CN116987736A - 自复制rna分子和新冠病毒多价疫苗 - Google Patents
自复制rna分子和新冠病毒多价疫苗 Download PDFInfo
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Abstract
本发明提出了一种自复制RNA分子，其包括：自复制核心序列，所述自复制核心序列编码N蛋白、P蛋白和L蛋白或其功能片段；和抗原序列，所述抗原序列编码选自新冠病毒原始株、新冠病毒拉姆达株、新冠病毒德尔塔株、新冠病毒贝塔株、新冠奥密克戎株至少一种的特异性抗原S蛋白的至少一部分。该自复制RNA分子能够复制或者翻译抗原蛋白，这些抗原蛋白可以在体内引起有效的免疫反应，产生抵抗至少两种新冠病毒的免疫反应和抗体。
Description
技术领域
本申请属于生物医药领域，具体的，本申请涉及自复制RNA分子和新冠病毒多价疫苗。
背景技术
人类冠状病毒是冠状病毒科的高度传染性包膜的，阳性单链RNA病毒。已知冠状病毒科的两个亚家族引起人类疾病。SARS-CoV-2病毒引起的大流行性疾病已被WHO命名为Covid-19(Coronavirus Disease 2019)。
然而，目前针对新冠病毒的多价疫苗仍有待进一步改进。
发明内容
本申请旨在至少在一定程度上解决相关技术中的技术问题之一。
在本发明的一个方面，本发明提出了一种自复制RNA分子，其包括：自复制核心序列，所述自复制核心序列编码N蛋白、P蛋白和L蛋白或其功能片段；和抗原序列，所述抗原序列编码选自新冠病毒原始株、新冠病毒拉姆达株、新冠病毒德尔塔株、新冠病毒贝塔株、新冠奥密克戎株至少一种的特异性抗原S蛋白的至少一部分。
发明人通过对各种RNA病毒的RNA在细胞内的翻译及自我扩增机制进行了深入研究，发现通过采用编码来自弹状病毒的N蛋白、P蛋白及L蛋白的RNA分子作为核心区域，可以实现RNA在动物细胞内的自我复制和翻译，并且该核心区域作为强大的“引擎”，可以提供高效转录扩增和启动大分子蛋白的“动能”，能够进一步搭载抗原序列来复制或者翻译抗原蛋白，这些抗原蛋白可以在体内引起有效的免疫反应，产生抵抗至少一种新冠病毒的免疫反应和抗体。
在本申请第二方面，本申请提出了一种新冠病毒多价疫苗，其包括前面所述的自复制RNA分子。
本申请的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本申请的实践了解到。
附图说明
本申请的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1显示了根据本发明一个实施例的自复制RNA分子的结构示意图；
图2显示了根据本发明一个实施例的含有自复制RNA分子的蛋白质-RNA复合物的结构示意图；
图3显示了根据本发明实施例1中原始株的疫苗疫苗设计的示意图；
图4显示了根据本发明实施例1中蛋白抗原特异性结合抗体滴度；
图5显示了根据本发明实施例1中S蛋白长效抗体及不同IgG亚型抗体滴度检测结果图；
图6显示了根据本发明实施例1中的中和抗体检测结果图；
图7显示了根据本发明实施例1中S蛋白特异性T细胞免疫应答的检测结果图；
图8显示了根据本发明实施例1中reRNATM新冠疫苗促进不同细胞因子的检测结果图；
图9显示了根据本发明实施例1中抗原特异性CD4 T细胞及CD8 T细胞免疫应答检测结果图；
图10显示了根据本发明实施例2中五价reRNATM新冠疫苗设计示意图；
图11显示了根据本发明实施例2中S蛋白特异性不同亚型结合抗体滴度检测结果图；
图12显示了根据本发明实施例2中的中和抗体滴度检测结果图。
具体实施方式
下面详细描述本申请的实施例，所述实施例的示例在附图中示出。下面通过参考附图描述的实施例是示例性的，旨在用于解释本申请，而不能理解为对本申请的限制。
自复制RNA分子
在本文中所使用的术语“自复制RNA分子”也可以称为“自我扩增RNA”，与普通mRNA相比，自复制RNA分子的重要的区别在于它可以使用自己的RNA序列作为模板进行自我复制。根据本申请的实施例，自复制RNA分子还可以在细胞质进行翻译和复制工作，不进入细胞核，可以避免与基因组发生整合所带来的潜在风险。通常mRNA编码需要表达的蛋白质，细胞内的核糖体用来完成翻译和蛋白质生产。根据本申请的实施例，自复制RNA分子会携带一个能够表达RNA聚合酶(RNA依赖RNA聚合酶)的序列，在该RNA分子在细胞质中通过翻译产生RNA聚合酶产生后，它可以用该自复制RNA分子作为模板来产生更多的自复制RNA分子。
发明人通过对各种RNA病毒的RNA在细胞内的翻译及自我扩增机制进行了深入研究，发现通过采用编码来自弹状病毒的N蛋白，P蛋白及L蛋白的RNA分子作为核心区域，可以实现RNA在动物细胞内的自我复制和翻译，并且该核心区域作为强大的“引擎”，可以提供高效转录扩增和启动大分子蛋白的“动能”，能够进一步搭载“货物区”来复制或者翻译靶分子。
传统mRNA不是很稳定，在细胞内几天内就会降解，导致不可持续的蛋白质表达水平。自扩增RNA因为其可以在细胞质内进行自我复制，因此可以在很低的剂量下达到与传统mRNA相同的蛋白表达水平。例如，自复制RNA分子比传统信使RNA的用量可以小数百倍甚至数千倍，同样能够发挥相同的免疫刺激作用。可以减少免疫刺激中使用的注射剂量和次数，从而延长治疗效果，减少药物和给药载体可能产生的毒副作用。另外，自复制RNA分子在复制过程中会形成双链RNA，这与病毒RNA在复制过程中非常相似，可能会刺激细胞的固有免疫反应，进一步增强疫苗的效果。
发明人在研究过程中发现，由于自复制RNA分子将至少编码RNA聚合酶的序列与表达靶蛋白的序列连接起来，因此整个mRNA分子的分子量比传统mRNA的分子量大得多，分子量过大可能导致递送效率、翻译效率以及复制效率显著降低。为了对这些效率进行改善，发明人进行了深入研究，希望能够寻找到最短的核酸片段，可以正常发挥自我复制和翻译的功能。
在本发明的一个方面，本发明提出了一种自复制RNA分子，其包括：自复制核心序列，所述自复制核心序列编码N蛋白、P蛋白和L蛋白或其功能片段；和抗原序列，所述抗原序列编码选自新冠病毒原始株、新冠病毒拉姆达株、新冠病毒德尔塔株、新冠病毒贝塔株、新冠奥密克戎株至少一种的特异性抗原S蛋白的至少一部分。
发明人通过对各种RNA病毒的RNA在细胞内的翻译及自我扩增机制进行了深入研究，发现通过采用编码来自弹状病毒的N蛋白、P蛋白及L蛋白的RNA分子作为核心区域，可以实现RNA在动物细胞内的自我复制和翻译，并且该核心区域作为强大的“引擎”，可以提供高效转录扩增和启动大分子蛋白的“动能”，能够进一步搭载抗原序列来复制或者翻译抗原蛋白，这些抗原蛋白可以在体内引起有效的免疫反应，产生抵抗至少两种新冠病毒的免疫反应和抗体。
