CN112226445B - 编码SARS-CoV-2病毒刺突蛋白的核酸及其的应用 - Google Patents

编码SARS-CoV-2病毒刺突蛋白的核酸及其的应用 Download PDF

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CN112226445B
CN112226445B CN202011127475.9A CN202011127475A CN112226445B CN 112226445 B CN112226445 B CN 112226445B CN 202011127475 A CN202011127475 A CN 202011127475A CN 112226445 B CN112226445 B CN 112226445B
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王子豪
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Jiachen Xihai Hangzhou Biotechnology Co ltd
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Abstract

本发明涉及生物医药技术领域，尤其涉及编码SARS‑CoV‑2病毒刺突蛋白的核酸及其的应用。通过密码子优化，本发明提供了基于α病毒家族中委内瑞拉马脑脊髓炎病毒(TC83Venezuelan Equine Encephalitis Virus，VEEV)的Spike高表达新冠mRNA疫苗，该疫苗在动物实验中，接种14天后即可检测到spike抗体。

Description

编码SARS-CoV-2病毒刺突蛋白的核酸及其的应用
技术领域
本发明涉及生物医药技术领域，尤其涉及编码SARS-CoV-2病毒刺突蛋白的核酸及其的应用。
背景技术
新型冠状病毒肺炎是一种急性感染性肺炎，2020年2月11日，世界卫生组织(WHO)将其英文名称定为Corona Virus Disease 2019(COVID-19)。SARS-CoV-2病毒的核衣壳有核衣壳蛋白(Nucleocapsid protein,N)包裹单股正链RNA形成，外有包膜，嵌有三种糖蛋白，刺突蛋白(spike protein，S)、包膜蛋白(envelope protein，E)和膜蛋白(membraneprotein，M)。其中，E蛋白和M蛋白主要参与病毒的装配过程，S蛋白主要通过与宿主细胞受体结合介导病毒的入侵，并决定病毒组织或宿主嗜性。
目前，仍缺乏针对新冠肺炎病原体的有效抗病毒药物，以隔离治疗、对症支持治疗为主。疫苗是防控传染病最有效的手段。目前在研的COVID-19疫苗种类主要有灭活疫苗、减毒活疫苗、核酸疫苗(DNA疫苗和mRNA疫苗)、蛋白亚单位疫苗及病毒载体疫苗。其中mRNA疫苗具有安全、有效、研发周期短等特点，在应对大规模流行病时更具有优势。
近年来Moderna和BioNTech公司开发的mRNA(信使核糖核酸)疫苗启动临床试验，提供了mRNA疫苗的安全性和有效性的前期数据。目前，mRNA研发的关键技术的研究主要集中在：①提高mRNA稳定性及翻译效率，②调节免疫原性，③开发高效无毒的递送载体。mRNA的组成包括5’帽子结构(Cap)、5'UTR区、编码抗原蛋白的开放阅读框(open readingframe,ORF)，3'UTR区和3'Poly(A)尾结构。mRNA的合成一般是以含靶蛋白ORF的质粒DNA为模板，经体外转录合成，最后再加上5'C ap和3'Poly(A)尾。通过对DNA模板上的UTR区、Poly(A)尾和体外转录时的Cap，NTP等合成mRNA的元件进行设计，可提高mRNA的稳定性和翻译效率。此外，可利用分离和纯化技术来优化mRNA。本领域认为，mRNA的翻译效率与ORF序列密切相关，因此，对编码刺突蛋白的核酸序列进行密码子优化是疫苗研发成功的关键。
发明内容
有鉴于此，本发明要解决的技术问题在于提供编码SARS-CoV-2病毒刺突蛋白的核酸及其的应用，进一步提供基于α病毒家族中委内瑞拉马脑脊髓炎病毒(TC83 VenezuelanEquine Encephalitis Virus，VEEV)的Spike蛋白高表达新冠mRNA疫苗。
