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CN112574997B - 一种fbxw7环状rna的改构体及其在肿瘤药物和新冠疫苗中的应用 - Google Patents

一种fbxw7环状rna的改构体及其在肿瘤药物和新冠疫苗中的应用 Download PDF

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CN112574997B
CN112574997B CN202110058902.0A CN202110058902A CN112574997B CN 112574997 B CN112574997 B CN 112574997B CN 202110058902 A CN202110058902 A CN 202110058902A CN 112574997 B CN112574997 B CN 112574997B
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何越峰
谭婧文
周倩
尹锦瑶
孙明军
毛一竹
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Kaituo Biotechnology Suzhou Co ltd
Shanghai Kaituo Biotechnology Co ltd
Wuhan Kaituo Biotechnology Co ltd
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Abstract

本发明涉及一种在天然的FBXW7环状RNA的基础上，进行大量的人工序列改造后的环状RNA改构体，该改构体具有优良的翻译以及翻译终止作用，在改构体的插入位点插入适当的蛋白质编码RNA序列之后，可以在体内表达该蛋白，蛋白的不同可以具有不同的效应，可以来制备肿瘤治疗药物也可以用来制备新冠疫苗。

Description

一种FBXW7环状RNA的改构体及其在肿瘤药物和新冠疫苗中的 应用
技术领域
本发明属于生物技术应用领域 ，具体地涉及种FBXW7环状RNA的改构体，进人工序列改造后的环状RNA改构体，该改构体具有优良的翻译以及翻译终止作蛋白质编码RNA序列之后，可以在体内表达该蛋白，蛋白的不同可以具有不同的效应，可以来制备肿瘤治疗药物也可以用来制备新冠疫苗。
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。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。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。例如circRNA以独立于IRES的方式经历滚环扩增，从而导致生产力比线性转录本高100倍。值得注意的是， circRNA还可以与剪接因子，转录因子和RNA结合蛋白（RBP）相互作用，并且某些circRNA可以与核糖体结合并进行翻译。
发明内容
本发明的目的在于提供一种FBXW7环状RNA的改构体及其肿瘤与新冠疫苗中的应用。
本发明的目的是这样实现的，通过人为的改造方式得到的改构体首尾相连的RNA序列为SEQ ID NO.7所示或其具有至少90％同源性的序列。在其序列第287-288之间插入蛋白质编码RNA序列即可表达该蛋白，我们插入的P53的表达RNA后，可以表达该蛋白，可以治疗肿瘤，序列为SEQ ID NO.4；我们插入的新冠的棘突蛋白的D614G突变后，可表达该蛋白与引导蛋白序列为SEQ ID NO.3，可用于在制备新冠病毒的疫苗（81860572）。
本发明（优点）：该改构体适应性强，表达活力高，不容易损伤机体，比mRNA类的药物更容易保存，不容易被RNA酶分解，翻译效率高，更低的剂量就可达到治疗和免疫的目标。
附图说明
图1转染了含棘突序列或者P53序列的细胞的检测结果。
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。
实施例
线性DNA模板定制：首先DNA合成制作的生物公司订购模板序列为SEQ ID NO.1和SEQ ID NO.2的互补双链DNA,2个模板中均包括T7启动子，FBXW7环状RNA的改构体RNA相应DNA序列，以及在287-288位点分别插入了新冠病毒棘突蛋白与引导蛋白的RNA相应DNA序列，RNA序列为SEQ ID NO.3,为D614G突变, 以及在287-288位点分别插入了p53蛋白RNA相应DNA序列,RNA序列为SEQ ID NO.4。
以线性DNA为扩增模板，采用赛默飞公司Invitrogen™ T7 RNA Polymerase试剂盒（货号：18033019）RNA扩增，使用DNA酶将剩余的模板进行降解，提纯RNA产物后进行环化，采用北京百奥莱博科技有限公司的RNA环化试剂盒产品（货号：YTB4302）进行RNA环化得到序列为SEQ ID NO.5和SEQ ID NO.6的环化RNA。
通过百代公司的转染试剂将上述2个环状RNA转染进入A549细胞中，转染后48小时检测，采用western blot的方法P53和棘突蛋白，抗体用santa公司和ABclonal的抗体，经过曝光显影，内参使用GAPDH,结果如图1所示。
采用MTS试剂检测：将含有P53的环状转入p53的细胞的活力，A549细胞接种于96孔板一孔大约3000个，转染后72小时检测.每个组6孔，对照组的活力为100.00±8.45，转入组为60.45±5.47。
免疫小鼠参考了专利号为202010125774 .2专利发明的过程，将含有棘突蛋白序列的环状RNA采用递送剂制成疫苗,肌肉注射小鼠腿部，每组4只小鼠，一共3组，对照组：仅为递送剂制， 低剂量组：含有2ug RNA，高剂量组：6ugRNA，免疫之后14天抽取血，分离血清通过ELISA法检测是否产生抗体，对照组全部为阴性，低剂量组为弱阳性，高剂量组为阳性,实验结果小鼠产生了抗体。将环状RNA中的U采取1-甲基-3伪尿酰代替，效果时一样的，可以减轻疫苗的副反应。
序列表
<110> 昆明医科大学
<120> 一种FBXW7环状RNA的改构体及其在肿瘤药物和新冠疫苗中的应用
<130> 1
<140> 1
<141> 2020-12-29
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<170> SIPOSequenceListing 1.0
