CN112608945B - Modified PPP1R12A circular RNA vector and application thereof - Google Patents

Abstract

The invention discloses a modified PPP1R12A circular RNA vector and application thereof, which is characterized in that: the modified PPP1R12A circular RNA vector has the ability of starting translation, for example, a section of RNA with translation potential is added in the middle, the PPP1R12A circular RNA vector can stably express the protein and enter an immune animal body to generate the protein antibody, and the PPP1R12A circular RNA can be used as a vector of a vaccine and can be used for the vaccine for prevention or treatment.

Description

Translated fromChinese

一种改造的PPP1R12A环状RNA载体及其应用A modified PPP1R12A circular RNA vector and its application

技术领域technical field

本发明属生物技术领域 ，具体地涉及改造的PPP1R12A环状RNA载体，该载体的适当位点插入一段翻译序列之后可以表达该蛋白，载体可以用于多种疫苗的载体。The invention belongs to the field of biotechnology, and specifically relates to a modified PPP1R12A circular RNA vector, which can express the protein after inserting a translation sequence into an appropriate site of the vector, and the vector can be used as a vector for various vaccines.

背景技术Background technique

疫苗接种是预防和控制传染病的有效手段，对人类健康产生了巨大的影响。疫苗的品种非常多，根据其性质主要分为减毒活疫苗、灭活疫苗、亚单位疫苗、树突状细胞疫苗、DNA疫苗及mRNA疫苗等。此外，我国还将疫苗分为免疫规划疫苗和非免疫规划疫苗。Vaccination is an effective means of preventing and controlling infectious diseases and has a huge impact on human health. There are many varieties of vaccines, which are mainly divided into live attenuated vaccines, inactivated vaccines, subunit vaccines, dendritic cell vaccines, DNA vaccines and mRNA vaccines according to their properties. In addition, my country also divides vaccines into immunization program vaccines and non-immunization program vaccines.

mRNA疫苗是将mRNA传递至细胞， 表达产生蛋白， 进而扩大机体的免疫能力。mRNA疫苗是具有免疫性、安全性及灵活性的基因疫苗，相比传统疫苗，mRNA疫苗不仅能够刺激免疫系统产生平衡、长效的保护，而且能被以Toll 样受体为主的受体识别，进入细胞在胞液中表达，通过刺激免疫系统产生 B和T 细胞等多种机制， 达到预防流行病及抗肿瘤等作用。mRNA vaccines deliver mRNA to cells, express and produce proteins, thereby expanding the body's immune capacity. mRNA vaccines are genetic vaccines with immunity, safety and flexibility. Compared with traditional vaccines, mRNA vaccines can not only stimulate the immune system to produce balanced and long-lasting protection, but also be recognized by receptors mainly toll-like receptors. , into the cells and expressed in the cytosol, through stimulating the immune system to produce B and T cells and other mechanisms, to prevent epidemics and anti-tumor effects.

mRNA容易受到细胞外普遍存在的核糖核苷酸酶影响而迅速降解，可能在被目标细胞摄取前就失去编码蛋白质的功能。因此，mRNA疫苗稳定性较差、极易被降解问题是其广泛应用限制条件。目前，虽然可以从成体系、密码子优化和修饰核苷等入手提高mRNA自身的稳定，但其稳定性依然是mRNA疫苗最大的挑战。mRNA is susceptible to rapid degradation by ribonucleases that are ubiquitous outside the cell, and may lose its protein-encoding function before being taken up by target cells. Therefore, the poor stability and easy degradation of mRNA vaccines are the limiting conditions for its wide application. At present, although the stability of mRNA itself can be improved from system formation, codon optimization and modification of nucleosides, its stability is still the biggest challenge of mRNA vaccines.

环状RNA (circRNA) 是一类广泛存在于各种生物中的特殊的非编码RNA，与正常线性RNA不同, 它的3'和5'末端连接在一起形成共价闭合环状结构, 致使缺乏典型的末端结构 (5'帽装结构和3'多聚腺苷酸化)，阻碍核酸外切酶对其降解作用，使得circRNA具有高度保守性和稳定性。研究显示，circRNAs可充当miRNA分子海绵, 竞争内源性RNA, 通过阻断病毒miRNA对其靶mRNA的抑制作用, 发挥抑制病毒miRNA的生物学功能, 从而调节病毒miRNA的活动。此外，circRNA也参与免疫系统的调节。有趣的是，一小部分circRNA可编码蛋白，发挥调控作用。因此，circRNA具有稳定性特点，为circ-mRNA疫苗的设计提供了较强的科学理论。Circular RNA (circRNA) is a kind of special non-coding RNA that widely exists in various organisms. Unlike normal linear RNA, its 3' and 5' ends are linked together to form a covalently closed circular structure, resulting in the lack of The typical terminal structure (5' capping structure and 3' polyadenylation) hinders its degradation by exonuclease, making circRNAs highly conserved and stable. Studies have shown that circRNAs can act as miRNA molecular sponges, compete with endogenous RNAs, and play a biological function of inhibiting viral miRNAs by blocking the inhibitory effect of viral miRNAs on their target mRNAs, thereby regulating the activities of viral miRNAs. In addition, circRNAs are also involved in the regulation of the immune system. Interestingly, a small fraction of circRNAs can encode proteins that play regulatory roles. Therefore, circRNA has the characteristics of stability, which provides a strong scientific theory for the design of circ-mRNA vaccine.

发明内容SUMMARY OF THE INVENTION

本发明的目的在于提供一种改造的PPP1R12A环状RNA载体和运用。The purpose of the present invention is to provide a modified PPP1R12A circular RNA vector and its application.

本发明的目的是这样实现的，通过合成该改建了最后建立包含PPP1R12A环状RNA载体序列以及在第555与556脱氧核酸之前插入编码RNA序列的完成体，可较好的表达插入序列的蛋白，该载体插入适当序列后，可以用于在制备疫苗中的载体，包括现在的制备新冠病毒的疫苗（81460491）。The purpose of the present invention is to achieve this, by synthesizing the reconstructed and finally establishing a complete body comprising the PPP1R12A circular RNA vector sequence and inserting the coding RNA sequence before the 555th and 556th deoxynucleic acid, the protein of the inserted sequence can be better expressed, After the vector is inserted into the appropriate sequence, it can be used in the preparation of vaccines, including the current vaccine for the preparation of new coronavirus (81460491).

