CN112725378B - 一种人工改造的akt3环状rna及其在新冠疫苗中的应用 - Google Patents
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Abstract
本发明公开了一种人工改造的AKT3环状RNA及其在新冠疫苗中的应用，其特征是：经过改造AKT3环状RNA具有启动转录功能，在中间加入一段蛋白开放阅读框，使该环状RNA可稳定的表达上述白开放阅读框的蛋白，如果加入相应的免疫原蛋白开放阅读框，可以作用circRNA疫苗的载体，使免疫动物产生抗体，可用于为预防或治疗的疫苗。
Description
技术领域
本发明属于生物医药生物技术领域，具体地涉及一种人工改造的AKT3环状RNA，该环状RNA的插入位点插入开放阅读框后可以表达该蛋白，可以作用多种疫苗的载体。
背景技术
RNA的结构和性质是非常不稳定的，这使得越来越多的人们开始注意它的治疗用途。尽管该分子对几乎无所不在的核糖核酸酶（RNases）极其敏感，但在广泛应用体外转染技术发展之后，1989年首次推广了mRNA作为治疗剂。仅在几年后，mRNA被推广作为一种疫苗平台的治疗药物。与DNA相比，将减毒活疫苗的免疫学特征（如内源性抗原表达和T细胞诱导）与灭活或亚单位疫苗的免疫学特征（如确定的成分和安全性）结合在一起，mRNA是更为理想的具有治疗作用的治疗剂。特别是作为疫苗，mRNA具有更强的安全性优势。作为最小的基因构建体，它仅包含表达编码蛋白直接需要的元素。此外，尽管在极少数情况下可能会发生单链RNA分子之间的重组，但mRNA不会与基因组相互作用。因此，排除了可能有害的基因组整合，缺乏基因组整合以及与mRNA无复制性和代谢性的结合,几天之内的衰变使mRNA被视作是信息的瞬时载体。
mRNA作为治疗剂和疫苗的技术基础，其特点是在生产和应用方面具有极大的灵活性。任何蛋白质都可以由mRNA编码和表达，原则上可以开发出预防和治疗性疫苗，以抗击各种疾病，例如感染和癌症以及蛋白质替代疗法。编码蛋白质的变化只会改变RNA分子的序列，而在很大程度上不影响其理化特性，因此可以使用相同的既定生产工艺生产各种产品，而无需进行任何调整，与其他疫苗平台相比可以节省时间并降低成本。就功效而言，基于mRNA的治疗方法受益于以下事实，即它们不需要越过核膜而不是DNA。与肽相反，mRNA疫苗缺乏MHC单倍型限制。此外，mRNA可以与模式识别受体结合，并且可以将mRNA疫苗设计为具有自佐剂性，这是基于肽和蛋白质的疫苗所缺乏的特性。总而言之，即使具有挑战性，mRNA还是有希望的一类可能成为“破坏性技术”基础的治疗性分子。
在首次成功体内施用mRNA后约二十年，基于mRNA的疫苗有望成为改变性的疫苗技术平台，用于治疗和预防应用。今天，科学界急切地等待着第一批临床疗效数据。但是，基于mRNA的疫苗的进一步开发改进仍存在广阔的领域。如所讨论的，mRNA的形式和摄取是有效抗原表达的关键参数，其可以受到新颖的RNA设计以及mRNA的配制和施用的影响。但是，这些参数的任何变化可能会对mRNA的产生或其与RNA传感器的相互作用产生重大影响，因此应尽早充分考虑。例如，除了先前提到的核苷酸修饰以外，新的递送方式可能严重影响疫苗佐剂。虽然直接递送到细胞质中肯定会增强抗原表达，但与内体RNA受体相互作用的缺乏可能会严重削弱疫苗的免疫刺激作用，可能必须解决这个问题。在治疗特别具有挑战性的情况下，将辅助mRNA分子包含在mRNA疫苗中可能是获得最佳效果的有趣选择。而且，与其他抗肿瘤疗法的组合最有可能产生最大的效力。但是，这将增加疫苗和治疗方案的复杂性，使开发更具挑战性。综上所述，鉴于其灵活，有效和安全性，mRNA提供了一种有前途的疫苗载体。因此，基于 mRNA 免疫策略的研究也越来越多, 人们在不同的临床前模型中证明了其特异性的免疫保护作用。鉴于 mRNA 疫苗在生产周期、生产成本、安全性等方面的多种优势, 随着人们对新冠病毒致病机理研究的深入以及分子生物学技术的不断发展, mRNA疫苗将会在预防新冠肺炎中发挥作用。
CircRNA最近被发现为具有共价闭合结构的非编码RNA。它们调节疾病的发生和发展。 CircRNA由RNA聚合酶II转录，无5'-3'极性或多腺嘌呤尾。它具有与线性RNA相同的转录效率。CircRNA长数百至数千个碱基，主要由外显子组成。研究表明，哺乳动物circRNA具有内源性，丰富性，保守性和稳定性。 CircRNA是控制基因转录的miRNA海绵。此外，circRNA中高度保守的ORF在体内和体外均以独立于5'帽结构的方式编码功能肽，例如内部核糖体进入位点（IRES）诱导，促进腺苷甲基化（N6-甲基腺苷； m6A），滚动循环放大等。由于circRNA具有独特的共价封闭结构，因此其中的ORF跨剪接位点甚至超过其长度循环。因此，它还可以生产长度> 100个氨基酸的蛋白质。circRNA是真核生物中普遍存在的一类完整的丰富的非编码RNA。circRNAs是由非经典剪接形式产生的，最常见的情况是一个外显子的剪接供体位点与上游外显子的剪接受体位点连接。与线性RNA不同，环状RNA具有特殊的环状共价键结构，这使它们对核酸外切酶的耐受性更高。由于其保守性，丰度和组织特异性，circRNA在某些疾病（包括肿瘤）中可能起着特殊分子标记的作用。尽管处理效率低下并且大部分以低水平表达，但一些环状rna来自与人类疾病相关的基因组位点，越来越多的证据表明它们在转录，转录后和翻译调控中的潜在作用。其作为疫苗的载体可能是一个很好的方向。