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<212> DNA
<213> 人工序列(Artificial sequence)
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taatgttttt caaacacgtg caggctgttt aataggggct gaacatgtca acaactcata 2340
tgagtgtgac atacccattg gtgcaggtat atgcgctagt tatcagactc agactaattc 2400
tcctcggcgg gcacgtagtg tagctagtca atccatcatt gcctacacta tgtcacttgg 2460
tgcagaaaat tcagttgctt actctaataa ctctattgcc atacccacaa attttactat 2520
tagtgttacc acagaaattc taccagtgtc tatgaccaag acatcagtag attgtacaat 2580
gtacatttgt ggtgattcaa ctgaatgcag caatcttttg ttgcaatatg gcagtttttg 2640
tacacaatta aaccgtgctt taactggaat agctgttgaa caagacaaaa acacccaaga 2700
agtttttgca caagtcaaac aaatttacaa aacaccacca attaaagatt ttggtggttt 2760
taatttttca caaatattac cagatccatc aaaaccaagc aagaggtcat ttattgaaga 2820
tctacttttc aacaaagtga cacttgcaga tgctggcttc atcaaacaat atggtgattg 2880
ccttggtgat attgctgcta gagacctcat ttgtgcacaa aagtttaacg gccttactgt 2940
tttgccacct ttgctcacag atgaaatgat tgctcaatac acttctgcac tgttagcggg 3000
tacaatcact tctggttgga cctttggtgc aggtgctgca ttacaaatac catttgctat 3060
gcaaatggct tataggttta atggtattgg agttacacag aatgttctct atgagaacca 3120
aaaattgatt gccaaccaat ttaatagtgc tattggcaaa attcaagact cactttcttc 3180
cacagcaagt gcacttggaa aacttcaaga tgtggtcaac caaaatgcac aagctttaaa 3240
cacgcttgtt aaacaactta gctccaattt tggtgcaatt tcaagtgttt taaatgatat 3300
cctttcacgt cttgacaaag ttgaggctga agtgcaaatt gataggttga tcacaggcag 3360
acttcaaagt ttgcagacat atgtgactca acaattaatt agagctgcag aaatcagagc 3420
ttctgctaat cttgctgcta ctaaaatgtc agagtgtgta cttggacaat caaaaagagt 3480
tgatttttgt ggaaagggct atcatcttat gtccttccct cagtcagcac ctcatggtgt 3540
agtcttcttg catgtgactt atgtccctgc acaagaaaag aacttcacaa ctgctcctgc 3600
catttgtcat gatggaaaag cacactttcc tcgtgaaggt gtctttgttt caaatggcac 3660
acactggttt gtaacacaaa ggaattttta tgaaccacaa atcattacta cagacaacac 3720
atttgtgtct ggtaactgtg atgttgtaat aggaattgtc aacaacacag tttatgatcc 3780
tttgcaacct gaattagact cattcaagga ggagttagat aaatatttta agaatcatac 3840
atcaccagat gttgatttag gtgacatctc tggcattaat gcttcagttg taaacattca 3900
aaaagaaatt gaccgcctca atgaggttgc caagaattta aatgaatctc tcatcgatct 3960
ccaagaactt ggaaagtatg agcagtatat aaaatggcca tggtacattt ggctaggttt 4020
tatagctggc ttgattgcca tagtaatggt gacaattatg ctttgctgta tgaccagttg 4080
ctgtagttgt ctcaagggct gttgttcttg tggatcctgc tgcaaatttg atgaagacga 4140
ctctgagcca gtgctcaaag gagtcaaatt acattacaca taagtagtta g 4191
<210> 2
<211> 1494
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 2
taatacgact cactatagat gaggaggagg aggagatcga ccaggagagt gacgattttg 60
atcagtctga tcatagtagc agagaagatc aacatacaca tactaacagt gtcacgaact 120
ccagtagtat tgtggacctg cccgttcacc aactctcctc cccattctat acaaaaacaa 180
caaaagatta cttccttagg atagattgcc agaagtggag ttactgggtc agaggaatgt 240
gaaaaccttt gcatcttctg atagtctagc caaggtccaa gaagtagcaa gctggctttt 300
