CN114315986A - 新型冠状病毒mRNA疫苗及其制备方法与应用 - Google Patents

新型冠状病毒mRNA疫苗及其制备方法与应用 Download PDF

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CN114315986A
CN114315986A CN202011056131.3A CN202011056131A CN114315986A CN 114315986 A CN114315986 A CN 114315986A CN 202011056131 A CN202011056131 A CN 202011056131A CN 114315986 A CN114315986 A CN 114315986A
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胡勇
刘陈立
李楠
张昊
艾亮霞
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Shenzhen Ruiji Biotechnology Co ltd
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Abstract

本发明提供了一种新型冠状病毒mRNA疫苗及其制备方法与应用。本发明提供了如下(a)或(b)的蛋白：(a)SEQ ID No.1所示氨基酸序列组成的蛋白；(b)由SEQ ID No.1所示氨基酸序列经过取代、缺失和/或添加一个或几个氨基酸且与SEQ ID No.1所示氨基酸序列组成的蛋白具有相同功能的衍生蛋白。本发明设计了新型冠状病毒(SARS‑CoV‑2)中融合前构象的S蛋白序列，实现了人源细胞高效表达增加免疫源性。本发明的mRNA可实现对新型冠状病毒的免疫，适用性广。

Description

新型冠状病毒mRNA疫苗及其制备方法与应用
技术领域
本发明属于基因工程技术领域，具体而言是关于一种新型冠状病毒(SARS-CoV-2)mRNA疫苗抗原设计、疫苗及其制备方法与应用。
背景技术
冠状病毒(Coronavirus)是自然界广泛存在的一类RNA病毒，因该病毒形态在电镜下观察类似王冠而得名。冠状病毒是一个成员众多的大家族，仅感染脊椎动物，可引起人和动物呼吸道、消化道和神经系统疾病。新型冠状病毒(SARS-CoV-2)是2019年发现的一种新型冠状病毒。在发现SARS-CoV-2之前，已知感染人的冠状病毒有6种，其中4种在人群中较为常见，致病性较低，一般仅引起类似普通感冒的轻微呼吸道症状；而另外两种病毒，SARS冠状病毒和MERS冠状病毒，致病性较高。SARS-CoV-2传染性强，可造成肺部炎症，严重的可引发呼吸衰竭，进而死亡。
新型冠状病毒的结构蛋白由刺突(S)、包膜(E)、膜(M)和核衣壳(N)蛋白组成。其中S蛋白以三聚体的形式呈现在病毒表面，形成“冠状”刺突结构，并介导病毒与细胞受体结合及膜融合。S蛋白具有很高的免疫原性，可以激活T细胞免疫应答，并能够诱导中和抗体的产生。S蛋白是目前冠状病毒疫苗和药物研发的重要靶点。已有多项研究表明针对SARS-CoV和MERS-CoV S蛋白研发的疫苗可提供良好的免疫保护。相对于其他新冠病毒结构蛋白，S蛋白分子量较大，细胞外泌难度较大，通过结合高效外泌型信号肽能有效提高S蛋白外泌效率。同时，经过序列优化后能提高S蛋白在细胞中的表达量，增强有效抗原的表达。此外，通过在S蛋白C端插入Foldon三聚化结构域获得翻译产物为融合前构象的S蛋白编码序列。融合前构象的S蛋白对应的mRNA序列，可以在体内诱导高滴度的中和抗体，安全性更高。
mRNA疫苗技术可以作为快速灵活的技术平台，有效应对各种新兴病毒的威胁。因此mRNA疫苗被认为是对抗新型冠状病毒最有潜力的一种疫苗。传统的灭活疫苗、减毒疫苗以及多肽疫苗，研发周期长，生产工艺复杂，难以满足突发公共卫生事件的需求。此外，与DNA疫苗相比，mRNA疫苗有着明显优势。首先，mRNA疫苗比DNA疫苗药效更加明显。DNA疫苗需要穿过细胞膜和核膜才能表达成抗原，因此当没有足够剂量的DNA到达细胞核时，疫苗将无法激活体内免疫系统以及诱导抗体的产生。而mRNA只需要穿过细胞膜在细胞质中就可以高效表达抗原蛋白。其次，mRNA疫苗比DNA疫苗生物安全性更高。外源DNA有一定几率整合到人体基因组中，因此DNA疫苗有引起基因组突变的风险。而mRNA没有基因整合到基因组中的风险。其次，mRNA被翻译成蛋白质后易被降解，其瞬时表达的特性不仅确保了mRNA药物的安全性，而且使其剂量可控，避免了疫苗药物长期暴露引起的抗原免疫耐受(对特定抗原无反应的状态)。另外，由于不需要动物来源的病毒参与到疫苗研发过程中，规避了病毒和动物使用风险，同时可以短期内获得有效抗原。
总体而言，跟传统的疫苗相比，mRNA疫苗生产周期短，生产工序更简单，成本更低。mRNA疫苗从抗原序列设计到生产只需要6周时间，而传统疫苗的生产最快也要5-6个月。mRNA疫苗可以冻干并在室温下保存6-18个月，而传统的疫苗需要保存在2-8℃。因此，mRNA疫苗保存时间更久，不需要冷链，运输成本更低。传统疫苗对很多新型病毒有心无力，而mRNA疫苗适用性更广，序列可以灵活设计，以应对不同的病原体。
发明内容
本发明的一个目的在于提供针对新型冠状病毒(SARS-CoV-2)的mRNA疫苗的抗原。
本发明的另一目的在于提供针对新型冠状病毒(SARS-CoV-2)的疫苗。
本发明的另一目的在于提供针对新型冠状病毒(SARS-CoV-2)的mRNA疫苗的制备方法。
本发明的另一目的在于提供用于制备针对新型冠状病毒(SARS-CoV-2)的疫苗的mRNA。
本发明的另一目的在于提供用于制备所述针对新型冠状病毒(SARS-CoV-2)的mRNA的DNA。
本发明的另一目的在于提供用于制备所述针对新型冠状病毒(SARS-CoV-2)的mRNA的载体。
本发明的另一目的在于提供针对新型冠状病毒(SARS-CoV-2)的mRNA疫苗的应用。
新型冠状病毒(SARS-CoV-2)呈现球形椭球形，直径在80-120nm。在电子显微镜下，病毒粒表面有球棒状的突出部分，由三聚体刺突糖蛋白(Spike，S)组成。病毒的包膜由膜糖蛋白(membrane glycoprotein，M)构成，通过三个跨膜结构域嵌入病毒包膜里中。此外，少量的小的跨膜蛋白-包膜(envelope，E)蛋白也出现在包膜中。最后，核衣壳(nucleocapsid，N)蛋白以串珠的形式结合到RNA基因组上，形成螺旋对称的核衣壳。研究结果显示，S蛋白是冠状病毒引起机体免疫反应的主要成分。新型冠状病毒能利用一种紧密糖基化的、同源三聚体I类融合凸起蛋白(S蛋白)来进入宿主细胞中，S蛋白能以一种相对稳定的融合前构象存在，并经历剧烈的结构重排来促进病毒膜结构与宿主细胞膜进行融合，这一过程是由病毒S1亚单位与宿主受体的结合所诱发，其能破坏融合前三聚体的稳定性，从而导致S1亚单位脱落，并促使S2亚单位转变为高度稳定的融合后构象。为了能与宿主受体相结合，S1的受体结合结构域会经历链状样的构象运动，这种运动方式会暂时隐藏或暴露受体结合的决定因素，这两种状态被认为是“向上”或“向下”构象，其中向下的构象对应的是受体不可及的状态，而向上构象则对应着受体可达的状态，后者被认为具有不稳定。
本发明中，为提供针对新型冠状病毒(SARS-CoV-2)的mRNA疫苗，在野生型S蛋白的基础上对其氨基酸序列进行了改造，设计了mRNA编码的病毒抗原片段，然后合成编码病毒抗原片段的mRNA，以实现对新型冠状病毒的免疫。具体而言，本发明采用基因工程技术来使S稳定在其融合前构象中，防止结构重排，并暴露出抗原性较佳的抗原表位可引起更高的中和抗体应答。通过生物信息学和基因工程学方法，本发明分别将S蛋白编码序列进行改造使其翻译产物稳定在融合前构象。相关抗原序列通过基因工程技术克隆到质粒中用于mRNA合成。
一方面，本发明提供了如下(a)或(b)的蛋白：
(a)SEQ ID No.1所示氨基酸序列组成的蛋白；
(b)由SEQ ID No.1所示氨基酸序列经过取代、缺失和/或添加一个或几个氨基酸且与SEQ ID No.1所示氨基酸序列组成的蛋白具有相同功能的衍生蛋白。
SEQ ID No.1所示氨基酸序列组成的蛋白，为本发明的在野生型S蛋白基础上修饰改进后的蛋白，本发明中亦称其为融合前构型S蛋白。本发明根据新型冠状病毒(SARS-CoV-2)的基因组测序结果，设计并优化S序列，使得所表达出的S蛋白处于融合前构型，以实现人源细胞高效表达增加免疫源性。与野生型S蛋白氨基酸序列相比，本发明的融合前构型S蛋白更易诱导中和抗体的产生。
根据本发明的具体实施方案，本发明中，所述由SEQ ID No.1所示氨基酸序列经过取代、缺失和/或添加一个或几个氨基酸且与SEQ ID No.1所示氨基酸序列组成的蛋白具有相同功能的衍生蛋白，至少包含SEQ ID No.1所示氨基酸序列C末端连续至少15个氨基酸的序列。
根据本发明的具体实施方案，所述与SEQ ID No.1所示氨基酸序列组成的蛋白具有相同功能是指与SEQ ID No.1所示氨基酸序列组成的蛋白具有“更易诱导中和抗体的产生”和/或“增加免疫源性”(与野生型S蛋白相比)的功能。
另一方面，本发明还提供了所述的改进的S蛋白(融合前构型S蛋白和/或其衍生蛋白)作为抗原在制备疫苗中的应用。
另一方面，本发明还提供了编码本发明所述的改进的S蛋白的DNA。根据本发明的优选具体实施方案，所述DNA的核苷酸序列包括SEQ ID No.10、SEQ ID No.11、SEQ IDNo.12或SEQ ID No.13所示序列。
另一方面，本发明还提供了一种表达载体，其中含有本发明的所述的DNA(编码本发明所述的改进的S蛋白的DNA)。
另一方面，本发明还提供了编码本发明所述的改进的S蛋白的mRNA。
根据本发明的优选具体实施方案，所述mRNA是本发明前述的DNA进行体外转录得到。
根据本发明的优选具体实施方案，本发明的mRNA，其核苷酸序列包括SEQ IDNo.2、SEQ ID No.3、SEQ ID No.4或SEQ ID No.5所示序列。
根据本发明的优选具体实施方案，本发明的mRNA，其核苷酸序列5’端还可包括佐剂序列。例如，所述佐剂序列可以为促进蛋白分泌表达的核苷酸序列。根据本发明的优选实施方案，所述佐剂序列包括SEQ ID No.6、SEQ ID No.7、SEQ ID No.8或SEQ ID No.9所示序列。根据本发明的更具体实施方案，所述的mRNA包括SEQ ID No.6、SEQ ID No.7、SEQ IDNo.8或SEQ ID No.9所示序列分别与SEQ ID No.2、SEQ ID No.3、SEQ ID No.4或SEQ IDNo.5组合序列所示的mRNA(SEQ ID No.6、SEQ ID No.7、SEQ ID No.8或SEQ ID No.9任一所示序列位于SEQ ID No.2、SEQ ID No.3、SEQ ID No.4或SEQ ID No.5任一所示序列的5’端)。根据本发明的更优选的实施方案，所述的mRNA包括SEQ ID No.6、SEQ ID No.7、SEQ IDNo.8或SEQ ID No.9所示序列与SEQ ID No.4组合序列所示的mRNA。
另一方面，本发明还提供了所述mRNA的制备方法。
根据本发明的具体实施方案，本发明的所述mRNA的制备方法包括：
编码病毒抗原片段mRNA的合成：合成编码融合前构象S蛋白的DNA片段(可自行或商业化合成)，并克隆到质粒作为模板进行体外转录(in vitro transcription,IVT)得到未加帽的mRNA；
mRNA的加帽：在加帽酶催化下，以给未加帽的mRNA的5'端加上帽子结构，得到加帽的mRNA分子；
mRNA的纯化：将所述加帽的mRNA进行氯化锂/乙醇沉淀、离心柱、氯萃取/乙醇沉淀或凝胶纯化，以获得纯化后的mRNA。
另一方面，本发明还提供了所述的mRNA抗原设计在制备新型冠状病毒mRNA疫苗中的应用。
另一方面，本发明还提供了一种疫苗，其中包括本发明所述的mRNA中的一种或多种。
根据本发明的具体实施方案，本发明的疫苗是针对新型冠状病毒(SARS-CoV-2)的预防性疫苗，其中还可包括用于溶解mRNA的PBS溶液。
本发明的有益效果：
本发明的技术，是先通过生物信息学方法设计新型冠状病毒(SARS-CoV-2)中融合前构象的S蛋白序列，以实现人源细胞高效表达增加免疫源性；然后通过体外转录方法合成编码病毒抗原片段的mRNA，以实现对新型冠状病毒的免疫。整个mRNA疫苗生产周期短、工艺操作简单、生产成本低，保存时间久，不需要冷链，便于运输。传统疫苗对很多新型病毒引起的公共卫生事件无法做到快速响应，而mRNA疫苗适用性更广，序列可以灵活设计，以应对不同的病原体，针对急性传染病疫苗快速研发有及其重要的作用。
附图说明
图1为本发明的mRNA疫苗示意图。
图2显示本发明实施例1中经序列优化后的编码融合前构型S蛋白抗原片段的不同mRNA疫苗表达结果。其中，M：蛋白marker，1#：SEQ ID No.2编码的融合前构型S蛋白，2#：SEQID No.3编码的融合前构型S蛋白，3#：SEQ ID No.4编码的融合前构型S(prefusion)蛋白，4#：SEQ ID No.5编码的融合前构型S(prefusion)蛋白。
图3显示本发明实施例2中带有不同佐剂序列(SEQ ID No.6-9)的mRNA(SEQ IDNo.4)分别转染293T细胞24小时后细胞外泌结果。其中，Control：对照组mRNA(SEQ IDNo.4)，1#：带有SEQ ID No.6佐剂序列的mRNA(SEQ ID No.4)，2#：带有SEQ ID No.7佐剂序列的mRNA(SEQ ID No.4)，3#：带有SEQ ID No.8佐剂序列的mRNA(SEQ ID No.4)，4#：带有SEQ ID No.9佐剂序列的mRNA(SEQ ID No.4)。
图4显示本发明实施例3中不同佐剂与实施例2中优选方案mRNA(SEQ ID No.4)接种小鼠后S特异性抗体滴度结果。
图5显示本发明实施例4中不同佐剂与实施例2中优选方案mRNA(SEQ ID No.4)接种小鼠后中和抗体滴度结果。图中每条曲线代表一只小鼠。
具体实施方式
下面将结合本发明实施例及其附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明的一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
且下述实施例中所涉及的仪器、试剂、材料等，若无特别说明，均为现有技术中已有的常规仪器、试剂、材料等，可通过正规商业途径获得。下述实施例中所涉及的实验方法，检测方法等，若无特别说明，均为现有技术中已有的常规实验方法、检测方法等。
下述各实施例中，所用DNA片段为委托合成。所用mRNA按照以下方法制备获得：
合成编码融合前构象S蛋白的DNA片段，并克隆到质粒作为模板通过T7 RNA聚合酶进行体外转录(in vitro transcription,IVT)得到未加帽的mRNA；其中，所述的DNA片段的具体序列优选包括SEQ ID No.10、SEQ ID No.11、SEQ ID No.12、SEQ ID No.13所示序列，所述DNA片段的5’端还可根据需要还包括对应佐剂序列(优选SEQ ID No.6、SEQ ID No.7、SEQ ID No.8或SEQ ID No.9所示序列)的DNA片段；
mRNA的加帽：在加帽酶催化下，以给未加帽的mRNA的5'端加上帽子结构，得到加帽的mRNA分子；所述加帽酶包括但不限于二氧基甲基化转移酶(2'-OMethyltransferase)或者RNA三磷酸酶(triphosphatase)、RNA谷氨酰转移酶(guanyltransferase)及鸟嘌呤-7-甲基转移酶(Guanine-7-methyltransferase)中的一种或其组合；
mRNA的纯化：将所述加帽的mRNA进行氯化锂/乙醇沉淀、离心柱、氯萃取/乙醇沉淀或凝胶纯化，以获得纯化后的mRNA。
SEQ ID No.1：融合前构型S蛋白序列
MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSAYVRKDGEWVFL*
SEQ ID No.2：编码融合前构型S蛋白序列的mRNA(优化方案1)
AAG CAC AAG GAC CAC GAC GAC GGG GAC CAC UCG UCG GUC ACG CAC UUG GACUGG UGG GCC UGG GUC GAC GGG GGG CGG AUG UGG UUG UCG AAG UGG GCC CCG CAC AUGAUG GGG CUG UUC CAC AAG GCC UCG UCG CAC GAC GUG UCG UGG GUC CUG GAC AAG GACGGG AAG AAG UCG UUG CAC UGG ACC AAG GUG CGG UAG GUG CAC UCG CCG UGG UUG CCGUGG UUC GCC AAG CUG UUG GGG CAC GAC GGG AAG UUG CUG CCG CAC AUG AAG CGG UCGUGG CUC UUC UCG UUG UAG UAG GCC CCG ACC UAG AAG CCG UGG UGG GAC CUG UCG UUCUGG GUC UCG GAC GAC UAG CAC UUG UUG CGG UGG UUG CAC CAC UAG UUC CAC ACG CUCAAG GUC AAG ACG UUG CUG GGG AAG GAC CCG CAC AUG AUG GUG UUC UUG UUG UUC UCGACC UAC CUC UCG CUC AAG GCC CAC AUG UCG UCG CGG UUG UUG ACG UGG AAG CUC AUGCAC UCG GUC GGG AAG GAC UAC CUG GAC CUC CCG UUC GUC CCG UUG AAG UUC UUG GACGCC CUC AAG CAC AAG UUC UUG UAG CUG CCG AUG AAG UUC UAG AUG UCG UUC GUG UGGGGG UAG UUG GAC CAC GCC CUG GAC GGG GUC CCG AAG UCG CGG GAC CUC GGG GAC CACCUG GAC GGG UAG CCG UAG UUG UAG UGG GCC AAG GUC UGG GAC GAC CGG GAC GUG GCCUCG AUG GAC UGG GGG CCG CUG UCG UCG UCG CCG ACC UGG CGG CCG CGG CGG CGG AUGAUG CAC CCG AUG GAC GUC GGG GCC UGG AAG GAC GAC UUC AUG UUG CUC UUG CCG UGGUAG UGG CUG CGG CAC CUG ACG CGG GAC CUG GGG GAC UCG CUC UGG UUC ACG UGG GACUUC UCG AAG UGG CAC CUC UUC CCG UAG AUG GUC UGG UCG UUG AAG GCC CAC GUC GGGUGG CUC UCG UAG CAC GCC AAG GGG UUG UAG UGG UUG GAC ACG GGG AAG CCG CUC CACAAG UUG CGG UGG GCC AAG CGG UCG CAC AUG CGG ACC UUG GCC UUC GCC UAG UCG UUGACG CAC CGG CUG AUG UCG CAC GAC AUG UUG UCG CGG UCG AAG UCG UGG AAG UUC ACGAUG CCG CAC UCG GGG UGG UUC GAC UUG CUG GAC ACG AAG UGG UUG CAC AUG CGG CUGUCG AAG CAC UAG GCC CCG CUG CUC CAC GCC GUC UAG CGG GGG CCG GUC UGG CCG UUCUAG CGG CUG AUG UUG AUG UUC GAC GGG CUG CUG AAG UGG CCG ACG CAC UAG CGG ACCUUG UCG UUG UUG GAC CUG UCG UUC CAC CCG CCG UUG AUG UUG AUG GAC AUG GCC GACAAG GCC UUC UCG UUG GAC UUC GGG AAG CUC GCC CUG UAG UCG UGG CUC UAG AUG GUCCGG CCG UCG UGG GGG ACG UUG CCG CAC CUC CCG AAG UUG ACG AUG AAG GGG GAC GUCUCG AUG CCG AAG GUC GGG UGG UUG CCG CAC CCG AUG GUC GGG AUG GCC CAC CAC CACGAC UCG AAG CUC GAC GAC GUG CGG GGG CGG UGG CAC ACG CCG GGG UUC UUC UCG UGGUUG GAC CAC UUC UUG UUC ACG CAC UUG AAG UUG AAG UUG CCG GAC UGG CCG UGG CCGCAC GAC UGG CUC UCG UUG UUC UUC AAG GAC GGG AAG GUC GUC AAG CCG GCC CUG UAGCGG CUG UGG UGG CUG CGG CAC GCC CUG GGG GUC UGG GAC CUC UAG GAC CUG UAG UGGGGG ACG UCG AAG CCG CCG CAC UCG CAC UAG UGG GGG CCG UGG UUG UGG UCG UUG GUCCAC CGG CAC GAC AUG GUC CUG CAC UUG ACG UGG CUC CAC GGG CAC CGG UAG GUG CGGCUG GUC GAC UGG GGG UGG ACC GCC CAC AUG UCG UGG CCG UCG UUG CAC AAG GUC UGGGCC CGG CCG ACG GAC UAG CCG CGG CUC GUG CAC UUG UUG UCG AUG CUC ACG CUG UAGGGG UAG CCG CGG CCG UAG ACG CGG UCG AUG GUC UGG GUC UGG UUG UCG GGG GCC GCCCGG GCC UCG CAC CGG UCG GUC UCG UAG UAG CGG AUG UGG UAC UCG GAC CCG CGG CUCUUG UCG CAC CGG AUG UCG UUG UUG UCG UAG CGG UAG GGG UGG UUG AAG UGG UAG UCGCAC UGG UGG CUC UAG GAC GGG CAC UCG UAC UGG UUC UGG UCG CAC CUG ACG UGG UACAUG UAG ACG CCG CUG UCG UGG CUC ACG UCG UUG GAC GAC GAC GUC AUG CCG UCG AAGACG UGG GUC GAC UUG GCC CGG GAC UGG CCG UAG CGG CAC CUC GUC CUG UUC UUG UGGGUC CUC CAC AAG CGG GUC CAC UUC GUC UAG AUG UUC UGG GGG GGG UAG UUC CUG AAGCCG CCG AAG UUG AAG UCG GUC UAG GAC GGG CUG GGG UCG UUC GGG UCG UUC GCC UCGAAG UAG CUC CUG GAC GAC AAG UUG UUC CAC UGG GAC CGG CUG CGG CCG AAG UAG UUCGUC AUG CCG CUG ACG GAC CCG CUG UAG CGG CGG GCC CUG GAC UAG ACG CGG GUC UUCAAG UUG CCG GAC UGG CAC GAC GGG GGG GAC GAC UGG CUG CUC UAC UAG CGG GUC AUGUGG UCG CGG GAC GAC CGG CCG UGG UAG UGG UCG CCG ACC UGG AAG CCG CGG CCG CGGCGG GAC GUC UAG GGG AAG CGG UAC GUC UAC CGG AUG GCC AAG UUG CCG UAG CCG CACUGG GUC UUG CAC GAC AUG CUC UUG GUC UUC GAC UAG CGG UUG GUC AAG UUG UCG CGGUAG CCG UUC UAG GUC CUG UCG GAC UCG UCG UGG CGG UCG CGG GAC CCG UUC GAC GUCCUG CAC CAC UUG GUC UUG CGG GUC CGG GAC UUG UGG GAC CAC UUC GUC GAC UCG UCGUUG AAG CCG CGG UAG UCG UCG CAC GAC UUG CUG UAG GAC UCG GCC GAC CUG UUC CACCUC CGG CUC CAC GUC UAG CUG GCC GAC UAG UGG CCG GCC GAC GUC UCG GAC GUC UGGAUG CAC UGG GUC GUC GAC UAG GCC CGG CGG CUC UAG GCC CGG UCG CGG UUG GAC CGGCGG UGG UUC UAC UCG CUC ACG CAC GAC CCG GUC UCG UUC GCC CAC CUG AAG ACG CCGUUC CCG AUG GUG GAC UAC UCG AAG GGG GUC UCG CGG GGG GUG CCG CAC CAC AAG GACGUG CAC UGG AUG CAC GGG CGG GUC CUC UUC UUG AAG UGG UGG CGG GGG CGG UAG ACGGUG CUG CCG UUC CGG GUG AAG GGG GCC CUC CCG CAC AAG CAC UCG UUG CCG UGG GUGACC AAG CAC UGG GUC GCC UUG AAG AUG CUC GGG GUC UAG UAG UGG UGG CUG UUG UGGAAG CAC UCG CCG UUG ACG CUG CAC CAC UAG CCG UAG CAC UUG UUG UGG CAC AUG CUGGGG GAC GUC GGG CUC GAC CUG UCG AAG UUC CUC CUC GAC CUG UUC AUG AAG UUC UUGGUG UGG UCG GGG CUG CAC CUG GAC CCG CUG UAG UCG CCG UAG UUG CGG UCG CAC CACUUG UAG GUC UUC CUC UAG CUG GCC GAC UUG CUC CAC CGG UUC UUG GAC UUG CUC UCGGAC UAG CUG GAC GUC CUC GAC CCG UUC AUG CUC GUC AUG UAG UUC ACC GGG ACC AUGUAG ACC GAC CCG AAG UAG CGG CCG GAC UAG CGG UAG CAC UAC CAC UGG UAG UAC GACACG ACG UAC UGG UCG ACG ACG UCG ACG GAC UUC CCG ACG ACG UCG CGG AUG CAC GCCUUC CUG CCG CUC ACC CAC AAG GAC ACU
