CN113736801B - mRNA及包含其的新冠病毒mRNA疫苗 - Google Patents

mRNA及包含其的新冠病毒mRNA疫苗 Download PDF

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CN113736801B
CN113736801B CN202010470599.0A CN202010470599A CN113736801B CN 113736801 B CN113736801 B CN 113736801B CN 202010470599 A CN202010470599 A CN 202010470599A CN 113736801 B CN113736801 B CN 113736801B
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王冰
俞航
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Abstract

本发明提供了mRNA，其包含编码来源于SARS‑CoV‑2病毒的S蛋白、E蛋白、M蛋白和N蛋白中的一种、两种、三种或四种蛋白或其片段的mRNA，编码S蛋白的mRNA的序列如SEQ ID NO.18、SEQ ID NO.19或SEQ ID NO.20所示；编码E蛋白的mRNA的序列如SEQ ID NO.21所示；编码M蛋白的mRNA的序列如SEQ ID NO.22所示；编码N蛋白的mRNA的序列如SEQ ID NO.23所示。还提供了一种包含所述mRNA的脂质体纳米颗粒、一种针对新冠病毒的mRNA疫苗等。本发明的mRNA在细胞水平高效产生病毒蛋白，或由产生的蛋白自组装成病毒样颗粒。将包含本发明的mRNA制备成疫苗时，安全性高、有效性好、不会产生非中和抗体而不会产生抗体依赖增强感染效应。

Description

mRNA及包含其的新冠病毒mRNA疫苗
技术领域
本发明涉及一种mRNA及包含其的新冠病毒mRNA疫苗，本发明还涉及所述mRNA 及包含其的新冠病毒mRNA疫苗、脂质体纳米颗粒、药物组合物和试剂盒等。
背景技术
近年来，基于体外转录(IVT)的信使RNA(mRNA)的治疗正显示出巨大的潜力。 它的原理是将体外制备的mRNA包裹成药物递送到体内组织被细胞内吞，外源mRNA到 达细胞后被核糖体识别并根据其编码信息合成相应的蛋白质。早在1990年Wolff等证明 注射到小鼠体内的mRNA能被翻译蛋白质[7]。1992年Jirikowski等显示注射到下丘脑部位的vasopressin mRNA缓解了老鼠的尿崩症状[8]。mRNA药物在理论上有诸多优势：与 DNA疗法相比，mRNA不需要入核，无基因组整合的***突变风险；相对于蛋白质药物， mRNA利用细胞自身的翻译***，可实现高效和剂量依赖性的活性蛋白表达，解决了一些蛋白的不可成药性问题。然而mRNA一直受到体外制备、稳定性和递送问题的困扰。 直到近几年IVT(invitro transcription，体外转录)技术配合化学和酶学加帽法、修饰核 苷酸的引入、HPLC纯化技术使得mRNA可以在体外进行大规模制备[9,10]。同时脂质 体和脂质纳米颗粒在递送siRNA上获得成功后被证明也能用于mRNA的包裹和递送[11]。 这些技术的突破使得mRNA成药性大大提高，目前有超过25个mRNA药物包括mRNA 疫苗和蛋白替代的临床研究正在展开[12]，市场上第一个mRNA产品的竞争已全面展开。 越来越多的研究人员在关注mRNA药物的应用，而中国在这一领域的研究才刚刚起步。
mRNA药物一个最有潜力的应用是疫苗，包括肿瘤疫苗和感染性疾病疫苗。编码抗原蛋白的mRNA分子经体外合成并形成制剂后即可用于人体免疫，这一过程不涉及活病 毒培养的相关操作，大大缩短了研发时间[13]。mRNA疫苗在近几年不断取得突破性进 展，在2013年的一项研究中，研究者针对H7N9流感病毒设计并制备了mRNA疫苗， 在小鼠实验中取得成效[14]。2015年，针对HIV的mRNA疫苗在非人灵长类动物中产生体液免疫应答。2017年，寨卡病毒的mRNA疫苗在有效的保护了接受病毒攻击的小鼠[15]， 并能降低孕期老鼠感染的风险[16]。除了在动物实验上取得成功以外，mRNA疫苗(如 流感疫苗和寨卡疫苗)已经开始了临床试验，Moderna公司的流感病毒mRNA疫苗I期 临床结果显示其免疫原性或优于传统疫苗[17]。同样是该公司的寨卡病毒疫苗 mRNA-1893去年进入了美国FDA的快速通道。IVT mRNA的技术优势能有效应对病毒的高突变率，使得新爆发流行病疫苗的快速开发成为可能，有望成为提高新发传染病的 防治效率的突破方向。
传统的预防病毒的疫苗有重组蛋白疫苗、灭活疫苗、减毒活疫苗以及体外重组的病 毒样颗粒(virus-like particle，VLP)。在以往的经验中灭活或减毒疫苗因形态和组成与真实病毒一样，能产生强烈的免疫反应，成为疫苗的首选。但是他们有不可避免的缺点：灭活或减毒疫苗生产周期较长、有些病毒如诺如病毒不能大规模培养、灭活病毒往往不 能诱导免疫反应而减毒疫苗亦有返祖的风险等。而体外重组病毒样颗粒疫苗是由病毒衣 壳蛋白或包膜蛋白自主包装形成的空衣壳结构，能快速刺激机体产生体液免疫和细胞免 疫应答，病毒样颗粒不含有病毒遗传物质和免疫抑制蛋白，目前安全性最高的一种新型 候选疫苗，已经有多种基于VLP的疫苗产品上市[18]。2002年SARS-CoV和2012年 MERS-CoV爆发后，多种疫苗方案被研究，包括灭活或减毒毒株、基于重组DNA的S 蛋白质以及体外重组的病毒样颗粒[19,20]。其中S蛋白质由于是介导病毒入侵的主要蛋 白质，也是中和抗体的主要靶点，受到疫苗开发的特别关注。动物试验表明这些疫苗均 有保护效果，但是安全性还是最大的担忧。例如基于全长S蛋白抗原的疫苗产生了大量 非中和抗体，这些非中和抗体在抗体依赖增强感染效应(antibody-dependent enhancement，ADE)中扮演了重要角色[21]，反而加速了疾病进展，造成疫苗安全性存在重大问题。由于机体在接受mRNA药物后能根据编码信息合成任何蛋白质，mRNA在疫苗抗原的选择 上极其灵活。但是考虑到病毒样颗粒的优势，目前在临床的病毒mRNA疫苗多以病毒样 颗粒为最终抗原展示形式，如寨卡病毒[15]。
mRNA疫苗虽有诸多优势，但大部分还是理论阶段，需要大量的基础和临床研究。有效的能在体内诱导合成病毒样颗粒的mRNA疫苗应满足两个条件，一是表达效率要高， 产生足够剂量的病毒样颗粒刺激机体产生免疫应答；二是产的病毒样颗粒应与真实病毒从形态和结构组成上一致，使机体获得免疫能力能应对真实病毒。但是由于冠状病毒自 身的特点，研发也面临着很多挑战。冠状病毒为正链单股RNA病毒，它有一层脂双分子 膜形成包膜(envelope)结构，包膜中***结构蛋白M(membrane)、E(envelope)、和S(spike)。 其中S棘突蛋白质是冠状病毒最重要的表面蛋白，决定了病毒的宿主范围和特异性。S 蛋白质是宿主中和抗体的重要作用位点，因此成为SARS-CoV和MERS-CoV的疫苗设计 中的关键靶点。冠状病毒还有核蛋白N(nucleoprotein)在内层包裹病毒的基因组。除了结 合基因组，N蛋白质对包膜的形态塑成也有作用，因此也被认为是结构蛋白之一。冠状 病毒一个特点是其形态和大小并不是完全固定的，事实上冠状病毒的直径在80-200nm之 间。因此即使是高分辨率冷冻电镜，也无法使用单颗粒分析来获得整个病毒的原子结构。 冠状病毒包膜内结构蛋白质的比例也不固定，取决于病毒在细胞内组装时各个结构蛋白质的含量。这和寨卡病毒不同，寨卡病毒虽然也是包膜病毒，但是其形态固定，成刚性 二十面体结构，其结构蛋白单一且拷贝数固定，没有棘突结构。因此同样是合成病毒样 颗粒，冠状病毒的mRNA疫苗设计要比寨卡病毒复杂的多。首先，寨卡病毒的mRNA 疫苗只包含了一条mRNA，编码prM-E融合蛋白，而冠状病毒的mRNA疫苗必须是个组 合(cocktail)，包含至少3条mRNA编码不同的结构蛋白质。其次，对冠状病毒包膜结 构的装配目前还存在诸多争议。根据对SARS-CoV的研究，M和E共表达已经足够形成 病毒样颗粒，但是没有棘突结构，共表达S与M和E可以引入S蛋白质，使得VLP带有棘突。但是尽管形成病毒样颗粒，蛋白质组成比例却和真实病毒存在很大差别。另外N蛋白质虽然在内层主要与病毒基因组相互作用，有研究并表明但是它的存在对病毒样 颗粒的表达和分泌有加强作用。目前已有几种新冠病毒疫苗进入临床试验，均以新冠病 毒S蛋白为主要抗原，安全性和有效性还未经证明，仍有失败的风险。因此，急需继续研发针对新冠病毒的多种抗原策略的新型冠状病毒疫苗。
发明内容
本发明所要解决的技术问题是为了克服现有技术中没有商品化的新冠病毒疫苗等缺 陷，提供了一种mRNA、一种DNA以及包含其的新冠病毒mRNA疫苗、脂质体纳米颗 粒、由其表达产生的病毒样颗粒、药物组合物和试剂盒。本发明的经过密码子优化后或 者进一步经过核苷酸修饰的组装新冠病毒所需的几种蛋白的mRNA，各自单独在细胞中 都可以高表达。并且经过本发明特定比例组成的mRNA，在细胞水平高效产生病毒蛋白， 或由产生的蛋白自组装成病毒样颗粒，能够实现病毒样颗粒的高表达，且大小和形态结 构均极为接近真实病毒，后续用于临床时可以使机体获得免疫能力能应对真实病毒。包 含本发明mRNA的纳米颗粒，多条mRNA同时被脂质纳米颗粒包装的效率/表达效率高 依旧很高，从而能够产生足够剂量的病毒样颗粒刺激机体产生免疫应答，免疫原性和稳 定性较高。将包含本发明的经过密码子优化后或者经过核苷酸修饰的组装新冠病毒所需的几种蛋白的mRNA制备成疫苗(例如以表达病毒样颗粒的形式、仅表达S蛋白的疫苗 的形式或以仅表达S蛋白中RBD区域的疫苗的形式)时，安全性高、有效性好、不会产 生非中和抗体从而不会产生抗体依赖增强感染效应。
本领域技术人员公知，冠状病毒的形态和大小并非完全固定，其包膜内结构蛋白质 的比例也不固定，因此同样是合成病毒样颗粒，冠状病毒的mRNA疫苗设计要比现有技术中的其他病毒复杂的多。而本发明人通过大量实验和摸索，意外发现对密码子进行特定优化后，能够实现病毒样颗粒的完整表达。本发明人在实验中还发现，体外转录生成 mRNA的翻译效率和稳定性受其不同的化学修饰(对每种mRNA使用不同的修饰核苷酸 在细胞内的命运大相径庭)，5’和3’非翻译区序列(UTRs)，5’加帽方式(使用不同的cap0 或cap1类似物)和3’poly(A)尾的长度影响。本发明人经过大量研究，发现通过选用特定 的核苷化学修饰、特定的UTR序列及特定的优化加帽方式，能够进一步实现mRNA转 染细胞半小时后即开始表达较高水平蛋白，并且表达可持续一周。同时本发明人通过大 量实验，最终发现多种修饰核苷酸通过特定的组合能够进一步取得较好的免疫原性和稳 定性。另外，本发明中S蛋白质是长达1273个氨基酸，属于较大蛋白质，结合5’和3’UTR， 最终mRNA总长超过4000nt。本发明人在实验中发现长链mRNA的合成一直是个挑战， 而通过优化编码蛋白质(例如S蛋白质)的mRNA序列，同时优化UTR序列和修饰核苷酸，进行蛋白质(例如S蛋白质)的表达筛选，还能够克服超长基因mRNA的制备与 纯化的问题。
为了解决上述技术问题，本发明第一方面提供了mRNA，其包含编码来源于 SARS-CoV-2病毒的S蛋白、E蛋白、M蛋白和N蛋白中的一种、两种、三种或四种蛋 白、其片段、变体或衍生物的mRNA，
其中，编码所述S蛋白的mRNA的序列如SEQ ID NO.18、SEQ ID NO.19或SEQ IDNO.20所示；编码所述E蛋白的mRNA的序列如SEQ ID NO.21所示；编码所述M蛋 白的mRNA的序列如SEQ ID NO.22所示；编码所述N蛋白的mRNA的序列如SEQ ID NO.23所示。
较佳地，所述片段为所述S蛋白的RBD结构域的片段，其mRNA的序列优选如SEQ IDNO.37所示。
较佳地，所述mRNA还包含5’-帽结构，优选为3′-O-Me-m7G(5')ppp(5')G、 m7G(5')ppp(5')(2'OMeA)pG或m7(3'OMeG)(5')ppp(5')(2'OMeA)pG。
本发明中，所述3′-O-Me-m7G(5')ppp(5')G的结构一般如下所示：
本发明中，所述m7G(5')ppp(5')(2'OMeA)pG的结构一般如下所示：
本发明中，所述m7(3‘OMeG)(5’)ppp(5‘)(2’OMeA)pG的结构一般如下所示：
较佳地，所述mRNA序列还包含3’-聚腺苷酸，其序列优选包含约25至约400个腺 苷核苷酸的序列，优选约50至约400个腺苷核苷酸的序列，更优选约50至约300个腺 苷核苷酸的序列，进一步更优选约50至约250个腺苷核苷酸的序列，进一步优选约60 至约250个腺苷核苷酸的序列，最优选120个聚腺苷酸组成的序列。
较佳地，所述mRNA序列还包含5’-UTR，所述5’-UTR的序列优选如SEQ ID NO. 15所示。
较佳地，所述mRNA序列还包含3’-UTR，所述3’-UTR的序列优选来源于提供 稳定的mRNA的基因的3’UTR或来源于其同源物、片段或变体，更优选如SEQ ID NO. 16或SEQ IDNO.17所示。
较佳地，所述mRNA序列还包含多核苷酸修饰，所述多核苷酸优选5-methyl-CTP、pseudo-UTP、N1-Methylpseudo-UTP和5-Methoxy-UTP中的一种或多种。本发明中，所 述的5-methyl-CTP可以购于ApexBio，#B7967。所述的pseudo-UTP可以购于ApexBio， #B7972。所述的N1-Methylpseudo-UTP可以购于ApexBio，#B8049。所述的5-Methoxy-UTP可以购于ApexBio，#B8061。
更佳地，所述N蛋白的mRNA包括5-methyl-CTP、pseudo-UTP、N1-Methylpseudo-UTP或5-Methoxy-UTP的修饰，或、包括5-methyl-CTP和pseudo-UTP共同的修饰。
更佳地，所述E蛋白的mRNA包括5-methyl-CTP、pseudo-UTP或 N1-Methylpseudo-UTP的修饰。
更佳地，当编码所述S蛋白的mRNA的序列如SEQ ID NO.18所示时，所述S蛋白 的mRNA包括5-methyl-CTP、pseudo-UTP或N1-Methylpseudo-UTP的修饰，或包括 5-methyl-CTP和pseudo-UTP的修饰，优选包括pseudo-UTP或N1-Methylpseudo-UTP的 修饰。
更佳地，当编码所述S蛋白的mRNA的序列如SEQ ID NO.19所示时，所述S蛋白 的mRNA包括pseudo-UTP的修饰，或包括5-methyl-CTP和pseudo-UTP的修饰。
更佳地，当编码所述S蛋白的mRNA的序列如SEQ ID NO.20所示时，所述S蛋白 的mRNA包括pseudo-UTP或N1-Methylpseudo-UTP的修饰。
较佳地，所述mRNA包含编码来源于SARS-CoV-2病毒的S蛋白、E蛋白和M蛋白 的mRNA，所述S蛋白、E蛋白、M蛋白由三条独立的mRNA分别表达得到，表达所述 S蛋白、E蛋白、M蛋白的mRNA的摩尔比优选为1：(2～0.5)：(2～0.5)，例如为1:1:1。
较佳地，所述mRNA包含编码来源于SARS-CoV-2病毒的M蛋白和E蛋白的mRNA， 所述M蛋白和E蛋白的mRNA优选连接后进行表达，所述连接优选通过编码2A肽段的 mRNA的序列(经过蛋白表达后，所得的2A肽经过“自剪切”，最后能够得到独立的M 蛋白和E蛋白)进行连接。其中，所述2A肽段的氨基酸序列优选如SEQ ID NO.42或 SEQ ID NO.43所示，编码所述2A肽段的DNA序列进一步优选如SEQ ID NO.38或SEQ ID NO.39所示，编码所述2A肽段的mRNA序列进一步优选如SEQ ID NO.40或SEQ ID NO.41所示。更佳地，所述连接后的mRNA的序列优选如SEQ ID NO.35或36所示，其 DNA序列优选如SEQ ID NO.28或29所示。
较佳地，所述mRNA包含编码来源于SARS-CoV-2病毒的所述的S蛋白的mRNA。
较佳地，所述mRNA包含编码来源于SARS-CoV-2病毒的所述的S蛋白的RBD结 构域的mRNA。
较佳地，所述mRNA包含编码来源于SARS-CoV-2病毒的M蛋白、E蛋白和S蛋白 的mRNA，所述M蛋白和E蛋白的mRNA连接后进行表达，所述连接优选通过编码2A 肽段的所述2A肽段的序列进行连接。其中，所述2A肽段的氨基酸序列优选如SEQ ID NO. 42或SEQ IDNO.43所示，编码所述2A肽段的DNA序列进一步优选如SEQ ID NO.38 或SEQ ID NO.39所示，编码所述2A肽段的mRNA序列进一步优选如SEQ ID NO.40或SEQ ID NO.41所示。更佳地，所述连接后的mRNA的序列优选如SEQ ID NO.35或36 所示，其DNA序列优选如SEQ ID NO.28或29所示。进一步更佳地，连接后的mRNA 与所述S蛋白的mRNA的摩尔比优选为1.5:1～3:1，例如为2:1。
较佳地，当所述的mRNA包含编码来源于SARS-CoV-2病毒的S蛋白、E蛋白、M 蛋白和N蛋白中的两种、三种或四种蛋白或其片段的mRNA时，所述mRNA编码的蛋 白自组装成病毒样颗粒。
为了解决上述技术问题，本发明第二方面提供了一种DNA，其包含编码来源于SARS-CoV-2病毒的S蛋白、E蛋白、M蛋白和N蛋白中的至少一种蛋白(例如一种、 两种、三种、四种)或其片段的DNA，
其中，编码所述S蛋白的DNA的序列如SEQ ID NO.3、SEQ ID NO.4或SEQ ID NO. 5所示；编码所述E蛋白的DNA的序列如SEQ ID NO.8所示；编码所述M蛋白的DNA 的序列如SEQID NO.11所示；编码所述N蛋白的DNA的序列如SEQ ID NO.13所示。
较佳地，所述片段为所述S蛋白的RBD结构域的片段，其DNA序列优选如SEQ IDNO.30所示。
为了解决上述技术问题，本发明第三方面提供了组合物，其包含多个或多于一个如 本发明第一方面所述的mRNA或如本发明第二方面所述的DNA。
为了解决上述技术问题，本发明第四方面提供了一种脂质体纳米颗粒，其包含本发 明第一方面所述的mRNA、如本发明第二方面所述的DNA或如本发明第三方面所述的组合物。
较佳地，所脂质体纳米颗粒还包括阳离子脂质和辅助脂质，所述阳离子脂质优选为 DLin-MC3-DMA或DOTMA，所述辅助脂质优选为DSPC和/或胆固醇。
本发明中，所述DLin-MC3-DMA的结构式一般如下所示：
本发明中，所述DOTMA的结构式一般如下所示：
本发明中，所述DSPC的结构式一般如下所示：
较佳地，所脂质体纳米颗粒为长循环阳离子脂质体纳米颗粒，优选为经PEG或其衍生物修饰的长循环阳离子脂质体纳米颗粒；所述PEG的相对分子质量优选为2000～5000，例如为2000、3000、4000或5000。在本发明某一较佳实施例中，所述脂质体纳米颗粒为 包括DMPE-PEG2000的长循环阳离子脂质体纳米颗粒。
为了解决上述技术问题，本发明第五方面提供了一种病毒样颗粒，其包含由本发明 第一方面所述的mRNA所表达的相应蛋白自组装而成、如本发明第二方面所述的DNA所表达的相应蛋白自组装而成和/或如本发明第三方面所述的组合物所表达的相应蛋白 自组装而成，优选是将所述的mRNA、所述的DNA和/或所述的组合物转入细胞后表达 相应的蛋白，所述细胞优选为293T和/或293A。
较佳地，所述的病毒样颗粒由编码SARS-CoV-2病毒的S蛋白、E蛋白、M蛋白和 N蛋白中的两种、三种或四种蛋白或其片段的mRNA所表达的蛋白自组装而成，优选在 细胞中表达所述蛋白，所述细胞优选为293T和/或293A。
更佳地，所述的病毒样颗粒由编码SARS-CoV-2病毒的S蛋白、E蛋白和M蛋白的mRNA，所述S蛋白、E蛋白、M蛋白由三条独立的mRNA分别表达的蛋白自组装得到， 表达所述S蛋白、E蛋白、M蛋白的mRNA的摩尔比优选为1：(2～0.5)：(2～0.5)，例如 为1:1:1。
更佳地，所述的病毒样颗粒由编码SARS-CoV-2病毒的M蛋白和E蛋白的mRNA 所表达的蛋白自组装而成，优选在细胞中表达所述蛋白，所述细胞优选为293T和/或293A。 其中，所述M蛋白和E蛋白的mRNA优选连接后进行表达，所述连接优选通过编码2A 肽段的mRNA的序列进行连接。其中，所述2A肽段的氨基酸序列优选如SEQ ID NO.42 或SEQ ID NO.43所示，编码所述2A肽段的DNA序列进一步优选如SEQ ID NO.38或 SEQ ID NO.39所示，编码所述2A肽段的mRNA序列进一步优选如SEQ ID NO.40或SEQ ID NO.41所示。更佳地，所述连接后的mRNA序的列优选如SEQ ID NO.35或36所示， 其DNA序列优选如SEQ ID NO.28或29所示。
更佳地，所述的病毒样颗粒由编码SARS-CoV-2病毒的M蛋白、E蛋白和S蛋白的mRNA所表达的蛋白自组装而成，优选在细胞中表达所述蛋白，所述细胞优选为293T 和/或293A。其中，所述M蛋白和E蛋白的mRNA连接后进行表达，所述连接优选通过 编码2A肽段的mRNA的序列进行连接。其中，所述2A肽段的氨基酸序列优选如SEQ ID NO.42或SEQ IDNO.43所示，编码所述2A肽段的DNA序列进一步优选如SEQ ID NO. 38或SEQ ID NO.39所示，编码所述2A肽段的RNA序列进一步优选如SEQ ID NO.40 或SEQ ID NO.41所示。更佳地，所述连接后的mRNA的序列优选如SEQ ID NO.35或 36所示，其DNA序列优选如SEQ ID NO.28或29所示。进一步更佳地，连接后的mRNA 与所述S蛋白的mRNA的摩尔比优选为1.5:1～3:1，例如为2:1。
为了解决上述技术问题，本发明第六方面提供了一种针对新冠病毒的mRNA疫苗，其包含如本发明第一方面所述的mRNA、如本发明第二方面所述的DNA、如本发明第三 方面所述的组合物和/或如本发明第四方面所述的脂质体纳米颗粒。
较佳地，所述mRNA疫苗诱导细胞产生病毒样颗粒，以激活免疫***。
较佳地，所述mRNA疫苗还包括本领域常规使用的佐剂。
为了解决上述技术问题，本发明第七方面提供了一种药物组合物，其包含如本发明 第一方面所述的mRNA、如本发明第二方面所述的DNA、如本发明第三方面所述的组合物、如本发明第四方面所述的脂质体纳米颗粒、如本发明第五方面所述的病毒样颗粒、 和/或如本发明第六方面所述的mRNA疫苗，和任选地药用载体。
为了解决上述技术问题，本发明第八方面提供了一种试剂盒，其包含如本发明第一 方面所述的mRNA、如本发明第二方面所述的DNA、如本发明第三方面所述的组合物、如本发明第四方面所述的脂质体纳米颗粒、如本发明第五方面所述的病毒样颗粒、如本 发明第六方面所述的mRNA疫苗和/或如本发明第七方面所述的药物组合物。
为了解决上述技术问题，本发明还提供了一种编码2A肽段的mRNA，其序列优选 如SEQ ID NO.40或SEQ ID NO.41所示。
为了解决上述技术问题，本发明还提供了一种编码2A肽段的DNA，其序列如SEQ IDNO.38或SEQ ID NO.39所示。
为了解决上述技术问题，本发明还提供了如本发明第一方面所述的mRNA或如本发明第二方面所述的DNA或如本发明第三方面所述的组合物在制备如本发明第四方面所 述的脂质体纳米颗粒、如本发明第五方面所述的病毒样颗粒、如本发明第六方面所述的 mRNA疫苗、如本发明第七方面所述的药物组合物、和/或如本发明第八方面所述的试剂 盒中的应用。
为了解决上述技术问题，本发明还提供了一种预防和/或治疗新冠病毒感染的方法， 其包括(任选地向有需要的受试者)施用如本发明第一方面所述的mRNA、如本发明第二方面所述的DNA、如本发明第三方面所述的组合物、如本发明第四方面所述的脂质体 纳米颗粒、如本发明第五方面所述的病毒样颗粒、如本发明第六方面所述的mRNA疫苗、 如本发明第七方面所述的药物组合物、和/或如本发明第八方面所述的试剂盒的步骤。
为了解决上述技术问题，本发明还提供了一种如本发明第一方面所述的mRNA、如本发明第二方面所述的DNA、如本发明第三方面所述的组合物、如本发明第四方面所述 的脂质体纳米颗粒、如本发明第五方面所述的病毒样颗粒、如本发明第六方面所述的 mRNA疫苗、如本发明第七方面所述的药物组合物、和/或如本发明第八方面所述的试剂 盒在预防和/或治疗新冠病毒感染中的应用。
本发明中，所示编码2A肽段的序列可以是编码天然病毒的2A肽段的序列，也可以是经过优化的序列(例如T2A和P2A，T2A的mRNA序列可以是如SEQ ID NO.40所示， P2A的mRNA序列可以是如SEQ ID NO.41所示。其对应的DNA序列可以是如SEQ ID NO.38和SEQ IDNO.39所示，经翻译后所得多肽的氨基酸序列可以是如SEQ ID NO.42 和SEQ ID NO.43所示)。这段多肽可高效“自剪切”成前后两个片段，可以使得这段序列前后两部分的序列独立表达成两个单独的蛋白，达到一段序列上协同表达两个独立蛋 白的目的。
术语解释
本发明中，所述的mRNA又称信使RNA，通常是由DNA的一条链作为模板转录而 来的、携带遗传信息的能指导蛋白质合成的一类单链核糖核酸。以细胞中基因为模板， 依据碱基互补配对原则转录生成mRNA后，mRNA就含有与DNA分子中某些功能片段 相对应的碱基序列，作为蛋白质生物合成的直接模板。
本发明中，所述的mRNA疫苗通常是通过将编码病毒抗原的mRNA直接导入到人 体内，在细胞内表达病毒蛋白抗原，从而激活人体免疫***，产生针对病毒的中和抗体。
本发明中，所述的抗原(antigen，缩写Ag)一般是指能引起抗体生成的物质，是任何可诱发免疫反应的物质。
本发明中，所述的抗体一般是指机体在抗原物质刺激下，由B细胞分化成的浆细胞所产生的、可与相应抗原发生特异性结合反应的免疫球蛋白。
本发明中，所述的中和抗体一般是指微生物入侵人体后，会刺激产生很多种抗体，但只有部分抗体能迅速识别微生物，并在其入侵人体细胞前将其“抓住”，保护人体不被 感染。这个过程就叫中和作用，发挥作用的抗体就是中和抗体。
本发明中，所述的脂质体纳米颗粒一般是指利用脂质体将药物分子(小分子化合物、 RNA、DNA或蛋白质药物)包装成百纳米大小的复合物，将药物递送到体内，具有增加 药物溶解度、延长药物在体内的滞留时间、增强药物的靶向性及降低毒性等优势。
本发明中，所述的病毒样颗粒(virus-like particles，VLPs)通常是含有某种病毒的一 个或多个结构蛋白的空心颗粒，没有病毒核酸，不能自主复制，在形态上与真正病毒粒 子相同或相似，俗称伪病毒。
本发明中，所述的新冠病毒S蛋白(Spike蛋白)又称棘突或刺突蛋白。S蛋白是冠状病毒最重要的致病靶点蛋白，包含两个亚基，S1和S2。其中，S1主要包含有受体结 合区(RBD结构域)，冠状病毒正是通过RBD结构域与细胞表面受体结合来感染细胞。 S蛋白也因此主要承担病毒与宿主细胞膜受体结合及膜融合功能。同时，也是宿主中和抗体的重要作用位点以及疫苗设计的关键靶点。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：
(1)本发明的经过密码子优化后或者进一步经过核苷酸修饰的组装新冠病毒所需的 几种蛋白的mRNA，各自单独在细胞中都可以高表达。此外，进一步经过本发明特定比 例组成的mRNA，在细胞水平高效产生病毒蛋白，或由产生的蛋白自组装成病毒样颗粒， 能够实现病毒样颗粒的高表达，且大小和形态结构均极为接近真实病毒，后续用于临床时可以使机体获得免疫能力能应对真实病毒。
(2)包含本发明mRNA的纳米颗粒，多条mRNA同时被脂质纳米颗粒包装的效率 /表达效率高依旧很高，从而能够产生足够剂量的病毒样颗粒刺激机体产生免疫应答，免 疫原性和稳定性较高。
(3)将包含本发明的经过密码子优化后或者进一步经过核苷酸修饰的组装新冠病毒 所需的几种蛋白的mRNA制备成疫苗(例如以表达病毒样颗粒的形式、仅表达S蛋白的 疫苗的形式或以仅表达S蛋白中RBD区域的疫苗的形式)时，安全性高、有效性好、不 会产生非中和抗体从而不会产生抗体依赖增强感染效应。
附图说明
图1显示了本发明实施例的概述。实施例中用mRNA表达新冠病毒结构蛋白S、M、 E和N以及S蛋白的RBD结构域。用脂质体将mRNA包被成纳米粒(LNP)，进行细胞 转染或动物免疫。体外细胞转染的多种mRNA可高表达病毒蛋白，并在合适比例下，自 组装成病毒样颗粒(VLP)。LNP免疫小鼠后，激活小鼠免疫***，产生抗体。
图2显示了mRNA用脂质体包被转染293A细胞后，Western Blot检测蛋白表达的 结果图。其中，泳道(lane)1为经cap1修饰的mRNA表达的蛋白，泳道2为经cap1+5mC+pseudoU修饰的mRNA表达的蛋白，泳道3为经cap1+pseudoU修饰的mRNA 表达的蛋白，泳道4为经cap1+5moU修饰的mRNA表达的蛋白，泳道5为经 cap1+N1-m-pseudoU修饰的mRNA表达的蛋白，泳道6为经cap1+5mC修饰的mRNA表 达的蛋白。A为N蛋白的mRNA和NBL mRNA表达蛋白的WB结果图，B为EBL mRNA 和MBL mRNA表达蛋白的WB结果图，C为SGS mRNA、STFmRNA、SBLmRNA表达蛋白的WB结果图，D为SDC50、SDC54、SDC58、SDC60表达蛋白的WB结果图，E 为SGS-RBD结构域表达蛋白的WB结果图，F为MP2AE和MT2AE表达蛋白的WB结 果图。
图3显示了VLP颗粒的电镜照片。
图4显示了mRNA脂质纳米颗粒包装示意图。
图5显示了用ZetaView检测LNP色谱分布图。A的上图是包被了表达S蛋白的SGSmRNA的LNP过滤前的粒径和分布图，下图是同一LNP过滤后的粒径分布图。B的上 图是表达S蛋白的RBD结构域的mRNA的LNP过滤前的粒径和分布图，下图是同一LNP 过滤后的粒径分布图。C的上图是包被了表达M、E和S蛋白mRNA的LNP过滤前的粒径和分布图，下图是同一LNP过滤后的粒径分布图。
图6显示了经过第一次免疫后一周，用酶联免疫吸附法测试血清中抗体滴度的结果 图。
图7显示小鼠血清中和抗体滴度实验结果。表达S蛋白全长的mRNA(Spike)和 表达病毒样颗粒的mRNA组合(SME)，诱导产生的抗体滴度均大于104，单独产生RBD 结构域的mRNA(RBD)诱导产生的中和抗体滴度略高于空白对照(Ctrl)。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施 例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
本发明针对新型冠状病毒开发的mRNA疫苗，主要采用(1)表达多种病毒蛋白， 在体内组装成病毒样颗粒；(2)表达S蛋白全长的mRNA；(3)表达S蛋白RBD结构 域这三种方式，如图1所示。
实施例1 mRNA制备
为新型冠状病毒(SARS-CoV-2)4个结构基因S、M、E、N进行了密码子优化，并 为每条基因设计了多条编码序列。每种序列将被克隆到mRNA合成载体上。对每种序列， 制备两种mRNA，一种编码不含标签的野生型蛋白质，一种编码C端带有Flag标签，用 于后期表达验证。具体步骤为：