ggaaatggag gagccgcagt cagatcctag cgtcgagccc cctctgagtc aggaaacatt 360
ttcagaccta tggaaactac ttcctgaaaa caacgttctg tcccccttgc cgtcccaagc 420
aatggatgat ttgatgctgt ccccggacga tattgaacaa tggttcactg aagacccagg 480
tccagatgaa gctcccagaa tgccagaggc tgctcccccc gtggcccctg caccagcagc 540
tcctacaccg gcggcccctg caccagcccc ctcctggccc ctgtcatctt ctgtcccttc 600
ccagaaaacc taccagggca gctacggttt ccgtctgggc ttcttgcatt ctgggacagc 660
caagtctgtg acttgcacgt actcccctgc cctcaacaag atgttttgcc aactggccaa 720
gacctgccct gtgcagctgt gggttgattc cacacccccg cccggcaccc gcgtccgcgc 780
catggccatc tacaagcagt cacagcacat gacggaggtt gtgaggcgct gcccccacca 840
tgagcgctgc tcagatagcg atggtctggc ccctcctcag catcttatcc gagtggaagg 900
aaatttgcgt gtggagtatt tggatgacag aaacactttt cgacatagtg tggtggtgcc 960
ctatgagccg cctgaggttg gctctgactg taccaccatc cactacaact acatgtgtaa 1020
cagttcctgc atgggcggca tgaaccggag gcccatcctc accatcatca cactggaaga 1080
ctccagtggt aatctactgg gacggaacag ctttgaggtg cgtgtttgtg cctgtcctgg 1140
gagagaccgg cgcacagagg aagagaatct ccgcaagaaa ggggagcctc accacgagct 1200
gcccccaggg agcactaagc gagcactgcc caacaacacc agctcctctc cccagccaaa 1260
gaagaaacca ctggatggag aatatttcac ccttcagatc cgtgggcgtg agcgcttcga 1320
gatgttccga gagctgaatg aggccttgga actcaaggat gcccaggctg ggaaggagcc 1380
aggggggagc agggctcact ccagccacct gaagtccaaa aagggtcagt ctacctcccg 1440
ccataaaaaa ctcatgttca agacagaagg gcctgactca gactgagtag ttag 1494
<210> 3
<211> 3879
<212> RNA
<213> 人工序列(Artificial sequence)
<400> 3
auggcguuuc uguggcugcu gagcugcugg gcgcugcugg gcaccaccuu uggcuaaaug 60
uuuccgccac uuguuuuauu gccacuaguc ucuagucagu guguuaaucu uacaaccaga 120
acucaauuac ccccugcaua cacuaauucu uucacacgug guguuuauua cccugacaaa 180
guuuucagau ccucaguuuu acauucaacu caggacuugu ucuuaccuuu cuuuuccaau 240
guuacuuggu uccaugcuau acaugucucu gggaccaaug guacuaagag guuugauaac 300
ccuguccuac cauuuaauga ugguguuuau uuugcuucca cugagaaguc uaacauaaua 360
agaggcugga uuuuugguac uacuuuagau ucgaagaccc agucccuacu uauuguuaau 420
aacgcuacua auguuguuau uaaagucugu gaauuucaau uuuguaauga uccauuuuug 480
gguguuuauu accacaaaaa caacaaaagu uggauggaaa gugaguucag aguuuauucu 540
agugcgaaua auugcacuuu ugaauauguc ucucagccuu uucuuaugga ccuugaagga 600
aaacagggua auuucaaaaa ucuuagggaa uuuguguuua agaauauuga ugguuauuuu 660
aaaauauauu cuaagcacac gccuauuaau uuagugcgug aucucccuca ggguuuuucg 720
gcuuuagaac cauugguaga uuugccaaua gguauuaaca ucacuagguu ucaaacuuua 780
cuugcuuuac auagaaguua uuugacuccu ggugauucuu cuucagguug gacagcuggu 840
gcugcagcuu auuauguggg uuaucuucaa ccuaggacuu uucuauuaaa auauaaugaa 900
aauggaacca uuacagaugc uguagacugu gcacuugacc cucucucaga aacaaagugu 960
acguugaaau ccuucacugu agaaaaagga aucuaucaaa cuucuaacuu uagaguccaa 1020
ccaacagaau cuauuguuag auuuccuaau auuacaaacu ugugcccuuu uggugaaguu 1080
uuuaacgcca ccagauuugc aucuguuuau gcuuggaaca ggaagagaau cagcaacugu 1140
