基因表达具有调控作用的遗传元素的集合,例如启动子和增强子;ii)转录成mRNA并翻译成蛋白质的结构或编码序列;以及iii)适当的转录和翻译起始和终止序列的转录亚单位。重组表达载体以任何合适的方式构建。载体的性质并不重要,并可以使用任何载体,包括质粒、病毒、噬菌体和转座子。用于本公开的可能载体包括但不限于染色体、非染色体和合成DNA序列,例如病毒质粒、细菌质粒、噬菌体DNA、酵母质粒以及从质粒和噬菌体DNA的组合中衍生的载体,来自如慢病毒、逆转录病毒、牛痘、腺病毒、鸡痘、杆状病毒、SV40和伪狂犬病等病毒的DNA。
如本公开所使用的,术语“抗原”是指引发免疫应答的分子。这种免疫应答可能涉及抗体产生或特异性免疫细胞的活化,或两者兼有。任何大分子,包括基本上所有的蛋白质或肽,都可以用作抗原。在本公开中,抗原包括病毒来源的抗原,例如新型冠状病毒(SARS-CoV-2)抗原,或肿瘤特异性抗原等。
如本公开所使用的,术语“抗体”,指免疫球蛋白或其片段或它们的衍生物,并且包括其包含的抗原结合位点的任何多肽,而不管其是否是在体外或体内产生。该术语包括,但不限于,多克隆、单克隆、单特异性的、多特异性的、非特异性的、人源化、单链的、嵌合的、合成的、重组的、杂合的、突变的、嫁接的抗体。术语“抗体”还包括抗体片段例如Fab、F(ab’)2、FV、scFv、Fd、dAb和其它保留抗原结合功能的抗体片段。通常情况下,这样的片段将包括抗原结合片段。
如本公开所使用的,术语“单链抗体”(scFv),是由抗体重链可变区和轻链可变区通过有限个氨基酸的短肽(也被称为连接子,linker)连接而成的抗体。
如本公开所使用的,术语“T细胞受体”(T cell receptor,TCR)是能够靶向异源性抗原的各类T细胞受体。大多数T细胞的TCR由α和β肽链组成,少数T细胞的TCR由γ和δ肽链组成。
如本公开所使用的,术语“嵌合抗原受体”(chimeric antigen receptor,CAR)是人工受体,其被改造为含有免疫球蛋白抗原结合结构域。目前,嵌合抗原受体可以包括抗原结合区、铰链区、跨膜区和细胞内结构区等结构域。
本公开中的术语“宿主细胞”意指易于用包含本公开的重组核酸分子、环状RNA或重组表达载体转化、转染、转导等的任何细胞类型。术语“重组宿主细胞”涵盖导入重组核酸分子、环状RNA或重组表达载体后不同于亲本细胞的宿主细胞,重组宿主细胞具体通过转化来实现。本公开的宿主细胞可以是原核细胞或真核细胞,只要是能够导入本公开的重组核酸分子、环状RNA或重组表达载体的细胞即可。在导入本公开的重组核酸分子、环状RNA或重组表达载体后,可以得到表达目标多肽的重组宿主细胞。
本公开中的术语“转化、转染、转导”具有本领域技术人员普遍理解的意思,即将40外源性的DNA导入宿主的过程。所述转化、转染、转导的方法包括任何将核酸导入细胞的方法,这些方法包括但不限于电穿孔法、磷酸钙(CaPO4)沉淀法、氯化钙(CaCl2)沉淀法、微注射法、聚乙二醇(PEG)法、DEAE-葡聚糖法、阳离子脂质体法以及乙酸锂-DMSO法。
如本公开所使用的,“治疗”是指:在罹患疾病之后,使受试者接触(例如给药)本发明的菌株和/或巨噬细胞或含者有其的药物组合物(以下也称为“本发明的药物组合物”),从而与不接触时相比使该疾病的症状减轻,并不意味着必需完全抑制疾病的症状。罹患疾病是指:身体出现了疾病症状。
如本公开所使用的,“预防”是指:在罹患疾病之前,通过使受试者接触(例如给药)本发明的药物组合物等,从而与不接触时相比减轻罹患疾病后的症状,并不意味着必需完全抑制患病。
如本公开所使用的,术语“个体”、“患者”或“受试者”包括哺乳动物。哺乳动物包括但不限于,家养动物(例如,牛,羊,猫,狗和马),灵长类动物(例如,人和非人灵长类动物如猴),兔,以及啮齿类动物(例如,小鼠和大鼠)。
如本公开所使用的,术语“高严格条件”是指,对于长度为至少100个核苷酸的探针而言,遵循标准DNA印迹程序,在42℃处在5X SSPE(saline sodium phosphate EDTA)、0.3%SDS、200微克/ml剪切并变性的鲑精DNA和50%甲酰胺中预杂交和杂交12至24小时。最后在65℃处使用2X SSC、0.2%SDS将载体材料洗涤三次,每次15分钟。
如本公开所使用的,术语“非常高严格条件”是指,对于长度为至少100个核苷酸的探针而言,遵循标准DNA印迹程序,在42℃处在5X SSPE(saline sodium phosphate EDTA)、0.3%SDS、200微克/ml剪切并变性的鲑精DNA和50%甲酰胺中预杂交和杂交12至24小时。最后在70℃处使用2X SSC、0.2%SDS将载体材料洗涤三次,每次15分钟。
除非另外定义或由背景清楚指示,否则在本公开中的全部技术与科学术语具有如本公开所属领域的普通技术人员通常理解的相同含义。
技术方案
在本公开的技术方案中,说明书核苷酸和氨基酸序列表的编号所代表的含义如下所示:
SEQ ID NO:1所示的序列是T7启动子的核苷酸序列;
SEQ ID NO:2所示的序列是5’同源臂序列1(H1)的核苷酸序列;
SEQ ID NO:3所示的序列是5’同源臂序列2(H2)的核苷酸序列;
SEQ ID NO:4所示的序列是I类PIE***的3’内含子的核苷酸序列;
SEQ ID NO:5所示的序列是I类PIE***的第二外显子(E2)的核苷酸序列;
SEQ ID NO:6所示的序列是5’间隔区序列1的核苷酸序列;
SEQ ID NO:7所示的序列是5’间隔区序列2的核苷酸序列;
SEQ ID NO:8所示的序列是CVB3 IRES的核苷酸序列;
SEQ ID NO:9所示的序列是EV24 IRES的核苷酸序列;
SEQ ID NO:10所示的序列是EV29 IRES的核苷酸序列;
SEQ ID NO:11所示的序列是EV33 IRES的核苷酸序列;
SEQ ID NO:12所示的序列是EV24与CVB3v嵌合IRES的核苷酸序列;
SEQ ID NO:13所示的序列是EV29与CVB3v嵌合IRES的核苷酸序列;
SEQ ID NO:14所示的序列是EV33与CVB3v嵌合IRES的核苷酸序列;
SEQ ID NO:15所示的序列是I类PIE***的第一外显子(E1)的核苷酸序列;
SEQ ID NO:16所示的序列是I类PIE***的5’内含子的核苷酸序列;
SEQ ID NO:17所示的序列是3’同源臂序列1的核苷酸序列;
SEQ ID NO:18所示的序列是3’同源臂序列2的核苷酸序列;
SEQ ID NO:19所示的序列是XbaI酶切位点的核苷酸序列;
SEQ ID NO:20所示的序列是EGFP编码DNA的核苷酸序列;
SEQ ID NO:21所示的序列是EGFP氨基酸序列;
SEQ ID NO:22所示的序列是EGFP环状RNA(CVB3 IRES)的核苷酸序列;
SEQ ID NO:23所示的序列是EGFP环状RNA(EV24 IRES)的核苷酸序列;
SEQ ID NO:24所示的序列是EGFP环状RNA(EV24+CVB3v IRES)的核苷酸序列;
SEQ ID NO:25所示的序列是EGFP环状RNA(EV29 IRES)的核苷酸序列
SEQ ID NO:26所示的序列是EGFP环状RNA(EV29+CVB3v IRES)的核苷酸序列;
SEQ ID NO:27所示的序列是EGFP环状RNA(EV33 IRES)的核苷酸序列;
SEQ ID NO:28所示的序列是EGFP环状RNA(EV33+CVB3v IRES)的核苷酸序列;
SEQ ID NO:29所示的序列是EGFP环状RNA(EV29 IRES+H1S1)的核苷酸序列;
SEQ ID NO:30所示的序列是EGFP环状RNA(EV29 IRES+H2S2)的核苷酸序列;
SEQ ID NO:31所示的序列是RBD编码DNA的核苷酸序列;
SEQ ID NO:32所示的序列是RBD蛋白的氨基酸序列
SEQ ID NO:33所示的序列是RBD环状RNA(EV29 IRES+H1S1)的核苷酸序列;
SEQ ID NO:34所示的序列是EPO编码DNA的核苷酸序列;
SEQ ID NO:35所示的序列是EPO蛋白的氨基酸序列;
SEQ ID NO:36所示的序列是EPO环状RNA(EV29 IRES+H1S1)的核苷酸序列;
SEQ ID NO:37所示的序列是PD-1单克隆抗体轻链编码DNA的核苷酸序列;
SEQ ID NO:38所示的序列是PD-1单克隆抗体轻链的氨基酸序列;
SEQ ID NO:39所示的序列是PD-1单克隆抗体轻链环状RNA(EV29 IRES+H1S1)的核苷酸序列;
SEQ ID NO:40所示的序列是PD-1单克隆抗体重链编码DNA的的核苷酸序列;
SEQ ID NO:41所示的序列是PD-1单克隆抗体重链蛋白的氨基酸序列;
SEQ ID NO:42所示的序列是PD-1单克隆抗体重链环状RNA(EV29 IRES+H1S1)的核苷酸序列;
SEQ ID NO:43所示的序列是IL-15编码DNA的核苷酸序列;
SEQ ID NO:44所示的序列是IL-15蛋白的氨基酸序列;
SEQ ID NO:45所示的序列是IL-15环状RNA(EV29 IRES+H1S1)的核苷酸序列;
SEQ ID NO:46所示的序列是PAP编码DNA的核苷酸序列;
SEQ ID NO:47所示的序列是PAP蛋白的氨基酸序列;
SEQ ID NO:48所示的序列是PAP环状RNA(EV29 IRES+H1S1)的核苷酸序列;
SEQ ID NO:49所示的序列是CD16 CAR编码DNA的核苷酸序列;
SEQ ID NO:50所示的序列是CD16 CAR蛋白的氨基酸序列;
SEQ ID NO:51所示的序列是CD16 CAR环状RNA(EV29 IRES+H1S1)的核苷酸序列;
SEQ ID NO:52所示的序列是3’间隔区序列1的核苷酸序列;
SEQ ID NO:53所示的序列是3’间隔区序列2的核苷酸序列。
本公开在研究中发现,现有技术中的线性mRNA虽然具有较高的蛋白表达量,但无法实现长期、持久性的蛋白表达。引用文献15中公开的环状RNA虽然在一定程度上提高了环状RNA的蛋白表达量和表达时间,但仍无法满足蛋白工业化生产的需求,目前需要兼具高的蛋白表达量,且可实现长时间蛋白表达的环状RNA分子。
在一些实施方案中,本公开提供了一种重组核酸分子,其转录形成环状RNA。重组核酸分子包含编码目标多肽的编码区,和连接于所述编码区上游的IRES元件。IRES元件能够提高所述目标多肽的表达水平,以上述重组核酸分子转录的环状RNA可以实现在真核细胞中高效、持久的蛋白表达。
IRES元件包含如SEQ ID NO:8-11任一序列所组成的组中的一种或多种序列的核苷酸序列具有至少80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、100%序列同一性的序列。在一些实施方案中,IRES元件为SEQ ID NO:8所示核苷酸序列的CVB3 IRES、SEQ ID NO:9所示核苷酸序列的EV24 IRES、SEQ ID NO:10所示核苷酸序列的EV29 IRES、SEQ ID NO:11所示核苷酸序列的EV33 IRES。在一些实施方案中,IRES元件包含CVB3v IRES与EV24 IRES、EV29 IRES和EV33IRES中任意一种的嵌合体序列。
在一些具体的实施方案中,本公开的重组核酸分子还包含位于所述IRES元件上游的5’同源臂,和位于所述编码区下游且与所述5’同源臂互补的3’同源臂。
在本公开中,5’同源臂包括5’同源臂1(H1)和5’同源臂2(H2),具体的,5’同源臂的核苷酸序列为与SEQ ID NO:2-3任一序列所示的核苷酸序列相比具有至少80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、100%序列同一性的序列。3’同源臂的核苷酸序列为与SEQ ID NO:17-18任一序列所示的核苷酸序列相比具有至少80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、100%序列同一性的序列。
在一些具体的实施方案中,本公开的重组核酸分子还包含分别位于所述5’同源臂与所述IRES元件之间,和位于所述编码区与所述3’同源臂之间的间隔区。
在本公开中,间隔区包括5’间隔区和3’间隔区,具体的,5’间隔区的核苷酸序列为与SEQ ID NO:6-7任一序列所示的核苷酸序列相比具有至少80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、100%序列同一性的序列。3’间隔区的核苷酸序列为与SEQ ID NO:52-53任一序列所示的核苷酸序列相比具有至少80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、100%序列同一性的序列。
本公开中的5’同源臂、3’同源臂以及间隔区的序列能够进一步提高以重组核酸分子形成环状RNA的环化效率,进而提高环状RNA的蛋白表达水平。
在一些具体的实施方案中,本公开的重组核酸分子还包含位于所述5’同源臂与所述IRES元件之间的3’内含子和第二外显子,以及位于所述编码区与所述3’同源臂之间的第一外显子和5’内含子。
在本公开中,3’内含子的核苷酸序列为与SEQ ID NO:4所示的核苷酸序列相比具有至少80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、100%序列同一性的序列。第二外显子(E2)的核苷酸序列为与SEQ ID NO:5所示的核苷酸序列相比具有至少80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、100%序列同一性的序列。5’内含子的核苷酸序列为与SEQ ID NO:16所示的核苷酸序列相比具有至少80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、100%序列同一性的序列。第一外显子(E1)的核苷酸序列为与SEQ ID NO:15所示的核苷酸序列相比具有至少80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%、99%、100%序列同一性的序列。
在一些优选的实施方案中,重组核酸分子的结构如下:
5’同源臂-3’内含子-第二外显子E2-5’间隔区-IRES元件-编码区-3’间隔区-第一外显子E1-5’内含子-3’同源臂。
为使重组核酸分子能够进一步转录形成RNA分子,在重组核酸分子中还可以包含调控序列。示例性的,调控序列为连接于5’同源臂上游的T7启动子,T7启动子序列为SEQ IDNO:1所示的核苷酸序列。
在一些实施方案中,本公开提供了一种重组表达载体,包含上述的重组核酸分子。其中,连接重组核酸分子的载体可以是本领域常用的各类载体,例如pUC57质粒等。进一步的重组核酸分子中包含限制性酶切位点,使重组表达载体经酶切后得到适于转录的线性化载体。
在一些实施方案中,本公开提供了一种线状RNA,其由重组核酸分子或线性化的重组表达载体转录后形成。作为优选,线状RNA具有如下的结构:
5’同源臂-3’内含子-第二外显子E2-5’间隔区-IRES元件-编码区-3’间隔区-第一外显子E1-5’内含子-3’同源臂。
图1显示以包含重组核酸分子的重组表达载体(DNA载体)得到环状RNA的过程:首先将DNA载体酶切后得到线性化载体,线性化的DNA载体转录后得到线状的RNA载体。最后,线状的RNA载体通过如下过程成环:利用内含子的核酶特性,在GTP的引发下,5’内含子与第一外显子的连接处发生断裂;第一外显子的核酶裂口进一步攻击3’内含子与第二外显子的连接处,使该处发生断裂,3’内含子解离,第一外显子和第二外显子连接形成环状RNA。
在一些实施方案中,本公开提供了一种环状RNA,其由上述的线状RNA环化形成,或由重组核酸分子、重组表达载体转录后环化形成。具体的,在重组核酸分子中调控序列的指导下,重组核酸分子转录产生线状的RNA分子。具体的,线状RNA分子中的5’同源臂与3’同源臂互补配对,利用内含子的核酶特性,使3’内含子与第二外显子E2之间,以及第一外显子E1与5’内含子之间发生断裂,E1与E2连接得到具有第二外显子E2-间隔区-IRES元件-编码区-间隔区-第一外显子E1的序列结构的环状RNA。
在一些实施方案中,表达选自抗原、抗体、抗原结合受体、配体、融合蛋白和重组蛋白中的一种或多种的目标多肽。
在一些实施方案中,环状RNA表达如SEQ ID NO:21所示氨基酸序列的EGFP蛋白,或SEQ ID NO:21所示氨基酸序列经过取代、重复、缺失或添加一个或多个氨基酸,且具有EGFP蛋白活性的多肽。编码EGFP蛋白的核苷酸序列如SEQ ID NO:20所示。表达EGFP蛋白的环状RNA包含如SEQ ID NO:22-30任一序列所示的核苷酸序列。
在一些实施方案中,环状RNA表达病毒抗原。示例性的,病毒抗原是具有如SEQ IDNO:32所示氨基酸序列的RBD蛋白,或SEQ ID NO:32所示氨基酸序列经过取代、重复、缺失或添加一个或多个氨基酸,且具有RBD蛋白活性的多肽。编码RBD蛋白的核苷酸序列如SEQ IDNO:31所示。表达RBD蛋白的环状RNA包含如SEQ ID NO:33所示的核苷酸序列。
在一些实施方案中,环状RNA表达重组人源化蛋白。示例性的,重组人源化蛋白具体为如SEQ ID NO:35所示氨基酸序列的EPO蛋白,或SEQ ID NO:35所示氨基酸序列经过取代、重复、缺失或添加一个或多个氨基酸,且具有EPO蛋白活性的多肽。编码EPO蛋白的核苷酸序列如SEQ ID NO:34所示。表达EPO蛋白的环状RNA包含如SEQ ID NO:36所示的核苷酸序列。
在一些实施方案中,环状RNA表达细胞因子。细胞因子具体为如SEQ ID NO:44所示氨基酸序列的IL-15蛋白,或SEQ ID NO:44所示氨基酸序列经过取代、重复、缺失或添加一个或多个氨基酸,且具有IL-15蛋白活性的多肽。编码IL-15蛋白的核苷酸序列如SEQ IDNO:43所示。表达IL-15蛋白的环状RNA包含如SEQ ID NO:45所示的核苷酸序列。
在一些实施方案中,环状RNA表达肿瘤特异性抗原,肿瘤特异性抗原包括CEA AFPPSA PSMA MAGE-A3 PAP蛋白等等。示例性的,肿瘤特异性抗原是具有如SEQ ID NO:47所示氨基酸序列的PAP蛋白,或SEQ ID NO:47所示氨基酸序列经过取代、重复、缺失或添加一个或多个氨基酸,且具有PAP蛋白活性的多肽。编码PAP蛋白的核苷酸序列如SEQ ID NO:46所示。表达PAP蛋白的环状RNA包含如SEQ ID NO:48所示的核苷酸序列。
在一些实施方案中,环状RNA表达嵌合抗原受体的相关蛋白,嵌合抗原受体的相关蛋白包括CD19、CD20、CD133、CD138、BCMA、CD16蛋白等等。示例性的,嵌合抗原受体的相关蛋白是具有如SEQ ID NO:50所示氨基酸序列的CD16蛋白,或SEQ ID NO:50所示氨基酸序列经过取代、重复、缺失或添加一个或多个氨基酸,且具有CD16蛋白活性的多肽。编码CD16蛋白的核苷酸序列如SEQ ID NO:49所示。表达CD16蛋白的环状RNA包含如SEQ ID NO:51所示的核苷酸序列。
在一些实施方案中,环状RNA表达单克隆抗体。示例性的,单克隆抗体为PD-1单克隆抗体。PD-1单克隆抗体的轻链为如SEQ ID NO:38所示氨基酸序列的多肽,或SEQ ID NO:38所示氨基酸序列经过取代、重复、缺失或添加一个或多个氨基酸,且具有轻链活性的多肽。编码轻链的核苷酸序列如SEQ ID NO:37所示。表达PD-1单克隆抗体轻链的环状RNA包含如SEQ ID NO:39所示的核苷酸序列。PD-1单克隆抗体的重链为如SEQ ID NO:41所示氨基酸序列的多肽,或SEQ ID NO:41所示氨基酸序列经过取代、重复、缺失或添加一个或多个氨基酸,且具有重链活性的多肽。编码重链的核苷酸序列如SEQ ID NO:40所示。表达PD-1单克隆抗体重链的环状RNA包含如SEQ ID NO:42所示的核苷酸序列。
以本公开的环状RNA分子表达上述的蛋白,由于环状RNA包含特定序列的IRES元件,5’间隔区、3’间隔区,以及5’同源臂、3’同源臂,通过各元件的协同配合,使环状RNA能够实现蛋白的高效表达,且持久性好,优于现有的线性mRNA和环状RNA等各类蛋白表达元件。
在一些实施方案中,本公开提供了一种重组宿主细胞,包含上述的重组核酸分子、重组表达载体、线状RNA或环状RNA。作为优选,重组宿主细胞为真核生物来源的细胞,本公开的IRES元件可以实现目标多肽在真核细胞中的高效、持久表达。
在一些实施方案中,本公开提供了一种药物组合物,包括上述重组核酸分子、重组表达载体、线状RNA、环状RNA、重组宿主细胞或其所表达的蛋白。本公开的环状RNA即可作为病毒抗原、重组人源化蛋白、肿瘤特异性抗原、嵌合抗原受体等的表达元件,也可作为核酸疫苗直接导入生物体内,在生物体内产生病毒抗原、肿瘤特异性抗原、嵌合抗原受体等。
实施例
本公开的其他目的、特征和优点将从以下详细描述中变得明显。但是,应当理解的是,详细描述和具体实施例(虽然表示本公开的具体实施方式)仅为解释性目的而给出,因为在阅读该详细说明后,在本公开的精神和范围内所作出的各种改变和修饰,对于本领域技术人员来说将变得显而易见。
本实施例中所用到的实验技术与实验方法,如无特殊说明均为常规技术方法,例如下列实施例中未注明具体条件的实验方法,通常按照常规条件如Sambrook等人,分子克隆:实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件,或按照制造厂商所建议的条件。实施例中所使用的材料、试剂等,如无特殊说明,均可通过正规商业渠道获得。
实施例1:探究不同IRES介导形成的环状mRNA在293T细胞中的表达
1.1实验方法与步骤
(1)质粒构建
构建含有不同元件的EGFP目的基因,该步骤委托苏州金唯智生物科技有限公司进行基因合成与克隆。此处所使用的构建环状RNA的DNA载体,包含T7启动子,5’同源臂,3’内含子,第二外显子E2,5’间隔区,IRES元件,EGFP编码区,下游间隔区,5’内含子,第一外显子E1,3’同源臂,以及可用于质粒线性化的酶切位点XbaI。所得基因片段连接到pUC57载体。
IRES元件信息如下:
其中,IRES元件v结构域的定义可参见参考文献(Proc Natl Acad Sci U SA.2009Jun 9;106(23):9197–9202.)
以上述IRES元件所得的表达EGFP的环状RNA序列分别如下所示:
IRES名称 |
编码区基因 |
SEQ ID NO: |
CVB3 IRES |
EGFP |
SEQ ID NO:22 |
EV24 IRES |
EGFP |
SEQ ID NO:23 |
EV24+CVB3v IRES |
EGFP |
SEQ ID NO:24 |
EV29 IRES |
EGFP |
SEQ ID NO:25 |
EV29+CVB3v IRES |
EGFP |
SEQ ID NO:26 |
EV33 IRES |
EGFP |
SEQ ID NO:27 |
EV33+CVB3v IRES |
EGFP |
SEQ ID NO:28 |
(2)线性质粒模板制备
1)质粒抽提
①将外部合成的穿刺菌活化,条件37℃/220rpm/3~4h
②取活化菌液扩大培养,培养条件:37℃/220rpm/过夜
③质粒抽提(天根无内毒素小量中提试剂盒),测定OD值
2)质粒酶切
采取XbaI单酶切的方法酶切上述1)制备质粒
酶切体系如下:
表1
试剂 |
体积 |
质粒 |
10μg |
酶(1000units) |
5μl |
10x cutsmart buffer |
50μl |
Nuclease free,H2O |
Total,500μl |
37℃酶切过夜。采用通用型DNA胶回收试剂盒(天根生化科技有限公司)回收酶切产物,测定OD值并采用1%琼脂糖凝胶电泳鉴定酶切产物。纯化的线性质粒模板用于体外转录。