发明内容
本发明的目的在于提供一种人工改造的AKT3环状RNA及其在新冠疫苗中的应用。
本发明的目的是这样实现的，通过合成转录构建序列为SEQ ID NO.1所示首尾相连的环状RNA或其具有至少80％同源性的首尾相连的环状RNA。在其序列第504个核苷酸与第505核苷酸之间插入蛋白开放阅读框即可表达该蛋白，如果插入的新冠病毒的棘突蛋白的编码序列，例如为SEQ ID NO.2，可表达该蛋白。因此该改造AKT3环状RNA可以用于在制备疫苗，包括现在的制备新冠病毒的疫苗。（81860572）。
本发明（优点）：该载体可以高效的表达蛋白，其蛋白可以引起机体的免疫，比mRNA疫苗更容易保存，翻译效率高，不容易被RNA酶降解，更低的剂量就可以引起免疫反应。整个RNA片段较短有利于插入蛋白。
附图说明
图1为棘突蛋白表达结果图，对照组不含棘突蛋白，转让组含有棘突蛋白，内参是GAPDH。
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
1、DNA模板片段的合成
首先通过全基因组合成技术，合成序列为SEQ ID NO.3所示的DNA模板，其中包括人工改造的AKT3环状RNA序列和新冠病毒的棘突蛋白的序列。
2、DNA模板片段pcr的扩增
通过序列为SEQ ID NO.4和SEQ ID NO.5的引物进行PCR扩增，得到大量的双链DNA模板，注意使用高保真酶进行扩增，扩增完毕后进行PCR产物提纯。
3、根据模板片段进行RNA合成
采用T7启动子的T7 RNA Polymerase进行扩增，模板采用上一个步骤中PCR产物，扩增完毕后，采用无RNA酶的DNA酶进行消化DNA，然后提纯所等到的RNA。
4、环化线性RNA得到环状RNA
将上一步骤提纯所得到的RNA ，按照Circular RNA Synthesis Kit试剂盒要求进行环化，环状后在适当的条件下利用RNA酶剪切掉线性RNA,提纯环状RNA,我们得到环化的RNA包含有SEQ ID NO.1和SEQ ID NO.2，SEQ ID NO.2插入位点为SEQ ID NO.1 的第504个核苷酸与第505核苷酸之间,最终环状RNA 序列为SEQ ID NO.6。
5、转染细胞表达棘突蛋白
将序列为SEQ ID NO.6环状RNA使用转染试剂转染A549细胞，转染试剂可以采用阳离子脂质体，或者其他转染材料例如百代公司的转染试剂，转染后48小时我们采用westernblot的方法进行了蛋白检测，发现细胞中表达了含有新冠病毒棘突蛋白的融合蛋白,结果如图1所示。
6、通过环状RNA免疫动物的试验
将序列为SEQ ID NO.6环状RNA分别和递送剂制成疫苗,将制得的疫苗免疫小鼠腿部肌肉注射递送，每只小鼠给药剂量为对照组：仅为递送剂制， A组：2.5微克环状RNA，B组：5微克环状RNA，给药后14天抽取血清通过ELISA法检测抗体信号，对照组为阴性，A组为弱阳性，B组为阳性,实验结果显示环状RNA疫苗能够使小鼠体内产生抗体。
在整个过程中我们参考了专利号为202010125774 .2和201911042634 .2的专利发明，特别是第5和6个步骤，本发明采用了环状RNA的新方式在载体选择上有了显著的进步和提高，也提供了一种新的方式。SEQ ID NO.1和SEQ ID NO.6都是首尾相接的环状RNA序列。1-甲基-3伪尿酰代替尿嘧啶，可以达到同样目的而且减轻副反应。
序列表
<110> 昆明医科大学
<120> 一种人工改造的AKT3环状RNA及其应用
<130> 1
<140> 1
<141> 2020-12-27
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 530
<212> RNA
<213> 人工序列(Artificial sequence)
<400> 1
gauucugaag aaagaaguca uuauugcaaa ggaagaagug gcacacacuc uaacugaaag 60
cagaguauua aagaacacua gacaucccuu uuuaacaucc uugaaauauu ccuuccagac 120
aaaagaccgu uuguguuuug ugaaggaaua aguuaaaggg ggcgagaaag ccaguuaaug 180
aaaacagaac gaccaaagcc aaacacauuu auaaucagau gucuccagug gacuacuguu 240
auagagagaa cauuucaugu agauacucca gaggaaaggg aagaauggac agaagcuauc 300
caggcuguag cagacagacu gcagaggcaa gaagaggaga gaaugaauug uaguccaacu 360
ucacaaauug auaauauagg agaggaagag auggaugccu cuacaaccca ucauaaaaga 420
aagacaaugg gcgugcugcu gacccagcgc acccugcuga gccuggugcu ggcgcugcug 480
uuuccgagca uggcgagcau guaaguaguu aggaacaacc ugcagcagac 530
<210> 2
<211> 3822
<212> RNA
<213> 人工序列(Artificial sequence)
<400> 2