本发明（优点）：该载体表达蛋白效率高，制作简单，保存容易，作用剂量低。编码SARS-CoV-2病毒抗原的mRNA和疫苗及疫苗的制备方法的专利给予了我们的专利很大参考，本发明在他们的基础上改进了载体在思路上有了新的突破。The present invention (advantage): the vector has high protein expression efficiency, simple production, easy storage and low dose. The patents on the mRNA encoding the SARS-CoV-2 virus antigen and the vaccine and the preparation method of the vaccine have given our patents a great reference. The present invention improves the vector on their basis and has a new breakthrough in thinking.

附图说明Description of drawings

图1所示转染后的细胞表达了棘突蛋白。Transfected cells shown in Figure 1 expressed spike protein.

具体实施方式Detailed ways

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。The present invention is further described below, but the present invention is not limited in any way, and any transformation or replacement based on the teaching of the present invention belongs to the protection scope of the present invention.

实施例Example

通过生物公司订购一段线性DNA，其序列为SEQ ID NO.1所示，该序列首尾包含PCR结合区扩增，T7启动子区以及，改造的PPP1R12A环状RNA载体，以及插入的编码新冠病毒棘突的改进后的RNA序列,插入位点为第555与556之间,为n501y突变，以及第三和第四氨基酸为脯氨酸。通过BeyoAmp™ Extra-long DNA Polymerase酶进行扩增，引物序列为AGAGGTAAGCTGTCTGTCGG与CTTTTCTAGCCTGCTGCTGT, 为SEQ ID NO.2和3所示。扩增后得到大量的DNA用于下一阶段试验。Order a piece of linear DNA through a biological company, and its sequence is shown in SEQ ID NO. The modified RNA sequence of the mutation, the insertion site is between 555th and 556th, is the n501y mutation, and the third and fourth amino acids are proline. Amplification was carried out by BeyoAmp™ Extra-long DNA Polymerase enzyme, and the primer sequences were AGAGGTAAGCTGTTCGG and CTTTTCTAGCCTGCTGCTGT, as shown in SEQ ID NO.2 and 3. After amplification, a large amount of DNA is obtained for the next stage of experiments.

通过DNA模板合成相应的线状RNA，合成使用诺唯赞公司的T7 High Yield RNATranscription Kit按说明书操作，合成结束后去除DNA模板，对产物回收提纯，过程中使用RNA酶抑制剂保护RNA合成。The corresponding linear RNA was synthesized from the DNA template, and the T7 High Yield RNA Transcription Kit of Novozymes was used for the synthesis according to the instructions. After the synthesis, the DNA template was removed, and the product was recovered and purified. During the process, an RNase inhibitor was used to protect the RNA synthesis.

采用诺唯赞VAHTS Circularization Kit For MGI环化试剂盒对上一步得到的RNA产物进行环化，对产物回收提纯去除剩余的线状RNA分子,得到了序列为SEQ ID NO.4的环状RNA。The RNA product obtained in the previous step was cyclized by Novozan VAHTS Circularization Kit For MGI Circularization Kit, the product was recovered and purified to remove the remaining linear RNA molecules, and the circular RNA with the sequence of SEQ ID NO.4 was obtained.

将该RNA利用转染试剂例如阳离子脂质体，纳米高分子转染试剂等，转染到A549细胞，48小时后采用western blot检测改环状RNA是否表达改进的棘突蛋白结果如图1所示，成功的表达了该蛋白。The RNA was transfected into A549 cells using transfection reagents such as cationic liposomes, nanopolymer transfection reagents, etc. After 48 hours, western blot was used to detect whether the modified circular RNA expressed the improved spike protein. The results are shown in Figure 1. showed that the protein was successfully expressed.

将该环状RNA与免疫递送剂制成疫苗，将该疫苗对小鼠进行免疫接种，接种后14天检测抗体，通过ELISA法检测小鼠的血清，每组5只，结果如下对照组仅注射递送剂制，结果均为阴性，低剂量组含2.5-3.0微克环状RNA的疫苗，5只均为弱阳性，高剂量组含5-6微克环状RNA的疫苗，3只阳性，2只弱阳性。U被1-甲基-3伪尿酰(ψ)所取代，可以提高安全性和降低免疫反应。The circular RNA and the immune delivery agent were prepared into a vaccine, the vaccine was immunized to mice, the antibody was detected 14 days after inoculation, and the serum of the mice was detected by ELISA method, 5 mice in each group, and the results were as follows: the control group was only injected The results of the delivery formulation were all negative, the low-dose group of the vaccine containing 2.5-3.0 micrograms of circular RNA, 5 were weakly positive, the high-dose group of the vaccine containing 5-6 micrograms of circular RNA, 3 positive, 2 Weakly positive. U is replaced by 1-methyl-3-pseudouramide (ψ), which can improve safety and reduce immune response.

序列表sequence listing

<110> 昆明医科大学<110> Kunming Medical University

<120> 一种改造的PPP1R12A环状RNA载体及其应用<120> A modified PPP1R12A circular RNA vector and its application

<130> 1<130> 1

<140> 1<140> 1

<141> 2002-12-28<141> 2002-12-28

<160> 6<160> 6

<170> SIPOSequenceListing 1.0<170> SIPOSequenceListing 1.0

<210> 1<210> 1

<211> 4496<211> 4496

<212> DNA<212> DNA

<213> 人工序列(Artificial sequence)<213> Artificial sequence

<400> 1<400> 1

agaggtaagc tgtctgtcgg taatacgact cactatagat cactcaactg actttaaaaa 60agaggtaagc tgtctgtcgg taatacgact cactatagat cactcaactg actttaaaaa 60

gctttatcaa caaattctag ctgaaaatca aaagctgaag gcacagctac atcatacaaa 120gctttatcaa caaattctag ctgaaaatca aaagctgaag gcacagctac atcatacaaa 120

tatcgaacta acagatctta aattacagtt ggaaaaggcc acccagccct gtaagaccag 180tatcgaacta acagatctta aattacagtt ggaaaaggcc acccagccct gtaagaccag 180

taataaattt ctgtctactg atcattctat tgaattgaaa tccagagcca atcgcatctt 240taataaattt ctgtctactg atcattctat tgaattgaaa tccagagcca atcgcatctt 240

tcagaaaagg taacctgaat ttcagaacca gataataaga gaattctcct ttaccagtaa 300tcagaaaagg taacctgaat ttcagaacca gataataaga gaattctcct ttaaccagtaa 300

gatatctttt tcctgcttag gacaaaccca ttacttgtca ctttggatat actttatttc 360gatatctttt tcctgcttag gacaaaccca ttacttgtca ctttggatat actttatttc 360