(3)体外转录制备线状mRNA
1)体外转录
采用T7体外转录试剂盒(APExBIO T7 High Yield RNA Synthesis Kit)合成mRNA
转录体系如下:
表2
试剂 |
体积 |
10xReaction Buffer |
2μl |
ATP(20mM) |
2μl |
CTP(20mM) |
2μl |
UTP(20mM) |
2μl |
GTP(20mM) |
2μl |
线性化DNA模板 |
1μg |
T7 RNA Polymerase Mix |
2μl |
RNA Nuclease free,H2O |
Total 20μl |
37℃孵育2h,然后用DNase I消化线性DNA模板。消化条件:37℃消化15min。
2)线状mRNA纯化
将上述1)所得转录产物,使用硅膜离心柱法纯化(Thermo,GeneJET RNAPurification Kit),测定OD值及1%变性琼脂糖凝胶电泳鉴定RNA大小。
1%变性琼脂糖凝胶配方如下:
①称取1g琼脂糖,至72ml nuclease-free,H2O中,微波炉加热溶解;
②上述琼脂糖冷却至55~60℃时,在通风橱加0.1%的gel red,10ml 10xMOPS,18ml甲醛,灌胶。
变性琼脂糖凝胶电泳流程如下:取等体积样本RNA与2x Loading buffer,65~70℃变性5~10min。上样,采用100V/30min条件进行电泳,其后采用凝胶成像***拍照。
(4)mRNA环化
1)环化试剂:
GTP Buffer:50mM Tris-HCl,10mM MgCl2,1mM DTT,pH 7.5左右
2)环化体系与条件:
表3
溶液 |
体积 |
mRNA |
25μg RNA溶液 |
GTP solution(20mM) |
50μl |
GTP buffer |
补足至500μl |
将上述溶液于55℃加热15min,环化RNA产物使用硅膜离心柱法纯化(Thermo,GeneJET RNA Purification Kit),测定OD值及1%变性琼脂糖凝胶电泳鉴定RNA大小。
3)环状RNA鉴定
①1%变性琼脂糖凝胶鉴定:
A.试剂配制:1g琼脂糖粉加入72ml无核酸酶水中,加热将琼脂塘融化,加入10ml10×MOPS缓冲液。然后在通风柜中加入18ml新鲜37%甲醛,充分混合,将凝胶倒入槽中。
B.mRNA检测:取500ng左右mRNA溶液,加入等体积的2×RNA loading buffer混匀,65℃加热5min,上样进行琼脂糖凝胶检测。
②环化mRNA RT-RCR与测序鉴定
A.mRNA逆转录体系与条件
表4
溶液 |
体积 |
mRNA |
1μg RNA溶液 |
RT primer Mix |
4.0μl |
Primerscript RT Enzye Mix I |
1.0μl |
5×Primerscript buffer 2 |
4.0μl |
无核酸酶水 |
补足至20μl |
将实验组:环化处理的mRNA、对照组:未环化处理的mRNA,按照上述体系进行配制,于37℃加热15min,85℃加热5s,4℃保存。
B.逆转录产物PCR扩增体系与条件
表5
溶液 |
体积 |
逆转录产物 |
1.0μl |
10×buffer |
2.0μl |
dNTP |
1.6μl |
primer-F(10μM) |
1.0μl |
primer-R(10μM) |
1.0μl |
Taq酶 |
0.5μl |
无核酸酶水 |
12.9μl |
PCR扩增程序:95℃,1min;95℃,30s;60℃,30s;72℃,30s;(35cycles)72℃,7min;4℃。
C.PCR产物切胶纯化
核酸电泳,选择特异性存在于实验组,不存在于对照组的RT-RCR DNA条带,切胶回收,通用型DNA纯化回收试剂盒纯化,取纯化DNA、引物EV29-EGFP-F:GTGACAGCAGCAGGAATCACA、引物EV29-EGFP-R:TGGGATCAACCCACAGGCT送金唯智公司进行正、反向测序。
(5)编码EGFP的环状mRNA转染293T细胞及荧光强度测定
1)细胞培养:
293T接种于含有10%胎牛血清,1%双抗的DMEM高糖培养基中,于37℃,5%CO2培养箱中培养。细胞每隔2-3天进行传代培养。
2)细胞转染:
转染前将293T细胞以1×105个/孔接种于24孔板中,于37℃,5%CO2培养箱中培养。待细胞达到70-90%融合度后,使用Lipofectamine MessengerMax(Invitrogen)转染试剂将mRNA以500ng/孔量转染293T细胞,具体操作如下:
①稀释Messenger MAXTM Reagent
表6
试剂 |
体积/孔 |
MEM无血清培养基 |
25μl |
Messenger MAX<sup>TM</sup> Reagent |
0.75μl |
稀释混合后,室温静置孵育10min。
②稀释mRNA
表7
试剂 |
体积/孔 |
mRNA |
1μg |
MEM无血清培养基 |
补足至25μl |
③取混合稀释后的Messenger MAXTM Reagent和mRNA(1:1)
表8
试剂 |
体积/孔 |
稀释的Messenger MAX<sup>TM</sup> Reagent |
25μl |
稀释的mRNA |
25μl |
稀释混合后,室温静置孵育5min。
④吸取上述混合液50ul贴壁缓缓加入24孔板中,37℃、5%CO2培养箱中孵育培养。
3)蛋白表达检测
①细胞荧光观察:将转染后1-10天293T细胞于200×荧光显微镜下观察EGFP的表达情况。
②流式细胞术检测细胞平均荧光强度:将转染后1-10天293T细胞用流式细胞仪检测细胞平均荧光强度。
1.2实验结果
1)DNA转录模板制备
①质粒提取浓度:pUC57-CVB3-EGFP:271.2ng/μl,pUC57-EV24-EGFP:245.4ng/ul,pUC57-EV24+CVB3v-EGFP:263.8ng/ul,pUC57-EV29-EGFP:277.9ng/μl,pUC57-EV29+CVB3v-EGFP:249.9ng/μl,pUC57-EV33-EGFP:273.0ng/μl,pUC57-EV33+CVB3v-EGFP:283.3ng/μl;
②质粒酶切线性化后DNA浓度:pUC57-CVB3-EGFP:120.6ng/μl,pUC57-EV24-EGFP:134.5ng/ul,pUC57-EV24+CVB3v-EGFP:125.8ng/ul,pUC57-EV29-EGFP:146.1ng/μl,pUC57-EV29+CVB3v-EGFP:119.2ng/μl,pUC57-EV33-EGFP:141.1ng/μl,pUC57-EV33+CVB3v-EGFP:137.9ng/μl;
2)mRNA转录与环化
①mRNA转录纯化后RNA浓度:CVB3-EGFP:1149.8ng/μl,EV24-EGFP:1168.5ng/ul,EV24+CVB3v-EGFP:1284.6ng/ul,EV29-EGFP:1245.5μg/μl,EV29+CVB3vEGFP:1111.8ng/μl,EV33-EGFP:1180.4ng/μl,EV33+CVB3v-EGFP:1148.5ng/μl;
②mRNA环化并纯化后RNA浓度:CVB3-EGFP:482.8ng/μl,EV24-EGFP:462.4ng/ul,EV24+CVB3v-EGFP:532.5ng/ul,EV29-EGFP:444.5μg/μl,EV29+CVB3-EGFP:447.2ng/μl,EV33-EGFP:452.0ng/μl,EV33+CVB3 EGFP:415.5ng/μl;
③RNA变性琼脂糖凝胶电泳。
如图2所示:变性琼脂糖凝胶电泳图显示环化处理组mRNA较线状mRNA,其在胶上的迁移速度更快。
3)RT-PCR以及基因测序鉴定mRNA成环
为鉴定环化反应所得RNA是否为环状RNA,此处采用RT-PCR及DNA测序法进行检测。根据RNA环化的基本原理,通过内含子的核酶特性,在GTP的引发下,5’内含子与第一外显子E1连接处将发生断裂,该第一外显子E1处核酸裂口将进攻3’内含子与第二外显子E2的连接处,导致该连接处发生断裂,3’内含子发生解离,第一外显子E1与第二外显子E2形成共价连接,最终形成环状RNA。因此,通过扩增包含第一外显子E1与第二外显子E2连接序列并进行测序,可作为RNA是否成环的最终判断依据。通过随机引物介导的RNA逆转录反应获得cDNA。以cDNA作为模板,采用特异性引物进行PCR扩增。实验结果显示,线状mRNA组未见特异性扩增条带,环状mRNA组可见特异性扩增条带。通过对该特异性条带进行切胶回收,纯化后进行DNA测序。如图3所示,测序结果显示,该DNA条带包含连接后的E1和E2序列。由此可见,环状RNA包含连接的E1-E2 RNA序列,说明该RNA已连接成环。
4)蛋白表达检测
细胞转染后1-3天荧光强度定量测试结果如图4所示,与包含CVB3 IRES的环状EGFP mRNA(Circ-RNA CVB3,引用文献15)相比,包含EV24 IRES,EV24与CVB3v嵌合型IRES(EV24+CVB3v),EV29 IRES,EV29与CVB3v嵌合型IRES(EV29+CVB3v),EV33 IRES,EV33与CVB3v嵌合型IRES(EV33+CVB3v)的环状mRNA介导的细胞荧光更强,说明本专利提供的包含不同IRES组合的环状mRNA能介导更强的蛋白表达。
为鉴定该系列环状mRNA介导蛋白表达的持久度,对细胞转染后1-5天的荧光强度进行定量。测试结果如图5和图6所示:与包含CVB3 IRES的环状EGFP mRNA(Circ-RNA CVB3,引用文献15)相比,包含EV24 IRES,EV24+CVB3v IRES,EV29IRES,EV29+CVB3v IRES,EV33IRES,EV33+CVB3v IRES的环状mRNA介导的EGFP的表达更强且更持久,同时也显著地比线性EGFP mRNA(为购自APExBio的标准品,Cap1帽子结构及PolyA尾)介导的EGFP的表达更强且更持久。
实施例2:EV29 IRES与不同的同源臂、间隔序列组合所得环状mRNA在293T细胞中的表达
2.1实验方法与步骤
在上述实施例1的基础上,采用与上述实施例相同的内含子与外显子元件,以EV29IRES(SEQ ID NO:10所示核苷酸序列),结构新颖的5’同源臂1(SEQ ID NO:2所示的核苷酸序列)、3’同源臂1(SEQ ID NO:17所示的核苷酸序列)和5’间隔区1序列(SEQ ID NO:6所示的核苷酸序列)及3’间隔区1序列(SEQ ID NO:52所示的核苷酸序列)作为环状mRNA的基础元件,构建编码绿色荧光蛋白(EGFP)的环状mRNA(Circ-RNA EV29 H1S1)。
采用与上述环状mRNA相同的内含子与外显子元件,以EV29 IRES,5’同源臂2(SEQID NO:3所示的核苷酸序列)、3’同源臂2(SEQ ID NO:18所示的核苷酸序列)和5’间隔区2序列(SEQ ID NO:7所示的核苷酸序列)及3’间隔区2序列(SEQ ID NO:53所示的核苷酸序列)作为环状mRNA的基础元件,构建编码绿色荧光蛋白(EGFP)的环状mRNA(Circ-RNA EV29H2S2)。
编码EGFP的DNA序列如SEQ ID NO:20所示。DNA合成委托苏州金唯智生物科技有限公司完成。最终将含有T7启动子、I类PIE元件、5’同源臂1、3’同源臂1、5’间隔区1、3’间隔区1、EV29 IRES元件、EGFP编码区(或者T7启动子、I类PIE元件、5’同源臂2、3’同源臂2、5’间隔区2、3’间隔区2、EV29 IRES元件、EGFP编码区)的完整DNA片段克隆至pUC57质粒。
质粒DNA线性化,线状mRNA体外转录,线状mRNA纯化,mRNA的成环反应,环状mRNA的纯化,细胞培养与转染等方法均与实施例1的1.1相同。
2.2实验结果
2.2.1实验结果
1)DNA线性化模板制备
①质粒提取浓度:pUC57-CVB3-EGFP:356.4ng/μl,pUC57-EV29-EGFP:481.9ng/μl,pUC57-EV29-EGFP H1S1:283.1ng/μl,pUC57-EV29-EGFP H2S2:303.1ng/μl;
②质粒酶切线性化浓度:pUC57-CVB3-EGFP:249.6ng/μl pUC57-EV29-EGFP:289ng/μl,pUC57-EV29-EGFP H1S1:293ng/μl,pUC57-EV29-EGFP H2S2:294ng/μl;
2)mRNA转录与环化
①mRNA转录纯化后浓度:CVB3-EGFP:506.3ng/ul,EV29-EGFP:527.5ng/μl,EV29-EGFP H1S1:573.2ng/μl,EV29-EGFP H2S2:564.9ng/μl;
②mRNA环化纯化后浓度:CVB3-EGFP:257.4ng/ul,EV29-EGFP:236.2ng/μl,EV29-EGFP H1S1:208.0ng/μl,EV29-EGFP H2S2:240.3ng/μl;
采用变性琼脂糖凝胶鉴定RNA成环。
实验结果如图7所示:变性琼脂糖凝胶电泳图中各组环化后mRNA较相应的环化前线状mRNA在胶上的迁移速度更快。
3)蛋白表达检测
细胞转染后1-3d荧光定量如图8所示:环状mRNA Circ-RNA EV29-EGFP H1S1所介导的荧光蛋白表达量显著高于Circ-RNA EV29-EGFP组所对应的量,也显著高于Circ-RNACVB3-EGFP组所对应的量。说明本发明提供的新颖的5’同源臂1、3’同源臂1以及5’间隔区1、3’间隔区1组合可有效提高环状mRNA介导的蛋白表达。环状mRNA Circ-RNA EV29-EGFPH2S2所介导的荧光蛋白表达量较Circ-RNA EV29-EGFP组所对应的量有提高,且显著高于Circ-RNA CVB3-EGFP组所对应的量。说明本发明提供的新颖的5’同源臂2、3’同源臂2以及5’间隔区2、3’间隔区2组合可提高环状mRNA介导的蛋白表达。
为鉴定不同的环状mRNA介导蛋白表达的持久度,对细胞转染1-5d后荧光进行定量。如图9所示,Circ-RNA EV29-EGFP H1S1介导的荧光蛋白,其表达的强度和持久度均高于Circ-RNA EV29-EGFP组以及Circ-RNA CVB3-EGFP组。说明本发明提供的新颖的5’同源臂1、3’同源臂1以及5’间隔区1、3’间隔区1组合可有效提高环状mRNA介导的蛋白表达的持久性。Circ-RNA EV29-EGFP H2S2所介导的荧光蛋白表达持久度与Circ-RNA EV29-EGFP组相当,但显著高于Circ-RNA CVB3-EGFP组。说明本发明提供的新颖的5’同源臂2、3’同源臂2以及5’间隔区2、3’间隔区2组合,其环状mRNA介导的蛋白表达显著优于专利引用文献15所提供的方法及设计。另外,上述所有环状mRNA的EGFP表达持久度均显著高于线性mRNA(为购自APExBio的标准品,包含Cap1帽子结构及PolyA尾)。
实施例3:环状mRNA编码新型冠状病毒Spike抗原RBD蛋白实现在293T细胞的蛋白表达
3.1实验方法与步骤
在上述实施例2的基础上,以EV29 IRES,5’同源臂1、3’同源臂1和5’间隔区1、3’间隔区1序列作为环状mRNA的基础元件,构建编码新型冠状病毒Spike抗原的RBD结构域(receptor binding domain)的环状mRNA。RBD蛋白序列如SEQ ID NO:32所示,编码RBD的DNA序列如SEQ ID NO:31所示。DNA合成委托苏州金唯智生物科技有限公司完成。
最终将含有T7启动子,I类PIE元件,5’同源臂1、3’同源臂1,5’间隔区1、3’间隔区1,EV29 IRES,RBD编码区的完整DNA片段克隆至pUC57质粒。质粒DNA线性化,线状mRNA体外转录,线状mRNA纯化,mRNA的成环反应,环状mRNA的纯化,得到如SEQ ID NO:33所示序列的环状mRNA。细胞培养与转染等方法均与实施例1的1.1相同。采用His-tag ELISA检测试剂盒对分泌的His-RBD蛋白进行定量检测(南京金斯瑞生物科技有限公司)。
3.2实验结果
表9
核酸名称 |
浓度 |
pUC57-EV29-RBD-H1S1质粒(纯化后) |
342.2ng/μl |
pUC57-EV29-RBD-H1S1线性化质粒(纯化后) |
264.3ng/μl |
EV29-RBD-H1S1线状mRNA(纯化后) |
508.5ng/μl |
EV29-RBD-H1S1环状mRNA(纯化后) |
236.2ng/μl |
经His-tag ELISA检测,RBD-His环状mRNA在293T表达1-5天所得蛋白的量分别为21.6,35.4,40.3,28.6,22.7ng/ml,说明以本公开的环状mRNA可实现RBD蛋白的高效且持久性的表达。
实施例4:环状mRNA编码EPO实现在293T细胞的蛋白表达
4.1实验方法与步骤
在上述实施例2的基础上,以EV33 IRES,5’同源臂1、3’同源臂1和5’间隔区1、3’间隔区1作为环状mRNA的基础元件,构建编码红细胞生成素(EPO)的环状mRNA。编码EPO的DNA和蛋白序列分别如SEQ ID NO:34和SEQ ID NO:35所示.。DNA合成委托苏州金唯智生物科技有限公司完成。最终将含有T7启动子,I类PIE元件,5’同源臂1、3’同源臂1,5’间隔区1、3’间隔区1,EV33 IRES,EPO编码区的完整DNA片段克隆到pUC57质粒。质粒DNA线性化,线状mRNA体外转录,线状mRNA纯化,mRNA的成环反应,环状mRNA的纯化,细胞培养与转染等方法均与实施例1的1.1相同。采用EPO ELISA检测试剂盒(Thermo Fisher)对293T表达的EPO蛋白进行定量。
4.2实验结果
表10
核酸名称 |
浓度 |
pUC57-EV33-EPO-H1S1质粒(纯化后) |
297.5ng/μl |
pUC57-EV33-EPO-H1S1线性化质粒(纯化后) |
275.4ng/μl |
EV33-EPO-H1S1线状mRNA(纯化后) |
375.3ng/μl |
EV33-EPO-H1S1环状mRNA(纯化后) |
286.7ng/μl |
经EPO ELISA检测,EPO环状mRNA在293T表达1-5天所得蛋白的量分别为35.6,42.8,56.4,50.3,25.7ng/ml,说明以本公开的环状mRNA可实现EPO蛋白的高效、持久性表达。
实施例5:环状mRNA编码PD-1单克隆抗体实现在293T细胞的蛋白表达
5.1实验方法和步骤
上述实施例2的基础上,以EV29 IRES,5’同源臂1、3’同源臂1和5’间隔区1、3’间隔区1作为环状mRNA的基础元件,构建编码抗程序性细胞死亡受体1(PD-1)单克隆抗体)的环状mRNA。编码抗PD1单抗轻链的DNA和蛋白序列分别如SEQ ID NO:37和SEQ ID NO:38所示,编码抗PD1单抗重链的DNA和蛋白序列分别如SEQ ID NO:40和SEQ ID NO:41所示。DNA合成委托苏州金唯智生物科技有限公司完成。最终将含有T7启动子,I类PIE元件,5’同源臂1、3’同源臂1,5’间隔区1、3’间隔区1,EV29 IRES,PD1轻链编码区的完整DNA片段克隆到pUC57质粒。类似地,将含有T7启动子,I类PIE元件,5’同源臂1、3’同源臂1,5’间隔区1、3’间隔区1,EV29 IRES,PD1重链编码区的完整DNA片段克隆到pUC57质粒。质粒DNA线性化,线状mRNA体外转录,线状mRNA纯化,mRNA的成环反应,环状mRNA的纯化,得到序列如SEQ ID NO:39或42所示的两种环状RNA,细胞培养等方法均与实施例1的1.1相同。将编码PD1单抗轻链和重链的两种mRNA以1:1的比例混合,共转染到293T细胞。转染方法同实施例1。采用PD1 ELISA检测试剂盒(Thermo Fisher)对293T表达的PD1蛋白进行定量。
5.2实验结果
表11
核酸名称 |
浓度 |
pUC57-EV29-PD1L-H1S1质粒(纯化后) |
279.3ng/μl |
pUC57-EV29-PD1L-H1S1线性化质粒(纯化后) |
204.2ng/μl |
EV29-PD1L-H1S1线状mRNA(纯化后) |
396.5ng/μl |
EV29-PD1L-H1S1环状mRNA(纯化后) |
247.2ng/μl |
核酸名称 |
浓度 |
pUC57-EV29-PD1H-H1S1质粒(纯化后) |
268.6ng/μl |
pUC57-EV29-PD1H-H1S1线性化质粒(纯化后) |
201.3ng/μl |
EV29-PD1H-H1S1线状mRNA(纯化后) |
304.7ng/μl |
EV29-PD1H-H1S1环状mRNA(纯化后) |
207.4ng/μl |
经PD1单抗ELISA检测,PD1单抗环状mRNA在293T表达1-5天所得蛋白的量分别为120.3,234.6,356.4,221.6,104.8ng/ml,说明以本公开的环状mRNA可实现PD1单抗的高效、持久性表达。
实施例6:环状mRNA编码细胞因子IL-15实现在293T细胞的蛋白表达
6.1实验方法和步骤
在上述实施例2的基础上,以EV29 IRES,5’同源臂1、3’同源臂1,5’间隔区1、3’间隔区1作为环状mRNA的基础元件,构建编码白介素15(IL-15)的环状mRNA。编码IL-15的DNA和蛋白序列分别分别如SEQ ID NO:43和SEQ ID NO:44所示。DNA合成委托苏州金唯智生物科技有限公司完成。最终将含有T7启动子,I类PIE元件,5’同源臂1、3’同源臂1,5’间隔区1、3’间隔区1,EV29 IRES,IL-15编码区的完整DNA片段克隆到pUC57质粒。质粒DNA线性化,线状mRNA体外转录,线状mRNA纯化,mRNA的成环反应,环状mRNA的纯化,得到序列如SEQ IDNO:45所示的环状RNA,细胞培养与转染等方法均与实施例1的1.1相同。采用IL-15 ELISA检测试剂盒(Thermo Fisher)对293T表达的IL-15蛋白进行定量。
6.2实验结果
表12
核酸名称 |
浓度 |
pUC57-EV29-IL15-H1S1质粒(纯化后) |
286.3ng/μl |
pUC57-EV29-IL15-H1S1线性化质粒(纯化后) |
251.5ng/μl |
EV29-IL15-H1S1线状mRNA(纯化后) |
311.3ng/μl |
EV29-IL15-H1S1环状mRNA(纯化后) |
274.3ng/μl |
经IL-15ELISA检测,IL-15环状mRNA在293T表达1-5天所得蛋白的量分别为38.9,47.3,68.4,51.6,26.4ng/ml,说明以本公开的环状mRNA可实现IL-15的高效、持久性表达。
实施例7:环状mRNA编码肿瘤特异性抗原***癌PAP蛋白实现在293T细胞的蛋白表达
7.1实验方法和步骤
在上述实施例2的基础上,以EV29 IRES,5’同源臂1、3’同源臂1,5’间隔区1、3’间隔区1作为环状mRNA的基础元件,构建编***癌肿瘤特异性抗原酸性磷酸酶蛋白PAP(prostate acid phosphatase)的环状mRNA。编码PAP的DNA和蛋白序列分别分别如SEQ IDNO:46和SEQ ID NO:47所示。DNA合成委托苏州金唯智生物科技有限公司完成。最终将含有T7启动子,I类PIE元件,5’同源臂1、3’同源臂1,5’间隔区1、3’间隔区1,EV29 IRES,PAP编码区的完整DNA片段克隆到pUC57质粒。质粒DNA线性化,线状mRNA体外转录,线状mRNA纯化,mRNA的成环反应,环状mRNA的纯化,得到序列如SEQ ID NO:48所示的环状RNA,细胞培养与转染等方法均与实施例1的1.1相同。采用PAP ELISA检测试剂盒(Thermo Fisher)对293T表达的PAP蛋白进行定量。