guugcugauu auucuguccu auauaauucc gcaucauuuu ccacuuuuaa guguuaugga 1200
gugucuccua cuaaauuaaa ugaucucugc uuuacuaaug ucuaugcaga uucauuugua 1260
auuagaggug augaagucag acaaaucgcu ccagggcaaa cuggaaagau ugcugauuau 1320
aauuauaaau uaccagauga uuuuacaggc ugcguuauag cuuggaauuc uaacaaucuu 1380
gauucuaagg uuggugguaa uuauaauuac cuguauagau uguuuaggaa gucuaaucuc 1440
aaaccuuuug agagagauau uucaacugaa aucuaucagg ccgguagcac accuuguaau 1500
gguguugaag guuuuaauug uuacuuuccu uuacaaucau augguuucca acccacuaau 1560
gguguugguu accaaccaua cagaguagua guacuuucuu uugaacuucu acaugcacca 1620
gcaacuguuu guggaccuaa aaagucuacu aauuugguua aaaacaaaug ugucaauuuc 1680
aacuucaaug guuuaacagg cacagguguu cuuacugagu cuaacaaaaa guuucugccu 1740
uuccaacaau uuggcagaga cauugcugac acuacugaug cuguccguga uccacagaca 1800
cuugagauuc uugacauuac accauguucu uuugguggug ucaguguuau aacaccagga 1860
acaaauacuu cuaaccaggu ugcuguucuu uaucagggug uuaacugcac agaagucccu 1920
guugcuauuc augcagauca acuuacuccu acuuggcgug uuuauucuac agguucuaau 1980
guuuuucaaa cacgugcagg cuguuuaaua ggggcugaac augucaacaa cucauaugag 2040
ugugacauac ccauuggugc agguauaugc gcuaguuauc agacucagac uaauucuccu 2100
cggcgggcac guaguguagc uagucaaucc aucauugccu acacuauguc acuuggugca 2160
gaaaauucag uugcuuacuc uaauaacucu auugccauac ccacaaauuu uacuauuagu 2220
guuaccacag aaauucuacc agugucuaug accaagacau caguagauug uacaauguac 2280
auuuguggug auucaacuga augcagcaau cuuuuguugc aauauggcag uuuuuguaca 2340
caauuaaacc gugcuuuaac uggaauagcu guugaacaag acaaaaacac ccaagaaguu 2400
uuugcacaag ucaaacaaau uuacaaaaca ccaccaauua aagauuuugg ugguuuuaau 2460
uuuucacaaa uauuaccaga uccaucaaaa ccaagcaaga ggucauuuau ugaagaucua 2520
cuuuucaaca aagugacacu ugcagaugcu ggcuucauca aacaauaugg ugauugccuu 2580
ggugauauug cugcuagaga ccucauuugu gcacaaaagu uuaacggccu uacuguuuug 2640
ccaccuuugc ucacagauga aaugauugcu caauacacuu cugcacuguu agcggguaca 2700
aucacuucug guuggaccuu uggugcaggu gcugcauuac aaauaccauu ugcuaugcaa 2760
auggcuuaua gguuuaaugg uauuggaguu acacagaaug uucucuauga gaaccaaaaa 2820
uugauugcca accaauuuaa uagugcuauu ggcaaaauuc aagacucacu uucuuccaca 2880
gcaagugcac uuggaaaacu ucaagaugug gucaaccaaa augcacaagc uuuaaacacg 2940
cuuguuaaac aacuuagcuc caauuuuggu gcaauuucaa guguuuuaaa ugauauccuu 3000
ucacgucuug acaaaguuga ggcugaagug caaauugaua gguugaucac aggcagacuu 3060
caaaguuugc agacauaugu gacucaacaa uuaauuagag cugcagaaau cagagcuucu 3120
gcuaaucuug cugcuacuaa aaugucagag uguguacuug gacaaucaaa aagaguugau 3180
uuuuguggaa agggcuauca ucuuaugucc uucccucagu cagcaccuca ugguguaguc 3240
uucuugcaug ugacuuaugu cccugcacaa gaaaagaacu ucacaacugc uccugccauu 3300
ugucaugaug gaaaagcaca cuuuccucgu gaaggugucu uuguuucaaa uggcacacac 3360
ugguuuguaa cacaaaggaa uuuuuaugaa ccacaaauca uuacuacaga caacacauuu 3420
gugucuggua acugugaugu uguaauagga auugucaaca acacaguuua ugauccuuug 3480
caaccugaau uagacucauu caaggaggag uuagauaaau auuuuaagaa ucauacauca 3540