本发明提供了编码SARS-CoV-2病毒刺突蛋白的核酸，包括I)～IV)中至少一种：
I)、具有如SEQ ID NO:1所示核苷酸序列的核酸；
II)、在I)所述的片段中取代、缺失或添加一个或多个核苷酸的核酸；
III)、与如I所示的核苷酸序列具有至少70％同源性的序列且编码SARS-CoV-2病毒刺突蛋白的核酸；
IV)、与I)～III)中任一项部分互补或完全互补的核酸。
一些具体实施例中，本发明提供的编码SARS-CoV-2病毒刺突蛋白的核酸序列如SEQ ID NO:1所示。
本发明还提供了一种重组载体，其包括骨架载体和所述的核酸。
所述重组载体包括subgenomic promoter、本发明所述的核酸、3’UTR、polyA_site、5’UTR。一些实施例中，本发明所述核酸插入骨架载体的位点是Apa I和Not I位点之间。一些具体实施例中，本发明所述骨架载体的核酸序列如SEQ ID NO:2所示。本发明所述重组载体的质粒图谱如图1所示。
本发明还提供了一种线性化mRNA，其核酸序列如SEQ ID NO:4。
本发明所述线性化mRNA的制备方法，其包括：将所述的重组载体线性化后，经体外转录、模板DNA降解、5’端加7-甲基化鸟苷酸帽，获得SEQ ID NO:4所示的线性化mRNA。一些实施例中，所述线性化采用的酶为BspQ I。所述体外转录在T7 RNA聚合酶存在下进行。所述模板DNA的降解采用Turbo Dnase。所述5’端加7-甲基化鸟苷酸帽在加帽酶(Vacciniacapping enzyme)存在下进行。
本发明所述的核酸、重组载体、或线性化mRNA，在制备新型冠状病毒mRNA疫苗中的应用。
一种mRNA-LNP复合物，其制备原料包括所述的线性化mRNA和脂类。
本发明所述复合物中所述脂类包括：DMG-PEG2000、胆固醇、二硬脂酰基磷脂酰胆碱和DLinDMA；其中DMG-PEG2000、胆固醇、二硬脂酰基磷脂酰胆碱和DLinDMA的摩尔比为2:48:10:40。
本发明所述复合物的制备方法为，将所述的线性化mRNA和脂类经快速混合，制成脂质体。
本发明还提供了一种新型冠状病毒疫苗，其包括本发明所述的复合物。
本发明所述的疫苗中还包括疫苗中可接受的辅料；其中所述的复合物的浓度为30μg/mL。
一种预防新型冠状病毒的方法，其为给予本发明所述的疫苗。
通过密码子优化，本发明提供了基于α病毒家族中委内瑞拉马脑脊髓炎病毒(TC83Venezuelan Equine Encephalitis Virus，VEEV)的Spike高表达新冠mRNA疫苗，该疫苗在动物实验中，接种14天后即可检测到spike抗体。
附图说明
图1是本发明的mRNA-Spike质粒结构图谱，含有卡那霉素抗性基因(Kan)、T7启动子基因、nsp基因、Spike基因、polyA基因；
图2是本发明的mRNA-Spike体外转录后的电泳结果图；泳道1为mRNA-Spike，泳道M为RNAladder；
图3是本发明通过Western检测，mRNA-Spike在体外转染BHK细胞后Spike蛋白表达图；
图4是本发明通过ELISA检测，mRNA-Spike免疫小鼠后所产生的抗Spike抗体滴度结果图，其中，A示未经过密码子优化的野生型序列制备疫苗后的抗体滴度，B示本发明经过密码子的序列制备疫苗后的抗体滴度，其中1#mock～3#mock示不给疫苗也不造模的空白对照组；1#0.5μg～5#0.5μg分别示给药剂量为0.5μg的5只小鼠；1#0.2μg～4#0.2μg分别示给药剂量为0.2μg的4只小鼠；1#1.0μg～5#1.0μg分别示给药剂量为1.0μg的5只小鼠。
具体实施方式
本发明提供了编码SARS-CoV-2病毒刺突蛋白的核酸及其的应用，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。以下结合附图和具体实施例对本发明作具体的介绍。
本发明所述SARS-CoV-2病毒刺突蛋白，即SARS-CoV-2病毒的spike蛋白，或称为S蛋白具体为SARS-CoV-2，Wuhan seafood market pneumonia virus isolate Wuhan-Hu-1株的Spike蛋白，其NCBI登录号为MN908947.3。编码核酸序列进行密码子优化。经优化后的DNA序列更有利于在人体内的表达，因此可以获得更高表达水平的spike蛋白，进而提高疫苗接种后spike抗体的产生量和产生速度，从而获得良好的免疫效果。