参考图1和图2，自复制核心序列包括：第一RNA序列，所述第一RNA序列编码N蛋白或其功能片段；第二RNA序列，所述第二RNA序列编码P蛋白或其功能片段；和第三RNA序列，所述第三RNA序列编码L蛋白或其功能片段，其中，所述自复制RNA分子在动物细胞内适于产生所述N蛋白、所述P蛋白、所述L蛋白或其功能片段。由此，这些第一、第二和第三RNA序列构成了自复制RNA分子的核心区区域，也称为“核心生命区”，可以作为能够在动物细胞内实现自我复制和翻译的最小区域，进一步可以搭载其他的编码序列来表达或者复制靶分子，从而发挥疫苗的功能。
在本文中所使用的术语“功能片段”是指蛋白质的全长序列的一部分，但仍能够发挥与RNA分子自我复制相关的功能，例如可以是全长序列的截断型的，也可以是蛋白质全长序列的氨基酸序列发生替换、突变或者删除等改变后的蛋白质。根据本申请的实施例，对于N蛋白的功能片段，可以结合RNA分子，保护RNA不受核酸酶的影响，对于P蛋白的功能片段，能够结合N蛋白，在模板上定位L聚合酶，同时也能够作为RNA聚合酶转录和复制复合体的基本组成部分，进一步L蛋白的功能片段能够发挥RNA聚合酶的功能，与RNA的转录及复制有关。
根据本发明的实施例，所述抗原序列编码选自新冠病毒原始株、新冠病毒拉姆达株、新冠病毒德尔塔株、新冠病毒贝塔株、新冠奥密克戎株至少两种的特异性抗原S蛋白的至少一部分。
根据本发明的实施例，所述抗原序列编码选自新冠病毒原始株、新冠病毒拉姆达株、新冠病毒德尔塔株、新冠病毒贝塔株和新冠奥密克戎株五种的特异性抗原S蛋白的至少一部分。
根据本发明的实施例，所述N蛋白、所述P蛋白、所述L蛋白的至少之一分别独立地来自弹状病毒科病毒。
根据本发明的实施例，水泡性病毒属包括但不限于Vesicular stomatitis NewJersey virus,Maraba virus,Vesicular stomatitis Alagoas virus Indiana 3,Cocalvirus Indiana 2,Isfahan virus,Chandipura virus,Spring viraemia of carp virus,American bat vesiculovirus,Carajas vesiculovirus,Jurona vesiculovirus,MalpaisSpring vesiculovirus,Morreton vesiculovirus,Perinet vesiculovirus,Piryvesiculovirus,Radi vesiculovirus,Yug Bogdanovac vesiculovirus。狂犬病病毒属包括但不限于West Caucasian bat virus,Aravan lyssavirus,Khujand lyssavirus,Rabies virus,Australian bat lyssavirus,European bat lyssavirus 1,European batlyssavirus2,Irkut virus,Shimoni bat virus,Lagos bat virus,Mokola virus,Lleidabat lyssavirus,Ikoma lyssavirus，Gannoruwa bat lyssavirus,Bokeloh batlyssavirus,Duvenhage lyssavirus。短暂热病毒属包括但不限于Bovine feverephemerovirus,Adelaide River ephemerovirus,Berrimah ephemerovirus,Kimberleyephemerovirus。
由此，根据本发明的实施例，所述弹状病毒科病毒包括选自水疱性口炎新泽西病毒、马拉巴病毒、水疱性口炎阿拉戈斯病毒印第安纳3号、科卡尔病毒印第安纳2号、伊斯法罕病毒、钱迪普拉病毒、鲤鱼春季病毒血症病毒、美国蝙蝠水疱病毒、卡拉哈斯水疱病毒、朱罗纳水疱病毒、马尔帕斯春季水疱病毒、莫雷顿水疱病毒、佩里特水疱病毒、，皮里水泡病毒、拉迪水泡病毒、尤格·博格达诺瓦克水泡病毒、西高加索蝙蝠病毒、Aravan狂犬病毒、Khujand狂犬病毒、狂犬病病毒、澳大利亚蝙蝠狂犬病毒、欧洲蝙蝠狂犬病毒1、欧洲蝙蝠狂犬病毒2、伊尔库特病毒、希莫尼蝙蝠病毒、拉各斯蝙蝠病毒、莫科拉病毒、莱伊达蝙蝠狂犬病毒、伊科马狂犬病毒、甘诺鲁瓦蝙蝠狂犬病毒、博克洛蝙蝠狂犬病毒、杜文哈格狂犬病毒、热蜉蝣病毒、阿德莱德河蜉蝣病毒、伯里玛蜉蝣病毒、金伯利蜉蝣病毒的至少之一。
根据本发明的实施例，N蛋白、P蛋白和L蛋白也可以分别独立地来自相同病毒种的不同的病毒血清型，例如包括但不限于水泡性口炎病毒(VSV)印第安那株、新泽西株和可卡尔株。
根据本申请的实施例，所述N蛋白的序列包括SEQ ID NO：1所示的氨基酸序列。
MSVTVKRIIDNTVVVPKLPANEDPVEYPADYFRKSKEIPLYINTTKSLSDLRGYVYQGLKSGNVSIIHVNSYLYGALKDIRGKLDKDWSSFGINIGKAGDTIGIFDLVSLKALDGVLPDGVSDASRTSADDKWLPLYLLGLYRVGRTQMPEYRKKLMDGLTNQCKMINEQFEPLVPEGRDIFDVWGNDSNYTKIVAAVDMFFHMFKKHECASFRYGTIVSRFKDCAALATFGHLCKITGMSTEDVTTWILNREVADEMVQMMLPGQEIDKADSYMPYLIDFGLSSKSPYSSVKNPAFHFWGQLTALLLRSTRARNARQPDDIEYTSLTTAGLLYAYAVGSSADLAQQFCVGDNKYTPDDSTGGLTTNAPPQGRDVVEWLGWFEDQNRKPTPDMMQYAKRAVMSLQGLREKTIGKYAKSEFDK(SEQ ID NO:1)。
根据本申请的实施例，所述P蛋白的序列包括SEQ ID NO：2所示的氨基酸序列。
MDNLTKVREYLKSYSRLDQAVGEIDEIEAQRAEKSNYELFQEDGVEEHTKPSYFQAADDSDTESEPEIEDNQGLYAPDPEAEQVEGFIQGPLDDYADEEVDVVFTSDWKQPELESDEHGKTLRLTSPEGLSGEQKSQWLSTIKAVVQSAKYWNLAECTFEASGEGVIMKERQITPDVYKVTPVMNTHPSQSEAVSDVWSLSKTSMTFQPKKASLQPLTISLDELFSSRGEFISVGGDGRMSHKEAILLGLRYKKLYNQARVKYSL(SEQ ID NO:2)。
根据本申请的实施例，所述L蛋白的序列包括SEQ ID NO：3所示的氨基酸序列。
