CN112608946B - 一种环状rna载体及其在疫苗中的应用 - Google Patents

一种环状rna载体及其在疫苗中的应用 Download PDF

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CN112608946B
CN112608946B CN202110058897.3A CN202110058897A CN112608946B CN 112608946 B CN112608946 B CN 112608946B CN 202110058897 A CN202110058897 A CN 202110058897A CN 112608946 B CN112608946 B CN 112608946B
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CN112608946A (zh
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何越峰
尹锦瑶
孙明军
毛一竹
蒋成兰
谭婧文
周倩
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Kaituo Biotechnology Suzhou Co ltd
Shanghai Kaituo Biotechnology Co ltd
Wuhan Kaituo Biotechnology Co ltd
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Abstract

本发明公开了一种环状RNA载体及其在疫苗中的应用，该载体中的结构为包括环状RNA翻译启动区，蛋白定位引导区，蛋白翻译RNA片段插入点，结构增强区，该环状RNA载体具有较强的翻译功能，可以将蛋白翻译RNA片段的蛋白质翻译成蛋白并转让到适当区域，可以作用RNA疫苗的载体，比mRNA疫苗具有若干优点，可用于为预防或治疗的疫苗。

Description

一种环状RNA载体及其在疫苗中的应用
技术领域
本发明属于生物制药领域 ，具体地涉及一种环状RNA载体及其在疫苗中，该一种环状RNA载体，可以作用多种疫苗的载体。
背景技术
疫苗是将病原微生物及其代谢产物，经过人工减毒、灭活或利用转基因等方法制成的用于预防传染病的自动免疫制剂。接种疫苗是预防传染病的有效方法。疫苗可增强免疫力，并使个体对病原体感染具有抵抗力。疫苗可能含有减毒活病原体，通常是病毒，灭活的完整病原体，类毒素（导致疾病的细菌产生的毒素的灭活形式或病原体的一部分（例如天然或重组蛋白，多糖，结合多糖或病毒-类粒子,也可能含有佐剂以刺激免疫反应。按其性质可分为减毒活疫苗、灭活疫苗、抗毒素、亚单位疫苗（含多肽疫苗）、载体疫苗、核酸疫苗等。
mRNA是介于蛋白质编码DNA的翻译和核糖体在细胞质中产生蛋白质之间的中间步骤。与基于pDNA和基于病毒载体的疫苗相比，基于mRNA的疫苗具有多个优势。mRNA疫苗不会产生感染性颗粒或不会整合到宿主细胞的基因组中。它们可以原位递送以表达抗原，而无需穿过核膜屏障进行蛋白质表达，并且可以表达复杂的抗原而无包装限制。使用完全合成的生产工艺，可以在获得基因序列信息的几天之内快速生产mRNA疫苗。该平台具有多功能性，可适应多个目标，因此非常适合对新兴病原体的快速反应。目前正在研究两种主要类型的RNA作为疫苗：非复制性mRNA和病毒衍生的自扩增RNA。常规的基于mRNA的疫苗编码目标抗原，并包含5'和3'非翻译区（UTR），而自扩增RNA不仅编码抗原，而且还编码能够使细胞内RNA扩增和丰富的蛋白质表达的病毒复制机制。mRNA疫苗具有强大的潜力，快速开发的能力以及低成本生产和安全管理的潜力，因此可以替代常规疫苗。然而，直到最近，由于mRNA的体内递送的不稳定性和低效，它们的应用受到了限制。现在，最新的技术进步已在很大程度上克服了这些问题，针对传染病和多种类型癌症的多种mRNA疫苗平台已在动物模型和人类中证明了令人鼓舞的结果。
环状RNA (circRNA)是一种封闭结构的内源性长链非编码RNA,是通过对前体mRNA(pre-mRNAs)片段的反向剪切后并以共价键形式首尾相连而形成，被认为是非编码RNA这一大家族中最为稳定的RNA分子。在过去，circRNA因其编码蛋白基因的起始密码子和/或终止密码子被删除故被认为是一种不能被编码为蛋白质的长链非编码RNA。但近年来研究发现，部分circRNA可以编码为特定的蛋白质来影响肿瘤的发生发展。尽管尚未完全了解circRNA的产生方式，但许多是通过反向剪接反应共价连接pre-mRNA特定片段的5'和3'末端（跨越外显子或外显子与内含子的组合）而产生的，而规范剪接有助于生成一些内含子circRNA。鉴于circRNA缺少暴露的5'和3'末端，因此可以保护它们免受核酸外切酶降解，因此通常认为它们比其线性宿主转录物更稳定。circRNA翻译时只能采用与帽无关的方式,除了M6A介导的翻译，人工和含有内部核糖体进入位点（IRES），其直接招募核糖体，也可译为蛋白质。这两种方法可以彼此耦合。circRNA可以影响重要mRNA的衰变，翻译和转录，从而影响控制健康和疾病的基因表达程序。
发明内容
本发明的目的一种环状RNA载体及其在疫苗中的应用。
一种环状RNA载体，载体中的结构为包括环状RNA翻译启动区，蛋白定位引导区，蛋白翻译RNA片段插入点。载体中还可以包括结构增强区位于蛋白翻译RNA片段插入点之后。环状RNA翻译启动区为RNA序列为SEQ ID NO.1和SEQ ID NO.2。蛋白定位引导区RNA序列为SEQ ID NO.3和SEQ ID NO.4。蛋白翻译RNA片段插入点在蛋白定位引导区与结构增强区之间。蛋白翻译RNA片段插入点插入的序列为SEQ ID NO.5结构增强区的序列为SEQ ID NO.6。环状RNA载体的运用制备疫苗中，包括制备新冠病毒的疫苗中的应用（81860572）。
本发明试验方面我们参考了202010125774 .2发明专利。
本发明与一般的mRNA疫苗采用了完全不同的载体，改造后circRNA载体具有翻译高效，而且不容易被RNA酶水解，具有更加优良的性能。
附图说明
图1 采用GAPDH作用内参，蛋白检测显示新冠病毒棘突蛋白在细胞中表达了,左边是C环右边是S环。
具体实施方式
下面对本发明作进一步的说明，但不以任何方式对本发明加以限制，基于本发明教导所作的任何变换或替换，均属于本发明的保护范围。
具体实施方式：
实施例
根据我们需要生产的circRNAs，我们设计出相应的线状DNA模板，由生物公司合成构建成互补双链的DNA，其序列分别为SEQ ID NO.7和SEQ ID NO.8所示.将生物公司制作合成的双链DNA作为RNA合成模板，采用新海基因的T7高产RNA合成试剂盒体外大量合成线状RNA，然后将模板DNA去除，纯化后得到线性RNA,然后通过Circular RNA Synthesis Kit试剂将线性RNA 首尾相连进行环化，分离正确环化的RNA,其RNA序列分别为SEQ ID NO.9首尾相连（命名为C环）和SEQ ID NO.10首尾相连（命名为S环）。将得到的环状RNA。将上述两个环状RNA分别转染到A549细胞中，采用通常的转染试剂， 48小时我们采用western blot的方法，采用GAPDH作用内参，蛋白检测显示新冠病毒棘突蛋白在细胞中表达了,结果如图1所示。
将C环和S环分别和递送剂制成以环状RNA为载体的新冠病毒疫苗,将制疫苗腿部肌肉注射家兔，对照组3只：仅为递送剂制， C环和S环低剂量组每组3只，每只含2.5微克环状RNA，C环和S环高剂量组每组3只组：每只含5微克环状RNA，给药后15天, 通过ELISA法检测是否产生抗体，对照组为3只阴性，C环和S环低剂量组6只均为弱阳性，C环和S环低剂量组组5只阳性，C环一只弱阳性,实验显示环状RNA疫苗能够使家兔体内产生抗体。RNA中的U采取1-甲基-3伪尿酰代替，可以减轻疫苗的副反应和体内非特异免疫反应。