委托上海生工进行合成携带针对SARS-Cov-2的S蛋白(Spike protein)、M蛋白、E蛋白、N蛋白(氨基酸序列分别如SEQ ID NO.1、SEQ ID NO.9、SEQ ID NO.6和SEQ ID NO.12所示，四种蛋白的天然基因序列分别如SEQ ID NO.2(连接有3’-UTR-2)、SEQ ID NO.10、SEQID NO.7和SEQ ID NO.14(连接有3’-UTR-2)所示)的经过密码子优 化的基因序列，其中：S蛋白基因优化后的序列分别如SEQ ID NO.3(SGS，连接有 3’-UTR-2)、SEQ ID NO.4(SBL或S-benchling，连接有3’-UTR-1)、SEQ ID NO.5(STF， 连接有3’-UTR-1)、SEQ ID NO.24(SDC50，连接有3’-UTR-2)、SEQ ID NO.25(SDC54，连接有3’-UTR-2)、SEQ ID NO.26(SDC58，连接有3’-UTR-2)、SEQ ID NO.27(SDC60， 连接有3’-UTR-2)所示；M蛋白基因优化后的序列如SEQ ID NO.11(MBL，连接有 3’-UTR-1)所示；E蛋白基因优化后的序列如SEQ ID NO.8(EBL，连接有3’-UTR-1) 所示；N蛋白基因优化后的序列如SEQ ID NO.13(NBL，连接有3’-UTR-2)所示。随 后将上述密码子优化过的基因序列亚克隆到含有T7启动子和5’非编码区(5’UTR，序列如SEQ ID NO.15所示)、3’非编码区(3’UTR，序列如SEQ ID NO.16(3’-UTR-1)或 SEQ ID NO.17所示(3’-UTR-2))的载体(载体有两种：一种是在pUC19基础上加入 5’-UTR和3’-UTR-1区域；一种是在pUC57基础上加入5’-UTR和3’-UTR-2区域)中。 S、E、N和M蛋白分别在C-末端被HA和Flag标记。扩增载体后，通过限制性内切酶 进行酶切使载体线性化(操作步骤均为本领域常规)。进一步纯化酶切片段，并将其用作体外转录(IVT)的模板，合成修饰的mRNA，具体为：使用具有1-2μg模板的HyperScribe T7高产率RNA合成试剂盒(ApexBio)和加帽cap0或cap1类似物(购于ApexBio)(每 个修饰的核苷酸7.5mM)进行IVT。将反应在37℃下孵育2-4小时，然后进行DNase (Thermo)处理。使用poly(A)加尾试剂盒(ApexBio)将3′poly(A)尾巴进一步添加到IVT RNA产品中。通过使用RNAClean andConcentrator试剂盒(ApexBio)纯化 mRNA。其中，所得S蛋白基因优化后的mRNA序列分别如SEQ ID NO.18(SGS mRNA)、 SEQ ID NO.19(SBL mRNA)和SEQ ID NO.20(STF mRNA)、以及SEQ ID NO.31 (SDC50)、SEQ ID NO.32(SDC54)、SEQ ID NO.33(SDC58)、SEQ ID NO.34(SDC60) 所示，所得M蛋白基因优化后的mRNA序列如SEQ ID NO.22(MBL mRNA)所示，所 得E蛋白基因优化后的mRNA序列如SEQ ID NO.21(EBL mRNA)所示，所得N蛋白 基因优化后的mRNA序列如SEQ ID NO.23(NBL mRNA)所示。
实施例2 体外转录中掺入的修饰核苷酸
在实施例1中所述的体外转录合成修饰的mRNA过程中，在反应体系中按照一定比例加入修饰核苷酸，修饰核苷酸随机***mRNA序列中。本实施例尝试使用的修饰核苷 酸包括5-methyl-CTP(简称5mC，ApexBio，#B7967)、pseudo-UTP(简称pseudoU， ApexBio，#B7972)、N1-Methylpseudo-UTP(简称N1-m-pseudo，ApexBio，#B8049)、5-Methoxy-UTP(5moU，ApexBio，#B8061)；用于mRNA的5’加帽的修饰核苷酸有 3′-O-Me-m7G(5')ppp(5')G(ARCA，cap0，APExBIO公司产品，#B8175)、 m7G(5')ppp(5')(2'OMeA)pG(APExBIO公司产品EZ Cap#B8176，cap1，)和m7(3'OMeG)(5')ppp(5')(2'OMeA)pG(APExBIO公司产品EZ Cap#B8178，cap1类似物)。
具体实验步骤为：
(1)体外mRNA序列中***多种上述修饰核苷酸；体外转录过程中随机***修饰 核苷酸，按照修饰核苷酸与未修饰核苷酸1：5的摩尔比，掺入反应体系，使用APExBIO 公司试剂盒#K1047。按照试剂盒使用说明配置反应体系，37℃反应2-4小时。
(2)转录过程如加入5’加帽核苷酸；则在转录反应体系中同时加入5’ m7(3'OMeG)(5')ppp(5')(2'OMeA)pG、m7G(5')ppp(5')(2'OMeA)pG或 3′-O-Me-m7G(5')ppp(5')G，与GTP的摩尔比为8：1。
(3)3’端加入120个聚腺苷酸的序列；使用poly(A)拖尾试剂盒(APExBIO，#K1053)将3′poly(A)尾巴添加到IVT RNA产品中，按照试剂盒使用说明配置反应体系，37℃ 反应1小时。
(4)DNase消化DNA模板；使用NEB公司DNase I(货号#M0303S)进行DNA 模板消化，37℃反应1小时。
(5)mRNA纯化；用Thermo Fisher RiboPure Kit(#AM1924)纯化转录后，DNA 模板已消化的mRNA，用1mM柠檬酸钠，pH6.4的溶液洗脱。进行琼脂糖胶核酸电泳 检测mRNA，并用NanoDrop测定浓度。
实施例3 mRNA转染细胞
使用lipofectamine 2000(lipo2K，ThermoFisher Scientific#11668019)以质量体积比 1：2(mRNA：lipo2K，1g mRNA+2L lipo2K)的比例将实施例1和2中所得S、M、E、 N的mRNA分别转染到293A细胞中，24hr后用Western Blot检测蛋白表达。所得结果如图2所示。
图2中，数字代表mRNA序列中***了不同的修饰核苷酸：1.cap1；2. cap1+5mC+pseudoU；3.cap1+pseudoU；4.cap1+5moU；5.cap1+N1-m-pseudo；6.cap1+5mC。 细胞表达的N蛋白和E蛋白均带有HA序列标签，用抗HA的抗体做western blot检测细 胞中蛋白表达情况，GAPDH蛋白作为阳性对照。其中：
如图2的A所示，N蛋白较小，各序列和修饰优化的mRNA，均可以在细胞内表达 N蛋白，其中cap1+5mC+pseudoU(lane 2)和cap1+5moU(lane 4)两种修饰时的mRNA 对于蛋白表达相对较低。
图2的B中，EBL序列表达很强，用抗HA标签肽的抗体检测，信号很强。MBL序 列接flag标签肽，用抗flag标签肽的抗体进行检测，发现cap1(lane 1)、cap1+pseudoU (lane3)以及cap1+N1-m-pseudo(lane 5)、cap1+5mC(lane 6)四种修饰组合表达较好， cap1+5mC+pseudoU(lane 2)和cap1+5moU(lane 4)两种修饰组合的mRNA，E蛋白表 达量很低。
图2的C和D中，表达S蛋白的序列分别连接HA标签肽或flag标签肽，用抗HA 或flag标签肽的抗体进行检测，蛋白表达差异极大。由图2的C中可以看出，未经优化 的天然S基因序列在293A细胞中几乎不表达，或表达量极低。STF和SBL两种优化序 列比天然S基因序列的蛋白表达略有提高，cap1+pseudoU(lane 3)和cap1+N1-m-pseudo(lane 5)修饰的STF表达的蛋白量相对较高，cap1+pseudo(lane 3)修饰的SBL表达的 蛋白量相对较高。SGS基因优化序列使得蛋白表达大为上升，表达量最好的是添加 cap+pseudoU(lane 3)和cap1+N1-m-pseudoU(lane 5)两种方式修饰的SGS序列，cap1 (lane 1)、cap1+5mC+pseudoU(lane2)、cap1+5mC(lane 6)修饰的SGS序列的表达量 也较高。由图2的D中可以看出，SDC50、SDC54、SDC58、SDC60这些优化后的序列 所表达的蛋白中，杂蛋白很多。
图2的E中，带有HA标签肽的SGS-RBD优化mRNA序列(mRNA序列如SEQ ID NO.37所示，其对应的DNA序列如SEQ ID NO.30所述，均采用pseudoU多核苷酸修 饰，5’加帽的结构为Cap1，3’加入120个聚腺苷酸，连接了序列如SEQ ID NO.15所 示的5’UTR和序列如SEQ IDNO.16或17所示的3’UTR)可以在细胞中高表达S蛋白 RBD结构域。
图2的F中，用一条mRNA序列串联表达两种蛋白M和E(即将M蛋白和E蛋白 的mRNA相连接后再进行表达，中间可使用不同的表达2A肽段的mRNA进行连接(蛋 白表达后，2A肽经过“自剪切”，最后能够得到独立的M和E蛋白。在天然病毒2A序 列基础上，经过优化所得的表达2A肽段的T2A和P2A多肽对应的DNA序列如SEQ ID NO.38和SEQ ID NO.39所示，T2AmRNA序列如SEQ ID NO.40所示，P2A mRNA序 列如SEQ ID NO.41所示，可翻译成多肽(对于序列为SEQ ID NO.42和SEQ ID NO.43))，mRNA相连接后所得MT2AE的mRNA序列如SEQ IDNO.35所示(对应的DNA序列 如SEQ ID NO.28所示)，mRNA相连接后所得MP2AE的mRNA序列如SEQ ID NO.36 所示(对应的DNA序列如SEQ ID NO.29所示)，采用pseudoU多核苷酸修饰，5’加帽的结构为Cap1，3’加入120个聚腺苷酸，连接了序列如SEQ ID NO.15所示的5’UTR 和序列如SEQ ID NO.16或17所示的3’UTR)，western blot显示位置接近的双带代表了 M和E蛋白。MP2AE优化mRNA序列得到的两个蛋白量更接近。
实施例4 病毒样颗粒的制备和观测
为了产生病毒样颗粒(VLP)，将表达的S、M、E蛋白的mRNA(SGS mRNA、MBL mRNA、EBLmRNA组成的SME mRNA，均采用pseudoU多核苷酸修饰，5’加帽的结 构为Cap1，3’加入120个聚腺苷酸，连接了序列如SEQ ID NO.15所示的5’UTR和序 列如SEQ ID NO.16或17所示的3’UTR)，按照摩尔比1:0.5:0.5的比例，用lipo2K包被， 共转染到293A细胞中，并在转染后48小时收集上清液。或者采用串联表达M蛋白、E 蛋白的mRNA(即将M蛋白和E蛋白的mRNA相连接后再进行后续步骤，中间可使用 不同的连接肽进行连接，mRNA相连接后的序列如SEQ IDNO.35或36所示)，与表达 S蛋白mRNA(SEQ ID NO.3)按照摩尔比2:1的比例，用lipo2K包被，共转染到293A 细胞中，并在转染后48小时收集上清液。
将所收集的上清液使用100kDa截止浓度的Amicon Ultra-15(Millipore)进行浓缩， 然后将其置于合适的溶液中(20mM HEPES，pH 7.4，120mM NaCl)。在4℃，31,000rpm(Beckman超速离心机，转子型号SW32)超速离心90分钟后，立即用5mL注射器提 取30-40％(w/v)之间的蔗糖溶液，该溶液包含病毒样颗粒(VLP)。使用100kDa截止 浓度的AmiconUltra-15离心管进行溶液交换，将含VLP的溶液置换成PBS缓冲液。为 了制备用于负染透射电子显微镜(TEM)的栅格，将5μL VLP溶液在辉光放电碳涂层的 栅格上吸收2分钟。将网格以逐滴滴加方式染色60秒，然后加载到Talos L120C显微镜 (Thermofisher)上以可视化VLP。结果如图3显示，图3的a中显示了由mRNA转录 出的S、E和M蛋白，自组装成的新冠病毒样颗粒电镜照片；图3的b中单个病毒样颗 粒放大照片，并测量表面棘突大小；图3的c为新冠病毒样颗粒的卡通模式图。由图3可知，电镜下VLP颗粒直径约90nm，表面形成与天然病毒类似的三聚体棘突，棘突大 小约12×13nm，与天然病毒的大小和结构都极为接近。
实施例5 mRNA包被方法
按照之前报道，将实施例2中所得含有修饰核苷酸的mRNA(分别为表达S蛋白RBD结构域的mRNA；可以表达S蛋白的SGS mRNA；表达S、M、E三种蛋白的SME mRNA， 按摩尔比为1:0.5:0.5混合表达；均采用pseudoU多核苷酸修饰，5’加帽的结构为Cap1， 3’加入120个聚腺苷酸，连接了序列如SEQ ID NO.15所示的5’UTR和序列如SEQ ID NO. 16或17所示的3’UTR)利用DLin-MC3-DMA(APExBIO，#A8791)，在低pH下可离 子化(阳离子)，两种辅助脂质(DSPC和胆固醇)和聚乙二醇化脂质(DMPE-PEG2000) 包被成纳米颗粒(示意图如图4所示)。通过将溶解在超纯水中的mRNA，与pH 3.0的 100μmM柠檬酸盐缓冲液1：1(v/v)混合，制备了mRNA的水溶液。调节四种脂质组 分[可离子化的脂质：胆固醇：DSPC：DMPE-PEG2000]比例(50:10:38.5:1.5)，在乙醇(99.5％) 中溶解为脂质溶液。将mRNA和脂质溶液在NanoAssemblr(Precision Nanosystems)微 流体混合***中以Aq：EtOH＝3：1的体积混合比和恒定的12mL/min的总流速混合， 得到包含mRNA的脂质体纳米颗粒(LNP)。
为了表征上述所制得的LNP，在制备后，将25μL的样品级分注入975的10μmM磷 酸盐缓冲液(pH 7.4)中，并用于在ZetaSizer(Malvern Instruments Inc.)上测量强度平 均粒径(Z平均值)。立即将样品馏分转移至Slide-a-lyzer G2透析盒(10000MWCO，ThermoFischer Scientific Inc.)中，并在4℃下针对PBS(pH7.4)透析过夜。PBS缓冲液的体积 为样品体积的650-800倍。收集样品部分，并从该体积中将25μL注入975μL 10μmM 磷酸盐缓冲液(pH 7.4)中，并再次测量粒径(透析后粒径)，LNP在透析前后，直径约 100nm，均处于均一稳定的状态，见图5和表1。透析过的样品用来进行小鼠注射免疫。 图5中显示了表达S蛋白的SGS mRNA对应的结果图，以及表达S蛋白RBD结构域的 mRNA和表达S、M、E三种蛋白的对应mRNA的包装结果。这些结果表面，mRNA经 过脂质体包装后，粒径在100-110nm之间，包装效率大于90％。
表1中显示了用ZetaView检测LNP包被后mRNA样品粒径及分布。S-RBD mRNA 可以表达S蛋白RBD结构域，SGS mRNA可以表达S蛋白，SME mRNA表达S、M、E 三种蛋白，可以形成病毒样颗粒。包被后的LNP粒径均在100-110nm之间，符合纳米颗 粒大小预期。稀释后数量在100-300之间，稀释比例适合。在用1xPBS透析并用0.22μM 或0.45μM滤膜过滤后，粒径和数量保持稳定，可用于后续动物实验。
表1
实施例6 小鼠免疫实验
将包被好的表达新冠病毒VLP(含有实施例5中所述的表达S蛋白的SGS mRNA，或表达S、M、E三种蛋白的SME mRNA)或RBD(含有实施例5中所述的表达S蛋白RBD结构域的mRNA)的上述脂质体纳米颗粒，配合免疫佐剂，进行Balb/c小鼠(肌肉(i.m.) 注射，具体信息如下表2所示。在第42天收集血液样品，并且在荧光抗体病毒中和检测中 分析血清，具体如以下实施例7所述。
表2
组别 品系 数量 途径体积 疫苗剂量 接种时间
1 Balb/c 8 i.m.50μl×3 Control PBS D0,致敏；D14，加强免疫；D35，加强免疫
2 Balb/c 8 i.m.50μl×1 mRNA 10μg D0,致敏
3 Balb/c 8 i.m.50μl×2 mRNA 10μg D0,致敏；D14，加强免疫；
4 Balb/c 8 i.m.50μl×3 mRNA 10μg D0,致敏；D14，加强免疫；D35，加强免疫
实施例7 酶联免疫吸附法测定血清中抗体效价
分别用2μg/ml抗原蛋白(溶于PBS)，100ng，包被96孔ELISA板，50μl/孔，4 度过夜避光。其中S蛋白抗原购自Sino Biological，货号40589-V08B1；S蛋白RBD结 构域，购自Novoprotein，货号DRA36。PBST(0.05％Tween)洗3次，200μl/孔，每次 倒扣ELISA板并轻击清洗干净。加100μl/孔2％BSA(溶于PBST)封闭，室温孵育1hr。 PBST(0.05％Tween)洗3次，200μl/孔，每次倒扣ELISA板并轻击清洗干净。加小鼠血清(稀释100倍作为起始浓度，之后5倍梯度稀释，共6个梯度)于PBS中，混合均 匀后取100μl分别加到ELISA板中，室温孵育2hr。将实施例6中的小鼠进行眼周采血 100μl约20μl血清。洗板后，分别加HRP-anti-mouse IgG(1:5000稀释于PBS)，100μl/ 孔，室温孵育1hr。洗板后，分别加TMB底物(Thermo Fisher，货号34022)，50μl/孔， 室温等待5-15min(避光)，将呈现蓝色。分别加1M硫酸终止反应，150μl/孔，蓝色将变黄。酶标仪读取OD450。
所得结果如图6所示，其中可见mRNA表达病毒样颗粒产生的抗体滴度最高，达到107。表达S蛋白的mRNA产生的抗体滴度达到106。表达S蛋白RBD结构域的mRNA， 产生的抗体滴度为104。可见使用本发明的mRNA表达的病毒样颗粒均可有效激活小鼠 免疫***，促使血清中产生抗体，有效发挥疫苗的作用。
实施例8 中和抗体检测实验
通过病毒中和测定进行病毒中和抗体应答(特异性B细胞免疫应答)的检测。所述测定 的结果称为病毒中和滴度(VNT)。按照WHO标准，如果各自的VNT是至少0.5IU/ml，则认为抗体滴度是保护性的。因此，在第42天从实施例6中所述接种的小鼠中采集血液样品，并且制备血清。这些血清用于荧光抗体滴度中和(FAVN)检测，所述检测使用人源CACO-2 细胞。培养细胞用假病毒颗粒(表达新冠病毒S蛋白，内核为EGFP DNA)进行感染。之后不久，以连续两倍稀释四次一式四份检测热灭活的血清，检测其以50μl的体积中和假 病毒颗粒的100TCID50(组织培养感染剂量50％)的潜力。因此，将血清稀释液在37℃(在具有5％CO2的潮湿的培养箱中)用病毒温育1小时，然后加入胰蛋白酶解的CACO-2细胞(4 ×105个细胞/ml；50μl/孔)。将感染的细胞培养物在潮湿的培养箱中在37℃和5％CO2培养 48小时。在室温用80％丙酮固定细胞后，用荧光检测EGFP表达，用量标志细胞的感染情况。
根据图7的结果可知，本发明的疫苗(以表达病毒样颗粒的形式、仅表达S蛋白的疫苗的形式或者以仅表达S蛋白中RBD区域的疫苗的形式)均可有效激活小鼠免疫***， 血清中产生抗体，且其安全性高、有效性好。其中表达病毒样颗粒的mRNA组合产生的 中和抗体滴度最高。不会产生非中和抗体从而不会产生抗体依赖增强感染效应。
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ccagcaactg tttgtggacc taaaaagtct actaatttgg ttaaaaacaa atgtgtcaat 1620
ttcaacttca atggtttaac aggcacaggt gttcttactg agtctaacaa aaagtttctg 1680
cctttccaac aatttggcag agacattgct gacactactg atgctgtccg tgatccacag 1740
acacttgaga ttcttgacat tacaccatgt tcttttggtg gtgtcagtgt tataacacca 1800
ggaacaaata cttctaacca ggttgctgtt ctttatcagg atgttaactg cacagaagtc 1860
cctgttgcta ttcatgcaga tcaacttact cctacttggc gtgtttattc tacaggttct 1920
aatgtttttc aaacacgtgc aggctgttta ataggggctg aacatgtcaa caactcatat 1980
gagtgtgaca tacccattgg tgcaggtata tgcgctagtt atcagactca gactaattct 2040
cctcggcggg cacgtagtgt agctagtcaa tccatcattg cctacactat gtcacttggt 2100
gcagaaaatt cagttgctta ctctaataac tctattgcca tacccacaaa ttttactatt 2160
agtgttacca cagaaattct accagtgtct atgaccaaga catcagtaga ttgtacaatg 2220
tacatttgtg gtgattcaac tgaatgcagc aatcttttgt tgcaatatgg cagtttttgt 2280
acacaattaa accgtgcttt aactggaata gctgttgaac aagacaaaaa cacccaagaa 2340
gtttttgcac aagtcaaaca aatttacaaa acaccaccaa ttaaagattt tggtggtttt 2400
aatttttcac aaatattacc agatccatca aaaccaagca agaggtcatt tattgaagat 2460
ctacttttca acaaagtgac acttgcagat gctggcttca tcaaacaata tggtgattgc 2520
cttggtgata ttgctgctag agacctcatt tgtgcacaaa agtttaacgg ccttactgtt 2580
ttgccacctt tgctcacaga tgaaatgatt gctcaataca cttctgcact gttagcgggt 2640
acaatcactt ctggttggac ctttggtgca ggtgctgcat tacaaatacc atttgctatg 2700
caaatggctt ataggtttaa tggtattgga gttacacaga atgttctcta tgagaaccaa 2760
aaattgattg ccaaccaatt taatagtgct attggcaaaa ttcaagactc actttcttcc 2820
acagcaagtg cacttggaaa acttcaagat gtggtcaacc aaaatgcaca agctttaaac 2880
acgcttgtta aacaacttag ctccaatttt ggtgcaattt caagtgtttt aaatgatatc 2940
ctttcacgtc ttgacaaagt tgaggctgaa gtgcaaattg ataggttgat cacaggcaga 3000
cttcaaagtt tgcagacata tgtgactcaa caattaatta gagctgcaga aatcagagct 3060
tctgctaatc ttgctgctac taaaatgtca gagtgtgtac ttggacaatc aaaaagagtt 3120
gatttttgtg gaaagggcta tcatcttatg tccttccctc agtcagcacc tcatggtgta 3180
gtcttcttgc atgtgactta tgtccctgca caagaaaaga acttcacaac tgctcctgcc 3240
atttgtcatg atggaaaagc acactttcct cgtgaaggtg tctttgtttc aaatggcaca 3300
cactggtttg taacacaaag gaatttttat gaaccacaaa tcattactac agacaacaca 3360
tttgtgtctg gtaactgtga tgttgtaata ggaattgtca acaacacagt ttatgatcct 3420
ttgcaacctg aattagactc attcaaggag gagttagata aatattttaa gaatcataca 3480
tcaccagatg ttgatttagg tgacatctct ggcattaatg cttcagttgt aaacattcaa 3540
aaagaaattg accgcctcaa tgaggttgcc aagaatttaa atgaatctct catcgatctc 3600
caagaacttg gaaagtatga gcagtatata aaatggccat ggtacatttg gctaggtttt 3660
atagctggct tgattgccat agtaatggtg acaattatgc tttgctgtat gaccagttgc 3720
tgtagttgtc tcaagggctg ttgttcttgt ggatcctgct gcaaatttga tgaagacgac 3780
tctgagccag tgctcaaagg agtcaaatta cattacaca 3819
<210> 3
<211> 3819
<212> DNA
<213> Artificial Sequence
<220>
<223>S蛋白基因优化后的序列（S-GS）
<400> 3
atgttcgtct tcctggtcct gctgcctctg gtctcctcac agtgcgtcaa tctgacaact 60
cggactcagc tgccacctgc ttatactaat agcttcacca gaggcgtgta ctatcctgac 120
aaggtgttta gaagctccgt gctgcactct acacaggatc tgtttctgcc attctttagc 180
aacgtgacct ggttccacgc catccacgtg agcggcacca atggcacaaa gcggttcgac 240
aatcccgtgc tgccttttaa cgatggcgtg tacttcgcct ctaccgagaa gagcaacatc 300
atcagaggct ggatctttgg caccacactg gactccaaga cacagtctct gctgatcgtg 360
aacaatgcca ccaacgtggt catcaaggtg tgcgagttcc agttttgtaa tgatcccttc 420
ctgggcgtgt actatcacaa gaacaataag agctggatgg agtccgagtt tagagtgtat 480
tctagcgcca acaactgcac atttgagtac gtgagccagc ctttcctgat ggacctggag 540
ggcaagcagg gcaatttcaa gaacctgagg gagttcgtgt ttaagaatat cgacggctac 600
ttcaaaatct actctaagca cacccccatc aacctggtgc gcgacctgcc tcagggcttc 660
agcgccctgg agcccctggt ggatctgcct atcggcatca acatcacccg gtttcagaca 720
ctgctggccc tgcacagaag ctacctgaca cccggcgact cctctagcgg atggaccgcc 780
ggcgctgccg cctactatgt gggctacctc cagccccgga ccttcctgct gaagtacaac 840
gagaatggca ccatcacaga cgcagtggat tgcgccctgg accccctgag cgagacaaag 900
tgtacactga agtcctttac cgtggagaag ggcatctatc agacatccaa tttcagggtg 960
cagccaaccg agtctatcgt gcgctttcct aatatcacaa acctgtgccc atttggcgag 1020
gtgttcaacg caacccgctt cgccagcgtg tacgcctgga ataggaagcg gatcagcaac 1080
tgcgtggccg actatagcgt gctgtacaac tccgcctctt tcagcacctt taagtgctat 1140
ggcgtgtccc ccacaaagct gaatgacctg tgctttacca acgtctacgc cgattctttc 1200
gtgatcaggg gcgacgaggt gcgccagatc gcccccggcc agacaggcaa gatcgcagac 1260
tacaattata agctgccaga cgatttcacc ggctgcgtga tcgcctggaa cagcaacaat 1320
ctggattcca aagtgggcgg caactacaat tatctgtacc ggctgtttag aaagagcaat 1380
ctgaagccct tcgagaggga catctctaca gaaatctacc aggccggcag caccccttgc 1440
aatggcgtgg agggctttaa ctgttatttc ccactccagt cctacggctt ccagcccaca 1500
aacggcgtgg gctatcagcc ttaccgcgtg gtggtgctga gctttgagct gctgcacgcc 1560
ccagcaacag tgtgcggccc caagaagtcc accaatctgg tgaagaacaa gtgcgtgaac 1620
ttcaacttca acggcctgac cggcacaggc gtgctgaccg agtccaacaa gaagttcctg 1680
ccatttcagc agttcggcag ggacatcgca gataccacag acgccgtgcg cgacccacag 1740
accctggaga tcctggacat cacaccctgc tctttcggcg gcgtgagcgt gatcacaccc 1800
ggcaccaata caagcaacca ggtggccgtg ctgtatcagg acgtgaattg taccgaggtg 1860
cccgtggcta tccacgccga tcagctgacc ccaacatggc gggtgtacag caccggctcc 1920
aacgtcttcc agacaagagc cggatgcctg atcggagcag agcacgtgaa caattcctat 1980
gagtgcgaca tcccaatcgg cgccggcatc tgtgcctctt accagaccca gacaaactct 2040
cccagaagag cccggagcgt ggcctcccag tctatcatcg cctataccat gtccctgggc 2100
gccgagaaca gcgtggccta ctctaacaat agcatcgcca tcccaaccaa cttcacaatc 2160
tctgtgacca cagagatcct gcccgtgtcc atgaccaaga catctgtgga ctgcacaatg 2220
tatatctgtg gcgattctac cgagtgcagc aacctgctgc tccagtacgg cagcttttgt 2280
acccagctga atagagccct gacaggcatc gccgtggagc aggataagaa cacacaggag 2340
gtgttcgccc aggtgaagca aatctacaag acccccccta tcaaggactt tggcggcttc 2400
aatttttccc agatcctgcc tgatccatcc aagccttcta agcggagctt tatcgaggac 2460
ctgctgttca acaaggtgac cctggccgat gccggcttca tcaagcagta tggcgattgc 2520