SEQ ID No.3：编码融合前构型S蛋白序列的mRNA(优化方案2)
AAG CAC AAG GAC CAC GAC GAC GGG GAC CAC UCG UCG GUC ACG CAC UUG GACUGG UGG UCC UGG GUC GAC GGG GGG CGG AUG UGG UUG UCG AAG UGG UCC CCG CAC AUGAUG GGG CUG UUC CAC AAG UCC UCG UCG CAC GAC GUG UCG UGG GUC CUG GAC AAG GACGGG AAG AAG UCG UUG CAC UGG ACC AAG GUG CGG UAG GUG CAC UCG CCG UGG UUG CCGUGG UUC UCC AAG CUG UUG GGG CAC GAC GGG AAG UUG CUG CCG CAC AUG AAG CGG UCGUGG CUC UUC UCG UUG UAG UAG UCC CCG ACC UAG AAG CCG UGG UGG GAC CUG UCG UUCUGG GUC UCG GAC GAC UAG CAC UUG UUG CGG UGG UUG CAC CAC UAG UUC CAC ACG CUCAAG GUC AAG ACG UUG CUG GGG AAG GAC CCG CAC AUG AUG GUG UUC UUG UUG UUC UCGACC UAC CUC UCG CUC AAG UCC CAC AUG UCG UCG CGG UUG UUG ACG UGG AAG CUC AUGCAC UCG GUC GGG AAG GAC UAC CUG GAC CUC CCG UUC GUC CCG UUG AAG UUC UUG GACUCC CUC AAG CAC AAG UUC UUG UAG CUG CCG AUG AAG UUC UAG AUG UCG UUC GUG UGGGGG UAG UUG GAC CAC UCC CUG GAC GGG GUC CCG AAG UCG CGG GAC CUC GGG GAC CACCUG GAC GGG UAG CCG UAG UUG UAG UGG UCC AAG GUC UGG GAC GAC CGG GAC GUG UCCUCG AUG GAC UGG GGG CCG CUG UCG UCG UCG CCG ACC UGG CGG CCG CGG CGG CGG AUGAUG CAC CCG AUG GAC GUC GGG UCC UGG AAG GAC GAC UUC AUG UUG CUC UUG CCG UGGUAG UGG CUG CGG CAC CUG ACG CGG GAC CUG GGG GAC UCG CUC UGG UUC ACG UGG GACUUC UCG AAG UGG CAC CUC UUC CCG UAG AUG GUC UGG UCG UUG AAG UCC CAC GUC GGGUGG CUC UCG UAG CAC UCC AAG GGG UUG UAG UGG UUG GAC ACG GGG AAG CCG CUC CACAAG UUG CGG UGG UCC AAG CGG UCG CAC AUG CGG ACC UUG UCC UUC UCC UAG UCG UUGACG CAC CGG CUG AUG UCG CAC GAC AUG UUG UCG CGG UCG AAG UCG UGG AAG UUC ACGAUG CCG CAC UCG GGG UGG UUC GAC UUG CUG GAC ACG AAG UGG UUG CAC AUG CGG CUGUCG AAG CAC UAG UCC CCG CUG CUC CAC UCC GUC UAG CGG GGG CCG GUC UGG CCG UUCUAG CGG CUG AUG UUG AUG UUC GAC GGG CUG CUG AAG UGG CCG ACG CAC UAG CGG ACCUUG UCG UUG UUG GAC CUG UCG UUC CAC CCG CCG UUG AUG UUG AUG GAC AUG UCC GACAAG UCC UUC UCG UUG GAC UUC GGG AAG CUC UCC CUG UAG UCG UGG CUC UAG AUG GUCCGG CCG UCG UGG GGG ACG UUG CCG CAC CUC CCG AAG UUG ACG AUG AAG GGG GAC GUCUCG AUG CCG AAG GUC GGG UGG UUG CCG CAC CCG AUG GUC GGG AUG UCC CAC CAC CACGAC UCG AAG CUC GAC GAC GUG CGG GGG CGG UGG CAC ACG CCG GGG UUC UUC UCG UGGUUG GAC CAC UUC UUG UUC ACG CAC UUG AAG UUG AAG UUG CCG GAC UGG CCG UGG CCGCAC GAC UGG CUC UCG UUG UUC UUC AAG GAC GGG AAG GUC GUC AAG CCG UCC CUG UAGCGG CUG UGG UGG CUG CGG CAC UCC CUG GGG GUC UGG GAC CUC UAG GAC CUG UAG UGGGGG ACG UCG AAG CCG CCG CAC UCG CAC UAG UGG GGG CCG UGG UUG UGG UCG UUG GUCCAC CGG CAC GAC AUG GUC CUG CAC UUG ACG UGG CUC CAC GGG CAC CGG UAG GUG CGGCUG GUC GAC UGG GGG UGG ACC UCC CAC AUG UCG UGG CCG UCG UUG CAC AAG GUC UGGUCC CGG CCG ACG GAC UAG CCG CGG CUC GUG CAC UUG UUG UCG AUG CUC ACG CUG UAGGGG UAG CCG CGG CCG UAG ACG CGG UCG AUG GUC UGG GUC UGG UUG UCG GGG UCC UCCCGG UCC UCG CAC CGG UCG GUC UCG UAG UAG CGG AUG UGG UAC UCG GAC CCG CGG CUCUUG UCG CAC CGG AUG UCG UUG UUG UCG UAG CGG UAG GGG UGG UUG AAG UGG UAG UCGCAC UGG UGG CUC UAG GAC GGG CAC UCG UAC UGG UUC UGG UCG CAC CUG ACG UGG UACAUG UAG ACG CCG CUG UCG UGG CUC ACG UCG UUG GAC GAC GAC GUC AUG CCG UCG AAGACG UGG GUC GAC UUG UCC CGG GAC UGG CCG UAG CGG CAC CUC GUC CUG UUC UUG UGGGUC CUC CAC AAG CGG GUC CAC UUC GUC UAG AUG UUC UGG GGG GGG UAG UUC CUG AAGCCG CCG AAG UUG AAG UCG GUC UAG GAC GGG CUG GGG UCG UUC GGG UCG UUC UCC UCGAAG UAG CUC CUG GAC GAC AAG UUG UUC CAC UGG GAC CGG CUG CGG CCG AAG UAG UUCGUC AUG CCG CUG ACG GAC CCG CUG UAG CGG CGG UCC CUG GAC UAG ACG CGG GUC UUCAAG UUG CCG GAC UGG CAC GAC GGG GGG GAC GAC UGG CUG CUC UAC UAG CGG GUC AUGUGG UCG CGG GAC GAC CGG CCG UGG UAG UGG UCG CCG ACC UGG AAG CCG CGG CCG CGGCGG GAC GUC UAG GGG AAG CGG UAC GUC UAC CGG AUG UCC AAG UUG CCG UAG CCG CACUGG GUC UUG CAC GAC AUG CUC UUG GUC UUC GAC UAG CGG UUG GUC AAG UUG UCG CGGUAG CCG UUC UAG GUC CUG UCG GAC UCG UCG UGG CGG UCG CGG GAC CCG UUC GAC GUCCUG CAC CAC UUG GUC UUG CGG GUC CGG GAC UUG UGG GAC CAC UUC GUC GAC UCG UCGUUG AAG CCG CGG UAG UCG UCG CAC GAC UUG CUG UAG GAC UCG UCC GAC CUG UUC CACCUC CGG CUC CAC GUC UAG CUG UCC GAC UAG UGG CCG UCC GAC GUC UCG GAC GUC UGGAUG CAC UGG GUC GUC GAC UAG UCC CGG CGG CUC UAG UCC CGG UCG CGG UUG GAC CGGCGG UGG UUC UAC UCG CUC ACG CAC GAC CCG GUC UCG UUC UCC CAC CUG AAG ACG CCGUUC CCG AUG GUG GAC UAC UCG AAG GGG GUC UCG CGG GGG GUG CCG CAC CAC AAG GACGUG CAC UGG AUG CAC GGG CGG GUC CUC UUC UUG AAG UGG UGG CGG GGG CGG UAG ACGGUG CUG CCG UUC CGG GUG AAG GGG UCC CUC CCG CAC AAG CAC UCG UUG CCG UGG GUGACC AAG CAC UGG GUC UCC UUG AAG AUG CUC GGG GUC UAG UAG UGG UGG CUG UUG UGGAAG CAC UCG CCG UUG ACG CUG CAC CAC UAG CCG UAG CAC UUG UUG UGG CAC AUG CUGGGG GAC GUC GGG CUC GAC CUG UCG AAG UUC CUC CUC GAC CUG UUC AUG AAG UUC UUGGUG UGG UCG GGG CUG CAC CUG GAC CCG CUG UAG UCG CCG UAG UUG CGG UCG CAC CACUUG UAG GUC UUC CUC UAG CUG UCC GAC UUG CUC CAC CGG UUC UUG GAC UUG CUC UCGGAC UAG CUG GAC GUC CUC GAC CCG UUC AUG CUC GUC AUG UAG UUC ACC GGG ACC AUGUAG ACC GAC CCG AAG UAG CGG CCG GAC UAG CGG UAG CAC UAC CAC UGG UAG UAC GACACG ACG UAC UGG UCG ACG ACG UCG ACG GAC UUC CCG ACG ACG UCG CGG AUG CAC UCCUUC CUG CCG CUC ACC CAC AAG GAC ACU
SEQ ID No.4：编码融合前构型S蛋白序列的mRNA(优化方案3)
AAG CAC AAG GAC CAC GAC GAC GGA GAC CAC UCG UCG GUC ACG CAC UUG GACUGG UGG UCC UGG GUC GAC GGA GGA CGG AUG UGG UUG UCG AAG UGG UCC CCG CAC AUGAUG GGA CUG UUC CAC AAG UCC UCG UCG CAC GAC GUG UCG UGG GUC CUG GAC AAG GACGGA AAG AAG UCG UUG CAC UGG ACC AAG GUG CGG UAG GUG CAC UCG CCG UGG UUG CCGUGG UUC UCC AAG CUG UUG GGA CAC GAC GGA AAG UUG CUG CCG CAC AUG AAG CGG UCGUGG CUC UUC UCG UUG UAG UAG UCC CCG ACC UAG AAG CCG UGG UGG GAC CUG UCG UUCUGG GUC UCG GAC GAC UAG CAC UUG UUG CGG UGG UUG CAC CAC UAG UUC CAC ACG CUCAAG GUC AAG ACG UUG CUG GGA AAG GAC CCG CAC AUG AUG GUG UUC UUG UUG UUC UCGACC UAC CUC UCG CUC AAG UCC CAC AUG UCG UCG CGG UUG UUG ACG UGG AAG CUC AUGCAC UCG GUC GGA AAG GAC UAC CUG GAC CUC CCG UUC GUC CCG UUG AAG UUC UUG GACUCC CUC AAG CAC AAG UUC UUG UAG CUG CCG AUG AAG UUC UAG AUG UCG UUC GUG UGGGGA UAG UUG GAC CAC UCC CUG GAC GGA GUC CCG AAG UCG CGG GAC CUC GGA GAC CACCUG GAC GGA UAG CCG UAG UUG UAG UGG UCC AAG GUC UGG GAC GAC CGG GAC GUG UCCUCG AUG GAC UGG GGA CCG CUG UCG UCG UCG CCG ACC UGG CGG CCG CGG CGG CGG AUGAUG CAC CCG AUG GAC GUC GGA UCC UGG AAG GAC GAC UUC AUG UUG CUC UUG CCG UGGUAG UGG CUG CGG CAC CUG ACG CGG GAC CUG GGA GAC UCG CUC UGG UUC ACG UGG GACUUC UCG AAG UGG CAC CUC UUC CCG UAG AUG GUC UGG UCG UUG AAG UCC CAC GUC GGAUGG CUC UCG UAG CAC UCC AAG GGA UUG UAG UGG UUG GAC ACG GGA AAG CCG CUC CACAAG UUG CGG UGG UCC AAG CGG UCG CAC AUG CGG ACC UUG UCC UUC UCC UAG UCG UUGACG CAC CGG CUG AUG UCG CAC GAC AUG UUG UCG CGG UCG AAG UCG UGG AAG UUC ACGAUG CCG CAC UCG GGA UGG UUC GAC UUG CUG GAC ACG AAG UGG UUG CAC AUG CGG CUGUCG AAG CAC UAG UCC CCG CUG CUC CAC UCC GUC UAG CGG GGA CCG GUC UGG CCG UUCUAG CGG CUG AUG UUG AUG UUC GAC GGA CUG CUG AAG UGG CCG ACG CAC UAG CGG ACCUUG UCG UUG UUG GAC CUG UCG UUC CAC CCG CCG UUG AUG UUG AUG GAC AUG UCC GACAAG UCC UUC UCG UUG GAC UUC GGA AAG CUC UCC CUG UAG UCG UGG CUC UAG AUG GUCCGG CCG UCG UGG GGA ACG UUG CCG CAC CUC CCG AAG UUG ACG AUG AAG GGA GAC GUCUCG AUG CCG AAG GUC GGA UGG UUG CCG CAC CCG AUG GUC GGA AUG UCC CAC CAC CACGAC UCG AAG CUC GAC GAC GUG CGG GGA CGG UGG CAC ACG CCG GGA UUC UUC UCG UGGUUG GAC CAC UUC UUG UUC ACG CAC UUG AAG UUG AAG UUG CCG GAC UGG CCG UGG CCGCAC GAC UGG CUC UCG UUG UUC UUC AAG GAC GGA AAG GUC GUC AAG CCG UCC CUG UAGCGG CUG UGG UGG CUG CGG CAC UCC CUG GGA GUC UGG GAC CUC UAG GAC CUG UAG UGGGGA ACG UCG AAG CCG CCG CAC UCG CAC UAG UGG GGA CCG UGG UUG UGG UCG UUG GUCCAC CGG CAC GAC AUG GUC CUG CAC UUG ACG UGG CUC CAC GGA CAC CGG UAG GUG CGGCUG GUC GAC UGG GGA UGG ACC UCC CAC AUG UCG UGG CCG UCG UUG CAC AAG GUC UGGUCC CGG CCG ACG GAC UAG CCG CGG CUC GUG CAC UUG UUG UCG AUG CUC ACG CUG UAGGGA UAG CCG CGG CCG UAG ACG CGG UCG AUG GUC UGG GUC UGG UUG UCG GGA UCC UCCCGG UCC UCG CAC CGG UCG GUC UCG UAG UAG CGG AUG UGG UAC UCG GAC CCG CGG CUCUUG UCG CAC CGG AUG UCG UUG UUG UCG UAG CGG UAG GGA UGG UUG AAG UGG UAG UCGCAC UGG UGG CUC UAG GAC GGA CAC UCG UAC UGG UUC UGG UCG CAC CUG ACG UGG UACAUG UAG ACG CCG CUG UCG UGG CUC ACG UCG UUG GAC GAC GAC GUC AUG CCG UCG AAGACG UGG GUC GAC UUG UCC CGG GAC UGG CCG UAG CGG CAC CUC GUC CUG UUC UUG UGGGUC CUC CAC AAG CGG GUC CAC UUC GUC UAG AUG UUC UGG GGA GGA UAG UUC CUG AAGCCG CCG AAG UUG AAG UCG GUC UAG GAC GGA CUG GGA UCG UUC GGA UCG UUC UCC UCGAAG UAG CUC CUG GAC GAC AAG UUG UUC CAC UGG GAC CGG CUG CGG CCG AAG UAG UUCGUC AUG CCG CUG ACG GAC CCG CUG UAG CGG CGG UCC CUG GAC UAG ACG CGG GUC UUCAAG UUG CCG GAC UGG CAC GAC GGA GGA GAC GAC UGG CUG CUC UAC UAG CGG GUC AUGUGG UCG CGG GAC GAC CGG CCG UGG UAG UGG UCG CCG ACC UGG AAG CCG CGG CCG CGGCGG GAC GUC UAG GGA AAG CGG UAC GUC UAC CGG AUG UCC AAG UUG CCG UAG CCG CACUGG GUC UUG CAC GAC AUG CUC UUG GUC UUC GAC UAG CGG UUG GUC AAG UUG UCG CGGUAG CCG UUC UAG GUC CUG UCG GAC UCG UCG UGG CGG UCG CGG GAC CCG UUC GAC GUCCUG CAC CAC UUG GUC UUG CGG GUC CGG GAC UUG UGG GAC CAC UUC GUC GAC UCG UCGUUG AAG CCG CGG UAG UCG UCG CAC GAC UUG CUG UAG GAC UCG UCC GAC CUG UUC CACCUC CGG CUC CAC GUC UAG CUG UCC GAC UAG UGG CCG UCC GAC GUC UCG GAC GUC UGGAUG CAC UGG GUC GUC GAC UAG UCC CGG CGG CUC UAG UCC CGG UCG CGG UUG GAC CGGCGG UGG UUC UAC UCG CUC ACG CAC GAC CCG GUC UCG UUC UCC CAC CUG AAG ACG CCGUUC CCG AUG GUG GAC UAC UCG AAG GGA GUC UCG CGG GGA GUG CCG CAC CAC AAG GACGUG CAC UGG AUG CAC GGA CGG GUC CUC UUC UUG AAG UGG UGG CGG GGA CGG UAG ACGGUG CUG CCG UUC CGG GUG AAG GGA UCC CUC CCG CAC AAG CAC UCG UUG CCG UGG GUGACC AAG CAC UGG GUC UCC UUG AAG AUG CUC GGA GUC UAG UAG UGG UGG CUG UUG UGGAAG CAC UCG CCG UUG ACG CUG CAC CAC UAG CCG UAG CAC UUG UUG UGG CAC AUG CUGGGA GAC GUC GGA CUC GAC CUG UCG AAG UUC CUC CUC GAC CUG UUC AUG AAG UUC UUGGUG UGG UCG GGA CUG CAC CUG GAC CCG CUG UAG UCG CCG UAG UUG CGG UCG CAC CACUUG UAG GUC UUC CUC UAG CUG UCC GAC UUG CUC CAC CGG UUC UUG GAC UUG CUC UCGGAC UAG CUG GAC GU C CUC GAC CCG UUC AUG CUC GUC AUG UAG UUC ACC GGA ACC AUGUAG ACC GAC CCG AAG UAG CGG CCG GAC UAG CGG UAG CAC UAC CAC UGG UAG UAC GACACG ACG UAC UGG UCG ACG ACG UCG ACG GAC UUC CCG ACG ACG UCG CGG AUG CAC UCCUUC CUG CCG CUC ACC CAC AAG GAC ACU
SEQ ID No.5：编码融合前构型S蛋白序列的mRNA(优化方案4)