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acaaucacuu cugguuggac cuuuggugca ggugcugcau uacaaauacc auuugcuaug 2700
caaauggcuu auagguuuaa ugguauugga guuacacaga auguucucua ugagaaccaa 2760
aaauugauug ccaaccaauu uaauagugcu auuggcaaaa uucaagacuc acuuucuucc 2820
acagcaagug cacuuggaaa acuucaagau guggucaacc aaaaugcaca agcuuuaaac 2880
acgcuuguua aacaacuuag cuccaauuuu ggugcaauuu caaguguuuu aaaugauauc 2940
cuuucacguc uugacaaagu ugaggcugaa gugcaaauug auagguugau cacaggcaga 3000
cuucaaaguu ugcagacaua ugugacucaa caauuaauua gagcugcaga aaucagagcu 3060
ucugcuaauc uugcugcuac uaaaauguca gaguguguac uuggacaauc aaaaagaguu 3120
gauuuuugug gaaagggcua ucaucuuaug uccuucccuc agucagcacc ucauggugua 3180
gucuucuugc augugacuua ugucccugca caagaaaaga acuucacaac ugcuccugcc 3240
auuugucaug auggaaaagc acacuuuccu cgugaaggug ucuuuguuuc aaauggcaca 3300
cacugguuug uaacacaaag gaauuuuuau gaaccacaaa ucauuacuac agacaacaca 3360
uuugugucug guaacuguga uguuguaaua ggaauuguca acaacacagu uuaugauccu 3420
uugcaaccug aauuagacuc auucaaggag gaguuagaua aauauuuuaa gaaucauaca 3480
ucaccagaug uugauuuagg ugacaucucu ggcauuaaug cuucaguugu aaacauucaa 3540
aaagaaauug accgccucaa ugagguugcc aagaauuuaa augaaucucu caucgaucuc 3600
caagaacuug gaaaguauga gcaguauaua aaauggccau gguacauuug gcuagguuuu 3660
auagcuggcu ugauugccau aguaauggug acaauuaugc uuugcuguau gaccaguugc 3720
uguaguuguc ucaagggcug uuguucuugu ggauccugcu gcaaauuuga ugaagacgac 3780
ucugagccag ugcucaaagg agucaaauua cauuacacau aa 3822
<210> 6
<211> 116
<212> RNA
<213> 人工序列(Artificial sequence)
<400> 6
guaguuaggc accucuguug cccaucgcau cggcagggca cgcauauacc uuuuuuuucg 60
cuuuuuaggu auaugcgugc ccugccgaug cgaugggcaa cagaggugca aaaaaa 116
<210> 7
<211> 4612
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 7
taatacgact cactatagtt caccagtgga ttctgtgttg ttttatccca ttacaactct 60
ccacaacctt ttattacatc aagaaggagc taaaatcgca gtgcgtttag ctggtgggct 120
gcagaaaatc gttgccttgc tcaacaaaac aaatcttaaa ttcttggcta ttacgacaga 180
ctgccttcaa attttagctt atcgcaacca agaaagcaag ctcatcatac tggctagtgg 240
tggaccccaa gctttagtaa atataatcag gacctatact tacgaaaaac tactgtggac 300
cacaagcaga gtgctgaagg tgctatctgt ctgctctagt aataagccgg ctattgtaga 360
agctgggtat ttgaagtata ccatacaact gttttgaaaa tccagcgtgg acaatggcta 420
ctcaagctga tttgatggag ttggacatgg ccatggaacc agacagaaaa gcggctgtta 480
gtcactggca gcaacagtct tacctggact ctggaatcca ttctggtgcc actaccacag 540
ctccttctct gagtggtaaa ggcaatcctg aggaagagga tgtggatacc tcccaagtcc 600
tgtatgagtg ggaacaggga ttttctatga acctgctgct gattctgacc tttgtggcgg 660
cggcggtggc gtaaatgttt ccgccacttg ttttattgcc actagtctct agtcagtgtg 720
ttaatcttac aaccagaact caattacccc ctgcatacac taattctttc acacgtggtg 780
tttattaccc tgacaaagtt ttcagatcct cagttttaca ttcaactcag gacttgttct 840
tacctttctt ttccaatgtt acttggttcc atgctataca tgtctctggg accaatggta 900
ctaagaggtt tgataaccct gtcctaccat ttaatgatgg tgtttatttt gcttccactg 960
agaagtctaa cataataaga ggctggattt ttggtactac tttagattcg aagacccagt 1020
ccctacttat tgttaataac gctactaatg ttgttattaa agtctgtgaa tttcaatttt 1080
gtaatgatcc atttttgggt gtttattacc acaaaaacaa caaaagttgg atggaaagtg 1140
agttcagagt ttattctagt gcgaataatt gcacttttga atatgtctct cagccttttc 1200
ttatggacct tgaaggaaaa cagggtaatt tcaaaaatct tagggaattt gtgtttaaga 1260
atattgatgg ttattttaaa atatattcta agcacacgcc tattaattta gtgcgtgatc 1320
tccctcaggg tttttcggct ttagaaccat tggtagattt gccaataggt attaacatca 1380