ctgggcgaca tcgcagccag ggacctgatc tgcgcccaga agtttaatgg cctgaccgtg 2580
ctgccacccc tgctgacaga tgagatgatc gcacagtaca caagcgccct gctggccggc 2640
accatcacat ccggatggac cttcggcgca ggagccgccc tccagatccc ctttgccatg 2700
cagatggcct ataggttcaa cggcatcggc gtgacccaga atgtgctgta cgagaaccag 2760
aagctgatcg ccaatcagtt taactccgcc atcggcaaga tccaggacag cctgtcctct 2820
acagccagcg ccctgggcaa gctccaggat gtggtgaatc agaacgccca ggccctgaat 2880
accctggtga agcagctgag cagcaacttc ggcgccatct ctagcgtgct gaatgacatc 2940
ctgagccggc tggacaaggt ggaggcagag gtgcagatcg accggctgat caccggccgg 3000
ctccagagcc tccagaccta tgtgacacag cagctgatca gggccgccga gatcagggcc 3060
agcgccaatc tggcagcaac caagatgtcc gagtgcgtgc tgggccagtc taagagagtg 3120
gacttttgtg gcaagggcta tcacctgatg tccttccctc agtctgcccc acacggcgtg 3180
gtgtttctgc acgtgaccta cgtgcccgcc caggagaaga acttcaccac agcccctgcc 3240
atctgccacg atggcaaggc ccactttcca agggagggcg tgttcgtgtc caacggcacc 3300
cactggtttg tgacacagcg caatttctac gagccccaga tcatcaccac agacaacacc 3360
ttcgtgagcg gcaactgtga cgtggtcatc ggcatcgtga acaataccgt gtatgatcca 3420
ctccagcccg agctggacag ctttaaggag gagctggata agtatttcaa gaatcacacc 3480
tcccctgacg tggatctggg cgacatcagc ggcatcaatg cctccgtggt gaacatccag 3540
aaggagatcg accgcctgaa cgaggtggct aagaatctga acgagagcct gatcgacctc 3600
caggagctgg gcaagtatga gcagtacatc aagtggccct ggtacatctg gctgggcttc 3660
atcgccggcc tgatcgccat cgtgatggtg accatcatgc tgtgctgtat gacatcctgc 3720
tgttcttgcc tgaagggctg ctgtagctgt ggctcctgct gtaagtttga cgaggatgac 3780
tctgaacctg tgctgaaggg cgtgaagctg cattacacc 3819
<210> 4
<211> 3819
<212> DNA
<213> Artificial Sequence
<220>
<223> S蛋白基因优化后的序列 （SBL）
<400> 4
atgttcgttt tcctcgttct gctgcctctt gtcagctctc agtgtgtgaa cctgacaact 60
agaacacaac tacctcccgc ctacacaaac tctttcaccc ggggcgtgta ctacccagac 120
aaagtgttca ggagctctgt gttgcacagc acccaagacc tgtttttgcc attctttagt 180
aatgtgacct ggtttcacgc tatccatgtg tcgggcacca acgggaccaa aagattcgac 240
aaccccgttc tgccgttcaa cgacggcgtg tacttcgcta gcactgagaa gtccaacatt 300
attcgcgggt ggatcttcgg aactaccttg gactccaaaa cacagtctct actcatcgtg 360
aacaacgcga ctaacgtggt gattaaggtg tgtgaatttc agttctgcaa tgatccattt 420
ttaggagtgt actaccacaa aaataataaa tcatggatgg agtctgaatt tcgcgtatac 480
agtagcgcta ataactgtac attcgaatat gttagccaac cctttttgat ggacttagag 540
gggaagcagg gaaattttaa gaatttgcga gaatttgtgt tcaaaaatat cgatgggtat 600
ttcaagatct actccaagca tactcccata aatctggtgc gcgacttacc tcaagggttc 660
agcgcactgg agccactggt agacctgcca atcggcatca acatcacccg attccagacc 720
ctgcttgctc tgcaccgttc atatctgaca ccaggagatt cgtcttccgg atggacagca 780
ggggccgctg cttactatgt tggttatctt cagcctcgga cctttctgct caagtataat 840
gagaatggga ccattaccga cgctgttgat tgtgctctcg atcccctgtc agaaaccaag 900
tgcacactaa aatctttcac agtcgaaaag gggatctacc agacttctaa ctttcgtgta 960
cagcccaccg agagcatcgt caggttccca aatatcacta acctgtgtcc ttttggcgag 1020
gtgttcaacg ctacaagatt tgctagcgtg tacgcctgga acagaaaaag aatatcaaat 1080
tgcgtagccg attacagcgt cttatataac tctgcatcct tctcaacttt caagtgttat 1140
ggagtgagcc cgactaagct gaatgatttg tgctttacaa atgtttatgc cgattcattc 1200
gtgatccggg gcgacgaggt cagacagatc gcccctggcc aaacaggtaa gattgctgat 1260
tacaactaca aattacctga cgattttaca ggatgcgtta tcgcttggaa ctctaacaat 1320
ctcgattcta aggtcggcgg caattacaat tatctttatc gccttttcag gaagtcaaat 1380
cttaagccat tcgagcgaga catcagtacc gagatatacc aggcggggtc caccccgtgt 1440
aacggtgtcg agggtttcaa ctgctacttt ccactgcagt cctatgggtt ccagcccacc 1500
aatggcgtgg gttaccagcc ctaccgagta gtcgtattgt cttttgagct cttgcacgcc 1560
cccgccacgg tgtgcggtcc aaagaaatca actaacttag ttaagaataa atgtgtgaat 1620
tttaacttta acggcctgac agggacagga gtcctgacag aatccaataa gaagttcctt 1680
ccctttcagc agtttggacg cgacatcgca gacaccacag acgccgtgcg tgacccccaa 1740
actctcgaaa ttctcgatat cacaccctgc agttttggcg gggtcagtgt cattacccct 1800
gggaccaata ctagtaacca ggtcgcagtg ctttaccaag atgtcaactg taccgaggtt 1860
cctgtggcta ttcacgcaga ccaactgact ccgacttggc gggtgtatag tacaggctcc 1920
aatgtgtttc agacccgggc aggctgcctg attggggccg agcatgtaaa taactcctac 1980
gagtgcgata tccccatagg tgctggaata tgtgccagtt atcagaccca gacgaactcg 2040
ccaagacgag ctaggtccgt agcctctcag agcataatcg cgtacactat gagcctgggg 2100
gccgaaaatt ccgtggcata tagcaacaac agcattgcta ttcctactaa ctttacaatt 2160
tcagtcacga cggagatcct gccagtctcc atgactaaaa cctccgtgga ctgtacgatg 2220
tacatttgtg gcgattcaac tgaatgctct aacctgctct tacagtacgg ttctttttgt 2280
acccagctga accgggcatt gacgggcatc gcagttgagc aggacaagaa tactcaggag 2340
gtgtttgcgc aagtgaagca aatttataaa actcctccca ttaaggactt tggcggtttc 2400
aacttctcgc agatcctacc tgacccatca aaacctagca agaggtcttt cattgaagac 2460
cttctgttca acaaggtcac actggctgac gccggcttca ttaaacagta cggagattgt 2520
ctaggtgata ttgcagcgcg cgatctgatt tgcgcacaga agtttaacgg cctgacggtc 2580
ttaccccctc tccttaccga cgaaatgatt gcccagtaca ccagcgccct gctcgctggc 2640
acgattacta gcggatggac atttggggcc ggcgctgccc tccagatacc atttgccatg 2700
cagatggcgt ataggtttaa cggcatagga gtaacccaga acgtgctgta cgagaaccaa 2760
aaactgatag ccaatcaatt caatagtgcc ataggaaaga tacaggacag tctcagcagc 2820
accgcgtccg ctctcggaaa gctacaagat gtggtcaacc agaacgcgca ggcattgaat 2880
acactggtga agcagctctc ctcgaatttt ggagcaatca gcagcgtgct gaatgatatc 2940
ctgtctcggc tggacaaggt tgaagccgaa gtccagatcg acaggttaat caccggtcgg 3000
ctgcagagtc tccagacata tgttacccag caactcatca gagctgccga aatacgcgcc 3060
agtgccaatc ttgcagccac taagatgtcc gagtgcgtgt tggggcaaag taaaagggtt 3120
gatttctgtg gaaaaggata tcatcttatg agtttccctc aatccgcccc tcacggagtt 3180
gtcttcctgc atgtgaccta cgtgccagcg caggagaaga acttcacgac cgcccccgcc 3240
atctgccatg atggcaaggc ccattttccc cgcgaaggag tgttcgtatc caatggcacc 3300
cactggttcg tgacgcagag aaatttttat gagccgcaaa ttatcactac cgacaacaca 3360
ttcgtttccg gcaattgcga tgtcgtaatc gggatcgtga ataatacagt ctatgatcct 3420
cttcagccag aactcgattc attcaaagag gagctggata aatatttcaa gaaccacacc 3480
tcccccgatg tggatctggg tgacatatca ggaattaacg caagcgtcgt gaacattcag 3540
aaggaaatcg acaggctcaa tgaagtagca aagaacttga atgagtctct catcgacttg 3600
caggaactcg gcaaatatga gcagtacatt aaatggccgt ggtatatctg gctaggcttt 3660
atcgccggtc tgattgcaat tgtgatggtt actatcatgt tgtgctgcat gacaagttgc 3720
tgttcatgcc ttaaaggctg ctgctcctgc gggtcatgtt gtaaattcga tgaggacgac 3780
tctgagcccg tgctgaaagg ggtgaaactg cactacacg 3819
<210> 5
<211> 3819
<212> DNA
<213> Artificial Sequence
<220>
<223> S蛋白基因优化序列3 (STF)
<400> 5
atgttcgtgt tcctggtgct gctgcctctg gtgtccagcc agtgtgtgaa cctgaccacc 60
agaacacagc tgcctccagc ctacaccaac agctttacca gaggcgtgta ctaccccgac 120
aaggtgttca gatccagcgt gctgcactct acccaggacc tgttcctgcc tttcttcagc 180
aacgtgacct ggttccacgc catccacgtg tccggcacca atggcaccaa gagattcgac 240
aaccccgtgc tgcccttcaa cgacggggtg tactttgcca gcaccgagaa gtccaacatc 300
atcagaggct ggatcttcgg caccacactg gacagcaaga cccagagcct gctgatcgtg 360
aacaacgcca ccaacgtggt catcaaagtg tgcgagttcc agttctgcaa cgaccccttc 420
ctgggcgtct actaccacaa gaacaacaag agctggatgg aaagcgagtt ccgggtgtac 480
agcagcgcca acaactgcac cttcgagtac gtgtcccagc ctttcctgat ggacctggaa 540
ggcaagcagg gcaacttcaa gaacctgcgc gagttcgtgt ttaagaacat cgacggctac 600
ttcaagatct acagcaagca cacccctatc aacctcgtgc gggatctgcc tcagggcttc 660
tctgctctgg aacccctggt ggatctgccc atcggcatca acatcacccg gtttcagaca 720
ctgctggccc tgcacagaag ctacctgaca cctggcgata gcagcagcgg atggacagct 780
ggtgccgccg cttactatgt gggctacctg cagcctagaa ccttcctgct gaagtacaac 840
gagaacggca ccatcaccga cgccgtggat tgtgctctgg atcctctgag cgagacaaag 900
tgcaccctga agtccttcac cgtggaaaag ggcatctacc agaccagcaa cttccgggtg 960
cagcccaccg aatccatcgt gcggttcccc aatatcacca atctgtgccc cttcggcgag 1020
gtgttcaatg ccaccagatt cgcctctgtg tacgcctgga accggaagcg gatcagcaat 1080
tgcgtggccg actactccgt gctgtacaac tccgccagct tcagcacctt caagtgctac 1140
ggcgtgtccc ctaccaagct gaacgacctg tgcttcacaa acgtgtacgc cgacagcttc 1200
gtgatccggg gagatgaagt gcggcagatt gcccctggac agacaggcaa gatcgccgac 1260
tacaactaca agctgcccga cgacttcacc ggctgtgtga ttgcctggaa cagcaacaac 1320
ctggactcca aagtcggcgg caactacaat tacctgtacc ggctgttccg gaagtccaat 1380
ctgaagccct tcgagcggga catctccacc gagatctatc aggccggcag caccccttgt 1440
aacggcgtgg aaggcttcaa ctgctacttc ccactgcagt cctacggctt tcagcccaca 1500
aatggcgtgg gctatcagcc ctacagagtg gtggtgctga gcttcgaact gctgcatgcc 1560
cctgccacag tgtgcggccc taagaaaagc accaatctcg tgaagaacaa atgcgtgaac 1620
ttcaacttca acggcctgac cggcaccggc gtgctgacag agagcaacaa gaagttcctg 1680
ccattccagc agtttggccg ggatatcgcc gataccacag acgccgttag agatccccag 1740
acactggaaa tcctggacat caccccttgc agcttcggcg gagtgtctgt gatcacccct 1800
ggcaccaaca ccagcaatca ggtggcagtg ctgtaccagg acgtgaactg taccgaagtg 1860
cccgtggcca ttcacgccga tcagctgaca cctacatggc gggtgtactc caccggcagc 1920
aatgtgtttc agaccagagc cggctgtctg atcggagccg agcacgtgaa caatagctac 1980
gagtgcgaca tccccatcgg cgctggcatc tgtgccagct accagacaca gacaaacagc 2040
cccagacggg ccagatctgt ggccagccag agcatcattg cctacacaat gtctctgggc 2100
gccgagaaca gcgtggccta ctccaacaac tctatcgcta tccccaccaa cttcaccatc 2160
agcgtgacca cagagatcct gcctgtgtcc atgaccaaga ccagcgtgga ctgcaccatg 2220
tacatctgcg gcgattccac cgagtgctcc aacctgctgc tgcagtacgg cagcttctgc 2280
acccagctga atagagccct gacagggatc gccgtggaac aggacaagaa cacccaagag 2340
gtgttcgccc aagtgaagca gatctacaag acccctccta tcaaggactt cggcggcttc 2400
aatttcagcc agattctgcc cgatcctagc aagcccagca agcggagctt catcgaggac 2460
ctgctgttca acaaagtgac actggccgac gccggcttca tcaagcagta tggcgattgt 2520
ctgggcgaca ttgccgccag ggatctgatt tgcgcccaga agtttaacgg actgacagtg 2580
ctgcctcctc tgctgaccga tgagatgatc gcccagtaca catctgccct gctggccggc 2640
acaatcacaa gcggctggac atttggagct ggcgccgctc tgcagatccc ctttgctatg 2700
cagatggcct accggttcaa cggcatcgga gtgacccaga atgtgctgta cgagaaccag 2760
aagctgatcg ccaaccagtt caacagcgcc atcggcaaga tccaggacag cctgagcagc 2820
acagcaagcg ccctgggaaa gctgcaggac gtggtcaacc agaatgccca ggcactgaac 2880
accctggtca agcagctgtc ctccaacttc ggcgccatca gctctgtgct gaacgatatc 2940
ctgagcagac tggacaaggt ggaagccgag gtgcagatcg acagactgat caccggaagg 3000
ctgcagtccc tgcagaccta cgttacccag cagctgatca gagccgccga gattagagcc 3060
tctgccaatc tggccgccac caagatgtct gagtgtgtgc tgggccagag caagagagtg 3120
gacttttgcg gcaagggcta ccacctgatg agcttccctc agtctgcccc tcacggcgtg 3180
gtgtttctgc acgtgacata cgtgcccgct caagagaaga atttcaccac cgctccagcc 3240
atctgccacg acggcaaagc ccactttcct agagaaggcg tgttcgtgtc caacggcacc 3300
cattggttcg tgacccagcg gaacttctac gagccccaga tcatcaccac cgacaacacc 3360
ttcgtgtctg gcaactgcga cgtcgtgatc ggcattgtga acaataccgt gtacgaccct 3420
ctgcagcccg agctggacag cttcaaagag gaactggata agtactttaa gaaccacaca 3480
agccccgacg tggacctggg cgatatcagc ggaatcaatg ccagcgtcgt gaacatccag 3540
aaagagatcg accggctgaa cgaggtggcc aagaatctga acgagagcct gatcgacctg 3600
caagaactgg ggaagtacga gcagtacatc aagtggccct ggtacatctg gctgggcttt 3660
atcgccggac tgattgccat cgtgatggtc acaatcatgc tgtgttgcat gaccagctgc 3720
tgtagctgcc tgaagggctg ttgtagctgt ggcagctgct gcaagttcga cgaggacgat 3780
tctgagcccg tgctgaaggg cgtgaaactg cactacaca 3819
<210> 6
<211> 75
<212> PRT
<213> SARS-COV-2
<400> 6
Met Tyr Ser Phe Val Ser Glu Glu Thr Gly Thr Leu Ile Val Asn Ser
1 5 10 15
Val Leu Leu Phe Leu Ala Phe Val Val Phe Leu Leu Val Thr Leu Ala
20 25 30
Ile Leu Thr Ala Leu Arg Leu Cys Ala Tyr Cys Cys Asn Ile Val Asn
35 40 45
Val Ser Leu Val Lys Pro Ser Phe Tyr Val Tyr Ser Arg Val Lys Asn
50 55 60
Leu Asn Ser Ser Arg Val Pro Asp Leu Leu Val
65 70 75
<210> 7
<211> 228
<212> DNA
<213> SARS-COV-2
<400> 7
atgtactcat tcgtttcgga agagacaggt acgttaatag ttaatagcgt acttcttttt 60
cttgctttcg tggtattctt gctagttaca ctagccatcc ttactgcgct tcgattgtgt 120
gcgtactgct gcaatattgt taacgtgagt cttgtaaaac cttcttttta cgtttactct 180
cgtgttaaaa atctgaattc ttctagagtt cctgatcttc tggtctaa 228
<210> 8
<211> 225
<212> DNA
<213> Artificial Sequence
<220>
<223> E蛋白基因优化序列（EBL）
<400> 8
atgtacagct ttgtctcaga ggaaaccggc acgctgattg taaacagcgt gttactattc 60
ctcgccttcg ttgtgtttct ccttgttaca ctggcaatac tgactgccct gcggttgtgc 120
gcttactgct gtaatatcgt gaacgtgtct ttggtgaagc ccagtttcta tgtatattcc 180
agagtcaaaa atctcaactc ctctagggtg cctgacctgc ttgtc 225
<210> 9
<211> 222
<212> PRT
<213> SARS-COV-2
<400> 9
Met Ala Asp Ser Asn Gly Thr Ile Thr Val Glu Glu Leu Lys Lys Leu
1 5 10 15
Leu Glu Gln Trp Asn Leu Val Ile Gly Phe Leu Phe Leu Thr Trp Ile
20 25 30
Cys Leu Leu Gln Phe Ala Tyr Ala Asn Arg Asn Arg Phe Leu Tyr Ile
35 40 45
Ile Lys Leu Ile Phe Leu Trp Leu Leu Trp Pro Val Thr Leu Ala Cys
50 55 60
Phe Val Leu Ala Ala Val Tyr Arg Ile Asn Trp Ile Thr Gly Gly Ile
65 70 75 80
Ala Ile Ala Met Ala Cys Leu Val Gly Leu Met Trp Leu Ser Tyr Phe
85 90 95
Ile Ala Ser Phe Arg Leu Phe Ala Arg Thr Arg Ser Met Trp Ser Phe
100 105 110
Asn Pro Glu Thr Asn Ile Leu Leu Asn Val Pro Leu His Gly Thr Ile
115 120 125
Leu Thr Arg Pro Leu Leu Glu Ser Glu Leu Val Ile Gly Ala Val Ile
130 135 140
Leu Arg Gly His Leu Arg Ile Ala Gly His His Leu Gly Arg Cys Asp
145 150 155 160
Ile Lys Asp Leu Pro Lys Glu Ile Thr Val Ala Thr Ser Arg Thr Leu
165 170 175
Ser Tyr Tyr Lys Leu Gly Ala Ser Gln Arg Val Ala Gly Asp Ser Gly
180 185 190
Phe Ala Ala Tyr Ser Arg Tyr Arg Ile Gly Asn Tyr Lys Leu Asn Thr
195 200 205
Asp His Ser Ser Ser Ser Asp Asn Ile Ala Leu Leu Val Gln
210 215 220
<210> 10
<211> 669
<212> DNA
<213> SARS-COV-2
<400> 10
atggcagatt ccaacggtac tattaccgtt gaagagctta aaaagctcct tgaacaatgg 60
aacctagtaa taggtttcct attccttaca tggatttgtc ttctacaatt tgcctatgcc 120
aacaggaata ggtttttgta tataattaag ttaattttcc tctggctgtt atggccagta 180
actttagctt gttttgtgct tgctgctgtt tacagaataa attggatcac cggtggaatt 240
gctatcgcaa tggcttgtct tgtaggcttg atgtggctca gctacttcat tgcttctttc 300
agactgtttg cgcgtacgcg ttccatgtgg tcattcaatc cagaaactaa cattcttctc 360
aacgtgccac tccatggcac tattctgacc agaccgcttc tagaaagtga actcgtaatc 420
ggagctgtga tccttcgtgg acatcttcgt attgctggac accatctagg acgctgtgac 480
atcaaggacc tgcctaaaga aatcactgtt gctacatcac gaacgctttc ttattacaaa 540
ttgggagctt cgcagcgtgt agcaggtgac tcaggttttg ctgcatacag tcgctacagg 600
attggcaact ataaattaaa cacagaccat tccagtagca gtgacaatat tgctttgctt 660
gtacagtaa 669
<210> 11
<211> 669
<212> DNA
<213> Artificial Sequence
<220>
<223> M蛋白基因优化序列MBL
<400> 11
