CN114729373A - 一种新型冠状病毒mRNA疫苗及其制备方法与应用 - Google Patents

一种新型冠状病毒mRNA疫苗及其制备方法与应用 Download PDF

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CN114729373A
CN114729373A CN202280001268.XA CN202280001268A CN114729373A CN 114729373 A CN114729373 A CN 114729373A CN 202280001268 A CN202280001268 A CN 202280001268A CN 114729373 A CN114729373 A CN 114729373A
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胡勇
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Shenzhen Ruiji Biotechnology Co ltd
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Abstract

提供了一种新型冠状病毒mRNA疫苗及其制备方法与应用。本发明提供一种能编码目标多肽的mRNA分子，其中，所述的目标多肽包括SARS‑CoV‑2刺突蛋白S中NTD‑RBD天然结构域，所述NTD‑RBD天然结构域包括NTD片段与RBD片段，NTD片段与RBD片段之间以来源于S蛋白的天然氨基酸序列作为连接子连接。本发明提供了编码SARS‑CoV‑2中刺突蛋白的NTD‑RBD天然结构域的mRNA，实现了对新型冠状病毒突变株的免疫效果，适用性广。

Description

一种新型冠状病毒mRNA疫苗及其制备方法与应用
技术领域
本发明是关于一种mRNA疫苗及其制备方法与应用，具体是一种针对新型冠状病毒(包括原始株和/或突变株)的mRNA疫苗、其制备方法与相关应用。
背景技术
mRNA疫苗技术可以作为快速灵活的技术平台，有效应对各种新兴病毒的威胁。因此mRNA疫苗被认为是对抗新型冠状病毒(SARS-CoV-2)最有潜力的一种疫苗。随着化学修饰mRNA和纳米脂质载体技术的发展和成熟，mRNA在多种病毒疫苗研发中的优势愈发凸显。
由于免疫反应的特异性，靶抗原表位序列(包括B细胞表位和T细胞表位)直接决定了免疫反应的种类。鉴于B细胞免疫和T细胞免疫都在SARS-CoV-2病毒的清除中不可或缺，尽可能去选择合适的免疫原性片段，是新型冠状病毒疫苗设计的关键一步。在设计针对SARS-CoV-2突变株的靶抗原时，需要考虑到有些抗原表位会诱导出非中和抗体，这类抗体不能起到有效的保护作用，反而有概率引起ADE效应，加重病毒的感染程度。因此在这种情况下，全长S蛋白能否继续作为疫苗抗原值得深入研究探讨。经过研究COVID-19病人体内的血清发现S蛋白的NTD片段和RBD片段含有多个B细胞和T细胞有效表位，并能够引发强烈的保护性抗病毒免疫，这也进一步证实了设计新型冠状病毒疫苗时进行抗原表位分析的重要性。
WO2021159040A9公开了一种编码包含新型冠状病毒S蛋白NTD和RBD区域的mRNA疫苗，在氨基酸序列设计中，使用了甘氨酸-丝氨酸连接子(glycine-serine linker)对NTD和RBD区域进行连接。
另一方面，现已发现新型冠状病毒新型变异株，并且世界范围内的流行毒株发生了明显变化，其不同程度的免疫逃逸对现有新型冠状病毒疫苗和后续疫苗研发工作带来了挑战。
发明内容
本发明的一个目的在于提供一种针对新型冠状病毒的mRNA疫苗。
本发明的另一个目的在于提供针对新型冠状病毒的mRNA疫苗的制备方法。
本发明的另一个目的在于提供针对新型冠状病毒的mRNA疫苗的DNA模板。
本发明的另一个目的在于提供针对新型冠状病毒的mRNA疫苗的应用。
一方面，本发明提供了一种能编码目标多肽的mRNA分子，其中，所述的目标多肽包括SARS-CoV-2刺突蛋白S中NTD-RBD天然结构域，所述NTD-RBD天然结构域包括NTD片段与RBD片段，NTD片段与RBD片段之间以来源于S蛋白的天然氨基酸序列作为连接子连接。
根据本发明的具体实施方案，本发明提供的mRNA分子，还编码NTD-RBD天然结构域N端的信号肽。
根据本发明的具体实施方案，本发明提供的mRNA分子编码的氨基酸序列从N端到C端依次包括信号肽、NTD片段、连接子、RBD片段。
在本发明的一些具体实施方案中，所述信号肽包括但不限于：如SEQ ID NO:1第1-12位氨基酸组成的序列(MFVFLVLLPLVS)。
根据本发明的具体实施方案，本发明提供的mRNA分子，所编码的NTD-RBD天然结构域中，所述的连接子的氨基酸序列为SEQ ID NO:50。
根据本发明的具体实施方案，本发明提供的mRNA分子，所编码的NTD-RBD天然结构域中，NTD片段的氨基酸序列选自：
(a)SEQ ID NO:49的第1至第289位组成的氨基酸序列；
(b)由(a)的氨基酸序列经过替换、增加和/或缺失一个或几个氨基酸且具有与(a)相同功能的衍生序列。
根据本发明的具体实施方案，本发明提供的mRNA分子，所编码的NTD-RBD天然结构域中，RBD片段的氨基酸序列选自：
(c)SEQ ID NO:49的第304至第526位组成的氨基酸序列；
(d)由(c)的氨基酸序列经过替换、增加和/或缺失一个或几个氨基酸且具有与(a)相同功能的衍生序列。
本发明中，所述的“相同功能”是指同样具有免疫原性。
根据本发明的具体实施方案，本发明提供的mRNA分子，所编码的NTD-RBD天然结构域的氨基酸序列为：
SEQ ID NO:44至SEQ ID NO:49中任一序列所示氨基酸序列；或
与SEQ ID NO:44至SEQ ID NO:49中任一序列同一性在92.78％以上的衍生的氨基酸序列。
根据本发明的一些具体实施方案，本发明提供的mRNA分子，所编码的NTD-RBD天然结构域的氨基酸序列为SEQ ID NO:49、SEQ ID NO:48、SEQ ID NO:47、SEQ ID NO:46、SEQID NO:45、SEQ ID NO:44中的一种。
根据本发明的具体实施方案，本发明提供的mRNA分子，所编码的氨基酸序列为SEQID NO:21、SEQ ID NO:17、SEQ ID NO:13、SEQ ID NO:9、SEQ ID NO:5或SEQ ID NO:1。
根据本发明的具体实施方案，本发明提供的mRNA分子，其蛋白质编码区序列包括如SEQ ID NO:25的第37-1623位核苷酸组成的序列、SEQ ID NO:26的第37-1623位核苷酸组成的序列、SEQ ID NO:27的第37-1623位核苷酸组成的序列、SEQ ID NO:28的第37-1614位核苷酸组成的序列、SEQ ID NO:29的第37-1614位核苷酸组成的序列、SEQ ID NO:30的第37-1614位核苷酸组成的序列、SEQ ID NO:31的第37-1617位核苷酸组成的序列、SEQ IDNO:32的第37-1617位核苷酸组成的序列、SEQ ID NO:33的第37-1617位核苷酸组成的序列、SEQ ID NO:34的第37-1623位核苷酸组成的序列、SEQ ID NO:35的第37-1623位核苷酸组成的序列、SEQ ID NO:36的第37-1623位核苷酸组成的序列、SEQ ID NO:37的第37-1623位核苷酸组成的序列、SEQ ID NO:38的第37-1623位核苷酸组成的序列、SEQ ID NO:39的第37-1623位核苷酸组成的序列、SEQ ID NO:40的第37-1614位核苷酸组成的序列、SEQ ID NO:41的第37-1614位核苷酸组成的序列或SEQ ID NO:42的第37-1614位核苷酸组成的序列。
根据本发明的具体实施方案，本发明提供的mRNA分子，其蛋白质编码区序列如SEQID NO:25、SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36、SEQID NO:37、SEQ ID NO:38、SEQ ID NO:39、SEQ ID NO:40、SEQ ID NO:41或SEQ ID NO:42所示。
根据本发明的具体实施方案，本发明提供的mRNA分子，经过1-甲基假尿苷修饰。
根据本发明的具体实施方案，本发明提供的mRNA分子，还包括5’-UTR序列和/或3’-UTR序列。
具体地，5’-UTR序列可包含或不包含Kozak序列。在本发明的一些优选实施方案中，所述5'-UTR具有SEQ ID NO:51所示序列。
在本发明的一些优选实施方案中，3’-UTR序列具有SEQ ID NO:52所示序列。
根据本发明的具体实施方案，本发明提供的mRNA分子，还经过3'加尾修饰和/或至少一个5'加帽修饰。
具体地，3'加尾修饰例如包含聚-A尾，所述聚A尾可以是中间插入或不插入连接子的多聚腺苷酸。
具体地，至少一个5'加帽修饰的帽结构可选自Cap0、Cap1、ARCA、肌苷、N1-甲基-鸟苷、2'氟-鸟苷、7-脱氮-鸟苷、8-氧代-鸟苷、2-氨基-鸟苷、LNA-鸟苷或2-叠氮基-鸟苷。
根据本发明的具体实施方案，本发明提供的mRNA分子，其是分离的mRNA；
根据本发明的具体实施方案，本发明提供的mRNA分子，是经纯化的。所述的纯化包括但不限于经过层析、氯化锂或乙醇沉淀、离心柱、氯萃取、乙醇沉淀或凝胶纯化。
另一方面，本发明还提供了一种DNA分子，其编码本发明前述任意一种mRNA分子。
另一方面，本发明还提供了一种重组质粒，其含有本发明前述的DNA分子。
另一方面，本发明还提供了一种脂质纳米颗粒，其负载有本发明前述任意一种mRNA分子。
另一方面，本发明还提供了一种药物组合物，其包含：本发明前述任意一种mRNA分子，以及药学上可接受的赋形剂。所述的赋形剂可选自溶剂、水溶剂、非水溶剂、分散介质、稀释剂、分散剂、悬浮助剂、表面活性剂、等渗剂、增稠剂或乳化剂、防腐剂、脂质、类脂质(lipidoid)脂质体、脂质纳米颗粒、核-壳纳米颗粒、聚合物、脂复合物(lipoplexe)肽、蛋白、细胞、透明质酸酶、及它们的混合物。
另一方面，本发明还提供了本发明所述的mRNA分子、所述的DNA分子、所述的重组质粒、所述的脂质纳米颗粒或所述的药物组合物在制备新型冠状病毒mRNA疫苗中的应用。
另一方面，本发明还提供了一种新型冠状病毒mRNA疫苗，其包含本发明所述的mRNA分子。在本发明的一些具体实施方案中，所述的疫苗为脂质纳米颗粒剂型。
根据本发明的具体实施方案，本发明所述的新型冠状病毒mRNA疫苗，其中，脂质纳米颗粒的粒径为50nm～200nm，优选为50nm～150nm。
根据本发明的具体实施方案，本发明所述的新型冠状病毒mRNA疫苗，其中，所述的脂质纳米颗粒包括mRNA和脂质，其中，所述脂质包括：
a)正电荷脂质和/或可离子化脂质中的一种或多种；
b)中性辅助脂质；
c)胆固醇；
d)PEG修饰的脂质。
根据本发明的具体实施方案，本发明所述的新型冠状病毒mRNA疫苗，其中，所述的正电荷脂质和/或可离子化脂质与mRNA的氮磷摩尔比为5:1～20:1。
根据本发明的具体实施方案，本发明所述的新型冠状病毒mRNA疫苗，所述的脂质纳米颗粒中，以脂质的总摩尔量为100％计，各脂质成分的摩尔比为：
正电荷脂质或可离子化脂质 46％～50％；
中性辅助脂质 5％～10％；
胆固醇 38.5％～48％；
PEG修饰的脂质 0～3％。
根据本发明的具体实施方案，本发明所述的新型冠状病毒mRNA疫苗，所述的脂质纳米颗粒中，所述的可离子化脂质包括但不限于：4-(N,N-二甲基氨基)丁酸(二亚油基)甲酯((Dlin-MC3-DMA)、SM-102、((4-羟基丁基)氮杂二烷基)双(己烷-6,1-二基)双(2-己基癸酸酯)(ALC-0315)中的一种或多种，所述的正电荷脂质包括但不限于：DOTMA、DOTAP中的一种或多种；所述的中性辅助脂质包括但不限于：DSPC、DOPE、DSPE中的一种或多种；所述的PEG修饰的脂质包括但不限于：甲氧基聚乙二醇双十四烷基乙酰胺(ALC-0159)、DMG-PEG中的一种或多种。
根据本发明的具体实施方案，本发明所述的新型冠状病毒mRNA疫苗，为冷冻干燥剂型或冷冻剂型。
另一方面，本发明还提供了一种新型冠状病毒mRNA疫苗的制备方法，其包括制备所述mRNA分子的过程。具体地，包括以下步骤：
合成编码NTD-RBD天然结构域肽段的DNA片段，并克隆到质粒作为模板，转录制备得到目标mRNA分子。
优选地，所述目标mRNA分子为本发明前述的任意一项mRNA分子。
在本发明的一些具体实施方案，本发明提供了制备所述mRNA分子的方法，其中，合成编码NTD-RBD天然结构域肽段的DNA片段的过程可自行合成或委托合成。将所述DNA片段克隆到质粒作为模板进行，可按照如下反应制备目标mRNA分子：
a.三步法：体外转录得到未加帽未加尾的mRNA，在RNA聚合酶和ATP的条件下，在mRNA尾部加上polyA结构(polyA结构中间可插入连接子或不插入连接子)，在加帽酶催化下，以给未加帽的mRNA的5'端加上帽子结构，得到加帽加尾的mRNA分子；
b.两步法：体外转录得到加尾未加帽的mRNA；在加帽酶催化下，以给未加帽的mRNA的5'端加上帽子结构，得到加帽加尾的mRNA分子；
c.一步法：进行体外转录和加帽，得到加帽加尾的mRNA分子。
在本发明的一些具体实施方案，制备所述mRNA分子的方法还包括mRNA的纯化过程：将所述的mRNA进行氯化锂/乙醇沉淀、离心柱、氯萃取/乙醇沉淀或凝胶纯化，以获得纯化后的mRNA。
根据本发明的具体实施方案，本发明的新型冠状病毒mRNA疫苗的制备方法，还包括：
将所制备的mRNA分子溶解在柠檬酸缓冲液组成的水相中，采用微流控或冲击式射流的方法与溶解在乙醇相中的脂质成分混合，制备负载有mRNA的脂质纳米颗粒。其中，所述的脂质成分如前所述可包括可电离的阳离子磷脂(ionizable lipids)，中性辅助磷脂，胆固醇，聚乙二醇修饰的磷脂(PEGylated lipid)等。
根据本发明的具体实施方案，本发明的新型冠状病毒mRNA疫苗的制备方法，还包括将所制备的脂质纳米颗粒制成冷冻制剂或冷冻干燥制剂的过程。
根据本发明的具体实施方案，本发明的新型冠状病毒mRNA疫苗的制备方法，将脂质纳米颗粒制成冷冻干燥制剂的过程包括：
a)配制含有脂质纳米颗粒和冷冻干燥保护剂的缓冲液；
b)降温进行预冻；
c)在真空条件下升温进行干燥，使体系含水量在3％以下，制备得到脂质纳米颗粒的干燥制剂。
根据本发明的一些具体实施方案，本发明的新型冠状病毒mRNA疫苗的制备方法中，所述的冷冻干燥保护剂包括但不限于以下保护剂1至保护剂3中的一种或多种：
保护剂1：蔗糖；
保护剂2：蔗糖和非离子表面活性剂；
保护剂3：蔗糖和海藻糖。
根据本发明的具体实施方案，本发明的新型冠状病毒mRNA疫苗的制备方法中，所配制的含有脂质纳米颗粒和冷冻干燥保护剂的缓冲液中，蔗糖的质量体积浓度为10％～20％(即，10～20g/100mL)，优选为12～18％；海藻糖的质量体积浓度为0％～20％，优选为0％～5％，更优选为0.5％～3％；非离子表面活性剂的质量体积浓度为0％～2％。在本发明的一些具体实施方案中，所述非离子表面活性剂包括但不限于泊洛沙姆，例如可以是Pluronic F-68。泊洛沙姆在所配制的含有脂质纳米颗粒和冷冻干燥保护剂的缓冲液中的质量体积浓度优选为0％～1％。
本发明中，SEQ ID NO:1、SEQ ID NO:5、SEQ ID NO:9、SEQ ID NO:13、SEQ ID NO:17、SEQ ID NO:21分别为新型冠状病毒原始株、Alpha株、Beta株、Gamma株、Delta株和Omicron株的NTD-RBD抗原的氨基酸序列。
SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4分别为新型冠状病毒原始株的人源、小鼠、大鼠密码子优化的DNA片段序列；SEQ ID NO:25、SEQ ID NO:26、SEQ ID NO:27为其对应的mRNA编码区序列。
SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8分别为新型冠状病毒Alpha株的人源、小鼠、大鼠密码子优化的DNA片段序列；SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30为其对应的mRNA编码区序列。
SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12分别为新型冠状病毒Beta株的人源、小鼠、大鼠密码子优化的DNA片段序列；SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33为其对应的mRNA编码区序列。
SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16分别为新型冠状病毒Gamma株的人源、小鼠、大鼠密码子优化的DNA片段设计；SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36为其对应的mRNA编码区序列。
SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20分别为新型冠状病毒Delta株的人源、小鼠、大鼠密码子优化的DNA片段设计；SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:39为其对应的mRNA编码区序列。
SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:24分别为新型冠状病毒Omicron株的人源、小鼠、大鼠密码子优化的DNA片段设计；SEQ ID NO:40、SEQ ID NO:41、SEQ ID NO:42为其对应的mRNA编码区序列。
SEQ ID NO:43为新型冠状病毒Delta株RBD抗原的mRNA序列。
SEQ ID NO:44、SEQ ID NO:45、SEQ ID NO:46、SEQ ID NO:47、SEQ ID NO:48、SEQID NO:49分别为原始株、Alpha株、Beta株、Gamma株、Delta株和Omicron株的NTD-RBD天然结构域的氨基酸序列。
SEQ ID NO:50是NTD-RBD天然结构域中连接子的氨基酸序列。
本发明中，除特别注明以及结合上下文能明确确定外，所描述的新型冠状病毒包括原始株和/或突变株。
在本发明的一些具体实施方案中，本发明先通过对新型冠状病毒(SARS-CoV-2)进行抗原表位分析，确定以NTD-RBD为抗原靶点，LNP为递送载体，免疫方式为肌肉注射的mRNA疫苗剂型，同时通过UTR和密码子优化、体外转录方法合成编码病毒抗原片段的mRNA，最终实现人源细胞高效表达，并且对目前流行的主要突变株均具有保护作用；整个mRNA疫苗生产周期短、工艺操作简单、生产成本低，保存时间久，不需要冷链，便于运输。传统疫苗对很多新型病毒引起的公共卫生事件无法做到快速响应，而mRNA疫苗适用性更广，序列可以灵活设计，以应对不同的病原体，针对快速编译的急性传染病疫苗快速研发有及其重要的作用。
本发明设计的以NTD-RBD为抗原的mRNA疫苗，能在相同接种量的条件下，引起更强的中和抗体效应。
附图说明
图1显示本发明一具体实施例中所制备得到的mRNA-LNP的粒径和PDI测试结果。
图2显示本发明一具体实施例中所制备得到的mRNA-LNP的包封率测试结果。
图3显示本发明一具体实施例中所制备得到的mRNA冻干疫苗外观。
图4显示本发明一具体实施例中所制备得到的mRNA冻干疫苗体外活性效果。
图5显示本发明一具体实施例中所制备得到的mRNA冻干疫苗体内活性效果。
图6显示本发明的mRNA疫苗的特异性抗体滴度测试结果。
图7显示RBD和NTD-RBD抗原的免疫原性对比。
图8显示本发明的NTD-RBD mRNA疫苗对不同毒株的交叉保护效果。
图9显示本发明的NTD-RBD疫苗诱导强烈的细胞免疫。
图10显示本发明的NTD-RBD疫苗对ACE2小鼠体内攻毒实验结果。
图11显示本发明的NTD-RBD疫苗作为加强针的实验结果。
图12为本发明一些具体实施例中的mRNA诱导表达的蛋白相对表达量实验结果。
具体实施方式
为了对本发明的技术特征、目的和有益效果有更加清楚的理解，现对本发明的技术方案进行以下详细说明，但不能理解为对本发明的可实施范围的限定。
通过下面的实施例可以对本发明进行进一步的描述，然而，本发明的范围并不限于下述实施例。本领域的专业人员能够理解，在不背离本发明的精神和范围的前提下，可以对本发明进行各种变化和修饰。本发明对试验中所使用到的材料以及试验方法进行一般性和/或具体的描述。虽然为实现本发明目的所使用的许多材料和操作方法是本领域公知的，但是本发明仍然在此作尽可能详细描述。未详细注明的方法操作，按照所属领域现有技术的常规操作或商厂说明书建议的操作进行。
实施例1：mRNA疫苗及其制备方法
本实施例提供了mRNA疫苗，其制备方法主要按照以下操作进行。
1、根据待合成的mRNA合成对应的DNA片段，并将所述DNA片段克隆至表达质粒获得重组质粒，将所述重组质粒转入宿主细胞获得重组细胞，从扩繁后的重组细胞中提取质粒。
对构建好的质粒进行酶切线性化，酶切反应体系如下(以酶切2μg的重组质粒为例)：
10×Digestion Buffer I 2μl
BspQI(10U/μl)(购买自诺唯赞) 1μl
Plasmid DNA 2μg
RNase-free Water 补水至20μl
将上述酶切体系置于50℃反应1h。待反应结束后，取酶切反应前后体系各1μl进行DNA琼脂糖凝胶电泳(1.5％琼脂糖凝胶，5V/min，40min)。根据电泳结果比较显示，重组质粒是否酶切完全。
合格标准：电泳检测出现单一条带；与酶切前的超螺旋质粒比较，条带位于超螺旋质粒上方；大小符合预期要求。
测定结果：条带单一；大小符合预期且条带位于超螺旋质粒上方。
2、DNA模板超滤
利用Millipore 30Kd超滤管浓缩上述获得的DNA模板。
3、DNA模板FPLC纯化
将上述超滤得到的DNA浓缩液，加入等体积的苯酚/氯仿/异戊醇混合液(苯酚/氯仿/异戊醇体积比＝25/24/1)，充分震荡后，12000g离心15min。
去掉沉淀，转移上清至新的离心管中，加入上清体积的1/10 3M NaAc(pH5.2)，混匀，然后加入2倍体积的无水乙醇，混匀，至于-20℃静置30min。
4℃，12000g离心10min，弃上清。
用70％乙醇洗涤沉淀，12000g离心5min，取上清，于超净台晾干5min。
用适当的RNase-free水溶解纯化后的DNA模板。
用NanoDrop检测纯化后模板的浓度，以及260/280、260/230的比值。取样进行DNA琼脂糖凝胶电泳检测(1.5％琼脂糖，5V/min，40min)。
合格标准：260/280介于1.8至2.1之间，260/230在1.6至2.2之间。
测定结果：浓度为500ng/μl，260/280＝1.90，260/230＝1.7。
4、FPLC纯化后模板超滤
Millipore 30Kd超滤管浓缩FPLC纯化后的DNA模板，用RNase-free水洗脱溶解。用NanoDrop检测超滤后模板的浓度，以及260/280、260/230的比值。最终用RNase-free水稀释至150ng/μl。
测定结果：浓度为150ng/μl，260/280＝1.95，260/230＝1.85。
5、mRNA的体外合成
在恒温反应器中，进行mRNA的体外合成。
按照如下合成体系进行(反应试剂按照先后顺序添加)：
反应体积，1600μl(置于2ml RNase-free Tube管中，为单个管的反应体积，一次同时反应多管)：RNA-free水440μl、7.5mM的ATP 160μl、7.5mM的N1-甲基-假尿苷160μl、7.5mM的CTP 160μl、7.5mM的GTP 160μl、7.5mM的M7G(2’OMeA)pG 160μl、150ng/μl的DNA模板40μl、10×Buffer 160μl和Enzyme Mix 160μl。
所述RNA体外合成的程序为37℃，10h。
经过体外转录和加帽，得到目标mRNA分子。
本发明各具体实验例中，目标mRNA分子除编码序列外，还包括5’UTR(AGGGAGAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCGCCACC，SEQ ID NO:51)和3’UTR(GCUGCCUUCUGCGGGGCUUGCCUUCUGGCCAUGCCCUUCUUCUCUCCCUUGCACCUGUACCUCUUGGUCUUUGAAUAAAGCCUGAGUAGGAAG，SEQ ID NO:52)，5’CAP为m7G+-5'-ppp-5'-Am2'-3'-p-(cap1)，3’加聚-A尾(SEQ IDNO:53)。
6、DNase I消化去除DNA模板
向mRNA体外合成后的每个Tube管中各加入120μl DNase I。
上下颠倒10次混匀，1000rpm离心10s。
重新置于恒温反应器中，37℃，1h。
反应结束后，将反应液合并到RNase-free 50ml Tube管中，检测DNA片段的残留。三次测量结果为0.013ng，0.016ng，0.017ng每100μg mRNA。
7、mRNA沉淀回收
向上一步骤中的每个50ml Tube管中，加入等体积的醋酸铵溶液。
上下颠倒10次混匀。
置于-20℃2h，沉淀。
17000g，4℃离心，30min。
去掉上清，用70％乙醇洗涤沉淀。
17000g，4℃离心，10min。
去掉70％乙醇，于超净台中蒸干，每管加入RNase-free水20ml。
静置10min后，用枪头轻吹混匀。
用NanoDrop检测回收后的mRNA浓度为5μg/μl，A260/A280为1.90、A260/A230为2.0。
取1μl，稀释10倍，进行RNA ScreenTape assay以及琼脂糖凝胶电泳检测其片段完整性。
检测结果为：条带符合大小，片段完整。
8、LiCl沉淀纯化mRNA
将上一步骤中回收的mRNA按照其1.5倍体积加入Rnase-free水，混匀。
加入原mRNA 1.5倍体积-20预冷的LiCl溶液，混匀。
然后于-20℃静至2h。
16000g离心20min。
弃上清，用70％乙醇洗涤沉淀，16000g离心15min。
取上清，于超净台晾干5min。
用适当的RNase-free水溶解纯化后的mRNA。
纯化后的mRNA用0.1M柠檬酸稀释至2μg/μl。
9、LNP制备
配制水相：将mRNA按照2μg/ul的终浓度稀释于柠檬酸buffer；
按照表1配制乙醇相溶液；
表1
组分 配制1mL所需组分/μL 配制0.5mL所需组分/μL
ALC-0315 559.7 279.8
ALC-0159 117.2 58.6
DSPC 260.5 130.2
Cholesterol 62.7 31.3
准备好PBS作为LNP稀释液；
注射泵仪器操作步骤：
(1).将A相(mRNA缓冲液)装入5mL注射器，B相(脂质化合物溶于无水乙醇)装入5mL注射器，安装于注射泵，夹紧；
(2).将芯片连接到注射器，设定注射泵流速；
(3).点击注射泵的开始按键，将料液注入芯片；
(4).观察芯片出口的产品颜色，弃去前5滴乳白色液滴(约为100ul)后，开始收集到60mL PBS中；
(5).收集完成的产品，轻柔地上下颠倒混匀，保存于4℃；
(6).取0.3mL作为包封率检测样品；
(7).裂解：取步骤(6)的0.1mL，加入2μL 10％Triton X-100，混匀，室温孵育10min；
(8).包封率检测：分别取64μL步骤(6)和步骤(7)的样品稀释5倍，作为裂解前后的LNP RNA样品；测定RNA浓度；用裂解前后浓度差值除以裂解后浓度，得出包封率；
(9).于马尔文公司的Zetasizer nano仪器上使用标准检测方法进行粒径与PDI检测、Zeta电位分析，上样体积为600μl，样品池为DTS1070，检测温度为25℃。
本实施例所制备得到的负载mRNA的LNP的粒径、PDI和包封率的检测结果如图1、图2所示。图1、图2中，横坐标编号分别对应各mRNA的序列编号，即，图中25对应的样品为SEQID NO:25的mRNA样品，图中26对应的样品为SEQ ID NO:26的mRNA样品，图中27对应的样品为SEQ ID NO:27的mRNA样品，以此类推，图中42对应的样品为SEQ ID NO:42的mRNA样品。
10.LNP冻干
LNP溶液加入的冻干保护剂，放入冷冻干燥机，质量体积分数(w/v％)为蔗糖15％、海藻糖2％。(新芝Scientz-10N)冷阱预冻4小时后，开启真空抽真空48小时，随后样品转移到冷冻干燥机上层，二次干燥16小时。在冷阱中干燥时样品温度探头显示-30℃左右，在上层干燥时样品温度探头显示为4℃左右。干燥结束后收集产物(即LNP冻干粉)，使用与冻干前等体积的超纯水复溶，加入超纯水后冻干粉迅速溶解，整个过程不超过20s，用RiboGreenTM测定纳米颗粒包封率，欧美克NS-90Z纳米粒度及电位分析仪测定纳米颗粒粒径及zeta电位。制备得到本实施例的mRNA冻干疫苗。所制备得到的疫苗的成品图及复溶后外观参见图3所示。分别将37#(对应SEQ ID NO:37的mRNA样品)2μg mRNA-LNP溶液、冻干复溶液和GFP mRNA-LNP孵育培养的293T细胞，24小时后取细胞沉淀，裂解细胞，收获可溶性蛋白，进行Westernblot(蛋白免疫印迹)，检测蛋白表达效率，以及将37#(对应SEQ ID NO:37的mRNA样品)100μg mRNA-LNP粉末在注射接种前用200μl注射用水复溶，在第1天和第14天两次注射接种6周龄balb/c小鼠，在第35天取小鼠血清后检测血清中抗S蛋白特异性抗体的滴度，结果如图4和图5所示，冻干工艺基本不影响mRNA脂质颗粒的生物学活性。
实施例2
本实施例中，分别将25#～42#(即分别对应SEQ ID NO:25～SEQ ID NO:42的mRNA样本)100μg mRNA-LNP粉末在注射接种前用200μl注射用水复溶，在第1天和第14天两次注射接种6周龄balb/c小鼠，在第35天取小鼠血清，检测血清中抗S蛋白特异性抗体的滴度。具体按照以下操作进行：
1、包被：将S1蛋白(义翘神州，40591-MM43)用coating buffer稀释成200ng/ml的溶液，加入到酶标板中，每孔加入的体积为100μl，每个稀释度重复3个孔，盖上封板膜，4℃过夜包被。
2、洗板：包被好的96孔板，倒出包被液，到扣吸水纸上，使劲扣板，直到孔中无残留为止。配制洗脱液，用去离子水稀释50x的Washing buffer，加入到洗板机的进液瓶中，设置程序，每孔洗板体积设为300μl，重复洗4次。
3、封闭：洗好的板，扣干里面的溶液，按每孔250μl的体积加入Blocking buffer，随后封上封板膜，室温封闭2h。
4、洗板：封闭好的酶标板按照步骤2完成洗板。
5、血清孵育：用dilution buffer将小鼠血清稀释成40x，400x，4000x，40000x，400000x，4000000x，40000000x，按照每孔100μl的体积加入洗好的96孔板中，随后封上封板膜室温下孵育1.5h。
6、洗板：按照步骤2完成洗板，其中洗板次数增加为6次。
7、加入二抗：用Dilution buffer稀释HRP标记山羊抗小鼠IgG，稀释倍数为10000x，稀释好的抗体按照每孔100μl的体积加入酶标板中，封上封板膜，室温下避光孵育1h。
8、洗板：按照步骤2完成洗板，此步骤务必洗干净板，扣干溶液。
9、显色：加入TMB buffer 100μl，避光显色20-30分钟，此时阳性样品显蓝色。
10、终止：加入Stop buffer 100μl，10分钟内在酶标板上读数，设置吸收波长450nm。
根据标准品孔光吸收值绘制标准曲线，线性回归的相关系数应大于0.0995。根据供试品吸光度的大小计算样品中S蛋白特异性抗体的残留含量。
特异性抗体滴度测试结果参见图6所示。结果显示，编码NTD-RBD的mRNA候选疫苗免疫效果具有良好的免疫原性，诱导产生了针对S蛋白的特异性抗体，对照组注射生理盐水的小鼠不能产生针对S蛋白的特异性抗体。
实施例3
本实施例中，通过实验考察了编码RBD的mRNA疫苗(43#，即对应SEQ ID NO:43的样本)和编码NTD-RBD疫苗(37#，即对应SEQ ID NO:37的样本)的中抗体滴度。具体按照以下操作进行：
接种疫苗，分别在0d、14d时，对6～8周龄的雌性小鼠接种5μg新型冠状病毒mRNA疫苗(溶解于PBS，200μl，肌肉注射)；28d时取小鼠外周血。
准备Vero E6细胞(24孔板)，血清热灭活，连续3倍梯度稀释。100μl血清与100μl病毒储存液(100PFU)等体积混匀，同时100μl DMEM(含2％FBS)与100μl病毒液等体积混匀，作为阴性对照。37℃孵育1h。
将上述血清-病毒混合液(共200μl)转移至24孔板Vero E6细胞，吸附1h。期间可轻柔混匀3-4次。移除上一步的吸附液，更换甲基纤维素培养基，培养3天。多聚甲醛固定、结晶紫染色，空斑计数。以阴性对照为基准，计算血清中和百分比。以JMP分析软件Probit方法进行拟合曲线，计算PRNT50值。
实验结果如图7所示，NTD-RBD诱导的中和抗体滴度高于RBD，因此NTD-RBD的免疫原性由于RBD。
实施例4
本实施例中，通过实验考察了NTD-RBD mRNA疫苗对2019-nCoV假病毒的交叉免疫保护。具体按照以下操作进行：
接种疫苗，分别在0d、14d时，对6～8周龄的雌性小鼠接种5μg新型冠状病毒mRNA疫苗(溶解于PBS，200μl，肌肉注射)；28d时取小鼠外周血，采用微量中和实验对热灭活血清进行测定，以检测抗体中和新型冠状假病毒侵染表达ACE2单层细胞的水平高低；于96孔板上稀释4孔，在第3天和第4天时检测细胞的病毒性病变作用(cpe)，通过Reed Muench公式计算出完全抑制cpe的血清在50％终点法中测定的血清稀释度；采用非参数双尾t检测(Mann-Whiteny)进行统计分析。
实验结果如图8所示(图中各图案的数字编号分别为对应相同SEQ ID NO编号的样本)，本发明所构建的mRNA疫苗对新型冠状病毒突变株均有不同程度的交叉保护效果。
实施例5：NTD-RBD新型冠状疫苗诱导强烈的细胞免疫
本实施例中，考察了NTD-RBD新型冠状疫苗诱导细胞免疫的作用。具体按照以下操作进行：
一、包被96孔板
用70％乙醇浸润96孔板中的PVDF膜30s。
加入捕获抗体(PBS稀释)，4℃过夜。
倒空板中包被液，轻轻在纸上拍干，用PBS洗涤，禁用洗板机。
加入100ul 2％的脱脂奶粉(或者BSA)室温孵育2小时，封闭板中空白位点。
PBS洗涤1次。
二、细胞刺激和细胞因子捕获
接种疫苗，分别在0d、14d时，对6～8周龄的雌性小鼠接种5μg新型冠状病毒mRNA疫苗RH109(40#)(溶解于PBS，200μl，肌肉注射)；28d时取小鼠外周血，从新鲜血液中用Ficoll分离PBMC并进行计后，将细胞用培养液稀释后加入96孔板中。通常所用细胞数量为1-2×105/孔。
将96孔板在37℃CO2孵箱中孵育过夜。禁止移动或晃动板子。
培养板中加入S蛋白pepmix刺激物孵育8小时；