7.2实验结果
表13
核酸名称 |
浓度 |
pUC57-EV29-PAP-H1S1质粒(纯化后) |
386.7ng/μl |
pUC57-EV29-PAP-H1S1线性化质粒(纯化后) |
294.5ng/μl |
EV29-PAP--H1S1线状mRNA(纯化后) |
317.2ng/μl |
EV29-PAP-H1S1环状mRNA(纯化后) |
268.9ng/μl |
PAP ELISA检测,EPO环状mRNA在293T表达1-5天所得蛋白的量分别为69.3,86.4,75.5,52.4,38.6ng/ml,说明以本公开的环状mRNA可实现EPO的高效、持久性表达。
实施例8:环状mRNA编码嵌合抗原受体CD16 CAR蛋白实现在293T细胞的表达
8.1实验方法和步骤
在上述实施例2的基础上,以EV29 IRES,5’同源臂1、3’同源臂1,5’间隔区1、3’间隔区1作为环状mRNA的基础元件,构建编嵌合抗原受体CD16 CAR的环状mRNA。编码CD16 CAR的DNA和蛋白序列分别如SEQ ID NO:49和SEQ ID NO:50所示。DNA合成委托苏州金唯智生物科技有限公司完成。最终将含有T7启动子,I类PIE元件,5’同源臂1、3’同源臂1,5’间隔区1、3’间隔区1,EV29 IRES,CD16 CAR编码区的完整DNA片段克隆到pUC57质粒。质粒DNA线性化,线状mRNA体外转录,线状mRNA纯化,mRNA的成环反应,环状mRNA的纯化,得到序列如SEQ IDNO:51所示的环状RNA,细胞培养与转染等方法均与实施例1的1.1相同。采用抗CD16单克隆抗体介导的流式细胞术(Thermo Fisher)对293T表达的CD16 CAR蛋白进行表达效率测试。
8.2实验结果
表14
核酸名称 |
浓度 |
pUC57-EV29-CD16CAR-H1S1质粒(纯化后) |
268.5ng/μl |
pUC57-EV29-CD16CAR-H1S1线性化质粒(纯化后) |
221.7ng/μl |
EV29-CD16CAR--H1S1线状mRNA(纯化后) |
375.4ng/μl |
EV29-CD16CAR-H1S1环状mRNA(纯化后) |
284.3ng/μl |
通过采用抗CD16抗体检测环状mRNA介导的CD16CAR在293T的表达阳性率。结果现实,在环状mRNA转染1-5天,CD16 CAR表达的阳性率分别为90.4%,85.6%,80.3%,78.4%以及60.5%,说明以本公开的环状mRNA可实现CD16抗体的高效、持久性表达。
本公开的上述实施例仅是为清楚地说明本公开所作的举例,而并非是对本公开的实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。凡在本公开的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本公开权利要求的保护范围之内。
序列表
<110> 江苏普瑞康生物医药科技有限公司
<120> 一种转录环状RNA的重组核酸分子及其在蛋白表达中的应用
<130> 6A23-2018183I
<141> 2020-12-04
<160> 53
<170> SIPOSequenceListing 1.0
<210> 1
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 1
taatacgact cactatagg 19
<210> 2
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 2
accgtcagtt gctcactgtg c 21
<210> 3
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 3
accgtgctat gtccacgtgt c 21
<210> 4
<211> 131
<212> DNA
<213> Artificial Sequence
<400> 4
aacaatagat gacttacaac taatcggaag gtgcagagac tcgacgggag ctaccctaac 60
gtcaagacga gggtaaagag agagtccaat tctcaaagcc aataggcagt agcgaaagct 120
gcaagagaat g 131
<210> 5
<211> 51
<212> DNA
<213> Artificial Sequence
<400> 5
aaaatccgtt gaccttaaac ggtcgtgtgg gttcaagtcc ctccaccccc a 51
<210> 6
<211> 50
<212> DNA
<213> Artificial Sequence
<400> 6
aaaaaacaaa aacaaaaaaa acaaaaaaac aaaaaaaaaa ccaaaacaca 50
<210> 7
<211> 50
<212> DNA
<213> Artificial Sequence
<400> 7
aaaaacaaaa aacaaaaaaa aaaccaaaaa aacaaaaaaa acaaaacaca 50
<210> 8
<211> 741
<212> DNA
<213> Artificial Sequence
<400> 8
ttaaaacagc ctgtgggttg atcccaccca caggcccatt gggcgctagc actctggtat 60
cacggtacct ttgtgcgcct gttttatacc ccctccccca actgtaactt agaagtaaca 120
cacaccgatc aacagtcagc gtggcacacc agccacgttt tgatcaagca cttctgttac 180
cccggactga gtatcaatag actgctcacg cggttgaagg agaaagcgtt cgttatccgg 240
ccaactactt cgaaaaacct agtaacaccg tggaagttgc agagtgtttc gctcagcact 300
accccagtgt agatcaggtc gatgagtcac cgcattcccc acgggcgacc gtggcggtgg 360
ctgcgttggc ggcctgccca tggggaaacc catgggacgc tctaatacag acatggtgcg 420
aagagtctat tgagctagtt ggtagtcctc cggcccctga atgcggctaa tcctaactgc 480
ggagcacaca ccctcaagcc agagggcagt gtgtcgtaac gggcaactct gcagcggaac 540
cgactacttt gggtgtccgt gtttcatttt attcctatac tggctgctta tggtgacaat 600
tgagagatcg ttaccatata gctattggat tggccatccg gtgactaata gagctattat 660
atatcccttt gttgggttta taccacttag cttgaaagag gttaaaacat tacaattcat 720
tgttaagttg aatacagcaa a 741
<210> 9
<211> 682
<212> DNA
<213> Artificial Sequence
<400> 9
ttaaaacagc ctgtgggttg cacccaccca cagggcccac agggcgctag cactctggta 60
tcacggtacc tttgtgcgcc tgttttatta ccccttcccc aattgaaaat tagaagcaat 120
gcacaccgat caacagcagg cgtggcgcac cagtcacgtc tcgatcaagc acttctgttt 180
ccccggaccg agtatcaata gactgctcac gcggttgaag gagaaagtgt tcgttatccg 240
gctaaccact tcgagaaacc cagtaacacc atgaaagttg cagggtgttt cgctcagcac 300
ttccccagtg tagatcaggt cgatgagtca ccgcgttccc cacgggcgac cgtggcggtg 360
gctgcgttgg cggcctgcct atgggttaac ccataggacg ctctaataca gacatggtgc 420
gaagagttta ttgagctggt tagtatccct ccggcccctg aatgcggcta atcctaactg 480
cggagcacgt gcctccaatc cagggggttg catgtcgtaa cgggtaactc tgcagcggaa 540
ccgactactt tgggtgtccg tgtttccttt tattcttata ctggctgctt atggtgacaa 600
tcgaggaatt gttaccatat agctattgga ttggccatcc ggtgtctaac agagcgatta 660
tatacctctt tgttggattt at 682
<210> 10
<211> 742
<212> DNA
<213> Artificial Sequence
<400> 10
ttaaaacagc ctgtgggttg atcccaccca cagggcccac tgggcgctag cactctggta 60
tcacggtacc tttgtgcgcc tgttttatac ttcctccccc aactgcaact tagaagtaac 120
acaaaccgat caacagtcag cgtggcacac cagccacgtt ttgatcaaac acttctgtta 180
ccccggactg agtatcaata gactgctcac gcggttgaag gagaaaacgt tcgttatccg 240
gccaactact tcgagaaacc tagtaacgcc atggaagttg tggagtgttt cgctcagcac 300
taccccagtg tagatcaggt tgatgagtca ccgcattccc cacgggtgac cgtggcggtg 360
gctgcgttgg cggcctgccc atggggaaac ccatgggacg ctcttataca gacatggtgc 420
gaagagtcta ttgagctagt tggtagtcct ccggcccctg aatgcggcta atcccaactg 480
cggagcatac actctcaagc cagagggtag tgtgtcgtaa tgggcaactc tgcagcggaa 540
ccgactactt tgggtgtccg tgtttcattt tattcctata ctggctgctt atggtgacaa 600
ttgagagatt gttaccatat agctattgga ttggccatcc ggtgactaac agagctatta 660
tatatctttt tgttgggttt ataccactta gcttgaaaga ggttaaaact ctacattaca 720
ttttaatact gaacaccgca aa 742
<210> 11
<211> 737
<212> DNA
<213> Artificial Sequence
<400> 11
ttaaaacagc ctgtgggttg atcccaccca cagggcccat tgggcgctag cactctggta 60
tcacggtacc cttgtgcgcc tgttttatgt cccttccctc aactgtaact tagaagtaac 120
gcacaccgat caacagtcag cgtggcacac cagccatgtt ttgatcaagc acttctgtta 180
ccccggaccg agtatcaaca gactgctcac gcggttgaag gagaaagtgt tcgttatccg 240
gccaactact tcgaaaaacc tagtaacacc atggaagttg cagagtgttt cgctcagcac 300
taccccagtg tagatcaggt cgatgagtca ccgcatcccc cacgggcgac cgtggcggtg 360
gctgcgttgg cggcctgcct atgggggaac ccataggacg ctctaataca gacatggtgc 420
gaagagtcca ttgagctagt tggtagtcct ccggcccctg aatgcggcta atcctaactg 480
cggagcacac accttcaagc cagagggcag tgtgtcgtaa cgggcaactc tgcagcggaa 540
ccgactactt tgggtgtccg tgtttcattt tattcttata ctggctgctt atggtgacaa 600
ttgagagatt gttaccatat agctattgga ttggccatcc agtgactagc agagctatta 660
tatacctctt tgttgggttt ataccaccta atttgaaaga agttaaaaca ttagaattca 720
ttattaaatt gaataca 737
<210> 12
<211> 683
<212> DNA
<213> Artificial Sequence
<400> 12
ttaaaacagc ctgtgggttg cacccaccca cagggcccac agggcgctag cactctggta 60
tcacggtacc tttgtgcgcc tgttttatta ccccttcccc aattgaaaat tagaagcaat 120
gcacaccgat caacagcagg cgtggcgcac cagtcacgtc tcgatcaagc acttctgttt 180
ccccggaccg agtatcaata gactgctcac gcggttgaag gagaaagtgt tcgttatccg 240
gctaaccact tcgagaaacc cagtaacacc atgaaagttg cagggtgttt cgctcagcac 300
ttccccagtg tagatcaggt cgatgagtca ccgcgttccc cacgggcgac cgtggcggtg 360
gctgcgttgg cggcctgcct atgggttaac ccataggacg ctctaataca gacatggtgc 420
gaagagttta ttgagctggt tagtatctcc tccggcccct gaatgcggct aatcctaact 480
gcggagcaca caccctcaag ccagagggca gtgtgtcgta acgggcaact ctgcagcgga 540
accgactact ttgggtgtcc gtgtttcctt ttattcttat actggctgct tatggtgaca 600
atcgaggaat tgttaccata tagctattgg attggccatc cggtgtctaa cagagcgatt 660
atatacctct ttgttggatt tat 683
<210> 13
<211> 742
<212> DNA
<213> Artificial Sequence
<400> 13
ttaaaacagc ctgtgggttg atcccaccca cagggcccac tgggcgctag cactctggta 60
tcacggtacc tttgtgcgcc tgttttatac ttcctccccc aactgcaact tagaagtaac 120
acaaaccgat caacagtcag cgtggcacac cagccacgtt ttgatcaaac acttctgtta 180
ccccggactg agtatcaata gactgctcac gcggttgaag gagaaaacgt tcgttatccg 240
gccaactact tcgagaaacc tagtaacgcc atggaagttg tggagtgttt cgctcagcac 300
taccccagtg tagatcaggt tgatgagtca ccgcattccc cacgggtgac cgtggcggtg 360
gctgcgttgg cggcctgccc atggggaaac ccatgggacg ctcttataca gacatggtgc 420
gaagagtcta ttgagctagt tggtagtcct ccggcccctg aatgcggcta atcctaactg 480
cggagcacac accctcaagc cagagggcag tgtgtcgtaa cgggcaactc tgcagcggaa 540
ccgactactt tgggtgtccg tgtttcattt tattcctata ctggctgctt atggtgacaa 600
ttgagagatt gttaccatat agctattgga ttggccatcc ggtgactaac agagctatta 660
tatatctttt tgttgggttt ataccactta gcttgaaaga ggttaaaact ctacattaca 720
ttttaatact gaacaccgca aa 742
<210> 14
<211> 737
<212> DNA
<213> Artificial Sequence
<400> 14
ttaaaacagc ctgtgggttg atcccaccca cagggcccat tgggcgctag cactctggta 60
tcacggtacc cttgtgcgcc tgttttatgt cccttccctc aactgtaact tagaagtaac 120
gcacaccgat caacagtcag cgtggcacac cagccatgtt ttgatcaagc acttctgtta 180
ccccggaccg agtatcaaca gactgctcac gcggttgaag gagaaagtgt tcgttatccg 240
gccaactact tcgaaaaacc tagtaacacc atggaagttg cagagtgttt cgctcagcac 300
taccccagtg tagatcaggt cgatgagtca ccgcatcccc cacgggcgac cgtggcggtg 360
gctgcgttgg cggcctgcct atgggggaac ccataggacg ctctaataca gacatggtgc 420
gaagagtcca ttgagctagt tggtagtcct ccggcccctg aatgcggcta atcctaactg 480
cggagcacac accctcaagc cagagggcag tgtgtcgtaa cgggcaactc tgcagcggaa 540
ccgactactt tgggtgtccg tgtttcattt tattcttata ctggctgctt atggtgacaa 600
ttgagagatt gttaccatat agctattgga ttggccatcc agtgactagc agagctatta 660
tatacctctt tgttgggttt ataccaccta atttgaaaga agttaaaaca ttagaattca 720
ttattaaatt gaataca 737
<210> 15
<211> 16
<212> DNA
<213> Artificial Sequence
<400> 15
agacgctacg gactta 16
<210> 16
<211> 114
<212> DNA
<213> Artificial Sequence
<400> 16
aataattgag ccttaaagaa gaaattcttt aagtggatgc tctcaaactc agggaaacct 60
aaatctagtt atagacaagg caatcctgag ccaagccgaa gtagtaatta gtaa 114
<210> 17
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 17
gcacagtgag caactgacgg a 21
<210> 18
<211> 21
<212> DNA
<213> Artificial Sequence
<400> 18
gacacgtgga catagcacgg a 21
<210> 19
<211> 6
<212> DNA
<213> Artificial Sequence
<400> 19
tctaga 6
<210> 20
<211> 720
<212> DNA
<213> Artificial Sequence
<400> 20
atggtgtcaa agggtgagga attattcacc ggcgtggtgc ctatccttgt ggaacttgat 60
ggagatgtga acggacacaa attcagtgta tcaggagaag gagaaggaga tgcaacatac 120
ggaaagctca ctcttaaatt tatctgcaca acaggaaagc tcccggtgcc ttggcctaca 180
cttgtgacaa cacttacata cggagtgcaa tgcttctcgc gttaccctga tcacatgaaa 240
caacacgatt tcttcaagag tgcaatgcct gaaggatacg tgcaagaaag aacaatcttc 300
ttcaaggacg atggaaacta caagactcgt gcagaagtga aatttgaagg agatacactt 360
gtgaacagaa tcgaacttaa aggaatcgat ttcaaggagg atggaaacat ccttggacac 420
aaacttgaat acaactacaa ctcacacaac gtgtacatca tggcagataa acagaagaat 480
ggtatcaaag tgaactttaa gattcgccac aacatcgaag atggatcagt gcaacttgca 540
gatcactacc aacagaatac gccgatagga gatggacctg tgcttcttcc tgataaccac 600
tacctttcaa cacaatcagc actttcaaag gacccaaacg agaagcgaga ccacatggtg 660
cttcttgaat ttgtgacagc agcaggaatc acacttggaa tggatgaact ttacaaatga 720
<210> 21
<211> 239
<212> PRT
<213> Artificial Sequence
<400> 21
Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
1 5 10 15
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
20 25 30