atggcagatt ccaacggtac aattaccgtc gaagagctga aaaagctcct tgagcagtgg 60
aacctggtca tagggttcct attcctgaca tggatttgcc tgctgcaatt tgcctatgcc 120
aacaggaata ggtttttgta tataatcaag ctgattttcc tctggctgtt atggccagtg 180
accctggcct gttttgtgct tgccgctgtt tacagaataa attggatcac cggcggaatc 240
gccatcgcaa tggcttgcct tgtaggcttg atgtggctca gctacttcat tgcttctttc 300
cggctgtttg cgcgaacgcg gtccatgtgg tctttcaatc cggagactaa catactcctc 360
aatgtgcccc tccatggcac tattctgacc agacccctgc tagagagtga actcgtcatc 420
ggagctgtga tcctgcgggg gcacctgaga atcgccggac accacttagg ccgctgtgac 480
atcaaggatc tgcctaaaga aatcactgtt gccacatcac gaaccctttc ttattacaag 540
ttgggggcct cgcagcgtgt ggcaggagac tcaggttttg cggcatacag tcgctacagg 600
attggcaact ataaattaaa cacagaccat tccagcagca gcgataatat tgctttgctt 660
gtgcagtga 669
<210> 12
<211> 419
<212> PRT
<213> SARS-COV-2
<400> 12
Met Ser Asp Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro Arg Ile Thr
1 5 10 15
Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly Glu Arg
20 25 30
Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn
35 40 45
Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Asp Leu
50 55 60
Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro
65 70 75 80
Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly
85 90 95
Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr
100 105 110
Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn Lys Asp
115 120 125
Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp
130 135 140
His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val Leu Gln
145 150 155 160
Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly Ser
165 170 175
Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg Asn
180 185 190
Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Thr Ser Pro Ala
195 200 205
Arg Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu Leu
210 215 220
Asp Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys Gly Gln Gln
225 230 235 240
Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys
245 250 255
Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr Gln
260 265 270
Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp
275 280 285
Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln Ile
290 295 300
Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile
305 310 315 320
Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr Gly Ala
325 330 335
Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln Val Ile Leu
340 345 350
Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu Pro
355 360 365
Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr Gln Ala Leu Pro Gln
370 375 380
Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala Ala Asp Leu
385 390 395 400
Asp Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser Ala Asp Ser
405 410 415
Thr Gln Ala
<210> 13
<211> 1257
<212> DNA
<213> Artificial Sequence
<220>
<223> N蛋白基因优化序列NBL
<400> 13
atgtcagata acggaccgca gaaccaaagg aacgcccctc ggatcacttt cgggggtcct 60
agcgacagca ctgggtctaa ccaaaatgga gaacgttccg gcgcaagatc caaacagagg 120
aggcctcagg ggcttcctaa caatacagcc tcctggttca cagctctcac acagcatggc 180
aaggaagacc tgaagtttcc tagaggccag ggggttccca tcaatactaa ctcctcccca 240
gacgatcaga ttggttatta tcggcgggct accaggcgga tccggggcgg agacggtaag 300
atgaaggacc tctctccccg ttggtacttt tactacctcg gtacaggccc cgaggctggg 360
cttccgtatg gcgccaataa ggatggaata atttgggtgg ctacggaagg ggccctcaac 420
acaccgaagg atcacattgg cacccgtaat cccgcgaata atgccgccat tgtcctgcag 480
ttgccccagg ggacgacgtt gcccaaaggc ttttacgcag aaggatcgcg cggaggatcc 540
caagcctcca gccgatcaag ctctcgatct cggaactcaa gtcgcaatag cacaccaggg 600
tcttctcgcg ggaccagccc tgcaaggatg gccggaaacg gcggtgatgc tgctttagcg 660
ctgctgctgc tggatagact gaaccaatta gagagtaaaa tgtcaggtaa aggccagcaa 720
cagcaggggc agacagtgac caaaaaaagt gcggccgagg ccagcaagaa accccgccag 780
aaacgaacag ccactaaagc ctacaacgta acccaagcat tcggaaggag aggaccagag 840
cagacccaag gcaattttgg cgatcaagag ctgatccgcc aggggacgga ctataagcat 900
tggccacaga tcgcccagtt cgcacccagt gcttcagcct tcttcggaat gtcgagaatc 960
ggtatggagg tcactccttc tggcacttgg ctgacttata ccggcgcaat aaagctagac 1020
gacaaagacc ctaactttaa ggatcaggtg atcctgctaa ataaacacat tgatgcgtac 1080
aaaacattcc caccaactga gccaaagaag gacaagaaga agaaggcaga tgaaacccag 1140
gctttgcccc agagacagaa aaagcagcag accgtgacct tgctgccagc agccgacctc 1200
gacgattttt caaagcaact tcagcagtcc atgagtagcg ctgacagcac ccaggct 1257
<210> 14
<211> 1257
<212> DNA
<213> SARS-COV-2
<400> 14
atgtctgata atggacccca aaatcagcga aatgcacccc gcattacgtt tggtggaccc 60
tcagattcaa ctggcagtaa ccagaatgga gaacgcagtg gggcgcgatc aaaacaacgt 120
cggccccaag gtttacccaa taatactgcg tcttggttca ccgctctcac tcaacatggc 180
aaggaagacc ttaaattccc tcgaggacaa ggcgttccaa ttaacaccaa tagcagtcca 240
gatgaccaaa ttggctacta ccgaagagct accagacgaa ttcgtggtgg tgacggtaaa 300
atgaaagatc tcagtccaag atggtatttc tactacctag gaactgggcc agaagctgga 360
cttccctatg gtgctaacaa agacggcatc atatgggttg caactgaggg agccttgaat 420
acaccaaaag atcacattgg cacccgcaat cctgctaaca atgctgcaat cgtgctacaa 480
cttcctcaag gaacaacatt gccaaaaggc ttctacgcag aagggagcag aggcggcagt 540
caagcctctt ctcgttcctc atcacgtagt cgcaacagtt caagaaattc aactccaggc 600
agcagtaggg gaacttctcc tgctagaatg gctggcaatg gcggtgatgc tgctcttgct 660
ttgctgctgc ttgacagatt gaaccagctt gagagcaaaa tgtctggtaa aggccaacaa 720
caacaaggcc aaactgtcac taagaaatct gctgctgagg cttctaagaa gcctcggcaa 780
aaacgtactg ccactaaagc atacaatgta acacaagctt tcggcagacg tggtccagaa 840
caaacccaag gaaattttgg ggaccaggaa ctaatcagac aaggaactga ttacaaacat 900
tggccgcaaa ttgcacaatt tgcccccagc gcttcagcgt tcttcggaat gtcgcgcatt 960
ggcatggaag tcacaccttc gggaacgtgg ttgacctaca caggtgccat caaattggat 1020
gacaaagatc caaatttcaa agatcaagtc attttgctga ataagcatat tgacgcatac 1080
aaaacattcc caccaacaga gcctaaaaag gacaaaaaga agaaggctga tgaaactcaa 1140
gccttaccgc agagacagaa gaaacagcaa actgtgactc ttcttcctgc tgcagatttg 1200
gatgatttct ccaaacaatt gcaacaatcc atgagcagtg ctgactcaac tcaggcc 1257
<210> 15
<211> 46
<212> DNA
<213> Artificial Sequence
<220>
<223> 5 'UTR
<400> 15
ggaaataaga gagaaaagaa gagtaagaag aaatataaga gccacc 46
<210> 16
<211> 110
<212> DNA
<213> Artificial Sequence
<220>
<223> 3'UTR-1
<400> 16
gctggagcct cggtggccat gcttcttgcc ccttgggcct ccccccagcc cctcctcccc 60
ttcctgcacc cgtacccccg tggtctttga ataaagtctg agtgggcggc 110
<210> 17
<211> 109
<212> DNA
<213> Artificial Sequence
<220>
<223> 3'UTR-2
<400> 17
gcggccgctt aattaagctg ccttctgcgg ggcttgcctt ctggccatgc ccttcttctc 60
tcccttgcac ctgtacctct tggtctttga ataaagcctg agtaggaag 109
<210> 18
<211> 3819
<212> RNA
<213> Artificial Sequence
<220>
<223> S蛋白基因优化后的mRNA序列1（S-GS mRNA）
<400> 18
auguucgucu uccugguccu gcugccucug gucuccucac agugcgucaa ucugacaacu 60
cggacucagc ugccaccugc uuauacuaau agcuucacca gaggcgugua cuauccugac 120
aagguguuua gaagcuccgu gcugcacucu acacaggauc uguuucugcc auucuuuagc 180
aacgugaccu gguuccacgc cauccacgug agcggcacca auggcacaaa gcgguucgac 240
aaucccgugc ugccuuuuaa cgauggcgug uacuucgccu cuaccgagaa gagcaacauc 300
aucagaggcu ggaucuuugg caccacacug gacuccaaga cacagucucu gcugaucgug 360
aacaaugcca ccaacguggu caucaaggug ugcgaguucc aguuuuguaa ugaucccuuc 420
cugggcgugu acuaucacaa gaacaauaag agcuggaugg aguccgaguu uagaguguau 480
ucuagcgcca acaacugcac auuugaguac gugagccagc cuuuccugau ggaccuggag 540
ggcaagcagg gcaauuucaa gaaccugagg gaguucgugu uuaagaauau cgacggcuac 600
uucaaaaucu acucuaagca cacccccauc aaccuggugc gcgaccugcc ucagggcuuc 660
agcgcccugg agccccuggu ggaucugccu aucggcauca acaucacccg guuucagaca 720
cugcuggccc ugcacagaag cuaccugaca cccggcgacu ccucuagcgg auggaccgcc 780
ggcgcugccg ccuacuaugu gggcuaccuc cagccccgga ccuuccugcu gaaguacaac 840
gagaauggca ccaucacaga cgcaguggau ugcgcccugg acccccugag cgagacaaag 900
uguacacuga aguccuuuac cguggagaag ggcaucuauc agacauccaa uuucagggug 960
cagccaaccg agucuaucgu gcgcuuuccu aauaucacaa accugugccc auuuggcgag 1020
guguucaacg caacccgcuu cgccagcgug uacgccugga auaggaagcg gaucagcaac 1080
ugcguggccg acuauagcgu gcuguacaac uccgccucuu ucagcaccuu uaagugcuau 1140
ggcguguccc ccacaaagcu gaaugaccug ugcuuuacca acgucuacgc cgauucuuuc 1200
gugaucaggg gcgacgaggu gcgccagauc gcccccggcc agacaggcaa gaucgcagac 1260
uacaauuaua agcugccaga cgauuucacc ggcugcguga ucgccuggaa cagcaacaau 1320
cuggauucca aagugggcgg caacuacaau uaucuguacc ggcuguuuag aaagagcaau 1380
cugaagcccu ucgagaggga caucucuaca gaaaucuacc aggccggcag caccccuugc 1440
aauggcgugg agggcuuuaa cuguuauuuc ccacuccagu ccuacggcuu ccagcccaca 1500
aacggcgugg gcuaucagcc uuaccgcgug guggugcuga gcuuugagcu gcugcacgcc 1560
ccagcaacag ugugcggccc caagaagucc accaaucugg ugaagaacaa gugcgugaac 1620
uucaacuuca acggccugac cggcacaggc gugcugaccg aguccaacaa gaaguuccug 1680
ccauuucagc aguucggcag ggacaucgca gauaccacag acgccgugcg cgacccacag 1740
acccuggaga uccuggacau cacacccugc ucuuucggcg gcgugagcgu gaucacaccc 1800
ggcaccaaua caagcaacca gguggccgug cuguaucagg acgugaauug uaccgaggug 1860
cccguggcua uccacgccga ucagcugacc ccaacauggc ggguguacag caccggcucc 1920
aacgucuucc agacaagagc cggaugccug aucggagcag agcacgugaa caauuccuau 1980
gagugcgaca ucccaaucgg cgccggcauc ugugccucuu accagaccca gacaaacucu 2040
cccagaagag cccggagcgu ggccucccag ucuaucaucg ccuauaccau gucccugggc 2100
gccgagaaca gcguggccua cucuaacaau agcaucgcca ucccaaccaa cuucacaauc 2160
ucugugacca cagagauccu gcccgugucc augaccaaga caucugugga cugcacaaug 2220
uauaucugug gcgauucuac cgagugcagc aaccugcugc uccaguacgg cagcuuuugu 2280
acccagcuga auagagcccu gacaggcauc gccguggagc aggauaagaa cacacaggag 2340
guguucgccc aggugaagca aaucuacaag acccccccua ucaaggacuu uggcggcuuc 2400
aauuuuuccc agauccugcc ugauccaucc aagccuucua agcggagcuu uaucgaggac 2460
cugcuguuca acaaggugac ccuggccgau gccggcuuca ucaagcagua uggcgauugc 2520
cugggcgaca ucgcagccag ggaccugauc ugcgcccaga aguuuaaugg ccugaccgug 2580
cugccacccc ugcugacaga ugagaugauc gcacaguaca caagcgcccu gcuggccggc 2640
accaucacau ccggauggac cuucggcgca ggagccgccc uccagauccc cuuugccaug 2700
cagauggccu auagguucaa cggcaucggc gugacccaga augugcugua cgagaaccag 2760
aagcugaucg ccaaucaguu uaacuccgcc aucggcaaga uccaggacag ccuguccucu 2820
acagccagcg cccugggcaa gcuccaggau guggugaauc agaacgccca ggcccugaau 2880
acccugguga agcagcugag cagcaacuuc ggcgccaucu cuagcgugcu gaaugacauc 2940
cugagccggc uggacaaggu ggaggcagag gugcagaucg accggcugau caccggccgg 3000
cuccagagcc uccagaccua ugugacacag cagcugauca gggccgccga gaucagggcc 3060
agcgccaauc uggcagcaac caagaugucc gagugcgugc ugggccaguc uaagagagug 3120
gacuuuugug gcaagggcua ucaccugaug uccuucccuc agucugcccc acacggcgug 3180
guguuucugc acgugaccua cgugcccgcc caggagaaga acuucaccac agccccugcc 3240
aucugccacg auggcaaggc ccacuuucca agggagggcg uguucguguc caacggcacc 3300
cacugguuug ugacacagcg caauuucuac gagccccaga ucaucaccac agacaacacc 3360
uucgugagcg gcaacuguga cguggucauc ggcaucguga acaauaccgu guaugaucca 3420
cuccagcccg agcuggacag cuuuaaggag gagcuggaua aguauuucaa gaaucacacc 3480
uccccugacg uggaucuggg cgacaucagc ggcaucaaug ccuccguggu gaacauccag 3540
aaggagaucg accgccugaa cgagguggcu aagaaucuga acgagagccu gaucgaccuc 3600
caggagcugg gcaaguauga gcaguacauc aaguggcccu gguacaucug gcugggcuuc 3660
aucgccggcc ugaucgccau cgugauggug accaucaugc ugugcuguau gacauccugc 3720
uguucuugcc ugaagggcug cuguagcugu ggcuccugcu guaaguuuga cgaggaugac 3780
ucugaaccug ugcugaaggg cgugaagcug cauuacacc 3819
<210> 19
<211> 3819
<212> RNA
<213> Artificial Sequence
<220>
<223> S蛋白基因优化后的mRNA序列（SBLmRNA）
<400> 19
auguucguuu uccucguucu gcugccucuu gucagcucuc agugugugaa ccugacaacu 60
agaacacaac uaccucccgc cuacacaaac ucuuucaccc ggggcgugua cuacccagac 120
aaaguguuca ggagcucugu guugcacagc acccaagacc uguuuuugcc auucuuuagu 180
aaugugaccu gguuucacgc uauccaugug ucgggcacca acgggaccaa aagauucgac 240
aaccccguuc ugccguucaa cgacggcgug uacuucgcua gcacugagaa guccaacauu 300
auucgcgggu ggaucuucgg aacuaccuug gacuccaaaa cacagucucu acucaucgug 360
aacaacgcga cuaacguggu gauuaaggug ugugaauuuc aguucugcaa ugauccauuu 420
uuaggagugu acuaccacaa aaauaauaaa ucauggaugg agucugaauu ucgcguauac 480
aguagcgcua auaacuguac auucgaauau guuagccaac ccuuuuugau ggacuuagag 540
gggaagcagg gaaauuuuaa gaauuugcga gaauuugugu ucaaaaauau cgauggguau 600
uucaagaucu acuccaagca uacucccaua aaucuggugc gcgacuuacc ucaaggguuc 660
agcgcacugg agccacuggu agaccugcca aucggcauca acaucacccg auuccagacc 720
cugcuugcuc ugcaccguuc auaucugaca ccaggagauu cgucuuccgg auggacagca 780
ggggccgcug cuuacuaugu ugguuaucuu cagccucgga ccuuucugcu caaguauaau 840
gagaauggga ccauuaccga cgcuguugau ugugcucucg auccccuguc agaaaccaag 900
ugcacacuaa aaucuuucac agucgaaaag gggaucuacc agacuucuaa cuuucgugua 960
cagcccaccg agagcaucgu cagguuccca aauaucacua accugugucc uuuuggcgag 1020
guguucaacg cuacaagauu ugcuagcgug uacgccugga acagaaaaag aauaucaaau 1080
ugcguagccg auuacagcgu cuuauauaac ucugcauccu ucucaacuuu caaguguuau 1140
ggagugagcc cgacuaagcu gaaugauuug ugcuuuacaa auguuuaugc cgauucauuc 1200
gugauccggg gcgacgaggu cagacagauc gccccuggcc aaacagguaa gauugcugau 1260
uacaacuaca aauuaccuga cgauuuuaca ggaugcguua ucgcuuggaa cucuaacaau 1320
cucgauucua aggucggcgg caauuacaau uaucuuuauc gccuuuucag gaagucaaau 1380
cuuaagccau ucgagcgaga caucaguacc gagauauacc aggcgggguc caccccgugu 1440
aacggugucg aggguuucaa cugcuacuuu ccacugcagu ccuauggguu ccagcccacc 1500
aauggcgugg guuaccagcc cuaccgagua gucguauugu cuuuugagcu cuugcacgcc 1560
cccgccacgg ugugcggucc aaagaaauca acuaacuuag uuaagaauaa augugugaau 1620
uuuaacuuua acggccugac agggacagga guccugacag aauccaauaa gaaguuccuu 1680
cccuuucagc aguuuggacg cgacaucgca gacaccacag acgccgugcg ugacccccaa 1740
acucucgaaa uucucgauau cacacccugc aguuuuggcg gggucagugu cauuaccccu 1800
gggaccaaua cuaguaacca ggucgcagug cuuuaccaag augucaacug uaccgagguu 1860
ccuguggcua uucacgcaga ccaacugacu ccgacuuggc ggguguauag uacaggcucc 1920
aauguguuuc agacccgggc aggcugccug auuggggccg agcauguaaa uaacuccuac 1980
gagugcgaua uccccauagg ugcuggaaua ugugccaguu aucagaccca gacgaacucg 2040
ccaagacgag cuagguccgu agccucucag agcauaaucg cguacacuau gagccugggg 2100
gccgaaaauu ccguggcaua uagcaacaac agcauugcua uuccuacuaa cuuuacaauu 2160
ucagucacga cggagauccu gccagucucc augacuaaaa ccuccgugga cuguacgaug 2220
uacauuugug gcgauucaac ugaaugcucu aaccugcucu uacaguacgg uucuuuuugu 2280