用含0.1％Tween 20的PBS孵育10分钟，去除细胞和未结合的细胞因子。然后用含0.1％Tween 20的PBS洗涤3次。
三、加入检测抗体检测
加入标记的检测抗体(用含1％BSA的PBS稀释)，室温孵育1-2小时。
加入底物显色，在加入底物之前用蒸馏水洗涤板子膜的两侧，以防止部分溶液漏出后导致的背景。监测斑点的形成，适时终止反应。
用蒸馏水洗涤终止反应。
四、结果分析
将96孔板干燥。(将板子放在4℃避光过夜，可使斑点边缘锐化，更易分辨)
使用读板仪进行结果分析，结果如图9所示，ELISPot实验所示，多肽刺激后，疫苗组小鼠能够产生大量的IFN-γ斑点，达到3000spots/100万细胞，而生理盐水组几乎没有斑点产生，说明RH109激发了强细胞免疫反应。
用流式细胞术胞内染色实验对CD4和CD8细胞进行进一步分析，发现IL2+/TNF-α+/IFN-γ+的CD4+细胞，以及TNF-α+/IFN-γ+的CD8+细胞明显升高，说明RH109疫苗激活了强Th1型和CD8杀伤性T细胞免疫反应。
实施例6
接种疫苗，分别在0d、21d时，对6～8周龄的雌性小鼠接种5μg或10ug新型冠状病毒mRNA疫苗(37#)(溶解于PBS，200μl，肌肉注射)；ivD为攻毒期，ivD0为攻毒当天，新型冠状病毒(Delta株)滴鼻感染hACE2转基因小鼠，感染剂量初步为105PFU。D42为攻毒时间。攻毒后ivD3和ivD5，分批将动物安乐死，动物安乐死后取肺(右肺)等组织器官，提取RNA，用Q-PCR法测定病毒载量或检测活病毒滴度。期间每天两次观察，每周称量一次体重。病毒载量及生存曲线如图10所示。由图10可知，接种疫苗后的小鼠在进行真病毒攻毒实验后，肺部病毒载量显著低于对照组。佐剂对照组小鼠于攻毒后第7天均达到安乐死标准。与佐剂对照组相比，疫苗低剂量组和疫苗高剂量组的小鼠全部存活，表明疫苗对SARS-CoV-2病毒Delta毒株感染小鼠有显著的保护作用。
实施例7
接种疫苗，分别在0d、21d时，对6～8周龄的雌性小鼠接种两针科兴灭活疫苗，42d时接种5μg新型冠状病毒mRNA疫苗RH109(溶解于PBS，200μl，肌肉注射)；56d时取小鼠外周血，采用微量中和实验对热灭活血清进行测定，以检测抗体中和新型冠状假病毒侵染表达ACE2单层细胞的水平高低；于96孔板上稀释4孔，在第3天和第4天时检测细胞的病毒性病变作用(cpe)，通过Reed Muench公式计算出完全抑制cpe的血清在50％终点法中测定的血清稀释度；采用非参数双尾t检测(Mann-Whiteny)进行统计分析。如图11所示，RH109免疫后的血清对Omicron，Delta,原始毒株均产生了高中和抗体水平，几何平均滴度分别是7970、9091、13998，其中针对Omicron比第二针后提高了39倍数；同时，相比于用科兴灭活苗作为第三针加强，对Omicron、Detla和原始毒株分别是后者的倍35、6.2、3.4倍，说明RH109非常适合作为灭活苗基础上的加强针使用。另，本发明还比较了本发明的不同mRNA不同剂量诱导表达蛋白实验，分别将SEQ ID NO:28、SEQ ID NO:54、SEQ ID NO:55的mRNA-LNP溶液以2μg、4μg孵育培养的293T细胞，24小时后取细胞沉淀，裂解细胞，平行检测得到的目的蛋白条带进行灰度分析24h胞内蛋白相对表达量。结果参见图12(图中编号28-1对应SEQ ID NO:28、编号30-1对应SEQ ID NO:54、编号30-3对应SEQ ID NO:55)。本发明的mRNA具有良好的蛋白表达效率。
应当指出的是，以上所述仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。
SEQUENCE LISTING
<110> 深圳市瑞吉生物科技有限公司
<120> 一种新型冠状病毒mRNA疫苗及其制备方法与应用
<130> GAI22CN0175P-CN
<150> CN202210101841.6
<151> 2022-01-27
<160> 55
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atgttcgtgt tcctggtgct gctgcctctg gtgagcagcc agtgcgtgaa cctgaccacc 60
agaacccagc tgcctcctgc ctacaccaac agcttcacca gaggcgtgta ctaccctgac 120
aaggtgttca gaagcagcgt gctgcacagc acccaggacc tgttcctgcc tttcttcagc 180
aacgtgacct ggttccacgc catccacgtg agcggcacca acggcaccaa gagattcgac 240
aaccctgtgc tgcctttcaa cgacggcgtg tacttcgcca gcaccgagaa gagcaacatc 300
atcagaggct ggatcttcgg caccaccctg gacagcaaga cccagagcct gctgatcgtg 360
aacaacgcca ccaacgtggt gatcaaggtg tgcgagttcc agttctgcaa cgaccctttc 420
ctgggcgtgt actaccacaa gaacaacaag agctggatgg agagcgagtt cagagtgtac 480
agcagcgcca acaactgcac cttcgagtac gtgagccagc ctttcctgat ggacctggag 540
ggcaagcagg gcaacttcaa gaacctgaga gagttcgtgt tcaagaacat cgacggctac 600
ttcaagatct acagcaagca cacccctatc aacctggtga gagacctgcc tcagggcttc 660
agcgccctgg agcctctggt ggacctgcct atcggcatca acatcaccag attccagacc 720
ctgctggccc tgcacagaag ctacctgacc cctggcgaca gcagcagcgg ctggaccgcc 780
ggcgccgccg cctactacgt gggctacctg cagcctagaa ccttcctgct gaagtacaac 840
gagaacggca ccatcaccga cgccgtggac tgcgccctgg accctctgag cgagaccaag 900
tgcaccctga agagcttcac cgtggagaag ggcatctacc agaccagcaa cttcagagtg 960
cagcctaccg agagcatcgt gagattccct aacatcacca acctgtgccc tttcggcgag 1020
gtgttcaacg ccaccagatt cgccagcgtg tacgcctgga acagaaagag aatcagcaac 1080
tgcgtggccg actacagcgt gctgtacaac agcgccagct tcagcacctt caagtgctac 1140
ggcgtgagcc ctaccaagct gaacgacctg tgcttcacca acgtgtacgc cgacagcttc 1200
gtgatcagag gcgacgaggt gagacagatc gcccctggcc agaccggcaa gatcgccgac 1260
tacaactaca agctgcctga cgacttcacc ggctgcgtga tcgcctggaa cagcaacaac 1320
ctggacagca aggtgggcgg caactacaac tacctgtaca gactgttcag aaagagcaac 1380
ctgaagcctt tcgagagaga catcagcacc gagatctacc aggccggcag caccccttgc 1440
aacggcgtgg agggcttcaa ctgctacttc cctctgcaga gctacggctt ccagcctacc 1500
aacggcgtgg gctaccagcc ttacagagtg gtggtgctga gcttcgagct gctgcacgcc 1560
cctgccaccg tgtgcggccc taagaagagc accaacctgg tgaagaacaa gtgcgtgaac 1620
ttc 1623
<210> 4
<211> 1623
<212> DNA
<213> Artificial Sequence
<220>
<223> 用于制备mRNA疫苗
<400> 4
atgttcgtgt tcctggtgct gctgcccctg gtgagcagcc agtgcgtgaa cctgaccacc 60
aggacccagc tgccccccgc ctacaccaac agcttcacca ggggcgtgta ctaccccgac 120
aaggtgttca ggagcagcgt gctgcacagc acccaggacc tgttcctgcc cttcttcagc 180
aacgtgacct ggttccacgc catccacgtg agcggcacca acggcaccaa gaggttcgac 240
aaccccgtgc tgcccttcaa cgacggcgtg tacttcgcca gcaccgagaa gagcaacatc 300
atcaggggct ggatcttcgg caccaccctg gacagcaaga cccagagcct gctgatcgtg 360
aacaacgcca ccaacgtggt gatcaaggtg tgcgagttcc agttctgcaa cgaccccttc 420
ctgggcgtgt actaccacaa gaacaacaag agctggatgg agagcgagtt cagggtgtac 480
agcagcgcca acaactgcac cttcgagtac gtgagccagc ccttcctgat ggacctggag 540
ggcaagcagg gcaacttcaa gaacctgagg gagttcgtgt tcaagaacat cgacggctac 600
ttcaagatct acagcaagca cacccccatc aacctggtga gggacctgcc ccagggcttc 660
agcgccctgg agcccctggt ggacctgccc atcggcatca acatcaccag gttccagacc 720
ctgctggccc tgcacaggag ctacctgacc cccggcgaca gcagcagcgg ctggaccgcc 780
ggcgccgccg cctactacgt gggctacctg cagcccagga ccttcctgct gaagtacaac 840
gagaacggca ccatcaccga cgccgtggac tgcgccctgg accccctgag cgagaccaag 900
tgcaccctga agagcttcac cgtggagaag ggcatctacc agaccagcaa cttcagggtg 960
cagcccaccg agagcatcgt gaggttcccc aacatcacca acctgtgccc cttcggcgag 1020
gtgttcaacg ccaccaggtt cgccagcgtg tacgcctgga acaggaagag gatcagcaac 1080
tgcgtggccg actacagcgt gctgtacaac agcgccagct tcagcacctt caagtgctac 1140
ggcgtgagcc ccaccaagct gaacgacctg tgcttcacca acgtgtacgc cgacagcttc 1200
gtgatcaggg gcgacgaggt gaggcagatc gcccccggcc agaccggcaa gatcgccgac 1260
tacaactaca agctgcccga cgacttcacc ggctgcgtga tcgcctggaa cagcaacaac 1320
ctggacagca aggtgggcgg caactacaac tacctgtaca ggctgttcag gaagagcaac 1380
ctgaagccct tcgagaggga catcagcacc gagatctacc aggccggcag caccccctgc 1440
aacggcgtgg agggcttcaa ctgctacttc cccctgcaga gctacggctt ccagcccacc 1500
aacggcgtgg gctaccagcc ctacagggtg gtggtgctga gcttcgagct gctgcacgcc 1560
cccgccaccg tgtgcggccc caagaagagc accaacctgg tgaagaacaa gtgcgtgaac 1620
ttc 1623
<210> 5
<211> 538
<212> PRT
<213> Artificial Sequence
<220>
<223> 抗原
<400> 5
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val
1 5 10 15
Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe
20 25 30
Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu
35 40 45
His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp
50 55 60
Phe His Ala Ile Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp Asn Pro
65 70 75 80
Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu Lys Ser
85 90 95
Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser Lys Thr
100 105 110
Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile Lys Val
115 120 125
Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr His Lys
130 135 140
Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr Ser Ser Ala
145 150 155 160
Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu Met Asp Leu
165 170 175
Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe Val Phe Lys
180 185 190
Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr Pro Ile Asn
195 200 205
Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu Pro Leu Val
210 215 220
Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr Leu Leu Ala
225 230 235 240
Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser Gly Trp Thr
245 250 255
Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro Arg Thr Phe
260 265 270
Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala Val Asp Cys
275 280 285
Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys Ser Phe Thr
290 295 300
Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val Gln Pro Thr
305 310 315 320
Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly
325 330 335
Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg
340 345 350
Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser
355 360 365
Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu
370 375 380
Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg
385 390 395 400
Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala
405 410 415
Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala
420 425 430
Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr
435 440 445
Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp
450 455 460
Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val
465 470 475 480
Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro
485 490 495
Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe
500 505 510
Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr
515 520 525
Asn Leu Val Lys Asn Lys Cys Val Asn Phe
530 535
<210> 6
<211> 1614
<212> DNA
<213> Artificial Sequence
<220>
<223> 用于制备mRNA疫苗
<400> 6
atgttcgtgt tcctggtgct gctgcccctg gtgagcagcc agtgcgtgaa cctgaccacc 60
agaacccagc tgccccccgc ctacaccaac agcttcacca gaggcgtgta ctaccccgac 120
aaggtgttca gaagcagcgt gctgcacagc acccaggacc tgttcctgcc cttcttcagc 180
aacgtgacct ggttccacgc catcagcggc accaacggca ccaagagatt cgacaacccc 240
gtgctgccct tcaacgacgg cgtgtacttc gccagcaccg agaagagcaa catcatcaga 300
ggctggatct tcggcaccac cctggacagc aagacccaga gcctgctgat cgtgaacaac 360
gccaccaacg tggtgatcaa ggtgtgcgag ttccagttct gcaacgaccc cttcctgggc 420
gtgtaccaca agaacaacaa gagctggatg gagagcgagt tcagagtgta cagcagcgcc 480
aacaactgca ccttcgagta cgtgagccag cccttcctga tggacctgga gggcaagcag 540
ggcaacttca agaacctgag agagttcgtg ttcaagaaca tcgacggcta cttcaagatc 600
tacagcaagc acacccccat caacctggtg agagacctgc cccagggctt cagcgccctg 660
gagcccctgg tggacctgcc catcggcatc aacatcacca gattccagac cctgctggcc 720
ctgcacagaa gctacctgac ccccggcgac agcagcagcg gctggaccgc cggcgccgcc 780
gcctactacg tgggctacct gcagcccaga accttcctgc tgaagtacaa cgagaacggc 840
accatcaccg acgccgtgga ctgcgccctg gaccccctga gcgagaccaa gtgcaccctg 900
aagagcttca ccgtggagaa gggcatctac cagaccagca acttcagagt gcagcccacc 960
gagagcatcg tgagattccc caacatcacc aacctgtgcc ccttcggcga ggtgttcaac 1020
gccaccagat tcgccagcgt gtacgcctgg aacagaaaga gaatcagcaa ctgcgtggcc 1080
gactacagcg tgctgtacaa cagcgccagc ttcagcacct tcaagtgcta cggcgtgagc 1140
cccaccaagc tgaacgacct gtgcttcacc aacgtgtacg ccgacagctt cgtgatcaga 1200
ggcgacgagg tgagacagat cgcccccggc cagaccggca agatcgccga ctacaactac 1260
aagctgcccg acgacttcac cggctgcgtg atcgcctgga acagcaacaa cctggacagc 1320
aaggtgggcg gcaactacaa ctacctgtac agactgttca gaaagagcaa cctgaagccc 1380
ttcgagagag acatcagcac cgagatctac caggccggca gcaccccctg caacggcgtg 1440
gagggcttca actgctactt ccccctgcag agctacggct tccagcccac ctacggcgtg 1500
ggctaccagc cctacagagt ggtggtgctg agcttcgagc tgctgcacgc ccccgccacc 1560
gtgtgcggcc ccaagaagag caccaacctg gtgaagaaca agtgcgtgaa cttc 1614
<210> 7
<211> 1614
<212> DNA
<213> Artificial Sequence
<220>
<223> 用于制备mRNA疫苗
<400> 7
atgttcgtgt tcctggtgct gctgcctctg gtgagcagcc agtgcgtgaa cctgaccacc 60
agaacccagc tgcctcctgc ctacaccaac agcttcacca gaggcgtgta ctaccctgac 120
aaggtgttca gaagcagcgt gctgcacagc acccaggacc tgttcctgcc tttcttcagc 180
aacgtgacct ggttccacgc catcagcggc accaacggca ccaagagatt cgacaaccct 240
gtgctgcctt tcaacgacgg cgtgtacttc gccagcaccg agaagagcaa catcatcaga 300
ggctggatct tcggcaccac cctggacagc aagacccaga gcctgctgat cgtgaacaac 360
gccaccaacg tggtgatcaa ggtgtgcgag ttccagttct gcaacgaccc tttcctgggc 420
gtgtaccaca agaacaacaa gagctggatg gagagcgagt tcagagtgta cagcagcgcc 480
aacaactgca ccttcgagta cgtgagccag cctttcctga tggacctgga gggcaagcag 540
ggcaacttca agaacctgag agagttcgtg ttcaagaaca tcgacggcta cttcaagatc 600
tacagcaagc acacccctat caacctggtg agagacctgc ctcagggctt cagcgccctg 660
gagcctctgg tggacctgcc tatcggcatc aacatcacca gattccagac cctgctggcc 720
ctgcacagaa gctacctgac ccctggcgac agcagcagcg gctggaccgc cggcgccgcc 780
gcctactacg tgggctacct gcagcctaga accttcctgc tgaagtacaa cgagaacggc 840
accatcaccg acgccgtgga ctgcgccctg gaccctctga gcgagaccaa gtgcaccctg 900
aagagcttca ccgtggagaa gggcatctac cagaccagca acttcagagt gcagcctacc 960
gagagcatcg tgagattccc taacatcacc aacctgtgcc ctttcggcga ggtgttcaac 1020
gccaccagat tcgccagcgt gtacgcctgg aacagaaaga gaatcagcaa ctgcgtggcc 1080
gactacagcg tgctgtacaa cagcgccagc ttcagcacct tcaagtgcta cggcgtgagc 1140
cctaccaagc tgaacgacct gtgcttcacc aacgtgtacg ccgacagctt cgtgatcaga 1200
ggcgacgagg tgagacagat cgcccctggc cagaccggca agatcgccga ctacaactac 1260
aagctgcctg acgacttcac cggctgcgtg atcgcctgga acagcaacaa cctggacagc 1320
aaggtgggcg gcaactacaa ctacctgtac agactgttca gaaagagcaa cctgaagcct 1380
ttcgagagag acatcagcac cgagatctac caggccggca gcaccccttg caacggcgtg 1440
gagggcttca actgctactt ccctctgcag agctacggct tccagcctac ctacggcgtg 1500
ggctaccagc cttacagagt ggtggtgctg agcttcgagc tgctgcacgc ccctgccacc 1560
gtgtgcggcc ctaagaagag caccaacctg gtgaagaaca agtgcgtgaa cttc 1614
<210> 8
<211> 1614
<212> DNA
<213> Artificial Sequence
<220>
<223> 用于制备mRNA疫苗
<400> 8
atgttcgtgt tcctggtgct gctgcccctg gtgagcagcc agtgcgtgaa cctgaccacc 60
aggacccagc tgccccccgc ctacaccaac agcttcacca ggggcgtgta ctaccccgac 120
aaggtgttca ggagcagcgt gctgcacagc acccaggacc tgttcctgcc cttcttcagc 180
aacgtgacct ggttccacgc catcagcggc accaacggca ccaagaggtt cgacaacccc 240
gtgctgccct tcaacgacgg cgtgtacttc gccagcaccg agaagagcaa catcatcagg 300
ggctggatct tcggcaccac cctggacagc aagacccaga gcctgctgat cgtgaacaac 360
gccaccaacg tggtgatcaa ggtgtgcgag ttccagttct gcaacgaccc cttcctgggc 420
gtgtaccaca agaacaacaa gagctggatg gagagcgagt tcagggtgta cagcagcgcc 480
aacaactgca ccttcgagta cgtgagccag cccttcctga tggacctgga gggcaagcag 540
ggcaacttca agaacctgag ggagttcgtg ttcaagaaca tcgacggcta cttcaagatc 600
tacagcaagc acacccccat caacctggtg agggacctgc cccagggctt cagcgccctg 660
gagcccctgg tggacctgcc catcggcatc aacatcacca ggttccagac cctgctggcc 720
ctgcacagga gctacctgac ccccggcgac agcagcagcg gctggaccgc cggcgccgcc 780
gcctactacg tgggctacct gcagcccagg accttcctgc tgaagtacaa cgagaacggc 840
accatcaccg acgccgtgga ctgcgccctg gaccccctga gcgagaccaa gtgcaccctg 900
aagagcttca ccgtggagaa gggcatctac cagaccagca acttcagggt gcagcccacc 960
gagagcatcg tgaggttccc caacatcacc aacctgtgcc ccttcggcga ggtgttcaac 1020
gccaccaggt tcgccagcgt gtacgcctgg aacaggaaga ggatcagcaa ctgcgtggcc 1080
gactacagcg tgctgtacaa cagcgccagc ttcagcacct tcaagtgcta cggcgtgagc 1140
cccaccaagc tgaacgacct gtgcttcacc aacgtgtacg ccgacagctt cgtgatcagg 1200
ggcgacgagg tgaggcagat cgcccccggc cagaccggca agatcgccga ctacaactac 1260
aagctgcccg acgacttcac cggctgcgtg atcgcctgga acagcaacaa cctggacagc 1320
aaggtgggcg gcaactacaa ctacctgtac aggctgttca ggaagagcaa cctgaagccc 1380
ttcgagaggg acatcagcac cgagatctac caggccggca gcaccccctg caacggcgtg 1440
gagggcttca actgctactt ccccctgcag agctacggct tccagcccac ctacggcgtg 1500
ggctaccagc cctacagggt ggtggtgctg agcttcgagc tgctgcacgc ccccgccacc 1560
gtgtgcggcc ccaagaagag caccaacctg gtgaagaaca agtgcgtgaa cttc 1614
<210> 9
<211> 539
<212> PRT
<213> Artificial Sequence
<220>
<223> 抗原
<400> 9
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val
1 5 10 15
Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe
20 25 30
Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu
35 40 45
His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp
50 55 60
Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Ala
65 70 75 80
Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu
85 90 95
Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser
100 105 110
Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile
115 120 125
Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr
130 135 140
Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr
145 150 155 160
Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu
165 170 175
Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe
180 185 190
Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr
195 200 205
Pro Ile Asn Leu Val Arg Gly Leu Pro Gln Gly Phe Ser Ala Leu Glu
210 215 220
Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr
225 230 235 240
Leu Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser Gly Trp
245 250 255
Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro Arg Thr
260 265 270
Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala Val Asp
275 280 285
Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys Ser Phe
290 295 300
Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val Gln Pro
305 310 315 320
Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe
325 330 335
Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn
340 345 350
Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn
355 360 365
Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys
370 375 380
Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile
385 390 395 400
Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Asn Ile
405 410 415
Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile
420 425 430
Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn
435 440 445
Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg
450 455 460
Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly
465 470 475 480
Val Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln
485 490 495
Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser
500 505 510
Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser
515 520 525
Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe
530 535
<210> 10
<211> 1617
<212> DNA
<213> Artificial Sequence
<220>
<223> 用于制备mRNA疫苗
<400> 10
atgttcgtgt tcctggtgct gctgcccctg gtgagcagcc agtgcgtgaa cctgaccacc 60
aggacccagc tgccccccgc ctacaccaac agcttcacca ggggcgtgta ctaccccgac 120
aaggtgttca ggagcagcgt gctgcacagc acccaggacc tgttcctgcc cttcttcagc 180
aacgtgacct ggttccacgc catccacgtg agcggcacca acggcaccaa gaggttcgcc 240
aaccccgtgc tgcccttcaa cgacggcgtg tacttcgcca gcaccgagaa gagcaacatc 300
atcaggggct ggatcttcgg caccaccctg gacagcaaga cccagagcct gctgatcgtg 360
aacaacgcca ccaacgtggt gatcaaggtg tgcgagttcc agttctgcaa cgaccccttc 420
ctgggcgtgt actaccacaa gaacaacaag agctggatgg agagcgagtt cagggtgtac 480
agcagcgcca acaactgcac cttcgagtac gtgagccagc ccttcctgat ggacctggag 540
ggcaagcagg gcaacttcaa gaacctgagg gagttcgtgt tcaagaacat cgacggctac 600
ttcaagatct acagcaagca cacccccatc aacctggtga ggggcctgcc ccagggcttc 660
agcgccctgg agcccctggt ggacctgccc atcggcatca acatcaccag gttccagacc 720
ctgctgcaca ggagctacct gacccccggc gacagcagca gcggctggac cgccggcgcc 780
gccgcctact acgtgggcta cctgcagccc aggaccttcc tgctgaagta caacgagaac 840
ggcaccatca ccgacgccgt ggactgcgcc ctggaccccc tgagcgagac caagtgcacc 900
ctgaagagct tcaccgtgga gaagggcatc taccagacca gcaacttcag ggtgcagccc 960
accgagagca tcgtgaggtt ccccaacatc accaacctgt gccccttcgg cgaggtgttc 1020
aacgccacca ggttcgccag cgtgtacgcc tggaacagga agaggatcag caactgcgtg 1080
gccgactaca gcgtgctgta caacagcgcc agcttcagca ccttcaagtg ctacggcgtg 1140
agccccacca agctgaacga cctgtgcttc accaacgtgt acgccgacag cttcgtgatc 1200
aggggcgacg aggtgaggca gatcgccccc ggccagaccg gcaacatcgc cgactacaac 1260
tacaagctgc ccgacgactt caccggctgc gtgatcgcct ggaacagcaa caacctggac 1320
agcaaggtgg gcggcaacta caactacctg tacaggctgt tcaggaagag caacctgaag 1380
cccttcgaga gggacatcag caccgagatc taccaggccg gcagcacccc ctgcaacggc 1440
gtgaagggct tcaactgcta cttccccctg cagagctacg gcttccagcc cacctacggc 1500
gtgggctacc agccctacag ggtggtggtg ctgagcttcg agctgctgca cgcccccgcc 1560
accgtgtgcg gccccaagaa gagcaccaac ctggtgaaga acaagtgcgt gaacttc 1617
<210> 11
<211> 1617
<212> DNA
<213> Artificial Sequence
<220>
<223> 用于制备mRNA疫苗
<400> 11
atgttcgtgt tcctggtgct gctgcctctg gtgagcagcc agtgcgtgaa cctgaccacc 60
aggacccagc tgcctcctgc ctacaccaac agcttcacca ggggcgtgta ctaccctgac 120
aaggtgttca ggagcagcgt gctgcacagc acccaggacc tgttcctgcc tttcttcagc 180
aacgtgacct ggttccacgc catccacgtg agcggcacca acggcaccaa gaggttcgcc 240
aaccctgtgc tgcctttcaa cgacggcgtg tacttcgcca gcaccgagaa gagcaacatc 300
atcaggggct ggatcttcgg caccaccctg gacagcaaga cccagagcct gctgatcgtg 360
aacaacgcca ccaacgtggt gatcaaggtg tgcgagttcc agttctgcaa cgaccctttc 420
ctgggcgtgt actaccacaa gaacaacaag agctggatgg agagcgagtt cagggtgtac 480
agcagcgcca acaactgcac cttcgagtac gtgagccagc ctttcctgat ggacctggag 540
ggcaagcagg gcaacttcaa gaacctgagg gagttcgtgt tcaagaacat cgacggctac 600
ttcaagatct acagcaagca cacccctatc aacctggtga ggggcctgcc tcagggcttc 660
agcgccctgg agcctctggt ggacctgcct atcggcatca acatcaccag gttccagacc 720
ctgctgcaca ggagctacct gacccctggc gacagcagca gcggctggac cgccggcgcc 780
gccgcctact acgtgggcta cctgcagcct aggaccttcc tgctgaagta caacgagaac 840
ggcaccatca ccgacgccgt ggactgcgcc ctggaccctc tgagcgagac caagtgcacc 900
ctgaagagct tcaccgtgga gaagggcatc taccagacca gcaacttcag ggtgcagcct 960
accgagagca tcgtgaggtt ccctaacatc accaacctgt gccctttcgg cgaggtgttc 1020
aacgccacca ggttcgccag cgtgtacgcc tggaacagga agaggatcag caactgcgtg 1080
gccgactaca gcgtgctgta caacagcgcc agcttcagca ccttcaagtg ctacggcgtg 1140
agccctacca agctgaacga cctgtgcttc accaacgtgt acgccgacag cttcgtgatc 1200
aggggcgacg aggtgaggca gatcgcccct ggccagaccg gcaacatcgc cgactacaac 1260
tacaagctgc ctgacgactt caccggctgc gtgatcgcct ggaacagcaa caacctggac 1320
agcaaggtgg gcggcaacta caactacctg tacaggctgt tcaggaagag caacctgaag 1380
cctttcgaga gggacatcag caccgagatc taccaggccg gcagcacccc ttgcaacggc 1440
gtgaagggct tcaactgcta cttccctctg cagagctacg gcttccagcc tacctacggc 1500
gtgggctacc agccttacag ggtggtggtg ctgagcttcg agctgctgca cgcccctgcc 1560
accgtgtgcg gccctaagaa gagcaccaac ctggtgaaga acaagtgcgt gaacttc 1617
<210> 12
<211> 1617
<212> DNA
<213> Artificial Sequence
<220>
<223> 用于制备mRNA疫苗
<400> 12
atgttcgtgt tcctggtgct gctgcccctg gtgagcagcc agtgcgtgaa cctgaccacc 60
aggacccagc tgccccccgc ctacaccaac agcttcacca ggggcgtgta ctaccccgac 120
aaggtgttca ggagcagcgt gctgcacagc acccaggacc tgttcctgcc cttcttcagc 180
aacgtgacct ggttccacgc catccacgtg agcggcacca acggcaccaa gaggttcgcc 240
aaccccgtgc tgcccttcaa cgacggcgtg tacttcgcca gcaccgagaa gagcaacatc 300
atcaggggct ggatcttcgg caccaccctg gacagcaaga cccagagcct gctgatcgtg 360
aacaacgcca ccaacgtggt gatcaaggtg tgcgagttcc agttctgcaa cgaccccttc 420
ctgggcgtgt actaccacaa gaacaacaag agctggatgg agagcgagtt cagggtgtac 480
agcagcgcca acaactgcac cttcgagtac gtgagccagc ccttcctgat ggacctggag 540
ggcaagcagg gcaacttcaa gaacctgagg gagttcgtgt tcaagaacat cgacggctac 600
ttcaagatct acagcaagca cacccccatc aacctggtga ggggcctgcc ccagggcttc 660
agcgccctgg agcccctggt ggacctgccc atcggcatca acatcaccag gttccagacc 720
ctgctgcaca ggagctacct gacccccggc gacagcagca gcggctggac cgccggcgcc 780
gccgcctact acgtgggcta cctgcagccc aggaccttcc tgctgaagta caacgagaac 840
ggcaccatca ccgacgccgt ggactgcgcc ctggaccccc tgagcgagac caagtgcacc 900
ctgaagagct tcaccgtgga gaagggcatc taccagacca gcaacttcag ggtgcagccc 960
accgagagca tcgtgaggtt ccccaacatc accaacctgt gccccttcgg cgaggtgttc 1020
aacgccacca ggttcgccag cgtgtacgcc tggaacagga agaggatcag caactgcgtg 1080
gccgactaca gcgtgctgta caacagcgcc agcttcagca ccttcaagtg ctacggcgtg 1140
agccccacca agctgaacga cctgtgcttc accaacgtgt acgccgacag cttcgtgatc 1200
aggggcgacg aggtgaggca gatcgccccc ggccagaccg gcaacatcgc cgactacaac 1260
tacaagctgc ccgacgactt caccggctgc gtgatcgcct ggaacagcaa caacctggac 1320
agcaaggtgg gcggcaacta caactacctg tacaggctgt tcaggaagag caacctgaag 1380
cccttcgaga gggacatcag caccgagatc taccaggccg gcagcacccc ctgcaacggc 1440
gtgaagggct tcaactgcta cttccccctg cagagctacg gcttccagcc cacctacggc 1500
gtgggctacc agccctacag ggtggtggtg ctgagcttcg agctgctgca cgcccccgcc 1560
accgtgtgcg gccccaagaa gagcaccaac ctggtgaaga acaagtgcgt gaacttc 1617
<210> 13
<211> 541
<212> PRT
<213> Artificial Sequence
<220>
<223> 抗原
<400> 13
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val
1 5 10 15
Asn Phe Thr Asn Arg Thr Gln Leu Pro Ser Ala Tyr Thr Asn Ser Phe
20 25 30
Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu
35 40 45
His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp
50 55 60
Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp
65 70 75 80
Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu
85 90 95
Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser
100 105 110
Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile
115 120 125
Lys Val Cys Glu Phe Gln Phe Cys Asn Tyr Pro Phe Leu Gly Val Tyr
130 135 140
Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr
145 150 155 160
Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu
165 170 175
Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Ser Glu Phe
180 185 190
Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr
195 200 205
Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu
210 215 220
Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr
225 230 235 240
Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser
245 250 255
Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro
260 265 270
Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala
275 280 285
Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys
290 295 300
Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val
305 310 315 320
Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys
325 330 335
Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala
340 345 350
Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu
355 360 365
Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro
370 375 380
Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe
385 390 395 400
Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly
405 410 415
Thr Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys
420 425 430
Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn
435 440 445
Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe
450 455 460
Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys
465 470 475 480
Asn Gly Val Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly
485 490 495
Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val
500 505 510
Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys
515 520 525
Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe
530 535 540
<210> 14
<211> 1623
<212> DNA
<213> Artificial Sequence
<220>
<223> 用于制备mRNA疫苗
<400> 14
atgttcgtgt tcctggtgct gctgcccctg gtgagcagcc agtgcgtgaa cttcaccaac 60
agaacccagc tgcccagcgc ctacaccaac agcttcacca gaggcgtgta ctaccccgac 120
aaggtgttca gaagcagcgt gctgcacagc acccaggacc tgttcctgcc cttcttcagc 180
aacgtgacct ggttccacgc catccacgtg agcggcacca acggcaccaa gagattcgac 240
aaccccgtgc tgcccttcaa cgacggcgtg tacttcgcca gcaccgagaa gagcaacatc 300
atcagaggct ggatcttcgg caccaccctg gacagcaaga cccagagcct gctgatcgtg 360
aacaacgcca ccaacgtggt gatcaaggtg tgcgagttcc agttctgcaa ctaccccttc 420
ctgggcgtgt actaccacaa gaacaacaag agctggatgg agagcgagtt cagagtgtac 480
agcagcgcca acaactgcac cttcgagtac gtgagccagc ccttcctgat ggacctggag 540
ggcaagcagg gcaacttcaa gaacctgagc gagttcgtgt tcaagaacat cgacggctac 600
ttcaagatct acagcaagca cacccccatc aacctggtga gagacctgcc ccagggcttc 660
agcgccctgg agcccctggt ggacctgccc atcggcatca acatcaccag attccagacc 720
ctgctggccc tgcacagaag ctacctgacc cccggcgaca gcagcagcgg ctggaccgcc 780
ggcgccgccg cctactacgt gggctacctg cagcccagaa ccttcctgct gaagtacaac 840
gagaacggca ccatcaccga cgccgtggac tgcgccctgg accccctgag cgagaccaag 900
tgcaccctga agagcttcac cgtggagaag ggcatctacc agaccagcaa cttcagagtg 960
cagcccaccg agagcatcgt gagattcccc aacatcacca acctgtgccc cttcggcgag 1020
gtgttcaacg ccaccagatt cgccagcgtg tacgcctgga acagaaagag aatcagcaac 1080
tgcgtggccg actacagcgt gctgtacaac agcgccagct tcagcacctt caagtgctac 1140
ggcgtgagcc ccaccaagct gaacgacctg tgcttcacca acgtgtacgc cgacagcttc 1200
gtgatcagag gcgacgaggt gagacagatc gcccccggcc agaccggcac catcgccgac 1260
tacaactaca agctgcccga cgacttcacc ggctgcgtga tcgcctggaa cagcaacaac 1320
ctggacagca aggtgggcgg caactacaac tacctgtaca gactgttcag aaagagcaac 1380
ctgaagccct tcgagagaga catcagcacc gagatctacc aggccggcag caccccctgc 1440
aacggcgtga agggcttcaa ctgctacttc cccctgcaga gctacggctt ccagcccacc 1500
tacggcgtgg gctaccagcc ctacagagtg gtggtgctga gcttcgagct gctgcacgcc 1560
cccgccaccg tgtgcggccc caagaagagc accaacctgg tgaagaacaa gtgcgtgaac 1620
ttc 1623
<210> 15
<211> 1623
<212> DNA
<213> Artificial Sequence
<220>
<223> 用于制备mRNA疫苗
<400> 15
atgttcgtgt tcctggtgct gctgcctctg gtgagcagcc agtgcgtgaa cttcaccaac 60
agaacccagc tgcctagcgc ctacaccaac agcttcacca gaggcgtgta ctaccctgac 120
aaggtgttca gaagcagcgt gctgcacagc acccaggacc tgttcctgcc tttcttcagc 180
aacgtgacct ggttccacgc catccacgtg agcggcacca acggcaccaa gagattcgac 240
aaccctgtgc tgcctttcaa cgacggcgtg tacttcgcca gcaccgagaa gagcaacatc 300
atcagaggct ggatcttcgg caccaccctg gacagcaaga cccagagcct gctgatcgtg 360
aacaacgcca ccaacgtggt gatcaaggtg tgcgagttcc agttctgcaa ctaccctttc 420
ctgggcgtgt actaccacaa gaacaacaag agctggatgg agagcgagtt cagagtgtac 480
agcagcgcca acaactgcac cttcgagtac gtgagccagc ctttcctgat ggacctggag 540
ggcaagcagg gcaacttcaa gaacctgagc gagttcgtgt tcaagaacat cgacggctac 600
ttcaagatct acagcaagca cacccctatc aacctggtga gagacctgcc tcagggcttc 660
agcgccctgg agcctctggt ggacctgcct atcggcatca acatcaccag attccagacc 720
ctgctggccc tgcacagaag ctacctgacc cctggcgaca gcagcagcgg ctggaccgcc 780
ggcgccgccg cctactacgt gggctacctg cagcctagaa ccttcctgct gaagtacaac 840
gagaacggca ccatcaccga cgccgtggac tgcgccctgg accctctgag cgagaccaag 900
tgcaccctga agagcttcac cgtggagaag ggcatctacc agaccagcaa cttcagagtg 960
cagcctaccg agagcatcgt gagattccct aacatcacca acctgtgccc tttcggcgag 1020
gtgttcaacg ccaccagatt cgccagcgtg tacgcctgga acagaaagag aatcagcaac 1080
tgcgtggccg actacagcgt gctgtacaac agcgccagct tcagcacctt caagtgctac 1140
ggcgtgagcc ctaccaagct gaacgacctg tgcttcacca acgtgtacgc cgacagcttc 1200
gtgatcagag gcgacgaggt gagacagatc gcccctggcc agaccggcac catcgccgac 1260
tacaactaca agctgcctga cgacttcacc ggctgcgtga tcgcctggaa cagcaacaac 1320
ctggacagca aggtgggcgg caactacaac tacctgtaca gactgttcag aaagagcaac 1380
ctgaagcctt tcgagagaga catcagcacc gagatctacc aggccggcag caccccttgc 1440
aacggcgtga agggcttcaa ctgctacttc cctctgcaga gctacggctt ccagcctacc 1500
tacggcgtgg gctaccagcc ttacagagtg gtggtgctga gcttcgagct gctgcacgcc 1560
cctgccaccg tgtgcggccc taagaagagc accaacctgg tgaagaacaa gtgcgtgaac 1620
ttc 1623
<210> 16
<211> 1623
<212> DNA
<213> Artificial Sequence
<220>
<223> 用于制备mRNA疫苗
<400> 16
atgttcgtgt tcctggtgct gctgcccctg gtgagcagcc agtgcgtgaa cttcaccaac 60
aggacccagc tgcccagcgc ctacaccaac agcttcacca ggggcgtgta ctaccccgac 120
aaggtgttca ggagcagcgt gctgcacagc acccaggacc tgttcctgcc cttcttcagc 180
aacgtgacct ggttccacgc catccacgtg agcggcacca acggcaccaa gaggttcgac 240
aaccccgtgc tgcccttcaa cgacggcgtg tacttcgcca gcaccgagaa gagcaacatc 300
atcaggggct ggatcttcgg caccaccctg gacagcaaga cccagagcct gctgatcgtg 360
aacaacgcca ccaacgtggt gatcaaggtg tgcgagttcc agttctgcaa ctaccccttc 420
ctgggcgtgt actaccacaa gaacaacaag agctggatgg agagcgagtt cagggtgtac 480
agcagcgcca acaactgcac cttcgagtac gtgagccagc ccttcctgat ggacctggag 540
ggcaagcagg gcaacttcaa gaacctgagc gagttcgtgt tcaagaacat cgacggctac 600
ttcaagatct acagcaagca cacccccatc aacctggtga gggacctgcc ccagggcttc 660
agcgccctgg agcccctggt ggacctgccc atcggcatca acatcaccag gttccagacc 720
ctgctggccc tgcacaggag ctacctgacc cccggcgaca gcagcagcgg ctggaccgcc 780
ggcgccgccg cctactacgt gggctacctg cagcccagga ccttcctgct gaagtacaac 840
gagaacggca ccatcaccga cgccgtggac tgcgccctgg accccctgag cgagaccaag 900
tgcaccctga agagcttcac cgtggagaag ggcatctacc agaccagcaa cttcagggtg 960
cagcccaccg agagcatcgt gaggttcccc aacatcacca acctgtgccc cttcggcgag 1020
gtgttcaacg ccaccaggtt cgccagcgtg tacgcctgga acaggaagag gatcagcaac 1080
tgcgtggccg actacagcgt gctgtacaac agcgccagct tcagcacctt caagtgctac 1140
ggcgtgagcc ccaccaagct gaacgacctg tgcttcacca acgtgtacgc cgacagcttc 1200