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile
35 40 45
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
50 55 60
Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys
65 70 75 80
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
85 90 95
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
100 105 110
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
115 120 125
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
130 135 140
Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn
145 150 155 160
Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser
165 170 175
Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly
180 185 190
Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu
195 200 205
Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe
210 215 220
Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys
225 230 235
<210> 22
<211> 1638
<212> RNA
<213> Artificial Sequence
<400> 22
aaaauccguu gaccuuaaac ggucgugugg guucaagucc cuccaccccc acgccggaaa 60
cgcaauagcc gaaaaacaaa aaacaaaaaa aacaaaaaaa aaaccaaaaa aacaaaacac 120
auuaaaacag ccuguggguu gaucccaccc acaggcccau ugggcgcuag cacucuggua 180
ucacgguacc uuugugcgcc uguuuuauac ccccuccccc aacuguaacu uagaaguaac 240
acacaccgau caacagucag cguggcacac cagccacguu uugaucaagc acuucuguua 300
ccccggacug aguaucaaua gacugcucac gcgguugaag gagaaagcgu ucguuauccg 360
gccaacuacu ucgaaaaacc uaguaacacc guggaaguug cagaguguuu cgcucagcac 420
uaccccagug uagaucaggu cgaugaguca ccgcauuccc cacgggcgac cguggcggug 480
gcugcguugg cggccugccc auggggaaac ccaugggacg cucuaauaca gacauggugc 540
gaagagucua uugagcuagu ugguaguccu ccggccccug aaugcggcua auccuaacug 600
cggagcacac acccucaagc cagagggcag ugugucguaa cgggcaacuc ugcagcggaa 660
ccgacuacuu uggguguccg uguuucauuu uauuccuaua cuggcugcuu auggugacaa 720
uugagagauc guuaccauau agcuauugga uuggccaucc ggugacuaau agagcuauua 780
uauaucccuu uguuggguuu auaccacuua gcuugaaaga gguuaaaaca uuacaauuca 840
uuguuaaguu gaauacagca aaaugguguc aaagggugag gaauuauuca ccggcguggu 900
gccuauccuu guggaacuug auggagaugu gaacggacac aaauucagug uaucaggaga 960
aggagaagga gaugcaacau acggaaagcu cacucuuaaa uuuaucugca caacaggaaa 1020
gcucccggug ccuuggccua cacuugugac aacacuuaca uacggagugc aaugcuucuc 1080
gcguuacccu gaucacauga aacaacacga uuucuucaag agugcaaugc cugaaggaua 1140
cgugcaagaa agaacaaucu ucuucaagga cgauggaaac uacaagacuc gugcagaagu 1200
gaaauuugaa ggagauacac uugugaacag aaucgaacuu aaaggaaucg auuucaagga 1260
ggauggaaac auccuuggac acaaacuuga auacaacuac aacucacaca acguguacau 1320
cauggcagau aaacagaaga augguaucaa agugaacuuu aagauucgcc acaacaucga 1380
agauggauca gugcaacuug cagaucacua ccaacagaau acgccgauag gagauggacc 1440
ugugcuucuu ccugauaacc acuaccuuuc aacacaauca gcacuuucaa aggacccaaa 1500
cgagaagcga gaccacaugg ugcuucuuga auuugugaca gcagcaggaa ucacacuugg 1560
aauggaugaa cuuuacaaau gaaaaaaaca aaaaacaaaa cggcuauuau gcguuaccgg 1620
cgagacgcua cggacuua 1638
<210> 23
<211> 1579
<212> RNA
<213> Artificial Sequence
<400> 23
aaaauccguu gaccuuaaac ggucgugugg guucaagucc cuccaccccc acgccggaaa 60
cgcaauagcc gaaaaacaaa aaacaaaaaa aacaaaaaaa aaaccaaaaa aacaaaacac 120
auuaaaacag ccuguggguu gcacccaccc acagggccca cagggcgcua gcacucuggu 180
aucacgguac cuuugugcgc cuguuuuauu accccuuccc caauugaaaa uuagaagcaa 240
ugcacaccga ucaacagcag gcguggcgca ccagucacgu cucgaucaag cacuucuguu 300
uccccggacc gaguaucaau agacugcuca cgcgguugaa ggagaaagug uucguuaucc 360
ggcuaaccac uucgagaaac ccaguaacac caugaaaguu gcaggguguu ucgcucagca 420
cuuccccagu guagaucagg ucgaugaguc accgcguucc ccacgggcga ccguggcggu 480
ggcugcguug gcggccugcc uauggguuaa cccauaggac gcucuaauac agacauggug 540
cgaagaguuu auugagcugg uuaguauccc uccggccccu gaaugcggcu aauccuaacu 600
gcggagcacg ugccuccaau ccaggggguu gcaugucgua acggguaacu cugcagcgga 660
accgacuacu uugggugucc guguuuccuu uuauucuuau acuggcugcu uauggugaca 720
aucgaggaau uguuaccaua uagcuauugg auuggccauc cggugucuaa cagagcgauu 780
auauaccucu uuguuggauu uauauggugu caaaggguga ggaauuauuc accggcgugg 840
ugccuauccu uguggaacuu gauggagaug ugaacggaca caaauucagu guaucaggag 900
aaggagaagg agaugcaaca uacggaaagc ucacucuuaa auuuaucugc acaacaggaa 960
agcucccggu gccuuggccu acacuuguga caacacuuac auacggagug caaugcuucu 1020
cgcguuaccc ugaucacaug aaacaacacg auuucuucaa gagugcaaug ccugaaggau 1080
acgugcaaga aagaacaauc uucuucaagg acgauggaaa cuacaagacu cgugcagaag 1140
ugaaauuuga aggagauaca cuugugaaca gaaucgaacu uaaaggaauc gauuucaagg 1200
aggauggaaa cauccuugga cacaaacuug aauacaacua caacucacac aacguguaca 1260
ucauggcaga uaaacagaag aaugguauca aagugaacuu uaagauucgc cacaacaucg 1320
aagauggauc agugcaacuu gcagaucacu accaacagaa uacgccgaua ggagauggac 1380
cugugcuucu uccugauaac cacuaccuuu caacacaauc agcacuuuca aaggacccaa 1440
acgagaagcg agaccacaug gugcuucuug aauuugugac agcagcagga aucacacuug 1500
gaauggauga acuuuacaaa ugaaaaaaac aaaaaacaaa acggcuauua ugcguuaccg 1560
gcgagacgcu acggacuua 1579
<210> 24
<211> 1580
<212> RNA
<213> Artificial Sequence
<400> 24
aaaauccguu gaccuuaaac ggucgugugg guucaagucc cuccaccccc acgccggaaa 60
cgcaauagcc gaaaaacaaa aaacaaaaaa aacaaaaaaa aaaccaaaaa aacaaaacac 120
auuaaaacag ccuguggguu gcacccaccc acagggccca cagggcgcua gcacucuggu 180
aucacgguac cuuugugcgc cuguuuuauu accccuuccc caauugaaaa uuagaagcaa 240
ugcacaccga ucaacagcag gcguggcgca ccagucacgu cucgaucaag cacuucuguu 300
uccccggacc gaguaucaau agacugcuca cgcgguugaa ggagaaagug uucguuaucc 360
ggcuaaccac uucgagaaac ccaguaacac caugaaaguu gcaggguguu ucgcucagca 420
cuuccccagu guagaucagg ucgaugaguc accgcguucc ccacgggcga ccguggcggu 480
ggcugcguug gcggccugcc uauggguuaa cccauaggac gcucuaauac agacauggug 540
cgaagaguuu auugagcugg uuaguaucuc cuccggcccc ugaaugcggc uaauccuaac 600
ugcggagcac acacccucaa gccagagggc agugugucgu aacgggcaac ucugcagcgg 660
aaccgacuac uuuggguguc cguguuuccu uuuauucuua uacuggcugc uuauggugac 720
aaucgaggaa uuguuaccau auagcuauug gauuggccau ccggugucua acagagcgau 780
uauauaccuc uuuguuggau uuauauggug ucaaagggug aggaauuauu caccggcgug 840
gugccuaucc uuguggaacu ugauggagau gugaacggac acaaauucag uguaucagga 900
gaaggagaag gagaugcaac auacggaaag cucacucuua aauuuaucug cacaacagga 960
aagcucccgg ugccuuggcc uacacuugug acaacacuua cauacggagu gcaaugcuuc 1020
ucgcguuacc cugaucacau gaaacaacac gauuucuuca agagugcaau gccugaagga 1080
uacgugcaag aaagaacaau cuucuucaag gacgauggaa acuacaagac ucgugcagaa 1140
gugaaauuug aaggagauac acuugugaac agaaucgaac uuaaaggaau cgauuucaag 1200
gaggauggaa acauccuugg acacaaacuu gaauacaacu acaacucaca caacguguac 1260
aucauggcag auaaacagaa gaaugguauc aaagugaacu uuaagauucg ccacaacauc 1320
gaagauggau cagugcaacu ugcagaucac uaccaacaga auacgccgau aggagaugga 1380
ccugugcuuc uuccugauaa ccacuaccuu ucaacacaau cagcacuuuc aaaggaccca 1440
aacgagaagc gagaccacau ggugcuucuu gaauuuguga cagcagcagg aaucacacuu 1500
ggaauggaug aacuuuacaa augaaaaaaa caaaaaacaa aacggcuauu augcguuacc 1560
ggcgagacgc uacggacuua 1580
<210> 25
<211> 1639
<212> RNA
<213> Artificial Sequence
<400> 25
aaaauccguu gaccuuaaac ggucgugugg guucaagucc cuccaccccc acgccggaaa 60
cgcaauagcc gaaaaacaaa aaacaaaaaa aacaaaaaaa aaaccaaaaa aacaaaacac 120
auuaaaacag ccuguggguu gaucccaccc acagggccca cugggcgcua gcacucuggu 180
aucacgguac cuuugugcgc cuguuuuaua cuuccucccc caacugcaac uuagaaguaa 240
cacaaaccga ucaacaguca gcguggcaca ccagccacgu uuugaucaaa cacuucuguu 300
accccggacu gaguaucaau agacugcuca cgcgguugaa ggagaaaacg uucguuaucc 360
ggccaacuac uucgagaaac cuaguaacgc cauggaaguu guggaguguu ucgcucagca 420
cuaccccagu guagaucagg uugaugaguc accgcauucc ccacggguga ccguggcggu 480
ggcugcguug gcggccugcc cauggggaaa cccaugggac gcucuuauac agacauggug 540
cgaagagucu auugagcuag uugguagucc uccggccccu gaaugcggcu aaucccaacu 600
gcggagcaua cacucucaag ccagagggua gugugucgua augggcaacu cugcagcgga 660
accgacuacu uugggugucc guguuucauu uuauuccuau acuggcugcu uauggugaca 720
auugagagau uguuaccaua uagcuauugg auuggccauc cggugacuaa cagagcuauu 780
auauaucuuu uuguuggguu uauaccacuu agcuugaaag agguuaaaac ucuacauuac 840
auuuuaauac ugaacaccgc aaaauggugu caaaggguga ggaauuauuc accggcgugg 900
ugccuauccu uguggaacuu gauggagaug ugaacggaca caaauucagu guaucaggag 960
aaggagaagg agaugcaaca uacggaaagc ucacucuuaa auuuaucugc acaacaggaa 1020
agcucccggu gccuuggccu acacuuguga caacacuuac auacggagug caaugcuucu 1080
cgcguuaccc ugaucacaug aaacaacacg auuucuucaa gagugcaaug ccugaaggau 1140
acgugcaaga aagaacaauc uucuucaagg acgauggaaa cuacaagacu cgugcagaag 1200
ugaaauuuga aggagauaca cuugugaaca gaaucgaacu uaaaggaauc gauuucaagg 1260
aggauggaaa cauccuugga cacaaacuug aauacaacua caacucacac aacguguaca 1320
ucauggcaga uaaacagaag aaugguauca aagugaacuu uaagauucgc cacaacaucg 1380
aagauggauc agugcaacuu gcagaucacu accaacagaa uacgccgaua ggagauggac 1440
cugugcuucu uccugauaac cacuaccuuu caacacaauc agcacuuuca aaggacccaa 1500
acgagaagcg agaccacaug gugcuucuug aauuugugac agcagcagga aucacacuug 1560
gaauggauga acuuuacaaa ugaaaaaaac aaaaaacaaa acggcuauua ugcguuaccg 1620
gcgagacgcu acggacuua 1639
<210> 26
<211> 1639
<212> RNA
<213> Artificial Sequence
<400> 26
aaaauccguu gaccuuaaac ggucgugugg guucaagucc cuccaccccc acgccggaaa 60
cgcaauagcc gaaaaacaaa aaacaaaaaa aacaaaaaaa aaaccaaaaa aacaaaacac 120
auuaaaacag ccuguggguu gaucccaccc acagggccca cugggcgcua gcacucuggu 180
aucacgguac cuuugugcgc cuguuuuaua cuuccucccc caacugcaac uuagaaguaa 240
cacaaaccga ucaacaguca gcguggcaca ccagccacgu uuugaucaaa cacuucuguu 300
accccggacu gaguaucaau agacugcuca cgcgguugaa ggagaaaacg uucguuaucc 360
ggccaacuac uucgagaaac cuaguaacgc cauggaaguu guggaguguu ucgcucagca 420
cuaccccagu guagaucagg uugaugaguc accgcauucc ccacggguga ccguggcggu 480
ggcugcguug gcggccugcc cauggggaaa cccaugggac gcucuuauac agacauggug 540
cgaagagucu auugagcuag uugguagucc uccggccccu gaaugcggcu aauccuaacu 600
gcggagcaca cacccucaag ccagagggca gugugucgua acgggcaacu cugcagcgga 660
accgacuacu uugggugucc guguuucauu uuauuccuau acuggcugcu uauggugaca 720
auugagagau uguuaccaua uagcuauugg auuggccauc cggugacuaa cagagcuauu 780
auauaucuuu uuguuggguu uauaccacuu agcuugaaag agguuaaaac ucuacauuac 840
auuuuaauac ugaacaccgc aaaauggugu caaaggguga ggaauuauuc accggcgugg 900
ugccuauccu uguggaacuu gauggagaug ugaacggaca caaauucagu guaucaggag 960
aaggagaagg agaugcaaca uacggaaagc ucacucuuaa auuuaucugc acaacaggaa 1020
agcucccggu gccuuggccu acacuuguga caacacuuac auacggagug caaugcuucu 1080
cgcguuaccc ugaucacaug aaacaacacg auuucuucaa gagugcaaug ccugaaggau 1140
acgugcaaga aagaacaauc uucuucaagg acgauggaaa cuacaagacu cgugcagaag 1200
ugaaauuuga aggagauaca cuugugaaca gaaucgaacu uaaaggaauc gauuucaagg 1260
aggauggaaa cauccuugga cacaaacuug aauacaacua caacucacac aacguguaca 1320
ucauggcaga uaaacagaag aaugguauca aagugaacuu uaagauucgc cacaacaucg 1380