MEVHDFETDEFNDFNEDDYATREFLNPDERMTYLNHADYNLNSPLISDDIDNLIRKFNSLPIPSMWDSKNWDGVLEMLTSCQANPIPTSQMHKWMGSWLMSDNHDASQGYSFLHEVDKEAEITFDVVETFIRGWGNKPIEYIKKERWTDSFKILAYLCQKFLDLHKLTLILNAVSEVELLNLARTFKGKVRRSSHGTNICRIRVPSLGPTFISEGWAYFKKLDILMDRNFLLMVKDVIIGRMQTVLSMVCRIDNLFSEQDIFSLLNIYRIGDKIVERQGNFSYDLIKMVEPICNLKLMKLARESRPLVPQFPHFENHIKTSVDEGAKIDRGIRFLHDQIMSVKTVDLTLVIYGSFRHWGHPFIDYYTGLEKLHSQVTMKKDIDVSYAKALASDLARIVLFQQFNDHKKWFVNGDLLPHDHPFKSHVKENTWPTAAQVQDFGDKWHELPLIKCFEIPDLLDPSIIYSDKSHSMNRSEVLKHVRMNPNTPIPSKKVLQTMLDTKATNWKEFLKEIDEKGLDDDDLIIGLKGKERELKLAGRFFSLMSWKLREYFVITEYLIKTHFVPMFKGLTMADDLTAVIKKMLDSSSGQGLKSYEAICIANHIDYEKWNNHQRKLSNGPVFRVMGQFLGYPSLIERTHEFFEKSLIYYNGRPDLMRVHNNTLINSTSQRVCWQGQEGGLEGLRQKGWSILNLLVIQREAKIRNTAVKVLAQGDNQVICTQYKTKKSRNVVELQGALNQMVSNNEKIMTAIKIGTGKLGLLINDDETMQSADYLNYGKIPIFRGVIRGLETKRWSRVTCVTNDQIPTCANIMSSVSTNALTVAHFAENPINAMIQYNYFGTFARLLLMMHDPALRQSLYEVQDKIPGLHSSTFKYAMLYLDPSIGGVSGMSLSRFLIRAFPDPVTESLSFWRFIHVHARSEHLKEMSAVFGNPEIAKFRITHIDKLVEDPTSLNIAMGMSPANLLKTEVKKCLIESRQTIRNQVIKDATIYLYHEEDRLRSFLWSINPLFPRFLSEFKSGTFLGVADGLISLFQNSRTIRNSFKKKYHRELDDLIVRSEVSSLTHLGKLHLRRGSCKMWTCSATHADTLRYKSWGRTVIGTTVPHPLEMLGPQHRKETPCAPCNTSGFNYVSVHCPDGIHDVFSSRGPLPAYLGSKTSESTSILQPWERESKVPLIKRATRLRDAISWFVEPDSKLAMTILSNIHSLTGEEWTKRQHGFKRTGSALHRFSTSRMSHGGFASQSTAALTRLMATTDTMRDLGDQNFDFLFQATLLYAQITTTVARDGWITSCTDHYHIACKSCLRPIEEITLDSSMDYTPPDVSHVLKTWRNGEGSWGQEIKQIYPLEGNWKNLAPAEQSYQVGRCIGFLYGDLAYRKSTHAEDSSLFPLSIQGRIRGRGFLKGLLDGLMRASCCQVIHRRSLAHLKRPANAVYGGLIYLIDKLSVSPPFLSLTRSGPIRDELETIPHKIPTSYPTSNRDMGVIVRNYFKYQCRLIEKGKYRSHYSQLWLFSDVLSIDFIGPFSISTTLLQILYKPFLSGKDKNELRELANLSSLLRSGEGWEDIHVKFFTKDILLCPEEIRHACKFGIAKDNNKDMSYPPWGRESRGTITTIPVYYTTTPYPKMLEMPPRIQNPLLSGIRLGQLPTGAHYKIRSILHGMGIHYRDFLSCGDGSGGMTAALLRENVHSRGIFNSLLELSGSVMRGASPEPPSALETLGGDKSRCVNGETCWEYPSDLCDPRTWDYFLRLKAGLGLQIDLIVMDMEVRDSSTSLKIETNVRNYVHRILDEQGVLIYKTYGTYICESEKNAVTILGPMFKTVDLVQTEFSSSQTSEVYMVCKGLKKLIDEPNPDWSSINESWKNLYAFQSSEQEFARAKKVSTYFTLTGIPSQFIPDPFVNIETMLQIFGVPTGVSHAAALKSSDRPADLLTISLFYMAIISYYNINHIRVGPIPPNPPSDGIAQNVGIAITGISFWLSLMEKDIPLYQQCLAVIQQSFPIRWEAVSVKGGYKQKWSTRGDGLPKDTRISDSLAPIGNWIRSLELVRNQVRLNPFNEILFNQLCRTVDNHLKWSNLRRNTGMIEWINRRISKEDRSILMLKSDLHEENSWRD(SEQ ID NO:3)。
根据本申请的实施例，所述新冠病毒拉姆达株的特异性抗原S蛋白的至少一部分具有SEQ ID NO：4所示的氨基酸序列。
MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNVIKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHNSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYQYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYSPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLNVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT(SEQ ID NO:4)。
根据本申请的实施例，所述新冠病毒原始株的特异性抗原S蛋白的至少一部分具有SEQ ID NO：5所示的氨基酸序列。
MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT(SEQ ID NO:5)。
根据本申请的实施例，所述新冠病毒德尔塔的特异性抗原S蛋白的至少一部分具有SEQ ID NO：6所示的氨基酸序列。
MFVFLVLLPLVSSQCVNLRTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESGVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYRYRLFRKSNLKPFERDISTEIYQAGSKPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSRRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQNVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT(SEQ ID NO:6)。