AAG CAC AAG GAC CAC GAC GAC GGA GAC CAC UCG UCG GUC ACA CAC UUG GACUGU UGU UCU UGU GUC GAC GGA GGA CGA AUG UGU UUG UCG AAG UGU UCU CCU CAC AUGAUG GGA CUG UUC CAC AAG UCU UCG UCG CAC GAC GUG UCG UGU GUC CUG GAC AAG GACGGA AAG AAG UCG UUG CAC UGU ACC AAG GUG CGA UAG GUG CAC UCG CCU UGU UUG CCUUGU UUC UCU AAG CUG UUG GGA CAC GAC GGA AAG UUG CUG CCU CAC AUG AAG CGA UCGUGU CUC UUC UCG UUG UAG UAG UCU CCU ACC UAG AAG CCU UGU UGU GAC CUG UCG UUCUGU GUC UCG GAC GAC UAG CAC UUG UUG CGA UGU UUG CAC CAC UAG UUC CAC ACA CUCAAG GUC AAG ACA UUG CUG GGA AAG GAC CCU CAC AUG AUG GUG UUC UUG UUG UUC UCGACC UAC CUC UCG CUC AAG UCU CAC AUG UCG UCG CGA UUG UUG ACA UGU AAG CUC AUGCAC UCG GUC GGA AAG GAC UAC CUG GAC CUC CCU UUC GUC CCU UUG AAG UUC UUG GACUCU CUC AAG CAC AAG UUC UUG UAG CUG CCU AUG AAG UUC UAG AUG UCG UUC GUG UGUGGA UAG UUG GAC CAC UCU CUG GAC GGA GUC CCU AAG UCG CGA GAC CUC GGA GAC CACCUG GAC GGA UAG CCU UAG UUG UAG UGU UCU AAG GUC UGU GAC GAC CGA GAC GUG UCUUCG AUG GAC UGU GGA CCU CUG UCG UCG UCG CCU ACC UGU CGA CCU CGA CGA CGA AUGAUG CAC CCU AUG GAC GUC GGA UCU UGU AAG GAC GAC UUC AUG UUG CUC UUG CCU UGUUAG UGU CUG CGA CAC CUG ACA CGA GAC CUG GGA GAC UCG CUC UGU UUC ACA UGU GACUUC UCG AAG UGU CAC CUC UUC CCU UAG AUG GUC UGU UCG UUG AAG UCU CAC GUC GGAUGU CUC UCG UAG CAC UCU AAG GGA UUG UAG UGU UUG GAC ACA GGA AAG CCU CUC CACAAG UUG CGA UGU UCU AAG CGA UCG CAC AUG CGA ACC UUG UCU UUC UCU UAG UCG UUGACA CAC CGA CUG AUG UCG CAC GAC AUG UUG UCG CGA UCG AAG UCG UGU AAG UUC ACAAUG CCU CAC UCG GGA UGU UUC GAC UUG CUG GAC ACA AAG UGU UUG CAC AUG CGA CUGUCG AAG CAC UAG UCU CCU CUG CUC CAC UCU GUC UAG CGA GGA CCU GUC UGU CCU UUCUAG CGA CUG AUG UUG AUG UUC GAC GGA CUG CUG AAG UGU CCU ACA CAC UAG CGA ACCUUG UCG UUG UUG GAC CUG UCG UUC CAC CCU CCU UUG AUG UUG AUG GAC AUG UCU GACAAG UCU UUC UCG UUG GAC UUC GGA AAG CUC UCU CUG UAG UCG UGU CUC UAG AUG GUCCGA CCU UCG UGU GGA ACA UUG CCU CAC CUC CCU AAG UUG ACA AUG AAG GGA GAC GUCUCG AUG CCU AAG GUC GGA UGU UUG CCU CAC CCU AUG GUC GGA AUG UCU CAC CAC CACGAC UCG AAG CUC GAC GAC GUG CGA GGA CGA UGU CAC ACA CCU GGA UUC UUC UCG UGUUUG GAC CAC UUC UUG UUC ACA CAC UUG AAG UUG AAG UUG CCU GAC UGU CCU UGU CCUCAC GAC UGU CUC UCG UUG UUC UUC AAG GAC GGA AAG GUC GUC AAG CCU UCU CUG UAGCGA CUG UGU UGU CUG CGA CAC UCU CUG GGA GUC UGU GAC CUC UAG GAC CUG UAG UGUGGA ACA UCG AAG CCU CCU CAC UCG CAC UAG UGU GGA CCU UGU UUG UGU UCG UUG GUCCAC CGA CAC GAC AUG GUC CUG CAC UUG ACA UGU CUC CAC GGA CAC CGA UAG GUG CGACUG GUC GAC UGU GGA UGU ACC UCU CAC AUG UCG UGU CCU UCG UUG CAC AAG GUC UGUUCU CGA CCU ACA GAC UAG CCU CGA CUC GUG CAC UUG UUG UCG AUG CUC ACA CUG UAGGGA UAG CCU CGA CCU UAG ACA CGA UCG AUG GUC UGU GUC UGU UUG UCG GGA UCU UCUCGA UCU UCG CAC CGA UCG GUC UCG UAG UAG CGA AUG UGU UAC UCG GAC CCU CGA CUCUUG UCG CAC CGA AUG UCG UUG UUG UCG UAG CGA UAG GGA UGU UUG AAG UGU UAG UCGCAC UGU UGU CUC UAG GAC GGA CAC UCG UAC UGU UUC UGU UCG CAC CUG ACA UGU UACAUG UAG ACA CCU CUG UCG UGU CUC ACA UCG UUG GAC GAC GAC GUC AUG CCU UCG AAGACA UGU GUC GAC UUG UCU CGA GAC UGU CCU UAG CGA CAC CUC GUC CUG UUC UUG UGUGUC CUC CAC AAG CGA GUC CAC UUC GUC UAG AUG UUC UGU GGA GGA UAG UUC CUG AAGCCU CCU AAG UUG AAG UCG GUC UAG GAC GGA CUG GGA UCG UUC GGA UCG UUC UCU UCGAAG UAG CUC CUG GAC GAC AAG UUG UUC CAC UGU GAC CGA CUG CGA CCU AAG UAG UUCGUC AUG CCU CUG ACA GAC CCU CUG UAG CGA CGA UCU CUG GAC UAG ACA CGA GUC UUCAAG UUG CCU GAC UGU CAC GAC GGA GGA GAC GAC UGU CUG CUC UAC UAG CGA GUC AUGUGU UCG CGA GAC GAC CGA CCU UGU UAG UGU UCG CCU ACC UGU AAG CCU CGA CCU CGACGA GAC GUC UAG GGA AAG CGA UAC GUC UAC CGA AUG UCU AAG UUG CCU UAG CCU CACUGU GUC UUG CAC GAC AUG CUC UUG GUC UUC GAC UAG CGA UUG GUC AAG UUG UCG CGAUAG CCU UUC UAG GUC CUG UCG GAC UCG UCG UGU CGA UCG CGA GAC CCU UUC GAC GUCCUG CAC CAC UUG GUC UUG CGA GUC CGA GAC UUG UGU GAC CAC UUC GUC GAC UCG UCGUUG AAG CCU CGA UAG UCG UCG CAC GAC UUG CUG UAG GAC UCG UCU GAC CUG UUC CACCUC CGA CUC CAC GUC UAG CUG UCU GAC UAG UGU CCU UCU GAC GUC UCG GAC GUC UGUAUG CAC UGU GUC GUC GAC UAG UCU CGA CGA CUC UAG UCU CGA UCG CGA UUG GAC CGACGA UGU UUC UAC UCG CUC ACA CAC GAC CCU GUC UCG UUC UCU CAC CUG AAG ACA CCUUUC CCU AUG GUG GAC UAC UCG AAG GGA GUC UCG CGA GGA GUG CCU CAC CAC AAG GACGUG CAC UGU AUG CAC GGA CGA GUC CUC UUC UUG AAG UGU UGU CGA GGA CGA UAG ACAGUG CUG CCU UUC CGA GUG AAG GGA UCU CUC CCU CAC AAG CAC UCG UUG CCU UGU GUGACC AAG CAC UGU GUC UCU UUG AAG AUG CUC GGA GUC UAG UAG UGU UGU CUG UUG UGUAAG CAC UCG CCU UUG ACA CUG CAC CAC UAG CCU UAG CAC UUG UUG UGU CAC AUG CUGGGA GAC GUC GGA CUC GAC CUG UCG AAG UUC CUC CUC GAC CUG UUC AUG AAG UUC UUGGUG UGU UCG GGA CUG CAC CUG GAC CCU CUG UAG UCG CCU UAG UUG CGA UCG CAC CACUUG UAG GUC UUC CUC UAG CUG UCU GAC UUG CUC CAC CGA UUC UUG GAC UUG CUC UCGGAC UAG CUG GAC GUC CUC GAC CCU UUC AUG CUC GUC AUG UAG UUC ACC GGA ACC AUGUAG ACC GAC CCU AAG UAG CGA CCU GAC UAG CGA UAG CAC UAC CAC UGU UAG UAC GACACA ACA UAC UGU UCG ACA ACA UCG ACA GAC UUC CCU ACA ACA UCG CGA AUG CAC UCUUUC CUG CCU CUC ACC CAC AAG GAC ACU
SEQ ID No.6：佐剂序列(1)
UAC CGC CAC UAC CGC GGC GCG UGG GAC CAC GAC GAC GAC UCG CCG CGC GACCGC GAC UGG GUC UGG ACC CGC
SEQ ID No.7：佐剂序列(2)
UAC CGC UAA UAC CGC GGC GCG UGG GAC CAC GAC GAC GAC UCG CCG CGC GACCGC GAC UGG
SEQ ID No.8：佐剂序列(3)
UAC GCG CAC UGG CGC GGC GCG UGG CAC GAC GAC GAC GAC UCG CGC CGC GACCGC GAC UGG CUU UGG ACC CGC
SEQ ID No.9：佐剂序列(4)
UAC CUA ACC UGG ACC UAA GAC AAA GAC CAC CGC CGC CGC UGG GCG
SEQ ID No.10：编码融合前构型S蛋白序列的DNA
AAG CAC AAG GAC CAC GAC GAC GGG GAC CAC TCG TCG GTC ACG CAC TTG GACTGG TGG GCC TGG GTC GAC GGG GGG CGG ATG TGG TTG TCG AAG TGG GCC CCG CAC ATGATG GGG CTG TTC CAC AAG GCC TCG TCG CAC GAC GTG TCG TGG GTC CTG GAC AAG GACGGG AAG AAG TCG TTG CAC TGG ACC AAG GTG CGG TAG GTG CAC TCG CCG TGG TTG CCGTGG TTC GCC AAG CTG TTG GGG CAC GAC GGG AAG TTG CTG CCG CAC ATG AAG CGG TCGTGG CTC TTC TCG TTG TAG TAG GCC CCG ACC TAG AAG CCG TGG TGG GAC CTG TCG TTCTGG GTC TCG GAC GAC TAG CAC TTG TTG CGG TGG TTG CAC CAC TAG TTC CAC ACG CTCAAG GTC AAG ACG TTG CTG GGG AAG GAC CCG CAC ATG ATG GTG TTC TTG TTG TTC TCGACC TAC CTC TCG CTC AAG GCC CAC ATG TCG TCG CGG TTG TTG ACG TGG AAG CTC ATGCAC TCG GTC GGG AAG GAC TAC CTG GAC CTC CCG TTC GTC CCG TTG AAG TTC TTG GACGCC CTC AAG CAC AAG TTC TTG TAG CTG CCG ATG AAG TTC TAG ATG TCG TTC GTG TGGGGG TAG TTG GAC CAC GCC CTG GAC GGG GTC CCG AAG TCG CGG GAC CTC GGG GAC CACCTG GAC GGG TAG CCG TAG TTG TAG TGG GCC AAG GTC TGG GAC GAC CGG GAC GTG GCCTCG ATG GAC TGG GGG CCG CTG TCG TCG TCG CCG ACC TGG CGG CCG CGG CGG CGG ATGATG CAC CCG ATG GAC GTC GGG GCC TGG AAG GAC GAC TTC ATG TTG CTC TTG CCG TGGTAG TGG CTG CGG CAC CTG ACG CGG GAC CTG GGG GAC TCG CTC TGG TTC ACG TGG GACTTC TCG AAG TGG CAC CTC TTC CCG TAG ATG GTC TGG TCG TTG AAG GCC CAC GTC GGGTGG CTC TCG TAG CAC GCC AAG GGG TTG TAG TGG TTG GAC ACG GGG AAG CCG CTC CACAAG TTG CGG TGG GCC AAG CGG TCG CAC ATG CGG ACC TTG GCC TTC GCC TAG TCG TTGACG CAC CGG CTG ATG TCG CAC GAC ATG TTG TCG CGG TCG AAG TCG TGG AAG TTC ACGATG CCG CAC TCG GGG TGG TTC GAC TTG CTG GAC ACG AAG TGG TTG CAC ATG CGG CTGTCG AAG CAC TAG GCC CCG CTG CTC CAC GCC GTC TAG CGG GGG CCG GTC TGG CCG TTCTAG CGG CTG ATG TTG ATG TTC GAC GGG CTG CTG AAG TGG CCG ACG CAC TAG CGG ACCTTG TCG TTG TTG GAC CTG TCG TTC CAC CCG CCG TTG ATG TTG ATG GAC ATG GCC GACAAG GCC TTC TCG TTG GAC TTC GGG AAG CTC GCC CTG TAG TCG TGG CTC TAG ATG GTCCGG CCG TCG TGG GGG ACG TTG CCG CAC CTC CCG AAG TTG ACG ATG AAG GGG GAC GTCTCG ATG CCG AAG GTC GGG TGG TTG CCG CAC CCG ATG GTC GGG ATG GCC CAC CAC CACGAC TCG AAG CTC GAC GAC GTG CGG GGG CGG TGG CAC ACG CCG GGG TTC TTC TCG TGGTTG GAC CAC TTC TTG TTC ACG CAC TTG AAG TTG AAG TTG CCG GAC TGG CCG TGG CCGCAC GAC TGG CTC TCG TTG TTC TTC AAG GAC GGG AAG GTC GTC AAG CCG GCC CTG TAGCGG CTG TGG TGG CTG CGG CAC GCC CTG GGG GTC TGG GAC CTC TAG GAC CTG TAG TGGGGG ACG TCG AAG CCG CCG CAC TCG CAC TAG TGG GGG CCG TGG TTG TGG TCG TTG GTCCAC CGG CAC GAC ATG GTC CTG CAC TTG ACG TGG CTC CAC GGG CAC CGG TAG GTG CGGCTG GTC GAC TGG GGG TGG ACC GCC CAC ATG TCG TGG CCG TCG TTG CAC AAG GTC TGGGCC CGG CCG ACG GAC TAG CCG CGG CTC GTG CAC TTG TTG TCG ATG CTC ACG CTG TAGGGG TAG CCG CGG CCG TAG ACG CGG TCG ATG GTC TGG GTC TGG TTG TCG GGG GCC GCCCGG GCC TCG CAC CGG TCG GTC TCG TAG TAG CGG ATG TGG TAC TCG GAC CCG CGG CTCTTG TCG CAC CGG ATG TCG TTG TTG TCG TAG CGG TAG GGG TGG TTG AAG TGG TAG TCGCAC TGG TGG CTC TAG GAC GGG CAC TCG TAC TGG TTC TGG TCG CAC CTG ACG TGG TACATG TAG ACG CCG CTG TCG TGG CTC ACG TCG TTG GAC GAC GAC GTC ATG CCG TCG AAGACG TGG GTC GAC TTG GCC CGG GAC TGG CCG TAG CGG CAC CTC GTC CTG TTC TTG TGGGTC CTC CAC AAG CGG GTC CAC TTC GTC TAG ATG TTC TGG GGG GGG TAG TTC CTG AAGCCG CCG AAG TTG AAG TCG GTC TAG GAC GGG CTG GGG TCG TTC GGG TCG TTC GCC TCGAAG TAG CTC CTG GAC GAC AAG TTG TTC CAC TGG GAC CGG CTG CGG CCG AAG TAG TTCGTC ATG CCG CTG ACG GAC CCG CTG TAG CGG CGG GCC CTG GAC TAG ACG CGG GTC TTCAAG TTG CCG GAC TGG CAC GAC GGG GGG GAC GAC TGG CTG CTC TAC TAG CGG GTC ATGTGG TCG CGG GAC GAC CGG CCG TGG TAG TGG TCG CCG ACC TGG AAG CCG CGG CCG CGGCGG GAC GTC TAG GGG AAG CGG TAC GTC TAC CGG ATG GCC AAG TTG CCG TAG CCG CACTGG GTC TTG CAC GAC ATG CTC TTG GTC TTC GAC TAG CGG TTG GTC AAG TTG TCG CGGTAG CCG TTC TAG GTC CTG TCG GAC TCG TCG TGG CGG TCG CGG GAC CCG TTC GAC GTCCTG CAC CAC TTG GTC TTG CGG GTC CGG GAC TTG TGG GAC CAC TTC GTC GAC TCG TCGTTG AAG CCG CGG TAG TCG TCG CAC GAC TTG CTG TAG GAC TCG GCC GAC CTG TTC CACCTC CGG CTC CAC GTC TAG CTG GCC GAC TAG TGG CCG GCC GAC GTC TCG GAC GTC TGGATG CAC TGG GTC GTC GAC TAG GCC CGG CGG CTC TAG GCC CGG TCG CGG TTG GAC CGGCGG TGG TTC TAC TCG CTC ACG CAC GAC CCG GTC TCG TTC GCC CAC CTG AAG ACG CCGTTC CCG ATG GTG GAC TAC TCG AAG GGG GTC TCG CGG GGG GTG CCG CAC CAC AAG GACGTG CAC TGG ATG CAC GGG CGG GTC CTC TTC TTG AAG TGG TGG CGG GGG CGG TAG ACGGTG CTG CCG TTC CGG GTG AAG GGG GCC CTC CCG CAC AAG CAC TCG TTG CCG TGG GTGACC AAG CAC TGG GTC GCC TTG AAG ATG CTC GGG GTC TAG TAG TGG TGG CTG TTG TGGAAG CAC TCG CCG TTG ACG CTG CAC CAC TAG CCG TAG CAC TTG TTG TGG CAC ATG CTGGGG GAC GTC GGG CTC GAC CTG TCG AAG TTC CTC CTC GAC CTG TTC ATG AAG TTC TTGGTG TGG TCG GGG CTG CAC CTG GAC CCG CTG TAG TCG CCG TAG TTG CGG TCG CAC CACTTG TAG GTC TTC CTC TAG CTG GCC GAC TTG CTC CAC CGG TTC TTG GAC TTG CTC TCGGAC TAG CTG GAC GTC CTC GAC CCG TTC ATG CTC GTC ATG TAG TTC ACC GGG ACC ATGTAG ACC GAC CCG AAG TAG CGG CCG GAC TAG CGG TAG CAC TAC CAC TGG TAG TAC GACACG ACG TAC TGG TCG ACG ACG TCG ACG GAC TTC CCG ACG ACG TCG CGG ATG CAC GCCTTC CTG CCG CTC ACC CAC AAG GAC ACT
SEQ ID No.11：编码融合前构型S蛋白序列的DNA
AAG CAC AAG GAC CAC GAC GAC GGG GAC CAC TCG TCG GTC ACG CAC TTG GACTGG TGG TCC TGG GTC GAC GGG GGG CGG ATG TGG TTG TCG AAG TGG TCC CCG CAC ATGATG GGG CTG TTC CAC AAG TCC TCG TCG CAC GAC GTG TCG TGG GTC CTG GAC AAG GACGGG AAG AAG TCG TTG CAC TGG ACC AAG GTG CGG TAG GTG CAC TCG CCG TGG TTG CCGTGG TTC TCC AAG CTG TTG GGG CAC GAC GGG AAG TTG CTG CCG CAC ATG AAG CGG TCGTGG CTC TTC TCG TTG TAG TAG TCC CCG ACC TAG AAG CCG TGG TGG GAC CTG TCG TTCTGG GTC TCG GAC GAC TAG CAC TTG TTG CGG TGG TTG CAC CAC TAG TTC CAC ACG CTCAAG GTC AAG ACG TTG CTG GGG AAG GAC CCG CAC ATG ATG GTG TTC TTG TTG TTC TCGACC TAC CTC TCG CTC AAG TCC CAC ATG TCG TCG CGG TTG TTG ACG TGG AAG CTC ATGCAC TCG GTC GGG AAG GAC TAC CTG GAC CTC CCG TTC GTC CCG TTG AAG TTC TTG GACTCC CTC AAG CAC AAG TTC TTG TAG CTG CCG ATG AAG TTC TAG ATG TCG TTC GTG TGGGGG TAG TTG GAC CAC TCC CTG GAC GGG GTC CCG AAG TCG CGG GAC CTC GGG GAC CACCTG GAC GGG TAG CCG TAG TTG TAG TGG TCC AAG GTC TGG GAC GAC CGG GAC GTG TCCTCG ATG GAC TGG GGG CCG CTG TCG TCG TCG CCG ACC TGG CGG CCG CGG CGG CGG ATGATG CAC CCG ATG GAC GTC GGG TCC TGG AAG GAC GAC TTC ATG TTG CTC TTG CCG TGGTAG TGG CTG CGG CAC CTG ACG CGG GAC CTG GGG GAC TCG CTC TGG TTC ACG TGG GACTTC TCG AAG TGG CAC CTC TTC CCG TAG ATG GTC TGG TCG TTG AAG TCC CAC GTC GGGTGG CTC TCG TAG CAC TCC AAG GGG TTG TAG TGG TTG GAC ACG GGG AAG CCG CTC CACAAG TTG CGG TGG TCC AAG CGG TCG CAC ATG CGG ACC TTG TCC TTC TCC TAG TCG TTGACG CAC CGG CTG ATG TCG CAC GAC ATG TTG TCG CGG TCG AAG TCG TGG AAG TTC ACGATG CCG CAC TCG GGG TGG TTC GAC TTG CTG GAC ACG AAG TGG TTG CAC ATG CGG CTGTCG AAG CAC TAG TCC CCG CTG CTC CAC TCC GTC TAG CGG GGG CCG GTC TGG CCG TTCTAG CGG CTG ATG TTG ATG TTC GAC GGG CTG CTG AAG TGG CCG ACG CAC TAG CGG ACCTTG TCG TTG TTG GAC CTG TCG TTC CAC CCG CCG TTG ATG TTG ATG GAC ATG TCC GACAAG TCC TTC TCG TTG GAC TTC GGG AAG CTC TCC CTG TAG TCG TGG CTC TAG ATG GTCCGG CCG TCG TGG GGG ACG TTG CCG CAC CTC CCG AAG TTG ACG ATG AAG GGG GAC GTCTCG ATG CCG AAG GTC GGG TGG TTG CCG CAC CCG ATG GTC GGG ATG TCC CAC CAC CACGAC TCG AAG CTC GAC GAC GTG CGG GGG CGG TGG CAC ACG CCG GGG TTC TTC TCG TGGTTG GAC CAC TTC TTG TTC ACG CAC TTG AAG TTG AAG TTG CCG GAC TGG CCG TGG CCGCAC GAC TGG CTC TCG TTG TTC TTC AAG GAC GGG AAG GTC GTC AAG CCG TCC CTG TAGCGG CTG TGG TGG CTG CGG CAC TCC CTG GGG GTC TGG GAC CTC TAG GAC CTG TAG TGGGGG ACG TCG AAG CCG CCG CAC TCG CAC TAG TGG GGG CCG TGG TTG TGG TCG TTG GTCCAC CGG CAC GAC ATG GTC CTG CAC TTG ACG TGG CTC CAC GGG CAC CGG TAG GTG CGGCTG GTC GAC TGG GGG TGG ACC TCC CAC ATG TCG TGG CCG TCG TTG CAC AAG GTC TGGTCC CGG CCG ACG GAC TAG CCG CGG CTC GTG CAC TTG TTG TCG ATG CTC ACG CTG TAGGGG TAG CCG CGG CCG TAG ACG CGG TCG ATG GTC TGG GTC TGG TTG TCG GGG TCC TCCCGG TCC TCG CAC CGG TCG GTC TCG TAG TAG CGG ATG TGG TAC TCG GAC CCG CGG CTCTTG TCG CAC CGG ATG TCG TTG TTG TCG TAG CGG TAG GGG TGG TTG AAG TGG TAG TCGCAC TGG TGG CTC TAG GAC GGG CAC TCG TAC TGG TTC TGG TCG CAC CTG ACG TGG TACATG TAG ACG CCG CTG TCG TGG CTC ACG TCG TTG GAC GAC GAC GTC ATG CCG TCG AAGACG TGG GTC GAC TTG TCC CGG GAC TGG CCG TAG CGG CAC CTC GTC CTG TTC TTG TGGGTC CTC CAC AAG CGG GTC CAC TTC GTC TAG ATG TTC TGG GGG GGG TAG TTC CTG AAGCCG CCG AAG TTG AAG TCG GTC TAG GAC GGG CTG GGG TCG TTC GGG TCG TTC TCC TCGAAG TAG CTC CTG GAC GAC AAG TTG TTC CAC TGG GAC CGG CTG CGG CCG AAG TAG TTCGTC ATG CCG CTG ACG GAC CCG CTG TAG CGG CGG TCC CTG GAC TAG ACG CGG GTC TTCAAG TTG CCG GAC TGG CAC GAC GGG GGG GAC GAC TGG CTG CTC TAC TAG CGG GTC ATGTGG TCG CGG GAC GAC CGG CCG TGG TAG TGG TCG CCG ACC TGG AAG CCG CGG CCG CGGCGG GAC GTC TAG GGG AAG CGG TAC GTC TAC CGG ATG TCC AAG TTG CCG TAG CCG CACTGG GTC TTG CAC GAC ATG CTC TTG GTC TTC GAC TAG CGG TTG GTC AAG TTG TCG CGGTAG CCG TTC TAG GTC CTG TCG GAC TCG TCG TGG CGG TCG CGG GAC CCG TTC GAC GTCCTG CAC CAC TTG GTC TTG CGG GTC CGG GAC TTG TGG GAC CAC TTC GTC GAC TCG TCGTTG AAG CCG CGG TAG TCG TCG CAC GAC TTG CTG TAG GAC TCG TCC GAC CTG TTC CACCTC CGG CTC CAC GTC TAG CTG TCC GAC TAG TGG CCG TCC GAC GTC TCG GAC GTC TGGATG CAC TGG GTC GTC GAC TAG TCC CGG CGG CTC TAG TCC CGG TCG CGG TTG GAC CGGCGG TGG TTC TAC TCG CTC ACG CAC GAC CCG GTC TCG TTC TCC CAC CTG AAG ACG CCGTTC CCG ATG GTG GAC TAC TCG AAG GGG GTC TCG CGG GGG GTG CCG CAC CAC AAG GACGTG CAC TGG ATG CAC GGG CGG GTC CTC TTC TTG AAG TGG TGG CGG GGG CGG TAG ACGGTG CTG CCG TTC CGG GTG AAG GGG TCC CTC CCG CAC AAG CAC TCG TTG CCG TGG GTGACC AAG CAC TGG GTC TCC TTG AAG ATG CTC GGG GTC TAG TAG TGG TGG CTG TTG TGGAAG CAC TCG CCG TTG ACG CTG CAC CAC TAG CCG TAG CAC TTG TTG TGG CAC ATG CTGGGG GAC GTC GGG CTC GAC CTG TCG AAG TTC CTC CTC GAC CTG TTC ATG AAG TTC TTGGTG TGG TCG GGG CTG CAC CTG GAC CCG CTG TAG TCG CCG TAG TTG CGG TCG CAC CACTTG TAG GTC TTC CTC TAG CTG TCC GAC TTG CTC CAC CGG TTC TTG GAC TTG CTC TCGGAC TAG CTG GAC GTC CTC GAC CCG TTC ATG CTC GTC ATG TAG TTC ACC GGG ACC ATGTAG ACC GAC CCG AAG TAG CGG CCG GAC TAG CGG TAG CAC TAC CAC TGG TAG TAC GACACG ACG TAC TGG TCG ACG ACG TCG ACG GAC TTC CCG ACG ACG TCG CGG ATG CAC TCCTTC CTG CCG CTC ACC CAC AAG GAC ACT