acccagcuga accgggcauu gacgggcauc gcaguugagc aggacaagaa uacucaggag 2340
guguuugcgc aagugaagca aauuuauaaa acuccuccca uuaaggacuu uggcgguuuc 2400
aacuucucgc agauccuacc ugacccauca aaaccuagca agaggucuuu cauugaagac 2460
cuucuguuca acaaggucac acuggcugac gccggcuuca uuaaacagua cggagauugu 2520
cuaggugaua uugcagcgcg cgaucugauu ugcgcacaga aguuuaacgg ccugacgguc 2580
uuacccccuc uccuuaccga cgaaaugauu gcccaguaca ccagcgcccu gcucgcuggc 2640
acgauuacua gcggauggac auuuggggcc ggcgcugccc uccagauacc auuugccaug 2700
cagauggcgu auagguuuaa cggcauagga guaacccaga acgugcugua cgagaaccaa 2760
aaacugauag ccaaucaauu caauagugcc auaggaaaga uacaggacag ucucagcagc 2820
accgcguccg cucucggaaa gcuacaagau guggucaacc agaacgcgca ggcauugaau 2880
acacugguga agcagcucuc cucgaauuuu ggagcaauca gcagcgugcu gaaugauauc 2940
cugucucggc uggacaaggu ugaagccgaa guccagaucg acagguuaau caccggucgg 3000
cugcagaguc uccagacaua uguuacccag caacucauca gagcugccga aauacgcgcc 3060
agugccaauc uugcagccac uaagaugucc gagugcgugu uggggcaaag uaaaaggguu 3120
gauuucugug gaaaaggaua ucaucuuaug aguuucccuc aauccgcccc ucacggaguu 3180
gucuuccugc augugaccua cgugccagcg caggagaaga acuucacgac cgcccccgcc 3240
aucugccaug auggcaaggc ccauuuuccc cgcgaaggag uguucguauc caauggcacc 3300
cacugguucg ugacgcagag aaauuuuuau gagccgcaaa uuaucacuac cgacaacaca 3360
uucguuuccg gcaauugcga ugucguaauc gggaucguga auaauacagu cuaugauccu 3420
cuucagccag aacucgauuc auucaaagag gagcuggaua aauauuucaa gaaccacacc 3480
ucccccgaug uggaucuggg ugacauauca ggaauuaacg caagcgucgu gaacauucag 3540
aaggaaaucg acaggcucaa ugaaguagca aagaacuuga augagucucu caucgacuug 3600
caggaacucg gcaaauauga gcaguacauu aaauggccgu gguauaucug gcuaggcuuu 3660
aucgccgguc ugauugcaau ugugaugguu acuaucaugu ugugcugcau gacaaguugc 3720
uguucaugcc uuaaaggcug cugcuccugc gggucauguu guaaauucga ugaggacgac 3780
ucugagcccg ugcugaaagg ggugaaacug cacuacacg 3819
<210> 20
<211> 3819
<212> RNA
<213> Artificial Sequence
<220>
<223> S蛋白基因优化mRNA序列3(STF mRNA)
<400> 20
auguucgugu uccuggugcu gcugccucug guguccagcc agugugugaa ccugaccacc 60
agaacacagc ugccuccagc cuacaccaac agcuuuacca gaggcgugua cuaccccgac 120
aagguguuca gauccagcgu gcugcacucu acccaggacc uguuccugcc uuucuucagc 180
aacgugaccu gguuccacgc cauccacgug uccggcacca auggcaccaa gagauucgac 240
aaccccgugc ugcccuucaa cgacggggug uacuuugcca gcaccgagaa guccaacauc 300
aucagaggcu ggaucuucgg caccacacug gacagcaaga cccagagccu gcugaucgug 360
aacaacgcca ccaacguggu caucaaagug ugcgaguucc aguucugcaa cgaccccuuc 420
cugggcgucu acuaccacaa gaacaacaag agcuggaugg aaagcgaguu ccggguguac 480
agcagcgcca acaacugcac cuucgaguac gugucccagc cuuuccugau ggaccuggaa 540
ggcaagcagg gcaacuucaa gaaccugcgc gaguucgugu uuaagaacau cgacggcuac 600
uucaagaucu acagcaagca caccccuauc aaccucgugc gggaucugcc ucagggcuuc 660
ucugcucugg aaccccuggu ggaucugccc aucggcauca acaucacccg guuucagaca 720
cugcuggccc ugcacagaag cuaccugaca ccuggcgaua gcagcagcgg auggacagcu 780
ggugccgccg cuuacuaugu gggcuaccug cagccuagaa ccuuccugcu gaaguacaac 840
gagaacggca ccaucaccga cgccguggau ugugcucugg auccucugag cgagacaaag 900
ugcacccuga aguccuucac cguggaaaag ggcaucuacc agaccagcaa cuuccgggug 960
cagcccaccg aauccaucgu gcgguucccc aauaucacca aucugugccc cuucggcgag 1020
guguucaaug ccaccagauu cgccucugug uacgccugga accggaagcg gaucagcaau 1080
ugcguggccg acuacuccgu gcuguacaac uccgccagcu ucagcaccuu caagugcuac 1140
ggcguguccc cuaccaagcu gaacgaccug ugcuucacaa acguguacgc cgacagcuuc 1200
gugauccggg gagaugaagu gcggcagauu gccccuggac agacaggcaa gaucgccgac 1260
uacaacuaca agcugcccga cgacuucacc ggcuguguga uugccuggaa cagcaacaac 1320
cuggacucca aagucggcgg caacuacaau uaccuguacc ggcuguuccg gaaguccaau 1380
cugaagcccu ucgagcggga caucuccacc gagaucuauc aggccggcag caccccuugu 1440
aacggcgugg aaggcuucaa cugcuacuuc ccacugcagu ccuacggcuu ucagcccaca 1500
aauggcgugg gcuaucagcc cuacagagug guggugcuga gcuucgaacu gcugcaugcc 1560
ccugccacag ugugcggccc uaagaaaagc accaaucucg ugaagaacaa augcgugaac 1620
uucaacuuca acggccugac cggcaccggc gugcugacag agagcaacaa gaaguuccug 1680
ccauuccagc aguuuggccg ggauaucgcc gauaccacag acgccguuag agauccccag 1740
acacuggaaa uccuggacau caccccuugc agcuucggcg gagugucugu gaucaccccu 1800
ggcaccaaca ccagcaauca gguggcagug cuguaccagg acgugaacug uaccgaagug 1860
cccguggcca uucacgccga ucagcugaca ccuacauggc ggguguacuc caccggcagc 1920
aauguguuuc agaccagagc cggcugucug aucggagccg agcacgugaa caauagcuac 1980
gagugcgaca uccccaucgg cgcuggcauc ugugccagcu accagacaca gacaaacagc 2040
cccagacggg ccagaucugu ggccagccag agcaucauug ccuacacaau gucucugggc 2100
gccgagaaca gcguggccua cuccaacaac ucuaucgcua uccccaccaa cuucaccauc 2160
agcgugacca cagagauccu gccugugucc augaccaaga ccagcgugga cugcaccaug 2220
uacaucugcg gcgauuccac cgagugcucc aaccugcugc ugcaguacgg cagcuucugc 2280
acccagcuga auagagcccu gacagggauc gccguggaac aggacaagaa cacccaagag 2340
guguucgccc aagugaagca gaucuacaag accccuccua ucaaggacuu cggcggcuuc 2400
aauuucagcc agauucugcc cgauccuagc aagcccagca agcggagcuu caucgaggac 2460
cugcuguuca acaaagugac acuggccgac gccggcuuca ucaagcagua uggcgauugu 2520
cugggcgaca uugccgccag ggaucugauu ugcgcccaga aguuuaacgg acugacagug 2580
cugccuccuc ugcugaccga ugagaugauc gcccaguaca caucugcccu gcuggccggc 2640
acaaucacaa gcggcuggac auuuggagcu ggcgccgcuc ugcagauccc cuuugcuaug 2700
cagauggccu accgguucaa cggcaucgga gugacccaga augugcugua cgagaaccag 2760
aagcugaucg ccaaccaguu caacagcgcc aucggcaaga uccaggacag ccugagcagc 2820
acagcaagcg cccugggaaa gcugcaggac guggucaacc agaaugccca ggcacugaac 2880
acccugguca agcagcuguc cuccaacuuc ggcgccauca gcucugugcu gaacgauauc 2940
cugagcagac uggacaaggu ggaagccgag gugcagaucg acagacugau caccggaagg 3000
cugcaguccc ugcagaccua cguuacccag cagcugauca gagccgccga gauuagagcc 3060
ucugccaauc uggccgccac caagaugucu gagugugugc ugggccagag caagagagug 3120
gacuuuugcg gcaagggcua ccaccugaug agcuucccuc agucugcccc ucacggcgug 3180
guguuucugc acgugacaua cgugcccgcu caagagaaga auuucaccac cgcuccagcc 3240
aucugccacg acggcaaagc ccacuuuccu agagaaggcg uguucguguc caacggcacc 3300
cauugguucg ugacccagcg gaacuucuac gagccccaga ucaucaccac cgacaacacc 3360
uucgugucug gcaacugcga cgucgugauc ggcauuguga acaauaccgu guacgacccu 3420
cugcagcccg agcuggacag cuucaaagag gaacuggaua aguacuuuaa gaaccacaca 3480
agccccgacg uggaccuggg cgauaucagc ggaaucaaug ccagcgucgu gaacauccag 3540
aaagagaucg accggcugaa cgagguggcc aagaaucuga acgagagccu gaucgaccug 3600
caagaacugg ggaaguacga gcaguacauc aaguggcccu gguacaucug gcugggcuuu 3660
aucgccggac ugauugccau cgugaugguc acaaucaugc uguguugcau gaccagcugc 3720
uguagcugcc ugaagggcug uuguagcugu ggcagcugcu gcaaguucga cgaggacgau 3780
ucugagcccg ugcugaaggg cgugaaacug cacuacaca 3819
<210> 21
<211> 225
<212> RNA
<213> Artificial Sequence
<220>
<223> E蛋白基因优化mRNA序列（EBL mRNA）
<400> 21
auguacagcu uugucucaga ggaaaccggc acgcugauug uaaacagcgu guuacuauuc 60
cucgccuucg uuguguuucu ccuuguuaca cuggcaauac ugacugcccu gcgguugugc 120
gcuuacugcu guaauaucgu gaacgugucu uuggugaagc ccaguuucua uguauauucc 180
agagucaaaa aucucaacuc cucuagggug ccugaccugc uuguc 225
<210> 22
<211> 669
<212> RNA
<213> Artificial Sequence
<220>
<223> M蛋白基因优化mRNA序列（MBL mRNA）
<400> 22
auggcagauu ccaacgguac aauuaccguc gaagagcuga aaaagcuccu ugagcagugg 60
aaccugguca uaggguuccu auuccugaca uggauuugcc ugcugcaauu ugccuaugcc 120
aacaggaaua gguuuuugua uauaaucaag cugauuuucc ucuggcuguu auggccagug 180
acccuggccu guuuugugcu ugccgcuguu uacagaauaa auuggaucac cggcggaauc 240
gccaucgcaa uggcuugccu uguaggcuug auguggcuca gcuacuucau ugcuucuuuc 300
cggcuguuug cgcgaacgcg guccaugugg ucuuucaauc cggagacuaa cauacuccuc 360
aaugugcccc uccauggcac uauucugacc agaccccugc uagagaguga acucgucauc 420
ggagcuguga uccugcgggg gcaccugaga aucgccggac accacuuagg ccgcugugac 480
aucaaggauc ugccuaaaga aaucacuguu gccacaucac gaacccuuuc uuauuacaag 540
uugggggccu cgcagcgugu ggcaggagac ucagguuuug cggcauacag ucgcuacagg 600
auuggcaacu auaaauuaaa cacagaccau uccagcagca gcgauaauau ugcuuugcuu 660
gugcaguga 669
<210> 23
<211> 1257
<212> RNA
<213> Artificial Sequence
<220>
<223> N蛋白基因优化mRNA序列（NBL mRNA）
<400> 23
augucagaua acggaccgca gaaccaaagg aacgccccuc ggaucacuuu cggggguccu 60
agcgacagca cugggucuaa ccaaaaugga gaacguuccg gcgcaagauc caaacagagg 120
aggccucagg ggcuuccuaa caauacagcc uccugguuca cagcucucac acagcauggc 180
aaggaagacc ugaaguuucc uagaggccag gggguuccca ucaauacuaa cuccucccca 240
gacgaucaga uugguuauua ucggcgggcu accaggcgga uccggggcgg agacgguaag 300
augaaggacc ucucuccccg uugguacuuu uacuaccucg guacaggccc cgaggcuggg 360
cuuccguaug gcgccaauaa ggauggaaua auuugggugg cuacggaagg ggcccucaac 420
acaccgaagg aucacauugg cacccguaau cccgcgaaua augccgccau uguccugcag 480
uugccccagg ggacgacguu gcccaaaggc uuuuacgcag aaggaucgcg cggaggaucc 540
caagccucca gccgaucaag cucucgaucu cggaacucaa gucgcaauag cacaccaggg 600
ucuucucgcg ggaccagccc ugcaaggaug gccggaaacg gcggugaugc ugcuuuagcg 660
cugcugcugc uggauagacu gaaccaauua gagaguaaaa ugucagguaa aggccagcaa 720
cagcaggggc agacagugac caaaaaaagu gcggccgagg ccagcaagaa accccgccag 780
aaacgaacag ccacuaaagc cuacaacgua acccaagcau ucggaaggag aggaccagag 840
cagacccaag gcaauuuugg cgaucaagag cugauccgcc aggggacgga cuauaagcau 900
uggccacaga ucgcccaguu cgcacccagu gcuucagccu ucuucggaau gucgagaauc 960
gguauggagg ucacuccuuc uggcacuugg cugacuuaua ccggcgcaau aaagcuagac 1020
gacaaagacc cuaacuuuaa ggaucaggug auccugcuaa auaaacacau ugaugcguac 1080
aaaacauucc caccaacuga gccaaagaag gacaagaaga agaaggcaga ugaaacccag 1140
gcuuugcccc agagacagaa aaagcagcag accgugaccu ugcugccagc agccgaccuc 1200
gacgauuuuu caaagcaacu ucagcagucc augaguagcg cugacagcac ccaggcu 1257
<210> 24
<211> 3819
<212> DNA
<213> Artificial Sequence
<220>
<223> SDC50
<400> 24
atgttcgtgt ttctggtgct gctgcctctg gtgtcttctc agtgtgtgaa tctgacaaca 60
agaacacagc tgcctcctgc ctacaccaac agctttacaa gaggagtgta ctaccctgac 120
aaggtgttca gaagcagcgt gctgcattct acacaggacc tgtttctgcc tttcttcagc 180
aacgtgacct ggtttcacgc cattcacgtg tctggcacaa atggaaccaa gaggttcgac 240
aatcctgtgc tgcctttcaa cgatggcgtg tactttgcct ctaccgagaa gagcaacatc 300
atcagaggct ggatctttgg caccacactg gatagcaaga cacagtctct gctgatcgtg 360
aacaatgcca ccaacgtggt gatcaaggtg tgtgagttcc agttctgcaa cgaccctttt 420
ctgggcgtgt actaccacaa gaacaacaag agctggatgg agagcgagtt cagagtgtac 480
agctctgcca acaattgcac ctttgagtac gtgagccagc ctttcctgat ggatctggaa 540
ggaaagcagg gcaatttcaa gaacctgcgg gagttcgtgt tcaagaacat cgacggctac 600
ttcaagatct acagcaagca cacccccatc aatctggtga gagatctgcc tcagggattt 660
tctgctctgg aacctctggt ggatctgcct attggcatca acatcaccag attccagaca 720
ctgctggctc tgcacagatc ttacctgaca cctggagatt cttcttctgg atggacagct 780
ggagctgctg cttattacgt gggctatctg cagcctagaa ccttcctgct gaagtacaac 840
gagaatggca ccatcacaga tgctgtggat tgtgctctgg atcctctgtc tgagaccaag 900
tgtacactga agagcttcac agtggagaag ggcatctacc agaccagcaa tttcagagtg 960
cagcctacag agagcatcgt gagattcccc aacatcacca atctgtgccc ttttggagag 1020
gtgttcaatg ccaccagatt tgcctctgtg tacgcctgga acagaaagag gatcagcaac 1080
tgtgtggccg attactctgt gctgtacaac tctgccagct ttagcacctt caagtgctac 1140
ggagtgtctc ctacaaagct gaacgacctg tgtttcacca acgtgtacgc cgatagcttc 1200
gtgattagag gcgatgaagt gagacagatt gctcctggcc agacaggaaa gatcgccgat 1260
tacaactaca agctgcctga tgacttcacc ggctgtgtga ttgcctggaa tagcaataac 1320
ctggacagca aagtgggcgg caactacaac tacctgtaca gactgttcag gaagagcaac 1380
ctgaagccct tcgagagaga catctctacc gagatttatc aggctggaag caccccttgt 1440
aatggcgtgg aaggcttcaa ctgttacttt cctctgcaga gctacggctt tcagcctacc 1500
aatggagtgg gatatcagcc ttatagagtg gtggtgctga gctttgaact gctgcatgct 1560
cctgctacag tgtgtggacc taagaagagc accaacctgg tgaagaacaa gtgcgtgaac 1620
ttcaacttca acggcctgac aggaacagga gtgctgacag agagcaataa gaagttcctg 1680
cccttccagc agtttggcag agacattgcc gatacaacag atgccgtgag agatcctcag 1740
acactggaga tcctggatat cacaccttgt agctttggcg gcgtgtctgt gattacacct 1800
ggaaccaata ccagcaatca ggtggctgtg ctgtaccagg atgtgaattg cacagaagtg 1860
cctgtggcca ttcatgctga tcagctgaca cctacatgga gagtgtacag caccggctct 1920
aatgtgtttc agaccagagc tggatgtctg attggagccg agcacgtgaa taacagctac 1980
gagtgtgaca tccctattgg agccggaatc tgtgcctctt atcagacaca gaccaactct 2040
cctagaagag ccagatctgt ggcctctcag tctatcatcg cctataccat gtctctggga 2100
gctgagaata gcgtggccta tagcaacaac agcattgcca tccctaccaa cttcaccatc 2160
agcgtgacaa cagagattct gcctgtgagc atgaccaaga catctgtgga ctgcaccatg 2220
tacatctgtg gcgattctac cgagtgtagc aatctgctgc tgcagtacgg ctctttttgt 2280
acccagctga atagagccct gacaggaatt gccgtggaac aggacaagaa tacccaggaa 2340
gtgtttgccc aggtgaagca gatctacaag acccctccta tcaaggactt tggcggcttc 2400
aacttctctc agattctgcc tgatcctagc aagcccagca agagaagttt catcgaggat 2460
ctgctgttca acaaggtgac actggccgat gccggattta tcaagcagta tggagattgt 2520
ctgggcgata tcgccgccag agatctgatt tgtgcccaga agtttaatgg actgaccgtg 2580
ctgcctcctc tgctgacaga tgagatgatt gctcagtata catctgccct gctggccgga 2640
acaatcacat ctggatggac atttggagct ggagctgctc tgcagattcc ttttgccatg 2700
cagatggcct acagattcaa tggcatcggc gtgacacaga atgtgctgta cgagaaccag 2760
aagctgattg ccaaccagtt caacagcgcc attggcaaga tccaggattc tctgtcttct 2820
acagcctctg ctctgggaaa actgcaggat gtggtgaatc agaatgccca ggccctgaat 2880
acactggtga agcagctgtc tagcaatttt ggcgccatct ctagcgtgct gaatgacatc 2940
ctgagcagac tggataaagt ggaggccgaa gtgcagatcg atagactgat cacaggcaga 3000
ctgcagtctc tgcagacata tgtgacacag cagctgatta gagctgccga gatcagagct 3060
tctgctaatc tggctgccac aaagatgtct gagtgtgtgc tgggacagtc taagagagtg 3120
gacttctgtg gcaaaggcta tcacctgatg agctttcctc agtctgctcc tcatggagtg 3180
gtgtttctgc atgtgacata tgtgcctgcc caggagaaga acttcacaac agctcctgcc 3240
atttgtcatg atggcaaggc ccactttcct agagaaggag tgttcgtgtc taatggcaca 3300
cactggttcg tgacacagag gaacttctac gagcctcaga tcatcaccac cgataacacc 3360
ttcgtgtctg gcaattgcga tgtggtgatc ggcatcgtga acaataccgt gtatgatcct 3420
ctgcagcctg agctggatag cttcaaggag gagctggaca agtacttcaa gaaccacacc 3480
tctcctgatg tggatctggg cgatatctct ggcatcaatg cctctgtggt gaacatccag 3540
aaggagatcg acagactgaa tgaggtggcc aagaacctga atgagagcct gatcgatctg 3600
caggaactgg gaaagtacga gcagtacatc aagtggcctt ggtacatctg gctgggattt 3660
attgccggac tgattgccat cgtgatggtg accatcatgc tgtgctgtat gaccagctgt 3720
tgtagctgtc tgaaaggctg ctgtagctgt ggcagctgtt gcaagtttga tgaggatgat 3780