根据本申请的实施例，所述新冠病毒贝塔株的特异性抗原S蛋白的至少一部分具有SEQ ID NO：7所示的氨基酸序列。
MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFANPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRGLPQGFSALEPLVDLPIGINITRFQTLHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFQLLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGVENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT(SEQ ID NO:7)。
所述新冠病毒奥密克戎株的特异性抗原具有SEQ ID NO：8所示的氨基酸序列。
MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHVISGTNGTKRFDNPVLPFNDGVYFASIEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLDHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPIIVREPEDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFDEVFNATRFASVYAWNRKRISNCVADYSVLYNLAPFFTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKVSGNYNYLYRLFRKSNLKPFERDISTEIYQAGNKPCNGVAGFNCYFPLRSYSFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLKGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEYVNNSYECDIPIGAGICASYQTQTKSHRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLKRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKYFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFKGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNHNAQALNTLVKQLSSKFGAISSVLNDIFSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT(SEQ ID NO:8)。
根据本申请的实施例，所述第一RNA序列具有SEQ ID NO：9所示的核苷酸序列。
AUGUCUGUUACAGUCAAGAGAAUCAUUGACAACACAGUCGUAGUUCCAAAACUUCCUGCAAAUGAGGAUCCAGUGGAAUACCCGGCAGAUUACUUCAGAAAAUCAAAGGAGAUUCCUCUUUACAUCAAUACUACAAAAAGUUUGUCAGAUCUAAGAGGAUAUGUCUACCAAGGCCUCAAAUCCGGAAAUGUAUCAAUCAUACAUGUCAACAGCUACUUGUAUGGAGCAUUAAAGGACAUCCGGGGUAAGUUGGAUAAAGAUUGGUCAAGUUUCGGAAUAAACAUCGGGAAAGCAGGGGAUACAAUCGGAAUAUUUGACCUUGUAUCCUUGAAAGCCCUGGACGGCGUACUUCCAGAUGGAGUAUCGGAUGCUUCCAGAACCAGCGCAGAUGACAAAUGGUUGCCUUUGUAUCUACUUGGCUUAUACAGAGUGGGCAGAACACAAAUGCCUGAAUACAGAAAAAAGCUCAUGGAUGGGCUGACAAAUCAAUGCAAAAUGAUCAAUGAACAGUUUGAACCUCUUGUGCCAGAAGGUCGUGACAUUUUUGAUGUGUGGGGAAAUGACAGUAAUUACACAAAAAUUGUCGCUGCAGUGGACAUGUUCUUCCACAUGUUCAAAAAACAUGAAUGUGCCUCGUUCAGAUACGGAACUAUUGUUUCCAGAUUCAAAGAUUGUGCUGCAUUGGCAACAUUUGGACACCUCUGCAAAAUAACCGGAAUGUCUACAGAAGAUGUAACGACCUGGAUCUUGAACCGAGAAGUUGCAGAUGAAAUGGUCCAAAUGAUGCUUCCAGGCCAAGAAAUUGACAAGGCCGAUUCAUACAUGCCUUAUUUGAUCGACUUUGGAUUGUCUUCUAAGUCUCCAUAUUCUUCCGUCAAAAACCCUGCCUUCCACUUCUGGGGGCAAUUGACAGCUCUUCUGCUCAGAUCCACCAGAGCAAGGAAUGCCCGACAGCCUGAUGACAUUGAGUAUACAUCUCUUACUACAGCAGGUUUGUUGUACGCUUAUGCAGUAGGAUCCUCUGCCGACUUGGCACAACAGUUUUGUGUUGGAGAUAACAAAUACACUCCAGAUGAUAGUACCGGAGGAUUGACGACUAAUGCACCGCCACAAGGCAGAGAUGUGGUCGAAUGGCUCGGAUGGUUUGAAGAUCAAAACAGAAAACCGACUCCUGAUAUGAUGCAGUAUGCGAAAAGAGCAGUCAUGUCACUGCAAGGCCUAAGAGAGAAGACAAUUGGCAAGUAUGCUAAGUCAGAAUUUGACAAAUGA(SEQ ID NO：9)。
根据本申请的实施例，所述第二RNA序列具有SEQ ID NO：10所示的核苷酸序列。
AUGGAUAAUCUCACAAAAGUUCGUGAGUAUCUCAAGUCCUAUUCUCGUCUGGAUCAGGCG