ctaggtttca aactttactt gctttacata gaagttattt gactcctggt gattcttctt 1440
caggttggac agctggtgct gcagcttatt atgtgggtta tcttcaacct aggacttttc 1500
tattaaaata taatgaaaat ggaaccatta cagatgctgt agactgtgca cttgaccctc 1560
tctcagaaac aaagtgtacg ttgaaatcct tcactgtaga aaaaggaatc tatcaaactt 1620
ctaactttag agtccaacca acagaatcta ttgttagatt tcctaatatt acaaacttgt 1680
gcccttttgg tgaagttttt aacgccacca gatttgcatc tgtttatgct tggaacagga 1740
agagaatcag caactgtgtt gctgattatt ctgtcctata taattccgca tcattttcca 1800
cttttaagtg ttatggagtg tctcctacta aattaaatga tctctgcttt actaatgtct 1860
atgcagattc atttgtaatt agaggtgatg aagtcagaca aatcgctcca gggcaaactg 1920
gaaagattgc tgattataat tataaattac cagatgattt tacaggctgc gttatagctt 1980
ggaattctaa caatcttgat tctaaggttg gtggtaatta taattacctg tatagattgt 2040
ttaggaagtc taatctcaaa ccttttgaga gagatatttc aactgaaatc tatcaggccg 2100
gtagcacacc ttgtaatggt gttgaaggtt ttaattgtta ctttccttta caatcatatg 2160
gtttccaacc cactaatggt gttggttacc aaccatacag agtagtagta ctttcttttg 2220
aacttctaca tgcaccagca actgtttgtg gacctaaaaa gtctactaat ttggttaaaa 2280
acaaatgtgt caatttcaac ttcaatggtt taacaggcac aggtgttctt actgagtcta 2340
acaaaaagtt tctgcctttc caacaatttg gcagagacat tgctgacact actgatgctg 2400
tccgtgatcc acagacactt gagattcttg acattacacc atgttctttt ggtggtgtca 2460
gtgttataac accaggaaca aatacttcta accaggttgc tgttctttat cagggtgtta 2520
actgcacaga agtccctgtt gctattcatg cagatcaact tactcctact tggcgtgttt 2580
attctacagg ttctaatgtt tttcaaacac gtgcaggctg tttaataggg gctgaacatg 2640
tcaacaactc atatgagtgt gacataccca ttggtgcagg tatatgcgct agttatcaga 2700
ctcagactaa ttctcctcgg cgggcacgta gtgtagctag tcaatccatc attgcctaca 2760
ctatgtcact tggtgcagaa aattcagttg cttactctaa taactctatt gccataccca 2820
caaattttac tattagtgtt accacagaaa ttctaccagt gtctatgacc aagacatcag 2880
tagattgtac aatgtacatt tgtggtgatt caactgaatg cagcaatctt ttgttgcaat 2940
atggcagttt ttgtacacaa ttaaaccgtg ctttaactgg aatagctgtt gaacaagaca 3000
aaaacaccca agaagttttt gcacaagtca aacaaattta caaaacacca ccaattaaag 3060
attttggtgg ttttaatttt tcacaaatat taccagatcc atcaaaacca agcaagaggt 3120
catttattga agatctactt ttcaacaaag tgacacttgc agatgctggc ttcatcaaac 3180
aatatggtga ttgccttggt gatattgctg ctagagacct catttgtgca caaaagttta 3240
acggccttac tgttttgcca cctttgctca cagatgaaat gattgctcaa tacacttctg 3300
cactgttagc gggtacaatc acttctggtt ggacctttgg tgcaggtgct gcattacaaa 3360
taccatttgc tatgcaaatg gcttataggt ttaatggtat tggagttaca cagaatgttc 3420
tctatgagaa ccaaaaattg attgccaacc aatttaatag tgctattggc aaaattcaag 3480
actcactttc ttccacagca agtgcacttg gaaaacttca agatgtggtc aaccaaaatg 3540
cacaagcttt aaacacgctt gttaaacaac ttagctccaa ttttggtgca atttcaagtg 3600
ttttaaatga tatcctttca cgtcttgaca aagttgaggc tgaagtgcaa attgataggt 3660
tgatcacagg cagacttcaa agtttgcaga catatgtgac tcaacaatta attagagctg 3720
cagaaatcag agcttctgct aatcttgctg ctactaaaat gtcagagtgt gtacttggac 3780
aatcaaaaag agttgatttt tgtggaaagg gctatcatct tatgtccttc cctcagtcag 3840
cacctcatgg tgtagtcttc ttgcatgtga cttatgtccc tgcacaagaa aagaacttca 3900
caactgctcc tgccatttgt catgatggaa aagcacactt tcctcgtgaa ggtgtctttg 3960
tttcaaatgg cacacactgg tttgtaacac aaaggaattt ttatgaacca caaatcatta 4020
ctacagacaa cacatttgtg tctggtaact gtgatgttgt aataggaatt gtcaacaaca 4080
cagtttatga tcctttgcaa cctgaattag actcattcaa ggaggagtta gataaatatt 4140
ttaagaatca tacatcacca gatgttgatt taggtgacat ctctggcatt aatgcttcag 4200
ttgtaaacat tcaaaaagaa attgaccgcc tcaatgaggt tgccaagaat ttaaatgaat 4260