tctgagcctg tgctgaaggg cgtgaagctg cactacacc 3819
<210> 25
<211> 3819
<212> DNA
<213> Artificial Sequence
<220>
<223> SDC54
<400> 25
atgttcgtgt tcctggtgct gctgcctctg gtgagctctc agtgtgtgaa tctgaccaca 60
agaacccagc tgcctcctgc ctacaccaac agctttacca gaggagtgta ctaccccgac 120
aaggtgttca gaagcagcgt gctgcatagc acacaggatc tgttcctgcc cttcttcagc 180
aacgtgacct ggtttcacgc catccatgtg tctggcacca atggcaccaa gagattcgac 240
aaccctgtgc tgcctttcaa cgatggcgtg tacttcgcct ctaccgagaa gagcaacatc 300
atcagaggct ggatcttcgg caccacactg gatagcaaga cccagtctct gctgatcgtg 360
aacaacgcca ccaacgtggt gatcaaggtg tgcgagttcc agttctgcaa cgaccccttc 420
ctgggcgtgt actaccacaa gaacaacaag agctggatgg agagcgagtt cagggtgtac 480
agcagcgcca acaattgcac cttcgagtac gtgagccagc ctttcctgat ggatctggag 540
ggaaagcagg gcaacttcaa gaacctgcgg gagttcgtgt tcaagaacat cgacggctac 600
ttcaagatct acagcaagca cacccccatc aacctggtga gagatctgcc tcagggattt 660
tctgctctgg agcctctggt ggatctgcct atcggcatca acatcaccag attccagaca 720
ctgctggccc tgcacagaag ctacctgaca cctggagatt cttcttctgg ctggacagct 780
ggagctgctg cctattacgt gggctatctg cagcccagaa ccttcctgct gaagtacaac 840
gagaacggca ccatcacaga tgccgtggat tgtgccctgg atcctctgtc tgagaccaag 900
tgtaccctga agagcttcac cgtggagaag ggcatctacc agaccagcaa cttcagagtg 960
cagcctaccg agagcatcgt gagattcccc aacatcacca acctgtgccc ttttggcgag 1020
gtgttcaatg ccaccagatt tgccagcgtg tacgcctgga acaggaagag gatcagcaac 1080
tgtgtggccg attacagcgt gctgtacaac tctgccagct tcagcacctt caagtgctac 1140
ggcgtgtctc ctacaaagct gaacgacctg tgcttcacca acgtgtacgc cgacagcttc 1200
gtgattagag gcgatgaggt gagacagatt gctcctggcc agacaggcaa gattgccgac 1260
tacaactaca agctgcctga cgacttcacc ggctgtgtga ttgcctggaa cagcaacaat 1320
ctggacagca aggtgggcgg caactacaac tacctgtaca ggctgttcag gaagagcaac 1380
ctgaagccct tcgagagaga catcagcacc gagatctatc aggctggaag caccccttgt 1440
aatggcgtgg agggcttcaa ctgttacttc cctctgcaga gctacggctt tcagcctacc 1500
aatggagtgg gctatcagcc ttacagagtg gtggtgctga gctttgaact gctgcatgct 1560
cctgctacag tgtgtggccc caagaagagc accaacctgg tgaagaacaa gtgcgtgaac 1620
ttcaacttca acggcctgac cggaacagga gtgctgacag agagcaacaa gaagttcctg 1680
cccttccagc agttcggcag agatatcgcc gataccacag atgccgtgag agatcctcag 1740
acactggaga tcctggacat cacaccttgc agctttggcg gagtgtctgt gatcacacct 1800
ggcaccaata ccagcaatca ggtggctgtg ctgtaccagg acgtgaattg caccgaagtg 1860
cctgtggcca ttcatgctga tcagctgacc cctacatgga gagtgtacag caccggctct 1920
aatgtgttcc agaccagagc cggatgtctg attggagccg agcacgtgaa taacagctac 1980
gagtgcgaca tccctattgg agccggcatc tgtgcctctt atcagaccca gaccaactct 2040
cctagaagag ccagaagcgt ggcctctcag agcatcattg cctacaccat gtctctggga 2100
gccgagaata gcgtggccta cagcaataac agcatcgcca tccccaccaa cttcaccatc 2160
agcgtgacca cagagattct gcctgtgagc atgaccaaga cctctgtgga ctgcaccatg 2220
tacatctgtg gcgactctac cgagtgcagc aatctgctgc tgcagtatgg cagcttttgt 2280
acccagctga acagagccct gacaggcatt gctgtggagc aggataagaa cacccaggag 2340
gtgtttgccc aggtgaagca gatctacaag acccctccca tcaaggactt cggcggcttt 2400
aacttcagcc agatcctgcc tgatcctagc aagcccagca agaggagctt tatcgaggac 2460
ctgctgttca acaaggtgac cctggccgat gctggcttta tcaagcagta cggagattgt 2520
ctgggcgata tcgccgccag agacctgatt tgtgcccaga agttcaatgg actgaccgtg 2580
ctgcctcctc tgctgacaga tgagatgatt gcccagtaca catctgccct gctggctggc 2640
acaatcacat ctggatggac atttggagct ggagctgccc tgcagatccc ttttgccatg 2700
cagatggcct acagattcaa cggcatcggc gtgacccaga atgtgctgta cgagaaccag 2760
aagctgatcg ccaaccagtt caacagcgcc atcggcaaga tccaggattc tctgtctagc 2820
acagcctctg ctctgggaaa gctgcaggat gtggtgaatc agaatgccca ggccctgaat 2880
acactggtga agcagctgag cagcaacttt ggcgccatca gctctgtgct gaatgacatc 2940
ctgagcagac tggacaaggt ggaggctgaa gtgcagatcg acagactgat cacaggcaga 3000
ctgcagtctc tgcagaccta cgtgacacag cagctgatta gagctgccga gatcagagct 3060
tctgccaatc tggctgccac caagatgtct gagtgtgtgc tgggacagag caagagagtg 3120
gacttctgtg gcaaaggcta ccacctgatg agcttccctc agtctgctcc tcatggagtg 3180
gtgtttctgc acgtgaccta tgtgcctgcc caggagaaga acttcaccac agctcctgcc 3240
atttgtcacg atggcaaggc ccactttcct agagaaggcg tgttcgtgag caatggcaca 3300
cactggttcg tgacccagag gaacttctac gagccccaga tcatcaccac cgataacacc 3360
ttcgtgagcg gcaattgcga cgtggtgatc ggcatcgtga acaataccgt gtacgatcct 3420
ctgcagcctg agctggacag cttcaaggag gagctggaca agtacttcaa gaaccacacc 3480
agccctgatg tggatctggg cgacatctct ggcatcaatg ccagcgtggt gaacatccag 3540
aaggagatcg acaggctgaa cgaggtggcc aagaacctga atgagagcct gatcgatctg 3600
caggagctgg gcaagtacga gcagtacatc aagtggcctt ggtacatctg gctgggcttt 3660
atcgccggac tgattgccat cgtgatggtg accatcatgc tgtgctgcat gaccagctgc 3720
tgtagctgtc tgaagggctg ttgtagctgt ggcagctgtt gcaagttcga cgaggatgat 3780
agcgagcctg tgctgaaagg cgtgaagctg cactacacc 3819
<210> 26
<211> 3819
<212> DNA
<213> Artificial Sequence
<220>
<223> SDC58
<400> 26
atgttcgtgt tcctggtgct gctgcccctg gtgagctctc agtgtgtgaa cctgaccacc 60
agaacccagc tgcctcctgc ctacaccaac agcttcacca gaggcgtgta ctaccccgac 120
aaggtgttca gaagcagcgt gctgcacagc acccaggacc tgttcctgcc cttcttcagc 180
aacgtgacct ggttccacgc catccacgtg tctggcacca atggcaccaa gaggttcgac 240
aaccctgtgc 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 ccgggtgtac 480
agcagcgcca acaactgcac cttcgagtac gtgagccagc ccttcctgat ggacctggag 540
ggcaagcagg gcaacttcaa gaacctgcgg gagttcgtgt tcaagaacat cgacggctac 600
ttcaagatct acagcaagca cacccccatc aacctggtga gagacctgcc tcagggcttt 660
tctgccctgg agcctctggt ggacctgcct atcggcatca acatcaccag gttccagacc 720
ctgctggccc tgcacagaag ctacctgaca cctggcgata gctcttctgg ctggacagct 780
ggagctgctg cctattacgt gggctacctg cagcccagga ccttcctgct gaagtacaac 840
gagaacggca ccatcaccga cgccgtggat tgtgccctgg atcctctgag cgagaccaag 900
tgcaccctga agagcttcac cgtggagaag ggcatctacc agaccagcaa cttccgggtg 960
cagcctaccg agagcatcgt gaggttcccc aacatcacca acctgtgccc tttcggcgag 1020
gtgttcaacg ccaccagatt cgcctctgtg tacgcctgga acaggaagcg gatcagcaac 1080
tgcgtggccg actacagcgt gctgtacaac agcgccagct tcagcacctt caagtgctac 1140
ggcgtgagcc ctaccaagct gaacgacctg tgcttcacca acgtgtacgc cgacagcttc 1200
gtgatcagag gcgatgaggt gagacagatc gcccctggac agaccggcaa gatcgccgac 1260
tacaactaca agctgcccga cgacttcacc ggctgtgtga tcgcctggaa cagcaacaac 1320
ctggacagca aggtgggcgg caactacaac tacctgtacc ggctgttccg gaagagcaac 1380
ctgaagccct tcgagaggga catcagcacc gagatctacc aggccggaag cacaccttgc 1440
aatggcgtgg agggcttcaa ctgctacttc cccctgcaga gctacggctt tcagcctacc 1500
aatggcgtgg gctaccagcc ctacagagtg gtggtgctga gctttgaact gctgcatgcc 1560
cctgccacag tgtgtggccc caagaagagc accaacctgg tgaagaacaa gtgcgtgaac 1620
ttcaacttca acggcctgac cggcacaggc gtgctgaccg agagcaacaa gaagttcctg 1680
cccttccagc agttcggcag agacatcgcc gataccaccg atgccgtgag agatcctcag 1740
accctggaga tcctggacat caccccttgc agctttggcg gagtgagcgt gatcacacct 1800
ggcaccaaca ccagcaatca ggtggccgtg ctgtaccagg acgtgaactg cacagaggtg 1860
cctgtggcca ttcatgccga tcagctgacc cctacctgga gagtgtacag caccggcagc 1920
aatgtgttcc agaccagagc cggctgtctg atcggagccg agcacgtgaa caacagctac 1980
gagtgcgaca tccctatcgg agccggcatc tgcgcctctt accagacaca gaccaacagc 2040
cccagaagag ccagaagcgt ggccagccag tctatcatcg cctacaccat gagcctggga 2100
gccgagaaca gcgtggccta cagcaacaac agcatcgcca tccccaccaa cttcaccatc 2160
agcgtgacca ccgagatcct gcccgtgagc atgaccaaga ccagcgtgga ctgcaccatg 2220
tacatctgcg gcgacagcac agagtgcagc aacctgctgc tgcagtacgg cagcttttgc 2280
acccagctga acagagccct gacaggcatt gccgtggagc aggacaagaa cacccaggag 2340
gtgttcgccc aggtgaagca gatctacaag acccccccca tcaaggactt cggcggcttc 2400
aacttcagcc agatcctgcc tgaccctagc aagcccagca agcggagctt catcgaggac 2460
ctgctgttca acaaggtgac cctggccgat gccggcttca tcaagcagta cggcgattgt 2520
ctgggcgata tcgccgccag agacctgatc tgtgcccaga agttcaacgg cctgaccgtg 2580
ctgcctcctc tgctgacaga tgagatgatc gcccagtaca cctctgccct gctggccgga 2640
accatcacat ctggctggac atttggagct ggagccgccc tgcagatccc tttcgccatg 2700
cagatggcct acaggttcaa cggcatcggc gtgacccaga acgtgctgta cgagaaccag 2760
aagctgatcg ccaaccagtt caacagcgcc atcggcaaga tccaggacag cctgtctagc 2820
acagcctctg ctctgggcaa gctgcaggat gtggtgaacc agaatgccca ggccctgaac 2880
accctggtga agcagctgag cagcaatttc ggcgccatca gcagcgtgct gaacgacatc 2940
ctgagcagac tggacaaggt ggaggccgag gtgcagatcg acagactgat caccggcaga 3000
ctgcagagcc tgcagaccta cgtgacacag cagctgatca gagccgccga gatcagagcc 3060
tctgccaatc tggctgccac caagatgagc gagtgtgtgc tgggccagag caagagagtg 3120
gacttctgcg gcaaaggcta ccacctgatg agcttccccc agtctgctcc tcatggcgtg 3180
gtgtttctgc acgtgaccta cgtgcctgcc caggagaaga acttcaccac agcccctgcc 3240
atctgtcacg atggcaaggc ccacttccct agagagggcg tgttcgtgag caatggcacc 3300
cactggttcg tgacccagcg gaacttctac gagccccaga tcatcaccac cgacaacacc 3360
ttcgtgagcg gcaactgcga cgtggtgatc ggcatcgtga acaacaccgt gtacgaccct 3420
ctgcagcccg agctggacag cttcaaggag gagctggaca agtacttcaa gaaccacacc 3480
agccccgacg tggatctggg cgacatcagc ggcatcaacg ccagcgtggt gaacatccag 3540
aaggagatcg accggctgaa cgaggtggcc aagaacctga acgagagcct gatcgacctg 3600
caggagctgg gcaagtacga gcagtacatc aagtggccct ggtacatctg gctgggcttt 3660
atcgccggcc tgatcgccat cgtgatggtg accatcatgc tgtgctgcat gaccagctgc 3720
tgcagctgcc tgaagggctg ttgtagctgt ggcagctgct gcaagttcga cgaggacgat 3780
agcgagcctg tgctgaaggg cgtgaagctg cactacacc 3819
<210> 27
<211> 3819
<212> DNA
<213> Artificial Sequence
<220>
<223> SDC60
<400> 27
atgttcgtgt tcctggtgct gctgcccctg gtgagcagcc agtgtgtgaa cctgaccacc 60
agaacccagc tgcctcccgc ctacaccaac agcttcacca ggggcgtgta ctaccccgac 120
aaggtgttca ggagcagcgt gctgcacagc acccaggacc tgttcctgcc cttcttcagc 180
aacgtgacct ggttccacgc catccacgtg agcggcacca atggcaccaa gcggttcgac 240
aaccctgtgc tgcccttcaa cgacggcgtg tacttcgcca gcaccgagaa gagcaacatc 300
atccggggct ggatcttcgg caccaccctg gacagcaaga cccagagcct gctgatcgtg 360
aacaacgcca ccaacgtggt gatcaaggtg tgcgagttcc agttctgcaa cgaccccttc 420
ctgggcgtgt actaccacaa gaacaacaag agctggatgg agagcgagtt ccgggtgtac 480
agcagcgcca acaactgcac cttcgagtac gtgagccagc ccttcctgat ggacctggag 540
ggcaagcagg gcaacttcaa gaacctgcgg gagttcgtgt tcaagaacat cgacggctac 600
ttcaagatct acagcaagca cacccccatc aacctggtga gggacctgcc tcagggcttt 660
tctgccctgg agcctctggt ggacctgccc atcggcatca acatcaccag gttccagacc 720
ctgctggccc tgcacaggag ctacctgaca cctggcgata gctcttctgg ctggacagcc 780
ggagctgctg cctactacgt gggctacctg cagccccgga ccttcctgct gaagtacaac 840
gagaacggca ccatcaccga cgccgtggat tgcgccctgg atcctctgag cgagaccaag 900
tgcaccctga agagcttcac cgtggagaag ggcatctacc agaccagcaa cttccgggtg 960
cagcccaccg agagcatcgt gaggttcccc aacatcacca acctgtgccc cttcggcgag 1020
gtgttcaacg ccaccagatt cgccagcgtg tacgcctgga accggaagcg gatcagcaac 1080
tgcgtggccg actacagcgt gctgtacaac agcgccagct tcagcacctt caagtgctac 1140
ggcgtgagcc ccaccaagct gaacgacctg tgcttcacca acgtgtacgc cgacagcttc 1200
gtgatcaggg gcgatgaggt gagacagatc gcccctggcc agaccggcaa gatcgccgac 1260
tacaactaca agctgcccga cgacttcacc ggctgcgtga tcgcctggaa cagcaacaac 1320
ctggacagca aggtgggcgg caactacaac tacctgtacc ggctgttccg gaagagcaac 1380
ctgaagccct tcgagcggga catcagcacc gagatctacc aggccggaag caccccttgc 1440
aacggcgtgg agggcttcaa ctgctacttc cccctgcaga gctacggctt ccagcctacc 1500
aatggcgtgg gctaccagcc ctacagggtg gtggtgctga gctttgagct gctgcatgct 1560
cctgccaccg tgtgcggccc caagaagagc accaacctgg tgaagaacaa gtgcgtgaac 1620
ttcaacttca acggcctgac cggcaccggc gtgctgaccg agagcaacaa gaagttcctg 1680
cccttccagc agttcggcag ggacatcgcc gataccaccg atgccgtgag agaccctcag 1740
accctggaga tcctggacat caccccttgc agcttcggcg gagtgagcgt gatcacacct 1800
ggcaccaaca ccagcaacca ggtggccgtg ctgtaccagg acgtgaactg caccgaggtg 1860
cctgtggcca ttcacgccga tcagctgacc cccacctgga gagtgtacag caccggcagc 1920
aacgtgttcc agaccagagc cggctgtctg atcggcgccg agcacgtgaa caacagctac 1980
gagtgcgaca tccccatcgg cgccggcatc tgtgccagct atcagaccca gaccaacagc 2040
cctaggaggg ccagaagcgt ggccagccag tctatcatcg cctacaccat gagcctgggc 2100
gccgagaaca gcgtggccta cagcaacaac agcatcgcca tccccaccaa cttcaccatc 2160
agcgtgacca ccgagatcct gcccgtgagc atgaccaaga ccagcgtgga ctgcaccatg 2220
tacatctgcg gcgacagcac cgagtgcagc aacctgctgc tgcagtacgg cagcttctgc 2280
acccagctga acagagccct gacaggcatc gccgtggagc aggacaagaa cacccaggag 2340
gtgttcgccc aggtgaagca gatctacaag acccccccca tcaaggactt cggcggcttc 2400
aacttcagcc agatcctgcc tgaccccagc aagcccagca agcggagctt catcgaggac 2460
ctgctgttca acaaggtgac cctggccgac gccggcttca tcaagcagta cggcgactgt 2520
ctgggcgaca tcgccgccag agacctgatc tgtgcccaga agttcaacgg cctgaccgtg 2580
ctgccccctc tgctgaccga tgagatgatc gcccagtaca cctctgccct gctggccggc 2640
accatcacat ctggctggac ctttggagct ggagccgccc tgcagatccc tttcgccatg 2700
cagatggcct accggttcaa cggcatcggc gtgacccaga acgtgctgta cgagaaccag 2760
aagctgatcg ccaaccagtt caacagcgcc atcggcaaga tccaggacag cctgagcagc 2820
accgcctctg ctctgggcaa actgcaggac gtggtgaacc agaacgccca ggccctgaac 2880
accctggtga agcagctgag cagcaacttc ggcgccatca gcagcgtgct gaacgacatc 2940
ctgagcaggc tggacaaggt ggaggccgag gtgcagatcg acaggctgat caccggcaga 3000
ctgcagagcc tgcagaccta cgtgacccag cagctgatca gagccgccga gatcagagcc 3060
tctgccaatc tggccgccac caagatgagc gagtgtgtgc tgggccagag caagagggtg 3120
gacttctgcg gcaagggcta ccacctgatg agcttccccc agtctgcccc tcatggcgtg 3180
gtgttcctgc acgtgaccta cgtgcctgcc caggagaaga acttcaccac cgcccctgcc 3240
atctgccacg atggcaaggc ccacttccct agagagggcg tgttcgtgag caacggcacc 3300
cactggttcg tgacccagcg gaacttctac gagccccaga tcatcaccac cgacaacacc 3360
ttcgtgagcg gcaactgcga cgtggtgatc ggcatcgtga acaacaccgt gtacgacccc 3420
ctgcagcccg agctggacag cttcaaggag gagctggaca agtacttcaa gaaccacacc 3480
agccccgacg tggacctggg cgacatcagc ggcatcaacg ccagcgtggt gaacatccag 3540
aaggagatcg accggctgaa cgaggtggcc aagaacctga acgagagcct gatcgacctg 3600
caggagctgg gcaagtacga gcagtacatc aagtggccct ggtacatctg gctgggcttc 3660
atcgccggcc tgatcgccat cgtgatggtg accatcatgc tgtgctgcat gaccagctgc 3720
tgcagctgcc tgaagggctg ctgcagctgt ggcagctgtt gcaagttcga cgaggacgac 3780
agcgagcccg tgctgaaggg cgtgaagctg cactacacc 3819
<210> 28
<211> 957
<212> DNA
<213> Artificial Sequence
<220>
<223> MT2AE
<400> 28
atggccgatt ctaatggcac catcaccgtg gaagagctga agaagctgct cgagcaatgg 60