atcaaatcta agacattact cattttaaaa caccattgtg ttacctacta ctgaaaaaaa 420atcaaatcta agacattact cattttaaaa caccattgtg ttacctacta ctgaaaaaaa 420

ggaaaggtgc taccaattaa actataatcc atcatcaatt gtaagccatt ttctggtttt 480ggaaaggtgc taccaattaa actataatcc atcatcaatt gtaagccatt ttctggtttt 480

ggaaatatta aaatctggga ggaagtgtac aatttaaaat attaatcatt atatgggctg 540ggaaatatta aaatctggga ggaagtgtac aatttaaaat attaatcatt atatgggctg 540

gagctgcatt attctgtttc tggtggcgac cgcgaccggc gtgcatagct aaatgtttcc 600gagctgcatt attctgtttc tggtggcgac cgcgaccggc gtgcatagct aaatgtttcc 600

gccacttgtt ttattgccac tagtctctag tcagtgtgtt aatcttacaa ccagaactca 660gccacttgtt ttattgccac tagtctctag tcagtgtgtt aatcttacaa ccagaactca 660

attaccccct gcatacacta attctttcac acgtggtgtt tattaccctg acaaagtttt 720attaccccct gcatacacta attctttcac acgtggtgtt tattaccctg acaaagtttt 720

cagatcctca gttttacatt caactcagga cttgttctta cctttctttt ccaatgttac 780cagatcctca gttttacatt caactcagga cttgttctta cctttctttt ccaatgttac 780

ttggttccat gctatacatg tctctgggac caatggtact aagaggtttg ataaccctgt 840ttggttccat gctatacatg tctctgggac caatggtact aagaggtttg ataaccctgt 840

cctaccattt aatgatggtg tttattttgc ttccactgag aagtctaaca taataagagg 900cctaccattt aatgatggtg ttttttttgc ttccactgag aagtctaaca taataagagg 900

ctggattttt ggtactactt tagattcgaa gacccagtcc ctacttattg ttaataacgc 960ctggattttt ggtactactt tagattcgaa gacccagtcc ctacttattg ttaataacgc 960

tactaatgtt gttattaaag tctgtgaatt tcaattttgt aatgatccat ttttgggtgt 1020tactaatgtt gttattaaag tctgtgaatt tcaattttgt aatgatccat ttttgggtgt 1020

ttattaccac aaaaacaaca aaagttggat ggaaagtgag ttcagagttt attctagtgc 1080ttattaccac aaaaacaaca aaagttggat ggaaagtgag ttcagagttt attctagtgc 1080

gaataattgc acttttgaat atgtctctca gccttttctt atggaccttg aaggaaaaca 1140gaataattgc acttttgaat atgtctctca gccttttctt atggaccttg aaggaaaaca 1140

gggtaatttc aaaaatctta gggaatttgt gtttaagaat attgatggtt attttaaaat 1200gggtaatttc aaaaatctta gggaatttgt gtttaagaat attgatggtt attttaaaat 1200

atattctaag cacacgccta ttaatttagt gcgtgatctc cctcagggtt tttcggcttt 1260atattctaag cacacgccta ttaatttagt gcgtgatctc cctcagggtt tttcggcttt 1260

agaaccattg gtagatttgc caataggtat taacatcact aggtttcaaa ctttacttgc 1320agaaccattg gtagatttgc caataggtat taacatcact aggtttcaaa ctttacttgc 1320

tttacataga agttatttga ctcctggtga ttcttcttca ggttggacag ctggtgctgc 1380tttacataga agttatttga ctcctggtga ttcttcttca ggttggacag ctggtgctgc 1380

agcttattat gtgggttatc ttcaacctag gacttttcta ttaaaatata atgaaaatgg 1440agcttattat gtgggttatc ttcaacctag gacttttcta ttaaaatata atgaaaatgg 1440

aaccattaca gatgctgtag actgtgcact tgaccctctc tcagaaacaa agtgtacgtt 1500aaccattaca gatgctgtag actgtgcact tgaccctctc tcagaaacaa agtgtacgtt 1500

gaaatccttc actgtagaaa aaggaatcta tcaaacttct aactttagag tccaaccaac 1560gaaatccttc actgtagaaa aaggaatcta tcaaacttct aactttagag tccaaccaac 1560

agaatctatt gttagatttc ctaatattac aaacttgtgc ccttttggtg aagtttttaa 1620agaatctatt gttagatttc ctaatattac aaacttgtgc ccttttggtg aagtttttaa 1620

cgccaccaga tttgcatctg tttatgcttg gaacaggaag agaatcagca actgtgttgc 1680cgccaccaga tttgcatctg tttatgcttg gaacaggaag agaatcagca actgtgttgc 1680

tgattattct gtcctatata attccgcatc attttccact tttaagtgtt atggagtgtc 1740tgattattct gtcctatata attccgcatc attttccact tttaagtgtt atggagtgtc 1740

tcctactaaa ttaaatgatc tctgctttac taatgtctat gcagattcat ttgtaattag 1800tcctactaaa ttaaatgatc tctgctttac taatgtctat gcagattcat ttgtaattag 1800

aggtgatgaa gtcagacaaa tcgctccagg gcaaactgga aagattgctg attataatta 1860aggtgatgaa gtcagacaaa tcgctccagg gcaaactgga aagattgctg attataatta 1860

taaattacca gatgatttta caggctgcgt tatagcttgg aattctaaca atcttgattc 1920taaattacca gatgatttta caggctgcgt tatagcttgg aattctaaca atcttgattc 1920

taaggttggt ggtaattata attacctgta tagattgttt aggaagtcta atctcaaacc 1980taaggttggt ggtaattata attacctgta tagattgttt aggaagtcta atctcaaacc 1980

ttttgagaga gatatttcaa ctgaaatcta tcaggccggt agcacacctt gtaatggtgt 2040ttttgagaga gatatttcaa ctgaaatcta tcaggccggt agcacacctt gtaatggtgt 2040

tgaaggtttt aattgttact ttcctttaca atcatatggt ttccaaccca cttatggtgt 2100tgaaggtttt aattgttact ttcctttaca atcatatggt ttccaaccca cttatggtgt 2100

tggttaccaa ccatacagag tagtagtact ttcttttgaa cttctacatg caccagcaac 2160tggttaccaa ccatacagag tagtagtact ttcttttgaa cttctacatg caccagcaac 2160

tgtttgtgga cctaaaaagt ctactaattt ggttaaaaac aaatgtgtca atttcaactt 2220tgtttgtgga cctaaaaagt ctactaattt ggttaaaaac aaatgtgtca atttcaactt 2220