gtgatcaggg gcgacgaggt gaggcagatc gcccccggcc agaccggcac catcgccgac 1260
tacaactaca agctgcccga cgacttcacc ggctgcgtga tcgcctggaa cagcaacaac 1320
ctggacagca aggtgggcgg caactacaac tacctgtaca ggctgttcag gaagagcaac 1380
ctgaagccct tcgagaggga catcagcacc gagatctacc aggccggcag caccccctgc 1440
aacggcgtga agggcttcaa ctgctacttc cccctgcaga gctacggctt ccagcccacc 1500
tacggcgtgg gctaccagcc ctacagggtg gtggtgctga gcttcgagct gctgcacgcc 1560
cccgccaccg tgtgcggccc caagaagagc accaacctgg tgaagaacaa gtgcgtgaac 1620
ttc 1623
<210> 17
<211> 541
<212> PRT
<213> Artificial Sequence
<220>
<223> 抗原
<400> 17
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val
1 5 10 15
Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe
20 25 30
Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu
35 40 45
His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp
50 55 60
Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp
65 70 75 80
Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu
85 90 95
Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser
100 105 110
Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile
115 120 125
Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr
130 135 140
Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr
145 150 155 160
Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu
165 170 175
Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe
180 185 190
Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr
195 200 205
Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu
210 215 220
Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr
225 230 235 240
Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser
245 250 255
Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro
260 265 270
Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala
275 280 285
Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys
290 295 300
Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val
305 310 315 320
Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys
325 330 335
Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala
340 345 350
Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu
355 360 365
Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro
370 375 380
Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe
385 390 395 400
Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly
405 410 415
Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys
420 425 430
Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn
435 440 445
Tyr Asn Tyr Arg Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe
450 455 460
Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys
465 470 475 480
Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly
485 490 495
Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val
500 505 510
Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys
515 520 525
Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe
530 535 540
<210> 18
<211> 1623
<212> DNA
<213> Artificial Sequence
<220>
<223> 用于制备mRNA疫苗
<400> 18
atgttcgtgt tcctggtgct gctgcccctg gtgagcagcc agtgcgtgaa cctgaccacc 60
agaacccagc tgccccccgc ctacaccaac agcttcacca gaggcgtgta ctaccccgac 120
aaggtgttca gaagcagcgt gctgcacagc acccaggacc tgttcctgcc cttcttcagc 180
aacgtgacct ggttccacgc catccacgtg agcggcacca acggcaccaa gagattcgac 240
aaccccgtgc tgcccttcaa cgacggcgtg tacttcgcca gcaccgagaa gagcaacatc 300
atcagaggct ggatcttcgg caccaccctg gacagcaaga cccagagcct gctgatcgtg 360
aacaacgcca ccaacgtggt gatcaaggtg tgcgagttcc agttctgcaa cgaccccttc 420
ctgggcgtgt actaccacaa gaacaacaag agctggatgg agagcgagtt cagagtgtac 480
agcagcgcca acaactgcac cttcgagtac gtgagccagc ccttcctgat ggacctggag 540
ggcaagcagg gcaacttcaa gaacctgaga gagttcgtgt tcaagaacat cgacggctac 600
ttcaagatct acagcaagca cacccccatc aacctggtga gagacctgcc ccagggcttc 660
agcgccctgg agcccctggt ggacctgccc atcggcatca acatcaccag attccagacc 720
ctgctggccc tgcacagaag ctacctgacc cccggcgaca gcagcagcgg ctggaccgcc 780
ggcgccgccg cctactacgt gggctacctg cagcccagaa ccttcctgct gaagtacaac 840
gagaacggca ccatcaccga cgccgtggac tgcgccctgg accccctgag cgagaccaag 900
tgcaccctga agagcttcac cgtggagaag ggcatctacc agaccagcaa cttcagagtg 960
cagcccaccg agagcatcgt gagattcccc aacatcacca acctgtgccc cttcggcgag 1020
gtgttcaacg ccaccagatt cgccagcgtg tacgcctgga acagaaagag aatcagcaac 1080
tgcgtggccg actacagcgt gctgtacaac agcgccagct tcagcacctt caagtgctac 1140
ggcgtgagcc ccaccaagct gaacgacctg tgcttcacca acgtgtacgc cgacagcttc 1200
gtgatcagag gcgacgaggt gagacagatc gcccccggcc agaccggcaa gatcgccgac 1260
tacaactaca agctgcccga cgacttcacc ggctgcgtga tcgcctggaa cagcaacaac 1320
ctggacagca aggtgggcgg caactacaac tacagataca gactgttcag aaagagcaac 1380
ctgaagccct tcgagagaga catcagcacc gagatctacc aggccggcag caccccctgc 1440
aacggcgtgg agggcttcaa ctgctacttc cccctgcaga gctacggctt ccagcccacc 1500
aacggcgtgg gctaccagcc ctacagagtg gtggtgctga gcttcgagct gctgcacgcc 1560
cccgccaccg tgtgcggccc caagaagagc accaacctgg tgaagaacaa gtgcgtgaac 1620
ttc 1623
<210> 19
<211> 1623
<212> DNA
<213> Artificial Sequence
<220>
<223> 用于制备mRNA疫苗
<400> 19
atgttcgtgt tcctggtgct gctgcctctg gtgagcagcc agtgcgtgaa cctgaccacc 60
agaacccagc tgcctcctgc ctacaccaac agcttcacca gaggcgtgta ctaccctgac 120
aaggtgttca gaagcagcgt gctgcacagc acccaggacc tgttcctgcc tttcttcagc 180
aacgtgacct ggttccacgc catccacgtg agcggcacca acggcaccaa gagattcgac 240
aaccctgtgc tgcctttcaa cgacggcgtg tacttcgcca gcaccgagaa gagcaacatc 300
atcagaggct ggatcttcgg caccaccctg gacagcaaga cccagagcct gctgatcgtg 360
aacaacgcca ccaacgtggt gatcaaggtg tgcgagttcc agttctgcaa cgaccctttc 420
ctgggcgtgt actaccacaa gaacaacaag agctggatgg agagcgagtt cagagtgtac 480
agcagcgcca acaactgcac cttcgagtac gtgagccagc ctttcctgat ggacctggag 540
ggcaagcagg gcaacttcaa gaacctgaga gagttcgtgt tcaagaacat cgacggctac 600
ttcaagatct acagcaagca cacccctatc aacctggtga gagacctgcc tcagggcttc 660
agcgccctgg agcctctggt ggacctgcct atcggcatca acatcaccag attccagacc 720
ctgctggccc tgcacagaag ctacctgacc cctggcgaca gcagcagcgg ctggaccgcc 780
ggcgccgccg cctactacgt gggctacctg cagcctagaa ccttcctgct gaagtacaac 840
gagaacggca ccatcaccga cgccgtggac tgcgccctgg accctctgag cgagaccaag 900
tgcaccctga agagcttcac cgtggagaag ggcatctacc agaccagcaa cttcagagtg 960
cagcctaccg agagcatcgt gagattccct aacatcacca acctgtgccc tttcggcgag 1020
gtgttcaacg ccaccagatt cgccagcgtg tacgcctgga acagaaagag aatcagcaac 1080
tgcgtggccg actacagcgt gctgtacaac agcgccagct tcagcacctt caagtgctac 1140
ggcgtgagcc ctaccaagct gaacgacctg tgcttcacca acgtgtacgc cgacagcttc 1200
gtgatcagag gcgacgaggt gagacagatc gcccctggcc agaccggcaa gatcgccgac 1260
tacaactaca agctgcctga cgacttcacc ggctgcgtga tcgcctggaa cagcaacaac 1320
ctggacagca aggtgggcgg caactacaac tacagataca gactgttcag aaagagcaac 1380
ctgaagcctt tcgagagaga catcagcacc gagatctacc aggccggcag caccccttgc 1440
aacggcgtgg agggcttcaa ctgctacttc cctctgcaga gctacggctt ccagcctacc 1500
aacggcgtgg gctaccagcc ttacagagtg gtggtgctga gcttcgagct gctgcacgcc 1560
cctgccaccg tgtgcggccc taagaagagc accaacctgg tgaagaacaa gtgcgtgaac 1620
ttc 1623
<210> 20
<211> 1623
<212> DNA
<213> Artificial Sequence
<220>
<223> 用于制备mRNA疫苗
<400> 20
atgttcgtgt tcctggtgct gctgcccctg gtgagcagcc agtgcgtgaa cctgaccacc 60
aggacccagc tgccccccgc ctacaccaac agcttcacca ggggcgtgta ctaccccgac 120
aaggtgttca ggagcagcgt gctgcacagc acccaggacc tgttcctgcc cttcttcagc 180
aacgtgacct ggttccacgc catccacgtg agcggcacca acggcaccaa gaggttcgac 240
aaccccgtgc tgcccttcaa cgacggcgtg tacttcgcca gcaccgagaa gagcaacatc 300
atcaggggct ggatcttcgg caccaccctg gacagcaaga cccagagcct gctgatcgtg 360
aacaacgcca ccaacgtggt gatcaaggtg tgcgagttcc agttctgcaa cgaccccttc 420
ctgggcgtgt actaccacaa gaacaacaag agctggatgg agagcgagtt cagggtgtac 480
agcagcgcca acaactgcac cttcgagtac gtgagccagc ccttcctgat ggacctggag 540
ggcaagcagg gcaacttcaa gaacctgagg gagttcgtgt tcaagaacat cgacggctac 600
ttcaagatct acagcaagca cacccccatc aacctggtga gggacctgcc ccagggcttc 660
agcgccctgg agcccctggt ggacctgccc atcggcatca acatcaccag gttccagacc 720
ctgctggccc tgcacaggag ctacctgacc cccggcgaca gcagcagcgg ctggaccgcc 780
ggcgccgccg cctactacgt gggctacctg cagcccagga ccttcctgct gaagtacaac 840
gagaacggca ccatcaccga cgccgtggac tgcgccctgg accccctgag cgagaccaag 900
tgcaccctga agagcttcac cgtggagaag ggcatctacc agaccagcaa cttcagggtg 960
cagcccaccg agagcatcgt gaggttcccc aacatcacca acctgtgccc cttcggcgag 1020
gtgttcaacg ccaccaggtt cgccagcgtg tacgcctgga acaggaagag gatcagcaac 1080
tgcgtggccg actacagcgt gctgtacaac agcgccagct tcagcacctt caagtgctac 1140
ggcgtgagcc ccaccaagct gaacgacctg tgcttcacca acgtgtacgc cgacagcttc 1200
gtgatcaggg gcgacgaggt gaggcagatc gcccccggcc agaccggcaa gatcgccgac 1260
tacaactaca agctgcccga cgacttcacc ggctgcgtga tcgcctggaa cagcaacaac 1320
ctggacagca aggtgggcgg caactacaac tacaggtaca ggctgttcag gaagagcaac 1380
ctgaagccct tcgagaggga catcagcacc gagatctacc aggccggcag caccccctgc 1440
aacggcgtgg agggcttcaa ctgctacttc cccctgcaga gctacggctt ccagcccacc 1500
aacggcgtgg gctaccagcc ctacagggtg gtggtgctga gcttcgagct gctgcacgcc 1560
cccgccaccg tgtgcggccc caagaagagc accaacctgg tgaagaacaa gtgcgtgaac 1620
ttc 1623
<210> 21
<211> 538
<212> PRT
<213> Artificial Sequence
<220>
<223> 抗原
<400> 21
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val
1 5 10 15
Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe
20 25 30
Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu
35 40 45
His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp
50 55 60
Phe His Val Ile Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp Asn Pro
65 70 75 80
Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Ile Glu Lys Ser
85 90 95
Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser Lys Thr
100 105 110
Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile Lys Val
115 120 125
Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Asp His Lys Asn Asn
130 135 140
Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr Ser Ser Ala Asn Asn
145 150 155 160
Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu Met Asp Leu Glu Gly
165 170 175
Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe Val Phe Lys Asn Ile
180 185 190
Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr Pro Ile Ile Val Arg
195 200 205
Glu Pro Glu Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu Pro Leu Val
210 215 220
Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr Leu Leu Ala
225 230 235 240
Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser Gly Trp Thr
245 250 255
Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro Arg Thr Phe
260 265 270
Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala Val Asp Cys
275 280 285
Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys Ser Phe Thr
290 295 300
Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val Gln Pro Thr
305 310 315 320
Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Asp
325 330 335
Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg
340 345 350
Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Leu
355 360 365
Ala Pro Phe Phe Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu
370 375 380
Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg
385 390 395 400
Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Asn Ile Ala
405 410 415
Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala
420 425 430
Trp Asn Ser Asn Lys Leu Asp Ser Lys Val Ser Gly Asn Tyr Asn Tyr
435 440 445
Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp
450 455 460
Ile Ser Thr Glu Ile Tyr Gln Ala Gly Asn Lys Pro Cys Asn Gly Val
465 470 475 480
Ala Gly Phe Asn Cys Tyr Phe Pro Leu Arg Ser Tyr Ser Phe Arg Pro
485 490 495
Thr Tyr Gly Val Gly His Gln Pro Tyr Arg Val Val Val Leu Ser Phe
500 505 510
Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr
515 520 525
Asn Leu Val Lys Asn Lys Cys Val Asn Phe
530 535
<210> 22
<211> 1620
<212> DNA
<213> Artificial Sequence
<220>
<223> 用于制备mRNA疫苗
<400> 22
atgtttgtgt tcctggtgct gctgcccctg gtgagcagcc agtgcgtgaa cctgaccacc 60
agaacacagc tgcctcccgc ctacaccaac tccttcaccc gcggcgtgta ctaccctgat 120
aaggtgttca ggagctccgt gctgcacagc acccaggacc tgttcctgcc tttcttctct 180
aacgtgacct ggttccacgt gattagcggc acaaacggca ccaagcggtt cgataacccc 240
gtgctgccct tcaacgacgg cgtgtacttc gcctccatcg aaaaaagcaa cattatcaga 300
ggctggatct tcggcaccac cctggattcc aagacccagt ccctgctgat cgtgaacaac 360
gccaccaacg tcgtgatcaa ggtgtgtgag ttccagttct gtaatgaccc cttcctggac 420
cacaagaaca acaagagctg gatggagagc gagttcaggg tgtacagctc cgccaacaac 480
tgcacattcg agtacgtgag ccagcccttc ctgatggacc tggagggcaa gcagggcaac 540
ttcaagaatc tgagagagtt cgtgttcaag aacatcgatg gatacttcaa gatctacagc 600
aagcacaccc ctatcatcgt gagggagcct gaggacctgc cccagggctt cagcgccctg 660
gagcccctcg tggacctgcc tatcggcatc aacatcacaa ggttccagac cctgctggcc 720
ctgcacaggt cctacctgac accaggcgac agcagcagcg gctggacagc cggcgccgcc 780
gcctactatg tgggctacct gcagcccaga acattcctgc tgaagtataa cgagaatggc 840
accatcacag acgccgtgga ctgtgccctg gatcccctga gcgagaccaa gtgcactctg 900
aagtccttta cagtggagaa gggaatctac cagacaagca atttcagggt gcagcctaca 960
gagagcattg tgagattccc taacatcacc aacctgtgcc cctttgacga agtgtttaac 1020
gctacacggt ttgcatcagt gtatgcctgg aacaggaaga gaatcagtaa ctgtgtggcc 1080
gactactctg tgctgtataa cctggctccc ttctttactt tcaaatgcta cggggtgagc 1140
cctactaagc tgaatgacct gtgtttcaca aacgtgtatg ccgatagctt tgtgatcagg 1200
ggcgacgaag tgagacagat cgctccaggc cagacaggca atattgccga ttacaactat 1260
aaactgcccg atgacttcac aggatgcgtg attgcctgga atagcaataa gctggattct 1320
aaagtgagcg gcaattacaa ttacctgtat aggctgttca gaaagagcaa cctgaagcct 1380
ttcgagagag acatctccac tgaaatctat caggccggca ataagccctg caacggcgtg 1440
gctggcttta attgttactt tcccctgaga tcctatagct ttcgccccac ctatggcgtg 1500
ggacaccagc cttacagagt ggtggtgctg tccttcgagc tgctgcacgc ccctgccaca 1560
gtgtgcggcc caaagaagtc caccaatctg gtgaagaata agtgcgtgaa cttctgataa 1620
<210> 23
<211> 1614
<212> DNA
<213> Artificial Sequence
<220>
<223> 用于制备mRNA疫苗
<400> 23
atgttcgtgt tcctggtgct gctgcctctg gtgagcagcc agtgcgtgaa cctgaccacc 60
aggacccagc tgcctcctgc ctacaccaac agcttcacca ggggcgtgta ctaccctgac 120
aaggtgttca ggagcagcgt gctgcacagc acccaggacc tgttcctgcc tttcttcagc 180
aacgtgacct ggttccacgt gatcagcggc accaacggca ccaagaggtt cgacaaccct 240
gtgctgcctt tcaacgacgg cgtgtacttc gccagcatcg agaagagcaa catcatcagg 300
ggctggatct tcggcaccac cctggacagc aagacccaga gcctgctgat cgtgaacaac 360
gccaccaacg tggtgatcaa ggtgtgcgag ttccagttct gcaacgaccc tttcctggac 420
cacaagaaca acaagagctg gatggagagc gagttcaggg tgtacagcag cgccaacaac 480
tgcaccttcg agtacgtgag ccagcctttc ctgatggacc tggagggcaa gcagggcaac 540
ttcaagaacc tgagggagtt cgtgttcaag aacatcgacg gctacttcaa gatctacagc 600
aagcacaccc ctatcctggt gagggagcct gaggacctgc ctcagggctt cagcgccctg 660
gagcctctgg tggacctgcc tatcggcatc aacatcacca ggttccagac cctgctggcc 720
ctgcacagga gctacctgac ccctggcgac agcagcagcg gctggaccgc cggcgccgcc 780
gcctactacg tgggctacct gcagcctagg accttcctgc tgaagtacaa cgagaacggc 840
accatcaccg acgccgtgga ctgcgccctg gaccctctga gcgagaccaa gtgcaccctg 900
aagagcttca ccgtggagaa gggcatctac cagaccagca acttcagggt gcagcctacc 960
gagagcatcg tgaggttccc taacatcacc aacctgtgcc ctttcgacga ggtgttcaac 1020
gccaccaggt tcgccagcgt gtacgcctgg aacaggaaga ggatcagcaa ctgcgtggcc 1080
gactacagcg tgctgtacaa cctggcccct ttcttcacct tcaagtgcta cggcgtgagc 1140
cctaccaagc tgaacgacct gtgcttcacc aacgtgtacg ccgacagctt cgtgatcagg 1200
ggcgacgagg tgaggcagat cgcccctggc cagaccggca acatcgccga ctacaactac 1260
aagctgcctg acgacttcac cggctgcgtg atcgcctgga acagcaacaa gctggacagc 1320