aagauggauc agugcaacuu gcagaucacu accaacagaa uacgccgaua ggagauggac 1440
cugugcuucu uccugauaac cacuaccuuu caacacaauc agcacuuuca aaggacccaa 1500
acgagaagcg agaccacaug gugcuucuug aauuugugac agcagcagga aucacacuug 1560
gaauggauga acuuuacaaa ugaaaaaaac aaaaaacaaa acggcuauua ugcguuaccg 1620
gcgagacgcu acggacuua 1639
<210> 27
<211> 1634
<212> RNA
<213> Artificial Sequence
<400> 27
aaaauccguu gaccuuaaac ggucgugugg guucaagucc cuccaccccc acgccggaaa 60
cgcaauagcc gaaaaacaaa aaacaaaaaa aacaaaaaaa aaaccaaaaa aacaaaacac 120
auuaaaacag ccuguggguu gaucccaccc acagggccca uugggcgcua gcacucuggu 180
aucacgguac ccuugugcgc cuguuuuaug ucccuucccu caacuguaac uuagaaguaa 240
cgcacaccga ucaacaguca gcguggcaca ccagccaugu uuugaucaag cacuucuguu 300
accccggacc gaguaucaac agacugcuca cgcgguugaa ggagaaagug uucguuaucc 360
ggccaacuac uucgaaaaac cuaguaacac cauggaaguu gcagaguguu ucgcucagca 420
cuaccccagu guagaucagg ucgaugaguc accgcauccc ccacgggcga ccguggcggu 480
ggcugcguug gcggccugcc uaugggggaa cccauaggac gcucuaauac agacauggug 540
cgaagagucc auugagcuag uugguagucc uccggccccu gaaugcggcu aauccuaacu 600
gcggagcaca caccuucaag ccagagggca gugugucgua acgggcaacu cugcagcgga 660
accgacuacu uugggugucc guguuucauu uuauucuuau acuggcugcu uauggugaca 720
auugagagau uguuaccaua uagcuauugg auuggccauc cagugacuag cagagcuauu 780
auauaccucu uuguuggguu uauaccaccu aauuugaaag aaguuaaaac auuagaauuc 840
auuauuaaau ugaauacaau ggugucaaag ggugaggaau uauucaccgg cguggugccu 900
auccuugugg aacuugaugg agaugugaac ggacacaaau ucaguguauc aggagaagga 960
gaaggagaug caacauacgg aaagcucacu cuuaaauuua ucugcacaac aggaaagcuc 1020
ccggugccuu ggccuacacu ugugacaaca cuuacauacg gagugcaaug cuucucgcgu 1080
uacccugauc acaugaaaca acacgauuuc uucaagagug caaugccuga aggauacgug 1140
caagaaagaa caaucuucuu caaggacgau ggaaacuaca agacucgugc agaagugaaa 1200
uuugaaggag auacacuugu gaacagaauc gaacuuaaag gaaucgauuu caaggaggau 1260
ggaaacaucc uuggacacaa acuugaauac aacuacaacu cacacaacgu guacaucaug 1320
gcagauaaac agaagaaugg uaucaaagug aacuuuaaga uucgccacaa caucgaagau 1380
ggaucagugc aacuugcaga ucacuaccaa cagaauacgc cgauaggaga uggaccugug 1440
cuucuuccug auaaccacua ccuuucaaca caaucagcac uuucaaagga cccaaacgag 1500
aagcgagacc acauggugcu ucuugaauuu gugacagcag caggaaucac acuuggaaug 1560
gaugaacuuu acaaaugaaa aaaacaaaaa acaaaacggc uauuaugcgu uaccggcgag 1620
acgcuacgga cuua 1634
<210> 28
<211> 1634
<212> RNA
<213> Artificial Sequence
<400> 28
aaaauccguu gaccuuaaac ggucgugugg guucaagucc cuccaccccc acgccggaaa 60
cgcaauagcc gaaaaacaaa aaacaaaaaa aacaaaaaaa aaaccaaaaa aacaaaacac 120
auuaaaacag ccuguggguu gaucccaccc acagggccca uugggcgcua gcacucuggu 180
aucacgguac ccuugugcgc cuguuuuaug ucccuucccu caacuguaac uuagaaguaa 240
cgcacaccga ucaacaguca gcguggcaca ccagccaugu uuugaucaag cacuucuguu 300
accccggacc gaguaucaac agacugcuca cgcgguugaa ggagaaagug uucguuaucc 360
ggccaacuac uucgaaaaac cuaguaacac cauggaaguu gcagaguguu ucgcucagca 420
cuaccccagu guagaucagg ucgaugaguc accgcauccc ccacgggcga ccguggcggu 480
ggcugcguug gcggccugcc uaugggggaa cccauaggac gcucuaauac agacauggug 540
cgaagagucc auugagcuag uugguagucc uccggccccu gaaugcggcu aauccuaacu 600
gcggagcaca cacccucaag ccagagggca gugugucgua acgggcaacu cugcagcgga 660
accgacuacu uugggugucc guguuucauu uuauucuuau acuggcugcu uauggugaca 720
auugagagau uguuaccaua uagcuauugg auuggccauc cagugacuag cagagcuauu 780
auauaccucu uuguuggguu uauaccaccu aauuugaaag aaguuaaaac auuagaauuc 840
auuauuaaau ugaauacaau ggugucaaag ggugaggaau uauucaccgg cguggugccu 900
auccuugugg aacuugaugg agaugugaac ggacacaaau ucaguguauc aggagaagga 960
gaaggagaug caacauacgg aaagcucacu cuuaaauuua ucugcacaac aggaaagcuc 1020
ccggugccuu ggccuacacu ugugacaaca cuuacauacg gagugcaaug cuucucgcgu 1080
uacccugauc acaugaaaca acacgauuuc uucaagagug caaugccuga aggauacgug 1140
caagaaagaa caaucuucuu caaggacgau ggaaacuaca agacucgugc agaagugaaa 1200
uuugaaggag auacacuugu gaacagaauc gaacuuaaag gaaucgauuu caaggaggau 1260
ggaaacaucc uuggacacaa acuugaauac aacuacaacu cacacaacgu guacaucaug 1320
gcagauaaac agaagaaugg uaucaaagug aacuuuaaga uucgccacaa caucgaagau 1380
ggaucagugc aacuugcaga ucacuaccaa cagaauacgc cgauaggaga uggaccugug 1440
cuucuuccug auaaccacua ccuuucaaca caaucagcac uuucaaagga cccaaacgag 1500
aagcgagacc acauggugcu ucuugaauuu gugacagcag caggaaucac acuuggaaug 1560
gaugaacuuu acaaaugaaa aaaacaaaaa acaaaacggc uauuaugcgu uaccggcgag 1620
acgcuacgga cuua 1634
<210> 29
<211> 1645
<212> RNA
<213> Artificial Sequence
<400> 29
aaaauccguu gaccuuaaac ggucgugugg guucaagucc cuccaccccc acgccggaaa 60
cgcaauagcc gaaaaaacaa aaacaaaaaa aacaaaaaaa caaaaaaaaa accaaaacac 120
auuaaaacag ccuguggguu gaucccaccc acagggccca cugggcgcua gcacucuggu 180
aucacgguac cuuugugcgc cuguuuuaua cuuccucccc caacugcaac uuagaaguaa 240
cacaaaccga ucaacaguca gcguggcaca ccagccacgu uuugaucaaa cacuucuguu 300
accccggacu gaguaucaau agacugcuca cgcgguugaa ggagaaaacg uucguuaucc 360
ggccaacuac uucgagaaac cuaguaacgc cauggaaguu guggaguguu ucgcucagca 420
cuaccccagu guagaucagg uugaugaguc accgcauucc ccacggguga ccguggcggu 480
ggcugcguug gcggccugcc cauggggaaa cccaugggac gcucuuauac agacauggug 540
cgaagagucu auugagcuag uugguagucc uccggccccu gaaugcggcu aaucccaacu 600
gcggagcaua cacucucaag ccagagggua gugugucgua augggcaacu cugcagcgga 660
accgacuacu uugggugucc guguuucauu uuauuccuau acuggcugcu uauggugaca 720
auugagagau uguuaccaua uagcuauugg auuggccauc cggugacuaa cagagcuauu 780
auauaucuuu uuguuggguu uauaccacuu agcuugaaag agguuaaaac ucuacauuac 840
auuuuaauac ugaacaccgc aaaauggugu caaaggguga ggaauuauuc accggcgugg 900
ugccuauccu uguggaacuu gauggagaug ugaacggaca caaauucagu guaucaggag 960
aaggagaagg agaugcaaca uacggaaagc ucacucuuaa auuuaucugc acaacaggaa 1020
agcucccggu gccuuggccu acacuuguga caacacuuac auacggagug caaugcuucu 1080
cgcguuaccc ugaucacaug aaacaacacg auuucuucaa gagugcaaug ccugaaggau 1140
acgugcaaga aagaacaauc uucuucaagg acgauggaaa cuacaagacu cgugcagaag 1200
ugaaauuuga aggagauaca cuugugaaca gaaucgaacu uaaaggaauc gauuucaagg 1260
aggauggaaa cauccuugga cacaaacuug aauacaacua caacucacac aacguguaca 1320
ucauggcaga uaaacagaag aaugguauca aagugaacuu uaagauucgc cacaacaucg 1380
aagauggauc agugcaacuu gcagaucacu accaacagaa uacgccgaua ggagauggac 1440
cugugcuucu uccugauaac cacuaccuuu caacacaauc agcacuuuca aaggacccaa 1500
acgagaagcg agaccacaug gugcuucuug aauuugugac agcagcagga aucacacuug 1560
gaauggauga acuuuacaaa ugaaaaaaaa caaaaaaaca aaacaaacgg cuauuaugcg 1620
uuaccggcga gacgcuacgg acuua 1645
<210> 30
<211> 1642
<212> RNA
<213> Artificial Sequence
<400> 30
aaaauccguu gaccuuaaac ggucgugugg guucaagucc cuccaccccc acgccggaaa 60
cgcaauagcc gaaaaacaaa aaacaaaaaa aaaaccaaaa aaacaaaaaa aacaaaacac 120
auuaaaacag ccuguggguu gaucccaccc acagggccca cugggcgcua gcacucuggu 180
aucacgguac cuuugugcgc cuguuuuaua cuuccucccc caacugcaac uuagaaguaa 240
cacaaaccga ucaacaguca gcguggcaca ccagccacgu uuugaucaaa cacuucuguu 300
accccggacu gaguaucaau agacugcuca cgcgguugaa ggagaaaacg uucguuaucc 360
ggccaacuac uucgagaaac cuaguaacgc cauggaaguu guggaguguu ucgcucagca 420
cuaccccagu guagaucagg uugaugaguc accgcauucc ccacggguga ccguggcggu 480
ggcugcguug gcggccugcc cauggggaaa cccaugggac gcucuuauac agacauggug 540
cgaagagucu auugagcuag uugguagucc uccggccccu gaaugcggcu aaucccaacu 600
gcggagcaua cacucucaag ccagagggua gugugucgua augggcaacu cugcagcgga 660
accgacuacu uugggugucc guguuucauu uuauuccuau acuggcugcu uauggugaca 720
auugagagau uguuaccaua uagcuauugg auuggccauc cggugacuaa cagagcuauu 780
auauaucuuu uuguuggguu uauaccacuu agcuugaaag agguuaaaac ucuacauuac 840
auuuuaauac ugaacaccgc aaaauggugu caaaggguga ggaauuauuc accggcgugg 900
ugccuauccu uguggaacuu gauggagaug ugaacggaca caaauucagu guaucaggag 960
aaggagaagg agaugcaaca uacggaaagc ucacucuuaa auuuaucugc acaacaggaa 1020
agcucccggu gccuuggccu acacuuguga caacacuuac auacggagug caaugcuucu 1080
cgcguuaccc ugaucacaug aaacaacacg auuucuucaa gagugcaaug ccugaaggau 1140
acgugcaaga aagaacaauc uucuucaagg acgauggaaa cuacaagacu cgugcagaag 1200
ugaaauuuga aggagauaca cuugugaaca gaaucgaacu uaaaggaauc gauuucaagg 1260
aggauggaaa cauccuugga cacaaacuug aauacaacua caacucacac aacguguaca 1320
ucauggcaga uaaacagaag aaugguauca aagugaacuu uaagauucgc cacaacaucg 1380
aagauggauc agugcaacuu gcagaucacu accaacagaa uacgccgaua ggagauggac 1440
cugugcuucu uccugauaac cacuaccuuu caacacaauc agcacuuuca aaggacccaa 1500
acgagaagcg agaccacaug gugcuucuug aauuugugac agcagcagga aucacacuug 1560
gaauggauga acuuuacaaa ugaaaaaaca aaaaacaaaa caaacggcua uuaugcguua 1620
ccggcgagac gcuacggacu ua 1642
<210> 31
<211> 822
<212> DNA
<213> Artificial Sequence
<400> 31
atgtttgtgt tcctggtgct gctgcctctg gtgtctaggg tgcagcctac agagagcatt 60
gtgaggttcc ctaacatcac caacctgtgc ccttttggag aggtgttcaa tgccacaagg 120
tttgcctctg tgtatgcctg gaataggaag aggatcagca actgtgtggc tgactactct 180
gtgctgtaca actctgctag cttcagcacc ttcaagtgct atggagtgag ccctaccaag 240
ctgaatgacc tgtgcttcac caatgtgtat gctgacagct ttgtgattag gggagatgag 300
gtgaggcaga ttgcccctgg acagactggc aagattgctg actacaacta caagctgcct 360
gatgacttca ctggctgtgt gattgcctgg aacagcaaca acctggacag caaggtggga 420
ggcaactaca actacctgta taggctgttt aggaagagca acctgaagcc ttttgagagg 480
gacatcagca cagagatcta ccaagctggc agcacccctt gcaatggagt ggagggcttc 540
aactgctact tccctctgca gagctatggc tttcagccta ccaatggagt gggctatcag 600
ccttataggg tggtggtgct gagctttgag ctgctgcatg cccctgccac agtgtgtggc 660
cctaagaaga gcaccaacct ggtgaagaac aagtgtgtga acttcagctc tggcctggtg 720
cctaccggct ctggctctgg ctacatccct gaggccccta gggatggcca agcctatgtg 780
aggaaggatg gagagtgggt gctgctgagc accttcctgt ga 822
<210> 32
<211> 273
<212> PRT
<213> Artificial Sequence
<400> 32
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Arg Val Gln Pro
1 5 10 15
Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe
20 25 30
Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn
35 40 45
Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn
50 55 60
Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys
65 70 75 80
Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile
85 90 95
Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile
100 105 110
Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile
115 120 125
Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn
130 135 140
Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg
145 150 155 160
Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly
165 170 175
Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln
180 185 190
Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser
195 200 205
Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser
210 215 220
Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Ser Ser Gly Leu Val
225 230 235 240
Pro Thr Gly Ser Gly Ser Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly
245 250 255
Gln Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe
260 265 270
Leu
<210> 33
<211> 1747
<212> RNA
<213> Artificial Sequence
<400> 33
aaaauccguu gaccuuaaac ggucgugugg guucaagucc cuccaccccc acgccggaaa 60
cgcaauagcc gaaaaaacaa aaacaaaaaa aacaaaaaaa caaaaaaaaa accaaaacac 120
auuaaaacag ccuguggguu gaucccaccc acagggccca cugggcgcua gcacucuggu 180
aucacgguac cuuugugcgc cuguuuuaua cuuccucccc caacugcaac uuagaaguaa 240
cacaaaccga ucaacaguca gcguggcaca ccagccacgu uuugaucaaa cacuucuguu 300
accccggacu gaguaucaau agacugcuca cgcgguugaa ggagaaaacg uucguuaucc 360
ggccaacuac uucgagaaac cuaguaacgc cauggaaguu guggaguguu ucgcucagca 420
cuaccccagu guagaucagg uugaugaguc accgcauucc ccacggguga ccguggcggu 480
ggcugcguug gcggccugcc cauggggaaa cccaugggac gcucuuauac agacauggug 540
cgaagagucu auugagcuag uugguagucc uccggccccu gaaugcggcu aaucccaacu 600
gcggagcaua cacucucaag ccagagggua gugugucgua augggcaacu cugcagcgga 660
accgacuacu uugggugucc guguuucauu uuauuccuau acuggcugcu uauggugaca 720
auugagagau uguuaccaua uagcuauugg auuggccauc cggugacuaa cagagcuauu 780
auauaucuuu uuguuggguu uauaccacuu agcuugaaag agguuaaaac ucuacauuac 840
auuuuaauac ugaacaccgc aaaauguuug uguuccuggu gcugcugccu cuggugucua 900
gggugcagcc uacagagagc auugugaggu ucccuaacau caccaaccug ugcccuuuug 960
gagagguguu caaugccaca agguuugccu cuguguaugc cuggaauagg aagaggauca 1020
gcaacugugu ggcugacuac ucugugcugu acaacucugc uagcuucagc accuucaagu 1080
gcuauggagu gagcccuacc aagcugaaug accugugcuu caccaaugug uaugcugaca 1140
gcuuugugau uaggggagau gaggugaggc agauugcccc uggacagacu ggcaagauug 1200
cugacuacaa cuacaagcug ccugaugacu ucacuggcug ugugauugcc uggaacagca 1260
acaaccugga cagcaaggug ggaggcaacu acaacuaccu guauaggcug uuuaggaaga 1320
gcaaccugaa gccuuuugag agggacauca gcacagagau cuaccaagcu ggcagcaccc 1380
cuugcaaugg aguggagggc uucaacugcu acuucccucu gcagagcuau ggcuuucagc 1440
cuaccaaugg agugggcuau cagccuuaua gggugguggu gcugagcuuu gagcugcugc 1500
augccccugc cacagugugu ggcccuaaga agagcaccaa ccuggugaag aacaagugug 1560
ugaacuucag cucuggccug gugccuaccg gcucuggcuc uggcuacauc ccugaggccc 1620
cuagggaugg ccaagccuau gugaggaagg auggagagug ggugcugcug agcaccuucc 1680
ugugaaaaaa aacaaaaaaa caaaacaaac ggcuauuaug cguuaccggc gagacgcuac 1740
ggacuua 1747
<210> 34
<211> 579
<212> DNA
<213> Artificial Sequence
<400> 34
atgggcgtgc acgagtgccc cgcctggctg tggctgctgc tgagcctgct gagcctgccc 60
ctgggcctgc ccgtgctggg cgcccccccc aggctgatct gcgacagcag ggtgctggag 120
aggtacctgc tggaggccaa ggaggccgag aacatcacca ccggctgcgc cgagcactgc 180
agcctgaacg agaacatcac cgtgcccgac accaaggtga acttctacgc ctggaagagg 240
atggaggtgg gccagcaggc cgtggaggtg tggcagggcc tggccctgct gagcgaggcc 300
gtgctgaggg gccaggccct gctggtgaac agcagccagc cctgggagcc cctgcagctg 360
cacgtggaca aggccgtgag cggcctgagg agcctgacca ccctgctgag ggccctgggc 420
gcccagaagg aggccatcag cccccccgac gccgccagcg ccgcccccct gaggaccatc 480
accgccgaca ccttcaggaa gctgttcagg gtgtacagca acttcctgag gggcaagctg 540
aagctgtaca ccggcgaggc ctgcaggacc ggcgacagg 579
<210> 35
<211> 193
<212> PRT
<213> Artificial Sequence
<400> 35
Met Gly Val His Glu Cys Pro Ala Trp Leu Trp Leu Leu Leu Ser Leu
1 5 10 15
Leu Ser Leu Pro Leu Gly Leu Pro Val Leu Gly Ala Pro Pro Arg Leu
20 25 30
Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu
35 40 45
Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu
50 55 60
Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg
65 70 75 80
Met Glu Val Gly Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu
85 90 95
Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser
100 105 110
Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly
115 120 125
Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Gly Ala Gln Lys Glu
130 135 140
Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile
145 150 155 160
Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu
165 170 175
Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp
180 185 190
Arg
<210> 36
<211> 1502
<212> RNA
<213> Artificial Sequence
<400> 36
aaaauccguu gaccuuaaac ggucgugugg guucaagucc cuccaccccc acgccggaaa 60
cgcaauagcc gaaaaacaaa aaacaaaaaa aacaaaaaaa aaaccaaaaa aacaaaacac 120
auuaaaacag ccuguggguu gaucccaccc acagggccca uugggcgcua gcacucuggu 180
aucacgguac ccuugugcgc cuguuuuaug ucccuucccu caacuguaac uuagaaguaa 240
cgcacaccga ucaacaguca gcguggcaca ccagccaugu uuugaucaag cacuucuguu 300
accccggacc gaguaucaac agacugcuca cgcgguugaa ggagaaagug uucguuaucc 360
ggccaacuac uucgaaaaac cuaguaacac cauggaaguu gcagaguguu ucgcucagca 420
cuaccccagu guagaucagg ucgaugaguc accgcauccc ccacgggcga ccguggcggu 480
ggcugcguug gcggccugcc uaugggggaa cccauaggac gcucuaauac agacauggug 540
cgaagagucc auugagcuag uugguagucc uccggccccu gaaugcggcu aauccuaacu 600
gcggagcaca caccuucaag ccagagggca gugugucgua acgggcaacu cugcagcgga 660
accgacuacu uugggugucc guguuucauu uuauucuuau acuggcugcu uauggugaca 720
auugagagau uguuaccaua uagcuauugg auuggccauc cagugacuag cagagcuauu 780
auauaccucu uuguuggguu uauaccaccu aauuugaaag aaguuaaaac auuagaauuc 840
auuauuaaau ugaauacaau gggcgugcac gagugccccg ccuggcugug gcugcugcug 900
agccugcuga gccugccccu gggccugccc gugcugggcg ccccccccag gcugaucugc 960
gacagcaggg ugcuggagag guaccugcug gaggccaagg aggccgagaa caucaccacc 1020
ggcugcgccg agcacugcag ccugaacgag aacaucaccg ugcccgacac caaggugaac 1080
uucuacgccu ggaagaggau ggaggugggc cagcaggccg uggaggugug gcagggccug 1140
gcccugcuga gcgaggccgu gcugaggggc caggcccugc uggugaacag cagccagccc 1200
ugggagcccc ugcagcugca cguggacaag gccgugagcg gccugaggag ccugaccacc 1260
cugcugaggg cccugggcgc ccagaaggag gccaucagcc cccccgacgc cgccagcgcc 1320
gccccccuga ggaccaucac cgccgacacc uucaggaagc uguucagggu guacagcaac 1380
uuccugaggg gcaagcugaa gcuguacacc ggcgaggccu gcaggaccgg cgacagguga 1440
aaaaaaacaa aaaaacaaaa caaacggcua uuaugcguua ccggcgagac gcuacggacu 1500
ua 1502
<210> 37
<211> 648
<212> DNA
<213> Artificial Sequence
<400> 37
atggagatcg tgctgaccca gagccccgcc accctgagcc tgagccccgg cgagagggcc 60
accctgagct gcagggccag ccagagcgtg agcagctacc tggcctggta ccagcagaag 120
cccggccagg cccccaggct gctgatctac gacgccagca acagggccac cggcatcccc 180
gccaggttca gcggcagcgg cagcggcacc gacttcaccc tgaccatcag cagcctggag 240
cccgaggact tcgccgtgta ctactgccag cagagcagca actggcccag gaccttcggc 300
cagggcacca aggtggagat caagaggacc gtggccgccc ccagcgtgtt catcttcccc 360
cccagcgacg agcagctgaa gagcggcacc gccagcgtgg tgtgcctgct gaacaacttc 420
taccccaggg aggccaaggt gcagtggaag gtggacaacg ccctgcagag cggcaacagc 480
caggagagcg tgaccgagca ggacagcaag gacagcacct acagcctgag cagcaccctg 540
accctgagca aggccgacta cgagaagcac aaggtgtacg cctgcgaggt gacccaccag 600
ggcctgagca gccccgtgac caagagcttc aacaggggcg agtgctga 648
<210> 38
<211> 215
<212> PRT
<213> Artificial Sequence
<400> 38
Met Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro
1 5 10 15
Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser
20 25 30
Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu
35 40 45
Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser
50 55 60
Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu
65 70 75 80
Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro
85 90 95
Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala
100 105 110
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
115 120 125
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
130 135 140
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
145 150 155 160
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
165 170 175
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
180 185 190
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
195 200 205
Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 39
<211> 1573
<212> RNA
<213> Artificial Sequence
<400> 39
aaaauccguu gaccuuaaac ggucgugugg guucaagucc cuccaccccc acgccggaaa 60
cgcaauagcc gaaaaaacaa aaacaaaaaa aacaaaaaaa caaaaaaaaa accaaaacac 120
auuaaaacag ccuguggguu gaucccaccc acagggccca cugggcgcua gcacucuggu 180
aucacgguac cuuugugcgc cuguuuuaua cuuccucccc caacugcaac uuagaaguaa 240
cacaaaccga ucaacaguca gcguggcaca ccagccacgu uuugaucaaa cacuucuguu 300
accccggacu gaguaucaau agacugcuca cgcgguugaa ggagaaaacg uucguuaucc 360
ggccaacuac uucgagaaac cuaguaacgc cauggaaguu guggaguguu ucgcucagca 420
cuaccccagu guagaucagg uugaugaguc accgcauucc ccacggguga ccguggcggu 480
ggcugcguug gcggccugcc cauggggaaa cccaugggac gcucuuauac agacauggug 540
cgaagagucu auugagcuag uugguagucc uccggccccu gaaugcggcu aaucccaacu 600
gcggagcaua cacucucaag ccagagggua gugugucgua augggcaacu cugcagcgga 660
accgacuacu uugggugucc guguuucauu uuauuccuau acuggcugcu uauggugaca 720
auugagagau uguuaccaua uagcuauugg auuggccauc cggugacuaa cagagcuauu 780
auauaucuuu uuguuggguu uauaccacuu agcuugaaag agguuaaaac ucuacauuac 840
auuuuaauac ugaacaccgc aaaauggaga ucgugcugac ccagagcccc gccacccuga 900
gccugagccc cggcgagagg gccacccuga gcugcagggc cagccagagc gugagcagcu 960
accuggccug guaccagcag aagcccggcc aggcccccag gcugcugauc uacgacgcca 1020
gcaacagggc caccggcauc cccgccaggu ucagcggcag cggcagcggc accgacuuca 1080
cccugaccau cagcagccug gagcccgagg acuucgccgu guacuacugc cagcagagca 1140
gcaacuggcc caggaccuuc ggccagggca ccaaggugga gaucaagagg accguggccg 1200
cccccagcgu guucaucuuc ccccccagcg acgagcagcu gaagagcggc accgccagcg 1260
uggugugccu gcugaacaac uucuacccca gggaggccaa ggugcagugg aagguggaca 1320
acgcccugca gagcggcaac agccaggaga gcgugaccga gcaggacagc aaggacagca 1380
ccuacagccu gagcagcacc cugacccuga gcaaggccga cuacgagaag cacaaggugu 1440
acgccugcga ggugacccac cagggccuga gcagccccgu gaccaagagc uucaacaggg 1500
gcgagugcug aaaaaaaaca aaaaaacaaa acaaacggcu auuaugcguu accggcgaga 1560
cgcuacggac uua 1573
<210> 40
<211> 1326
<212> DNA
<213> Artificial Sequence
<400> 40
atgcaggtgc agctggtgga gagcggcggc ggcgtggtgc agcccggcag gagcctgagg 60
ctggactgca aggccagcgg catcaccttc agcaacagcg gcatgcactg ggtgaggcag 120
gcccccggca agggcctgga gtgggtggcc gtgatctggt acgacggcag caagaggtac 180
tacgccgaca gcgtgaaggg caggttcacc atcagcaggg acaacagcaa gaacaccctg 240
ttcctgcaga tgaacagcct gagggccgag gacaccgccg tgtactactg cgccaccaac 300
gacgactact ggggccaggg caccctggtg accgtgagca gcgccagcac caagggcccc 360
agcgtgttcc ccctggcccc ctgcagcagg agcaccagcg agagcaccgc cgccctgggc 420
tgcctggtga aggactactt ccccgagccc gtgaccgtga gctggaacag cggcgccctg 480
accagcggcg tgcacacctt ccccgccgtg ctgcagagca gcggcctgta cagcctgagc 540