SEQ ID No.12：编码融合前构型S蛋白序列的DNA
AAG CAC AAG GAC CAC GAC GAC GGA GAC CAC TCG TCG GTC ACG CAC TTG GACTGG TGG TCC TGG GTC GAC GGA GGA CGG ATG TGG TTG TCG AAG TGG TCC CCG CAC ATGATG GGA CTG TTC CAC AAG TCC TCG TCG CAC GAC GTG TCG TGG GTC CTG GAC AAG GACGGA AAG AAG TCG TTG CAC TGG ACC AAG GTG CGG TAG GTG CAC TCG CCG TGG TTG CCGTGG TTC TCC AAG CTG TTG GGA CAC GAC GGA AAG TTG CTG CCG CAC ATG AAG CGG TCGTGG CTC TTC TCG TTG TAG TAG TCC CCG ACC TAG AAG CCG TGG TGG GAC CTG TCG TTCTGG GTC TCG GAC GAC TAG CAC TTG TTG CGG TGG TTG CAC CAC TAG TTC CAC ACG CTCAAG GTC AAG ACG TTG CTG GGA AAG GAC CCG CAC ATG ATG GTG TTC TTG TTG TTC TCGACC TAC CTC TCG CTC AAG TCC CAC ATG TCG TCG CGG TTG TTG ACG TGG AAG CTC ATGCAC TCG GTC GGA AAG GAC TAC CTG GAC CTC CCG TTC GTC CCG TTG AAG TTC TTG GACTCC CTC AAG CAC AAG TTC TTG TAG CTG CCG ATG AAG TTC TAG ATG TCG TTC GTG TGGGGA TAG TTG GAC CAC TCC CTG GAC GGA GTC CCG AAG TCG CGG GAC CTC GGA GAC CACCTG GAC GGA TAG CCG TAG TTG TAG TGG TCC AAG GTC TGG GAC GAC CGG GAC GTG TCCTCG ATG GAC TGG GGA CCG CTG TCG TCG TCG CCG ACC TGG CGG CCG CGG CGG CGG ATGATG CAC CCG ATG GAC GTC GGA TCC TGG AAG GAC GAC TTC ATG TTG CTC TTG CCG TGGTAG TGG CTG CGG CAC CTG ACG CGG GAC CTG GGA GAC TCG CTC TGG TTC ACG TGG GACTTC TCG AAG TGG CAC CTC TTC CCG TAG ATG GTC TGG TCG TTG AAG TCC CAC GTC GGATGG CTC TCG TAG CAC TCC AAG GGA TTG TAG TGG TTG GAC ACG GGA AAG CCG CTC CACAAG TTG CGG TGG TCC AAG CGG TCG CAC ATG CGG ACC TTG TCC TTC TCC TAG TCG TTGACG CAC CGG CTG ATG TCG CAC GAC ATG TTG TCG CGG TCG AAG TCG TGG AAG TTC ACGATG CCG CAC TCG GGA TGG TTC GAC TTG CTG GAC ACG AAG TGG TTG CAC ATG CGG CTGTCG AAG CAC TAG TCC CCG CTG CTC CAC TCC GTC TAG CGG GGA CCG GTC TGG CCG TTCTAG CGG CTG ATG TTG ATG TTC GAC GGA CTG CTG AAG TGG CCG ACG CAC TAG CGG ACCTTG TCG TTG TTG GAC CTG TCG TTC CAC CCG CCG TTG ATG TTG ATG GAC ATG TCC GACAAG TCC TTC TCG TTG GAC TTC GGA AAG CTC TCC CTG TAG TCG TGG CTC TAG ATG GTCCGG CCG TCG TGG GGA ACG TTG CCG CAC CTC CCG AAG TTG ACG ATG AAG GGA GAC GTCTCG ATG CCG AAG GTC GGA TGG TTG CCG CAC CCG ATG GTC GGA ATG TCC CAC CAC CACGAC TCG AAG CTC GAC GAC GTG CGG GGA CGG TGG CAC ACG CCG GGA TTC TTC TCG TGGTTG GAC CAC TTC TTG TTC ACG CAC TTG AAG TTG AAG TTG CCG GAC TGG CCG TGG CCGCAC GAC TGG CTC TCG TTG TTC TTC AAG GAC GGA AAG GTC GTC AAG CCG TCC CTG TAGCGG CTG TGG TGG CTG CGG CAC TCC CTG GGA GTC TGG GAC CTC TAG GAC CTG TAG TGGGGA ACG TCG AAG CCG CCG CAC TCG CAC TAG TGG GGA CCG TGG TTG TGG TCG TTG GTCCAC CGG CAC GAC ATG GTC CTG CAC TTG ACG TGG CTC CAC GGA CAC CGG TAG GTG CGGCTG GTC GAC TGG GGA TGG ACC TCC CAC ATG TCG TGG CCG TCG TTG CAC AAG GTC TGGTCC CGG CCG ACG GAC TAG CCG CGG CTC GTG CAC TTG TTG TCG ATG CTC ACG CTG TAGGGA TAG CCG CGG CCG TAG ACG CGG TCG ATG GTC TGG GTC TGG TTG TCG GGA TCC TCCCGG TCC TCG CAC CGG TCG GTC TCG TAG TAG CGG ATG TGG TAC TCG GAC CCG CGG CTCTTG TCG CAC CGG ATG TCG TTG TTG TCG TAG CGG TAG GGA TGG TTG AAG TGG TAG TCGCAC TGG TGG CTC TAG GAC GGA CAC TCG TAC TGG TTC TGG TCG CAC CTG ACG TGG TACATG TAG ACG CCG CTG TCG TGG CTC ACG TCG TTG GAC GAC GAC GTC ATG CCG TCG AAGACG TGG GTC GAC TTG TCC CGG GAC TGG CCG TAG CGG CAC CTC GTC CTG TTC TTG TGGGTC CTC CAC AAG CGG GTC CAC TTC GTC TAG ATG TTC TGG GGA GGA TAG TTC CTG AAGCCG CCG AAG TTG AAG TCG GTC TAG GAC GGA CTG GGA TCG TTC GGA TCG TTC TCC TCGAAG TAG CTC CTG GAC GAC AAG TTG TTC CAC TGG GAC CGG CTG CGG CCG AAG TAG TTCGTC ATG CCG CTG ACG GAC CCG CTG TAG CGG CGG TCC CTG GAC TAG ACG CGG GTC TTCAAG TTG CCG GAC TGG CAC GAC GGA GGA GAC GAC TGG CTG CTC TAC TAG CGG GTC ATGTGG TCG CGG GAC GAC CGG CCG TGG TAG TGG TCG CCG ACC TGG AAG CCG CGG CCG CGGCGG GAC GTC TAG GGA AAG CGG TAC GTC TAC CGG ATG TCC AAG TTG CCG TAG CCG CACTGG GTC TTG CAC GAC ATG CTC TTG GTC TTC GAC TAG CGG TTG GTC AAG TTG TCG CGGTAG CCG TTC TAG GTC CTG TCG GAC TCG TCG TGG CGG TCG CGG GAC CCG TTC GAC GTCCTG CAC CAC TTG GTC TTG CGG GTC CGG GAC TTG TGG GAC CAC TTC GTC GAC TCG TCGTTG AAG CCG CGG TAG TCG TCG CAC GAC TTG CTG TAG GAC TCG TCC GAC CTG TTC CACCTC CGG CTC CAC GTC TAG CTG TCC GAC TAG TGG CCG TCC GAC GTC TCG GAC GTC TGGATG CAC TGG GTC GTC GAC TAG TCC CGG CGG CTC TAG TCC CGG TCG CGG TTG GAC CGGCGG TGG TTC TAC TCG CTC ACG CAC GAC CCG GTC TCG TTC TCC CAC CTG AAG ACG CCGTTC CCG ATG GTG GAC TAC TCG AAG GGA GTC TCG CGG GGA GTG CCG CAC CAC AAG GACGTG CAC TGG ATG CAC GGA CGG GTC CTC TTC TTG AAG TGG TGG CGG GGA CGG TAG ACGGTG CTG CCG TTC CGG GTG AAG GGA TCC CTC CCG CAC AAG CAC TCG TTG CCG TGG GTGACC AAG CAC TGG GTC TCC TTG AAG ATG CTC GGA GTC TAG TAG TGG TGG CTG TTG TGGAAG CAC TCG CCG TTG ACG CTG CAC CAC TAG CCG TAG CAC TTG TTG TGG CAC ATG CTGGGA GAC GTC GGA CTC GAC CTG TCG AAG TTC CTC CTC GAC CTG TTC ATG AAG TTC TTGGTG TGG TCG GGA CTG CAC CTG GAC CCG CTG TAG TCG CCG TAG TTG CGG TCG CAC CACTTG TAG GTC TTC CTC TAG CTG TCC GAC TTG CTC CAC CGG TTC TTG GAC TTG CTC TCGGAC TAG CTG GAC GTC CTC GAC CCG TTC ATG CTC GTC ATG TAG TTC ACC GGA ACC ATGTAG ACC GAC CCG AAG TAG CGG CCG GAC TAG CGG TAG CAC TAC CAC TGG TAG TAC GACACG ACG TAC TGG TCG ACG ACG TCG ACG GAC TTC CCG ACG ACG TCG CGG ATG CAC TCCTTC CTG CCG CTC ACC CAC AAG GAC ACT
SEQ ID No.13：编码融合前构型S蛋白序列的DNA
AAG CAC AAG GAC CAC GAC GAC GGA GAC CAC TCG TCG GTC ACA CAC TTG GACTGT TGT TCT TGT GTC GAC GGA GGA CGA ATG TGT TTG TCG AAG TGT TCT CCT CAC ATGATG GGA CTG TTC CAC AAG TCT TCG TCG CAC GAC GTG TCG TGT GTC CTG GAC AAG GACGGA AAG AAG TCG TTG CAC TGT ACC AAG GTG CGA TAG GTG CAC TCG CCT TGT TTG CCTTGT TTC TCT AAG CTG TTG GGA CAC GAC GGA AAG TTG CTG CCT CAC ATG AAG CGA TCGTGT CTC TTC TCG TTG TAG TAG TCT CCT ACC TAG AAG CCT TGT TGT GAC CTG TCG TTCTGT GTC TCG GAC GAC TAG CAC TTG TTG CGA TGT TTG CAC CAC TAG TTC CAC ACA CTCAAG GTC AAG ACA TTG CTG GGA AAG GAC CCT CAC ATG ATG GTG TTC TTG TTG TTC TCGACC TAC CTC TCG CTC AAG TCT CAC ATG TCG TCG CGA TTG TTG ACA TGT AAG CTC ATGCAC TCG GTC GGA AAG GAC TAC CTG GAC CTC CCT TTC GTC CCT TTG AAG TTC TTG GACTCT CTC AAG CAC AAG TTC TTG TAG CTG CCT ATG AAG TTC TAG ATG TCG TTC GTG TGTGGA TAG TTG GAC CAC TCT CTG GAC GGA GTC CCT AAG TCG CGA GAC CTC GGA GAC CACCTG GAC GGA TAG CCT TAG TTG TAG TGT TCT AAG GTC TGT GAC GAC CGA GAC GTG TCTTCG ATG GAC TGT GGA CCT CTG TCG TCG TCG CCT ACC TGT CGA CCT CGA CGA CGA ATGATG CAC CCT ATG GAC GTC GGA TCT TGT AAG GAC GAC TTC ATG TTG CTC TTG CCT TGTTAG TGT CTG CGA CAC CTG ACA CGA GAC CTG GGA GAC TCG CTC TGT TTC ACA TGT GACTTC TCG AAG TGT CAC CTC TTC CCT TAG ATG GTC TGT TCG TTG AAG TCT CAC GTC GGATGT CTC TCG TAG CAC TCT AAG GGA TTG TAG TGT TTG GAC ACA GGA AAG CCT CTC CACAAG TTG CGA TGT TCT AAG CGA TCG CAC ATG CGA ACC TTG TCT TTC TCT TAG TCG TTGACA CAC CGA CTG ATG TCG CAC GAC ATG TTG TCG CGA TCG AAG TCG TGT AAG TTC ACAATG CCT CAC TCG GGA TGT TTC GAC TTG CTG GAC ACA AAG TGT TTG CAC ATG CGA CTGTCG AAG CAC TAG TCT CCT CTG CTC CAC TCT GTC TAG CGA GGA CCT GTC TGT CCT TTCTAG CGA CTG ATG TTG ATG TTC GAC GGA CTG CTG AAG TGT CCT ACA CAC TAG CGA ACCTTG TCG TTG TTG GAC CTG TCG TTC CAC CCT CCT TTG ATG TTG ATG GAC ATG TCT GACAAG TCT TTC TCG TTG GAC TTC GGA AAG CTC TCT CTG TAG TCG TGT CTC TAG ATG GTCCGA CCT TCG TGT GGA ACA TTG CCT CAC CTC CCT AAG TTG ACA ATG AAG GGA GAC GTCTCG ATG CCT AAG GTC GGA TGT TTG CCT CAC CCT ATG GTC GGA ATG TCT CAC CAC CACGAC TCG AAG CTC GAC GAC GTG CGA GGA CGA TGT CAC ACA CCT GGA TTC TTC TCG TGTTTG GAC CAC TTC TTG TTC ACA CAC TTG AAG TTG AAG TTG CCT GAC TGT CCT TGT CCTCAC GAC TGT CTC TCG TTG TTC TTC AAG GAC GGA AAG GTC GTC AAG CCT TCT CTG TAGCGA CTG TGT TGT CTG CGA CAC TCT CTG GGA GTC TGT GAC CTC TAG GAC CTG TAG TGTGGA ACA TCG AAG CCT CCT CAC TCG CAC TAG TGT GGA CCT TGT TTG TGT TCG TTG GTCCAC CGA CAC GAC ATG GTC CTG CAC TTG ACA TGT CTC CAC GGA CAC CGA TAG GTG CGACTG GTC GAC TGT GGA TGT ACC TCT CAC ATG TCG TGT CCT TCG TTG CAC AAG GTC TGTTCT CGA CCT ACA GAC TAG CCT CGA CTC GTG CAC TTG TTG TCG ATG CTC ACA CTG TAGGGA TAG CCT CGA CCT TAG ACA CGA TCG ATG GTC TGT GTC TGT TTG TCG GGA TCT TCTCGA TCT TCG CAC CGA TCG GTC TCG TAG TAG CGA ATG TGT TAC TCG GAC CCT CGA CTCTTG TCG CAC CGA ATG TCG TTG TTG TCG TAG CGA TAG GGA TGT TTG AAG TGT TAG TCGCAC TGT TGT CTC TAG GAC GGA CAC TCG TAC TGT TTC TGT TCG CAC CTG ACA TGT TACATG TAG ACA CCT CTG TCG TGT CTC ACA TCG TTG GAC GAC GAC GTC ATG CCT TCG AAGACA TGT GTC GAC TTG TCT CGA GAC TGT CCT TAG CGA CAC CTC GTC CTG TTC TTG TGTGTC CTC CAC AAG CGA GTC CAC TTC GTC TAG ATG TTC TGT GGA GGA TAG TTC CTG AAGCCT CCT AAG TTG AAG TCG GTC TAG GAC GGA CTG GGA TCG TTC GGA TCG TTC TCT TCGAAG TAG CTC CTG GAC GAC AAG TTG TTC CAC TGT GAC CGA CTG CGA CCT AAG TAG TTCGTC ATG CCT CTG ACA GAC CCT CTG TAG CGA CGA TCT CTG GAC TAG ACA CGA GTC TTCAAG TTG CCT GAC TGT CAC GAC GGA GGA GAC GAC TGT CTG CTC TAC TAG CGA GTC ATGTGT TCG CGA GAC GAC CGA CCT TGT TAG TGT TCG CCT ACC TGT AAG CCT CGA CCT CGACGA GAC GTC TAG GGA AAG CGA TAC GTC TAC CGA ATG TCT AAG TTG CCT TAG CCT CACTGT GTC TTG CAC GAC ATG CTC TTG GTC TTC GAC TAG CGA TTG GTC AAG TTG TCG CGATAG CCT TTC TAG GTC CTG TCG GAC TCG TCG TGT CGA TCG CGA GAC CCT TTC GAC GTCCTG CAC CAC TTG GTC TTG CGA GTC CGA GAC TTG TGT GAC CAC TTC GTC GAC TCG TCGTTG AAG CCT CGA TAG TCG TCG CAC GAC TTG CTG TAG GAC TCG TCT GAC CTG TTC CACCTC CGA CTC CAC GTC TAG CTG TCT GAC TAG TGT CCT TCT GAC GTC TCG GAC GTC TGTATG CAC TGT GTC GTC GAC TAG TCT CGA CGA CTC TAG TCT CGA TCG CGA TTG GAC CGACGA TGT TTC TAC TCG CTC ACA CAC GAC CCT GTC TCG TTC TCT CAC CTG AAG ACA CCTTTC CCT ATG GTG GAC TAC TCG AAG GGA GTC TCG CGA GGA GTG CCT CAC CAC AAG GACGTG CAC TGT ATG CAC GGA CGA GTC CTC TTC TTG AAG TGT TGT CGA GGA CGA TAG ACAGTG CTG CCT TTC CGA GTG AAG GGA TCT CTC CCT CAC AAG CAC TCG TTG CCT TGT GTGACC AAG CAC TGT GTC TCT TTG AAG ATG CTC GGA GTC TAG TAG TGT TGT CTG TTG TGTAAG CAC TCG CCT TTG ACA CTG CAC CAC TAG CCT TAG CAC TTG TTG TGT CAC ATG CTGGGA GAC GTC GGA CTC GAC CTG TCG AAG TTC CTC CTC GAC CTG TTC ATG AAG TTC TTGGTG TGT TCG GGA CTG CAC CTG GAC CCT CTG TAG TCG CCT TAG TTG CGA TCG CAC CACTTG TAG GTC TTC CTC TAG CTG TCT GAC TTG CTC CAC CGA TTC TTG GAC TTG CTC TCGGAC TAG CTG GAC GTC CTC GAC CCT TTC ATG CTC GTC ATG TAG TTC ACC GGA ACC ATGTAG ACC GAC CCT AAG TAG CGA CCT GAC TAG CGA TAG CAC TAC CAC TGT TAG TAC GACACA ACA TAC TGT TCG ACA ACA TCG ACA GAC TTC CCT ACA ACA TCG CGA ATG CAC TCTTTC CTG CCT CTC ACC CAC AAG GAC ACT
实施例1