caatggttta acaggcacag gtgttcttac tgagtctaac aaaaagtttc tgcctttcca 2280caatggttta acaggcacag gtgttcttac tgagtctaac aaaaagtttc tgcctttcca 2280

acaatttggc agagacattg ctgacactac tgatgctgtc cgtgatccac agacacttga 2340acaatttggc agagacattg ctgacactac tgatgctgtc cgtgatccac agacacttga 2340

gattcttgac attacaccat gttcttttgg tggtgtcagt gttataacac caggaacaaa 2400gattcttgac attacaccat gttcttttgg tggtgtcagt gttataacac caggaacaaa 2400

tacttctaac caggttgctg ttctttatca ggatgttaac tgcacagaag tccctgttgc 2460tacttctaac caggttgctg ttctttatca ggatgttaac tgcacagaag tccctgttgc 2460

tattcatgca gatcaactta ctcctacttg gcgtgtttat tctacaggtt ctaatgtttt 2520tattcatgca gatcaactta ctcctacttg gcgtgtttat tctacaggtt ctaatgtttt 2520

tcaaacacgt gcaggctgtt taataggggc tgaacatgtc aacaactcat atgagtgtga 2580tcaaacacgt gcaggctgtt taataggggc tgaacatgtc aacaactcat atgagtgtga 2580

catacccatt ggtgcaggta tatgcgctag ttatcagact cagactaatt ctcctcggcg 2640catacccatt ggtgcaggta tatgcgctag ttatcagact cagactaatt ctcctcggcg 2640

ggcacgtagt gtagctagtc aatccatcat tgcctacact atgtcacttg gtgcagaaaa 2700ggcacgtagt gtagctagtc aatccatcat tgcctacact atgtcacttg gtgcagaaaa 2700

ttcagttgct tactctaata actctattgc catacccaca aattttacta ttagtgttac 2760ttcagttgct tactctaata actctattgc catacccaca aattttacta ttagtgttac 2760

cacagaaatt ctaccagtgt ctatgaccaa gacatcagta gattgtacaa tgtacatttg 2820cacagaaatt ctaccagtgt ctatgaccaa gacatcagta gattgtacaa tgtacatttg 2820

tggtgattca actgaatgca gcaatctttt gttgcaatat ggcagttttt gtacacaatt 2880tggtgattca actgaatgca gcaatctttt gttgcaatat ggcagttttt gtacacaatt 2880

aaaccgtgct ttaactggaa tagctgttga acaagacaaa aacacccaag aagtttttgc 2940aaaccgtgct ttaactggaa tagctgttga acaagacaaa aacacccaag aagtttttgc 2940

acaagtcaaa caaatttaca aaacaccacc aattaaagat tttggtggtt ttaatttttc 3000acaagtcaaa caaatttaca aaacaccacc aattaaagat tttggtggtt ttaatttttc 3000

acaaatatta ccagatccat caaaaccaag caagaggtca tttattgaag atctactttt 3060acaaatatta ccagatccat caaaaccaag caagaggtca tttattgaag atctactttt 3060

caacaaagtg acacttgcag atgctggctt catcaaacaa tatggtgatt gccttggtga 3120caacaaagtg acacttgcag atgctggctt catcaaacaa tatggtgatt gccttggtga 3120

tattgctgct agagacctca tttgtgcaca aaagtttaac ggccttactg ttttgccacc 3180tattgctgct agagacctca tttgtgcaca aaagtttaac ggccttactg ttttgccacc 3180

tttgctcaca gatgaaatga ttgctcaata cacttctgca ctgttagcgg gtacaatcac 3240tttgctcaca gatgaaatga ttgctcaata cacttctgca ctgttagcgg gtacaatcac 3240

ttctggttgg acctttggtg caggtgctgc attacaaata ccatttgcta tgcaaatggc 3300ttctggttgg acctttggtg caggtgctgc attacaaata ccatttgcta tgcaaatggc 3300

ttataggttt aatggtattg gagttacaca gaatgttctc tatgagaacc aaaaattgat 3360ttataggttt aatggtattg gagttacaca gaatgttctc tatgagaacc aaaaattgat 3360

tgccaaccaa tttaatagtg ctattggcaa aattcaagac tcactttctt ccacagcaag 3420tgccaaccaa tttaatagtg ctattggcaa aattcaagac tcactttctt ccacagcaag 3420

tgcacttgga aaacttcaag atgtggtcaa ccaaaatgca caagctttaa acacgcttgt 3480tgcacttgga aaacttcaag atgtggtcaa ccaaaatgca caagctttaa acacgcttgt 3480

taaacaactt agctccaatt ttggtgcaat ttcaagtgtt ttaaatgata tcctttcacg 3540taaacaactt agctccaatt ttggtgcaat ttcaagtgtt ttaaatgata tcctttcacg 3540

tcttgacaaa gttgaggctg aagtgcaaat tgataggttg atcacaggca gacttcaaag 3600tcttgacaaa gttgaggctg aagtgcaaat tgataggttg atcacaggca gacttcaaag 3600

tttgcagaca tatgtgactc aacaattaat tagagctgca gaaatcagag cttctgctaa 3660tttgcagaca tatgtgactc aacaattaat tagagctgca gaaatcagag cttctgctaa 3660

tcttgctgct actaaaatgt cagagtgtgt acttggacaa tcaaaaagag ttgatttttg 3720tcttgctgct actaaaatgt cagagtgtgt acttggacaa tcaaaaagag ttgatttttg 3720

tggaaagggc tatcatctta tgtccttccc tcagtcagca cctcatggtg tagtcttctt 3780tggaaagggc tatcatctta tgtccttccc tcagtcagca cctcatggtg tagtcttctt 3780

gcatgtgact tatgtccctg cacaagaaaa gaacttcaca actgctcctg ccatttgtca 3840gcatgtgact tatgtccctg cacaagaaaa gaacttcaca actgctcctg ccatttgtca 3840

tgatggaaaa gcacactttc ctcgtgaagg tgtctttgtt tcaaatggca cacactggtt 3900tgatggaaaa gcacactttc ctcgtgaagg tgtctttgtt tcaaatggca cacactggtt 3900

tgtaacacaa aggaattttt atgaaccaca aatcattact acagacaaca catttgtgtc 3960tgtaacacaa aggaattttt atgaaccaca aatcattact acagacaaca catttgtgtc 3960

tggtaactgt gatgttgtaa taggaattgt caacaacaca gtttatgatc ctttgcaacc 4020tggtaactgt gatgttgtaa taggaattgt caacaacaca gtttatgatc ctttgcaacc 4020