ccagauguug auuuagguga caucucuggc auuaaugcuu caguuguaaa cauucaaaaa 3600
gaaauugacc gccucaauga gguugccaag aauuuaaaug aaucucucau cgaucuccaa 3660
gaacuuggaa aguaugagca guauauaaaa uggccauggu acauuuggcu agguuuuaua 3720
gcuggcuuga uugccauagu aauggugaca auuaugcuuu gcuguaugac caguugcugu 3780
aguugucuca agggcuguug uucuugugga uccugcugca aauuugauga agacgacucu 3840
gagccagugc ucaaaggagu caaauuacau uacacauaa 3879
<210> 4
<211> 1182
<212> RNA
<213> 人工序列(Artificial sequence)
<400> 4
auggaggagc cgcagucaga uccuagcguc gagcccccuc ugagucagga aacauuuuca 60
gaccuaugga aacuacuucc ugaaaacaac guucuguccc ccuugccguc ccaagcaaug 120
gaugauuuga ugcugucccc ggacgauauu gaacaauggu ucacugaaga cccaggucca 180
gaugaagcuc ccagaaugcc agaggcugcu ccccccgugg ccccugcacc agcagcuccu 240
acaccggcgg ccccugcacc agcccccucc uggccccugu caucuucugu cccuucccag 300
aaaaccuacc agggcagcua cgguuuccgu cugggcuucu ugcauucugg gacagccaag 360
ucugugacuu gcacguacuc cccugcccuc aacaagaugu uuugccaacu ggccaagacc 420
ugcccugugc agcugugggu ugauuccaca cccccgcccg gcacccgcgu ccgcgccaug 480
gccaucuaca agcagucaca gcacaugacg gagguuguga ggcgcugccc ccaccaugag 540
cgcugcucag auagcgaugg ucuggccccu ccucagcauc uuauccgagu ggaaggaaau 600
uugcgugugg aguauuugga ugacagaaac acuuuucgac auaguguggu ggugcccuau 660
gagccgccug agguuggcuc ugacuguacc accauccacu acaacuacau guguaacagu 720
uccugcaugg gcggcaugaa ccggaggccc auccucacca ucaucacacu ggaagacucc 780
agugguaauc uacugggacg gaacagcuuu gaggugcgug uuugugccug uccugggaga 840
gaccggcgca cagaggaaga gaaucuccgc aagaaagggg agccucacca cgagcugccc 900
ccagggagca cuaagcgagc acugcccaac aacaccagcu ccucucccca gccaaagaag 960
aaaccacugg auggagaaua uuucacccuu cagauccgug ggcgugagcg cuucgagaug 1020
uuccgagagc ugaaugaggc cuuggaacuc aaggaugccc aggcugggaa ggagccaggg 1080
gggagcaggg cucacuccag ccaccugaag uccaaaaagg gucagucuac cucccgccau 1140
aaaaaacuca uguucaagac agaagggccu gacucagacu ga 1182
<210> 5
<211> 4174
<212> RNA
<213> 人工序列(Artificial sequence)
<400> 5
gaugaggagg aggaggagau cgaccaggag agugacgauu uugaucaguc ugaucauagu 60
agcagagaag aucaacauac acauacuaac agugucacga acuccaguag uauuguggac 120
cugcccguuc accaacucuc cuccccauuc uauacaaaaa caacaaaaga uuacuuccuu 180
aggauagauu gccagaagug gaguuacugg gucagaggaa ugugaaaacc uuugcaucuu 240
cugauagucu agccaagguc caagaaguag caagcuggcu uuuggaaaug gcguuucugu 300
ggcugcugag cugcugggcg cugcugggca ccaccuuugg cuaaauguuu ccgccacuug 360
uuuuauugcc acuagucucu agucagugug uuaaucuuac aaccagaacu caauuacccc 420
cugcauacac uaauucuuuc acacguggug uuuauuaccc ugacaaaguu uucagauccu 480
caguuuuaca uucaacucag gacuuguucu uaccuuucuu uuccaauguu acuugguucc 540
augcuauaca ugucucuggg accaauggua cuaagagguu ugauaacccu guccuaccau 600
uuaaugaugg uguuuauuuu gcuuccacug agaagucuaa cauaauaaga ggcuggauuu 660
uugguacuac uuuagauucg aagacccagu cccuacuuau uguuaauaac gcuacuaaug 720
uuguuauuaa agucugugaa uuucaauuuu guaaugaucc auuuuugggu guuuauuacc 780