293T细胞传代后约24小时，观察6孔板内的细胞状态，汇合度在90％左右即可进行转染。取200μl opti-MEM，加入10μg经密码子优化后编码S蛋白抗原片段的不同mRNA，用枪头轻轻吹打混匀，再加入60μl PEI(浓度1mg/ml)，立即置于漩涡振荡器上振荡10次，每次1s，充分混匀，静置10min。将配制好的转染体系，直接均匀滴加进入培养的细胞中，再前后左右摇匀，使得转染体系均匀分布于细胞上。转染后6h换液，吸掉旧的培养基，每孔换为2ml新鲜培养基(90％DMEM+10％FBS)。转染后30h收获。吸掉旧的培养基，用1ml PBS清洗一遍。吸掉PBS，继续用1ml PBS将细胞吹打下来，收集于1.5ml 离心管中，300g离心5min。将离心后的上清尽量吸去干净，沉淀的细胞进行裂解，提取总蛋白进行Western blot检测。电泳转膜后进行抗体孵育，S蛋白一抗孵育：取3μL SARS-CoV-2(2019-nCoV)Spike Antibody(义翘神州，P/N 40591-MM42)稀释到3ml 5％脱脂牛奶中(稀释比1:1000)，置于4℃摇床过夜孵育,内参蛋白抗体为GAPDH(proteintech，P/N 60004-1-Ig)；二抗孵育：S蛋白和GAPDH蛋白的抗体均是Mouse来源的，因此用相同的二抗：取1μLHRP-conjugated Affinipure GoatAnti-Mouse IgG(H+L)(proteintech，P/N SA00001-1)稀释到5ml 5％脱脂牛奶中(稀释比1:5000)，常温孵育1h；
实验结果如图2所示，其中，M：蛋白marker，1#：SEQ ID No.2编码的融合前构型S蛋白，2#：SEQ ID No.3编码的融合前构型S蛋白，3#：SEQ ID No.4编码的融合前构型S(prefusion)蛋白，4#：SEQ ID No.5编码的融合前构型S(prefusion)蛋白。
结果显示，采用SEQ ID No.4编码的融合前构型S蛋白表达量明显高于SEQ IDNo.2、SEQ ID No.3和SEQ ID No.5编码的融合前构型S蛋白。
实施例2
本实施例中，带有不同佐剂序列(SEQ ID No.6-9)的mRNA(SEQ ID No.4)分别转染293T细胞24小时后提取细胞上清，见此上清中S蛋白外泌水平。具体步骤如下：
1、包被：将鼠抗S蛋白单抗SARS-CoV-2(2019-nCoV)Spike Antibody(义翘神州，P/N 40591-MM42)用coating buffer稀释成1000x的溶液，加入到酶标板中，每孔加入的体积为100ul，每个稀释度重复3个孔，盖上封板膜，4℃过夜包被。
2、洗板：包被好的96孔板，倒出包被液，到扣吸水纸上，使劲扣板，直到孔中无残留为止。配制洗脱液，用去离子水稀释50x的Washing buffer，加入到洗板机的进液瓶中，设置程序，每孔洗板体积设为300ul，重复洗4次。
3、封闭：洗好的板，扣干里面的溶液，按每孔250ul的体积加入Blocking buffer，随后封上封板膜，室温封闭2h。
4、洗板：封闭好的酶标板按照步骤3完成洗板。
5、血清或标准品孵育：将血清或标准品(义翘神州，40591-V08H)梯度稀释，按照每孔100ul的体积加入洗好的96孔板中，随后封上封板膜室温下孵育1.5h。
6、洗板：按照步骤3完成洗板其中洗板次数增加为6次。
7、一抗孵育：用dilution buffer稀释兔抗S蛋白抗体SARS-CoV-2(2019-nCoV)Spike Antibody(义翘神州，P/N 40589-T62)(1：5000)，按照每孔100ul的体积加入洗好的96孔板中，随后封上封板膜室温下孵育1.5h。
8、洗板：按照步骤3完成洗板其中洗板次数增加为6次。
9、加入二抗：用Dilution buffer 稀释HRP标记山羊抗兔IgG(abcam，ab6721)，稀释倍数为10000x，稀释好的抗体按照每孔100ul的体积加入酶标板中，封上封板膜，室温下避光孵育1h。
10、洗板：按照步骤8完成洗板，此步骤务必洗干净板，扣干溶液。
11、显色：加入TMB buffer 100ul，避光显色20-30分钟，此时阳性样品显蓝色。
12、终止：加入Stop buffer 100ul，10分钟内在酶标板上读数，设置吸收波长450nm。
13、根据标准品孔光吸收值绘制标准曲线，线性回归的相关系数应大于0.0995。根据供试品吸光度的大小计算样品中S蛋白的含量。
实验结果如图3所示。其中，Control：对照组mRNA(SEQ ID No.4)，1#：带有SEQ IDNo.6佐剂序列的mRNA(SEQ ID No.4)，2#：带有SEQ ID No.7佐剂序列的mRNA(SEQ IDNo.4)，3#：带有SEQ ID No.8佐剂序列的mRNA(SEQ ID No.4)，4#：带有SEQ ID No.9佐剂序列的mRNA(SEQ ID No.4)。带有SEQ ID No.7佐剂序列的mRNA(SEQ ID No.4)所编码的融合前构型S蛋白外泌效果明显高于带有SEQ ID No.6/SEQ ID No.8/SEQ ID No.9佐剂序列的mRNA(SEQ ID No.4)所编码的融合前构型S蛋白。
实施例3
本实施例中，将分别带有SEQ ID No.5-9佐剂序列的mRNA(SEQ ID No.4)分别在第1天和第14天两次注射接种6周龄balb/c小鼠，在第14天和第35天取小鼠血清，检测血清中抗S蛋白特异性抗体的滴度。
1、包被：将S蛋白(义翘神州，40591-V08H)用coating buffer稀释成200ng/ml的溶液，加入到酶标板中，每孔加入的体积为100ul，每个稀释度重复3个孔，盖上封板膜，4℃过夜包被。
2、洗板：包被好的96孔板，倒出包被液，到扣吸水纸上，使劲扣板，直到孔中无残留为止。配制洗脱液，用去离子水稀释50x的Washing buffer，加入到洗板机的进液瓶中，设置程序，每孔洗板体积设为300ul，重复洗4次。
3、封闭：洗好的板，扣干里面的溶液，按每孔250ul的体积加入Blocking buffer，随后封上封板膜，室温封闭2h。
4、洗板：封闭好的酶标板按照步骤3完成洗板。
5、血清孵育：用dilution buffer将小鼠血清稀释成40x，400x，4000x，40000x，400000x，4000000x，40000000x，按照每孔100ul的体积加入洗好的96孔板中，随后封上封板膜室温下孵育1.5h。
6、洗板：按照步骤3完成洗板其中洗板次数增加为6次。
7、加入二抗：用Dilution buffer 稀释HRP标记山羊抗小鼠IgG，稀释倍数为10000x，稀释好的抗体按照每孔100ul的体积加入酶标板中，封上封板膜，室温下避光孵育1h。
8、洗板：按照步骤8完成洗板，此步骤务必洗干净板，扣干溶液。
9、显色：加入TMB buffer 100ul，避光显色20-30分钟，此时阳性样品显蓝色。
10、终止：加入Stop buffer 100ul，10分钟内在酶标板上读数，设置吸收波长450nm。
根据标准品孔光吸收值绘制标准曲线，线性回归的相关系数应大于0.0995。根据供试品吸光度的大小计算样品中S蛋白特异性抗体的残留含量。
实验结果如图4所示，其中Control：对照组mRNA(SEQ ID No.4)，1#：带有SEQ IDNo.6佐剂序列的mRNA(SEQ ID No.4)，2#：带有SEQ ID No.7佐剂序列的mRNA(SEQ IDNo.4)，3#：带有SEQ ID No.8佐剂序列的mRNA(SEQ ID No.4)，4#：带有SEQ ID No.9佐剂序列的mRNA(SEQ ID No.4)。
结果显示，带有SEQ ID No.9佐剂序列的mRNA(SEQ ID No.4)候选疫苗免疫效果优于带有SEQ ID No.6/SEQ ID No.7/SEQ ID No.8佐剂序列的mRNA(SEQ ID No.4)的候选疫苗。
实施例4
在本实施例中，将带有SEQ ID No.9佐剂序列的mRNA(SEQ ID No.4)分别以低剂量(1ug/只)和高剂量(30ug/只)在第1天和第14天两次注射接种6周龄balb/c小鼠，在第35天取小鼠血清，通过focus reduction neutralization tests(FRNT)实验检测中和抗体滴度。通过将标准剂量的新冠病毒2019-nCoV与56℃灭活血清的两倍连续稀释液混合在37℃下孵育1小时。然后将病毒-血清混合物(100μl)接种到培养的293T ACE2过表达细胞。孵育3天后，通过与生物素偶联的S蛋白单克隆抗体，抗生蛋白链菌素-HRP和TrueBlue过氧化物酶底物(KPL)进行连续孵育，将板进行福尔马林固定，透化，封闭和染色。未加血清的阳性对照组焦点数目最高，实验组焦点计数≤50％时做为中和临界值，并且计算了几何平均值作为技术重复。
结果如图5显示，其中，图片A：带有SEQ ID No.9佐剂序列的mRNA(SEQ ID No.4)低剂量组(1ug/只)，图片B：带有SEQ ID No.9佐剂序列的mRNA(SEQ ID No.4)高剂量组(30ug/只)。中和抗体滴度与候选疫苗接种剂量成正比例关系，高剂量(30ug/只)组中和抗体保护效果ID50＝260.4明显优于低剂量(1ug/只)组中和抗体保护效果ID50＝41.03。
序列表
<110> 深圳先进技术研究院
<120> 新型冠状病毒mRNA疫苗及其制备方法与应用
<130> GAI20CN2857
<160> 13
<170> PatentIn version 3.5
<210> 1
<211> 1261
<212> PRT
<213> Artificial Sequence
<220>
<223> 针对新型冠状病毒（SARS-CoV-2）的抗原
<400> 1
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val
1 5 10 15
Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe
20 25 30
Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu
35 40 45
His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp
50 55 60
Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp
65 70 75 80
Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu
85 90 95
Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser
100 105 110
Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile
115 120 125
Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr
130 135 140
Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr
145 150 155 160
Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu
165 170 175
Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe
180 185 190
Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr
195 200 205
Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu
210 215 220
Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr
225 230 235 240
Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser
245 250 255
Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro
260 265 270
Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala
275 280 285
Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys
290 295 300
Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val
305 310 315 320
Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys
325 330 335
Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala
340 345 350
Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu
355 360 365
Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro
370 375 380
Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe
385 390 395 400
Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly
405 410 415
Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys
420 425 430
Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn
435 440 445
Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe
450 455 460
Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys
465 470 475 480
Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly
485 490 495
Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val
500 505 510
Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys
515 520 525
Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn
530 535 540
Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu
545 550 555 560
Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val
565 570 575
Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe
580 585 590
Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val
595 600 605
Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile
610 615 620
His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser
625 630 635 640
Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val
645 650 655
Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala
660 665 670
Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala
675 680 685
Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser
690 695 700
Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile
705 710 715 720
Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val
725 730 735
Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu
740 745 750
Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr
755 760 765
Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln
770 775 780
Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe
785 790 795 800
Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser
805 810 815
Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly
820 825 830
Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp
835 840 845
Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu
850 855 860
Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly
865 870 875 880
Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile
885 890 895
Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr
900 905 910
Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn
915 920 925
Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala
930 935 940
Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn
945 950 955 960
Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val
965 970 975
Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln
980 985 990
Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val
995 1000 1005
Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn
1010 1015 1020
Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys
1025 1030 1035
Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro
1040 1045 1050
Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val
1055 1060 1065
Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His
1070 1075 1080
Asp Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn
1085 1090 1095
Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln
1100 1105 1110
Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val
1115 1120 1125
Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro
1130 1135 1140
Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn
1145 1150 1155
His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn
1160 1165 1170
Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu
1175 1180 1185
Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu
1190 1195 1200
Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu
1205 1210 1215
Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met
1220 1225 1230
Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys
1235 1240 1245
Ser Ala Tyr Val Arg Lys Asp Gly Glu Trp Val Phe Leu
1250 1255 1260
<210> 2
<211> 3783
<212> RNA
<213> Artificial Sequence
<220>
<223> 编码融合前构型S蛋白序列的mRNA
<400> 2
aagcacaagg accacgacga cggggaccac ucgucgguca cgcacuugga cuggugggcc 60
ugggucgacg gggggcggau gugguugucg aagugggccc cgcacaugau ggggcuguuc 120
cacaaggccu cgucgcacga cgugucgugg guccuggaca aggacgggaa gaagucguug 180
cacuggacca aggugcggua ggugcacucg ccgugguugc cgugguucgc caagcuguug 240
gggcacgacg ggaaguugcu gccgcacaug aagcggucgu ggcucuucuc guuguaguag 300
gccccgaccu agaagccgug gugggaccug ucguucuggg ucucggacga cuagcacuug 360
uugcgguggu ugcaccacua guuccacacg cucaagguca agacguugcu ggggaaggac 420
ccgcacauga ugguguucuu guuguucucg accuaccucu cgcucaaggc ccacaugucg 480
ucgcgguugu ugacguggaa gcucaugcac ucggucggga aggacuaccu ggaccucccg 540
uucgucccgu ugaaguucuu ggacgcccuc aagcacaagu ucuuguagcu gccgaugaag 600
uucuagaugu cguucgugug gggguaguug gaccacgccc uggacggggu cccgaagucg 660
cgggaccucg gggaccaccu ggacggguag ccguaguugu agugggccaa ggucugggac 720
gaccgggacg uggccucgau ggacuggggg ccgcugucgu cgucgccgac cuggcggccg 780
cggcggcgga ugaugcaccc gauggacguc ggggccugga aggacgacuu cauguugcuc 840
uugccguggu aguggcugcg gcaccugacg cgggaccugg gggacucgcu cugguucacg 900
ugggacuucu cgaaguggca ccucuucccg uagauggucu ggucguugaa ggcccacguc 960
ggguggcucu cguagcacgc caagggguug uagugguugg acacggggaa gccgcuccac 1020
aaguugcggu gggccaagcg gucgcacaug cggaccuugg ccuucgccua gucguugacg 1080
caccggcuga ugucgcacga cauguugucg cggucgaagu cguggaaguu cacgaugccg 1140
cacucggggu gguucgacuu gcuggacacg aagugguugc acaugcggcu gucgaagcac 1200
uaggccccgc ugcuccacgc cgucuagcgg gggccggucu ggccguucua gcggcugaug 1260
uugauguucg acgggcugcu gaaguggccg acgcacuagc ggaccuuguc guuguuggac 1320
cugucguucc acccgccguu gauguugaug gacauggccg acaaggccuu cucguuggac 1380
uucgggaagc ucgcccugua gucguggcuc uagauggucc ggccgucgug ggggacguug 1440
ccgcaccucc cgaaguugac gaugaagggg gacgucucga ugccgaaggu cgggugguug 1500
ccgcacccga uggucgggau ggcccaccac cacgacucga agcucgacga cgugcggggg 1560
cgguggcaca cgccgggguu cuucucgugg uuggaccacu ucuuguucac gcacuugaag 1620
uugaaguugc cggacuggcc guggccgcac gacuggcucu cguuguucuu caaggacggg 1680
aaggucguca agccggcccu guagcggcug ugguggcugc ggcacgcccu gggggucugg 1740
gaccucuagg accuguagug ggggacgucg aagccgccgc acucgcacua gugggggccg 1800
ugguuguggu cguuggucca ccggcacgac augguccugc acuugacgug gcuccacggg 1860
caccgguagg ugcggcuggu cgacuggggg uggaccgccc acaugucgug gccgucguug 1920