auguuuccgc cacuuguuuu auugccacua gucucuaguc aguguguuaa ucuuacaacc 60
agaacucaau uacccccugc auacacuaau ucuuucacac gugguguuua uuacccugac 120
aaaguuuuca gauccucagu uuuacauuca acucaggacu uguucuuacc uuucuuuucc 180
aauguuacuu gguuccaugc uauacauguc ucugggacca augguacuaa gagguuugau 240
aacccugucc uaccauuuaa ugaugguguu uauuuugcuu ccacugagaa gucuaacaua 300
auaagaggcu ggauuuuugg uacuacuuua gauucgaaga cccagucccu acuuauuguu 360
aauaacgcua cuaauguugu uauuaaaguc ugugaauuuc aauuuuguaa ugauccauuu 420
uuggguguuu auuaccacaa aaacaacaaa aguuggaugg aaagugaguu cagaguuuau 480
ucuagugcga auaauugcac uuuugaauau gucucucagc cuuuucuuau ggaccuugaa 540
ggaaaacagg guaauuucaa aaaucuuagg gaauuugugu uuaagaauau ugaugguuau 600
uuuaaaauau auucuaagca cacgccuauu aauuuagugc gugaucuccc ucaggguuuu 660
ucggcuuuag aaccauuggu agauuugcca auagguauua acaucacuag guuucaaacu 720
uuacuugcuu uacauagaag uuauuugacu ccuggugauu cuucuucagg uuggacagcu 780
ggugcugcag cuuauuaugu ggguuaucuu caaccuagga cuuuucuauu aaaauauaau 840
gaaaauggaa ccauuacaga ugcuguagac ugugcacuug acccucucuc agaaacaaag 900
uguacguuga aauccuucac uguagaaaaa ggaaucuauc aaacuucuaa cuuuagaguc 960
caaccaacag aaucuauugu uagauuuccu aauauuacaa acuugugccc uuuuggugaa 1020
guuuuuaacg ccaccagauu ugcaucuguu uaugcuugga acaggaagag aaucagcaac 1080
uguguugcug auuauucugu ccuauauaau uccgcaucau uuuccacuuu uaaguguuau 1140
ggagugucuc cuacuaaauu aaaugaucuc ugcuuuacua augucuaugc agauucauuu 1200
guaauuagag gugaugaagu cagacaaauc gcuccagggc aaacuggaaa gauugcugau 1260
uauaauuaua aauuaccaga ugauuuuaca ggcugcguua uagcuuggaa uucuaacaau 1320
cuugauucua agguuggugg uaauuauaau uaccuguaua gauuguuuag gaagucuaau 1380
cucaaaccuu uugagagaga uauuucaacu gaaaucuauc aggccgguag cacaccuugu 1440
aaugguguug aagguuuuaa uuguuacuuu ccuuuacaau cauaugguuu ccaacccacu 1500
aaugguguug guuaccaacc auacagagua guaguacuuu cuuuugaacu ucuacaugca 1560
ccagcaacug uuuguggacc uaaaaagucu acuaauuugg uuaaaaacaa augugucaau 1620
uucaacuuca augguuuaac aggcacaggu guucuuacug agucuaacaa aaaguuucug 1680
ccuuuccaac aauuuggcag agacauugcu gacacuacug augcuguccg ugauccacag 1740
acacuugaga uucuugacau uacaccaugu ucuuuuggug gugucagugu uauaacacca 1800
ggaacaaaua cuucuaacca gguugcuguu cuuuaucagg auguuaacug cacagaaguc 1860
ccuguugcua uucaugcaga ucaacuuacu ccuacuuggc guguuuauuc uacagguucu 1920
aauguuuuuc aaacacgugc aggcuguuua auaggggcug aacaugucaa caacucauau 1980