GUAGGAGAGAUAGAUGAGAUCGAAGCACAACGAGCUGAAAAGUCCAAUUAUGAGUUGUUCCA
AGAGGAUGGAGUGGAAGAGCAUACUAAGCCCUCUUAUUUUCAGGCAGCAGAUGAUUCUGACA
CAGAAUCUGAACCAGAAAUUGAAGACAAUCAAGGCUUGUAUGCACCAGAUCCAGAAGCUGAGC
AAGUUGAAGGCUUUAUACAGGGGCCUUUAGAUGACUAUGCAGAUGAGGAAGUGGAUGUUGUA
UUUACUUCGGACUGGAAACAGCCUGAGCUUGAAUCUGACGAGCAUGGAAAGACCUUACGGUUG
ACAUCGCCAGAGGGUUUAAGUGGAGAGCAGAAAUCCCAGUGGCUUUCGACGAUUAAAGCAGUC
GUGCAAAGUGCCAAAUACUGGAAUCUGGCAGAGUGCACAUUUGAAGCAUCGGGAGAAGGGGU
CAUUAUGAAGGAGCGCCAGAUAACUCCGGAUGUAUAUAAGGUCACUCCAGUGAUGAACACACA
UCCGUCCCAAUCAGAAGCAGUAUCAGAUGUUUGGUCUCUCUCAAAGACAUCCAUGACUUUCCA
ACCCAAGAAAGCAAGUCUUCAGCCUCUCACCAUAUCCUUGGAUGAAUUGUUCUCAUCUAGAGG
AGAGUUCAUCUCUGUCGGAGGUGACGGACGAAUGUCUCAUAAAGAGGCCAUCCUGCUCGGCCUGAGAUACAAAAAGUUGUACAAUCAGGCGAGAGUCAAAUAUUCUCUGUAG(SEQ ID NO：10)。
根据本申请的实施例，所述第三RNA序列具有SEQ ID NO：11所示的核苷酸序列。
AUGGAAGUCCACGAUUUUGAGACCGACGAGUUCAAUGAUUUCAAUGAAGAUGACUAUGCC
ACAAGAGAAUUCCUGAAUCCCGAUGAGCGCAUGACGUACUUGAAUCAUGCUGAUUACAACCUG
AAUUCUCCUCUAAUUAGUGAUGAUAUUGACAAUUUAAUCAGGAAAUUCAAUUCUCUUCCAAU
UCCCUCGAUGUGGGAUAGUAAGAACUGGGAUGGAGUUCUUGAGAUGUUAACGUCAUGUCAAG
CCAAUCCCAUCCCAACAUCUCAGAUGCAUAAAUGGAUGGGAAGUUGGUUAAUGUCUGAUAAUC
AUGAUGCCAGUCAAGGGUAUAGUUUUUUACAUGAAGUGGACAAAGAGGCAGAAAUAACAUUU
GACGUGGUGGAGACCUUCAUCCGCGGCUGGGGCAACAAACCAAUUGAAUACAUCAAAAAGGAA
AGAUGGACUGACUCAUUCAAAAUUCUCGCUUAUUUGUGUCAAAAGUUUUUGGACUUACACAA
GUUGACAUUAAUCUUAAAUGCUGUCUCUGAGGUGGAAUUGCUCAACUUGGCGAGGACUUUCA
AAGGCAAAGUCAGAAGAAGUUCUCAUGGAACGAACAUAUGCAGGAUUAGGGUUCCCAGCUUG
GGUCCUACUUUUAUUUCAGAAGGAUGGGCUUACUUCAAGAAACUUGAUAUUCUAAUGGACCG
AAACUUUCUGUUAAUGGUCAAAGAUGUGAUUAUAGGGAGGAUGCAAACGGUGCUAUCCAUGG
UAUGUAGAAUAGACAACCUGUUCUCAGAGCAAGACAUCUUCUCCCUUCUAAAUAUCUACAGAA
UUGGAGAUAAAAUUGUGGAGAGGCAGGGAAAUUUUUCUUAUGACUUGAUUAAAAUGGUGGAA
CCGAUAUGCAACUUGAAGCUGAUGAAAUUAGCAAGAGAAUCAAGGCCUUUAGUCCCACAAUUC
CCUCAUUUUGAAAAUCAUAUCAAGACUUCUGUUGAUGAAGGGGCAAAAAUUGACCGAGGUAU
AAGAUUCCUCCAUGAUCAGAUAAUGAGUGUGAAAACAGUGGAUCUCACACUGGUGAUUUAUG
GAUCGUUCAGACAUUGGGGUCAUCCUUUUAUAGAUUAUUACACUGGACUAGAAAAAUUACAU
UCCCAAGUAACCAUGAAGAAAGAUAUUGAUGUGUCAUAUGCAAAAGCACUUGCAAGUGAUUU
AGCUCGGAUUGUUCUAUUUCAACAGUUCAAUGAUCAUAAAAAGUGGUUCGUGAAUGGAGACU
UGCUCCCUCAUGAUCAUCCCUUUAAAAGUCAUGUUAAAGAAAAUACAUGGCCCACAGCUGCUC
AAGUUCAAGAUUUUGGAGAUAAAUGGCAUGAACUUCCGCUGAUUAAAUGUUUUGAAAUACCC
GACUUACUAGACCCAUCGAUAAUAUACUCUGACAAAAGUCAUUCAAUGAAUAGGUCAGAGGU
GUUGAAACAUGUCCGAAUGAAUCCGAACACUCCUAUCCCUAGUAAAAAGGUGUUGCAGACUAU
GUUGGACACAAAGGCUACCAAUUGGAAAGAAUUUCUUAAAGAGAUUGAUGAGAAGGGCUUAG
AUGAUGAUGAUCUAAUUAUUGGUCUUAAAGGAAAGGAGAGGGAACUGAAGUUGGCAGGUAGA
UUUUUCUCCCUAAUGUCUUGGAAAUUGCGAGAAUACUUUGUAAUUACCGAAUAUUUGAUAAA
GACUCAUUUCGUCCCUAUGUUUAAAGGCCUGACAAUGGCGGACGAUCUAACUGCAGUCAUUAA
AAAGAUGUUAGAUUCCUCAUCCGGCCAAGGAUUGAAGUCAUAUGAGGCAAUUUGCAUAGCCA
AUCACAUUGAUUACGAAAAAUGGAAUAACCACCAAAGGAAGUUAUCAAACGGCCCAGUGUUCC
GAGUUAUGGGCCAGUUCUUAGGUUAUCCAUCCUUAAUCGAGAGAACUCAUGAAUUUUUUGAG
AAAAGUCUUAUAUACUACAAUGGAAGACCAGACUUGAUGCGUGUUCACAACAACACACUGAUC
AAUUCAACCUCCCAACGAGUUUGUUGGCAAGGACAAGAGGGUGGACUGGAAGGUCUACGGCAA