cacaaggucu gggcccggcc gacggacuag ccgcggcucg ugcacuuguu gucgaugcuc 1980
acgcuguagg gguagccgcg gccguagacg cggucgaugg ucugggucug guugucgggg 2040
gccgcccggg ccucgcaccg gucggucucg uaguagcgga ugugguacuc ggacccgcgg 2100
cucuugucgc accggauguc guuguugucg uagcgguagg ggugguugaa gugguagucg 2160
cacugguggc ucuaggacgg gcacucguac ugguucuggu cgcaccugac gugguacaug 2220
uagacgccgc ugucguggcu cacgucguug gacgacgacg ucaugccguc gaagacgugg 2280
gucgacuugg cccgggacug gccguagcgg caccucgucc uguucuugug gguccuccac 2340
aagcgggucc acuucgucua gauguucugg gggggguagu uccugaagcc gccgaaguug 2400
aagucggucu aggacgggcu ggggucguuc gggucguucg ccucgaagua gcuccuggac 2460
gacaaguugu uccacuggga ccggcugcgg ccgaaguagu ucgucaugcc gcugacggac 2520
ccgcuguagc ggcgggcccu ggacuagacg cgggucuuca aguugccgga cuggcacgac 2580
gggggggacg acuggcugcu cuacuagcgg gucauguggu cgcgggacga ccggccgugg 2640
uaguggucgc cgaccuggaa gccgcggccg cggcgggacg ucuaggggaa gcgguacguc 2700
uaccggaugg ccaaguugcc guagccgcac ugggucuugc acgacaugcu cuuggucuuc 2760
gacuagcggu uggucaaguu gucgcgguag ccguucuagg uccugucgga cucgucgugg 2820
cggucgcggg acccguucga cguccugcac cacuuggucu ugcggguccg ggacuugugg 2880
gaccacuucg ucgacucguc guugaagccg cgguagucgu cgcacgacuu gcuguaggac 2940
ucggccgacc uguuccaccu ccggcuccac gucuagcugg ccgacuagug gccggccgac 3000
gucucggacg ucuggaugca cugggucguc gacuaggccc ggcggcucua ggcccggucg 3060
cgguuggacc ggcggugguu cuacucgcuc acgcacgacc cggucucguu cgcccaccug 3120
aagacgccgu ucccgauggu ggacuacucg aagggggucu cgcggggggu gccgcaccac 3180
aaggacgugc acuggaugca cgggcggguc cucuucuuga agugguggcg ggggcgguag 3240
acggugcugc cguuccgggu gaagggggcc cucccgcaca agcacucguu gccgugggug 3300
accaagcacu gggucgccuu gaagaugcuc ggggucuagu agugguggcu guuguggaag 3360
cacucgccgu ugacgcugca ccacuagccg uagcacuugu uguggcacau gcugggggac 3420
gucgggcucg accugucgaa guuccuccuc gaccuguuca ugaaguucuu gguguggucg 3480
gggcugcacc uggacccgcu guagucgccg uaguugcggu cgcaccacuu guaggucuuc 3540
cucuagcugg ccgacuugcu ccaccgguuc uuggacuugc ucucggacua gcuggacguc 3600
cucgacccgu ucaugcucgu cauguaguuc accgggacca uguagaccga cccgaaguag 3660
cggccggacu agcgguagca cuaccacugg uaguacgaca cgacguacug gucgacgacg 3720
ucgacggacu ucccgacgac gucgcggaug cacgccuucc ugccgcucac ccacaaggac 3780
acu 3783
<210> 3
<211> 3783
<212> RNA
<213> Artificial Sequence
<220>
<223> 编码融合前构型S蛋白序列的mRNA
<400> 3
aagcacaagg accacgacga cggggaccac ucgucgguca cgcacuugga cugguggucc 60
ugggucgacg gggggcggau gugguugucg aagugguccc cgcacaugau ggggcuguuc 120
cacaaguccu cgucgcacga cgugucgugg guccuggaca aggacgggaa gaagucguug 180
cacuggacca aggugcggua ggugcacucg ccgugguugc cgugguucuc caagcuguug 240
gggcacgacg ggaaguugcu gccgcacaug aagcggucgu ggcucuucuc guuguaguag 300
uccccgaccu agaagccgug gugggaccug ucguucuggg ucucggacga cuagcacuug 360
uugcgguggu ugcaccacua guuccacacg cucaagguca agacguugcu ggggaaggac 420
ccgcacauga ugguguucuu guuguucucg accuaccucu cgcucaaguc ccacaugucg 480
ucgcgguugu ugacguggaa gcucaugcac ucggucggga aggacuaccu ggaccucccg 540
uucgucccgu ugaaguucuu ggacucccuc aagcacaagu ucuuguagcu gccgaugaag 600
uucuagaugu cguucgugug gggguaguug gaccacuccc uggacggggu cccgaagucg 660
cgggaccucg gggaccaccu ggacggguag ccguaguugu agugguccaa ggucugggac 720
gaccgggacg uguccucgau ggacuggggg ccgcugucgu cgucgccgac cuggcggccg 780
cggcggcgga ugaugcaccc gauggacguc ggguccugga aggacgacuu cauguugcuc 840
uugccguggu aguggcugcg gcaccugacg cgggaccugg gggacucgcu cugguucacg 900
ugggacuucu cgaaguggca ccucuucccg uagauggucu ggucguugaa gucccacguc 960
ggguggcucu cguagcacuc caagggguug uagugguugg acacggggaa gccgcuccac 1020
aaguugcggu gguccaagcg gucgcacaug cggaccuugu ccuucuccua gucguugacg 1080
caccggcuga ugucgcacga cauguugucg cggucgaagu cguggaaguu cacgaugccg 1140
cacucggggu gguucgacuu gcuggacacg aagugguugc acaugcggcu gucgaagcac 1200
uaguccccgc ugcuccacuc cgucuagcgg gggccggucu ggccguucua gcggcugaug 1260
uugauguucg acgggcugcu gaaguggccg acgcacuagc ggaccuuguc guuguuggac 1320
cugucguucc acccgccguu gauguugaug gacauguccg acaaguccuu cucguuggac 1380
uucgggaagc ucucccugua gucguggcuc uagauggucc ggccgucgug ggggacguug 1440
ccgcaccucc cgaaguugac gaugaagggg gacgucucga ugccgaaggu cgggugguug 1500
ccgcacccga uggucgggau gucccaccac cacgacucga agcucgacga cgugcggggg 1560
cgguggcaca cgccgggguu cuucucgugg uuggaccacu ucuuguucac gcacuugaag 1620
uugaaguugc cggacuggcc guggccgcac gacuggcucu cguuguucuu caaggacggg 1680
aaggucguca agccgucccu guagcggcug ugguggcugc ggcacucccu gggggucugg 1740
gaccucuagg accuguagug ggggacgucg aagccgccgc acucgcacua gugggggccg 1800
ugguuguggu cguuggucca ccggcacgac augguccugc acuugacgug gcuccacggg 1860
caccgguagg ugcggcuggu cgacuggggg uggaccuccc acaugucgug gccgucguug 1920
cacaaggucu ggucccggcc gacggacuag ccgcggcucg ugcacuuguu gucgaugcuc 1980
acgcuguagg gguagccgcg gccguagacg cggucgaugg ucugggucug guugucgggg 2040
uccucccggu ccucgcaccg gucggucucg uaguagcgga ugugguacuc ggacccgcgg 2100
cucuugucgc accggauguc guuguugucg uagcgguagg ggugguugaa gugguagucg 2160
cacugguggc ucuaggacgg gcacucguac ugguucuggu cgcaccugac gugguacaug 2220
uagacgccgc ugucguggcu cacgucguug gacgacgacg ucaugccguc gaagacgugg 2280
gucgacuugu cccgggacug gccguagcgg caccucgucc uguucuugug gguccuccac 2340
aagcgggucc acuucgucua gauguucugg gggggguagu uccugaagcc gccgaaguug 2400
aagucggucu aggacgggcu ggggucguuc gggucguucu ccucgaagua gcuccuggac 2460
gacaaguugu uccacuggga ccggcugcgg ccgaaguagu ucgucaugcc gcugacggac 2520
ccgcuguagc ggcggucccu ggacuagacg cgggucuuca aguugccgga cuggcacgac 2580
gggggggacg acuggcugcu cuacuagcgg gucauguggu cgcgggacga ccggccgugg 2640
uaguggucgc cgaccuggaa gccgcggccg cggcgggacg ucuaggggaa gcgguacguc 2700
uaccggaugu ccaaguugcc guagccgcac ugggucuugc acgacaugcu cuuggucuuc 2760
gacuagcggu uggucaaguu gucgcgguag ccguucuagg uccugucgga cucgucgugg 2820
cggucgcggg acccguucga cguccugcac cacuuggucu ugcggguccg ggacuugugg 2880
gaccacuucg ucgacucguc guugaagccg cgguagucgu cgcacgacuu gcuguaggac 2940
ucguccgacc uguuccaccu ccggcuccac gucuagcugu ccgacuagug gccguccgac 3000
gucucggacg ucuggaugca cugggucguc gacuaguccc ggcggcucua gucccggucg 3060
cgguuggacc ggcggugguu cuacucgcuc acgcacgacc cggucucguu cucccaccug 3120
aagacgccgu ucccgauggu ggacuacucg aagggggucu cgcggggggu gccgcaccac 3180
aaggacgugc acuggaugca cgggcggguc cucuucuuga agugguggcg ggggcgguag 3240
acggugcugc cguuccgggu gaaggggucc cucccgcaca agcacucguu gccgugggug 3300
accaagcacu gggucuccuu gaagaugcuc ggggucuagu agugguggcu guuguggaag 3360
cacucgccgu ugacgcugca ccacuagccg uagcacuugu uguggcacau gcugggggac 3420
gucgggcucg accugucgaa guuccuccuc gaccuguuca ugaaguucuu gguguggucg 3480
gggcugcacc uggacccgcu guagucgccg uaguugcggu cgcaccacuu guaggucuuc 3540
cucuagcugu ccgacuugcu ccaccgguuc uuggacuugc ucucggacua gcuggacguc 3600
cucgacccgu ucaugcucgu cauguaguuc accgggacca uguagaccga cccgaaguag 3660
cggccggacu agcgguagca cuaccacugg uaguacgaca cgacguacug gucgacgacg 3720
ucgacggacu ucccgacgac gucgcggaug cacuccuucc ugccgcucac ccacaaggac 3780
acu 3783
<210> 4
<211> 3783
<212> RNA
<213> Artificial Sequence
<220>
<223> 编码融合前构型S蛋白序列的mRNA
<400> 4
aagcacaagg accacgacga cggagaccac ucgucgguca cgcacuugga cugguggucc 60
ugggucgacg gaggacggau gugguugucg aagugguccc cgcacaugau gggacuguuc 120
cacaaguccu cgucgcacga cgugucgugg guccuggaca aggacggaaa gaagucguug 180
cacuggacca aggugcggua ggugcacucg ccgugguugc cgugguucuc caagcuguug 240
ggacacgacg gaaaguugcu gccgcacaug aagcggucgu ggcucuucuc guuguaguag 300
uccccgaccu agaagccgug gugggaccug ucguucuggg ucucggacga cuagcacuug 360
uugcgguggu ugcaccacua guuccacacg cucaagguca agacguugcu gggaaaggac 420
ccgcacauga ugguguucuu guuguucucg accuaccucu cgcucaaguc ccacaugucg 480
ucgcgguugu ugacguggaa gcucaugcac ucggucggaa aggacuaccu ggaccucccg 540
uucgucccgu ugaaguucuu ggacucccuc aagcacaagu ucuuguagcu gccgaugaag 600
uucuagaugu cguucgugug gggauaguug gaccacuccc uggacggagu cccgaagucg 660
cgggaccucg gagaccaccu ggacggauag ccguaguugu agugguccaa ggucugggac 720
gaccgggacg uguccucgau ggacugggga ccgcugucgu cgucgccgac cuggcggccg 780
cggcggcgga ugaugcaccc gauggacguc ggauccugga aggacgacuu cauguugcuc 840
uugccguggu aguggcugcg gcaccugacg cgggaccugg gagacucgcu cugguucacg 900
ugggacuucu cgaaguggca ccucuucccg uagauggucu ggucguugaa gucccacguc 960
ggauggcucu cguagcacuc caagggauug uagugguugg acacgggaaa gccgcuccac 1020
aaguugcggu gguccaagcg gucgcacaug cggaccuugu ccuucuccua gucguugacg 1080
caccggcuga ugucgcacga cauguugucg cggucgaagu cguggaaguu cacgaugccg 1140
cacucgggau gguucgacuu gcuggacacg aagugguugc acaugcggcu gucgaagcac 1200
uaguccccgc ugcuccacuc cgucuagcgg ggaccggucu ggccguucua gcggcugaug 1260
uugauguucg acggacugcu gaaguggccg acgcacuagc ggaccuuguc guuguuggac 1320
cugucguucc acccgccguu gauguugaug gacauguccg acaaguccuu cucguuggac 1380
uucggaaagc ucucccugua gucguggcuc uagauggucc ggccgucgug gggaacguug 1440
ccgcaccucc cgaaguugac gaugaaggga gacgucucga ugccgaaggu cggaugguug 1500
ccgcacccga uggucggaau gucccaccac cacgacucga agcucgacga cgugcgggga 1560
cgguggcaca cgccgggauu cuucucgugg uuggaccacu ucuuguucac gcacuugaag 1620
uugaaguugc cggacuggcc guggccgcac gacuggcucu cguuguucuu caaggacgga 1680
aaggucguca agccgucccu guagcggcug ugguggcugc ggcacucccu gggagucugg 1740
gaccucuagg accuguagug gggaacgucg aagccgccgc acucgcacua guggggaccg 1800
ugguuguggu cguuggucca ccggcacgac augguccugc acuugacgug gcuccacgga 1860
caccgguagg ugcggcuggu cgacugggga uggaccuccc acaugucgug gccgucguug 1920
cacaaggucu ggucccggcc gacggacuag ccgcggcucg ugcacuuguu gucgaugcuc 1980
acgcuguagg gauagccgcg gccguagacg cggucgaugg ucugggucug guugucggga 2040
uccucccggu ccucgcaccg gucggucucg uaguagcgga ugugguacuc ggacccgcgg 2100
cucuugucgc accggauguc guuguugucg uagcgguagg gaugguugaa gugguagucg 2160
cacugguggc ucuaggacgg acacucguac ugguucuggu cgcaccugac gugguacaug 2220
uagacgccgc ugucguggcu cacgucguug gacgacgacg ucaugccguc gaagacgugg 2280
gucgacuugu cccgggacug gccguagcgg caccucgucc uguucuugug gguccuccac 2340
aagcgggucc acuucgucua gauguucugg ggaggauagu uccugaagcc gccgaaguug 2400
aagucggucu aggacggacu gggaucguuc ggaucguucu ccucgaagua gcuccuggac 2460
gacaaguugu uccacuggga ccggcugcgg ccgaaguagu ucgucaugcc gcugacggac 2520
ccgcuguagc ggcggucccu ggacuagacg cgggucuuca aguugccgga cuggcacgac 2580
ggaggagacg acuggcugcu cuacuagcgg gucauguggu cgcgggacga ccggccgugg 2640
uaguggucgc cgaccuggaa gccgcggccg cggcgggacg ucuagggaaa gcgguacguc 2700
uaccggaugu ccaaguugcc guagccgcac ugggucuugc acgacaugcu cuuggucuuc 2760
gacuagcggu uggucaaguu gucgcgguag ccguucuagg uccugucgga cucgucgugg 2820
cggucgcggg acccguucga cguccugcac cacuuggucu ugcggguccg ggacuugugg 2880
gaccacuucg ucgacucguc guugaagccg cgguagucgu cgcacgacuu gcuguaggac 2940
ucguccgacc uguuccaccu ccggcuccac gucuagcugu ccgacuagug gccguccgac 3000
gucucggacg ucuggaugca cugggucguc gacuaguccc ggcggcucua gucccggucg 3060
cgguuggacc ggcggugguu cuacucgcuc acgcacgacc cggucucguu cucccaccug 3120
aagacgccgu ucccgauggu ggacuacucg aagggagucu cgcggggagu gccgcaccac 3180
aaggacgugc acuggaugca cggacggguc cucuucuuga agugguggcg gggacgguag 3240
acggugcugc cguuccgggu gaagggaucc cucccgcaca agcacucguu gccgugggug 3300
accaagcacu gggucuccuu gaagaugcuc ggagucuagu agugguggcu guuguggaag 3360
cacucgccgu ugacgcugca ccacuagccg uagcacuugu uguggcacau gcugggagac 3420
gucggacucg accugucgaa guuccuccuc gaccuguuca ugaaguucuu gguguggucg 3480
ggacugcacc uggacccgcu guagucgccg uaguugcggu cgcaccacuu guaggucuuc 3540
cucuagcugu ccgacuugcu ccaccgguuc uuggacuugc ucucggacua gcuggacguc 3600
cucgacccgu ucaugcucgu cauguaguuc accggaacca uguagaccga cccgaaguag 3660
cggccggacu agcgguagca cuaccacugg uaguacgaca cgacguacug gucgacgacg 3720
ucgacggacu ucccgacgac gucgcggaug cacuccuucc ugccgcucac ccacaaggac 3780
acu 3783
<210> 5
<211> 3783
<212> RNA
<213> Artificial Sequence
<220>
<223> 编码融合前构型S蛋白序列的mRNA
<400> 5
aagcacaagg accacgacga cggagaccac ucgucgguca cacacuugga cuguuguucu 60
ugugucgacg gaggacgaau guguuugucg aaguguucuc cucacaugau gggacuguuc 120
cacaagucuu cgucgcacga cgugucgugu guccuggaca aggacggaaa gaagucguug 180
cacuguacca aggugcgaua ggugcacucg ccuuguuugc cuuguuucuc uaagcuguug 240
ggacacgacg gaaaguugcu gccucacaug aagcgaucgu gucucuucuc guuguaguag 300
ucuccuaccu agaagccuug uugugaccug ucguucugug ucucggacga cuagcacuug 360
uugcgauguu ugcaccacua guuccacaca cucaagguca agacauugcu gggaaaggac 420