tgaattagac tcattcaagg aggagttaga taaatatttt aagaatcata catcaccaga 4080tgaattagac tcattcaagg aggagttaga taaatatttt aagaatcata catcaccaga 4080

tgttgattta ggtgacatct ctggcattaa tgcttcagtt gtaaacattc aaaaagaaat 4140tgttgattta ggtgacatct ctggcattaa tgcttcagtt gtaaacattc aaaaagaaat 4140

tgaccgcctc aatgaggttg ccaagaattt aaatgaatct ctcatcgatc tccaagaact 4200tgaccgcctc aatgaggttg ccaagaattt aaatgaatct ctcatcgatc tccaagaact 4200

tggaaagtat gagcagtata taaaatggcc atggtacatt tggctaggtt ttatagctgg 4260tggaaagtat gagcagtata taaaatggcc atggtacatt tggctaggtt ttatagctgg 4260

cttgattgcc atagtaatgg tgacaattat gctttgctgt atgaccagtt gctgtagttg 4320cttgattgcc atagtaatgg tgacaattat gctttgctgt atgaccagtt gctgtagttg 4320

tctcaagggc tgttgttctt gtggatcctg ctgcaaattt gatgaagacg actctgagcc 4380tctcaagggc tgttgttctt gtggatcctg ctgcaaattt gatgaagacg actctgagcc 4380

agtgctcaaa ggagtcaaat tacattacac ataagtagtt agattccttg ctgggtcgct 4440agtgctcaaa ggagtcaaat tacattacac ataagtagtt agattccttg ctgggtcgct 4440

ctggatcata cagttactta gaagaaagaa aaccttacag cagcaggcta gaaaag 4496ctggatcata cagttactta gaagaaagaa aaccttacag cagcaggcta gaaaag 4496

<210> 2<210> 2

<211> 20<211> 20

<212> DNA<212> DNA

<213> 人工序列(Artificial sequence)<213> Artificial sequence

<400> 2<400> 2

agaggtaagc tgtctgtcgg 20agaggtaagc tgtctgtcgg 20

<210> 3<210> 3

<211> 20<211> 20

<212> DNA<212> DNA

<213> 人工序列(Artificial sequence)<213> Artificial sequence

<400> 3<400> 3

cttttctagc ctgctgctgt 20cttttctagc ctgctgctgt 20

<210> 4<210> 4

<211> 4459<211> 4459

<212> RNA<212> RNA

<213> 人工序列(Artificial sequence)<213> Artificial sequence

<400> 4<400> 4

gaucacucaa cugacuuuaa aaagcuuuau caacaaauuc uagcugaaaa ucaaaagcug 60gaucacucaa cugacuuuaa aaagcuuuau caacaaauuc uagcugaaaa ucaaaagcug 60

aaggcacagc uacaucauac aaauaucgaa cuaacagauc uuaaauuaca guuggaaaag 120aaggcacagc uacaucauac aaauaucgaa cuaacagauc uuaaauuaca guuggaaaag 120

gccacccagc ccuguaagac caguaauaaa uuucugucua cugaucauuc uauugaauug 180gccacccagc ccuguaagac caguaauaaa uuucugucua cugaucauuc uauugaauug 180

aaauccagag ccaaucgcau cuuucagaaa agguaaccug aauuucagaa ccagauaaua 240aaauccagag ccaaucgcau cuuucagaaa agguaaccug aauuucagaa ccagauaaua 240

agagaauucu ccuuuaccag uaagauaucu uuuuccugcu uaggacaaac ccauuacuug 300agagaauucu ccuuuaccag uaagauaucu uuuuccugcu uaggacaaac ccauuacuug 300

ucacuuugga uauacuuuau uucaucaaau cuaagacauu acucauuuua aaacaccauu 360ucacuuugga uauacuuuau uucaucaaau cuaagacauu acucauuuua aaacaccauu 360

guguuaccua cuacugaaaa aaaggaaagg ugcuaccaau uaaacuauaa uccaucauca 420guguuaccua cuacugaaaa aaaggaaagg ugcuaccaau uaaacuauaa uccaucauca 420

auuguaagcc auuuucuggu uuuggaaaua uuaaaaucug ggaggaagug uacaauuuaa 480auuguaagcc auuuucuggu uuuggaaaua uuaaaaucug ggaggaagug uacaauuuaa 480

aauauuaauc auuauauggg cuggagcugc auuauucugu uucugguggc gaccgcgacc 540aauauuaauc auuauauggg cuggagcugc auuauucugu uucugguggc gaccgcgacc 540

ggcgugcaua gcuaaauguu uccgccacuu guuuuauugc cacuagucuc uagucagugu 600ggcgugcaua gcuaaauguu uccgccacuu guuuuauugc cacuagucuc uagucagugu 600

guuaaucuua caaccagaac ucaauuaccc ccugcauaca cuaauucuuu cacacguggu 660guuaaucuua caaccagaac ucaauuaccc ccugcauaca cuaauucuuu cacacguggu 660

guuuauuacc cugacaaagu uuucagaucc ucaguuuuac auucaacuca ggacuuguuc 720guuuauuacc cugacaaagu uuucagaucc ucaguuuuac auucaacuca ggacuuguuc 720

uuaccuuucu uuuccaaugu uacuugguuc caugcuauac augucucugg gaccaauggu 780uuaccuuucu uuuccaaugu uacuugguuc caugcuauac augucucugg gaccaauggu 780

acuaagaggu uugauaaccc uguccuacca uuuaaugaug guguuuauuu ugcuuccacu 840acuaagaggu uugauaaccc uguccuacca uuuaaugaug guguuuauuu ugcuuccacu 840

gagaagucua acauaauaag aggcuggauu uuugguacua cuuuagauuc gaagacccag 900gagaagucua acauaauaag aggcuggauu uuugguacua cuuuagauuc gaagacccag 900

ucccuacuua uuguuaauaa cgcuacuaau guuguuauua aagucuguga auuucaauuu 960ucccuacuua uuguuaauaa cgcuacuaau guuguuauua aagucuguga auuucaauuu 960

uguaaugauc cauuuuuggg uguuuauuac cacaaaaaca acaaaaguug gauggaaagu 1020uguaaugauc cauuuuuggg uguuuauuac cacaaaaaca acaaaaguug gauggaaagu 1020

gaguucagag uuuauucuag ugcgaauaau ugcacuuuug aauaugucuc ucagccuuuu 1080gaguucagag uuuauucuag ugcgaauaau ugcacuuuug aauaugucuc ucagccuuuu 1080