gagugugaca uacccauugg ugcagguaua ugcgcuaguu aucagacuca gacuaauucu 2040
ccucggcggg cacguagugu agcuagucaa uccaucauug ccuacacuau gucacuuggu 2100
gcagaaaauu caguugcuua cucuaauaac ucuauugcca uacccacaaa uuuuacuauu 2160
aguguuacca cagaaauucu accagugucu augaccaaga caucaguaga uuguacaaug 2220
uacauuugug gugauucaac ugaaugcagc aaucuuuugu ugcaauaugg caguuuuugu 2280
acacaauuaa accgugcuuu aacuggaaua gcuguugaac aagacaaaaa cacccaagaa 2340
guuuuugcac aagucaaaca aauuuacaaa acaccaccaa uuaaagauuu uggugguuuu 2400
aauuuuucac aaauauuacc agauccauca aaaccaagca agaggucauu uauugaagau 2460
cuacuuuuca acaaagugac acuugcagau gcuggcuuca ucaaacaaua uggugauugc 2520
cuuggugaua uugcugcuag agaccucauu ugugcacaaa aguuuaacgg ccuuacuguu 2580
uugccaccuu ugcucacaga ugaaaugauu gcucaauaca cuucugcacu guuagcgggu 2640
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> 3
<211> 4390
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 3
gtcaacttca gataccactg gtaatacgac tcactataga ttctgaagaa agaagtcatt 60
attgcaaagg aagaagtggc acacactcta actgaaagca gagtattaaa gaacactaga 120
catccctttt taacatcctt gaaatattcc ttccagacaa aagaccgttt gtgttttgtg 180
aaggaataag ttaaaggggg cgagaaagcc agttaatgaa aacagaacga ccaaagccaa 240
acacatttat aatcagatgt ctccagtgga ctactgttat agagagaaca tttcatgtag 300
atactccaga ggaaagggaa gaatggacag aagctatcca ggctgtagca gacagactgc 360
agaggcaaga agaggagaga atgaattgta gtccaacttc acaaattgat aatataggag 420
aggaagagat ggatgcctct acaacccatc ataaaagaaa gacaatgggc gtgctgctga 480
cccagcgcac cctgctgagc ctggtgctgg cgctgctgtt tccgagcatg gcgagcatgt 540
aaatgtttcc gccacttgtt ttattgccac tagtctctag tcagtgtgtt aatcttacaa 600
ccagaactca attaccccct gcatacacta attctttcac acgtggtgtt tattaccctg 660
acaaagtttt cagatcctca gttttacatt caactcagga cttgttctta cctttctttt 720
ccaatgttac ttggttccat gctatacatg tctctgggac caatggtact aagaggtttg 780
ataaccctgt cctaccattt aatgatggtg tttattttgc ttccactgag aagtctaaca 840
taataagagg ctggattttt ggtactactt tagattcgaa gacccagtcc ctacttattg 900
ttaataacgc tactaatgtt gttattaaag tctgtgaatt tcaattttgt aatgatccat 960
ttttgggtgt ttattaccac aaaaacaaca aaagttggat ggaaagtgag ttcagagttt 1020
attctagtgc gaataattgc acttttgaat atgtctctca gccttttctt atggaccttg 1080
aaggaaaaca gggtaatttc aaaaatctta gggaatttgt gtttaagaat attgatggtt 1140
attttaaaat atattctaag cacacgccta ttaatttagt gcgtgatctc cctcagggtt 1200
tttcggcttt agaaccattg gtagatttgc caataggtat taacatcact aggtttcaaa 1260