aaggtgagcg gcaactacaa ctacctgtac aggctgttca ggaagagcaa cctgaagcct 1380
ttcgagaggg acatcagcac cgagatctac caggccggca acaagccttg caacggcgtg 1440
gccggcttca actgctactt ccctctgagg agctacagct tcaggcctac ctacggcgtg 1500
ggccaccagc cttacagggt ggtggtgctg agcttcgagc tgctgcacgc ccctgccacc 1560
gtgtgcggcc ctaagaagag caccaacctg gtgaagaaca agtgcgtgaa cttc 1614
<210> 24
<211> 1614
<212> DNA
<213> Artificial Sequence
<220>
<223> 用于制备mRNA疫苗
<400> 24
atgttcgtgt tcctggtgct gctgcccctg gtgagcagcc agtgcgtgaa cctgaccacc 60
aggacccagc tgccccccgc ctacaccaac agcttcacca ggggcgtgta ctaccccgac 120
aaggtgttca ggagcagcgt gctgcacagc acccaggacc tgttcctgcc cttcttcagc 180
aacgtgacct ggttccacgt gatcagcggc accaacggca ccaagaggtt cgacaacccc 240
gtgctgccct tcaacgacgg cgtgtacttc gccagcatcg agaagagcaa catcatcagg 300
ggctggatct tcggcaccac cctggacagc aagacccaga gcctgctgat cgtgaacaac 360
gccaccaacg tggtgatcaa ggtgtgcgag ttccagttct gcaacgaccc cttcctggac 420
cacaagaaca acaagagctg gatggagagc gagttcaggg tgtacagcag cgccaacaac 480
tgcaccttcg agtacgtgag ccagcccttc ctgatggacc tggagggcaa gcagggcaac 540
ttcaagaacc tgagggagtt cgtgttcaag aacatcgacg gctacttcaa gatctacagc 600
aagcacaccc ccatcctggt gagggagccc gaggacctgc cccagggctt cagcgccctg 660
gagcccctgg tggacctgcc catcggcatc aacatcacca ggttccagac cctgctggcc 720
ctgcacagga gctacctgac ccccggcgac agcagcagcg gctggaccgc cggcgccgcc 780
gcctactacg tgggctacct gcagcccagg accttcctgc tgaagtacaa cgagaacggc 840
accatcaccg acgccgtgga ctgcgccctg gaccccctga gcgagaccaa gtgcaccctg 900
aagagcttca ccgtggagaa gggcatctac cagaccagca acttcagggt gcagcccacc 960
gagagcatcg tgaggttccc caacatcacc aacctgtgcc ccttcgacga ggtgttcaac 1020
gccaccaggt tcgccagcgt gtacgcctgg aacaggaaga ggatcagcaa ctgcgtggcc 1080
gactacagcg tgctgtacaa cctggccccc ttcttcacct tcaagtgcta cggcgtgagc 1140
cccaccaagc tgaacgacct gtgcttcacc aacgtgtacg ccgacagctt cgtgatcagg 1200
ggcgacgagg tgaggcagat cgcccccggc cagaccggca acatcgccga ctacaactac 1260
aagctgcccg acgacttcac cggctgcgtg atcgcctgga acagcaacaa gctggacagc 1320
aaggtgagcg gcaactacaa ctacctgtac aggctgttca ggaagagcaa cctgaagccc 1380
ttcgagaggg acatcagcac cgagatctac caggccggca acaagccctg caacggcgtg 1440
gccggcttca actgctactt ccccctgagg agctacagct tcaggcccac ctacggcgtg 1500
ggccaccagc cctacagggt ggtggtgctg agcttcgagc tgctgcacgc ccccgccacc 1560
gtgtgcggcc ccaagaagag caccaacctg gtgaagaaca agtgcgtgaa cttc 1614
<210> 25
<211> 1623
<212> RNA
<213> Artificial Sequence
<220>
<223> mRNA疫苗
<400> 25
auguucgugu uccuggugcu gcugccccug gugagcagcc agugcgugaa ccugaccacc 60
aggacccagc ugccccccgc cuacaccaac agcuucacca ggggcgugua cuaccccgac 120
aagguguuca ggagcagcgu gcugcacagc acccaggacc uguuccugcc cuucuucagc 180
aacgugaccu gguuccacgc cauccacgug agcggcacca acggcaccaa gagguucgac 240
aaccccgugc ugcccuucaa cgacggcgug uacuucgcca gcaccgagaa gagcaacauc 300
aucaggggcu ggaucuucgg caccacccug gacagcaaga cccagagccu gcugaucgug 360
aacaacgcca ccaacguggu gaucaaggug ugcgaguucc aguucugcaa cgaccccuuc 420
cugggcgugu acuaccacaa gaacaacaag agcuggaugg agagcgaguu caggguguac 480
agcagcgcca acaacugcac cuucgaguac gugagccagc ccuuccugau ggaccuggag 540
ggcaagcagg gcaacuucaa gaaccugagg gaguucgugu ucaagaacau cgacggcuac 600
uucaagaucu acagcaagca cacccccauc aaccugguga gggaccugcc ccagggcuuc 660
agcgcccugg agccccuggu ggaccugccc aucggcauca acaucaccag guuccagacc 720
cugcuggccc ugcacaggag cuaccugacc cccggcgaca gcagcagcgg cuggaccgcc 780
ggcgccgccg ccuacuacgu gggcuaccug cagcccagga ccuuccugcu gaaguacaac 840
gagaacggca ccaucaccga cgccguggac ugcgcccugg acccccugag cgagaccaag 900
ugcacccuga agagcuucac cguggagaag ggcaucuacc agaccagcaa cuucagggug 960
cagcccaccg agagcaucgu gagguucccc aacaucacca accugugccc cuucggcgag 1020
guguucaacg ccaccagguu cgccagcgug uacgccugga acaggaagag gaucagcaac 1080
ugcguggccg acuacagcgu gcuguacaac agcgccagcu ucagcaccuu caagugcuac 1140
ggcgugagcc ccaccaagcu gaacgaccug ugcuucacca acguguacgc cgacagcuuc 1200
gugaucaggg gcgacgaggu gaggcagauc gcccccggcc agaccggcaa gaucgccgac 1260
uacaacuaca agcugcccga cgacuucacc ggcugcguga ucgccuggaa cagcaacaac 1320
cuggacagca aggugggcgg caacuacaac uaccuguaca ggcuguucag gaagagcaac 1380
cugaagcccu ucgagaggga caucagcacc gagaucuacc aggccggcag cacccccugc 1440
aacggcgugg agggcuucaa cugcuacuuc ccccugcaga gcuacggcuu ccagcccacc 1500
aacggcgugg gcuaccagcc cuacagggug guggugcuga gcuucgagcu gcugcacgcc 1560
cccgccaccg ugugcggccc caagaagagc accaaccugg ugaagaacaa gugcgugaac 1620
uuc 1623
<210> 26
<211> 1623
<212> RNA
<213> Artificial Sequence
<220>
<223> mRNA疫苗
<400> 26
auguucgugu uccuggugcu gcugccucug gugagcagcc agugcgugaa ccugaccacc 60
agaacccagc ugccuccugc cuacaccaac agcuucacca gaggcgugua cuacccugac 120
aagguguuca gaagcagcgu gcugcacagc acccaggacc uguuccugcc uuucuucagc 180
aacgugaccu gguuccacgc cauccacgug agcggcacca acggcaccaa gagauucgac 240
aacccugugc ugccuuucaa cgacggcgug uacuucgcca gcaccgagaa gagcaacauc 300
aucagaggcu ggaucuucgg caccacccug gacagcaaga cccagagccu gcugaucgug 360
aacaacgcca ccaacguggu gaucaaggug ugcgaguucc aguucugcaa cgacccuuuc 420
cugggcgugu acuaccacaa gaacaacaag agcuggaugg agagcgaguu cagaguguac 480
agcagcgcca acaacugcac cuucgaguac gugagccagc cuuuccugau ggaccuggag 540
ggcaagcagg gcaacuucaa gaaccugaga gaguucgugu ucaagaacau cgacggcuac 600
uucaagaucu acagcaagca caccccuauc aaccugguga gagaccugcc ucagggcuuc 660
agcgcccugg agccucuggu ggaccugccu aucggcauca acaucaccag auuccagacc 720
cugcuggccc ugcacagaag cuaccugacc ccuggcgaca gcagcagcgg cuggaccgcc 780
ggcgccgccg ccuacuacgu gggcuaccug cagccuagaa ccuuccugcu gaaguacaac 840
gagaacggca ccaucaccga cgccguggac ugcgcccugg acccucugag cgagaccaag 900
ugcacccuga agagcuucac cguggagaag ggcaucuacc agaccagcaa cuucagagug 960
cagccuaccg agagcaucgu gagauucccu aacaucacca accugugccc uuucggcgag 1020
guguucaacg ccaccagauu cgccagcgug uacgccugga acagaaagag aaucagcaac 1080
ugcguggccg acuacagcgu gcuguacaac agcgccagcu ucagcaccuu caagugcuac 1140
ggcgugagcc cuaccaagcu gaacgaccug ugcuucacca acguguacgc cgacagcuuc 1200
gugaucagag gcgacgaggu gagacagauc gccccuggcc agaccggcaa gaucgccgac 1260
uacaacuaca agcugccuga cgacuucacc ggcugcguga ucgccuggaa cagcaacaac 1320
cuggacagca aggugggcgg caacuacaac uaccuguaca gacuguucag aaagagcaac 1380
cugaagccuu ucgagagaga caucagcacc gagaucuacc aggccggcag caccccuugc 1440
aacggcgugg agggcuucaa cugcuacuuc ccucugcaga gcuacggcuu ccagccuacc 1500
aacggcgugg gcuaccagcc uuacagagug guggugcuga gcuucgagcu gcugcacgcc 1560
ccugccaccg ugugcggccc uaagaagagc accaaccugg ugaagaacaa gugcgugaac 1620
uuc 1623
<210> 27
<211> 1623
<212> RNA
<213> Artificial Sequence
<220>
<223> mRNA疫苗
<400> 27
auguucgugu uccuggugcu gcugccccug gugagcagcc agugcgugaa ccugaccacc 60
aggacccagc ugccccccgc cuacaccaac agcuucacca ggggcgugua cuaccccgac 120
aagguguuca ggagcagcgu gcugcacagc acccaggacc uguuccugcc cuucuucagc 180
aacgugaccu gguuccacgc cauccacgug agcggcacca acggcaccaa gagguucgac 240
aaccccgugc ugcccuucaa cgacggcgug uacuucgcca gcaccgagaa gagcaacauc 300
aucaggggcu ggaucuucgg caccacccug gacagcaaga cccagagccu gcugaucgug 360
aacaacgcca ccaacguggu gaucaaggug ugcgaguucc aguucugcaa cgaccccuuc 420
cugggcgugu acuaccacaa gaacaacaag agcuggaugg agagcgaguu caggguguac 480
agcagcgcca acaacugcac cuucgaguac gugagccagc ccuuccugau ggaccuggag 540
ggcaagcagg gcaacuucaa gaaccugagg gaguucgugu ucaagaacau cgacggcuac 600
uucaagaucu acagcaagca cacccccauc aaccugguga gggaccugcc ccagggcuuc 660
agcgcccugg agccccuggu ggaccugccc aucggcauca acaucaccag guuccagacc 720
cugcuggccc ugcacaggag cuaccugacc cccggcgaca gcagcagcgg cuggaccgcc 780
ggcgccgccg ccuacuacgu gggcuaccug cagcccagga ccuuccugcu gaaguacaac 840
gagaacggca ccaucaccga cgccguggac ugcgcccugg acccccugag cgagaccaag 900
ugcacccuga agagcuucac cguggagaag ggcaucuacc agaccagcaa cuucagggug 960
cagcccaccg agagcaucgu gagguucccc aacaucacca accugugccc cuucggcgag 1020
guguucaacg ccaccagguu cgccagcgug uacgccugga acaggaagag gaucagcaac 1080
ugcguggccg acuacagcgu gcuguacaac agcgccagcu ucagcaccuu caagugcuac 1140
ggcgugagcc ccaccaagcu gaacgaccug ugcuucacca acguguacgc cgacagcuuc 1200
gugaucaggg gcgacgaggu gaggcagauc gcccccggcc agaccggcaa gaucgccgac 1260
uacaacuaca agcugcccga cgacuucacc ggcugcguga ucgccuggaa cagcaacaac 1320
cuggacagca aggugggcgg caacuacaac uaccuguaca ggcuguucag gaagagcaac 1380
cugaagcccu ucgagaggga caucagcacc gagaucuacc aggccggcag cacccccugc 1440
aacggcgugg agggcuucaa cugcuacuuc ccccugcaga gcuacggcuu ccagcccacc 1500
aacggcgugg gcuaccagcc cuacagggug guggugcuga gcuucgagcu gcugcacgcc 1560
cccgccaccg ugugcggccc caagaagagc accaaccugg ugaagaacaa gugcgugaac 1620
uuc 1623
<210> 28
<211> 1614
<212> RNA
<213> Artificial Sequence
<220>
<223> mRNA疫苗
<400> 28
auguucgugu uccuggugcu gcugccccug gugagcagcc agugcgugaa ccugaccacc 60
agaacccagc ugccccccgc cuacaccaac agcuucacca gaggcgugua cuaccccgac 120
aagguguuca gaagcagcgu gcugcacagc acccaggacc uguuccugcc cuucuucagc 180
aacgugaccu gguuccacgc caucagcggc accaacggca ccaagagauu cgacaacccc 240
gugcugcccu ucaacgacgg cguguacuuc gccagcaccg agaagagcaa caucaucaga 300
ggcuggaucu ucggcaccac ccuggacagc aagacccaga gccugcugau cgugaacaac 360
gccaccaacg uggugaucaa ggugugcgag uuccaguucu gcaacgaccc cuuccugggc 420
guguaccaca agaacaacaa gagcuggaug gagagcgagu ucagagugua cagcagcgcc 480
aacaacugca ccuucgagua cgugagccag cccuuccuga uggaccugga gggcaagcag 540
ggcaacuuca agaaccugag agaguucgug uucaagaaca ucgacggcua cuucaagauc 600
uacagcaagc acacccccau caaccuggug agagaccugc cccagggcuu cagcgcccug 660
gagccccugg uggaccugcc caucggcauc aacaucacca gauuccagac ccugcuggcc 720
cugcacagaa gcuaccugac ccccggcgac agcagcagcg gcuggaccgc cggcgccgcc 780
gccuacuacg ugggcuaccu gcagcccaga accuuccugc ugaaguacaa cgagaacggc 840
accaucaccg acgccgugga cugcgcccug gacccccuga gcgagaccaa gugcacccug 900
aagagcuuca ccguggagaa gggcaucuac cagaccagca acuucagagu gcagcccacc 960
gagagcaucg ugagauuccc caacaucacc aaccugugcc ccuucggcga gguguucaac 1020
gccaccagau ucgccagcgu guacgccugg aacagaaaga gaaucagcaa cugcguggcc 1080
gacuacagcg ugcuguacaa cagcgccagc uucagcaccu ucaagugcua cggcgugagc 1140
cccaccaagc ugaacgaccu gugcuucacc aacguguacg ccgacagcuu cgugaucaga 1200
ggcgacgagg ugagacagau cgcccccggc cagaccggca agaucgccga cuacaacuac 1260
aagcugcccg acgacuucac cggcugcgug aucgccugga acagcaacaa ccuggacagc 1320
aaggugggcg gcaacuacaa cuaccuguac agacuguuca gaaagagcaa ccugaagccc 1380
uucgagagag acaucagcac cgagaucuac caggccggca gcacccccug caacggcgug 1440
gagggcuuca acugcuacuu cccccugcag agcuacggcu uccagcccac cuacggcgug 1500
ggcuaccagc ccuacagagu gguggugcug agcuucgagc ugcugcacgc ccccgccacc 1560
gugugcggcc ccaagaagag caccaaccug gugaagaaca agugcgugaa cuuc 1614
<210> 29
<211> 1614
<212> RNA
<213> Artificial Sequence
<220>
<223> mRNA疫苗
<400> 29
auguucgugu uccuggugcu gcugccucug gugagcagcc agugcgugaa ccugaccacc 60
agaacccagc ugccuccugc cuacaccaac agcuucacca gaggcgugua cuacccugac 120
aagguguuca gaagcagcgu gcugcacagc acccaggacc uguuccugcc uuucuucagc 180
aacgugaccu gguuccacgc caucagcggc accaacggca ccaagagauu cgacaacccu 240
gugcugccuu ucaacgacgg cguguacuuc gccagcaccg agaagagcaa caucaucaga 300
ggcuggaucu ucggcaccac ccuggacagc aagacccaga gccugcugau cgugaacaac 360
gccaccaacg uggugaucaa ggugugcgag uuccaguucu gcaacgaccc uuuccugggc 420
guguaccaca agaacaacaa gagcuggaug gagagcgagu ucagagugua cagcagcgcc 480
aacaacugca ccuucgagua cgugagccag ccuuuccuga uggaccugga gggcaagcag 540
ggcaacuuca agaaccugag agaguucgug uucaagaaca ucgacggcua cuucaagauc 600
uacagcaagc acaccccuau caaccuggug agagaccugc cucagggcuu cagcgcccug 660
gagccucugg uggaccugcc uaucggcauc aacaucacca gauuccagac ccugcuggcc 720
cugcacagaa gcuaccugac cccuggcgac agcagcagcg gcuggaccgc cggcgccgcc 780
gccuacuacg ugggcuaccu gcagccuaga accuuccugc ugaaguacaa cgagaacggc 840
accaucaccg acgccgugga cugcgcccug gacccucuga gcgagaccaa gugcacccug 900
aagagcuuca ccguggagaa gggcaucuac cagaccagca acuucagagu gcagccuacc 960
gagagcaucg ugagauuccc uaacaucacc aaccugugcc cuuucggcga gguguucaac 1020
gccaccagau ucgccagcgu guacgccugg aacagaaaga gaaucagcaa cugcguggcc 1080
gacuacagcg ugcuguacaa cagcgccagc uucagcaccu ucaagugcua cggcgugagc 1140
ccuaccaagc ugaacgaccu gugcuucacc aacguguacg ccgacagcuu cgugaucaga 1200
ggcgacgagg ugagacagau cgccccuggc cagaccggca agaucgccga cuacaacuac 1260
aagcugccug acgacuucac cggcugcgug aucgccugga acagcaacaa ccuggacagc 1320
aaggugggcg gcaacuacaa cuaccuguac agacuguuca gaaagagcaa ccugaagccu 1380
uucgagagag acaucagcac cgagaucuac caggccggca gcaccccuug caacggcgug 1440
gagggcuuca acugcuacuu cccucugcag agcuacggcu uccagccuac cuacggcgug 1500
ggcuaccagc cuuacagagu gguggugcug agcuucgagc ugcugcacgc cccugccacc 1560
gugugcggcc cuaagaagag caccaaccug gugaagaaca agugcgugaa cuuc 1614
<210> 30
<211> 1614
<212> RNA
<213> Artificial Sequence
<220>
<223> mRNA疫苗
<400> 30
auguucgugu uccuggugcu gcugccccug gugagcagcc agugcgugaa ccugaccacc 60
aggacccagc ugccccccgc cuacaccaac agcuucacca ggggcgugua cuaccccgac 120
aagguguuca ggagcagcgu gcugcacagc acccaggacc uguuccugcc cuucuucagc 180
aacgugaccu gguuccacgc caucagcggc accaacggca ccaagagguu cgacaacccc 240
gugcugcccu ucaacgacgg cguguacuuc gccagcaccg agaagagcaa caucaucagg 300
ggcuggaucu ucggcaccac ccuggacagc aagacccaga gccugcugau cgugaacaac 360
gccaccaacg uggugaucaa ggugugcgag uuccaguucu gcaacgaccc cuuccugggc 420
guguaccaca agaacaacaa gagcuggaug gagagcgagu ucagggugua cagcagcgcc 480
aacaacugca ccuucgagua cgugagccag cccuuccuga uggaccugga gggcaagcag 540
ggcaacuuca agaaccugag ggaguucgug uucaagaaca ucgacggcua cuucaagauc 600
uacagcaagc acacccccau caaccuggug agggaccugc cccagggcuu cagcgcccug 660
gagccccugg uggaccugcc caucggcauc aacaucacca gguuccagac ccugcuggcc 720
cugcacagga gcuaccugac ccccggcgac agcagcagcg gcuggaccgc cggcgccgcc 780
gccuacuacg ugggcuaccu gcagcccagg accuuccugc ugaaguacaa cgagaacggc 840
accaucaccg acgccgugga cugcgcccug gacccccuga gcgagaccaa gugcacccug 900
aagagcuuca ccguggagaa gggcaucuac cagaccagca acuucagggu gcagcccacc 960
gagagcaucg ugagguuccc caacaucacc aaccugugcc ccuucggcga gguguucaac 1020
gccaccaggu ucgccagcgu guacgccugg aacaggaaga ggaucagcaa cugcguggcc 1080
gacuacagcg ugcuguacaa cagcgccagc uucagcaccu ucaagugcua cggcgugagc 1140
cccaccaagc ugaacgaccu gugcuucacc aacguguacg ccgacagcuu cgugaucagg 1200
ggcgacgagg ugaggcagau cgcccccggc cagaccggca agaucgccga cuacaacuac 1260
aagcugcccg acgacuucac cggcugcgug aucgccugga acagcaacaa ccuggacagc 1320
aaggugggcg gcaacuacaa cuaccuguac aggcuguuca ggaagagcaa ccugaagccc 1380
uucgagaggg acaucagcac cgagaucuac caggccggca gcacccccug caacggcgug 1440
gagggcuuca acugcuacuu cccccugcag agcuacggcu uccagcccac cuacggcgug 1500
ggcuaccagc ccuacagggu gguggugcug agcuucgagc ugcugcacgc ccccgccacc 1560
gugugcggcc ccaagaagag caccaaccug gugaagaaca agugcgugaa cuuc 1614
<210> 31
<211> 1617
<212> RNA
<213> Artificial Sequence
<220>
<223> mRNA疫苗
<400> 31
auguucgugu uccuggugcu gcugccccug gugagcagcc agugcgugaa ccugaccacc 60
aggacccagc ugccccccgc cuacaccaac agcuucacca ggggcgugua cuaccccgac 120
aagguguuca ggagcagcgu gcugcacagc acccaggacc uguuccugcc cuucuucagc 180
aacgugaccu gguuccacgc cauccacgug agcggcacca acggcaccaa gagguucgcc 240
aaccccgugc ugcccuucaa cgacggcgug uacuucgcca gcaccgagaa gagcaacauc 300
aucaggggcu ggaucuucgg caccacccug gacagcaaga cccagagccu gcugaucgug 360
aacaacgcca ccaacguggu gaucaaggug ugcgaguucc aguucugcaa cgaccccuuc 420
cugggcgugu acuaccacaa gaacaacaag agcuggaugg agagcgaguu caggguguac 480
agcagcgcca acaacugcac cuucgaguac gugagccagc ccuuccugau ggaccuggag 540
ggcaagcagg gcaacuucaa gaaccugagg gaguucgugu ucaagaacau cgacggcuac 600
uucaagaucu acagcaagca cacccccauc aaccugguga ggggccugcc ccagggcuuc 660
agcgcccugg agccccuggu ggaccugccc aucggcauca acaucaccag guuccagacc 720
cugcugcaca ggagcuaccu gacccccggc gacagcagca gcggcuggac cgccggcgcc 780