cuuauggacc uugaaggaaa acaggguaau uucaaaaauc uuagggaauu uguguuuaag 1140cuuauggacc uugaaggaaa acaggguaau uucaaaaauc uuagggaauu uguguuuaag 1140

aauauugaug guuauuuuaa aauauauucu aagcacacgc cuauuaauuu agugcgugau 1200aauauugaug guuauuuuaa aauauauucu aagcacacgc cuauuaauuu agugcgugau 1200

cucccucagg guuuuucggc uuuagaacca uugguagauu ugccaauagg uauuaacauc 1260cucccucagg guuuuucggc uuuagaacca uugguagauu ugccaauagg uauuaacauc 1260

acuagguuuc aaacuuuacu ugcuuuacau agaaguuauu ugacuccugg ugauucuucu 1320acuagguuuc aaacuuuacu ugcuuuacau agaaguuauu ugacuccugg ugauucuucu 1320

ucagguugga cagcuggugc ugcagcuuau uauguggguu aucuucaacc uaggacuuuu 1380ucagguugga cagcuggugc ugcagcuuau uauguggguu aucuucaacc uaggacuuuu 1380

cuauuaaaau auaaugaaaa uggaaccauu acagaugcug uagacugugc acuugacccu 1440cuauuaaaau auaaugaaaa uggaaccauu acagaugcug uagacugugc acuugacccu 1440

cucucagaaa caaaguguac guugaaaucc uucacuguag aaaaaggaau cuaucaaacu 1500cucucagaaa caaaguguac guugaaaucc uucacuguag aaaaaggaau cuaucaaacu 1500

ucuaacuuua gaguccaacc aacagaaucu auuguuagau uuccuaauau uacaaacuug 1560ucuaacuuua gaguccaacc aacagaaucu auuguuagau uuccuaauau uacaaacuug 1560

ugcccuuuug gugaaguuuu uaacgccacc agauuugcau cuguuuaugc uuggaacagg 1620ugcccuuuug gugaaguuuu uaacgccacc agauuugcau cuguuuaugc uuggaacagg 1620

aagagaauca gcaacugugu ugcugauuau ucuguccuau auaauuccgc aucauuuucc 1680aagagaauca gcaacugugu ugcugauuau ucuguccuau auaauuccgc aucauuuucc 1680

acuuuuaagu guuauggagu gucuccuacu aaauuaaaug aucucugcuu uacuaauguc 1740acuuuuaagu guuauggagu gucuccuacu aaauuaaaug aucucugcuu uacuaauguc 1740

uaugcagauu cauuuguaau uagaggugau gaagucagac aaaucgcucc agggcaaacu 1800uaugcagauu cauuuguaau uagagggau gaagucagac aaaucgcucc agggcaaacu 1800

ggaaagauug cugauuauaa uuauaaauua ccagaugauu uuacaggcug cguuauagcu 1860ggaaagauug cugauuauaa uuauaaauua ccagaugauu uuacaggcug cguuauagcu 1860

uggaauucua acaaucuuga uucuaagguu ggugguaauu auaauuaccu guauagauug 1920uggaauucua acaaucuuga uucuaagguu ggugguaauu auaauuaccu guauagauug 1920

uuuaggaagu cuaaucucaa accuuuugag agagauauuu caacugaaau cuaucaggcc 1980uuuaggaagu cuaaucucaa accuuuugag agagauauuu caacugaaau cuaucaggcc 1980

gguagcacac cuuguaaugg uguugaaggu uuuaauuguu acuuuccuuu acaaucauau 2040gguagcacac cuuguaaugg uguugaaggu uuuaauuguu acuuuccuuu acaaucauau 2040

gguuuccaac ccacuuaugg uguugguuac caaccauaca gaguaguagu acuuucuuuu 2100gguuuccaac ccacuuaugg uguugguuac caaccauaca gaguaguagu acuuucuuuu 2100

gaacuucuac augcaccagc aacuguuugu ggaccuaaaa agucuacuaa uuugguuaaa 2160gaacuucuac augcaccagc aacuguuugu ggaccuaaaa agucuacuaa uuugguuaaa 2160

aacaaaugug ucaauuucaa cuucaauggu uuaacaggca cagguguucu uacugagucu 2220aacaaaugug ucaauuucaa cuucaauggu uuaacaggca cagguguucu uacugagucu 2220

aacaaaaagu uucugccuuu ccaacaauuu ggcagagaca uugcugacac uacugaugcu 2280aacaaaaagu uucugccuuu ccaacaauuu ggcagagaca uugcugacac uacugaugcu 2280

guccgugauc cacagacacu ugagauucuu gacauuacac cauguucuuu uggugguguc 2340guccgugauc cacagacacu ugagauucuu gacauuacac cauguucuuu uggugguguc 2340

aguguuauaa caccaggaac aaauacuucu aaccagguug cuguucuuua ucaggauguu 2400aguguuauaa caccaggaac aaauacuucu aaccagguug cuguucuuua ucaggauguu 2400

aacugcacag aagucccugu ugcuauucau gcagaucaac uuacuccuac uuggcguguu 2460aacugcacag aagucccugu ugcuauucau gcagaucaac uuacuccuac uuggcguguu 2460

uauucuacag guucuaaugu uuuucaaaca cgugcaggcu guuuaauagg ggcugaacau 2520uauucuacag guucuaaugu uuuucaaaca cgugcaggcu guuuaauagg ggcugaacau 2520

gucaacaacu cauaugagug ugacauaccc auuggugcag guauaugcgc uaguuaucag 2580gucaacaacu cauauugagug ugacauaccc auuggugcag guauaugcgc uaguuaucag 2580

acucagacua auucuccucg gcgggcacgu aguguagcua gucaauccau cauugccuac 2640acucagacua auucucccucg gcgggcacgu aguguagcua gucaauccau cauugccuac 2640

acuaugucac uuggugcaga aaauucaguu gcuuacucua auaacucuau ugccauaccc 2700acuaugucac uuggugcaga aaauucaguu gcuuacucua auaacucuau ugccauaccc 2700

acaaauuuua cuauuagugu uaccacagaa auucuaccag ugucuaugac caagacauca 2760acaaauuuua cuauuagugu uaccacagaa auucuaccag ugucuaugac caagacauca 2760

guagauugua caauguacau uuguggugau ucaacugaau gcagcaaucu uuuguugcaa 2820guagauugua caauguacau uuguggugau ucaacugaau gcagcaaucu uuuguugcaa 2820

uauggcaguu uuuguacaca auuaaaccgu gcuuuaacug gaauagcugu ugaacaagac 2880uauggcaguu uuuguacaca auuaaaccgu gcuuuaacug gaauagcugu ugaacaagac 2880