ctttacttgc tttacataga agttatttga ctcctggtga ttcttcttca ggttggacag 1320
ctggtgctgc agcttattat gtgggttatc ttcaacctag gacttttcta ttaaaatata 1380
atgaaaatgg aaccattaca gatgctgtag actgtgcact tgaccctctc tcagaaacaa 1440
agtgtacgtt gaaatccttc actgtagaaa aaggaatcta tcaaacttct aactttagag 1500
tccaaccaac agaatctatt gttagatttc ctaatattac aaacttgtgc ccttttggtg 1560
aagtttttaa cgccaccaga tttgcatctg tttatgcttg gaacaggaag agaatcagca 1620
actgtgttgc tgattattct gtcctatata attccgcatc attttccact tttaagtgtt 1680
atggagtgtc tcctactaaa ttaaatgatc tctgctttac taatgtctat gcagattcat 1740
ttgtaattag aggtgatgaa gtcagacaaa tcgctccagg gcaaactgga aagattgctg 1800
attataatta taaattacca gatgatttta caggctgcgt tatagcttgg aattctaaca 1860
atcttgattc taaggttggt ggtaattata attacctgta tagattgttt aggaagtcta 1920
atctcaaacc ttttgagaga gatatttcaa ctgaaatcta tcaggccggt agcacacctt 1980
gtaatggtgt tgaaggtttt aattgttact ttcctttaca atcatatggt ttccaaccca 2040
ctaatggtgt tggttaccaa ccatacagag tagtagtact ttcttttgaa cttctacatg 2100
caccagcaac tgtttgtgga cctaaaaagt ctactaattt ggttaaaaac aaatgtgtca 2160
atttcaactt caatggttta acaggcacag gtgttcttac tgagtctaac aaaaagtttc 2220
tgcctttcca acaatttggc agagacattg ctgacactac tgatgctgtc cgtgatccac 2280
agacacttga gattcttgac attacaccat gttcttttgg tggtgtcagt gttataacac 2340
caggaacaaa tacttctaac caggttgctg ttctttatca ggatgttaac tgcacagaag 2400
tccctgttgc tattcatgca gatcaactta ctcctacttg gcgtgtttat tctacaggtt 2460
ctaatgtttt tcaaacacgt gcaggctgtt taataggggc tgaacatgtc aacaactcat 2520
atgagtgtga catacccatt ggtgcaggta tatgcgctag ttatcagact cagactaatt 2580
ctcctcggcg ggcacgtagt gtagctagtc aatccatcat tgcctacact atgtcacttg 2640
gtgcagaaaa ttcagttgct tactctaata actctattgc catacccaca aattttacta 2700
ttagtgttac cacagaaatt ctaccagtgt ctatgaccaa gacatcagta gattgtacaa 2760
tgtacatttg tggtgattca actgaatgca gcaatctttt gttgcaatat ggcagttttt 2820
gtacacaatt aaaccgtgct ttaactggaa tagctgttga acaagacaaa aacacccaag 2880
aagtttttgc acaagtcaaa caaatttaca aaacaccacc aattaaagat tttggtggtt 2940
ttaatttttc acaaatatta ccagatccat caaaaccaag caagaggtca tttattgaag 3000
atctactttt caacaaagtg acacttgcag atgctggctt catcaaacaa tatggtgatt 3060
gccttggtga tattgctgct agagacctca tttgtgcaca aaagtttaac ggccttactg 3120
ttttgccacc tttgctcaca gatgaaatga ttgctcaata cacttctgca ctgttagcgg 3180
gtacaatcac ttctggttgg acctttggtg caggtgctgc attacaaata ccatttgcta 3240