acaaaaacaa caaaaguugg auggaaagug aguucagagu uuauucuagu gcgaauaauu 840
gcacuuuuga auaugucucu cagccuuuuc uuauggaccu ugaaggaaaa caggguaauu 900
ucaaaaaucu uagggaauuu guguuuaaga auauugaugg uuauuuuaaa auauauucua 960
agcacacgcc uauuaauuua gugcgugauc ucccucaggg uuuuucggcu uuagaaccau 1020
ugguagauuu gccaauaggu auuaacauca cuagguuuca aacuuuacuu gcuuuacaua 1080
gaaguuauuu gacuccuggu gauucuucuu cagguuggac agcuggugcu gcagcuuauu 1140
auguggguua ucuucaaccu aggacuuuuc uauuaaaaua uaaugaaaau ggaaccauua 1200
cagaugcugu agacugugca cuugacccuc ucucagaaac aaaguguacg uugaaauccu 1260
ucacuguaga aaaaggaauc uaucaaacuu cuaacuuuag aguccaacca acagaaucua 1320
uuguuagauu uccuaauauu acaaacuugu gcccuuuugg ugaaguuuuu aacgccacca 1380
gauuugcauc uguuuaugcu uggaacagga agagaaucag caacuguguu gcugauuauu 1440
cuguccuaua uaauuccgca ucauuuucca cuuuuaagug uuauggagug ucuccuacua 1500
aauuaaauga ucucugcuuu acuaaugucu augcagauuc auuuguaauu agaggugaug 1560
aagucagaca aaucgcucca gggcaaacug gaaagauugc ugauuauaau uauaaauuac 1620
cagaugauuu uacaggcugc guuauagcuu ggaauucuaa caaucuugau ucuaagguug 1680
gugguaauua uaauuaccug uauagauugu uuaggaaguc uaaucucaaa ccuuuugaga 1740
gagauauuuc aacugaaauc uaucaggccg guagcacacc uuguaauggu guugaagguu 1800
uuaauuguua cuuuccuuua caaucauaug guuuccaacc cacuaauggu guugguuacc 1860
aaccauacag aguaguagua cuuucuuuug aacuucuaca ugcaccagca acuguuugug 1920
gaccuaaaaa gucuacuaau uugguuaaaa acaaaugugu caauuucaac uucaaugguu 1980
uaacaggcac agguguucuu acugagucua acaaaaaguu ucugccuuuc caacaauuug 2040
gcagagacau ugcugacacu acugaugcug uccgugaucc acagacacuu gagauucuug 2100
acauuacacc auguucuuuu ggugguguca guguuauaac accaggaaca aauacuucua 2160
accagguugc uguucuuuau caggguguua acugcacaga agucccuguu gcuauucaug 2220
cagaucaacu uacuccuacu uggcguguuu auucuacagg uucuaauguu uuucaaacac 2280
gugcaggcug uuuaauaggg gcugaacaug ucaacaacuc auaugagugu gacauaccca 2340
uuggugcagg uauaugcgcu aguuaucaga cucagacuaa uucuccucgg cgggcacgua 2400
guguagcuag ucaauccauc auugccuaca cuaugucacu uggugcagaa aauucaguug 2460
cuuacucuaa uaacucuauu gccauaccca caaauuuuac uauuaguguu accacagaaa 2520
uucuaccagu gucuaugacc aagacaucag uagauuguac aauguacauu uguggugauu 2580
caacugaaug cagcaaucuu uuguugcaau auggcaguuu uuguacacaa uuaaaccgug 2640
cuuuaacugg aauagcuguu gaacaagaca aaaacaccca agaaguuuuu gcacaaguca 2700
aacaaauuua caaaacacca ccaauuaaag auuuuggugg uuuuaauuuu ucacaaauau 2760
uaccagaucc aucaaaacca agcaagaggu cauuuauuga agaucuacuu uucaacaaag 2820
ugacacuugc agaugcuggc uucaucaaac aauaugguga uugccuuggu gauauugcug 2880
cuagagaccu cauuugugca caaaaguuua acggccuuac uguuuugcca ccuuugcuca 2940
cagaugaaau gauugcucaa uacacuucug cacuguuagc ggguacaauc acuucugguu 3000
ggaccuuugg ugcaggugcu gcauuacaaa uaccauuugc uaugcaaaug gcuuauaggu 3060
uuaaugguau uggaguuaca cagaauguuc ucuaugagaa ccaaaaauug auugccaacc 3120
aauuuaauag ugcuauuggc aaaauucaag acucacuuuc uuccacagca agugcacuug 3180