ccucacauga ugguguucuu guuguucucg accuaccucu cgcucaaguc ucacaugucg 480
ucgcgauugu ugacauguaa gcucaugcac ucggucggaa aggacuaccu ggaccucccu 540
uucgucccuu ugaaguucuu ggacucucuc aagcacaagu ucuuguagcu gccuaugaag 600
uucuagaugu cguucgugug uggauaguug gaccacucuc uggacggagu cccuaagucg 660
cgagaccucg gagaccaccu ggacggauag ccuuaguugu aguguucuaa ggucugugac 720
gaccgagacg ugucuucgau ggacugugga ccucugucgu cgucgccuac cugucgaccu 780
cgacgacgaa ugaugcaccc uauggacguc ggaucuugua aggacgacuu cauguugcuc 840
uugccuuguu agugucugcg acaccugaca cgagaccugg gagacucgcu cuguuucaca 900
ugugacuucu cgaaguguca ccucuucccu uagauggucu guucguugaa gucucacguc 960
ggaugucucu cguagcacuc uaagggauug uaguguuugg acacaggaaa gccucuccac 1020
aaguugcgau guucuaagcg aucgcacaug cgaaccuugu cuuucucuua gucguugaca 1080
caccgacuga ugucgcacga cauguugucg cgaucgaagu cguguaaguu cacaaugccu 1140
cacucgggau guuucgacuu gcuggacaca aaguguuugc acaugcgacu gucgaagcac 1200
uagucuccuc ugcuccacuc ugucuagcga ggaccugucu guccuuucua gcgacugaug 1260
uugauguucg acggacugcu gaaguguccu acacacuagc gaaccuuguc guuguuggac 1320
cugucguucc acccuccuuu gauguugaug gacaugucug acaagucuuu cucguuggac 1380
uucggaaagc ucucucugua gucgugucuc uagauggucc gaccuucgug uggaacauug 1440
ccucaccucc cuaaguugac aaugaaggga gacgucucga ugccuaaggu cggauguuug 1500
ccucacccua uggucggaau gucucaccac cacgacucga agcucgacga cgugcgagga 1560
cgaugucaca caccuggauu cuucucgugu uuggaccacu ucuuguucac acacuugaag 1620
uugaaguugc cugacugucc uuguccucac gacugucucu cguuguucuu caaggacgga 1680
aaggucguca agccuucucu guagcgacug uguugucugc gacacucucu gggagucugu 1740
gaccucuagg accuguagug uggaacaucg aagccuccuc acucgcacua guguggaccu 1800
uguuuguguu cguuggucca ccgacacgac augguccugc acuugacaug ucuccacgga 1860
caccgauagg ugcgacuggu cgacugugga uguaccucuc acaugucgug uccuucguug 1920
cacaaggucu guucucgacc uacagacuag ccucgacucg ugcacuuguu gucgaugcuc 1980
acacuguagg gauagccucg accuuagaca cgaucgaugg ucugugucug uuugucggga 2040
ucuucucgau cuucgcaccg aucggucucg uaguagcgaa uguguuacuc ggacccucga 2100
cucuugucgc accgaauguc guuguugucg uagcgauagg gauguuugaa guguuagucg 2160
cacuguuguc ucuaggacgg acacucguac uguuucuguu cgcaccugac auguuacaug 2220
uagacaccuc ugucgugucu cacaucguug gacgacgacg ucaugccuuc gaagacaugu 2280
gucgacuugu cucgagacug uccuuagcga caccucgucc uguucuugug uguccuccac 2340
aagcgagucc acuucgucua gauguucugu ggaggauagu uccugaagcc uccuaaguug 2400
aagucggucu aggacggacu gggaucguuc ggaucguucu cuucgaagua gcuccuggac 2460
gacaaguugu uccacuguga ccgacugcga ccuaaguagu ucgucaugcc ucugacagac 2520
ccucuguagc gacgaucucu ggacuagaca cgagucuuca aguugccuga cugucacgac 2580
ggaggagacg acugucugcu cuacuagcga gucauguguu cgcgagacga ccgaccuugu 2640
uaguguucgc cuaccuguaa gccucgaccu cgacgagacg ucuagggaaa gcgauacguc 2700
uaccgaaugu cuaaguugcc uuagccucac ugugucuugc acgacaugcu cuuggucuuc 2760
gacuagcgau uggucaaguu gucgcgauag ccuuucuagg uccugucgga cucgucgugu 2820
cgaucgcgag acccuuucga cguccugcac cacuuggucu ugcgaguccg agacuugugu 2880
gaccacuucg ucgacucguc guugaagccu cgauagucgu cgcacgacuu gcuguaggac 2940
ucgucugacc uguuccaccu ccgacuccac gucuagcugu cugacuagug uccuucugac 3000
gucucggacg ucuguaugca cugugucguc gacuagucuc gacgacucua gucucgaucg 3060
cgauuggacc gacgauguuu cuacucgcuc acacacgacc cugucucguu cucucaccug 3120
aagacaccuu ucccuauggu ggacuacucg aagggagucu cgcgaggagu gccucaccac 3180
aaggacgugc acuguaugca cggacgaguc cucuucuuga aguguugucg aggacgauag 3240
acagugcugc cuuuccgagu gaagggaucu cucccucaca agcacucguu gccuugugug 3300
accaagcacu gugucucuuu gaagaugcuc ggagucuagu aguguugucu guuguguaag 3360
cacucgccuu ugacacugca ccacuagccu uagcacuugu ugugucacau gcugggagac 3420
gucggacucg accugucgaa guuccuccuc gaccuguuca ugaaguucuu gguguguucg 3480
ggacugcacc uggacccucu guagucgccu uaguugcgau cgcaccacuu guaggucuuc 3540
cucuagcugu cugacuugcu ccaccgauuc uuggacuugc ucucggacua gcuggacguc 3600
cucgacccuu ucaugcucgu cauguaguuc accggaacca uguagaccga cccuaaguag 3660
cgaccugacu agcgauagca cuaccacugu uaguacgaca caacauacug uucgacaaca 3720
ucgacagacu ucccuacaac aucgcgaaug cacucuuucc ugccucucac ccacaaggac 3780
acu 3783
<210> 6
<211> 72
<212> RNA
<213> Artificial Sequence
<220>
<223> 佐剂序列
<400> 6
uaccgccacu accgcggcgc gugggaccac gacgacgacu cgccgcgcga ccgcgacugg 60
gucuggaccc gc 72
<210> 7
<211> 60
<212> RNA
<213> Artificial Sequence
<220>
<223> 佐剂序列
<400> 7
uaccgcuaau accgcggcgc gugggaccac gacgacgacu cgccgcgcga ccgcgacugg 60
<210> 8
<211> 72
<212> RNA
<213> Artificial Sequence
<220>
<223> 佐剂序列
<400> 8
uacgcgcacu ggcgcggcgc guggcacgac gacgacgacu cgcgccgcga ccgcgacugg 60
cuuuggaccc gc 72
<210> 9
<211> 45
<212> RNA
<213> Artificial Sequence
<220>
<223> 佐剂序列
<400> 9
uaccuaaccu ggaccuaaga caaagaccac cgccgccgcu gggcg 45
<210> 10
<211> 3783
<212> DNA
<213> Artificial Sequence
<220>
<223> 编码融合前构型S蛋白序列的DNA序列
<400> 10
aagcacaagg accacgacga cggggaccac tcgtcggtca cgcacttgga ctggtgggcc 60
tgggtcgacg gggggcggat gtggttgtcg aagtgggccc cgcacatgat ggggctgttc 120
cacaaggcct cgtcgcacga cgtgtcgtgg gtcctggaca aggacgggaa gaagtcgttg 180
cactggacca aggtgcggta ggtgcactcg ccgtggttgc cgtggttcgc caagctgttg 240
gggcacgacg ggaagttgct gccgcacatg aagcggtcgt ggctcttctc gttgtagtag 300
gccccgacct agaagccgtg gtgggacctg tcgttctggg tctcggacga ctagcacttg 360
ttgcggtggt tgcaccacta gttccacacg ctcaaggtca agacgttgct ggggaaggac 420
ccgcacatga tggtgttctt gttgttctcg acctacctct cgctcaaggc ccacatgtcg 480
tcgcggttgt tgacgtggaa gctcatgcac tcggtcggga aggactacct ggacctcccg 540
ttcgtcccgt tgaagttctt ggacgccctc aagcacaagt tcttgtagct gccgatgaag 600
ttctagatgt cgttcgtgtg ggggtagttg gaccacgccc tggacggggt cccgaagtcg 660
cgggacctcg gggaccacct ggacgggtag ccgtagttgt agtgggccaa ggtctgggac 720
gaccgggacg tggcctcgat ggactggggg ccgctgtcgt cgtcgccgac ctggcggccg 780
cggcggcgga tgatgcaccc gatggacgtc ggggcctgga aggacgactt catgttgctc 840
ttgccgtggt agtggctgcg gcacctgacg cgggacctgg gggactcgct ctggttcacg 900
tgggacttct cgaagtggca cctcttcccg tagatggtct ggtcgttgaa ggcccacgtc 960
gggtggctct cgtagcacgc caaggggttg tagtggttgg acacggggaa gccgctccac 1020
aagttgcggt gggccaagcg gtcgcacatg cggaccttgg ccttcgccta gtcgttgacg 1080
caccggctga tgtcgcacga catgttgtcg cggtcgaagt cgtggaagtt cacgatgccg 1140
cactcggggt ggttcgactt gctggacacg aagtggttgc acatgcggct gtcgaagcac 1200
taggccccgc tgctccacgc cgtctagcgg gggccggtct ggccgttcta gcggctgatg 1260
ttgatgttcg acgggctgct gaagtggccg acgcactagc ggaccttgtc gttgttggac 1320
ctgtcgttcc acccgccgtt gatgttgatg gacatggccg acaaggcctt ctcgttggac 1380
ttcgggaagc tcgccctgta gtcgtggctc tagatggtcc ggccgtcgtg ggggacgttg 1440
ccgcacctcc cgaagttgac gatgaagggg gacgtctcga tgccgaaggt cgggtggttg 1500
ccgcacccga tggtcgggat ggcccaccac cacgactcga agctcgacga cgtgcggggg 1560
cggtggcaca cgccggggtt cttctcgtgg ttggaccact tcttgttcac gcacttgaag 1620
ttgaagttgc cggactggcc gtggccgcac gactggctct cgttgttctt caaggacggg 1680
aaggtcgtca agccggccct gtagcggctg tggtggctgc ggcacgccct gggggtctgg 1740
gacctctagg acctgtagtg ggggacgtcg aagccgccgc actcgcacta gtgggggccg 1800
tggttgtggt cgttggtcca ccggcacgac atggtcctgc acttgacgtg gctccacggg 1860
caccggtagg tgcggctggt cgactggggg tggaccgccc acatgtcgtg gccgtcgttg 1920
cacaaggtct gggcccggcc gacggactag ccgcggctcg tgcacttgtt gtcgatgctc 1980
acgctgtagg ggtagccgcg gccgtagacg cggtcgatgg tctgggtctg gttgtcgggg 2040
gccgcccggg cctcgcaccg gtcggtctcg tagtagcgga tgtggtactc ggacccgcgg 2100
ctcttgtcgc accggatgtc gttgttgtcg tagcggtagg ggtggttgaa gtggtagtcg 2160
cactggtggc tctaggacgg gcactcgtac tggttctggt cgcacctgac gtggtacatg 2220
tagacgccgc tgtcgtggct cacgtcgttg gacgacgacg tcatgccgtc gaagacgtgg 2280
gtcgacttgg cccgggactg gccgtagcgg cacctcgtcc tgttcttgtg ggtcctccac 2340
aagcgggtcc acttcgtcta gatgttctgg ggggggtagt tcctgaagcc gccgaagttg 2400
aagtcggtct aggacgggct ggggtcgttc gggtcgttcg cctcgaagta gctcctggac 2460
gacaagttgt tccactggga ccggctgcgg ccgaagtagt tcgtcatgcc gctgacggac 2520
ccgctgtagc ggcgggccct ggactagacg cgggtcttca agttgccgga ctggcacgac 2580
gggggggacg actggctgct ctactagcgg gtcatgtggt cgcgggacga ccggccgtgg 2640
tagtggtcgc cgacctggaa gccgcggccg cggcgggacg tctaggggaa gcggtacgtc 2700
taccggatgg ccaagttgcc gtagccgcac tgggtcttgc acgacatgct cttggtcttc 2760
gactagcggt tggtcaagtt gtcgcggtag ccgttctagg tcctgtcgga ctcgtcgtgg 2820
cggtcgcggg acccgttcga cgtcctgcac cacttggtct tgcgggtccg ggacttgtgg 2880
gaccacttcg tcgactcgtc gttgaagccg cggtagtcgt cgcacgactt gctgtaggac 2940
tcggccgacc tgttccacct ccggctccac gtctagctgg ccgactagtg gccggccgac 3000
gtctcggacg tctggatgca ctgggtcgtc gactaggccc ggcggctcta ggcccggtcg 3060
cggttggacc ggcggtggtt ctactcgctc acgcacgacc cggtctcgtt cgcccacctg 3120
aagacgccgt tcccgatggt ggactactcg aagggggtct cgcggggggt gccgcaccac 3180
aaggacgtgc actggatgca cgggcgggtc ctcttcttga agtggtggcg ggggcggtag 3240
acggtgctgc cgttccgggt gaagggggcc ctcccgcaca agcactcgtt gccgtgggtg 3300
accaagcact gggtcgcctt gaagatgctc ggggtctagt agtggtggct gttgtggaag 3360
cactcgccgt tgacgctgca ccactagccg tagcacttgt tgtggcacat gctgggggac 3420
gtcgggctcg acctgtcgaa gttcctcctc gacctgttca tgaagttctt ggtgtggtcg 3480
gggctgcacc tggacccgct gtagtcgccg tagttgcggt cgcaccactt gtaggtcttc 3540
ctctagctgg ccgacttgct ccaccggttc ttggacttgc tctcggacta gctggacgtc 3600
ctcgacccgt tcatgctcgt catgtagttc accgggacca tgtagaccga cccgaagtag 3660
cggccggact agcggtagca ctaccactgg tagtacgaca cgacgtactg gtcgacgacg 3720
tcgacggact tcccgacgac gtcgcggatg cacgccttcc tgccgctcac ccacaaggac 3780
act 3783
<210> 11
<211> 3783
<212> DNA
<213> Artificial Sequence
<220>
<223> 编码融合前构型S蛋白序列的DNA序列
<400> 11
aagcacaagg accacgacga cggggaccac tcgtcggtca cgcacttgga ctggtggtcc 60
tgggtcgacg gggggcggat gtggttgtcg aagtggtccc cgcacatgat ggggctgttc 120
cacaagtcct cgtcgcacga cgtgtcgtgg gtcctggaca aggacgggaa gaagtcgttg 180
cactggacca aggtgcggta ggtgcactcg ccgtggttgc cgtggttctc caagctgttg 240
gggcacgacg ggaagttgct gccgcacatg aagcggtcgt ggctcttctc gttgtagtag 300
tccccgacct agaagccgtg gtgggacctg tcgttctggg tctcggacga ctagcacttg 360
ttgcggtggt tgcaccacta gttccacacg ctcaaggtca agacgttgct ggggaaggac 420
ccgcacatga tggtgttctt gttgttctcg acctacctct cgctcaagtc ccacatgtcg 480
tcgcggttgt tgacgtggaa gctcatgcac tcggtcggga aggactacct ggacctcccg 540
ttcgtcccgt tgaagttctt ggactccctc aagcacaagt tcttgtagct gccgatgaag 600
ttctagatgt cgttcgtgtg ggggtagttg gaccactccc tggacggggt cccgaagtcg 660
cgggacctcg gggaccacct ggacgggtag ccgtagttgt agtggtccaa ggtctgggac 720
gaccgggacg tgtcctcgat ggactggggg ccgctgtcgt cgtcgccgac ctggcggccg 780
cggcggcgga tgatgcaccc gatggacgtc gggtcctgga aggacgactt catgttgctc 840
ttgccgtggt agtggctgcg gcacctgacg cgggacctgg gggactcgct ctggttcacg 900
tgggacttct cgaagtggca cctcttcccg tagatggtct ggtcgttgaa gtcccacgtc 960
gggtggctct cgtagcactc caaggggttg tagtggttgg acacggggaa gccgctccac 1020
aagttgcggt ggtccaagcg gtcgcacatg cggaccttgt ccttctccta gtcgttgacg 1080
caccggctga tgtcgcacga catgttgtcg cggtcgaagt cgtggaagtt cacgatgccg 1140
cactcggggt ggttcgactt gctggacacg aagtggttgc acatgcggct gtcgaagcac 1200
tagtccccgc tgctccactc cgtctagcgg gggccggtct ggccgttcta gcggctgatg 1260
ttgatgttcg acgggctgct gaagtggccg acgcactagc ggaccttgtc gttgttggac 1320
ctgtcgttcc acccgccgtt gatgttgatg gacatgtccg acaagtcctt ctcgttggac 1380
ttcgggaagc tctccctgta gtcgtggctc tagatggtcc ggccgtcgtg ggggacgttg 1440
ccgcacctcc cgaagttgac gatgaagggg gacgtctcga tgccgaaggt cgggtggttg 1500
ccgcacccga tggtcgggat gtcccaccac cacgactcga agctcgacga cgtgcggggg 1560
cggtggcaca cgccggggtt cttctcgtgg ttggaccact tcttgttcac gcacttgaag 1620
ttgaagttgc cggactggcc gtggccgcac gactggctct cgttgttctt caaggacggg 1680
aaggtcgtca agccgtccct gtagcggctg tggtggctgc ggcactccct gggggtctgg 1740
gacctctagg acctgtagtg ggggacgtcg aagccgccgc actcgcacta gtgggggccg 1800
tggttgtggt cgttggtcca ccggcacgac atggtcctgc acttgacgtg gctccacggg 1860
caccggtagg tgcggctggt cgactggggg tggacctccc acatgtcgtg gccgtcgttg 1920
cacaaggtct ggtcccggcc gacggactag ccgcggctcg tgcacttgtt gtcgatgctc 1980
acgctgtagg ggtagccgcg gccgtagacg cggtcgatgg tctgggtctg gttgtcgggg 2040
tcctcccggt cctcgcaccg gtcggtctcg tagtagcgga tgtggtactc ggacccgcgg 2100
ctcttgtcgc accggatgtc gttgttgtcg tagcggtagg ggtggttgaa gtggtagtcg 2160
cactggtggc tctaggacgg gcactcgtac tggttctggt cgcacctgac gtggtacatg 2220
tagacgccgc tgtcgtggct cacgtcgttg gacgacgacg tcatgccgtc gaagacgtgg 2280