tgcaaatggc ttataggttt aatggtattg gagttacaca gaatgttctc tatgagaacc 3300
aaaaattgat tgccaaccaa tttaatagtg ctattggcaa aattcaagac tcactttctt 3360
ccacagcaag tgcacttgga aaacttcaag atgtggtcaa ccaaaatgca caagctttaa 3420
acacgcttgt taaacaactt agctccaatt ttggtgcaat ttcaagtgtt ttaaatgata 3480
tcctttcacg tcttgacaaa gttgaggctg aagtgcaaat tgataggttg atcacaggca 3540
gacttcaaag tttgcagaca tatgtgactc aacaattaat tagagctgca gaaatcagag 3600
cttctgctaa tcttgctgct actaaaatgt cagagtgtgt acttggacaa tcaaaaagag 3660
ttgatttttg tggaaagggc tatcatctta tgtccttccc tcagtcagca cctcatggtg 3720
tagtcttctt gcatgtgact tatgtccctg cacaagaaaa gaacttcaca actgctcctg 3780
ccatttgtca tgatggaaaa gcacactttc ctcgtgaagg tgtctttgtt tcaaatggca 3840
cacactggtt tgtaacacaa aggaattttt atgaaccaca aatcattact acagacaaca 3900
catttgtgtc tggtaactgt gatgttgtaa taggaattgt caacaacaca gtttatgatc 3960
ctttgcaacc tgaattagac tcattcaagg aggagttaga taaatatttt aagaatcata 4020
catcaccaga tgttgattta ggtgacatct ctggcattaa tgcttcagtt gtaaacattc 4080
aaaaagaaat tgaccgcctc aatgaggttg ccaagaattt aaatgaatct ctcatcgatc 4140
tccaagaact tggaaagtat gagcagtata taaaatggcc atggtacatt tggctaggtt 4200
ttatagctgg cttgattgcc atagtaatgg tgacaattat gctttgctgt atgaccagtt 4260
gctgtagttg tctcaagggc tgttgttctt gtggatcctg ctgcaaattt gatgaagacg 4320
actctgagcc agtgctcaaa ggagtcaaat tacattacac ataagtagtt aggaacaacc 4380
tgcagcagac 4390
<210> 4
<211> 21
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 4
gtcaacttca gataccactg g 21
<210> 5
<211> 18
<212> DNA
<213> 人工序列(Artificial sequence)
<400> 5
gtctgctgca ggttgttc 18
<210> 6
<211> 4334
<212> RNA
<213> 人工序列(Artificial sequence)
<400> 6
gauucugaag aaagaaguca uuauugcaaa ggaagaagug gcacacacuc uaacugaaag 60
cagaguauua aagaacacua gacaucccuu uuuaacaucc uugaaauauu ccuuccagac 120
aaaagaccgu uuguguuuug ugaaggaaua aguuaaaggg ggcgagaaag ccaguuaaug 180
aaaacagaac gaccaaagcc aaacacauuu auaaucagau gucuccagug gacuacuguu 240
auagagagaa cauuucaugu agauacucca gaggaaaggg aagaauggac agaagcuauc 300
caggcuguag cagacagacu gcagaggcaa gaagaggaga gaaugaauug uaguccaacu 360
ucacaaauug auaauauagg agaggaagag auggaugccu cuacaaccca ucauaaaaga 420
aagacaaugg gcgugcugcu gacccagcgc acccugcuga gccuggugcu ggcgcugcug 480
uuuccgagca uggcgagcau guaaauguuu ccgccacuug uuuuauugcc acuagucucu 540
agucagugug uuaaucuuac aaccagaacu caauuacccc cugcauacac uaauucuuuc 600