ctctcatcga tctccaagaa cttggaaagt atgagcagta tataaaatgg ccatggtaca 4320
tttggctagg ttttatagct ggcttgattg ccatagtaat ggtgacaatt atgctttgct 4380
gtatgaccag ttgctgtagt tgtctcaagg gctgttgttc ttgtggatcc tgctgcaaat 4440
ttgatgaaga cgactctgag ccagtgctca aaggagtcaa attacattac acataagtag 4500
ttaggcacct ctgttgccca tcgcatcggc agggcacgca tatacctttt ttttcgcttt 4560
ttaggtatat gcgtgccctg ccgatgcgat gggcaacaga ggtgcaaaaa aa 4612
<210> 8
<211> 4458
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 8
taatacgact cactataggt tctaatcgat taactatcat tgaagcaact catcttctct 60
tggtggagcc catattgaac cctgcccatc agcttcaggc catagggagg gtgcaccgaa 120
ttggacagac aaaacctact attgtacaca gattcttaat taaagcaaca atagaagaaa 180
gaatccaggc aatcctgaaa actgctgaga gaagggcagc cattctacaa aagtggaagc 240
tgtggtcaga actctgatga aaatacagct tagagatcca ggggccaaag cactcgtttt 300
ctcaacgtgg caagatgtat tagatattat ttcaaaagct cttactgaca acaacatgga 360
atttgcacaa atcagtcgtg ttaagacatt tcaggagaac ctttcagcat ttaaacgtga 420
tccccaaatc aatattttgc tgctgcccct gcacacaatg tatcgcatgc agctgctgag 480
ctgcattgcg ctgagcctgg cgctggtgac caacagctaa atgtttccgc cacttgtttt 540
attgccacta gtctctagtc agtgtgttaa tcttacaacc agaactcaat taccccctgc 600
atacactaat tctttcacac gtggtgttta ttaccctgac aaagttttca gatcctcagt 660
tttacattca actcaggact tgttcttacc tttcttttcc aatgttactt ggttccatgc 720
tatacatgtc tctgggacca atggtactaa gaggtttgat aaccctgtcc taccatttaa 780
tgatggtgtt tattttgctt ccactgagaa gtctaacata ataagaggct ggatttttgg 840
tactacttta gattcgaaga cccagtccct acttattgtt aataacgcta ctaatgttgt 900
tattaaagtc tgtgaatttc aattttgtaa tgatccattt ttgggtgttt attaccacaa 960
aaacaacaaa agttggatgg aaagtgagtt cagagtttat tctagtgcga ataattgcac 1020
ttttgaatat gtctctcagc cttttcttat ggaccttgaa ggaaaacagg gtaatttcaa 1080
aaatcttagg gaatttgtgt ttaagaatat tgatggttat tttaaaatat attctaagca 1140
cacgcctatt aatttagtgc gtgatctccc tcagggtttt tcggctttag aaccattggt 1200
agatttgcca ataggtatta acatcactag gtttcaaact ttacttgctt tacatagaag 1260
ttatttgact cctggtgatt cttcttcagg ttggacagct ggtgctgcag cttattatgt 1320
gggttatctt caacctagga cttttctatt aaaatataat gaaaatggaa ccattacaga 1380
tgctgtagac tgtgcacttg accctctctc agaaacaaag tgtacgttga aatccttcac 1440
tgtagaaaaa ggaatctatc aaacttctaa ctttagagtc caaccaacag aatctattgt 1500
tagatttcct aatattacaa acttgtgccc ttttggtgaa gtttttaacg ccaccagatt 1560
tgcatctgtt tatgcttgga acaggaagag aatcagcaac tgtgttgctg attattctgt 1620
cctatataat tccgcatcat tttccacttt taagtgttat ggagtgtctc ctactaaatt 1680
aaatgatctc tgctttacta atgtctatgc agattcattt gtaattagag gtgatgaagt 1740
cagacaaatc gctccagggc aaactggaaa gattgctgat tataattata aattaccaga 1800
tgattttaca ggctgcgtta tagcttggaa ttctaacaat cttgattcta aggttggtgg 1860
taattataat tacctgtata gattgtttag gaagtctaat ctcaaacctt ttgagagaga 1920
tatttcaact gaaatctatc aggccggtag cacaccttgt aatggtgttg aaggttttaa 1980
ttgttacttt cctttacaat catatggttt ccaacccact aatggtgttg gttaccaacc 2040
atacagagta gtagtacttt cttttgaact tctacatgca ccagcaactg tttgtggacc 2100
taaaaagtct actaatttgg ttaaaaacaa atgtgtcaat ttcaacttca atggtttaac 2160
aggcacaggt gttcttactg agtctaacaa aaagtttctg cctttccaac aatttggcag 2220
agacattgct gacactactg atgctgtccg tgatccacag acacttgaga ttcttgacat 2280
tacaccatgt tcttttggtg gtgtcagtgt tataacacca ggaacaaata cttctaacca 2340
ggttgctgtt ctttatcagg gtgttaactg cacagaagtc cctgttgcta ttcatgcaga 2400
tcaacttact cctacttggc gtgtttattc tacaggttct aatgtttttc aaacacgtgc 2460