gtcgacttgt cccgggactg gccgtagcgg cacctcgtcc tgttcttgtg ggtcctccac 2340
aagcgggtcc acttcgtcta gatgttctgg ggggggtagt tcctgaagcc gccgaagttg 2400
aagtcggtct aggacgggct ggggtcgttc gggtcgttct cctcgaagta gctcctggac 2460
gacaagttgt tccactggga ccggctgcgg ccgaagtagt tcgtcatgcc gctgacggac 2520
ccgctgtagc ggcggtccct ggactagacg cgggtcttca agttgccgga ctggcacgac 2580
gggggggacg actggctgct ctactagcgg gtcatgtggt cgcgggacga ccggccgtgg 2640
tagtggtcgc cgacctggaa gccgcggccg cggcgggacg tctaggggaa gcggtacgtc 2700
taccggatgt ccaagttgcc gtagccgcac tgggtcttgc acgacatgct cttggtcttc 2760
gactagcggt tggtcaagtt gtcgcggtag ccgttctagg tcctgtcgga ctcgtcgtgg 2820
cggtcgcggg acccgttcga cgtcctgcac cacttggtct tgcgggtccg ggacttgtgg 2880
gaccacttcg tcgactcgtc gttgaagccg cggtagtcgt cgcacgactt gctgtaggac 2940
tcgtccgacc tgttccacct ccggctccac gtctagctgt ccgactagtg gccgtccgac 3000
gtctcggacg tctggatgca ctgggtcgtc gactagtccc ggcggctcta gtcccggtcg 3060
cggttggacc ggcggtggtt ctactcgctc acgcacgacc cggtctcgtt ctcccacctg 3120
aagacgccgt tcccgatggt ggactactcg aagggggtct cgcggggggt gccgcaccac 3180
aaggacgtgc actggatgca cgggcgggtc ctcttcttga agtggtggcg ggggcggtag 3240
acggtgctgc cgttccgggt gaaggggtcc ctcccgcaca agcactcgtt gccgtgggtg 3300
accaagcact gggtctcctt gaagatgctc ggggtctagt agtggtggct gttgtggaag 3360
cactcgccgt tgacgctgca ccactagccg tagcacttgt tgtggcacat gctgggggac 3420
gtcgggctcg acctgtcgaa gttcctcctc gacctgttca tgaagttctt ggtgtggtcg 3480
gggctgcacc tggacccgct gtagtcgccg tagttgcggt cgcaccactt gtaggtcttc 3540
ctctagctgt ccgacttgct ccaccggttc ttggacttgc tctcggacta gctggacgtc 3600
ctcgacccgt tcatgctcgt catgtagttc accgggacca tgtagaccga cccgaagtag 3660
cggccggact agcggtagca ctaccactgg tagtacgaca cgacgtactg gtcgacgacg 3720
tcgacggact tcccgacgac gtcgcggatg cactccttcc tgccgctcac ccacaaggac 3780
act 3783
<210> 12
<211> 3783
<212> DNA
<213> Artificial Sequence
<220>
<223> 编码融合前构型S蛋白序列的DNA序列
<400> 12
aagcacaagg accacgacga cggagaccac tcgtcggtca cgcacttgga ctggtggtcc 60
tgggtcgacg gaggacggat gtggttgtcg aagtggtccc cgcacatgat gggactgttc 120
cacaagtcct cgtcgcacga cgtgtcgtgg gtcctggaca aggacggaaa gaagtcgttg 180
cactggacca aggtgcggta ggtgcactcg ccgtggttgc cgtggttctc caagctgttg 240
ggacacgacg gaaagttgct gccgcacatg aagcggtcgt ggctcttctc gttgtagtag 300
tccccgacct agaagccgtg gtgggacctg tcgttctggg tctcggacga ctagcacttg 360
ttgcggtggt tgcaccacta gttccacacg ctcaaggtca agacgttgct gggaaaggac 420
ccgcacatga tggtgttctt gttgttctcg acctacctct cgctcaagtc ccacatgtcg 480
tcgcggttgt tgacgtggaa gctcatgcac tcggtcggaa aggactacct ggacctcccg 540
ttcgtcccgt tgaagttctt ggactccctc aagcacaagt tcttgtagct gccgatgaag 600
ttctagatgt cgttcgtgtg gggatagttg gaccactccc tggacggagt cccgaagtcg 660
cgggacctcg gagaccacct ggacggatag ccgtagttgt agtggtccaa ggtctgggac 720
gaccgggacg tgtcctcgat ggactgggga ccgctgtcgt cgtcgccgac ctggcggccg 780
cggcggcgga tgatgcaccc gatggacgtc ggatcctgga aggacgactt catgttgctc 840
ttgccgtggt agtggctgcg gcacctgacg cgggacctgg gagactcgct ctggttcacg 900
tgggacttct cgaagtggca cctcttcccg tagatggtct ggtcgttgaa gtcccacgtc 960
ggatggctct cgtagcactc caagggattg tagtggttgg acacgggaaa gccgctccac 1020
aagttgcggt ggtccaagcg gtcgcacatg cggaccttgt ccttctccta gtcgttgacg 1080
caccggctga tgtcgcacga catgttgtcg cggtcgaagt cgtggaagtt cacgatgccg 1140
cactcgggat ggttcgactt gctggacacg aagtggttgc acatgcggct gtcgaagcac 1200
tagtccccgc tgctccactc cgtctagcgg ggaccggtct ggccgttcta gcggctgatg 1260
ttgatgttcg acggactgct gaagtggccg acgcactagc ggaccttgtc gttgttggac 1320
ctgtcgttcc acccgccgtt gatgttgatg gacatgtccg acaagtcctt ctcgttggac 1380
ttcggaaagc tctccctgta gtcgtggctc tagatggtcc ggccgtcgtg gggaacgttg 1440
ccgcacctcc cgaagttgac gatgaaggga gacgtctcga tgccgaaggt cggatggttg 1500
ccgcacccga tggtcggaat gtcccaccac cacgactcga agctcgacga cgtgcgggga 1560
cggtggcaca cgccgggatt cttctcgtgg ttggaccact tcttgttcac gcacttgaag 1620
ttgaagttgc cggactggcc gtggccgcac gactggctct cgttgttctt caaggacgga 1680
aaggtcgtca agccgtccct gtagcggctg tggtggctgc ggcactccct gggagtctgg 1740
gacctctagg acctgtagtg gggaacgtcg aagccgccgc actcgcacta gtggggaccg 1800
tggttgtggt cgttggtcca ccggcacgac atggtcctgc acttgacgtg gctccacgga 1860
caccggtagg tgcggctggt cgactgggga tggacctccc acatgtcgtg gccgtcgttg 1920
cacaaggtct ggtcccggcc gacggactag ccgcggctcg tgcacttgtt gtcgatgctc 1980
acgctgtagg gatagccgcg gccgtagacg cggtcgatgg tctgggtctg gttgtcggga 2040
tcctcccggt cctcgcaccg gtcggtctcg tagtagcgga tgtggtactc ggacccgcgg 2100
ctcttgtcgc accggatgtc gttgttgtcg tagcggtagg gatggttgaa gtggtagtcg 2160
cactggtggc tctaggacgg acactcgtac tggttctggt cgcacctgac gtggtacatg 2220
tagacgccgc tgtcgtggct cacgtcgttg gacgacgacg tcatgccgtc gaagacgtgg 2280
gtcgacttgt cccgggactg gccgtagcgg cacctcgtcc tgttcttgtg ggtcctccac 2340
aagcgggtcc acttcgtcta gatgttctgg ggaggatagt tcctgaagcc gccgaagttg 2400
aagtcggtct aggacggact gggatcgttc ggatcgttct cctcgaagta gctcctggac 2460
gacaagttgt tccactggga ccggctgcgg ccgaagtagt tcgtcatgcc gctgacggac 2520
ccgctgtagc ggcggtccct ggactagacg cgggtcttca agttgccgga ctggcacgac 2580
ggaggagacg actggctgct ctactagcgg gtcatgtggt cgcgggacga ccggccgtgg 2640
tagtggtcgc cgacctggaa gccgcggccg cggcgggacg tctagggaaa gcggtacgtc 2700
taccggatgt ccaagttgcc gtagccgcac tgggtcttgc acgacatgct cttggtcttc 2760
gactagcggt tggtcaagtt gtcgcggtag ccgttctagg tcctgtcgga ctcgtcgtgg 2820
cggtcgcggg acccgttcga cgtcctgcac cacttggtct tgcgggtccg ggacttgtgg 2880
gaccacttcg tcgactcgtc gttgaagccg cggtagtcgt cgcacgactt gctgtaggac 2940
tcgtccgacc tgttccacct ccggctccac gtctagctgt ccgactagtg gccgtccgac 3000
gtctcggacg tctggatgca ctgggtcgtc gactagtccc ggcggctcta gtcccggtcg 3060
cggttggacc ggcggtggtt ctactcgctc acgcacgacc cggtctcgtt ctcccacctg 3120
aagacgccgt tcccgatggt ggactactcg aagggagtct cgcggggagt gccgcaccac 3180
aaggacgtgc actggatgca cggacgggtc ctcttcttga agtggtggcg gggacggtag 3240
acggtgctgc cgttccgggt gaagggatcc ctcccgcaca agcactcgtt gccgtgggtg 3300
accaagcact gggtctcctt gaagatgctc ggagtctagt agtggtggct gttgtggaag 3360
cactcgccgt tgacgctgca ccactagccg tagcacttgt tgtggcacat gctgggagac 3420
gtcggactcg acctgtcgaa gttcctcctc gacctgttca tgaagttctt ggtgtggtcg 3480
ggactgcacc tggacccgct gtagtcgccg tagttgcggt cgcaccactt gtaggtcttc 3540
ctctagctgt ccgacttgct ccaccggttc ttggacttgc tctcggacta gctggacgtc 3600
ctcgacccgt tcatgctcgt catgtagttc accggaacca tgtagaccga cccgaagtag 3660
cggccggact agcggtagca ctaccactgg tagtacgaca cgacgtactg gtcgacgacg 3720
tcgacggact tcccgacgac gtcgcggatg cactccttcc tgccgctcac ccacaaggac 3780
act 3783
<210> 13
<211> 3783
<212> DNA
<213> Artificial Sequence
<220>
<223> 编码融合前构型S蛋白序列的DNA序列
<400> 13
aagcacaagg accacgacga cggagaccac tcgtcggtca cacacttgga ctgttgttct 60
tgtgtcgacg gaggacgaat gtgtttgtcg aagtgttctc ctcacatgat gggactgttc 120
cacaagtctt cgtcgcacga cgtgtcgtgt gtcctggaca aggacggaaa gaagtcgttg 180
cactgtacca aggtgcgata ggtgcactcg ccttgtttgc cttgtttctc taagctgttg 240
ggacacgacg gaaagttgct gcctcacatg aagcgatcgt gtctcttctc gttgtagtag 300
tctcctacct agaagccttg ttgtgacctg tcgttctgtg tctcggacga ctagcacttg 360
ttgcgatgtt tgcaccacta gttccacaca ctcaaggtca agacattgct gggaaaggac 420
cctcacatga tggtgttctt gttgttctcg acctacctct cgctcaagtc tcacatgtcg 480
tcgcgattgt tgacatgtaa gctcatgcac tcggtcggaa aggactacct ggacctccct 540
ttcgtccctt tgaagttctt ggactctctc aagcacaagt tcttgtagct gcctatgaag 600
ttctagatgt cgttcgtgtg tggatagttg gaccactctc tggacggagt ccctaagtcg 660
cgagacctcg gagaccacct ggacggatag ccttagttgt agtgttctaa ggtctgtgac 720
gaccgagacg tgtcttcgat ggactgtgga cctctgtcgt cgtcgcctac ctgtcgacct 780
cgacgacgaa tgatgcaccc tatggacgtc ggatcttgta aggacgactt catgttgctc 840
ttgccttgtt agtgtctgcg acacctgaca cgagacctgg gagactcgct ctgtttcaca 900
tgtgacttct cgaagtgtca cctcttccct tagatggtct gttcgttgaa gtctcacgtc 960
ggatgtctct cgtagcactc taagggattg tagtgtttgg acacaggaaa gcctctccac 1020
aagttgcgat gttctaagcg atcgcacatg cgaaccttgt ctttctctta gtcgttgaca 1080
caccgactga tgtcgcacga catgttgtcg cgatcgaagt cgtgtaagtt cacaatgcct 1140
cactcgggat gtttcgactt gctggacaca aagtgtttgc acatgcgact gtcgaagcac 1200
tagtctcctc tgctccactc tgtctagcga ggacctgtct gtcctttcta gcgactgatg 1260
ttgatgttcg acggactgct gaagtgtcct acacactagc gaaccttgtc gttgttggac 1320
ctgtcgttcc accctccttt gatgttgatg gacatgtctg acaagtcttt ctcgttggac 1380
ttcggaaagc tctctctgta gtcgtgtctc tagatggtcc gaccttcgtg tggaacattg 1440
cctcacctcc ctaagttgac aatgaaggga gacgtctcga tgcctaaggt cggatgtttg 1500
cctcacccta tggtcggaat gtctcaccac cacgactcga agctcgacga cgtgcgagga 1560
cgatgtcaca cacctggatt cttctcgtgt ttggaccact tcttgttcac acacttgaag 1620
ttgaagttgc ctgactgtcc ttgtcctcac gactgtctct cgttgttctt caaggacgga 1680
aaggtcgtca agccttctct gtagcgactg tgttgtctgc gacactctct gggagtctgt 1740
gacctctagg acctgtagtg tggaacatcg aagcctcctc actcgcacta gtgtggacct 1800
tgtttgtgtt cgttggtcca ccgacacgac atggtcctgc acttgacatg tctccacgga 1860
caccgatagg tgcgactggt cgactgtgga tgtacctctc acatgtcgtg tccttcgttg 1920
cacaaggtct gttctcgacc tacagactag cctcgactcg tgcacttgtt gtcgatgctc 1980
acactgtagg gatagcctcg accttagaca cgatcgatgg tctgtgtctg tttgtcggga 2040
tcttctcgat cttcgcaccg atcggtctcg tagtagcgaa tgtgttactc ggaccctcga 2100
ctcttgtcgc accgaatgtc gttgttgtcg tagcgatagg gatgtttgaa gtgttagtcg 2160
cactgttgtc tctaggacgg acactcgtac tgtttctgtt cgcacctgac atgttacatg 2220
tagacacctc tgtcgtgtct cacatcgttg gacgacgacg tcatgccttc gaagacatgt 2280
gtcgacttgt ctcgagactg tccttagcga cacctcgtcc tgttcttgtg tgtcctccac 2340
aagcgagtcc acttcgtcta gatgttctgt ggaggatagt tcctgaagcc tcctaagttg 2400
aagtcggtct aggacggact gggatcgttc ggatcgttct cttcgaagta gctcctggac 2460
gacaagttgt tccactgtga ccgactgcga cctaagtagt tcgtcatgcc tctgacagac 2520
cctctgtagc gacgatctct ggactagaca cgagtcttca agttgcctga ctgtcacgac 2580
ggaggagacg actgtctgct ctactagcga gtcatgtgtt cgcgagacga ccgaccttgt 2640
tagtgttcgc ctacctgtaa gcctcgacct cgacgagacg tctagggaaa gcgatacgtc 2700
taccgaatgt ctaagttgcc ttagcctcac tgtgtcttgc acgacatgct cttggtcttc 2760
gactagcgat tggtcaagtt gtcgcgatag cctttctagg tcctgtcgga ctcgtcgtgt 2820
cgatcgcgag accctttcga cgtcctgcac cacttggtct tgcgagtccg agacttgtgt 2880
gaccacttcg tcgactcgtc gttgaagcct cgatagtcgt cgcacgactt gctgtaggac 2940
tcgtctgacc tgttccacct ccgactccac gtctagctgt ctgactagtg tccttctgac 3000
gtctcggacg tctgtatgca ctgtgtcgtc gactagtctc gacgactcta gtctcgatcg 3060
cgattggacc gacgatgttt ctactcgctc acacacgacc ctgtctcgtt ctctcacctg 3120
aagacacctt tccctatggt ggactactcg aagggagtct cgcgaggagt gcctcaccac 3180
aaggacgtgc actgtatgca cggacgagtc ctcttcttga agtgttgtcg aggacgatag 3240
acagtgctgc ctttccgagt gaagggatct ctccctcaca agcactcgtt gccttgtgtg 3300
accaagcact gtgtctcttt gaagatgctc ggagtctagt agtgttgtct gttgtgtaag 3360
cactcgcctt tgacactgca ccactagcct tagcacttgt tgtgtcacat gctgggagac 3420
gtcggactcg acctgtcgaa gttcctcctc gacctgttca tgaagttctt ggtgtgttcg 3480
ggactgcacc tggaccctct gtagtcgcct tagttgcgat cgcaccactt gtaggtcttc 3540
ctctagctgt ctgacttgct ccaccgattc ttggacttgc tctcggacta gctggacgtc 3600
ctcgaccctt tcatgctcgt catgtagttc accggaacca tgtagaccga ccctaagtag 3660
cgacctgact agcgatagca ctaccactgt tagtacgaca caacatactg ttcgacaaca 3720
tcgacagact tccctacaac atcgcgaatg cactctttcc tgcctctcac ccacaaggac 3780
act 3783

Claims (10)

1.如下(a)或(b)的蛋白：
(a)SEQ ID No.1所示氨基酸序列组成的蛋白；
(b)由SEQ ID No.1所示氨基酸序列经过取代、缺失和/或添加一个或几个氨基酸且与SEQ ID No.1所示氨基酸序列组成的蛋白具有相同功能的衍生蛋白。
2.根据权利要求1所述的蛋白，其中，所述由SEQ ID No.1所示氨基酸序列经过取代、缺失和/或添加一个或几个氨基酸且与SEQ ID No.1所示氨基酸序列组成的蛋白具有相同功能的蛋白，至少包含SEQ ID No.1所示氨基酸序列C末端连续至少15个氨基酸的序列。
3.权利要求1或2所述的蛋白作为抗原在制备疫苗中的应用。
4.编码权利要求1或2所述的蛋白的DNA；优选地，所述DNA的核苷酸序列包括SEQ IDNo.10、SEQ ID No.11、SEQ ID No.12或SEQ ID No.13所示序列。
5.一种表达载体，其中含有权利要求4所述的DNA。
6.编码权利要求1或2所述的蛋白的mRNA；
优选地，所述mRNA是权利要求4所述的DNA进行体外转录得到。
7.根据权利要求6所述的mRNA，其核苷酸序列包括SEQ ID No.2、SEQ ID No.3、SEQ IDNo.4或SEQ ID No.5所示序列。
8.根据权利要求6或7所述的mRNA，其核苷酸序列5’端还包括佐剂序列；
优选地，所述佐剂序列为促进蛋白分泌表达的核苷酸序列；
进一步优选地，所述佐剂序列包括SEQ ID No.6、SEQ ID No.7、SEQ ID No.8或SEQ IDNo.9所示序列；
更优选地，所述的mRNA包括SEQ ID No.6、SEQ ID No.7、SEQ ID No.8或SEQ ID No.9所示序列与SEQ ID No.4组合序列所示的mRNA。
9.一种预防性疫苗，其中包括权利要求6所述的mRNA中的一种或多种。
10.根据权利要求9所述的疫苗，其中还包括用于溶解mRNA的PBS溶液。
CN202011056131.3A 2020-09-29 2020-09-29 新型冠状病毒mRNA疫苗及其制备方法与应用 Pending CN114315986A (zh)

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