AAAGGAUGGAGUAUCCUCAAUCUACUGGUUAUUCAAAGAGAGGCUAAAAUCAGAAACACUGC
UGUCAAAGUCUUGGCACAAGGUGAUAAUCAAGUUAUUUGCACACAGUAUAAAACGAAGAAAU
CGAGAAACGUUGUAGAAUUACAGGGUGCUCUCAAUCAAAUGGUUUCUAAUAAUGAGAAAAUU
AUGACUGCAAUCAAAAUAGGGACAGGGAAGUUAGGACUUUUGAUAAAUGACGAUGAGACUAU
GCAAUCUGCAGAUUACUUGAAUUAUGGAAAAAUACCGAUUUUCCGUGGAGUGAUUAGAGGGU
UAGAGACCAAGAGAUGGUCACGAGUGACUUGUGUCACCAAUGACCAAAUACCCACUUGUGCUA
AUAUAAUGAGCUCAGUUUCCACAAAUGCUCUCACCGUAGCUCAUUUUGCUGAGAACCCAAUCA
AUGCCAUGAUACAGUACAAUUAUUUUGGGACAUUUGCUAGACUCUUGUUGAUGAUGCAUGAU
CCUGCUCUUCGUCAAUCAUUGUAUGAAGUUCAAGAUAAGAUACCGGGCUUGCACAGUUCUACU
UUCAAAUACGCCAUGUUGUAUUUGGACCCUUCCAUUGGAGGAGUGUCGGGCAUGUCUUUGUCC
AGGUUUUUGAUUAGAGCCUUCCCAGAUCCCGUAACAGAAAGUCUCUCAUUCUGGAGAUUCAUC
CAUGUACAUGCUCGAAGUGAGCAUCUGAAGGAGAUGAGUGCAGUAUUUGGAAACCCCGAGAU
AGCCAAGUUUCGAAUAACUCACAUAGACAAGCUAGUAGAAGAUCCAACCUCUCUGAACAUCGC
UAUGGGAAUGAGUCCAGCGAACUUGUUAAAGACUGAGGUUAAAAAAUGCUUAAUCGAAUCAA
GACAAACCAUCAGGAACCAGGUGAUUAAGGAUGCAACCAUAUAUUUGUAUCAUGAAGAGGAU
CGGCUCAGAAGUUUCUUAUGGUCAAUAAAUCCUCUGUUCCCUAGAUUUUUAAGUGAAUUCAA
AUCAGGCACUUUUUUGGGAGUCGCAGACGGGCUCAUCAGUCUAUUUCAAAAUUCUCGUACUAU
UCGGAACUCCUUUAAGAAAAAGUAUCAUAGGGAAUUGGAUGAUUUGAUUGUGAGGAGUGAGG
UAUCCUCUUUGACACAUUUAGGGAAACUUCAUUUGAGAAGGGGAUCAUGUAAAAUGUGGACA
UGUUCAGCUACUCAUGCUGACACAUUAAGAUACAAAUCCUGGGGCCGUACAGUUAUUGGGACA
ACUGUACCCCAUCCAUUAGAAAUGUUGGGUCCACAACAUCGAAAAGAGACUCCUUGUGCACCA
UGUAACACAUCAGGGUUCAAUUAUGUUUCUGUGCAUUGUCCAGACGGGAUCCAUGACGUCUUU
AGUUCACGGGGACCAUUGCCUGCUUAUCUAGGGUCUAAAACAUCUGAAUCUACAUCUAUUUUG
CAGCCUUGGGAAAGGGAAAGCAAAGUCCCACUGAUUAAAAGAGCUACACGUCUUAGAGAUGCU
AUCUCUUGGUUUGUUGAACCCGACUCUAAACUAGCAAUGACUAUACUUUCUAACAUCCACUCU
UUAACAGGCGAAGAAUGGACCAAAAGGCAGCAUGGGUUCAAAAGAACAGGGUCUGCCCUUCAU
AGGUUUUCGACAUCUCGGAUGAGCCAUGGUGGGUUCGCAUCUCAGAGCACUGCAGCAUUGACC
AGGUUGAUGGCAACUACAGACACCAUGAGGGAUCUGGGAGAUCAGAAUUUCGACUUUUUAUU
CCAAGCAACGUUGCUCUAUGCUCAAAUUACCACCACUGUUGCAAGAGACGGAUGGAUCACCAG
UUGUACAGAUCAUUAUCAUAUUGCCUGUAAGUCCUGUUUGAGACCCAUAGAAGAGAUCACCCU
GGACUCAAGUAUGGACUACACGCCCCCAGAUGUAUCCCAUGUGCUGAAGACAUGGAGGAAUGG
GGAAGGUUCGUGGGGACAAGAGAUAAAACAGAUCUAUCCUUUAGAAGGGAAUUGGAAGAAUU
UAGCACCUGCUGAGCAAUCCUAUCAAGUCGGCAGAUGUAUAGGUUUUCUAUAUGGAGACUUG
GCGUAUAGAAAAUCUACUCAUGCCGAGGACAGUUCUCUAUUUCCUCUAUCUAUACAAGGUCGU
AUUAGAGGUCGAGGUUUCUUAAAAGGGUUGCUAGACGGAUUAAUGAGAGCAAGUUGCUGCCA
AGUAAUACACCGGAGAAGUCUGGCUCAUUUGAAGAGGCCGGCCAACGCAGUGUACGGAGGUUU
GAUUUACUUGAUUGAUAAAUUGAGUGUAUCACCUCCAUUCCUUUCUCUUACUAGAUCAGGACC
UAUUAGAGACGAAUUAGAAACGAUUCCCCACAAGAUCCCAACCUCCUAUCCGACAAGCAACCG
UGAUAUGGGGGUGAUUGUCAGAAAUUACUUCAAAUACCAAUGCCGUCUAAUUGAAAAGGGAA
AAUACAGAUCACAUUAUUCACAAUUAUGGUUAUUCUCAGAUGUCUUAUCCAUAGACUUCAUU
GGACCAUUCUCUAUUUCCACCACCCUCUUGCAAAUCCUAUACAAGCCAUUUUUAUCUGGGAAA
GAUAAGAAUGAGUUGAGAGAGCUGGCAAAUCUUUCUUCAUUGCUAAGAUCAGGAGAGGGGUG
GGAAGACAUACAUGUGAAAUUCUUCACCAAGGACAUAUUAUUGUGUCCAGAGGAAAUCAGAC
AUGCUUGCAAGUUCGGGAUUGCUAAGGAUAAUAAUAAAGACAUGAGCUAUCCCCCUUGGGGA
AGGGAAUCCAGAGGGACAAUUACAACAAUCCCUGUUUAUUAUACGACCACCCCUUACCCAAAG
AUGCUAGAGAUGCCUCCAAGAAUCCAAAAUCCCCUGCUGUCCGGAAUCAGGUUGGGCCAAUUA