aggctgttta ataggggctg aacatgtcaa caactcatat gagtgtgaca tacccattgg 2520
tgcaggtata tgcgctagtt atcagactca gactaattct cctcggcggg cacgtagtgt 2580
agctagtcaa tccatcattg cctacactat gtcacttggt gcagaaaatt cagttgctta 2640
ctctaataac tctattgcca tacccacaaa ttttactatt agtgttacca cagaaattct 2700
accagtgtct atgaccaaga catcagtaga ttgtacaatg tacatttgtg gtgattcaac 2760
tgaatgcagc aatcttttgt tgcaatatgg cagtttttgt acacaattaa accgtgcttt 2820
aactggaata gctgttgaac aagacaaaaa cacccaagaa gtttttgcac aagtcaaaca 2880
aatttacaaa acaccaccaa ttaaagattt tggtggtttt aatttttcac aaatattacc 2940
agatccatca aaaccaagca agaggtcatt tattgaagat ctacttttca acaaagtgac 3000
acttgcagat gctggcttca tcaaacaata tggtgattgc cttggtgata ttgctgctag 3060
agacctcatt tgtgcacaaa agtttaacgg ccttactgtt ttgccacctt tgctcacaga 3120
tgaaatgatt gctcaataca cttctgcact gttagcgggt acaatcactt ctggttggac 3180
ctttggtgca ggtgctgcat tacaaatacc atttgctatg caaatggctt ataggtttaa 3240
tggtattgga gttacacaga atgttctcta tgagaaccaa aaattgattg ccaaccaatt 3300
taatagtgct attggcaaaa ttcaagactc actttcttcc acagcaagtg cacttggaaa 3360
acttcaagat gtggtcaacc aaaatgcaca agctttaaac acgcttgtta aacaacttag 3420
ctccaatttt ggtgcaattt caagtgtttt aaatgatatc ctttcacgtc ttgacaaagt 3480
tgaggctgaa gtgcaaattg ataggttgat cacaggcaga cttcaaagtt tgcagacata 3540
tgtgactcaa caattaatta gagctgcaga aatcagagct tctgctaatc ttgctgctac 3600
taaaatgtca gagtgtgtac ttggacaatc aaaaagagtt gatttttgtg gaaagggcta 3660
tcatcttatg tccttccctc agtcagcacc tcatggtgta gtcttcttgc atgtgactta 3720
tgtccctgca caagaaaaga acttcacaac tgctcctgcc atttgtcatg atggaaaagc 3780
acactttcct cgtgaaggtg tctttgtttc aaatggcaca cactggtttg taacacaaag 3840
gaatttttat gaaccacaaa tcattactac agacaacaca tttgtgtctg gtaactgtga 3900
tgttgtaata ggaattgtca acaacacagt ttatgatcct ttgcaacctg aattagactc 3960
attcaaggag gagttagata aatattttaa gaatcataca tcaccagatg ttgatttagg 4020
tgacatctct ggcattaatg cttcagttgt aaacattcaa aaagaaattg accgcctcaa 4080
tgaggttgcc aagaatttaa atgaatctct catcgatctc caagaacttg gaaagtatga 4140
gcagtatata aaatggccat ggtacatttg gctaggtttt atagctggct tgattgccat 4200
agtaatggtg acaattatgc tttgctgtat gaccagttgc tgtagttgtc tcaagggctg 4260
ttgttcttgt ggatcctgct gcaaatttga tgaagacgac tctgagccag tgctcaaagg 4320
agtcaaatta cattacacat aagtagttag gcacctctgt tgcccatcgc atcggcaggg 4380
cacgcatata cctttttttt cgctttttag gtatatgcgt gccctgccga tgcgatgggc 4440
aacagaggtg caaaaaaa 4458
<210> 9
<211> 4595
<212> RNA
<213> 人工序列(Artificial sequence)
<400> 9
guucaccagu ggauucugug uuguuuuauc ccauuacaac ucuccacaac cuuuuauuac 60
aucaagaagg agcuaaaauc gcagugcguu uagcuggugg gcugcagaaa aucguugccu 120
ugcucaacaa aacaaaucuu aaauucuugg cuauuacgac agacugccuu caaauuuuag 180
cuuaucgcaa ccaagaaagc aagcucauca uacuggcuag ugguggaccc caagcuuuag 240
uaaauauaau caggaccuau acuuacgaaa aacuacugug gaccacaagc agagugcuga 300
aggugcuauc ugucugcucu aguaauaagc cggcuauugu agaagcuggg uauuugaagu 360
auaccauaca acuguuuuga aaauccagcg uggacaaugg cuacucaagc ugauuugaug 420
gaguuggaca uggccaugga accagacaga aaagcggcug uuagucacug gcagcaacag 480
ucuuaccugg acucuggaau ccauucuggu gccacuacca cagcuccuuc ucugaguggu 540
aaaggcaauc cugaggaaga ggauguggau accucccaag uccuguauga gugggaacag 600
ggauuuucua ugaaccugcu gcugauucug accuuugugg cggcggcggu ggcguaaaug 660
uuuccgccac uuguuuuauu gccacuaguc ucuagucagu guguuaaucu uacaaccaga 720
acucaauuac ccccugcaua cacuaauucu uucacacgug guguuuauua cccugacaaa 780
guuuucagau ccucaguuuu acauucaacu caggacuugu ucuuaccuuu cuuuuccaau 840