aacctggtga tcggatttct gttcctgacc tggatctgtc tgttgcagtt cgcctacgcc 120
aaccggaaca gattcctgta catcatcaaa ctgatcttcc tgtggctgct gtggcctgtg 180
accctggcct gcttcgtgct ggccgccgtg taccggatta actggatcac cggaggcatc 240
gctatcgcca tggcatgcct ggtcggactt atgtggctgt cttatttcat cgccagcttc 300
agactgttcg ctagaaccag aagcatgtgg tcctttaacc ctgagacaaa catcctgctg 360
aacgtgcctc tgcacggcac aatcctgaca cggccactgc tggaaagcga gctggtcatc 420
ggcgccgtga tcctgcgggg ccatctgcgc attgccggac accacctggg cagatgcgac 480
atcaaggacc tgcccaagga aatcaccgtg gccaccagca gaacactgtc ctactacaaa 540
ctgggcgcta gtcagagagt ggccggcgac agcggcttcg ccgcttattc tagatacaga 600
atcggcaact acaagctgaa taccgatcac agcagcagca gcgacaacat cgccctgctg 660
gtgcagggca gcggcgaggg cagaggaagc ctgctgacat gtggcgatgt ggaagagaac 720
cccggccctg ccatgtacag ctttgtgtct gaggaaaccg gcaccctgat cgtgaacagc 780
gtgctgctgt ttctggcctt cgtcgtgttc ctgctggtga cactggctat cctgaccgcc 840
ctgaggctgt gcgcctactg ctgcaacatc gtgaatgtat ccctggtgaa gccttccttc 900
tacgtgtaca gccgggtgaa gaaccttaat agctctagag tgcccgacct gctcgtt 957
<210> 29
<211> 960
<212> DNA
<213> Artificial Sequence
<220>
<223> MP2AE
<400> 29
atggccgaca gcaacggcac aatcacagtg gaagagctga agaagctgct ggagcagtgg 60
aacctggtga ttggatttct tttcctcacc tggatctgcc tgctgcagtt cgcctatgcc 120
aaccggaaca gattcctgta catcatcaag ctgatcttcc tgtggctgct gtggcccgtg 180
accctggcct gttttgtgct ggccgccgtg taccggatca actggatcac cggcggaatc 240
gctatcgcca tggcctgcct ggtgggcctg atgtggctga gctacttcat cgcctccttt 300
agactgttcg ccagaaccag aagcatgtgg tccttcaacc ctgagacaaa tatcctgctc 360
aacgtgcccc tgcacggcac catcctgacc cggcctctgc tcgagagcga gctggtgatc 420
ggcgccgtga tcctgagagg ccacctgaga atcgccggac accacctggg cagatgcgac 480
atcaaggacc tgccaaagga aatcaccgtt gctacaagca gaacactgtc ctactacaag 540
ctgggcgctt ctcaaagagt cgccggcgac agcggcttcg ctgcttatag ccgctacagg 600
attggaaatt acaagctgaa caccgatcat tcttctagca gcgacaacat cgccctgctg 660
gtccagggca gcggcgccac aaacttcagc ctgcttaaac aggccggcga tgtggaagag 720
aaccccggcc ctgccatgta cagcttcgtg tccgaggaaa ccggcaccct gatcgtgaac 780
agcgtgctgc tgttccttgc ttttgtggtg ttcctgctgg tcaccctggc catcctgacc 840
gccctgagac tgtgtgccta ctgctgcaac atcgtgaatg tgtctctggt gaagcctagc 900
ttctacgtgt acagccgggt gaaaaacctg aactctagcc gggtgcctga tctgctggtg 960
<210> 30
<211> 798
<212> DNA
<213> Artificial Sequence
<220>
<223> SGS-RBD
<400> 30
atggagacag acacactcct gctatgggta ctgctgctct gggttccagg ttccaccgga 60
gactgcccat ttggcgaggt gttcaacgca acccgcttcg ccagcgtgta cgcctggaat 120
aggaagcgga tcagcaactg cgtggccgac tatagcgtgc tgtacaactc cgcctctttc 180
agcaccttta agtgctatgg cgtgtccccc acaaagctga atgacctgtg ctttaccaac 240
gtctacgccg attctttcgt gatcaggggc gacgaggtgc gccagatcgc ccccggccag 300
acaggcaaga tcgcagacta caattataag ctgccagacg atttcaccgg ctgcgtgatc 360
gcctggaaca gcaacaatct ggattccaaa gtgggcggca actacaatta tctgtaccgg 420
ctgtttagaa agagcaatct gaagcccttc gagagggaca tctctacaga aatctaccag 480
gccggcagca ccccttgcaa tggcgtggag ggctttaact gttatttccc actccagtcc 540
tacggcttcc agcccacaaa cggcgtgggc tatcagcctt accgcgtggt ggtgctgagc 600
tttgagctgc tgcacgccta cccgtacgac gtgccggact acgccaatgc tgtgggccag 660
gacacgcagg aggtcatcgt ggtgccacac tccttgccct ttaaggtggt ggtgatctca 720
gccatcctgg ccctggtggt gctcaccatc atctccctta tcatcctcat catgctttgg 780
cagaagaagc cacgttag 798
<210> 31
<211> 3819
<212> RNA
<213> Artificial Sequence
<220>
<223> SDC-50 mRNA
<400> 31
auguucgugu uccuggugcu gcugccucug gugucuucuc agugugugaa ucugacaacc 60
agaacccagc ugccuccugc cuauaccaac agcuucacaa gaggcgugua cuacccugac 120
aagguguuca ggucuucugu gcugcacucu acccaggauc uguuccugcc uuucuucagc 180
aacgugaccu gguuucacgc cauccaugug ucuggcacca auggcaccaa gagauucgac 240
aauccugugc ugccuuucaa cgauggcgug uacuucgccu cuaccgagaa gagcaacauc 300
aucagaggcu ggaucuuugg cacaacccug gauagcaaga cccagucucu gcugaucgug 360
aacaaugcca ccaacguggu gaucaaggug ugcgaguucc aguucugcaa ugacccuuuc 420
cugggcgugu acuaccacaa gaacaacaag agcuggaugg agagcgaguu caggguguac 480
agcucugcca acaauugcac cuucgaguac gugagccagc cuuuccugau ggaucuggaa 540
ggaaagcagg gcaacuucaa gaaccugcgg gaguucgugu ucaagaacau cgacggcuac 600
uucaagaucu acagcaagca cacccccauc aaucugguga gagaucugcc ucagggauuu 660
ucugcucugg aaccucuggu ggaucugccu aucggcauca acaucaccag auuccagaca 720
cugcuggcuc ugcacagaag cuaucugaca ccuggcgauu cuucuucugg auggacagcu 780
ggagcugcug cuuauuacgu gggcuaccug cagccuagaa ccuuccugcu gaaguacaac 840
gagaauggca ccaucaccga ugcuguggau ugugcccugg auccucuguc ugagacaaag 900
uguacccuga agagcuucac cguggagaag ggcaucuacc agaccagcaa uuucagagug 960
cagccuaccg agagcaucgu gagauucccc aacaucacca aucugugccc uuuuggcgag 1020
guguucaaug ccaccagauu ugccagcgug uaugccugga acaggaagag aaucagcaac 1080
uguguggccg acuacagcgu gcuguacaau ucugccagcu uuagcaccuu caagugcuac 1140
ggcgugucuc cuaccaagcu gaaugaccug uguuucacca acguguacgc cgacagcuuc 1200
gugaucagag gagaugaagu gagacagauu gcuccuggcc agacaggcaa gaucgccgau 1260
uacaacuaca agcugccuga ugacuucacc ggcuguguga ucgccuggaa uagcaacaau 1320
cuggacagca aagugggcgg caacuacaac uaccuguaca ggcuguucag gaagagcaac 1380
cugaagcccu ucgagagaga caucucuacc gagaucuauc aggcuggaag caccccuugu 1440
aauggcgugg aaggcuucaa cuguuacuuc ccucugcaga gcuacggcuu ucagccuacc 1500
aauggagugg gauaucagcc uuacagagug guggugcuga gcuuugaacu gcugcaugcu 1560
ccugcuacag uguguggccc uaagaagagc accaaccugg ugaagaacaa gugcgugaac 1620
uucaacuuca acggccugac cggaacagga gugcugacag agagcaacaa gaaguuccug 1680
cccuuccagc aguuuggcag agacauugcc gauaccacag augccgugag agauccucag 1740
acacuggaga uccuggauau cacaccuugc agcuuuggcg gagugucugu gaucacaccu 1800
ggaaccaaua ccagcaauca gguggcugug cuguaccagg acgugaauug cacagaagug 1860
ccuguggcca uucaugcuga ucagcugacc ccuacaugga gaguguacag cacaggcagc 1920
aauguguuuc agaccagagc cggaugucug auuggagcug agcacgugaa caacagcuac 1980
gagugugaca ucccuauugg agccggaauc ugugccagcu aucagacaca gaccaacucu 2040
ccuagaagag ccagaucugu ggccagccag ucuaucaucg ccuauaccau gucucuggga 2100
gcugagaaua gcguggccua cagcaacaac agcaucgcca ucccuaccaa cuucaccauc 2160
agcgugacaa cagagauccu gccugugagc augaccaaga caucugugga cugcaccaug 2220
uacaucugug gcgacagcac agaguguagc aaucugcugc ugcaguacgg cagcuuuugu 2280
acccagcuga auagagcccu gacaggaauu gccguggagc aggauaagaa uacccaggag 2340
guguuugccc aggugaagca gaucuacaag accccuccua ucaaggacuu cggcggcuuc 2400
aacuucagcc agauucugcc ugauccuagc aagcccagca agagaaguuu caucgaggau 2460
cugcuguuca acaaggugac ccuggccgau gccggauuua ucaagcagua uggcgauugu 2520
cugggcgaua ucgccgccag agaucugauu ugugcccaga aguucaaugg acugaccgug 2580
cugccuccuc ugcugacaga ugagaugauu gcucaguaca caucugcccu gcuggcuggc 2640
acaaucacau cuggauggac auuuggagcu ggagcugcuc ugcagauccc uuuugccaug 2700
cagauggccu acagauucaa cggcaucgga gugacccaga augugcugua cgagaaccag 2760
aagcugaucg ccaaccaguu caacagcgcc auuggcaaga uccaggauuc ucugucuagc 2820
acagcuucug cucugggcaa acugcaggau guggugaauc agaaugcuca ggcccugaau 2880
acccugguga agcagcuguc uagcaauuuu ggcgccauca gcagcgugcu gaaugacauc 2940
cugagcagac uggauaaagu ggaggccgaa gugcagaucg acagacugau cacaggaaga 3000
cugcagucuc ugcagaccua cgugacacag cagcugauua gagcugccga gauuagagcc 3060
ucugcuaauc uggcugccac caagaugucu gagugugugc ugggacaguc uaagagagug 3120
gacuucugug gcaaaggcua ccaccugaug agcuuuccuc agucugcucc ucauggagug 3180
guguuucugc acgugacaua ugugccugcc caggagaaga acuucaccac agcuccugcc 3240
auuugucacg auggcaaagc ccacuuuccu agagaaggcg uguucgugag caauggaacc 3300
cacugguuug ugacccagag aaacuucuac gagccccaga ucaucaccac cgacaauacc 3360
uucgugucug gcaauugcga cguggugauc ggcaucguga acaauaccgu guaugauccu 3420
cugcagccug agcuggacag cuucaaggag gagcuggaca aguacuucaa gaaccacacc 3480
agcccugaug uggaucuggg cgauaucucu ggcaucaaug ccucuguggu gaacauccag 3540
aaggagaucg acaggcugaa ugagguggcc aagaaccuga augagagccu gaucgaucug 3600
caggagcugg gaaaguacga gcaguacauc aaguggccuu gguacaucug gcugggcuuu 3660
auugccggac ugauugccau cgugauggug accaucaugc ugugcugcau gacaagcugu 3720
uguagcuguc ugaagggcug cuguucuugu ggcagcuguu gcaaguucga ugaggaugau 3780
agcgagccug ugcugaaagg cgugaagcug cacuacacc 3819
<210> 32
<211> 3819
<212> RNA
<213> Artificial Sequence
<220>
<223> SDC-54 mRNA
<400> 32
auguucgugu uccuggugcu gcugccucug gugagcucuc agugugugaa ucugaccaca 60
agaacccagc ugccuccugc cuacaccaac agcuuuacca gaggagugua cuaccccgac 120
aagguguuca gaagcagcgu gcugcauagc acacaggauc uguuccugcc cuucuucagc 180
aacgugaccu gguuucacgc cauccaugug ucuggcacca auggcaccaa gagauucgac 240
aacccugugc ugccuuucaa cgauggcgug uacuucgccu cuaccgagaa gagcaacauc 300
aucagaggcu ggaucuucgg caccacacug gauagcaaga cccagucucu gcugaucgug 360
aacaacgcca ccaacguggu gaucaaggug ugcgaguucc aguucugcaa cgaccccuuc 420
cugggcgugu acuaccacaa gaacaacaag agcuggaugg agagcgaguu caggguguac 480
agcagcgcca acaauugcac cuucgaguac gugagccagc cuuuccugau ggaucuggag 540
ggaaagcagg gcaacuucaa gaaccugcgg gaguucgugu ucaagaacau cgacggcuac 600
uucaagaucu acagcaagca cacccccauc aaccugguga gagaucugcc ucagggauuu 660
ucugcucugg agccucuggu ggaucugccu aucggcauca acaucaccag auuccagaca 720
cugcuggccc ugcacagaag cuaccugaca ccuggagauu cuucuucugg cuggacagcu 780
ggagcugcug ccuauuacgu gggcuaucug cagcccagaa ccuuccugcu gaaguacaac 840
gagaacggca ccaucacaga ugccguggau ugugcccugg auccucuguc ugagaccaag 900
uguacccuga agagcuucac cguggagaag ggcaucuacc agaccagcaa cuucagagug 960
cagccuaccg agagcaucgu gagauucccc aacaucacca accugugccc uuuuggcgag 1020
guguucaaug ccaccagauu ugccagcgug uacgccugga acaggaagag gaucagcaac 1080
uguguggccg auuacagcgu gcuguacaac ucugccagcu ucagcaccuu caagugcuac 1140
ggcgugucuc cuacaaagcu gaacgaccug ugcuucacca acguguacgc cgacagcuuc 1200
gugauuagag gcgaugaggu gagacagauu gcuccuggcc agacaggcaa gauugccgac 1260
uacaacuaca agcugccuga cgacuucacc ggcuguguga uugccuggaa cagcaacaau 1320
cuggacagca aggugggcgg caacuacaac uaccuguaca ggcuguucag gaagagcaac 1380
cugaagcccu ucgagagaga caucagcacc gagaucuauc aggcuggaag caccccuugu 1440
aauggcgugg agggcuucaa cuguuacuuc ccucugcaga gcuacggcuu ucagccuacc 1500
aauggagugg gcuaucagcc uuacagagug guggugcuga gcuuugaacu gcugcaugcu 1560
ccugcuacag uguguggccc caagaagagc accaaccugg ugaagaacaa gugcgugaac 1620
uucaacuuca acggccugac cggaacagga gugcugacag agagcaacaa gaaguuccug 1680
cccuuccagc aguucggcag agauaucgcc gauaccacag augccgugag agauccucag 1740
acacuggaga uccuggacau cacaccuugc agcuuuggcg gagugucugu gaucacaccu 1800
ggcaccaaua ccagcaauca gguggcugug cuguaccagg acgugaauug caccgaagug 1860
ccuguggcca uucaugcuga ucagcugacc ccuacaugga gaguguacag caccggcucu 1920
aauguguucc agaccagagc cggaugucug auuggagccg agcacgugaa uaacagcuac 1980
gagugcgaca ucccuauugg agccggcauc ugugccucuu aucagaccca gaccaacucu 2040
ccuagaagag ccagaagcgu ggccucucag agcaucauug ccuacaccau gucucuggga 2100
gccgagaaua gcguggccua cagcaauaac agcaucgcca uccccaccaa cuucaccauc 2160
agcgugacca cagagauucu gccugugagc augaccaaga ccucugugga cugcaccaug 2220
uacaucugug gcgacucuac cgagugcagc aaucugcugc ugcaguaugg cagcuuuugu 2280
acccagcuga acagagcccu gacaggcauu gcuguggagc aggauaagaa cacccaggag 2340
guguuugccc aggugaagca gaucuacaag accccuccca ucaaggacuu cggcggcuuu 2400
aacuucagcc agauccugcc ugauccuagc aagcccagca agaggagcuu uaucgaggac 2460
cugcuguuca acaaggugac ccuggccgau gcuggcuuua ucaagcagua cggagauugu 2520
cugggcgaua ucgccgccag agaccugauu ugugcccaga aguucaaugg acugaccgug 2580
cugccuccuc ugcugacaga ugagaugauu gcccaguaca caucugcccu gcuggcuggc 2640
acaaucacau cuggauggac auuuggagcu ggagcugccc ugcagauccc uuuugccaug 2700
cagauggccu acagauucaa cggcaucggc gugacccaga augugcugua cgagaaccag 2760
aagcugaucg ccaaccaguu caacagcgcc aucggcaaga uccaggauuc ucugucuagc 2820
acagccucug cucugggaaa gcugcaggau guggugaauc agaaugccca ggcccugaau 2880
acacugguga agcagcugag cagcaacuuu ggcgccauca gcucugugcu gaaugacauc 2940
cugagcagac uggacaaggu ggaggcugaa gugcagaucg acagacugau cacaggcaga 3000
cugcagucuc ugcagaccua cgugacacag cagcugauua gagcugccga gaucagagcu 3060
ucugccaauc uggcugccac caagaugucu gagugugugc ugggacagag caagagagug 3120
gacuucugug gcaaaggcua ccaccugaug agcuucccuc agucugcucc ucauggagug 3180
guguuucugc acgugaccua ugugccugcc caggagaaga acuucaccac agcuccugcc 3240
auuugucacg auggcaaggc ccacuuuccu agagaaggcg uguucgugag caauggcaca 3300
cacugguucg ugacccagag gaacuucuac gagccccaga ucaucaccac cgauaacacc 3360
uucgugagcg gcaauugcga cguggugauc ggcaucguga acaauaccgu guacgauccu 3420
cugcagccug agcuggacag cuucaaggag gagcuggaca aguacuucaa gaaccacacc 3480
agcccugaug uggaucuggg cgacaucucu ggcaucaaug ccagcguggu gaacauccag 3540
aaggagaucg acaggcugaa cgagguggcc aagaaccuga augagagccu gaucgaucug 3600
caggagcugg gcaaguacga gcaguacauc aaguggccuu gguacaucug gcugggcuuu 3660
aucgccggac ugauugccau cgugauggug accaucaugc ugugcugcau gaccagcugc 3720
uguagcuguc ugaagggcug uuguagcugu ggcagcuguu gcaaguucga cgaggaugau 3780
agcgagccug ugcugaaagg cgugaagcug cacuacacc 3819
<210> 33
<211> 3819
<212> RNA
<213> Artificial Sequence
<220>
<223> SDC-58 mRNA
<400> 33
auguucgugu uccuggugcu gcugccccug gugagcucuc agugugugaa ccugaccacc 60
agaacccagc ugccuccugc cuacaccaac agcuucacca gaggcgugua cuaccccgac 120
aagguguuca gaagcagcgu gcugcacagc acccaggacc uguuccugcc cuucuucagc 180
aacgugaccu gguuccacgc cauccacgug ucuggcacca auggcaccaa gagguucgac 240
aacccugugc 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 ccggguguac 480
agcagcgcca acaacugcac cuucgaguac gugagccagc ccuuccugau ggaccuggag 540
ggcaagcagg gcaacuucaa gaaccugcgg gaguucgugu ucaagaacau cgacggcuac 600
uucaagaucu acagcaagca cacccccauc aaccugguga gagaccugcc ucagggcuuu 660
ucugcccugg agccucuggu ggaccugccu aucggcauca acaucaccag guuccagacc 720
cugcuggccc ugcacagaag cuaccugaca ccuggcgaua gcucuucugg cuggacagcu 780
ggagcugcug ccuauuacgu gggcuaccug cagcccagga ccuuccugcu gaaguacaac 840
gagaacggca ccaucaccga cgccguggau ugugcccugg auccucugag cgagaccaag 900
ugcacccuga agagcuucac cguggagaag ggcaucuacc agaccagcaa cuuccgggug 960
cagccuaccg agagcaucgu gagguucccc aacaucacca accugugccc uuucggcgag 1020
guguucaacg ccaccagauu cgccucugug uacgccugga acaggaagcg gaucagcaac 1080
ugcguggccg acuacagcgu gcuguacaac agcgccagcu ucagcaccuu caagugcuac 1140
ggcgugagcc cuaccaagcu gaacgaccug ugcuucacca acguguacgc cgacagcuuc 1200
gugaucagag gcgaugaggu gagacagauc gccccuggac agaccggcaa gaucgccgac 1260