CCAACUGGCGCUCAUUAUAAAAUUCGGAGUAUAUUACAUGGAAUGGGAAUCCAUUACAGGGA
CUUCUUGAGUUGUGGAGACGGCUCCGGAGGGAUGACUGCUGCAUUACUACGAGAAAAUGUGC
AUAGCAGAGGAAUAUUCAAUAGUCUGUUAGAAUUAUCAGGGUCAGUCAUGCGAGGCGCCUCU
CCUGAGCCCCCCAGUGCCCUAGAAACUUUAGGAGGAGAUAAAUCGAGAUGUGUAAAUGGUGAA
ACAUGUUGGGAAUAUCCAUCUGACUUAUGUGACCCAAGGACUUGGGACUAUUUCCUCCGACUC
AAAGCAGGCUUGGGGCUUCAAAUUGAUUUAAUUGUAAUGGAUAUGGAAGUUCGGGAUUCUUC
UACUAGCCUGAAAAUUGAGACGAAUGUUAGAAAUUAUGUGCACCGGAUUUUGGAUGAGCAAG
GAGUUUUAAUCUACAAGACUUAUGGAACAUAUAUUUGUGAGAGCGAAAAGAAUGCAGUAACA
AUCCUUGGUCCCAUGUUCAAGACGGUCGACUUAGUUCAAACAGAAUUUAGUAGUUCUCAAACG
UCUGAAGUAUAUAUGGUAUGUAAAGGUUUGAAGAAAUUAAUCGAUGAACCCAAUCCCGAUUG
GUCUUCCAUCAAUGAAUCCUGGAAAAACCUGUACGCAUUCCAGUCAUCAGAACAGGAAUUUGC
CAGAGCAAAGAAGGUUAGUACAUACUUUACCUUGACAGGUAUUCCCUCCCAAUUCAUUCCUGA
UCCUUUUGUAAACAUUGAGACUAUGCUACAAAUAUUCGGAGUACCCACGGGUGUGUCUCAUGC
GGCUGCCUUAAAAUCAUCUGAUAGACCUGCAGAUUUAUUGACCAUUAGCCUUUUUUAUAUGGC
GAUUAUAUCGUAUUAUAACAUCAAUCAUAUCAGAGUAGGACCGAUACCUCCGAACCCCCCAUC
AGAUGGAAUUGCACAAAAUGUGGGGAUCGCUAUAACUGGUAUAAGCUUUUGGCUGAGUUUGA
UGGAGAAAGACAUUCCACUAUAUCAACAGUGUUUAGCAGUUAUCCAGCAAUCAUUCCCGAUUA
GGUGGGAGGCUGUUUCAGUAAAAGGAGGAUACAAGCAGAAGUGGAGUACUAGAGGUGAUGGG
CUCCCAAAAGAUACCCGAAUUUCAGACUCCUUGGCCCCAAUCGGGAACUGGAUCAGAUCUCUGGAAUUGGUCCGAAACCAAGUUCGUCUAAAUCCAUUCAAUGAGAUCUUGUUCAAUCAGCUAUGUCGUACAGUGGAUAAUCAUUUGAAAUGGUCAAAUUUGCGAAGAAACACAGGAAUGAUUGAAUGGAUCAAUAGACGAAUUUCAAAAGAAGACCGGUCUAUACUGAUGUUGAAGAGUGACCUACACGAGGAAAACUCUUGGAGAGAUUAA(SEQ ID NO：11)。
在本申请的第二方面，本申请提出了一种新冠病毒多价疫苗，其包括前面所述的自复制RNA分子。
根据本申请的实施例，这里需要说明的是，前述四种特异性抗原S蛋白的至少一部分的编码序列可以在同一自复制RNA分子上，也可以分别负载于两个或者更多个自复制RNA分子上。
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1：reRNATM新冠疫苗的设计及效果评价
1、reRNATM新冠疫苗的设计
新冠疫苗设计上采用自扩增RNA(reRNATM)核心复合体来表达新冠病毒的刺突蛋白S，原始株的疫苗疫苗设计如图3。具体方法为：采用本领域常规方法设计并合成SEQ ID NO：9、SEQ ID NO：10、SEQ ID NO：11以及SEQ ID NO：5(S蛋白)等核苷酸序列，并将上述核苷酸序列合成至pcDNA3.1，得到reRNATM疫苗质粒。将构建好的reRNATM疫苗质粒与辅助质粒(pCI-VSVG质粒以及T7RNA聚合酶质粒pCAG-T7pol)共转入293T细胞，48小时后收获上清进行纯化，即可获得reRNATM疫苗药物。
2、reRNATM新冠疫苗能够激活抗原特异性体液免疫应答及长效抗原特异性结合抗体的实验验证
采用6-8周龄大小的BALB/c小鼠作为实验动物，设计高、中、低三种不同的给药剂量(分别为14.2ng、7.1ng、1.4ng)，使用肌肉注射的给药方式及不同给药频次的方案。分别在第0天及第21天对每只小鼠注射相应剂量的reRNATM新冠疫苗，其中，空白对照组(Blank)小鼠不注射疫苗。分别在初次免疫后14天，及二次免疫后14天，利用Elisa检测方法对小鼠血浆内抗原特异性IgG抗体进行检测，具体参见图4。由图4可知，在初次免疫后第14天，最高剂量组产生的抗体滴度最高，同时也伴随着一定的剂量依赖关系。在二次免疫后14天检测小鼠血清后发现，三种不同剂量下抗原特异性抗体滴度均有显著升高，不同给药剂量之间，无显著性差异。因此，结果表明，reRNATM新冠疫苗能够有效的激活机体内抗原特异性的体液免疫应答并产生高水平的抗原特异性IgG抗体。
为了研究reRNATM新冠疫苗是否能够激活长效的抗原特异性体液免疫应答。发明人在上述实验中的二次给药后第60天，对小鼠血清内抗原特异性IgG抗体及不同IgG亚型抗体进行检测，具体参见图5。由图5可知，三种给药，均能够激活长效的IgG抗体，抗体滴度均维持在较高水平。通过检测不同的IgG抗体亚型发现，reRNATM新冠疫苗能够诱导机体产生IgG1及IgG2a两种不同的IgG亚型抗体。通过比较IgG1和IgG2a两种抗体水平得知，reRNATM新冠疫苗更倾向于激活机体内IgG2a为主的体液免疫应答。
由上可知，reRNATM新冠疫苗不仅能激活机体内抗原特异性体液免疫应答还能激活长效体液免疫应答。根据不同给药方式及不同给药剂量研究，其中采用肌肉给药方式、1.4ng给药剂量及二次免疫为最佳方案。
3、reRNATM新冠疫苗能够诱导机体产生高水平中和抗体的实验验证
通过对reRNATM新冠疫苗激活的体液免疫应答的研究，发现该疫苗能够激活较高水平的抗原特异性结合抗体。为探究reRNATM新冠疫苗所激活的体内抗原特异性抗体是否具有中和新冠病毒的作用，采用假病毒评价体系进行相关研究。该假病毒是一类表达有S蛋白的慢病毒，其感染细胞的机制与新冠病毒相似，因此该体系可以用来进行中和抗体研究。