gaaaacuuca agaugugguc aaccaaaaug cacaagcuuu aaacacgcuu guuaaacaac 3240
uuagcuccaa uuuuggugca auuucaagug uuuuaaauga uauccuuuca cgucuugaca 3300
aaguugaggc ugaagugcaa auugauaggu ugaucacagg cagacuucaa aguuugcaga 3360
cauaugugac ucaacaauua auuagagcug cagaaaucag agcuucugcu aaucuugcug 3420
cuacuaaaau gucagagugu guacuuggac aaucaaaaag aguugauuuu uguggaaagg 3480
gcuaucaucu uauguccuuc ccucagucag caccucaugg uguagucuuc uugcauguga 3540
cuuauguccc ugcacaagaa aagaacuuca caacugcucc ugccauuugu caugauggaa 3600
aagcacacuu uccucgugaa ggugucuuug uuucaaaugg cacacacugg uuuguaacac 3660
aaaggaauuu uuaugaacca caaaucauua cuacagacaa cacauuugug ucugguaacu 3720
gugauguugu aauaggaauu gucaacaaca caguuuauga uccuuugcaa ccugaauuag 3780
acucauucaa ggaggaguua gauaaauauu uuaagaauca uacaucacca gauguugauu 3840
uaggugacau cucuggcauu aaugcuucag uuguaaacau ucaaaaagaa auugaccgcc 3900
ucaaugaggu ugccaagaau uuaaaugaau cucucaucga ucuccaagaa cuuggaaagu 3960
augagcagua uauaaaaugg ccaugguaca uuuggcuagg uuuuauagcu ggcuugauug 4020
ccauaguaau ggugacaauu augcuuugcu guaugaccag uugcuguagu ugucucaagg 4080
gcuguuguuc uuguggaucc ugcugcaaau uugaugaaga cgacucugag ccagugcuca 4140
aaggagucaa auuacauuac acauaaguag uuag 4174
<210> 6
<211> 1477
<212> RNA
<213> 人工序列(Artificial sequence)
<400> 6
gaugaggagg aggaggagau cgaccaggag agugacgauu uugaucaguc ugaucauagu 60
agcagagaag aucaacauac acauacuaac agugucacga acuccaguag uauuguggac 120
cugcccguuc accaacucuc cuccccauuc uauacaaaaa caacaaaaga uuacuuccuu 180
aggauagauu gccagaagug gaguuacugg gucagaggaa ugugaaaacc uuugcaucuu 240
cugauagucu agccaagguc caagaaguag caagcuggcu uuuggaaaug gaggagccgc 300
agucagaucc uagcgucgag cccccucuga gucaggaaac auuuucagac cuauggaaac 360
uacuuccuga aaacaacguu cugucccccu ugccguccca agcaauggau gauuugaugc 420
uguccccgga cgauauugaa caaugguuca cugaagaccc agguccagau gaagcuccca 480
gaaugccaga ggcugcuccc cccguggccc cugcaccagc agcuccuaca ccggcggccc 540
cugcaccagc ccccuccugg ccccugucau cuucuguccc uucccagaaa accuaccagg 600
gcagcuacgg uuuccgucug ggcuucuugc auucugggac agccaagucu gugacuugca 660
cguacucccc ugcccucaac aagauguuuu gccaacuggc caagaccugc ccugugcagc 720
uguggguuga uuccacaccc ccgcccggca cccgcguccg cgccauggcc aucuacaagc 780
agucacagca caugacggag guugugaggc gcugccccca ccaugagcgc ugcucagaua 840
gcgauggucu ggccccuccu cagcaucuua uccgagugga aggaaauuug cguguggagu 900
auuuggauga cagaaacacu uuucgacaua gugugguggu gcccuaugag ccgccugagg 960
uuggcucuga cuguaccacc auccacuaca acuacaugug uaacaguucc ugcaugggcg 1020
gcaugaaccg gaggcccauc cucaccauca ucacacugga agacuccagu gguaaucuac 1080
ugggacggaa cagcuuugag gugcguguuu gugccugucc ugggagagac cggcgcacag 1140
aggaagagaa ucuccgcaag aaaggggagc cucaccacga gcugccccca gggagcacua 1200
agcgagcacu gcccaacaac accagcuccu cuccccagcc aaagaagaaa ccacuggaug 1260
gagaauauuu cacccuucag auccgugggc gugagcgcuu cgagauguuc cgagagcuga 1320