gccgccuacu acgugggcua ccugcagccc aggaccuucc ugcugaagua caacgagaac 840
ggcaccauca ccgacgccgu ggacugcgcc cuggaccccc ugagcgagac caagugcacc 900
cugaagagcu ucaccgugga gaagggcauc uaccagacca gcaacuucag ggugcagccc 960
accgagagca ucgugagguu ccccaacauc accaaccugu gccccuucgg cgagguguuc 1020
aacgccacca gguucgccag cguguacgcc uggaacagga agaggaucag caacugcgug 1080
gccgacuaca gcgugcugua caacagcgcc agcuucagca ccuucaagug cuacggcgug 1140
agccccacca agcugaacga ccugugcuuc accaacgugu acgccgacag cuucgugauc 1200
aggggcgacg aggugaggca gaucgccccc ggccagaccg gcaacaucgc cgacuacaac 1260
uacaagcugc ccgacgacuu caccggcugc gugaucgccu ggaacagcaa caaccuggac 1320
agcaaggugg gcggcaacua caacuaccug uacaggcugu ucaggaagag caaccugaag 1380
cccuucgaga gggacaucag caccgagauc uaccaggccg gcagcacccc cugcaacggc 1440
gugaagggcu ucaacugcua cuucccccug cagagcuacg gcuuccagcc caccuacggc 1500
gugggcuacc agcccuacag ggugguggug cugagcuucg agcugcugca cgcccccgcc 1560
accgugugcg gccccaagaa gagcaccaac cuggugaaga acaagugcgu gaacuuc 1617
<210> 32
<211> 1617
<212> RNA
<213> Artificial Sequence
<220>
<223> mRNA疫苗
<400> 32
auguucgugu uccuggugcu gcugccucug gugagcagcc agugcgugaa ccugaccacc 60
aggacccagc ugccuccugc cuacaccaac agcuucacca ggggcgugua cuacccugac 120
aagguguuca ggagcagcgu gcugcacagc acccaggacc uguuccugcc uuucuucagc 180
aacgugaccu gguuccacgc cauccacgug agcggcacca acggcaccaa gagguucgcc 240
aacccugugc ugccuuucaa cgacggcgug uacuucgcca gcaccgagaa gagcaacauc 300
aucaggggcu ggaucuucgg caccacccug gacagcaaga cccagagccu gcugaucgug 360
aacaacgcca ccaacguggu gaucaaggug ugcgaguucc aguucugcaa cgacccuuuc 420
cugggcgugu acuaccacaa gaacaacaag agcuggaugg agagcgaguu caggguguac 480
agcagcgcca acaacugcac cuucgaguac gugagccagc cuuuccugau ggaccuggag 540
ggcaagcagg gcaacuucaa gaaccugagg gaguucgugu ucaagaacau cgacggcuac 600
uucaagaucu acagcaagca caccccuauc aaccugguga ggggccugcc ucagggcuuc 660
agcgcccugg agccucuggu ggaccugccu aucggcauca acaucaccag guuccagacc 720
cugcugcaca ggagcuaccu gaccccuggc gacagcagca gcggcuggac cgccggcgcc 780
gccgccuacu acgugggcua ccugcagccu aggaccuucc ugcugaagua caacgagaac 840
ggcaccauca ccgacgccgu ggacugcgcc cuggacccuc ugagcgagac caagugcacc 900
cugaagagcu ucaccgugga gaagggcauc uaccagacca gcaacuucag ggugcagccu 960
accgagagca ucgugagguu cccuaacauc accaaccugu gcccuuucgg cgagguguuc 1020
aacgccacca gguucgccag cguguacgcc uggaacagga agaggaucag caacugcgug 1080
gccgacuaca gcgugcugua caacagcgcc agcuucagca ccuucaagug cuacggcgug 1140
agcccuacca agcugaacga ccugugcuuc accaacgugu acgccgacag cuucgugauc 1200
aggggcgacg aggugaggca gaucgccccu ggccagaccg gcaacaucgc cgacuacaac 1260
uacaagcugc cugacgacuu caccggcugc gugaucgccu ggaacagcaa caaccuggac 1320
agcaaggugg gcggcaacua caacuaccug uacaggcugu ucaggaagag caaccugaag 1380
ccuuucgaga gggacaucag caccgagauc uaccaggccg gcagcacccc uugcaacggc 1440
gugaagggcu ucaacugcua cuucccucug cagagcuacg gcuuccagcc uaccuacggc 1500
gugggcuacc agccuuacag ggugguggug cugagcuucg agcugcugca cgccccugcc 1560
accgugugcg gcccuaagaa gagcaccaac cuggugaaga acaagugcgu gaacuuc 1617
<210> 33
<211> 1617
<212> RNA
<213> Artificial Sequence
<220>
<223> mRNA疫苗
<400> 33
auguucgugu uccuggugcu gcugccccug gugagcagcc agugcgugaa ccugaccacc 60
aggacccagc ugccccccgc cuacaccaac agcuucacca ggggcgugua cuaccccgac 120
aagguguuca ggagcagcgu gcugcacagc acccaggacc uguuccugcc cuucuucagc 180
aacgugaccu gguuccacgc cauccacgug agcggcacca acggcaccaa gagguucgcc 240
aaccccgugc ugcccuucaa cgacggcgug uacuucgcca gcaccgagaa gagcaacauc 300
aucaggggcu ggaucuucgg caccacccug gacagcaaga cccagagccu gcugaucgug 360
aacaacgcca ccaacguggu gaucaaggug ugcgaguucc aguucugcaa cgaccccuuc 420
cugggcgugu acuaccacaa gaacaacaag agcuggaugg agagcgaguu caggguguac 480
agcagcgcca acaacugcac cuucgaguac gugagccagc ccuuccugau ggaccuggag 540
ggcaagcagg gcaacuucaa gaaccugagg gaguucgugu ucaagaacau cgacggcuac 600
uucaagaucu acagcaagca cacccccauc aaccugguga ggggccugcc ccagggcuuc 660
agcgcccugg agccccuggu ggaccugccc aucggcauca acaucaccag guuccagacc 720
cugcugcaca ggagcuaccu gacccccggc gacagcagca gcggcuggac cgccggcgcc 780
gccgccuacu acgugggcua ccugcagccc aggaccuucc ugcugaagua caacgagaac 840
ggcaccauca ccgacgccgu ggacugcgcc cuggaccccc ugagcgagac caagugcacc 900
cugaagagcu ucaccgugga gaagggcauc uaccagacca gcaacuucag ggugcagccc 960
accgagagca ucgugagguu ccccaacauc accaaccugu gccccuucgg cgagguguuc 1020
aacgccacca gguucgccag cguguacgcc uggaacagga agaggaucag caacugcgug 1080
gccgacuaca gcgugcugua caacagcgcc agcuucagca ccuucaagug cuacggcgug 1140
agccccacca agcugaacga ccugugcuuc accaacgugu acgccgacag cuucgugauc 1200
aggggcgacg aggugaggca gaucgccccc ggccagaccg gcaacaucgc cgacuacaac 1260
uacaagcugc ccgacgacuu caccggcugc gugaucgccu ggaacagcaa caaccuggac 1320
agcaaggugg gcggcaacua caacuaccug uacaggcugu ucaggaagag caaccugaag 1380
cccuucgaga gggacaucag caccgagauc uaccaggccg gcagcacccc cugcaacggc 1440
gugaagggcu ucaacugcua cuucccccug cagagcuacg gcuuccagcc caccuacggc 1500
gugggcuacc agcccuacag ggugguggug cugagcuucg agcugcugca cgcccccgcc 1560
accgugugcg gccccaagaa gagcaccaac cuggugaaga acaagugcgu gaacuuc 1617
<210> 34
<211> 1623
<212> RNA
<213> Artificial Sequence
<220>
<223> mRNA疫苗
<400> 34
auguucgugu uccuggugcu gcugccccug gugagcagcc agugcgugaa cuucaccaac 60
agaacccagc ugcccagcgc cuacaccaac agcuucacca gaggcgugua cuaccccgac 120
aagguguuca gaagcagcgu gcugcacagc acccaggacc uguuccugcc cuucuucagc 180
aacgugaccu gguuccacgc cauccacgug agcggcacca acggcaccaa gagauucgac 240
aaccccgugc ugcccuucaa cgacggcgug uacuucgcca gcaccgagaa gagcaacauc 300
aucagaggcu ggaucuucgg caccacccug gacagcaaga cccagagccu gcugaucgug 360
aacaacgcca ccaacguggu gaucaaggug ugcgaguucc aguucugcaa cuaccccuuc 420
cugggcgugu acuaccacaa gaacaacaag agcuggaugg agagcgaguu cagaguguac 480
agcagcgcca acaacugcac cuucgaguac gugagccagc ccuuccugau ggaccuggag 540
ggcaagcagg gcaacuucaa gaaccugagc gaguucgugu ucaagaacau cgacggcuac 600
uucaagaucu acagcaagca cacccccauc aaccugguga gagaccugcc ccagggcuuc 660
agcgcccugg agccccuggu ggaccugccc aucggcauca acaucaccag auuccagacc 720
cugcuggccc ugcacagaag cuaccugacc cccggcgaca gcagcagcgg cuggaccgcc 780
ggcgccgccg ccuacuacgu gggcuaccug cagcccagaa ccuuccugcu gaaguacaac 840
gagaacggca ccaucaccga cgccguggac ugcgcccugg acccccugag cgagaccaag 900
ugcacccuga agagcuucac cguggagaag ggcaucuacc agaccagcaa cuucagagug 960
cagcccaccg agagcaucgu gagauucccc aacaucacca accugugccc cuucggcgag 1020
guguucaacg ccaccagauu cgccagcgug uacgccugga acagaaagag aaucagcaac 1080
ugcguggccg acuacagcgu gcuguacaac agcgccagcu ucagcaccuu caagugcuac 1140
ggcgugagcc ccaccaagcu gaacgaccug ugcuucacca acguguacgc cgacagcuuc 1200
gugaucagag gcgacgaggu gagacagauc gcccccggcc agaccggcac caucgccgac 1260
uacaacuaca agcugcccga cgacuucacc ggcugcguga ucgccuggaa cagcaacaac 1320
cuggacagca aggugggcgg caacuacaac uaccuguaca gacuguucag aaagagcaac 1380
cugaagcccu ucgagagaga caucagcacc gagaucuacc aggccggcag cacccccugc 1440
aacggcguga agggcuucaa cugcuacuuc ccccugcaga gcuacggcuu ccagcccacc 1500
uacggcgugg gcuaccagcc cuacagagug guggugcuga gcuucgagcu gcugcacgcc 1560
cccgccaccg ugugcggccc caagaagagc accaaccugg ugaagaacaa gugcgugaac 1620
uuc 1623
<210> 35
<211> 1623
<212> RNA
<213> Artificial Sequence
<220>
<223> mRNA疫苗
<400> 35
auguucgugu uccuggugcu gcugccucug gugagcagcc agugcgugaa cuucaccaac 60
agaacccagc ugccuagcgc cuacaccaac agcuucacca gaggcgugua cuacccugac 120
aagguguuca gaagcagcgu gcugcacagc acccaggacc uguuccugcc uuucuucagc 180
aacgugaccu gguuccacgc cauccacgug agcggcacca acggcaccaa gagauucgac 240
aacccugugc ugccuuucaa cgacggcgug uacuucgcca gcaccgagaa gagcaacauc 300
aucagaggcu ggaucuucgg caccacccug gacagcaaga cccagagccu gcugaucgug 360
aacaacgcca ccaacguggu gaucaaggug ugcgaguucc aguucugcaa cuacccuuuc 420
cugggcgugu acuaccacaa gaacaacaag agcuggaugg agagcgaguu cagaguguac 480
agcagcgcca acaacugcac cuucgaguac gugagccagc cuuuccugau ggaccuggag 540
ggcaagcagg gcaacuucaa gaaccugagc gaguucgugu ucaagaacau cgacggcuac 600
uucaagaucu acagcaagca caccccuauc aaccugguga gagaccugcc ucagggcuuc 660
agcgcccugg agccucuggu ggaccugccu aucggcauca acaucaccag auuccagacc 720
cugcuggccc ugcacagaag cuaccugacc ccuggcgaca gcagcagcgg cuggaccgcc 780
ggcgccgccg ccuacuacgu gggcuaccug cagccuagaa ccuuccugcu gaaguacaac 840
gagaacggca ccaucaccga cgccguggac ugcgcccugg acccucugag cgagaccaag 900
ugcacccuga agagcuucac cguggagaag ggcaucuacc agaccagcaa cuucagagug 960
cagccuaccg agagcaucgu gagauucccu aacaucacca accugugccc uuucggcgag 1020
guguucaacg ccaccagauu cgccagcgug uacgccugga acagaaagag aaucagcaac 1080
ugcguggccg acuacagcgu gcuguacaac agcgccagcu ucagcaccuu caagugcuac 1140
ggcgugagcc cuaccaagcu gaacgaccug ugcuucacca acguguacgc cgacagcuuc 1200
gugaucagag gcgacgaggu gagacagauc gccccuggcc agaccggcac caucgccgac 1260
uacaacuaca agcugccuga cgacuucacc ggcugcguga ucgccuggaa cagcaacaac 1320
cuggacagca aggugggcgg caacuacaac uaccuguaca gacuguucag aaagagcaac 1380
cugaagccuu ucgagagaga caucagcacc gagaucuacc aggccggcag caccccuugc 1440
aacggcguga agggcuucaa cugcuacuuc ccucugcaga gcuacggcuu ccagccuacc 1500
uacggcgugg gcuaccagcc uuacagagug guggugcuga gcuucgagcu gcugcacgcc 1560
ccugccaccg ugugcggccc uaagaagagc accaaccugg ugaagaacaa gugcgugaac 1620
uuc 1623
<210> 36
<211> 1623
<212> RNA
<213> Artificial Sequence
<220>
<223> mRNA疫苗
<400> 36
auguucgugu uccuggugcu gcugccccug gugagcagcc agugcgugaa cuucaccaac 60
aggacccagc ugcccagcgc cuacaccaac agcuucacca ggggcgugua cuaccccgac 120
aagguguuca ggagcagcgu gcugcacagc acccaggacc uguuccugcc cuucuucagc 180
aacgugaccu gguuccacgc cauccacgug agcggcacca acggcaccaa gagguucgac 240
aaccccgugc ugcccuucaa cgacggcgug uacuucgcca gcaccgagaa gagcaacauc 300
aucaggggcu ggaucuucgg caccacccug gacagcaaga cccagagccu gcugaucgug 360
aacaacgcca ccaacguggu gaucaaggug ugcgaguucc aguucugcaa cuaccccuuc 420
cugggcgugu acuaccacaa gaacaacaag agcuggaugg agagcgaguu caggguguac 480
agcagcgcca acaacugcac cuucgaguac gugagccagc ccuuccugau ggaccuggag 540
ggcaagcagg gcaacuucaa gaaccugagc gaguucgugu ucaagaacau cgacggcuac 600
uucaagaucu acagcaagca cacccccauc aaccugguga gggaccugcc ccagggcuuc 660
agcgcccugg agccccuggu ggaccugccc aucggcauca acaucaccag guuccagacc 720
cugcuggccc ugcacaggag cuaccugacc cccggcgaca gcagcagcgg cuggaccgcc 780
ggcgccgccg ccuacuacgu gggcuaccug cagcccagga ccuuccugcu gaaguacaac 840
gagaacggca ccaucaccga cgccguggac ugcgcccugg acccccugag cgagaccaag 900
ugcacccuga agagcuucac cguggagaag ggcaucuacc agaccagcaa cuucagggug 960
cagcccaccg agagcaucgu gagguucccc aacaucacca accugugccc cuucggcgag 1020
guguucaacg ccaccagguu cgccagcgug uacgccugga acaggaagag gaucagcaac 1080
ugcguggccg acuacagcgu gcuguacaac agcgccagcu ucagcaccuu caagugcuac 1140
ggcgugagcc ccaccaagcu gaacgaccug ugcuucacca acguguacgc cgacagcuuc 1200
gugaucaggg gcgacgaggu gaggcagauc gcccccggcc agaccggcac caucgccgac 1260
uacaacuaca agcugcccga cgacuucacc ggcugcguga ucgccuggaa cagcaacaac 1320
cuggacagca aggugggcgg caacuacaac uaccuguaca ggcuguucag gaagagcaac 1380
cugaagcccu ucgagaggga caucagcacc gagaucuacc aggccggcag cacccccugc 1440
aacggcguga agggcuucaa cugcuacuuc ccccugcaga gcuacggcuu ccagcccacc 1500
uacggcgugg gcuaccagcc cuacagggug guggugcuga gcuucgagcu gcugcacgcc 1560
cccgccaccg ugugcggccc caagaagagc accaaccugg ugaagaacaa gugcgugaac 1620
uuc 1623
<210> 37
<211> 1623
<212> RNA
<213> Artificial Sequence
<220>
<223> mRNA疫苗
<400> 37
auguucgugu uccuggugcu gcugccccug gugagcagcc agugcgugaa ccugaccacc 60
agaacccagc ugccccccgc cuacaccaac agcuucacca gaggcgugua cuaccccgac 120
aagguguuca gaagcagcgu gcugcacagc acccaggacc uguuccugcc cuucuucagc 180
aacgugaccu gguuccacgc cauccacgug agcggcacca acggcaccaa gagauucgac 240
aaccccgugc ugcccuucaa cgacggcgug uacuucgcca gcaccgagaa gagcaacauc 300
aucagaggcu ggaucuucgg caccacccug gacagcaaga cccagagccu gcugaucgug 360
aacaacgcca ccaacguggu gaucaaggug ugcgaguucc aguucugcaa cgaccccuuc 420
cugggcgugu acuaccacaa gaacaacaag agcuggaugg agagcgaguu cagaguguac 480
agcagcgcca acaacugcac cuucgaguac gugagccagc ccuuccugau ggaccuggag 540
ggcaagcagg gcaacuucaa gaaccugaga gaguucgugu ucaagaacau cgacggcuac 600
uucaagaucu acagcaagca cacccccauc aaccugguga gagaccugcc ccagggcuuc 660
agcgcccugg agccccuggu ggaccugccc aucggcauca acaucaccag auuccagacc 720
cugcuggccc ugcacagaag cuaccugacc cccggcgaca gcagcagcgg cuggaccgcc 780
ggcgccgccg ccuacuacgu gggcuaccug cagcccagaa ccuuccugcu gaaguacaac 840
gagaacggca ccaucaccga cgccguggac ugcgcccugg acccccugag cgagaccaag 900
ugcacccuga agagcuucac cguggagaag ggcaucuacc agaccagcaa cuucagagug 960
cagcccaccg agagcaucgu gagauucccc aacaucacca accugugccc cuucggcgag 1020
guguucaacg ccaccagauu cgccagcgug uacgccugga acagaaagag aaucagcaac 1080
ugcguggccg acuacagcgu gcuguacaac agcgccagcu ucagcaccuu caagugcuac 1140
ggcgugagcc ccaccaagcu gaacgaccug ugcuucacca acguguacgc cgacagcuuc 1200
gugaucagag gcgacgaggu gagacagauc gcccccggcc agaccggcaa gaucgccgac 1260
uacaacuaca agcugcccga cgacuucacc ggcugcguga ucgccuggaa cagcaacaac 1320
cuggacagca aggugggcgg caacuacaac uacagauaca gacuguucag aaagagcaac 1380
cugaagcccu ucgagagaga caucagcacc gagaucuacc aggccggcag cacccccugc 1440
aacggcgugg agggcuucaa cugcuacuuc ccccugcaga gcuacggcuu ccagcccacc 1500
aacggcgugg gcuaccagcc cuacagagug guggugcuga gcuucgagcu gcugcacgcc 1560
cccgccaccg ugugcggccc caagaagagc accaaccugg ugaagaacaa gugcgugaac 1620
uuc 1623
<210> 38
<211> 1623
<212> RNA
<213> Artificial Sequence
<220>
<223> mRNA疫苗
<400> 38
auguucgugu uccuggugcu gcugccucug gugagcagcc agugcgugaa ccugaccacc 60
agaacccagc ugccuccugc cuacaccaac agcuucacca gaggcgugua cuacccugac 120
aagguguuca gaagcagcgu gcugcacagc acccaggacc uguuccugcc uuucuucagc 180
aacgugaccu gguuccacgc cauccacgug agcggcacca acggcaccaa gagauucgac 240
aacccugugc ugccuuucaa cgacggcgug uacuucgcca gcaccgagaa gagcaacauc 300
aucagaggcu ggaucuucgg caccacccug gacagcaaga cccagagccu gcugaucgug 360
aacaacgcca ccaacguggu gaucaaggug ugcgaguucc aguucugcaa cgacccuuuc 420
cugggcgugu acuaccacaa gaacaacaag agcuggaugg agagcgaguu cagaguguac 480
agcagcgcca acaacugcac cuucgaguac gugagccagc cuuuccugau ggaccuggag 540
ggcaagcagg gcaacuucaa gaaccugaga gaguucgugu ucaagaacau cgacggcuac 600
uucaagaucu acagcaagca caccccuauc aaccugguga gagaccugcc ucagggcuuc 660
agcgcccugg agccucuggu ggaccugccu aucggcauca acaucaccag auuccagacc 720
cugcuggccc ugcacagaag cuaccugacc ccuggcgaca gcagcagcgg cuggaccgcc 780
ggcgccgccg ccuacuacgu gggcuaccug cagccuagaa ccuuccugcu gaaguacaac 840
gagaacggca ccaucaccga cgccguggac ugcgcccugg acccucugag cgagaccaag 900
ugcacccuga agagcuucac cguggagaag ggcaucuacc agaccagcaa cuucagagug 960
cagccuaccg agagcaucgu gagauucccu aacaucacca accugugccc uuucggcgag 1020
guguucaacg ccaccagauu cgccagcgug uacgccugga acagaaagag aaucagcaac 1080
ugcguggccg acuacagcgu gcuguacaac agcgccagcu ucagcaccuu caagugcuac 1140
ggcgugagcc cuaccaagcu gaacgaccug ugcuucacca acguguacgc cgacagcuuc 1200
gugaucagag gcgacgaggu gagacagauc gccccuggcc agaccggcaa gaucgccgac 1260
uacaacuaca agcugccuga cgacuucacc ggcugcguga ucgccuggaa cagcaacaac 1320
cuggacagca aggugggcgg caacuacaac uacagauaca gacuguucag aaagagcaac 1380
cugaagccuu ucgagagaga caucagcacc gagaucuacc aggccggcag caccccuugc 1440
aacggcgugg agggcuucaa cugcuacuuc ccucugcaga gcuacggcuu ccagccuacc 1500
aacggcgugg gcuaccagcc uuacagagug guggugcuga gcuucgagcu gcugcacgcc 1560
ccugccaccg ugugcggccc uaagaagagc accaaccugg ugaagaacaa gugcgugaac 1620
uuc 1623
<210> 39
<211> 1623
<212> RNA
<213> Artificial Sequence
<220>
<223> mRNA疫苗
<400> 39
auguucgugu uccuggugcu gcugccccug gugagcagcc agugcgugaa ccugaccacc 60
aggacccagc ugccccccgc cuacaccaac agcuucacca ggggcgugua cuaccccgac 120
aagguguuca ggagcagcgu gcugcacagc acccaggacc uguuccugcc cuucuucagc 180