首先，设计高、中、低三种不同的给药剂量(具体参见本实施例的步骤2的给药量)，采用肌肉给药方式，在第0天及第21天进行小鼠免疫，其中，空白对照组(Blank)小鼠不注射疫苗。分别在初次免疫后14天和二次免疫后14天对小鼠血清内中和抗体效价进行检测。研究发现，在初次给药后reRNATM新冠疫苗能够诱导产生一定水平的中和抗体，但在二次给药后reRNATM新冠疫苗能够诱导更高水平的中和抗体。相比于初次免疫，在二次免疫后中和抗体效价提高近100倍。如图6所示，reRNATM新冠疫苗不仅具有免疫原性还能够激活机体产生高水平的中和抗体，也能够说明reRNATM新冠疫苗能够有效保护机体不被新冠病毒感染。
4、reRNATM新冠疫苗能够引发机体产生较强水平的T细胞免疫应答的实验验证
本实施例采用肌肉给药方式，每只小鼠注射7.2ng reRNATM新冠疫苗，其中，空白对照组(Blank)小鼠不注射疫苗。在免疫后第7天，体外利用IFN-γElispot检测技术对脾脏内抗原特异性T细胞免疫应答进行检测，其中，该实施例中主要使用Spike蛋白刺激CD4和CD8T细胞，进而激活T细胞分泌IFN-γ细胞因子，具体检测结果参见图7。
如图7所示，根据IFN-γElispot实验结果显示，通过是用肌肉给药方式能够激活较强水平的抗原特异性T细胞免疫应答。因此，结果表明，reRNATM新冠疫苗不仅能够激活体液免疫应答还能够有效的激活T细胞免疫应答。
5、reRNATM新冠疫苗能够促进Th1和CD8T细胞免疫应答的实验验证
为探究reRNATM新冠疫苗诱导激活的Spike蛋白特异性T细胞亚群种类及功能，将reRNATM新冠疫苗免疫小鼠后第七天取出脾脏细胞，使用spike抗原蛋白刺激脾脏细胞24小时，检测细胞培养上清中细胞因子种类，其中，空白对照组(Blank)小鼠不注射疫苗，具体参见图8，细胞因子检测结果表明，IFN、TNF及IL-2细胞因子较高，IL-4水平较低。同时，利用流式检测技术检测细胞胞内因子分泌情况，检测CD4及CD8T细胞所分泌的细胞因子种类，具体参见图9，研究发现，CD4T细胞主要分泌TNF-a及IFN-γ细胞因子，CD8T细胞主要分泌IFN-γ细胞因子。因此，可以得知，该疫苗均能够有效的激活CD4和CD8T细胞免疫应答。
实施例2：五价新冠疫苗的设计及效果评价
1、五价reRNATM新冠疫苗的设计如图10所示，都是基于自扩增RNA(reRNATM)核心复合体在完成S蛋白的表达。具体方法为：采用本领域常规方法设计并合成SEQ ID NO：16、SEQID NO：17、SEQ ID NO：18以及编码修饰后的S蛋白(分别为：SEQ ID NO：5、SEQ ID NO：6、SEQID NO：7、SEQ ID NO：8或SEQ ID NO：9)的核苷酸序列，并将上述核苷酸序列合成至pcDNA3.1，得到reRNATM疫苗质粒(pcDNA3.1)。将构建好的reRNATM疫苗质粒与辅助质粒(pCI-VSVG质粒以及T7RNA聚合酶质粒pCAG-T7pol)共转入293T细胞，48小时后收获上清进行纯化，即可获得reRNATM疫苗药物。
2、五价reRNATM新冠疫苗能够在小鼠体内引发较强的结合抗体及中和抗体滴度的实验验证。
分别在第0天及第14天进行采用肌肉给药方式将一定剂量(总共7ng，其中新冠病毒原始株、新冠病毒拉姆达株、新冠病毒德尔塔株、新冠病毒贝塔株和新冠奥密克戎株的特异性抗原S蛋白含量相同)注射至小鼠体内，在最后一次给药后第14天，对血浆内抗原特异性不同亚型的结合抗体及不同病毒株的中和抗体滴度进行检测，其中，空白对照组(Blank)小鼠不注射疫苗，具体参见图11所示，结果显示，经过疫苗给药后，五价新冠疫苗能够有效的激活机体内体液免疫应答。同时，利用不同的新冠病毒假病毒株进行中和抗体滴度检测，具体参见图12，结果显示，五价reRNATM新冠疫苗在针对不同突变毒株上均能够展现出一定的中和效果。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本申请的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本申请的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本申请的限制，本领域的普通技术人员在本申请的范围内可以对上述实施例进行变化、修改、替换和变型。
Claims (11)
1.一种自复制RNA分子，其特征在于，包括：
自复制核心序列，所述自复制核心序列编码N蛋白、P蛋白和L蛋白或其功能片段；和
抗原序列，所述抗原序列编码选自新冠病毒原始株、新冠病毒拉姆达株、新冠病毒德尔塔株、新冠病毒贝塔株、新冠奥密克戎株至少一种的特异性抗原S蛋白的至少一部分。
2.根据权利要求1所述的自复制RNA分子，所述N蛋白的序列包括SEQ ID NO：1所示的氨基酸序列。
3.根据权利要求1所述的自复制RNA分子，所述P蛋白的序列包括SEQ ID NO：2所示的氨基酸序列。
4.根据权利要求1所述的自复制RNA分子，所述L蛋白的序列包括SEQ ID NO：3所示的氨基酸序列。
5.根据权利要求1所述的自复制RNA分子，其特征在于，所述新冠病毒拉姆达株的特异性抗原具有SEQ ID NO：4所示的氨基酸序列。
6.根据权利要求1所述的自复制RNA分子，其特征在于，所述新冠病毒原始株的特异性抗原具有SEQ ID NO：5所示的氨基酸序列。
7.根据权利要求1所述的自复制RNA分子，其特征在于，所述新冠病毒德尔塔的特异性抗原具有SEQ ID NO：6所示的氨基酸序列。
8.根据权利要求1所述的自复制RNA分子，其特征在于，所述新冠病毒贝塔株的特异性抗原具有SEQ ID NO：7所示的氨基酸序列。
9.根据权利要求1所述的自复制RNA分子，其特征在于，所述新冠病毒奥密克戎株的特异性抗原具有SEQ ID NO：8所示的氨基酸序列。
10.根据权利要求1所述的自复制RNA分子，其特征在于，所述自复制核心序列包括：第一RNA序列，所述第一RNA序列编码N蛋白或其功能片段；第二RNA序列，所述第二RNA序列编码P蛋白或其功能片段；和第三RNA序列，所述第三RNA序列编码L蛋白或其功能片段；
任选地，所述第一RNA序列具有SEQ ID NO：9所示的核苷酸序列；
任选地，所述第二RNA序列具有SEQ ID NO：10所示的核苷酸序列；
任选地，所述第三RNA序列具有SEQ ID NO：11所示的核苷酸序列。
11.一种新冠病毒多价疫苗，其特征在于，包括权利要求1～10任一项所述的自复制RNA分子。
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