agcgtggtga ccgtgcccag cagcagcctg ggcaccaaga cctacacctg caacgtggac 600
cacaagccca gcaacaccaa ggtggacaag agggtggaga gcaagtacgg ccccccctgc 660
cccccctgcc ccgcccccga gttcctgggc ggccccagcg tgttcctgtt cccccccaag 720
cccaaggaca ccctgatgat cagcaggacc cccgaggtga cctgcgtggt ggtggacgtg 780
agccaggagg accccgaggt gcagttcaac tggtacgtgg acggcgtgga ggtgcacaac 840
gccaagacca agcccaggga ggagcagttc aacagcacct acagggtggt gagcgtgctg 900
accgtgctgc accaggactg gctgaacggc aaggagtaca agtgcaaggt gagcaacaag 960
ggcctgccca gcagcatcga gaagaccatc agcaaggcca agggccagcc cagggagccc 1020
caggtgtaca ccctgccccc cagccaggag gagatgacca agaaccaggt gagcctgacc 1080
tgcctggtga agggcttcta ccccagcgac atcgccgtgg agtgggagag caacggccag 1140
cccgagaaca actacaagac cacccccccc gtgctggaca gcgacggcag cttcttcctg 1200
tacagcaggc tgaccgtgga caagagcagg tggcaggagg gcaacgtgtt cagctgcagc 1260
gtgatgcacg aggccctgca caaccactac acccagaaga gcctgagcct gagcctgggc 1320
aagtga 1326
<210> 41
<211> 440
<212> PRT
<213> Artificial Sequence
<400> 41
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe Ser Asn Ser
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Trp Tyr Asp Gly Ser Lys Arg Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
100 105 110
Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser
115 120 125
Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
130 135 140
Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr
145 150 155 160
Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr
165 170 175
Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys
180 185 190
Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp
195 200 205
Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala
210 215 220
Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
225 230 235 240
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
245 250 255
Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val
260 265 270
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
275 280 285
Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
290 295 300
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly
305 310 315 320
Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
325 330 335
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr
340 345 350
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
355 360 365
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
370 375 380
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
385 390 395 400
Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe
405 410 415
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
420 425 430
Ser Leu Ser Leu Ser Leu Gly Lys
435 440
<210> 42
<211> 2251
<212> RNA
<213> Artificial Sequence
<400> 42
aaaauccguu gaccuuaaac ggucgugugg guucaagucc cuccaccccc acgccggaaa 60
cgcaauagcc gaaaaaacaa aaacaaaaaa aacaaaaaaa caaaaaaaaa accaaaacac 120
auuaaaacag ccuguggguu gaucccaccc acagggccca cugggcgcua gcacucuggu 180
aucacgguac cuuugugcgc cuguuuuaua cuuccucccc caacugcaac uuagaaguaa 240
cacaaaccga ucaacaguca gcguggcaca ccagccacgu uuugaucaaa cacuucuguu 300
accccggacu gaguaucaau agacugcuca cgcgguugaa ggagaaaacg uucguuaucc 360
ggccaacuac uucgagaaac cuaguaacgc cauggaaguu guggaguguu ucgcucagca 420
cuaccccagu guagaucagg uugaugaguc accgcauucc ccacggguga ccguggcggu 480
ggcugcguug gcggccugcc cauggggaaa cccaugggac gcucuuauac agacauggug 540
cgaagagucu auugagcuag uugguagucc uccggccccu gaaugcggcu aaucccaacu 600
gcggagcaua cacucucaag ccagagggua gugugucgua augggcaacu cugcagcgga 660
accgacuacu uugggugucc guguuucauu uuauuccuau acuggcugcu uauggugaca 720
auugagagau uguuaccaua uagcuauugg auuggccauc cggugacuaa cagagcuauu 780
auauaucuuu uuguuggguu uauaccacuu agcuugaaag agguuaaaac ucuacauuac 840
auuuuaauac ugaacaccgc aaaaugcagg ugcagcuggu ggagagcggc ggcggcgugg 900
ugcagcccgg caggagccug aggcuggacu gcaaggccag cggcaucacc uucagcaaca 960
gcggcaugca cugggugagg caggcccccg gcaagggccu ggagugggug gccgugaucu 1020
gguacgacgg cagcaagagg uacuacgccg acagcgugaa gggcagguuc accaucagca 1080
gggacaacag caagaacacc cuguuccugc agaugaacag ccugagggcc gaggacaccg 1140
ccguguacua cugcgccacc aacgacgacu acuggggcca gggcacccug gugaccguga 1200
gcagcgccag caccaagggc cccagcgugu ucccccuggc ccccugcagc aggagcacca 1260
gcgagagcac cgccgcccug ggcugccugg ugaaggacua cuuccccgag cccgugaccg 1320
ugagcuggaa cagcggcgcc cugaccagcg gcgugcacac cuuccccgcc gugcugcaga 1380
gcagcggccu guacagccug agcagcgugg ugaccgugcc cagcagcagc cugggcacca 1440
agaccuacac cugcaacgug gaccacaagc ccagcaacac caagguggac aagagggugg 1500
agagcaagua cggccccccc ugcccccccu gccccgcccc cgaguuccug ggcggcccca 1560
gcguguuccu guuccccccc aagcccaagg acacccugau gaucagcagg acccccgagg 1620
ugaccugcgu ggugguggac gugagccagg aggaccccga ggugcaguuc aacugguacg 1680
uggacggcgu ggaggugcac aacgccaaga ccaagcccag ggaggagcag uucaacagca 1740
ccuacagggu ggugagcgug cugaccgugc ugcaccagga cuggcugaac ggcaaggagu 1800
acaagugcaa ggugagcaac aagggccugc ccagcagcau cgagaagacc aucagcaagg 1860
ccaagggcca gcccagggag ccccaggugu acacccugcc ccccagccag gaggagauga 1920
ccaagaacca ggugagccug accugccugg ugaagggcuu cuaccccagc gacaucgccg 1980
uggaguggga gagcaacggc cagcccgaga acaacuacaa gaccaccccc cccgugcugg 2040
acagcgacgg cagcuucuuc cuguacagca ggcugaccgu ggacaagagc agguggcagg 2100
agggcaacgu guucagcugc agcgugaugc acgaggcccu gcacaaccac uacacccaga 2160
agagccugag ccugagccug ggcaagugaa aaaaaacaaa aaaacaaaac aaacggcuau 2220
uaugcguuac cggcgagacg cuacggacuu a 2251
<210> 43
<211> 489
<212> DNA
<213> Artificial Sequence
<400> 43
atgaggatca gcaagcccca cctgaggagc atcagcatcc agtgctacct gtgcctgctg 60
ctgaacagcc acttcctgac cgaggccggc atccacgtgt tcatcctggg ctgcttcagc 120
gccggcctgc ccaagaccga ggccaactgg gtgaacgtga tcagcgacct gaagaagatc 180
gaggacctga tccagagcat gcacatcgac gccaccctgt acaccgagag cgacgtgcac 240
cccagctgca aggtgaccgc catgaagtgc ttcctgctgg agctgcaggt gatcagcctg 300
gagagcggcg acgccagcat ccacgacacc gtggagaacc tgatcatcct ggccaacaac 360
agcctgagca gcaacggcaa cgtgaccgag agcggctgca aggagtgcga ggagctggag 420
gagaagaaca tcaaggagtt cctgcagagc ttcgtgcaca tcgtgcagat gttcatcaac 480
accagctga 489
<210> 44
<211> 162
<212> PRT
<213> Artificial Sequence
<400> 44
Met Arg Ile Ser Lys Pro His Leu Arg Ser Ile Ser Ile Gln Cys Tyr
1 5 10 15
Leu Cys Leu Leu Leu Asn Ser His Phe Leu Thr Glu Ala Gly Ile His
20 25 30
Val Phe Ile Leu Gly Cys Phe Ser Ala Gly Leu Pro Lys Thr Glu Ala
35 40 45
Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile
50 55 60
Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His
65 70 75 80
Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln
85 90 95
Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu
100 105 110
Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val
115 120 125
Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile
130 135 140
Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn
145 150 155 160
Thr Ser
<210> 45
<211> 1414
<212> RNA
<213> Artificial Sequence
<400> 45
aaaauccguu gaccuuaaac ggucgugugg guucaagucc cuccaccccc acgccggaaa 60
cgcaauagcc gaaaaaacaa aaacaaaaaa aacaaaaaaa caaaaaaaaa accaaaacac 120
auuaaaacag ccuguggguu gaucccaccc acagggccca cugggcgcua gcacucuggu 180
aucacgguac cuuugugcgc cuguuuuaua cuuccucccc caacugcaac uuagaaguaa 240
cacaaaccga ucaacaguca gcguggcaca ccagccacgu uuugaucaaa cacuucuguu 300
accccggacu gaguaucaau agacugcuca cgcgguugaa ggagaaaacg uucguuaucc 360
ggccaacuac uucgagaaac cuaguaacgc cauggaaguu guggaguguu ucgcucagca 420
cuaccccagu guagaucagg uugaugaguc accgcauucc ccacggguga ccguggcggu 480
ggcugcguug gcggccugcc cauggggaaa cccaugggac gcucuuauac agacauggug 540
cgaagagucu auugagcuag uugguagucc uccggccccu gaaugcggcu aaucccaacu 600
gcggagcaua cacucucaag ccagagggua gugugucgua augggcaacu cugcagcgga 660
accgacuacu uugggugucc guguuucauu uuauuccuau acuggcugcu uauggugaca 720
auugagagau uguuaccaua uagcuauugg auuggccauc cggugacuaa cagagcuauu 780
auauaucuuu uuguuggguu uauaccacuu agcuugaaag agguuaaaac ucuacauuac 840
auuuuaauac ugaacaccgc aaaaugagga ucagcaagcc ccaccugagg agcaucagca 900
uccagugcua ccugugccug cugcugaaca gccacuuccu gaccgaggcc ggcauccacg 960
uguucauccu gggcugcuuc agcgccggcc ugcccaagac cgaggccaac ugggugaacg 1020
ugaucagcga ccugaagaag aucgaggacc ugauccagag caugcacauc gacgccaccc 1080
uguacaccga gagcgacgug caccccagcu gcaaggugac cgccaugaag ugcuuccugc 1140
uggagcugca ggugaucagc cuggagagcg gcgacgccag cauccacgac accguggaga 1200
accugaucau ccuggccaac aacagccuga gcagcaacgg caacgugacc gagagcggcu 1260
gcaaggagug cgaggagcug gaggagaaga acaucaagga guuccugcag agcuucgugc 1320
acaucgugca gauguucauc aacaccagcu gaaaaaaaac aaaaaaacaa aacaaacggc 1380
uauuaugcgu uaccggcgag acgcuacgga cuua 1414
<210> 46
<211> 1161
<212> DNA
<213> Artificial Sequence
<400> 46
atgagggccg cccctctgct gctggctagg gccgctagcc tgagcctggg cttcctgttc 60
ctgctgttct tctggctgga taggagcgtg ctggccaagg agctgaagtt cgtgaccctg 120
gtgtttaggc acggcgatag gagccctatc gacaccttcc ctaccgaccc tatcaaggag 180
agcagctggc ctcaaggctt cggacagctg acacagctgg gcatggagca gcactacgag 240
ctgggcgagt acattaggaa gaggtatagg aagttcctga acgagagcta caagcacgag 300
caagtgtaca ttaggagcac cgacgtggat aggaccctga tgagcgccat gaccaacctg 360
gccgccctgt tccctcctga gggcgtgagc atctggaacc ctatcctgct gtggcagcct 420
atccctgtgc acaccgtgcc tctgagcgag gatcagctgc tgtacctgcc ttttaggaac 480
tgccctaggt tccaagagct ggagagcgag accctgaaga gcgaggagtt tcagaagagg 540
ctgcaccctt acaaggactt catcgccacc ctgggcaagc tctccggcct gcacggccaa 600
gacctgttcg gcatctggag caaggtgtac gaccctctgt actgcgagag cgtgcacaac 660
ttcaccctgc ctagctgggc caccgaggac accatgacca agctgaggga gctgagcgag 720
ctgagcctgc tgagcctgta cggcatccac aagcagaagg agaagtctag gctgcaaggc 780
ggcgtgctgg tgaacgagat cctgaaccac atgaagaggg ccacacagat ccctagctac 840
aagaagctga tcatgtacag cgcccacgac accaccgtca gcggcctgca gatggccctg 900
gacgtgtaca acggcctgct gcctccttac gctagctgcc acctgaccga gctgtacttc 960
gagaagggcg agtacttcgt ggagatgtac tataggaacg agacacagca cgagccttac 1020
cctctgatgc tgcctggctg cagccctagc tgccctctgg agaggttcgc cgagctggtg 1080
ggccctgtga tccctcaaga ctggagcacc gagtgcatga ccaccaacag ccaccaaggc 1140
accgaggaca gcaccgactg a 1161
<210> 47
<211> 386
<212> PRT
<213> Artificial Sequence
<400> 47
Met Arg Ala Ala Pro Leu Leu Leu Ala Arg Ala Ala Ser Leu Ser Leu
1 5 10 15
Gly Phe Leu Phe Leu Leu Phe Phe Trp Leu Asp Arg Ser Val Leu Ala
20 25 30
Lys Glu Leu Lys Phe Val Thr Leu Val Phe Arg His Gly Asp Arg Ser
35 40 45
Pro Ile Asp Thr Phe Pro Thr Asp Pro Ile Lys Glu Ser Ser Trp Pro
50 55 60
Gln Gly Phe Gly Gln Leu Thr Gln Leu Gly Met Glu Gln His Tyr Glu
65 70 75 80