aacgugaccu gguuccacgc cauccacgug agcggcacca acggcaccaa gagguucgac 240
aaccccgugc ugcccuucaa cgacggcgug uacuucgcca gcaccgagaa gagcaacauc 300
aucaggggcu ggaucuucgg caccacccug gacagcaaga cccagagccu gcugaucgug 360
aacaacgcca ccaacguggu gaucaaggug ugcgaguucc aguucugcaa cgaccccuuc 420
cugggcgugu acuaccacaa gaacaacaag agcuggaugg agagcgaguu caggguguac 480
agcagcgcca acaacugcac cuucgaguac gugagccagc ccuuccugau ggaccuggag 540
ggcaagcagg gcaacuucaa gaaccugagg gaguucgugu ucaagaacau cgacggcuac 600
uucaagaucu acagcaagca cacccccauc aaccugguga gggaccugcc ccagggcuuc 660
agcgcccugg agccccuggu ggaccugccc aucggcauca acaucaccag guuccagacc 720
cugcuggccc ugcacaggag cuaccugacc cccggcgaca gcagcagcgg cuggaccgcc 780
ggcgccgccg ccuacuacgu gggcuaccug cagcccagga ccuuccugcu gaaguacaac 840
gagaacggca ccaucaccga cgccguggac ugcgcccugg acccccugag cgagaccaag 900
ugcacccuga agagcuucac cguggagaag ggcaucuacc agaccagcaa cuucagggug 960
cagcccaccg agagcaucgu gagguucccc aacaucacca accugugccc cuucggcgag 1020
guguucaacg ccaccagguu cgccagcgug uacgccugga acaggaagag gaucagcaac 1080
ugcguggccg acuacagcgu gcuguacaac agcgccagcu ucagcaccuu caagugcuac 1140
ggcgugagcc ccaccaagcu gaacgaccug ugcuucacca acguguacgc cgacagcuuc 1200
gugaucaggg gcgacgaggu gaggcagauc gcccccggcc agaccggcaa gaucgccgac 1260
uacaacuaca agcugcccga cgacuucacc ggcugcguga ucgccuggaa cagcaacaac 1320
cuggacagca aggugggcgg caacuacaac uacagguaca ggcuguucag gaagagcaac 1380
cugaagcccu ucgagaggga caucagcacc gagaucuacc aggccggcag cacccccugc 1440
aacggcgugg agggcuucaa cugcuacuuc ccccugcaga gcuacggcuu ccagcccacc 1500
aacggcgugg gcuaccagcc cuacagggug guggugcuga gcuucgagcu gcugcacgcc 1560
cccgccaccg ugugcggccc caagaagagc accaaccugg ugaagaacaa gugcgugaac 1620
uuc 1623
<210> 40
<211> 1620
<212> RNA
<213> Artificial Sequence
<220>
<223> mRNA疫苗
<400> 40
auguuugugu uccuggugcu gcugccccug gugagcagcc agugcgugaa ccugaccacc 60
agaacacagc ugccucccgc cuacaccaac uccuucaccc gcggcgugua cuacccugau 120
aagguguuca ggagcuccgu gcugcacagc acccaggacc uguuccugcc uuucuucucu 180
aacgugaccu gguuccacgu gauuagcggc acaaacggca ccaagcgguu cgauaacccc 240
gugcugcccu ucaacgacgg cguguacuuc gccuccaucg aaaaaagcaa cauuaucaga 300
ggcuggaucu ucggcaccac ccuggauucc aagacccagu cccugcugau cgugaacaac 360
gccaccaacg ucgugaucaa ggugugugag uuccaguucu guaaugaccc cuuccuggac 420
cacaagaaca acaagagcug gauggagagc gaguucaggg uguacagcuc cgccaacaac 480
ugcacauucg aguacgugag ccagcccuuc cugauggacc uggagggcaa gcagggcaac 540
uucaagaauc ugagagaguu cguguucaag aacaucgaug gauacuucaa gaucuacagc 600
aagcacaccc cuaucaucgu gagggagccu gaggaccugc cccagggcuu cagcgcccug 660
gagccccucg uggaccugcc uaucggcauc aacaucacaa gguuccagac ccugcuggcc 720
cugcacaggu ccuaccugac accaggcgac agcagcagcg gcuggacagc cggcgccgcc 780
gccuacuaug ugggcuaccu gcagcccaga acauuccugc ugaaguauaa cgagaauggc 840
accaucacag acgccgugga cugugcccug gauccccuga gcgagaccaa gugcacucug 900
aaguccuuua caguggagaa gggaaucuac cagacaagca auuucagggu gcagccuaca 960
gagagcauug ugagauuccc uaacaucacc aaccugugcc ccuuugacga aguguuuaac 1020
gcuacacggu uugcaucagu guaugccugg aacaggaaga gaaucaguaa cuguguggcc 1080
gacuacucug ugcuguauaa ccuggcuccc uucuuuacuu ucaaaugcua cggggugagc 1140
ccuacuaagc ugaaugaccu guguuucaca aacguguaug ccgauagcuu ugugaucagg 1200
ggcgacgaag ugagacagau cgcuccaggc cagacaggca auauugccga uuacaacuau 1260
aaacugcccg augacuucac aggaugcgug auugccugga auagcaauaa gcuggauucu 1320
aaagugagcg gcaauuacaa uuaccuguau aggcuguuca gaaagagcaa ccugaagccu 1380
uucgagagag acaucuccac ugaaaucuau caggccggca auaagcccug caacggcgug 1440
gcuggcuuua auuguuacuu uccccugaga uccuauagcu uucgccccac cuauggcgug 1500
ggacaccagc cuuacagagu gguggugcug uccuucgagc ugcugcacgc cccugccaca 1560
gugugcggcc caaagaaguc caccaaucug gugaagaaua agugcgugaa cuucugauaa 1620
<210> 41
<211> 1614
<212> RNA
<213> Artificial Sequence
<220>
<223> mRNA疫苗
<400> 41
auguucgugu uccuggugcu gcugccccug gugagcagcc agugcgugaa ccugaccacc 60
aggacccagc ugccccccgc cuacaccaac agcuucacca ggggcgugua cuaccccgac 120
aagguguuca ggagcagcgu gcugcacagc acccaggacc uguuccugcc cuucuucagc 180
aacgugaccu gguuccacgu gaucagcggc accaacggca ccaagagguu cgacaacccc 240
gugcugcccu ucaacgacgg cguguacuuc gccagcaucg agaagagcaa caucaucagg 300
ggcuggaucu ucggcaccac ccuggacagc aagacccaga gccugcugau cgugaacaac 360
gccaccaacg uggugaucaa ggugugcgag uuccaguucu gcaacgaccc cuuccuggac 420
cacaagaaca acaagagcug gauggagagc gaguucaggg uguacagcag cgccaacaac 480
ugcaccuucg aguacgugag ccagcccuuc cugauggacc uggagggcaa gcagggcaac 540
uucaagaacc ugagggaguu cguguucaag aacaucgacg gcuacuucaa gaucuacagc 600
aagcacaccc ccauccuggu gagggagccc gaggaccugc cccagggcuu cagcgcccug 660
gagccccugg uggaccugcc caucggcauc aacaucacca gguuccagac ccugcuggcc 720
cugcacagga gcuaccugac ccccggcgac agcagcagcg gcuggaccgc cggcgccgcc 780
gccuacuacg ugggcuaccu gcagcccagg accuuccugc ugaaguacaa cgagaacggc 840
accaucaccg acgccgugga cugcgcccug gacccccuga gcgagaccaa gugcacccug 900
aagagcuuca ccguggagaa gggcaucuac cagaccagca acuucagggu gcagcccacc 960
gagagcaucg ugagguuccc caacaucacc aaccugugcc ccuucgacga gguguucaac 1020
gccaccaggu ucgccagcgu guacgccugg aacaggaaga ggaucagcaa cugcguggcc 1080
gacuacagcg ugcuguacaa ccuggccccc uucuucaccu ucaagugcua cggcgugagc 1140
cccaccaagc ugaacgaccu gugcuucacc aacguguacg ccgacagcuu cgugaucagg 1200
ggcgacgagg ugaggcagau cgcccccggc cagaccggca acaucgccga cuacaacuac 1260
aagcugcccg acgacuucac cggcugcgug aucgccugga acagcaacaa gcuggacagc 1320
aaggugagcg gcaacuacaa cuaccuguac aggcuguuca ggaagagcaa ccugaagccc 1380
uucgagaggg acaucagcac cgagaucuac caggccggca acaagcccug caacggcgug 1440
gccggcuuca acugcuacuu cccccugagg agcuacagcu ucaggcccac cuacggcgug 1500
ggccaccagc ccuacagggu gguggugcug agcuucgagc ugcugcacgc ccccgccacc 1560
gugugcggcc ccaagaagag caccaaccug gugaagaaca agugcgugaa cuuc 1614
<210> 42
<211> 1614
<212> RNA
<213> Artificial Sequence
<220>
<223> mRNA疫苗
<400> 42
auguucgugu uccuggugcu gcugccccug gugagcagcc agugcgugaa ccugaccacc 60
aggacccagc ugccccccgc cuacaccaac agcuucacca ggggcgugua cuaccccgac 120
aagguguuca ggagcagcgu gcugcacagc acccaggacc uguuccugcc cuucuucagc 180
aacgugaccu gguuccacgu gaucagcggc accaacggca ccaagagguu cgacaacccc 240
gugcugcccu ucaacgacgg cguguacuuc gccagcaucg agaagagcaa caucaucagg 300
ggcuggaucu ucggcaccac ccuggacagc aagacccaga gccugcugau cgugaacaac 360
gccaccaacg uggugaucaa ggugugcgag uuccaguucu gcaacgaccc cuuccuggac 420
cacaagaaca acaagagcug gauggagagc gaguucaggg uguacagcag cgccaacaac 480
ugcaccuucg aguacgugag ccagcccuuc cugauggacc uggagggcaa gcagggcaac 540
uucaagaacc ugagggaguu cguguucaag aacaucgacg gcuacuucaa gaucuacagc 600
aagcacaccc ccauccuggu gagggagccc gaggaccugc cccagggcuu cagcgcccug 660
gagccccugg uggaccugcc caucggcauc aacaucacca gguuccagac ccugcuggcc 720
cugcacagga gcuaccugac ccccggcgac agcagcagcg gcuggaccgc cggcgccgcc 780
gccuacuacg ugggcuaccu gcagcccagg accuuccugc ugaaguacaa cgagaacggc 840
accaucaccg acgccgugga cugcgcccug gacccccuga gcgagaccaa gugcacccug 900
aagagcuuca ccguggagaa gggcaucuac cagaccagca acuucagggu gcagcccacc 960
gagagcaucg ugagguuccc caacaucacc aaccugugcc ccuucgacga gguguucaac 1020
gccaccaggu ucgccagcgu guacgccugg aacaggaaga ggaucagcaa cugcguggcc 1080
gacuacagcg ugcuguacaa ccuggccccc uucuucaccu ucaagugcua cggcgugagc 1140
cccaccaagc ugaacgaccu gugcuucacc aacguguacg ccgacagcuu cgugaucagg 1200
ggcgacgagg ugaggcagau cgcccccggc cagaccggca acaucgccga cuacaacuac 1260
aagcugcccg acgacuucac cggcugcgug aucgccugga acagcaacaa gcuggacagc 1320
aaggugagcg gcaacuacaa cuaccuguac aggcuguuca ggaagagcaa ccugaagccc 1380
uucgagaggg acaucagcac cgagaucuac caggccggca acaagcccug caacggcgug 1440
gccggcuuca acugcuacuu cccccugagg agcuacagcu ucaggcccac cuacggcgug 1500
ggccaccagc ccuacagggu gguggugcug agcuucgagc ugcugcacgc ccccgccacc 1560
gugugcggcc ccaagaagag caccaaccug gugaagaaca agugcgugaa cuuc 1614
<210> 43
<211> 672
<212> RNA
<213> Artificial Sequence
<220>
<223> mRNA疫苗
<400> 43
augagggugc agcccaccga gagcaucgug agguucccca acaucaccaa ccugugcccc 60
uucggcgagg uguucaacgc caccagguuc gccagcgugu acgccuggaa caggaagagg 120
aucagcaacu gcguggccga cuacagcgug cuguacaaca gcgccagcuu cagcaccuuc 180
aagugcuacg gcgugagccc caccaagcug aacgaccugu gcuucaccaa cguguacgcc 240
gacagcuucg ugaucagggg cgacgaggug aggcagaucg cccccggcca gaccggcaag 300
aucgccgacu acaacuacaa gcugcccgac gacuucaccg gcugcgugau cgccuggaac 360
agcaacaacc uggacagcaa ggugggcggc aacuacaacu accuguacag gcuguucagg 420
aagagcaacc ugaagcccuu cgagagggac aucagcaccg agaucuacca ggccggcagc 480
acccccugca acggcgugga gggcuucaac ugcuacuucc cccugcagag cuacggcuuc 540
cagcccacca acggcguggg cuaccagccc uacagggugg uggugcugag cuucgagcug 600
cugcacgccc ccgccaccgu gugcggcccc aagaagagca ccaaccuggu gaagaacaag 660
ugcgugaacu uc 672
<210> 44
<211> 529
<212> PRT
<213> Artificial Sequence
<220>
<223> NTD-RBD
<400> 44
Ser Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr
1 5 10 15
Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg
20 25 30
Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser
35 40 45
Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr
50 55 60
Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe
65 70 75 80
Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr
85 90 95
Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr
100 105 110
Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe
115 120 125
Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu
130 135 140
Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser
145 150 155 160
Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn
165 170 175
Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr
180 185 190
Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe
195 200 205
Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr
210 215 220
Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly
225 230 235 240
Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly
245 250 255
Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr
260 265 270
Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys
275 280 285
Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser
290 295 300
Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile
305 310 315 320
Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala
325 330 335
Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp
340 345 350
Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr
355 360 365
Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr
370 375 380
Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro
385 390 395 400
Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp
405 410 415
Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys
420 425 430
Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn
435 440 445
Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly
450 455 460
Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu
465 470 475 480
Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr
485 490 495
Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val
500 505 510
Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn
515 520 525
Phe
<210> 45
<211> 526
<212> PRT
<213> Artificial Sequence
<220>
<223> NTD-RBD
<400> 45
Ser Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr
1 5 10 15
Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg
20 25 30
Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser
35 40 45
Asn Val Thr Trp Phe His Ala Ile Ser Gly Thr Asn Gly Thr Lys Arg
50 55 60
Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser
65 70 75 80
Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu
85 90 95
Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val
100 105 110
Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly
115 120 125
Val Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val
130 135 140
Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe
145 150 155 160
Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu
165 170 175
Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His
180 185 190
Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu
195 200 205
Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln
210 215 220
Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser
225 230 235 240
Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln
245 250 255
Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp
260 265 270
Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu
275 280 285
Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg
290 295 300
Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu
305 310 315 320
Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr
325 330 335
Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val
340 345 350
Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser
355 360 365
Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser
370 375 380
Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr
385 390 395 400
Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly
405 410 415
Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly
420 425 430
Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro
435 440 445
Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro
450 455 460
Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr
465 470 475 480
Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val Val
485 490 495
Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro
500 505 510
Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe
515 520 525
<210> 46
<211> 527
<212> PRT
<213> Artificial Sequence
<220>
<223> NTD-RBD
<400> 46
Ser Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr
1 5 10 15
Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg
20 25 30
Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser
35 40 45
Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr
50 55 60
Lys Arg Phe Ala Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe
65 70 75 80
Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr
85 90 95
Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr
100 105 110
Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe
115 120 125
Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu
130 135 140
Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser
145 150 155 160
Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn
165 170 175
Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr
180 185 190
Ser Lys His Thr Pro Ile Asn Leu Val Arg Gly Leu Pro Gln Gly Phe
195 200 205
Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr
210 215 220
Arg Phe Gln Thr Leu Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser
225 230 235 240
Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu
245 250 255
Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr
260 265 270
Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr
275 280 285
Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe
290 295 300
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
305 310 315 320
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
325 330 335
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
340 345 350
Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
355 360 365
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
370 375 380
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
385 390 395 400
Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
405 410 415
Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly
420 425 430
Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
435 440 445
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr
450 455 460