guuacuuggu uccaugcuau acaugucucu gggaccaaug guacuaagag guuugauaac 900
ccuguccuac cauuuaauga ugguguuuau uuugcuucca cugagaaguc uaacauaaua 960
agaggcugga uuuuugguac uacuuuagau ucgaagaccc agucccuacu uauuguuaau 1020
aacgcuacua auguuguuau uaaagucugu gaauuucaau uuuguaauga uccauuuuug 1080
gguguuuauu accacaaaaa caacaaaagu uggauggaaa gugaguucag aguuuauucu 1140
agugcgaaua auugcacuuu ugaauauguc ucucagccuu uucuuaugga ccuugaagga 1200
aaacagggua auuucaaaaa ucuuagggaa uuuguguuua agaauauuga ugguuauuuu 1260
aaaauauauu cuaagcacac gccuauuaau uuagugcgug aucucccuca ggguuuuucg 1320
gcuuuagaac cauugguaga uuugccaaua gguauuaaca ucacuagguu ucaaacuuua 1380
cuugcuuuac auagaaguua uuugacuccu ggugauucuu cuucagguug gacagcuggu 1440
gcugcagcuu auuauguggg uuaucuucaa ccuaggacuu uucuauuaaa auauaaugaa 1500
aauggaacca uuacagaugc uguagacugu gcacuugacc cucucucaga aacaaagugu 1560
acguugaaau ccuucacugu agaaaaagga aucuaucaaa cuucuaacuu uagaguccaa 1620
ccaacagaau cuauuguuag auuuccuaau auuacaaacu ugugcccuuu uggugaaguu 1680
uuuaacgcca ccagauuugc aucuguuuau gcuuggaaca ggaagagaau cagcaacugu 1740
guugcugauu auucuguccu auauaauucc gcaucauuuu ccacuuuuaa guguuaugga 1800
gugucuccua cuaaauuaaa ugaucucugc uuuacuaaug ucuaugcaga uucauuugua 1860
auuagaggug augaagucag acaaaucgcu ccagggcaaa cuggaaagau ugcugauuau 1920
aauuauaaau uaccagauga uuuuacaggc ugcguuauag cuuggaauuc uaacaaucuu 1980
gauucuaagg uuggugguaa uuauaauuac cuguauagau uguuuaggaa gucuaaucuc 2040
aaaccuuuug agagagauau uucaacugaa aucuaucagg ccgguagcac accuuguaau 2100
gguguugaag guuuuaauug uuacuuuccu uuacaaucau augguuucca acccacuaau 2160
gguguugguu accaaccaua cagaguagua guacuuucuu uugaacuucu acaugcacca 2220
gcaacuguuu guggaccuaa aaagucuacu aauuugguua aaaacaaaug ugucaauuuc 2280
aacuucaaug guuuaacagg cacagguguu cuuacugagu cuaacaaaaa guuucugccu 2340
uuccaacaau uuggcagaga cauugcugac acuacugaug cuguccguga uccacagaca 2400
cuugagauuc uugacauuac accauguucu uuugguggug ucaguguuau aacaccagga 2460
acaaauacuu cuaaccaggu ugcuguucuu uaucagggug uuaacugcac agaagucccu 2520
guugcuauuc augcagauca acuuacuccu acuuggcgug uuuauucuac agguucuaau 2580
guuuuucaaa cacgugcagg cuguuuaaua ggggcugaac augucaacaa cucauaugag 2640
ugugacauac ccauuggugc agguauaugc gcuaguuauc agacucagac uaauucuccu 2700
cggcgggcac guaguguagc uagucaaucc aucauugccu acacuauguc acuuggugca 2760
gaaaauucag uugcuuacuc uaauaacucu auugccauac ccacaaauuu uacuauuagu 2820
guuaccacag aaauucuacc agugucuaug accaagacau caguagauug uacaauguac 2880
auuuguggug auucaacuga augcagcaau cuuuuguugc aauauggcag uuuuuguaca 2940
caauuaaacc gugcuuuaac uggaauagcu guugaacaag acaaaaacac ccaagaaguu 3000
uuugcacaag ucaaacaaau uuacaaaaca ccaccaauua aagauuuugg ugguuuuaau 3060
uuuucacaaa uauuaccaga uccaucaaaa ccaagcaaga ggucauuuau ugaagaucua 3120
cuuuucaaca aagugacacu ugcagaugcu ggcuucauca aacaauaugg ugauugccuu 3180
ggugauauug cugcuagaga ccucauuugu gcacaaaagu uuaacggccu uacuguuuug 3240
ccaccuuugc ucacagauga aaugauugcu caauacacuu cugcacuguu agcggguaca 3300
aucacuucug guuggaccuu uggugcaggu gcugcauuac aaauaccauu ugcuaugcaa 3360
auggcuuaua gguuuaaugg uauuggaguu acacagaaug uucucuauga gaaccaaaaa 3420
uugauugcca accaauuuaa uagugcuauu ggcaaaauuc aagacucacu uucuuccaca 3480
gcaagugcac uuggaaaacu ucaagaugug gucaaccaaa augcacaagc uuuaaacacg 3540
cuuguuaaac aacuuagcuc caauuuuggu gcaauuucaa guguuuuaaa ugauauccuu 3600
ucacgucuug acaaaguuga ggcugaagug caaauugaua gguugaucac aggcagacuu 3660
caaaguuugc agacauaugu gacucaacaa uuaauuagag cugcagaaau cagagcuucu 3720