Leu Gly Glu Tyr Ile Arg Lys Arg Tyr Arg Lys Phe Leu Asn Glu Ser
85 90 95
Tyr Lys His Glu Gln Val Tyr Ile Arg Ser Thr Asp Val Asp Arg Thr
100 105 110
Leu Met Ser Ala Met Thr Asn Leu Ala Ala Leu Phe Pro Pro Glu Gly
115 120 125
Val Ser Ile Trp Asn Pro Ile Leu Leu Trp Gln Pro Ile Pro Val His
130 135 140
Thr Val Pro Leu Ser Glu Asp Gln Leu Leu Tyr Leu Pro Phe Arg Asn
145 150 155 160
Cys Pro Arg Phe Gln Glu Leu Glu Ser Glu Thr Leu Lys Ser Glu Glu
165 170 175
Phe Gln Lys Arg Leu His Pro Tyr Lys Asp Phe Ile Ala Thr Leu Gly
180 185 190
Lys Leu Ser Gly Leu His Gly Gln Asp Leu Phe Gly Ile Trp Ser Lys
195 200 205
Val Tyr Asp Pro Leu Tyr Cys Glu Ser Val His Asn Phe Thr Leu Pro
210 215 220
Ser Trp Ala Thr Glu Asp Thr Met Thr Lys Leu Arg Glu Leu Ser Glu
225 230 235 240
Leu Ser Leu Leu Ser Leu Tyr Gly Ile His Lys Gln Lys Glu Lys Ser
245 250 255
Arg Leu Gln Gly Gly Val Leu Val Asn Glu Ile Leu Asn His Met Lys
260 265 270
Arg Ala Thr Gln Ile Pro Ser Tyr Lys Lys Leu Ile Met Tyr Ser Ala
275 280 285
His Asp Thr Thr Val Ser Gly Leu Gln Met Ala Leu Asp Val Tyr Asn
290 295 300
Gly Leu Leu Pro Pro Tyr Ala Ser Cys His Leu Thr Glu Leu Tyr Phe
305 310 315 320
Glu Lys Gly Glu Tyr Phe Val Glu Met Tyr Tyr Arg Asn Glu Thr Gln
325 330 335
His Glu Pro Tyr Pro Leu Met Leu Pro Gly Cys Ser Pro Ser Cys Pro
340 345 350
Leu Glu Arg Phe Ala Glu Leu Val Gly Pro Val Ile Pro Gln Asp Trp
355 360 365
Ser Thr Glu Cys Met Thr Thr Asn Ser His Gln Gly Thr Glu Asp Ser
370 375 380
Thr Asp
385
<210> 48
<211> 2086
<212> RNA
<213> Artificial Sequence
<400> 48
aaaauccguu gaccuuaaac ggucgugugg guucaagucc cuccaccccc acgccggaaa 60
cgcaauagcc gaaaaaacaa aaacaaaaaa aacaaaaaaa caaaaaaaaa accaaaacac 120
auuaaaacag ccuguggguu gaucccaccc acagggccca cugggcgcua gcacucuggu 180
aucacgguac cuuugugcgc cuguuuuaua cuuccucccc caacugcaac uuagaaguaa 240
cacaaaccga ucaacaguca gcguggcaca ccagccacgu uuugaucaaa cacuucuguu 300
accccggacu gaguaucaau agacugcuca cgcgguugaa ggagaaaacg uucguuaucc 360
ggccaacuac uucgagaaac cuaguaacgc cauggaaguu guggaguguu ucgcucagca 420
cuaccccagu guagaucagg uugaugaguc accgcauucc ccacggguga ccguggcggu 480
ggcugcguug gcggccugcc cauggggaaa cccaugggac gcucuuauac agacauggug 540
cgaagagucu auugagcuag uugguagucc uccggccccu gaaugcggcu aaucccaacu 600
gcggagcaua cacucucaag ccagagggua gugugucgua augggcaacu cugcagcgga 660
accgacuacu uugggugucc guguuucauu uuauuccuau acuggcugcu uauggugaca 720
auugagagau uguuaccaua uagcuauugg auuggccauc cggugacuaa cagagcuauu 780
auauaucuuu uuguuggguu uauaccacuu agcuugaaag agguuaaaac ucuacauuac 840
auuuuaauac ugaacaccgc aaaaugaggg ccgccccucu gcugcuggcu agggccgcua 900
gccugagccu gggcuuccug uuccugcugu ucuucuggcu ggauaggagc gugcuggcca 960
aggagcugaa guucgugacc cugguguuua ggcacggcga uaggagcccu aucgacaccu 1020
ucccuaccga cccuaucaag gagagcagcu ggccucaagg cuucggacag cugacacagc 1080
ugggcaugga gcagcacuac gagcugggcg aguacauuag gaagagguau aggaaguucc 1140
ugaacgagag cuacaagcac gagcaagugu acauuaggag caccgacgug gauaggaccc 1200
ugaugagcgc caugaccaac cuggccgccc uguucccucc ugagggcgug agcaucugga 1260
acccuauccu gcuguggcag ccuaucccug ugcacaccgu gccucugagc gaggaucagc 1320
ugcuguaccu gccuuuuagg aacugcccua gguuccaaga gcuggagagc gagacccuga 1380
agagcgagga guuucagaag aggcugcacc cuuacaagga cuucaucgcc acccugggca 1440
agcucuccgg ccugcacggc caagaccugu ucggcaucug gagcaaggug uacgacccuc 1500
uguacugcga gagcgugcac aacuucaccc ugccuagcug ggccaccgag gacaccauga 1560
ccaagcugag ggagcugagc gagcugagcc ugcugagccu guacggcauc cacaagcaga 1620
aggagaaguc uaggcugcaa ggcggcgugc uggugaacga gauccugaac cacaugaaga 1680
gggccacaca gaucccuagc uacaagaagc ugaucaugua cagcgcccac gacaccaccg 1740
ucagcggccu gcagauggcc cuggacgugu acaacggccu gcugccuccu uacgcuagcu 1800
gccaccugac cgagcuguac uucgagaagg gcgaguacuu cguggagaug uacuauagga 1860
acgagacaca gcacgagccu uacccucuga ugcugccugg cugcagcccu agcugcccuc 1920
uggagagguu cgccgagcug gugggcccug ugaucccuca agacuggagc accgagugca 1980
ugaccaccaa cagccaccaa ggcaccgagg acagcaccga cugaaaaaaa acaaaaaaac 2040
aaaacaaacg gcuauuaugc guuaccggcg agacgcuacg gacuua 2086
<210> 49
<211> 1260
<212> DNA
<213> Artificial Sequence
<400> 49
atgggcggcg gcgccggcga gaggctgttc accagcagct gcctggtggg cctggtgccc 60
ctgggcctga ggatcagcct ggtgacctgc cccctgcagt gcggcatcat gtggcagctg 120
ctgctgccca ccgccctgct gctgctcgtg agcgccggca tgaggaccga ggacctgccc 180
aaggccgtgg tgttcctgga gccccagtgg tacagggtgc tggagaagga cagcgtgacc 240
ctgaagtgcc agggcgccta cagccccgag gacaacagca cccagtggtt ccacaacgag 300
agcctgatca gcagccaggc cagcagctac ttcatcgacg ccgccacagt ggacgactct 360
ggagagtaca ggtgccagac aaacctgagc accctgtctg accccgtgca gcttgaagtg 420
catatcggct ggctgttgct gcaggcccct aggtgggtgt tcaaggagga ggaccctatt 480
cacctgaggt gtcacagctg gaagaacacc gccctgcaca aggtgaccta cctgcagaac 540
ggcaagggca ggaagtactt ccaccacaac agcgacttct acatccccaa ggccaccctg 600
aaggacagcg gcagctactt ctgcaggggc gtgttcggca gcaagaacgt gagcagcgag 660
accgtgaaca tcaccattac ccagggcctg gccgtgagca ccatcagcag cttcttccct 720
cccggctacc agatctacat ctgggccccc ttggccggca cctgcggcgt gctgctgctg 780
agcctggtga tcaccaagag gggcagaaag aagctgctgt acatcttcaa gcagcccttc 840
atgagacccg tgcagaccac ccaggaggag gacggctgca gctgccggtt ccccgaagag 900
gaggagggcg gctgcgagct gagagtgaag ttcagcagga gcgccgacgc ccccgcctac 960
cagcagggcc agaaccagct gtacaacgag ctgaacctgg gcaggagaga ggagtacgac 1020
gtgctggaca agagaagggg cagggacccc gagatgggcg gcaagcccag aaggaagaac 1080
ccccaggagg gcctgtacaa cgagctgcag aaggacaaga tggccgaggc ctacagcgag 1140
atcggcatga agggcgagag gaggaggggc aagggccacg acggcctgta ccagggcctg 1200
agcaccgcca ccaaggacac ctacgacgcc ctgcacatgc aggccctgcc ccctaggtga 1260
<210> 50
<211> 419
<212> PRT
<213> Artificial Sequence
<400> 50
Met Gly Gly Gly Ala Gly Glu Arg Leu Phe Thr Ser Ser Cys Leu Val
1 5 10 15
Gly Leu Val Pro Leu Gly Leu Arg Ile Ser Leu Val Thr Cys Pro Leu
20 25 30
Gln Cys Gly Ile Met Trp Gln Leu Leu Leu Pro Thr Ala Leu Leu Leu
35 40 45
Leu Val Ser Ala Gly Met Arg Thr Glu Asp Leu Pro Lys Ala Val Val
50 55 60
Phe Leu Glu Pro Gln Trp Tyr Arg Val Leu Glu Lys Asp Ser Val Thr
65 70 75 80
Leu Lys Cys Gln Gly Ala Tyr Ser Pro Glu Asp Asn Ser Thr Gln Trp
85 90 95
Phe His Asn Glu Ser Leu Ile Ser Ser Gln Ala Ser Ser Tyr Phe Ile
100 105 110
Asp Ala Ala Thr Val Asp Asp Ser Gly Glu Tyr Arg Cys Gln Thr Asn
115 120 125
Leu Ser Thr Leu Ser Asp Pro Val Gln Leu Glu Val His Ile Gly Trp
130 135 140
Leu Leu Leu Gln Ala Pro Arg Trp Val Phe Lys Glu Glu Asp Pro Ile
145 150 155 160
His Leu Arg Cys His Ser Trp Lys Asn Thr Ala Leu His Lys Val Thr
165 170 175
Tyr Leu Gln Asn Gly Lys Gly Arg Lys Tyr Phe His His Asn Ser Asp
180 185 190
Phe Tyr Ile Pro Lys Ala Thr Leu Lys Asp Ser Gly Ser Tyr Phe Cys
195 200 205
Arg Gly Val Phe Gly Ser Lys Asn Val Ser Ser Glu Thr Val Asn Ile
210 215 220
Thr Ile Thr Gln Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro
225 230 235 240
Pro Gly Tyr Gln Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly
245 250 255
Val Leu Leu Leu Ser Leu Val Ile Thr Lys Arg Gly Arg Lys Lys Leu
260 265 270
Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln
275 280 285
Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly
290 295 300
Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr
305 310 315 320
Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg
325 330 335
Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met
340 345 350
Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu
355 360 365
Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys
370 375 380
Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu
385 390 395 400
Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu
405 410 415
Pro Pro Arg
<210> 51
<211> 2185
<212> RNA
<213> Artificial Sequence
<400> 51
aaaauccguu gaccuuaaac ggucgugugg guucaagucc cuccaccccc acgccggaaa 60
cgcaauagcc gaaaaaacaa aaacaaaaaa aacaaaaaaa caaaaaaaaa accaaaacac 120
auuaaaacag ccuguggguu gaucccaccc acagggccca cugggcgcua gcacucuggu 180
aucacgguac cuuugugcgc cuguuuuaua cuuccucccc caacugcaac uuagaaguaa 240
cacaaaccga ucaacaguca gcguggcaca ccagccacgu uuugaucaaa cacuucuguu 300
accccggacu gaguaucaau agacugcuca cgcgguugaa ggagaaaacg uucguuaucc 360
ggccaacuac uucgagaaac cuaguaacgc cauggaaguu guggaguguu ucgcucagca 420
cuaccccagu guagaucagg uugaugaguc accgcauucc ccacggguga ccguggcggu 480
ggcugcguug gcggccugcc cauggggaaa cccaugggac gcucuuauac agacauggug 540
cgaagagucu auugagcuag uugguagucc uccggccccu gaaugcggcu aaucccaacu 600
gcggagcaua cacucucaag ccagagggua gugugucgua augggcaacu cugcagcgga 660
accgacuacu uugggugucc guguuucauu uuauuccuau acuggcugcu uauggugaca 720
auugagagau uguuaccaua uagcuauugg auuggccauc cggugacuaa cagagcuauu 780
auauaucuuu uuguuggguu uauaccacuu agcuugaaag agguuaaaac ucuacauuac 840
auuuuaauac ugaacaccgc aaaaugggcg gcggcgccgg cgagaggcug uucaccagca 900
gcugccuggu gggccuggug ccccugggcc ugaggaucag ccuggugacc ugcccccugc 960
agugcggcau cauguggcag cugcugcugc ccaccgcccu gcugcugcuc gugagcgccg 1020
gcaugaggac cgaggaccug cccaaggccg ugguguuccu ggagccccag ugguacaggg 1080
ugcuggagaa ggacagcgug acccugaagu gccagggcgc cuacagcccc gaggacaaca 1140
gcacccagug guuccacaac gagagccuga ucagcagcca ggccagcagc uacuucaucg 1200
acgccgccac aguggacgac ucuggagagu acaggugcca gacaaaccug agcacccugu 1260
cugaccccgu gcagcuugaa gugcauaucg gcuggcuguu gcugcaggcc ccuagguggg 1320
uguucaagga ggaggacccu auucaccuga ggugucacag cuggaagaac accgcccugc 1380
acaaggugac cuaccugcag aacggcaagg gcaggaagua cuuccaccac aacagcgacu 1440
ucuacauccc caaggccacc cugaaggaca gcggcagcua cuucugcagg ggcguguucg 1500
gcagcaagaa cgugagcagc gagaccguga acaucaccau uacccagggc cuggccguga 1560
gcaccaucag cagcuucuuc ccucccggcu accagaucua caucugggcc cccuuggccg 1620
gcaccugcgg cgugcugcug cugagccugg ugaucaccaa gaggggcaga aagaagcugc 1680
uguacaucuu caagcagccc uucaugagac ccgugcagac cacccaggag gaggacggcu 1740
gcagcugccg guuccccgaa gaggaggagg gcggcugcga gcugagagug aaguucagca 1800
ggagcgccga cgcccccgcc uaccagcagg gccagaacca gcuguacaac gagcugaacc 1860
ugggcaggag agaggaguac gacgugcugg acaagagaag gggcagggac cccgagaugg 1920
gcggcaagcc cagaaggaag aacccccagg agggccugua caacgagcug cagaaggaca 1980
agauggccga ggccuacagc gagaucggca ugaagggcga gaggaggagg ggcaagggcc 2040
acgacggccu guaccagggc cugagcaccg ccaccaagga caccuacgac gcccugcaca 2100
ugcaggcccu gcccccuagg ugaaaaaaaa caaaaaaaca aaacaaacgg cuauuaugcg 2160
uuaccggcga gacgcuacgg acuua 2185
<210> 52
<211> 25
<212> DNA
<213> Artificial Sequence
<400> 52
aaaaaaacaa aaaaacaaaa caaac 25
<210> 53
<211> 22
<212> DNA
<213> Artificial Sequence
<400> 53
aaaaacaaaa aacaaaacaa ac 22