Pro Cys Asn Gly Val Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser
465 470 475 480
Tyr Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val
485 490 495
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
500 505 510
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe
515 520 525
<210> 47
<211> 529
<212> PRT
<213> Artificial Sequence
<220>
<223> NTD-RBD
<400> 47
Ser Gln Cys Val Asn Phe Thr Asn Arg Thr Gln Leu Pro Ser Ala Tyr
1 5 10 15
Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg
20 25 30
Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser
35 40 45
Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr
50 55 60
Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe
65 70 75 80
Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr
85 90 95
Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr
100 105 110
Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Tyr Pro Phe
115 120 125
Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu
130 135 140
Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser
145 150 155 160
Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn
165 170 175
Leu Ser Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr
180 185 190
Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe
195 200 205
Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr
210 215 220
Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly
225 230 235 240
Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly
245 250 255
Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr
260 265 270
Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys
275 280 285
Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser
290 295 300
Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile
305 310 315 320
Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala
325 330 335
Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp
340 345 350
Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr
355 360 365
Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr
370 375 380
Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro
385 390 395 400
Gly Gln Thr Gly Thr Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp
405 410 415
Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys
420 425 430
Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn
435 440 445
Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly
450 455 460
Ser Thr Pro Cys Asn Gly Val Lys Gly Phe Asn Cys Tyr Phe Pro Leu
465 470 475 480
Gln Ser Tyr Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr
485 490 495
Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val
500 505 510
Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn
515 520 525
Phe
<210> 48
<211> 529
<212> PRT
<213> Artificial Sequence
<220>
<223> NTD-RBD
<400> 48
Ser Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr
1 5 10 15
Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg
20 25 30
Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser
35 40 45
Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr
50 55 60
Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe
65 70 75 80
Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr
85 90 95
Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr
100 105 110
Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe
115 120 125
Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu
130 135 140
Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser
145 150 155 160
Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn
165 170 175
Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr
180 185 190
Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe
195 200 205
Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr
210 215 220
Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly
225 230 235 240
Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly
245 250 255
Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr
260 265 270
Ile Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys
275 280 285
Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser
290 295 300
Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile
305 310 315 320
Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala
325 330 335
Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp
340 345 350
Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr
355 360 365
Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr
370 375 380
Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro
385 390 395 400
Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp
405 410 415
Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys
420 425 430
Val Gly Gly Asn Tyr Asn Tyr Arg Tyr Arg Leu Phe Arg Lys Ser Asn
435 440 445
Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly
450 455 460
Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu
465 470 475 480
Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr
485 490 495
Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val
500 505 510
Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn
515 520 525
Phe
<210> 49
<211> 526
<212> PRT
<213> Artificial Sequence
<220>
<223> NTD-RBD
<400> 49
Ser Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr
1 5 10 15
Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg
20 25 30
Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser
35 40 45
Asn Val Thr Trp Phe His Val Ile Ser Gly Thr Asn Gly Thr Lys Arg
50 55 60
Phe Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser
65 70 75 80
Ile Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu
85 90 95
Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val
100 105 110
Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Asp
115 120 125
His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr Ser
130 135 140
Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu Met
145 150 155 160
Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe Val
165 170 175
Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr Pro
180 185 190
Ile Ile Val Arg Glu Pro Glu Asp Leu Pro Gln Gly Phe Ser Ala Leu
195 200 205
Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln
210 215 220
Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser
225 230 235 240
Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln
245 250 255
Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp
260 265 270
Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu
275 280 285
Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg
290 295 300
Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu
305 310 315 320
Cys Pro Phe Asp Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr
325 330 335
Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val
340 345 350
Leu Tyr Asn Leu Ala Pro Phe Phe Thr Phe Lys Cys Tyr Gly Val Ser
355 360 365
Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser
370 375 380
Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr
385 390 395 400
Gly Asn Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly
405 410 415
Cys Val Ile Ala Trp Asn Ser Asn Lys Leu Asp Ser Lys Val Ser Gly
420 425 430
Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro
435 440 445
Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Asn Lys Pro
450 455 460
Cys Asn Gly Val Ala Gly Phe Asn Cys Tyr Phe Pro Leu Arg Ser Tyr
465 470 475 480
Ser Phe Arg Pro Thr Tyr Gly Val Gly His Gln Pro Tyr Arg Val Val
485 490 495
Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro
500 505 510
Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe
515 520 525
<210> 50
<211> 14
<212> PRT
<213> Artificial Sequence
<220>
<223> 连接肽
<400> 50
Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe
1 5 10
<210> 51
<211> 51
<212> RNA
<213> Artificial Sequence
<220>
<223> 5'UTR
<400> 51
agggagauaa gagagaaaag aagaguaaga agaaauauaa gagccgccac c 51
<210> 52
<211> 93
<212> RNA
<213> Artificial Sequence
<220>
<223> 3'UTR
<400> 52
gcugccuucu gcggggcuug ccuucuggcc augcccuucu ucucucccuu gcaccuguac 60
cucuuggucu uugaauaaag ccugaguagg aag 93
<210> 53
<211> 112
<212> RNA
<213> Artificial Sequence
<220>
<223> 聚-A尾
<400> 53
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa agcauaugac uaaaaaaaaa aaaaaaaaaa 60
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aa 112
<210> 54
<211> 1614
<212> RNA
<213> Artificial Sequence
<220>
<223> mRNA疫苗
<400> 54
auguucgugu uccuggugcu gcugccguua gugaguaguc agugugugaa ccugacuacu 60
cgcacgcagc ugccaccagc guacacgaac agcuucacgc ggggcgugua uuaucccgac 120
aaaguauuuc gcagcagcgu ccugcacucc acccaggacc ucuuccuccc guucuucucc 180
aaugugacgu gguuucacgu cauaucgggg acgaacggga cgaagagguu cgacaauccg 240
guccugccuu uuaacgacgg gguguauuuc gcgucaauag agaagaguaa uauuauccga 300
ggguggaucu ucgggaccac ccuggauagu aagacucagu cucuauugau cgugaauaac 360
gccaccaacg ucguuauaaa ggucugcgag uuucaguucu gcaaugaucc auucuuggau 420
cauaagaaca auaagucgug gauggagagc gaguuucgag uguacucgag ugcaaacaau 480
ugcacuuucg aguacgucuc gcagccguuc uugauggacc uggaggguaa gcaggggaau 540
uucaagaauc uuagagaauu cgucuuuaag aauauugacg gauacuuuaa gauuuauucc 600
aagcauaccc cuauuaucgu ccgggagccg gaggaccugc cgcagggcuu cagcgcucug 660
gagccgcugg uugaccuccc gauagggauc aacaucacgc gguuccagac gcuguuagcc 720
cugcacaggu ccuaccugac ucccggcgau aguaguucug gguggacugc aggcgcugcu 780
gcguacuaug ucggguacuu acagccccgc acguucuugu ugaaguacaa cgagaacggc 840
acaaucacgg acgccguuga uugugcccua gauccguuau cggagaccaa guguacucuc 900
aagucguuca ccguggagaa gggcaucuac cagacgucca acuucagggu gcagccgacu 960
gagaguauag uucgguuucc gaacauaacg aaucuaugcc cguuugacga gguguucaau 1020
gcgacgcgcu ucgccuccgu guaugcaugg aaccgaaaac gcauaagcaa uugugucgca 1080
gauuauucag uuuuguacaa ucucgccccg uucuucacgu ucaaguguua cggggugagu 1140
ccgacaaagc ugaaugaucu guguuucaca aauguuuaug cggauucguu cgugauacgc 1200
ggagacgaag ugcgucagau ugcuccuggu cagacgggca auauagccga cuauaacuac 1260
aagcuaccag acgacuucac ugguugugug auagcuugga auucgaacaa gcuggacagc 1320
aagguaucag guaauuacaa uuaccuguac cggcuguuca ggaagucgaa uuugaagccu 1380
uucgagcgcg acaucucgac cgagaucuac caggccggaa acaagccgug caacggagua 1440
gcaggcuuca acugcuacuu uccguugcgc agcuacaguu uccggccuac cuacggggua 1500
gggcaccagc ccuaccgcgu aguggugcuc uccuucgagc ugcuccacgc accggccacg 1560
gugugugggc cgaagaagag caccaaucug gucaagaaca agugcgugaa cuuu 1614
<210> 55
<211> 1614
<212> RNA
<213> Artificial Sequence
<220>
<223> mRNA疫苗
<400> 55
auguucgucu uccuggugcu gcuuccgcug gucaguucac agugugugaa cuugaccacg 60
cggacgcagc uaccccccgc guauacgaac ucguuuacgc gggggguaua uuacccggau 120
aagguguuuc guaguuccgu ucuccacagu acucaggacc ucuuccuccc guucuucucc 180
aaugugacgu gguuucacgu cauaucgggg acgaacggga cgaagagguu cgacaacccc 240
guguugcccu ucaacgacgg gguuuauuuc gcgucaauag agaagaguaa uauuauccga 300
ggguggaucu ucgggaccac ccuggauagu aagacucagu cucuauugau cgugaacaac 360
gccacuaaug uggugaucaa ggugugcgag uuucaguucu gcaaugaucc auucuuggau 420
cauaagaaca acaagaguug gauggagagu gaauuccgug uuuacucuuc agccaacaau 480
uguaccuucg aguaugucag ucagccguuu cugauggauc uugaaggcaa gcagggcaau 540
uucaagaauu ugcgcgaguu ugucuucaag aacaucgacg gcuacuucaa gauauacucg 600
aagcacacgc cgauaauugu gcgcgagccg gaagaucuac cacagggcuu cagugcucug 660
gagcccuugg uagaucuucc gaucggcaua aauaucacgc guuuccagac gcugcuggcg 720
uugcaccgga guuaccuuac uccgggcgac uccagcagcg gcuggacggc aggggcugca 780
gcguacuaug uaggguaucu gcaaccgcgc acguucuugu ugaaguacaa cgagaacggc 840
acgauuacag augccguaga uugugcgcug gacccccugu cggagacuaa gugcaccuug 900
aagaguuuua cuguggagaa gggaaucuac cagacaucua acuuccgggu gcagccgaca 960
gagaguauug uacguuuccc gaacaucacg aaucuaugcc cguuugacga gguguuuaac 1020
gccacgcggu uugcuagugu guaugcgugg aaucgcaagc gcauuagcaa cugcguggcg 1080
gauuauucag uuuuguauaa ucucgccccg uucuucacgu ucaaguguua cggggugagu 1140
ccuacaaagc ugaaugaucu cugcuucacc aacgucuacg cggauagcuu uguuauccgc 1200
ggagacgagg ugaggcagau agcaccuggu cagacgggca auauugcuga cuacaacuau 1260
aagcucccgg augacuucac cgggugcguu auagcgugga acagcaauaa guuagauucg 1320
aaggugucgg gaaacuacaa uuaucucuau cggcuguuca ggaagucgaa cuuaaagccu 1380
uuugagcgug auauaucuac cgagauauau caggcuggga auaagccgug caacggggua 1440
gcaggcuuca acugcuacuu cccguugcgc agcuauuccu uucggcccac cuacggggua 1500
gggcaccagc ccuaccgcgu aguuguacug ucguuugagc ugcugcaugc uccagcgaca 1560
guaugugggc cgaagaaguc gacgaacuug gugaagaaca agugcgucaa cuuc 1614

Claims (31)

1.一种能编码目标多肽的mRNA分子，其中，所述的目标多肽包括SARS-CoV-2刺突蛋白S中NTD-RBD天然结构域，所述NTD-RBD天然结构域包括NTD片段与RBD片段，NTD片段与RBD片段之间以来源于S蛋白的天然氨基酸序列作为连接子连接。
2.根据权利要求1所述的mRNA分子，该mRNA还编码NTD-RBD天然结构域N端的信号肽。
3.根据权利要求1或2所述的mRNA分子，该mRNA分子编码的氨基酸序列从N端到C端依次包括信号肽、NTD片段、连接子、RBD片段。
4.根据权利要求1至3任意一项所述的mRNA分子，其中，所述的连接子的氨基酸序列为SEQ ID NO:50。
5.根据权利要求1至4中任意一项所述的mRNA分子，其中，NTD片段的氨基酸序列选自：
(a)SEQ ID NO:49的第1至第289位组成的氨基酸序列；
(b)由(a)的氨基酸序列经过替换、增加和/或缺失一个或几个氨基酸且具有与(a)相同功能的衍生序列。
6.根据权利要求1至5中任意一项所述的mRNA分子，其中，RBD片段的氨基酸序列选自：
(c)SEQ ID NO:49的第304至第526位组成的氨基酸序列；
(d)由(c)的氨基酸序列经过替换、增加和/或缺失一个或几个氨基酸且具有与(a)相同功能的衍生序列。
7.根据权利要求1至6中任意一项所述的mRNA分子，其中，所述的NTD-RBD天然结构域的氨基酸序列为：
SEQ ID NO:44至SEQ ID NO:49中任一序列所示氨基酸序列；或
与SEQ ID NO:44至SEQ ID NO:49中任一序列同一性在94.3％以上的衍生的氨基酸序列。
8.根据权利要求7所述的mRNA分子，其中，所述的NTD-RBD天然结构域的氨基酸序列为SEQ ID NO:49、SEQ ID NO:48、SEQ ID NO:47、SEQ ID NO:46、SEQ ID NO:45、SEQ ID NO:44中的一种。
9.根据权利要求3至7中任意一项所述的mRNA分子，其中，所述的mRNA编码的氨基酸序列为SEQ ID NO:21、SEQ ID NO:17、SEQ ID NO:13、SEQ ID NO:9、SEQ ID NO:5或SEQ IDNO:1。
10.根据权利要求1至9中任意一项所述的mRNA分子，其蛋白质编码区序列如SEQ IDNO:25、SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ IDNO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36、SEQ IDNO:37、SEQ ID NO:38、SEQ ID NO:39、SEQ ID NO:40、SEQ ID NO:41或SEQ ID NO:42所示。
11.根据权利要求1至10中任意一项所述的mRNA分子，其中，该mRNA经过1-甲基假尿苷修饰。
12.根据权利要求1至11中任意一项所述的mRNA分子，其中，该mRNA还包括5’-UTR序列和/或3’-UTR序列。
13.根据权利要求12所述的mRNA分子，其中，5’-UTR序列包含或不包含Kozak序列；
优选地，所述5'-UTR具有SEQ ID NO:51所示序列。
14.根据权利要求12所述的mRNA分子，其中，3’-UTR序列具有SEQ ID NO:52所示序列。
15.根据权利要求1至14中任意一项所述的mRNA分子，其中，该mRNA还经过3'加尾修饰和/或至少一个5'加帽修饰；
优选地，3'加尾修饰包含聚-A尾，所述聚-A尾为中间插入或不插入连接子的多聚腺苷酸；
优选地，至少一个5'加帽修饰的帽结构选自Cap0、Cap1、ARCA、肌苷、N1-甲基-鸟苷、2'氟-鸟苷、7-脱氮-鸟苷、8-氧代-鸟苷、2-氨基-鸟苷、LNA-鸟苷或2-叠氮基-鸟苷。
16.根据权利要求1至15中任一项所述的mRNA分子，其是分离的mRNA；
优选地，所述mRNA分子是经纯化的。
17.一种DNA分子，其编码权利要求1至16中任意一项所述的mRNA分子。
18.一种重组质粒，其含有权利要求17所述的DNA分子。
19.一种脂质纳米颗粒，其负载有权利要求1至16中任意一项所述的mRNA分子。
20.一种药物组合物，其包含：权利要求1至16中任一项所述的mRNA分子，以及药学上可接受的赋形剂。
21.权利要求1至16中任意一项所述的mRNA分子、权利要求17所述的DNA分子、权利要求18所述的重组质粒、权利要求19所述的脂质纳米颗粒或权利要求20所述的药物组合物在制备新型冠状病毒mRNA疫苗中的应用。
22.一种新型冠状病毒mRNA疫苗，其包含权利要求1至16中任意一项所述的mRNA分子，所述的疫苗为脂质纳米颗粒剂型。
23.根据权利要求22所述的新型冠状病毒mRNA疫苗，其中，所述的脂质纳米颗粒包括mRNA和脂质，其中，所述脂质包括：
a)正电荷脂质和/或可离子化脂质中的一种或多种；
b)中性辅助脂质；
c)胆固醇；
d)PEG修饰的脂质。
24.根据权利要求23所述的新型冠状病毒mRNA疫苗，其中，所述的正电荷脂质和/或可离子化脂质与mRNA的氮磷摩尔比为5:1～20:1。
25.根据权利要求23或24所述的新型冠状病毒mRNA疫苗，其中，所述的脂质纳米颗粒中，以脂质的总摩尔量为100％计，各脂质成分的摩尔比为：
正电荷脂质或可离子化脂质 46％～50％；
中性辅助脂质 5％～10％；
胆固醇 38.5％～48％；
PEG修饰的脂质 0～3％。
26.根据权利要求22至25中任意一项所述的新型冠状病毒mRNA疫苗，其中，所述的可离子化脂质包括但不限于：4-(N,N-二甲基氨基)丁酸(二亚油基)甲酯、SM-102、((4-羟基丁基)氮杂二烷基)双(己烷-6,1-二基)双(2-己基癸酸酯)中的一种或多种，所述的正电荷脂质包括但不限于：DOTMA、DOTAP中的一种或多种；所述的中性辅助脂质包括但不限于：DSPC、DOPE、DSPE中的一种或多种；所述的PEG修饰的脂质包括但不限于：甲氧基聚乙二醇双十四烷基乙酰胺、DMG-PEG中的一种或多种。
27.根据权利要求22至25中任意一项所述的新型冠状病毒mRNA疫苗，其中，疫苗剂型为冷冻干燥剂型或冷冻剂型。
28.一种新型冠状病毒mRNA疫苗的制备方法，其包括以下步骤：
合成编码NTD-RBD天然结构域肽段的DNA片段，并克隆到质粒作为模板，转录制备得到目标mRNA分子；
优选地，所述目标mRNA分子为权利要求1至16中任意一项所述的mRNA分子。
29.根据权利要求28所述的制备方法，该方法还包括：
将所制备的mRNA分子溶解在柠檬酸缓冲液组成的水相中，采用冲击式射流或微流控等方法与溶解在乙醇相中的脂质成分混合，制备负载有mRNA的脂质纳米颗粒。
30.根据权利要求29所述的制备方法，该方法还包括将所制备的脂质纳米颗粒制成冷冻制剂或冷冻干燥制剂的过程。
31.根据权利要求30所述的制备方法，其中，将脂质纳米颗粒制成冷冻干燥制剂的过程包括：
a)配制含有脂质纳米颗粒和冷冻干燥保护剂的缓冲液；
b)降温进行预冻；
c)在真空条件下升温进行干燥，使体系含水量在3％以下，制备得到脂质纳米颗粒的干燥制剂。
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