gcuaaucuug cugcuacuaa aaugucagag uguguacuug gacaaucaaa aagaguugau 3780
uuuuguggaa agggcuauca ucuuaugucc uucccucagu cagcaccuca ugguguaguc 3840
uucuugcaug ugacuuaugu cccugcacaa gaaaagaacu ucacaacugc uccugccauu 3900
ugucaugaug gaaaagcaca cuuuccucgu gaaggugucu uuguuucaaa uggcacacac 3960
ugguuuguaa cacaaaggaa uuuuuaugaa ccacaaauca uuacuacaga caacacauuu 4020
gugucuggua acugugaugu uguaauagga auugucaaca acacaguuua ugauccuuug 4080
caaccugaau uagacucauu caaggaggag uuagauaaau auuuuaagaa ucauacauca 4140
ccagauguug auuuagguga caucucuggc auuaaugcuu caguuguaaa cauucaaaaa 4200
gaaauugacc gccucaauga gguugccaag aauuuaaaug aaucucucau cgaucuccaa 4260
gaacuuggaa aguaugagca guauauaaaa uggccauggu acauuuggcu agguuuuaua 4320
gcuggcuuga uugccauagu aauggugaca auuaugcuuu gcuguaugac caguugcugu 4380
aguugucuca agggcuguug uucuugugga uccugcugca aauuugauga agacgacucu 4440
gagccagugc ucaaaggagu caaauuacau uacacauaag uaguuaggca ccucuguugc 4500
ccaucgcauc ggcagggcac gcauauaccu uuuuuuucgc uuuuuaggua uaugcgugcc 4560
cugccgaugc gaugggcaac agaggugcaa aaaaa 4595
<210> 10
<211> 4441
<212> RNA
<213> 人工序列(Artificial sequence)
<400> 10
gguucuaauc gauuaacuau cauugaagca acucaucuuc ucuuggugga gcccauauug 60
aacccugccc aucagcuuca ggccauaggg agggugcacc gaauuggaca gacaaaaccu 120
acuauuguac acagauucuu aauuaaagca acaauagaag aaagaaucca ggcaauccug 180
aaaacugcug agagaagggc agccauucua caaaagugga agcugugguc agaacucuga 240
ugaaaauaca gcuuagagau ccaggggcca aagcacucgu uuucucaacg uggcaagaug 300
uauuagauau uauuucaaaa gcucuuacug acaacaacau ggaauuugca caaaucaguc 360
guguuaagac auuucaggag aaccuuucag cauuuaaacg ugauccccaa aucaauauuu 420
ugcugcugcc ccugcacaca auguaucgca ugcagcugcu gagcugcauu gcgcugagcc 480
uggcgcuggu gaccaacagc uaaauguuuc cgccacuugu uuuauugcca cuagucucua 540
gucagugugu uaaucuuaca accagaacuc aauuaccccc ugcauacacu aauucuuuca 600
cacguggugu uuauuacccu gacaaaguuu ucagauccuc aguuuuacau ucaacucagg 660
acuuguucuu accuuucuuu uccaauguua cuugguucca ugcuauacau gucucuggga 720
ccaaugguac uaagagguuu gauaacccug uccuaccauu uaaugauggu guuuauuuug 780
cuuccacuga gaagucuaac auaauaagag gcuggauuuu ugguacuacu uuagauucga 840
agacccaguc ccuacuuauu guuaauaacg cuacuaaugu uguuauuaaa gucugugaau 900
uucaauuuug uaaugaucca uuuuugggug uuuauuacca caaaaacaac aaaaguugga 960
uggaaaguga guucagaguu uauucuagug cgaauaauug cacuuuugaa uaugucucuc 1020
agccuuuucu uauggaccuu gaaggaaaac aggguaauuu caaaaaucuu agggaauuug 1080
uguuuaagaa uauugauggu uauuuuaaaa uauauucuaa gcacacgccu auuaauuuag 1140
ugcgugaucu cccucagggu uuuucggcuu uagaaccauu gguagauuug ccaauaggua 1200
uuaacaucac uagguuucaa acuuuacuug cuuuacauag aaguuauuug acuccuggug 1260
auucuucuuc agguuggaca gcuggugcug cagcuuauua uguggguuau cuucaaccua 1320
ggacuuuucu auuaaaauau aaugaaaaug gaaccauuac agaugcugua gacugugcac 1380
uugacccucu cucagaaaca aaguguacgu ugaaauccuu cacuguagaa aaaggaaucu 1440
aucaaacuuc uaacuuuaga guccaaccaa cagaaucuau uguuagauuu ccuaauauua 1500
caaacuugug cccuuuuggu gaaguuuuua acgccaccag auuugcaucu guuuaugcuu 1560
ggaacaggaa gagaaucagc aacuguguug cugauuauuc uguccuauau aauuccgcau 1620
cauuuuccac uuuuaagugu uauggagugu cuccuacuaa auuaaaugau cucugcuuua 1680
cuaaugucua ugcagauuca uuuguaauua gaggugauga agucagacaa aucgcuccag 1740
ggcaaacugg aaagauugcu gauuauaauu auaaauuacc agaugauuuu acaggcugcg 1800
uuauagcuug gaauucuaac aaucuugauu cuaagguugg ugguaauuau aauuaccugu 1860
auagauuguu uaggaagucu aaucucaaac cuuuugagag agauauuuca acugaaaucu 1920