uacaacuaca agcugcccga cgacuucacc ggcuguguga ucgccuggaa cagcaacaac 1320
cuggacagca aggugggcgg caacuacaac uaccuguacc ggcuguuccg gaagagcaac 1380
cugaagcccu ucgagaggga caucagcacc gagaucuacc aggccggaag cacaccuugc 1440
aauggcgugg agggcuucaa cugcuacuuc ccccugcaga gcuacggcuu ucagccuacc 1500
aauggcgugg gcuaccagcc cuacagagug guggugcuga gcuuugaacu gcugcaugcc 1560
ccugccacag uguguggccc caagaagagc accaaccugg ugaagaacaa gugcgugaac 1620
uucaacuuca acggccugac cggcacaggc gugcugaccg agagcaacaa gaaguuccug 1680
cccuuccagc aguucggcag agacaucgcc gauaccaccg augccgugag agauccucag 1740
acccuggaga uccuggacau caccccuugc agcuuuggcg gagugagcgu gaucacaccu 1800
ggcaccaaca ccagcaauca gguggccgug cuguaccagg acgugaacug cacagaggug 1860
ccuguggcca uucaugccga ucagcugacc ccuaccugga gaguguacag caccggcagc 1920
aauguguucc agaccagagc cggcugucug aucggagccg agcacgugaa caacagcuac 1980
gagugcgaca ucccuaucgg agccggcauc ugcgccucuu accagacaca gaccaacagc 2040
cccagaagag ccagaagcgu ggccagccag ucuaucaucg ccuacaccau gagccuggga 2100
gccgagaaca gcguggccua cagcaacaac agcaucgcca uccccaccaa cuucaccauc 2160
agcgugacca ccgagauccu gcccgugagc augaccaaga ccagcgugga cugcaccaug 2220
uacaucugcg gcgacagcac agagugcagc aaccugcugc ugcaguacgg cagcuuuugc 2280
acccagcuga acagagcccu gacaggcauu gccguggagc aggacaagaa cacccaggag 2340
guguucgccc aggugaagca gaucuacaag acccccccca ucaaggacuu cggcggcuuc 2400
aacuucagcc agauccugcc ugacccuagc aagcccagca agcggagcuu caucgaggac 2460
cugcuguuca acaaggugac ccuggccgau gccggcuuca ucaagcagua cggcgauugu 2520
cugggcgaua ucgccgccag agaccugauc ugugcccaga aguucaacgg ccugaccgug 2580
cugccuccuc ugcugacaga ugagaugauc gcccaguaca ccucugcccu gcuggccgga 2640
accaucacau cuggcuggac auuuggagcu ggagccgccc ugcagauccc uuucgccaug 2700
cagauggccu acagguucaa cggcaucggc gugacccaga acgugcugua cgagaaccag 2760
aagcugaucg ccaaccaguu caacagcgcc aucggcaaga uccaggacag ccugucuagc 2820
acagccucug cucugggcaa gcugcaggau guggugaacc agaaugccca ggcccugaac 2880
acccugguga agcagcugag cagcaauuuc ggcgccauca gcagcgugcu gaacgacauc 2940
cugagcagac uggacaaggu ggaggccgag gugcagaucg acagacugau caccggcaga 3000
cugcagagcc ugcagaccua cgugacacag cagcugauca gagccgccga gaucagagcc 3060
ucugccaauc uggcugccac caagaugagc gagugugugc ugggccagag caagagagug 3120
gacuucugcg gcaaaggcua ccaccugaug agcuuccccc agucugcucc ucauggcgug 3180
guguuucugc acgugaccua cgugccugcc caggagaaga acuucaccac agccccugcc 3240
aucugucacg auggcaaggc ccacuucccu agagagggcg uguucgugag caauggcacc 3300
cacugguucg ugacccagcg gaacuucuac gagccccaga ucaucaccac cgacaacacc 3360
uucgugagcg gcaacugcga cguggugauc ggcaucguga acaacaccgu guacgacccu 3420
cugcagcccg agcuggacag cuucaaggag gagcuggaca aguacuucaa gaaccacacc 3480
agccccgacg uggaucuggg cgacaucagc ggcaucaacg ccagcguggu gaacauccag 3540
aaggagaucg accggcugaa cgagguggcc aagaaccuga acgagagccu gaucgaccug 3600
caggagcugg gcaaguacga gcaguacauc aaguggcccu gguacaucug gcugggcuuu 3660
aucgccggcc ugaucgccau cgugauggug accaucaugc ugugcugcau gaccagcugc 3720
ugcagcugcc ugaagggcug uuguagcugu ggcagcugcu gcaaguucga cgaggacgau 3780
agcgagccug ugcugaaggg cgugaagcug cacuacacc 3819
<210> 34
<211> 3819
<212> RNA
<213> Artificial Sequence
<220>
<223> SDC-60 mRNA
<400> 34
auguucgugu uccuggugcu gcugccccug gugagcagcc agugugugaa ccugaccacc 60
agaacccagc ugccucccgc cuacaccaac agcuucacca ggggcgugua cuaccccgac 120
aagguguuca ggagcagcgu gcugcacagc acccaggacc uguuccugcc cuucuucagc 180
aacgugaccu gguuccacgc cauccacgug agcggcacca auggcaccaa gcgguucgac 240
aacccugugc ugcccuucaa cgacggcgug uacuucgcca gcaccgagaa gagcaacauc 300
auccggggcu ggaucuucgg caccacccug gacagcaaga cccagagccu gcugaucgug 360
aacaacgcca ccaacguggu gaucaaggug ugcgaguucc aguucugcaa cgaccccuuc 420
cugggcgugu acuaccacaa gaacaacaag agcuggaugg agagcgaguu ccggguguac 480
agcagcgcca acaacugcac cuucgaguac gugagccagc ccuuccugau ggaccuggag 540
ggcaagcagg gcaacuucaa gaaccugcgg gaguucgugu ucaagaacau cgacggcuac 600
uucaagaucu acagcaagca cacccccauc aaccugguga gggaccugcc ucagggcuuu 660
ucugcccugg agccucuggu ggaccugccc aucggcauca acaucaccag guuccagacc 720
cugcuggccc ugcacaggag cuaccugaca ccuggcgaua gcucuucugg cuggacagcc 780
ggagcugcug ccuacuacgu gggcuaccug cagccccgga ccuuccugcu gaaguacaac 840
gagaacggca ccaucaccga cgccguggau ugcgcccugg auccucugag cgagaccaag 900
ugcacccuga agagcuucac cguggagaag ggcaucuacc agaccagcaa cuuccgggug 960
cagcccaccg agagcaucgu gagguucccc aacaucacca accugugccc cuucggcgag 1020
guguucaacg ccaccagauu cgccagcgug uacgccugga accggaagcg gaucagcaac 1080
ugcguggccg acuacagcgu gcuguacaac agcgccagcu ucagcaccuu caagugcuac 1140
ggcgugagcc ccaccaagcu gaacgaccug ugcuucacca acguguacgc cgacagcuuc 1200
gugaucaggg gcgaugaggu gagacagauc gccccuggcc agaccggcaa gaucgccgac 1260
uacaacuaca agcugcccga cgacuucacc ggcugcguga ucgccuggaa cagcaacaac 1320
cuggacagca aggugggcgg caacuacaac uaccuguacc ggcuguuccg gaagagcaac 1380
cugaagcccu ucgagcggga caucagcacc gagaucuacc aggccggaag caccccuugc 1440
aacggcgugg agggcuucaa cugcuacuuc ccccugcaga gcuacggcuu ccagccuacc 1500
aauggcgugg gcuaccagcc cuacagggug guggugcuga gcuuugagcu gcugcaugcu 1560
ccugccaccg ugugcggccc caagaagagc accaaccugg ugaagaacaa gugcgugaac 1620
uucaacuuca acggccugac cggcaccggc gugcugaccg agagcaacaa gaaguuccug 1680
cccuuccagc aguucggcag ggacaucgcc gauaccaccg augccgugag agacccucag 1740
acccuggaga uccuggacau caccccuugc agcuucggcg gagugagcgu gaucacaccu 1800
ggcaccaaca ccagcaacca gguggccgug cuguaccagg acgugaacug caccgaggug 1860
ccuguggcca uucacgccga ucagcugacc cccaccugga gaguguacag caccggcagc 1920
aacguguucc agaccagagc cggcugucug aucggcgccg agcacgugaa caacagcuac 1980
gagugcgaca uccccaucgg cgccggcauc ugugccagcu aucagaccca gaccaacagc 2040
ccuaggaggg ccagaagcgu ggccagccag ucuaucaucg ccuacaccau gagccugggc 2100
gccgagaaca gcguggccua cagcaacaac agcaucgcca uccccaccaa cuucaccauc 2160
agcgugacca ccgagauccu gcccgugagc augaccaaga ccagcgugga cugcaccaug 2220
uacaucugcg gcgacagcac cgagugcagc aaccugcugc ugcaguacgg cagcuucugc 2280
acccagcuga acagagcccu gacaggcauc gccguggagc aggacaagaa cacccaggag 2340
guguucgccc aggugaagca gaucuacaag acccccccca ucaaggacuu cggcggcuuc 2400
aacuucagcc agauccugcc ugaccccagc aagcccagca agcggagcuu caucgaggac 2460
cugcuguuca acaaggugac ccuggccgac gccggcuuca ucaagcagua cggcgacugu 2520
cugggcgaca ucgccgccag agaccugauc ugugcccaga aguucaacgg ccugaccgug 2580
cugcccccuc ugcugaccga ugagaugauc gcccaguaca ccucugcccu gcuggccggc 2640
accaucacau cuggcuggac cuuuggagcu ggagccgccc ugcagauccc uuucgccaug 2700
cagauggccu accgguucaa cggcaucggc gugacccaga acgugcugua cgagaaccag 2760
aagcugaucg ccaaccaguu caacagcgcc aucggcaaga uccaggacag ccugagcagc 2820
accgccucug cucugggcaa acugcaggac guggugaacc agaacgccca ggcccugaac 2880
acccugguga agcagcugag cagcaacuuc ggcgccauca gcagcgugcu gaacgacauc 2940
cugagcaggc uggacaaggu ggaggccgag gugcagaucg acaggcugau caccggcaga 3000
cugcagagcc ugcagaccua cgugacccag cagcugauca gagccgccga gaucagagcc 3060
ucugccaauc uggccgccac caagaugagc gagugugugc ugggccagag caagagggug 3120
gacuucugcg gcaagggcua ccaccugaug agcuuccccc agucugcccc ucauggcgug 3180
guguuccugc acgugaccua cgugccugcc caggagaaga acuucaccac cgccccugcc 3240
aucugccacg auggcaaggc ccacuucccu agagagggcg uguucgugag caacggcacc 3300
cacugguucg ugacccagcg gaacuucuac gagccccaga ucaucaccac cgacaacacc 3360
uucgugagcg gcaacugcga cguggugauc ggcaucguga acaacaccgu guacgacccc 3420
cugcagcccg agcuggacag cuucaaggag gagcuggaca aguacuucaa gaaccacacc 3480
agccccgacg uggaccuggg cgacaucagc ggcaucaacg ccagcguggu gaacauccag 3540
aaggagaucg accggcugaa cgagguggcc aagaaccuga acgagagccu gaucgaccug 3600
caggagcugg gcaaguacga gcaguacauc aaguggcccu gguacaucug gcugggcuuc 3660
aucgccggcc ugaucgccau cgugauggug accaucaugc ugugcugcau gaccagcugc 3720
ugcagcugcc ugaagggcug cugcagcugu ggcagcuguu gcaaguucga cgaggacgac 3780
agcgagcccg ugcugaaggg cgugaagcug cacuacacc 3819
<210> 35
<211> 957
<212> RNA
<213> Artificial Sequence
<220>
<223> MT2AE mRNA
<400> 35
auggccgauu cuaauggcac caucaccgug gaagagcuga agaagcugcu cgagcaaugg 60
aaccugguga ucggauuucu guuccugacc uggaucuguc uguugcaguu cgccuacgcc 120
aaccggaaca gauuccugua caucaucaaa cugaucuucc uguggcugcu guggccugug 180
acccuggccu gcuucgugcu ggccgccgug uaccggauua acuggaucac cggaggcauc 240
gcuaucgcca uggcaugccu ggucggacuu auguggcugu cuuauuucau cgccagcuuc 300
agacuguucg cuagaaccag aagcaugugg uccuuuaacc cugagacaaa cauccugcug 360
aacgugccuc ugcacggcac aauccugaca cggccacugc uggaaagcga gcuggucauc 420
ggcgccguga uccugcgggg ccaucugcgc auugccggac accaccuggg cagaugcgac 480
aucaaggacc ugcccaagga aaucaccgug gccaccagca gaacacuguc cuacuacaaa 540
cugggcgcua gucagagagu ggccggcgac agcggcuucg ccgcuuauuc uagauacaga 600
aucggcaacu acaagcugaa uaccgaucac agcagcagca gcgacaacau cgcccugcug 660
gugcagggca gcggcgaggg cagaggaagc cugcugacau guggcgaugu ggaagagaac 720
cccggcccug ccauguacag cuuugugucu gaggaaaccg gcacccugau cgugaacagc 780
gugcugcugu uucuggccuu cgucguguuc cugcugguga cacuggcuau ccugaccgcc 840
cugaggcugu gcgccuacug cugcaacauc gugaauguau cccuggugaa gccuuccuuc 900
uacguguaca gccgggugaa gaaccuuaau agcucuagag ugcccgaccu gcucguu 957
<210> 36
<211> 960
<212> RNA
<213> Artificial Sequence
<220>
<223> MP2AE mRNA
<400> 36
auggccgaca gcaacggcac aaucacagug gaagagcuga agaagcugcu ggagcagugg 60
aaccugguga uuggauuucu uuuccucacc uggaucugcc ugcugcaguu cgccuaugcc 120
aaccggaaca gauuccugua caucaucaag cugaucuucc uguggcugcu guggcccgug 180
acccuggccu guuuugugcu ggccgccgug uaccggauca acuggaucac cggcggaauc 240
gcuaucgcca uggccugccu ggugggccug auguggcuga gcuacuucau cgccuccuuu 300
agacuguucg ccagaaccag aagcaugugg uccuucaacc cugagacaaa uauccugcuc 360
aacgugcccc ugcacggcac cauccugacc cggccucugc ucgagagcga gcuggugauc 420
ggcgccguga uccugagagg ccaccugaga aucgccggac accaccuggg cagaugcgac 480
aucaaggacc ugccaaagga aaucaccguu gcuacaagca gaacacuguc cuacuacaag 540
cugggcgcuu cucaaagagu cgccggcgac agcggcuucg cugcuuauag ccgcuacagg 600
auuggaaauu acaagcugaa caccgaucau ucuucuagca gcgacaacau cgcccugcug 660
guccagggca gcggcgccac aaacuucagc cugcuuaaac aggccggcga uguggaagag 720
aaccccggcc cugccaugua cagcuucgug uccgaggaaa ccggcacccu gaucgugaac 780
agcgugcugc uguuccuugc uuuuguggug uuccugcugg ucacccuggc cauccugacc 840
gcccugagac ugugugccua cugcugcaac aucgugaaug ugucucuggu gaagccuagc 900
uucuacgugu acagccgggu gaaaaaccug aacucuagcc gggugccuga ucugcuggug 960
<210> 37
<211> 798
<212> RNA
<213> Artificial Sequence
<220>
<223> SGS-RBD mRNA
<400> 37
auggagacag acacacuccu gcuaugggua cugcugcucu ggguuccagg uuccaccgga 60
gacugcccau uuggcgaggu guucaacgca acccgcuucg ccagcgugua cgccuggaau 120
aggaagcgga ucagcaacug cguggccgac uauagcgugc uguacaacuc cgccucuuuc 180
agcaccuuua agugcuaugg cguguccccc acaaagcuga augaccugug cuuuaccaac 240
gucuacgccg auucuuucgu gaucaggggc gacgaggugc gccagaucgc ccccggccag 300
acaggcaaga ucgcagacua caauuauaag cugccagacg auuucaccgg cugcgugauc 360
gccuggaaca gcaacaaucu ggauuccaaa gugggcggca acuacaauua ucuguaccgg 420
cuguuuagaa agagcaaucu gaagcccuuc gagagggaca ucucuacaga aaucuaccag 480
gccggcagca ccccuugcaa uggcguggag ggcuuuaacu guuauuuccc acuccagucc 540
uacggcuucc agcccacaaa cggcgugggc uaucagccuu accgcguggu ggugcugagc 600
uuugagcugc ugcacgccua cccguacgac gugccggacu acgccaaugc ugugggccag 660
gacacgcagg aggucaucgu ggugccacac uccuugcccu uuaagguggu ggugaucuca 720
gccauccugg cccugguggu gcucaccauc aucucccuua ucauccucau caugcuuugg 780
cagaagaagc cacguuag 798
<210> 38
<211> 66
<212> DNA
<213> Artificial Sequence
<220>
<223> T2A DNA
<400> 38
ggcagcggcg agggcagagg aagcctgctg acatgtggcg atgtggaaga gaaccccggc 60
cctgcc 66
<210> 39
<211> 69
<212> DNA
<213> Artificial Sequence
<220>
<223> P2A DNA
<400> 39
ggcagcggcg ccacaaactt cagcctgctt aaacaggccg gcgatgtgga agagaacccc 60
ggccctgcc 69
<210> 40
<211> 66
<212> RNA
<213> Artificial Sequence
<220>
<223> T2A mRNA
<400> 40
ggcagcggcg agggcagagg aagccugcug acauguggcg auguggaaga gaaccccggc 60
ccugcc 66
<210> 41
<211> 69
<212> RNA
<213> Artificial Sequence
<220>
<223> P2A mRNA
<400> 41
ggcagcggcg ccacaaacuu cagccugcuu aaacaggccg gcgaugugga agagaacccc 60
ggcccugcc 69
<210> 42
<211> 22
<212> PRT
<213> thosea asigna virus 2A
<400> 42
Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu
1 5 10 15
Glu Asn Pro Gly Pro Ala
20
<210> 43
<211> 23
<212> PRT
<213> porcine teschovirus-1 2A
<400> 43
Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val
1 5 10 15
Glu Glu Asn Pro Gly Pro Ala
20

Claims (18)

1.mRNA，其特征在于，其包含编码来源于SARS-CoV-2病毒的S蛋白的mRNA，且编码所述S蛋白的mRNA的编码区序列如SEQ ID NO: 18所示；
或，其包含编码来源于SARS-CoV-2病毒的S蛋白、E蛋白和M蛋白的mRNA，且编码所述S蛋白的mRNA的编码区序列如SEQ ID NO: 18所示；编码所述E蛋白的mRNA的编码区序列如SEQID NO: 21所示；编码所述M蛋白的mRNA的编码区序列如SEQ ID NO: 22所示；所述mRNA还包含以下(a)~(e)：
(a) 5’-帽结构，为m7G(5')ppp(5')(2'OMeA)pG；
(b) 3’-聚腺苷酸，其序列包含120个聚腺苷酸组成的序列；
(c) 5’-UTR，所述5’-UTR的序列如SEQ ID NO: 15所示；
(d) 3’-UTR，所述3’-UTR的序列如SEQ ID NO: 16或SEQ ID NO: 17所示；
(e) 多核苷酸修饰，所述多核苷酸采用pseudo-UTP。
2.如权利要求1所述的mRNA，其特征在于，所述S蛋白、E蛋白、M蛋白由三条独立的mRNA分别表达得到，表达所述S蛋白、E蛋白、M蛋白的mRNA的摩尔比为1:1:1。
3.如权利要求1或2所述的mRNA，其特征在于，所述mRNA编码的蛋白自组装成病毒样颗粒。
4. DNA，其特征在于，其包含编码来源于SARS-CoV-2病毒的S蛋白、E蛋白和M蛋白中的至少一种蛋白的DNA，其中，编码所述S蛋白的DNA的序列如SEQ ID NO: 3所示；编码所述E蛋白的DNA的序列如SEQ ID NO: 8所示；编码所述M蛋白的DNA的序列如SEQ ID NO: 11所示。
5.一种组合物，其特征在于，其包含如权利要求1~3任一项所述的mRNA。
6.一种脂质体纳米颗粒，其特征在于，其包含如权利要求1~3任一项所述的mRNA和/或如权利要求5所述的组合物。
7.如权利要求6所述的脂质体纳米颗粒，其特征在于，所述脂质体纳米颗粒还包括阳离子脂质和辅助脂质，和/或，所述脂质体纳米颗粒为长循环阳离子脂质体纳米颗粒。
8.如权利要求7所述的脂质体纳米颗粒，其特征在于，所述阳离子脂质为DLin-MC3-DMA或DOTMA。
9.如权利要求7或8所述的脂质体纳米颗粒，其特征在于，所述辅助脂质为DSPC和/或胆固醇。
10.如权利要求7所述的脂质体纳米颗粒，其特征在于，所述长循环阳离子脂质体纳米颗粒为经PEG或其衍生物修饰的长循环阳离子脂质体纳米颗粒。
11.如权利要求10所述的脂质体纳米颗粒，其特征在于，所述PEG的相对分子质量为2000~5000。
12.如权利要求11所述的脂质体纳米颗粒，其特征在于，所述长循环阳离子脂质体纳米颗粒为包括DMPE-PEG2000的长循环阳离子脂质体纳米颗粒。
13.一种病毒样颗粒，其由包含如权利要求1~3任一项所述的mRNA和/或如权利要求5所述的组合物所表达的蛋白自组装而成，并且在细胞中表达所述蛋白。
14.如权利要求13所述的病毒样颗粒，其特征在于，所述细胞为293T和/或293A。
15.一种针对新冠病毒的mRNA疫苗，其特征在于，其包含如权利要求1~3任一项所述的mRNA、如权利要求5所述的组合物和/或如权利要6~12任一项所述的脂质体纳米颗粒。
16.如权利要求15所述的mRNA疫苗，其特征在于，所述mRNA疫苗诱导细胞产生病毒样颗粒；和/或，所述mRNA疫苗还包括佐剂。
17.一种药物组合物，其包含如权利要求1~3任一项所述的mRNA、如权利要求5所述的组合物、如权利要求6~12任一项所述的脂质体纳米颗粒、如权利要求13或14所述的病毒样颗粒和/或如权利要求15或16所述的mRNA疫苗，和任选地药用载体。
18.一种试剂盒，其包含如权利要求1~3任一项所述的mRNA、如权利要求4所述的DNA、如权利要求5所述的组合物、如权利要求6~12任一项所述的脂质体纳米颗粒、如权利要求13或14所述的病毒样颗粒、如权利要求15或16所述的mRNA疫苗和/或如权利要求17所述的药物组合物。
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