augaggccuu ggaacucaag gaugcccagg cugggaagga gccagggggg agcagggcuc 1380
acuccagcca ccugaagucc aaaaaggguc agucuaccuc ccgccauaaa aaacucaugu 1440
ucaagacaga agggccugac ucagacugag uaguuag 1477
<210> 7
<211> 295
<212> RNA
<213> 人工序列(Artificial sequence)
<400> 7
gaugaggagg aggaggagau cgaccaggag agugacgauu uugaucaguc ugaucauagu 60
agcagagaag aucaacauac acauacuaac agugucacga acuccaguag uauuguggac 120
cugcccguuc accaacucuc cuccccauuc uauacaaaaa caacaaaaga uuacuuccuu 180
aggauagauu gccagaagug gaguuacugg gucagaggaa ugugaaaacc uuugcaucuu 240
cugauagucu agccaagguc caagaaguag caagcuggcu uuuggaagua guuag 295

Claims (6)

1.一种FBXW7环状RNA的改构体，其特征在于：该改构体首尾相连的RNA序列为SEQ IDNO.7所示。
2.根据权利要求1所述的FBXW7环状RNA的改构体，其特征在于插入的蛋白质编码RNA序列为新冠病毒的棘突蛋白与引导蛋白的RNA序列， 序列为SEQ ID NO.3所示， RNA序列的插入位置为第287-288位之间。
3.根据权利要求1所述的FBXW7环状RNA的改构体，其特征在于所述的蛋白质编码RNA序列为p53编码序列， 其RNA序列为SEQ ID NO.4所示， RNA序列的插入位置为第287-288位之间。
4.根据权利要求1所述的FBXW7环状RNA改构体的应用，其特征在于在制备疫苗中的应用。
5.根据权利要求2所述的FBXW7环状RNA的改构体的应用，其特征在于在制备新冠病毒的疫苗中的应用。
6.根据权利要求3所述的FBXW7环状RNA的改构体的应用，其特征在于在制备肿瘤药物中的应用。
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019067092A1 (en) * 2017-08-07 2019-04-04 The Johns Hopkins University METHODS AND SUBSTANCES FOR THE EVALUATION AND TREATMENT OF CANCER
CN111378686A (zh) * 2020-04-16 2020-07-07 山东维真生物科技有限公司 一种高效形成环状RNA的过表达载体pCircleVG及其构建方法
CN111454950A (zh) * 2020-04-01 2020-07-28 广州医科大学附属第五医院 与rsv病毒感染相关的环状rna及其应用
CN112126647A (zh) * 2020-09-29 2020-12-25 中国科学院微生物研究所 一种流感病毒的环状rna疫苗

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100484570C (zh) * 2003-06-16 2009-05-06 中国科学院动物研究所 一种抗sars病毒的基因疫苗及其构建和应用
AU2014265608B2 (en) * 2013-05-15 2018-12-20 Robert Kruse Intracellular translation of circular RNA
RU2020103379A (ru) * 2017-07-04 2021-08-04 Куревак Аг Новые молекулы нуклеиновых кислот
CN111630173A (zh) * 2017-10-19 2020-09-04 库瑞瓦格股份公司 新型人工核酸分子
WO2019135701A1 (en) * 2018-01-05 2019-07-11 Nilsson Rolf Jonas Andreas Endogenous tumor-derived circular rna and proteins thereof for use as vaccine
CN108384783B (zh) * 2018-02-28 2019-09-10 中南大学 一种环状rna成环序列及应用
US11680109B2 (en) * 2019-05-29 2023-06-20 Duke University Compositions and methods for detection and measurement of RNA modifications through targeted RNA editing
CN111218508A (zh) * 2020-03-18 2020-06-02 昆明医科大学 一种环状rna作为肝衰竭标志物及其应用
CN111500587A (zh) * 2020-04-15 2020-08-07 湖南省科域生物医药科技有限公司 Pgr作为治疗子宫内膜异位症的产品中的用途及其检测pgr的试剂盒

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019067092A1 (en) * 2017-08-07 2019-04-04 The Johns Hopkins University METHODS AND SUBSTANCES FOR THE EVALUATION AND TREATMENT OF CANCER
CN111454950A (zh) * 2020-04-01 2020-07-28 广州医科大学附属第五医院 与rsv病毒感染相关的环状rna及其应用
CN111378686A (zh) * 2020-04-16 2020-07-07 山东维真生物科技有限公司 一种高效形成环状RNA的过表达载体pCircleVG及其构建方法
CN112126647A (zh) * 2020-09-29 2020-12-25 中国科学院微生物研究所 一种流感病毒的环状rna疫苗

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