aucaggccgg uagcacaccu uguaauggug uugaagguuu uaauuguuac uuuccuuuac 1980
aaucauaugg uuuccaaccc acuaauggug uugguuacca accauacaga guaguaguac 2040
uuucuuuuga acuucuacau gcaccagcaa cuguuugugg accuaaaaag ucuacuaauu 2100
ugguuaaaaa caaauguguc aauuucaacu ucaaugguuu aacaggcaca gguguucuua 2160
cugagucuaa caaaaaguuu cugccuuucc aacaauuugg cagagacauu gcugacacua 2220
cugaugcugu ccgugaucca cagacacuug agauucuuga cauuacacca uguucuuuug 2280
guggugucag uguuauaaca ccaggaacaa auacuucuaa ccagguugcu guucuuuauc 2340
aggguguuaa cugcacagaa gucccuguug cuauucaugc agaucaacuu acuccuacuu 2400
ggcguguuua uucuacaggu ucuaauguuu uucaaacacg ugcaggcugu uuaauagggg 2460
cugaacaugu caacaacuca uaugagugug acauacccau uggugcaggu auaugcgcua 2520
guuaucagac ucagacuaau ucuccucggc gggcacguag uguagcuagu caauccauca 2580
uugccuacac uaugucacuu ggugcagaaa auucaguugc uuacucuaau aacucuauug 2640
ccauacccac aaauuuuacu auuaguguua ccacagaaau ucuaccagug ucuaugacca 2700
agacaucagu agauuguaca auguacauuu guggugauuc aacugaaugc agcaaucuuu 2760
uguugcaaua uggcaguuuu uguacacaau uaaaccgugc uuuaacugga auagcuguug 2820
aacaagacaa aaacacccaa gaaguuuuug cacaagucaa acaaauuuac aaaacaccac 2880
caauuaaaga uuuugguggu uuuaauuuuu cacaaauauu accagaucca ucaaaaccaa 2940
gcaagagguc auuuauugaa gaucuacuuu ucaacaaagu gacacuugca gaugcuggcu 3000
ucaucaaaca auauggugau ugccuuggug auauugcugc uagagaccuc auuugugcac 3060
aaaaguuuaa cggccuuacu guuuugccac cuuugcucac agaugaaaug auugcucaau 3120
acacuucugc acuguuagcg gguacaauca cuucugguug gaccuuuggu gcaggugcug 3180
cauuacaaau accauuugcu augcaaaugg cuuauagguu uaaugguauu ggaguuacac 3240
agaauguucu cuaugagaac caaaaauuga uugccaacca auuuaauagu gcuauuggca 3300
aaauucaaga cucacuuucu uccacagcaa gugcacuugg aaaacuucaa gaugugguca 3360
accaaaaugc acaagcuuua aacacgcuug uuaaacaacu uagcuccaau uuuggugcaa 3420
uuucaagugu uuuaaaugau auccuuucac gucuugacaa aguugaggcu gaagugcaaa 3480
uugauagguu gaucacaggc agacuucaaa guuugcagac auaugugacu caacaauuaa 3540
uuagagcugc agaaaucaga gcuucugcua aucuugcugc uacuaaaaug ucagagugug 3600
uacuuggaca aucaaaaaga guugauuuuu guggaaaggg cuaucaucuu auguccuucc 3660
cucagucagc accucauggu guagucuucu ugcaugugac uuaugucccu gcacaagaaa 3720
agaacuucac aacugcuccu gccauuuguc augauggaaa agcacacuuu ccucgugaag 3780
gugucuuugu uucaaauggc acacacuggu uuguaacaca aaggaauuuu uaugaaccac 3840
aaaucauuac uacagacaac acauuugugu cugguaacug ugauguugua auaggaauug 3900
ucaacaacac aguuuaugau ccuuugcaac cugaauuaga cucauucaag gaggaguuag 3960
auaaauauuu uaagaaucau acaucaccag auguugauuu aggugacauc ucuggcauua 4020
augcuucagu uguaaacauu caaaaagaaa uugaccgccu caaugagguu gccaagaauu 4080
uaaaugaauc ucucaucgau cuccaagaac uuggaaagua ugagcaguau auaaaauggc 4140
caugguacau uuggcuaggu uuuauagcug gcuugauugc cauaguaaug gugacaauua 4200
ugcuuugcug uaugaccagu ugcuguaguu gucucaaggg cuguuguucu uguggauccu 4260
gcugcaaauu ugaugaagac gacucugagc cagugcucaa aggagucaaa uuacauuaca 4320
cauaaguagu uaggcaccuc uguugcccau cgcaucggca gggcacgcau auaccuuuuu 4380
uuucgcuuuu uagguauaug cgugcccugc cgaugcgaug ggcaacagag gugcaaaaaa 4440
a 4441

Claims (3)

1.一种环状RNA载体，其特征在于：载体中的结构为包括环状RNA翻译启动区、蛋白定位引导区、可翻译蛋白的RNA片段插入点和结构增强区，并首尾连接形成环状，各个部分的序列为环状RNA翻译启动区RNA序列为SEQ ID NO.1或者SEQ ID NO.2，蛋白定位引导区RNA序列为SEQ ID NO.3或者SEQ ID NO.4，结构增强区其序列为SEQ ID NO.6.。
2.根据权利要求1所述的一种环状RNA载体，其特征在于可翻译蛋白的RNA片段插入点插入的序列为SEQ ID NO.5，即形成序列为SEQ ID NO.9或者SEQ ID NO.10的环状RNA。
3.一种环状RNA载体的运用，其特征在于将序列为SEQ ID NO.9或者SEQ ID NO.10的环状RNA在制备新冠病毒的疫苗中的应用。
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