acacguggug uuuauuaccc ugacaaaguu uucagauccu caguuuuaca uucaacucag 660
gacuuguucu uaccuuucuu uuccaauguu acuugguucc augcuauaca ugucucuggg 720
accaauggua cuaagagguu ugauaacccu guccuaccau uuaaugaugg uguuuauuuu 780
gcuuccacug agaagucuaa cauaauaaga ggcuggauuu uugguacuac uuuagauucg 840
aagacccagu cccuacuuau uguuaauaac gcuacuaaug uuguuauuaa agucugugaa 900
uuucaauuuu guaaugaucc auuuuugggu guuuauuacc acaaaaacaa caaaaguugg 960
auggaaagug aguucagagu uuauucuagu gcgaauaauu gcacuuuuga auaugucucu 1020
cagccuuuuc uuauggaccu ugaaggaaaa caggguaauu ucaaaaaucu uagggaauuu 1080
guguuuaaga auauugaugg uuauuuuaaa auauauucua agcacacgcc uauuaauuua 1140
gugcgugauc ucccucaggg uuuuucggcu uuagaaccau ugguagauuu gccaauaggu 1200
auuaacauca cuagguuuca aacuuuacuu gcuuuacaua gaaguuauuu gacuccuggu 1260
gauucuucuu cagguuggac agcuggugcu gcagcuuauu auguggguua ucuucaaccu 1320
aggacuuuuc uauuaaaaua uaaugaaaau ggaaccauua cagaugcugu agacugugca 1380
cuugacccuc ucucagaaac aaaguguacg uugaaauccu ucacuguaga aaaaggaauc 1440
uaucaaacuu cuaacuuuag aguccaacca acagaaucua uuguuagauu uccuaauauu 1500
acaaacuugu gcccuuuugg ugaaguuuuu aacgccacca gauuugcauc uguuuaugcu 1560
uggaacagga agagaaucag caacuguguu gcugauuauu cuguccuaua uaauuccgca 1620
ucauuuucca cuuuuaagug uuauggagug ucuccuacua aauuaaauga ucucugcuuu 1680
acuaaugucu augcagauuc auuuguaauu agaggugaug aagucagaca aaucgcucca 1740
gggcaaacug gaaagauugc ugauuauaau uauaaauuac cagaugauuu uacaggcugc 1800
guuauagcuu ggaauucuaa caaucuugau ucuaagguug gugguaauua uaauuaccug 1860
uauagauugu uuaggaaguc uaaucucaaa ccuuuugaga gagauauuuc aacugaaauc 1920
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Claims (5)
1.一种人工改造的AKT3环状RNA，其特征在于：其序列为SEQ ID NO.1所示首尾相连的环状RNA, SEQ ID NO.1序列中蛋白开放阅读框的插入位点为第504个核苷酸与第505核苷酸之间。
2.根据权利要求1所述的人工改造的AKT3环状RNA，其特征在于：其插入的蛋白开放阅读框为新冠病毒的棘突蛋白的编码序列。
3.根据权利要求1所述的人工改造的AKT3环状RNA，其特征在于：所述的棘突蛋白的编码序列RNA为SEQ ID NO.2所示。
4.根据权利要求1所述的人工改造的AKT3环状RNA，其特征在于在制备疫苗中的应用。
5.根据权利要求2或者3所述的人工改造的AKT3环状RNA，其特征在于在制备新冠病毒的疫苗中的应用。
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| Title |
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| Circular RNA AKT3 upregulates PIK3R1 to enhance cisplatin resistance in gastric cancer via miR-198 suppression;Huang，X.X.等;《Molecular Cancer》;20191230;第18卷;第71篇第1-20页 * |
| 登录号：NM_001370074;Li Y等;《GENBANK》;20201212;第1页 * |
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