aaaaacaccc aagaaguuuu ugcacaaguc aaacaaauuu acaaaacacc accaauuaaa 2940aaaaacaccc aagaaguuuu ugcacaaguc aaacaaauuu acaaaacacc accaauuaaa 2940

gauuuuggug guuuuaauuu uucacaaaua uuaccagauc caucaaaacc aagcaagagg 3000gauuuuggug guuuuaauuu uucacaaaua uuaccagauc caucaaaacc aagcaagagg 3000

ucauuuauug aagaucuacu uuucaacaaa gugacacuug cagaugcugg cuucaucaaa 3060ucauuuauug aagaucuacu uuucaacaaa gugacacuug cagaugcugg cuucaucaaa 3060

caauauggug auugccuugg ugauauugcu gcuagagacc ucauuugugc acaaaaguuu 3120caauauggug auugccuugg ugauauugcu gcuagagacc ucauuugugc acaaaaguuu 3120

aacggccuua cuguuuugcc accuuugcuc acagaugaaa ugauugcuca auacacuucu 3180aacggccuua cuguuuugcc accuuugcuc acagaugaaa ugauugcuca auacacuucu 3180

gcacuguuag cggguacaau cacuucuggu uggaccuuug gugcaggugc ugcauuacaa 3240gcacuguuag cggguacaau cacuucuggu uggaccuuug gugcaggugc ugcauuacaa 3240

auaccauuug cuaugcaaau ggcuuauagg uuuaauggua uuggaguuac acagaauguu 3300auaccauuug cuaugcaaau ggcuuauagg uuuaauggua uuggaguuac acagaauguu 3300

cucuaugaga accaaaaauu gauugccaac caauuuaaua gugcuauugg caaaauucaa 3360cucuauugaga accaaaaauu gauugccaac caauuuaaua gugcuauugg caaaauucaa 3360

gacucacuuu cuuccacagc aagugcacuu ggaaaacuuc aagauguggu caaccaaaau 3420gacucacuuu cuuccacagc aagugcacuu ggaaaacuuc aagauguggu caaccaaaau 3420

gcacaagcuu uaaacacgcu uguuaaacaa cuuagcucca auuuuggugc aauuucaagu 3480gcacaagcuu uaaacacgcu uguuaaacaa cuuagcucca auuuuggugc aauuucaagu 3480

guuuuaaaug auauccuuuc acgucuugac aaaguugagg cugaagugca aauugauagg 3540guuuuaaaug auauccuuuc acgucuugac aaaguugagg cugaagugca aauugauagg 3540

uugaucacag gcagacuuca aaguuugcag acauauguga cucaacaauu aauuagagcu 3600uugaucacag gcagacuuca aaguuugcag acauauguga cucaacaauu aauuagagcu 3600

gcagaaauca gagcuucugc uaaucuugcu gcuacuaaaa ugucagagug uguacuugga 3660gcagaaauca gagcuucugc uaaucuugcu gcuacuaaaa ugucagagug uguacuugga 3660

caaucaaaaa gaguugauuu uuguggaaag ggcuaucauc uuauguccuu cccucaguca 3720caaucaaaaa gaguugauuu uuguggaaag ggcuaucauc uuauguccuu cccucaguca 3720

gcaccucaug guguagucuu cuugcaugug acuuaugucc cugcacaaga aaagaacuuc 3780gcaccucaug guguagucuu cuugcaugug acuuaugucc cugcacaaga aaagaacuuc 3780

acaacugcuc cugccauuug ucaugaugga aaagcacacu uuccucguga aggugucuuu 3840acaacugcuc cugccauuug ucaugaugga aaagcacacu uuccucguga aggugucuuu 3840

guuucaaaug gcacacacug guuuguaaca caaaggaauu uuuaugaacc acaaaucauu 3900guuucaaaug gcacacacug guuuguaaca caaaggaauu uuuaugaacc acaaaucauu 3900

acuacagaca acacauuugu gucugguaac ugugauguug uaauaggaau ugucaacaac 3960acuacagaca acacauuugu gucugguaac ugugauguug uaauaggaau ugucaacaac 3960

acaguuuaug auccuuugca accugaauua gacucauuca aggaggaguu agauaaauau 4020acaguuuaug auccuuugca accugaauua gacucauuca aggaggaguu agauaaauau 4020

uuuaagaauc auacaucacc agauguugau uuaggugaca ucucuggcau uaaugcuuca 4080uuuaagaauc auacaucacc agauguugau uuaggugaca ucucuggcau uaaugcuuca 4080

guuguaaaca uucaaaaaga aauugaccgc cucaaugagg uugccaagaa uuuaaaugaa 4140guuguaaaca uucaaaaaga aauugaccgc cucaaugagg uugccaagaa uuuaaaugaa 4140

ucucucaucg aucuccaaga acuuggaaag uaugagcagu auauaaaaug gccaugguac 4200ucucucaucg aucuccaaga acuuggaaag uaugagcagu auauaaaaug gccaugguac 4200

auuuggcuag guuuuauagc uggcuugauu gccauaguaa uggugacaau uaugcuuugc 4260auuuggcuag guuuuauagc uggcuugauu gccauaguaa uggugacaau uaugcuuugc 4260

uguaugacca guugcuguag uugucucaag ggcuguuguu cuuguggauc cugcugcaaa 4320uguaugacca guugcuguag uugucucaag ggcuguuguu cuuguggauc cugcugcaaa 4320

uuugaugaag acgacucuga gccagugcuc aaaggaguca aauuacauua cacauaagua 4380uuugaugaag acgacucuga gccagugcuc aaaggaguca aauuacauua cacauaagua 4380

guuagauucc uugcuggguc gcucuggauc auacaguuac uuagaagaaa gaaaaccuua 4440guuagauucc uugcuggguc gcucuggauc auacaguuac uuagaagaaa gaaaaccuua 4440

cagcagcagg cuagaaaag 4459cagcagcagg cuagaaaag 4459

<210> 5<210> 5

<211> 637<211> 637

<212> RNA<212> RNA

<213> 人工序列(Artificial sequence)<213> Artificial sequence

<400> 5<400> 5

gaucacucaa cugacuuuaa aaagcuuuau caacaaauuc uagcugaaaa ucaaaagcug 60gaucacucaa cugacuuuaa aaagcuuuau caacaaauuc uagcugaaaa ucaaaagcug 60

aaggcacagc uacaucauac aaauaucgaa cuaacagauc uuaaauuaca guuggaaaag 120aaggcacagc uacaucauac aaauaucgaa cuaacagauc uuaaauuaca guuggaaaag 120

gccacccagc ccuguaagac caguaauaaa uuucugucua cugaucauuc uauugaauug 180gccacccagc ccuguaagac caguaauaaa uuucugucua cugaucauuc uauugaauug 180

aaauccagag ccaaucgcau cuuucagaaa agguaaccug aauuucagaa ccagauaaua 240aaauccagag ccaaucgcau cuuucagaaa agguaaccug aauuucagaa ccagauaaua 240

agagaauucu ccuuuaccag uaagauaucu uuuuccugcu uaggacaaac ccauuacuug 300agagaauucu ccuuuaccag uaagauaucu uuuuccugcu uaggacaaac ccauuacuug 300

ucacuuugga uauacuuuau uucaucaaau cuaagacauu acucauuuua aaacaccauu 360ucacuuugga uauacuuuau uucaucaaau cuaagacauu acucauuuua aaacaccauu 360

guguuaccua cuacugaaaa aaaggaaagg ugcuaccaau uaaacuauaa uccaucauca 420guguuaccua cuacugaaaa aaaggaaagg ugcuaccaau uaaacuauaa uccaucauca 420

auuguaagcc auuuucuggu uuuggaaaua uuaaaaucug ggaggaagug uacaauuuaa 480auuguaagcc auuuucuggu uuuggaaaua uuaaaaucug ggaggaagug uacaauuuaa 480

aauauuaauc auuauauggg cuggagcugc auuauucugu uucugguggc gaccgcgacc 540aauauuaauc auuauauggg cuggagcugc auuauucugu uucugguggc gaccgcgacc 540

ggcgugcaua gcuaaguagu uagauuccuu gcugggucgc ucuggaucau acaguuacuu 600ggcgugcaua gcuaaguagu uagauuccuu gcugggucgc ucuggaucau acaguuacuu 600

agaagaaaga aaaccuuaca gcagcaggcu agaaaag 637agaagaaaga aaaccuuaca gcagcaggcu agaaaag 637

<210> 6<210> 6

<211> 3822<211> 3822

<212> RNA<212> RNA

<213> 人工序列(Artificial sequence)<213> Artificial sequence

<400> 6<400> 6

auguuuccgc cacuuguuuu auugccacua gucucuaguc aguguguuaa ucuuacaacc 60auguuuccgc cacuuguuuu auugccacua gucucuaguc aguguguuaa ucuuacaacc 60

agaacucaau uacccccugc auacacuaau ucuuucacac gugguguuua uuacccugac 120agaacucaau uacccccugc auacacuaau ucuuucacac gugguguuua uuacccugac 120

aaaguuuuca gauccucagu uuuacauuca acucaggacu uguucuuacc uuucuuuucc 180aaaguuuuca gauccucagu uuuacauuca acucaggacu uguucuuacc uuucuuuucc 180

aauguuacuu gguuccaugc uauacauguc ucugggacca augguacuaa gagguuugau 240aauguuacuu gguuccaugc uauacauguc ucugggacca augguacuaa gagguuugau 240

aacccugucc uaccauuuaa ugaugguguu uauuuugcuu ccacugagaa gucuaacaua 300aacccugucc uaccauuuaa ugaugguguu uauuuugcuu ccacugagaa gucuaacaua 300

auaagaggcu ggauuuuugg uacuacuuua gauucgaaga cccagucccu acuuauuguu 360auaagaggcu ggauuuuugg uacuacuuua gauucgaaga cccagucccu acuuauuguu 360

aauaacgcua cuaauguugu uauuaaaguc ugugaauuuc aauuuuguaa ugauccauuu 420aauaacgcua cuaauguugu uauuaaaguc ugugaauuuc aauuuuguaa ugauccauuu 420

uuggguguuu auuaccacaa aaacaacaaa aguuggaugg aaagugaguu cagaguuuau 480uuggguguuu auuaccacaa aaacaacaaa aguuggaugg aaagugaguu cagaguuuau 480

ucuagugcga auaauugcac uuuugaauau gucucucagc cuuuucuuau ggaccuugaa 540ucuaagugcga auaauugcac uuuugaauau gucucucagc cuuuucuuau ggaccuugaa 540

ggaaaacagg guaauuucaa aaaucuuagg gaauuugugu uuaagaauau ugaugguuau 600ggaaaacagg guaauuucaa aaaucuuagg gaauuugugu uuaagaauau ugaugguuau 600

uuuaaaauau auucuaagca cacgccuauu aauuuagugc gugaucuccc ucaggguuuu 660uuuaaaauau auucuaagca cacgccuauu aauuuagugc gugaucuccc ucaggguuuu 660

ucggcuuuag aaccauuggu agauuugcca auagguauua acaucacuag guuucaaacu 720ucggcuuuag aaccauuggu agauuugcca auagguauua acaucacuag guuucaaacu 720

uuacuugcuu uacauagaag uuauuugacu ccuggugauu cuucuucagg uuggacagcu 780uuacuugcuu uacauagaag uuauuugacu ccuggugauu cuucuucagg uuggacagcu 780

ggugcugcag cuuauuaugu ggguuaucuu caaccuagga cuuuucuauu aaaauauaau 840ggugcugcag cuuauuaugu ggguuaucuu caaccuagga cuuuucuauu aaaauauaau 840

gaaaauggaa ccauuacaga ugcuguagac ugugcacuug acccucucuc agaaacaaag 900gaaaauggaa ccauuacaga ugcuguagac ugugcacuug acccucucuc agaaacaaag 900

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ucugagccag ugcucaaagg agucaaauua cauuacacau aa 3822ucugagccag ugcucaaagg agucaaauua cauuacacau aa 3822

Claims (5)

1. An engineered PPP1R12A circular RNA vector, comprising: the RNA carrier is connected end to end in a ring shape, and the sequence of the RNA carrier is shown in SEQ ID NO. 5.

2. The engineered PPP1R12A circular RNA vector according to claim 1, characterized in that the insertion site of the RNA sequence translatable into a protein is between 555 and 556 nucleotides.

3. The engineered PPP1R12A circular RNA vector of claim 2, wherein: the sequence of RNA which can be translated into protein at the insertion site is shown as SEQ ID NO. 6.

4. Use of the engineered PPP1R12A circular RNA vector according to claim 1, characterized by the use in the preparation of a vaccine.

5. Use of the engineered PPP1R12A circular RNA vector according to claim 3, characterized by the use in the preparation of a vaccine for a new coronavirus.

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