本发明中，所述核酸是指由多个核苷酸聚合成的生物大分子化合物，本发明所述的核酸可以是DNA分子、RNA分子、PNA分子或LNA的形式。其可以为单链、双链形式存在，可为线性或环状。所述DNA形式包括cDNA、基因组DNA或人工合成的DNA。所述DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。本发明中，经优化后的编码SARS-CoV-2病毒的S蛋白的DNA序列如SEQ ID NO:1所示，具体的，SEQ ID NO:1所示核苷酸序列为：atgttcgtgttcctcgtgctgctgcccctggtgtctagccagtgcgtgaacctgaccaccagaacccagctgcctccagcctacaccaacagcttcaccaggggagtgtactaccccgacaaggtgttccggagcagcgtgctgcatagcacccaggacctgttcctgcccttcttcagcaacgtgacttggttccacgccatccacgtcagcggaaccaacggcaccaagcgctttgacaacccagtgctgcccttcaacgacggagtgtacttcgccagcaccgagaagtccaacatcatccggggttggatcttcggcaccacactggacagcaagacccagagcctgctgatcgtgaacaacgccaccaacgtcgtgatcaaggtctgcgagttccagttttgcaacgaccccttcctgggcgtgtactaccacaagaacaacaagtcttggatggagagcgagttccgggtgtacagcagcgccaacaattgcaccttcgagtacgtgtcccagcccttcctgatggacctggaaggcaagcagggcaacttcaagaacctgcgggagttcgtgttcaagaacatcgacggctacttcaagatctacagcaagcacacccccatcaacctcgtgagagacctgccacagggcttcagcgctctggaacctctggtggatctgccaatcggcatcaacatcacccggttccagaccctgctggctctgcacagaagctacctgaccccaggcgattcttctagcggctggacagcaggagccgccgcttattacgtgggctatctgcagccccggaccttcctgctgaagtacaacgagaacggcaccatcaccgacgcagtggattgcgctctggatcctctgagcgagaccaagtgcaccctgaagtccttcaccgtggagaagggcatctaccagaccagcaacttccgggtgcagcctacagagagcatcgtgcggttccctaacatcaccaacctctgccccttcggcgaggtgttcaacgccaccagattcgccagcgtctacgcctggaaccggaagcggatcagcaattgcgtggccgactacagcgtgctgtacaacagcgccagcttcagcaccttcaagtgctacggcgtgtcccctaccaagctgaacgatctgtgcttcaccaacgtgtacgccgacagcttcgtgatcaggggcgacgaagtgcgacagatcgccccaggacagacaggaaagatcgccgattacaactacaagctgcccgacgacttcaccggttgcgtgatcgcttggaacagcaacaacctggacagcaaggtcggcggcaactacaactacctgtaccggctgttccggaagtccaacctgaagcccttcgagcgggacatcagcacagagatctaccaggccggcagcacaccttgtaacggcgtggagggcttcaactgctacttccctctgcagagctacggcttccagcctacaaacggcgtgggataccagccttacagggtggtggtgctgagcttcgaactgctgcacgctccagccaccgtctgcggacctaaaaagagcaccaacctggtgaagaacaagtgcgtgaatttcaacttcaacggcctgaccggaaccggcgtgctgaccgagagcaacaagaagttcctgcccttccagcagttcggcagagacatcgcagacaccacagacgccgtgagagatcctcagaccctggagatcctggacatcaccccttgcagctttggcggagtgtccgtgatcaccccaggaaccaacaccagcaatcaggtggccgtgctgtaccaggacgtgaattgcaccgaggtgccagtggccattcacgcagatcagctgacccctacctggagagtgtacagcaccggaagcaacgtgttccagaccagggcaggttgcctgatcggagccgagcacgtgaacaacagctacgagtgcgatatccctatcggcgccggcatttgcgcctcttaccagacccagaccaacagccctaggagagccagaagcgtggcctctcagagcatcatcgcctacaccatgagcctgggagccgagaacagcgtggcctacagcaacaacagcatcgccatccccaccaacttcaccatcagcgtgaccaccgagattctgcccgtgtctatgaccaagaccagcgtcgactgcaccatgtacatctgcggcgacagcaccgagtgctctaacctgctgctgcagtacggctccttttgcacccagctgaacagggctctgacaggcatcgcagtggagcaggacaagaacacccaggaggtcttcgcccaggtcaagcagatctacaagaccccccctatcaaggacttcggcggctttaacttcagccagatcctgcccgatcccagcaagcctagcaagcggagcttcatcgaggacctgctgttcaacaaggtcaccctggccgacgcaggcttcatcaagcagtacggcgattgcctgggcgacattgccgccagagacctgatttgcgcccagaagttcaacggcctgacagtgctgcctcctctgctgaccgacgagatgatcgcccagtatacaagcgctctgctggcaggaaccatcaccagcggttggacctttggagccggagccgctctgcagatcccttttgccatgcagatggcctaccgcttcaacggaatcggcgtgacccagaacgtgctctacgagaaccagaagctgatcgccaaccagttcaacagcgccatcggcaagatccaggacagcctgagcagcacagctagcgctctgggcaagctgcaggatgtggtgaaccagaacgcccaggctctgaacaccctggtgaagcagctgagcagcaacttcggagccatcagcagcgtgctgaacgacatcctgagcaggctggacaaggtggaagccgaagtgcagatcgacaggctgatcaccggcagactgcagtctctgcagacctacgtgacccagcagctgatcagagccgccgagatcagagctagcgctaatctggccgccaccaagatgagcgagtgcgtgctgggacagagcaagagagtggacttctgcggcaagggctaccacctgatgagctttcctcagagcgctcctcacggagtggtgtttctgcacgtgacctacgtgccagcccaggagaagaacttcaccacagccccagccatttgccacgacggaaaggcccacttccccagagaaggcgtgttcgtgtccaacggcacccattggttcgtgacccagcggaacttctacgagccccagatcatcaccaccgacaacaccttcgtgagcggcaattgcgacgtggtcatcggcatcgtgaacaacaccgtgtacgaccccctgcagccagagctggactccttcaaggaggagctggacaagtacttcaagaaccacaccagcccagacgtggacctgggagatatcagcggcatcaacgccagcgtggtgaacatccagaaggagatcgacaggctgaacgaggtggccaagaacctgaacgagagcctgatcgacctgcaggagctgggcaagtacgagcagtacatcaagtggccttggtacatttggctgggcttcatcgccggcctgatcgcaatcgtcatggtgaccatcatgctctgttgcatgaccagttgctgcagctgcctgaagggttgctgcagttgcgggagttgctgcaagttcgacgaggacgacagcgagccagtgctgaaaggcgtgaagctgcactacacatga。本发明所述的核酸，还可以以RNA形式存在，即将DNA序列中，所有的胸腺嘧啶(T)替换为尿嘧啶(U)。一些实施例中，所述编码SARS-CoV-2病毒S蛋白的核酸与SEQ ID NO:3至少具有85％的同源性。另一些实施例中，所述编码SARS-CoV-2病毒的S蛋白的核酸与SEQID NO:3至少具有90％的同源性。另一些实施例中，所述编码SARS-CoV-2病毒的S蛋白的核酸与SEQ ID NO:3至少具有95％的同源性。另一些实施例中，所述编码SARS-CoV-2病毒的S蛋白的核酸与SEQ ID NO:3至少具有98％的同源性。另一些实施例中，所述编码SARS-CoV-2病毒的S蛋白的核酸与SEQ ID NO:3至少具有99％的同源性。
本发明中，所述重组载体，是指利用基因工程重组DNA技术，将DNA片段连接入骨架载体形成的基因表达载体。本发明提供的重组载体包括骨架载体和所述的核酸。其中，所述骨架载体基于α病毒家族中委内瑞拉马脑脊髓炎病毒(TC83 Venezuelan EquineEncephalitis Virus，VEEV)进行设计。其包含了可编码α病毒自复制组件的基因，但是缺乏编码用于制造具有传染性的α病毒颗粒的结构蛋白，并且构建到的质粒中做为模板来扩增，将设计好的序列通过合成直接获得，基因序列为序列表中SEQ ID NO.2所示。
本发明中，所述线性化的mRNA是由重组载体经线性化后，在体外经转录、模板DNA降解、5’端加7-甲基化鸟苷酸帽获得的，其核酸序列如SEQ ID NO:4所示。本发明中，所述线性化是指在限制性内切酶的作用下，将环状的JCXH-102质粒酶切形成双链线性结构的过程。本发明实施例中，线性化使用的限制性内切酶为BspQ I。所述的转录是指T7 RNA聚合酶存在下的体外转录反应(IVT)，该步骤在无细胞系统中，以线性化的质粒DNA为模板，模仿体内转录反应发生的过程，将载体上DNA形式的编码S蛋白的核酸片段转录生成相应的RNA片段。将产物中的DNA模板以Turbo DNase酶降解去除后，在转录产生的RNA片段的5’端添加7-甲基化鸟苷酸帽结构。本发明具体实施例中，本发明制得的线性化的mRNA核酸序列如SEQID NO:4所示。
本发明所述的核酸、重组载体、或线性化mRNA都是疫苗制备过程中的原料或中间产物。在疫苗的生产、制备过程中，所述核酸、重组载体或线性化的mRNA可以在每次制备开始前，重新合成，也可以合成后于适宜的条件下存储备用。所述的适宜条件包括-80℃～0℃，优选在-80℃～-40℃保存。
本发明中，所述的mRNA-LNP复合物是由脂类包裹mRNA形成。制备获得的mRNA-LNP复合物通过浓缩及换液制得所述疫苗所得疫苗的溶液为PBS缓冲液，其中mRNA-LNP复合物的浓度为30μg/mL。本发明所述疫苗能够用于防治因SARS-CoV-2病毒感染引起的新型冠状肺炎。该疫苗的应用方法采用疫苗的初免-加强给药应用方式；疫苗的初免-加强给药使用相同的mRNA-Spike疫苗。疫苗的给药方式为肌肉注射。
本发明所述的Spike高表达新冠mRNA疫苗的制备方法，制备mRNA-Spike的步骤包括：
步骤一，合成Spike基因片段；
查询Spike氨基酸序列，然后根据人偏好密码子表进行密码子优化得到优化后的序列，再在其上游加入ApaI酶切位点(GGGCCC)和启动子(subgenomic promoter)，下游加入NotI酶切位点(GCGGCCGC)，将设计好的序列通过合成直接获得，基因序列为序列表中SEQID NO.1所示；
步骤二，构建TC-83自复制载体；
自复制mRNA是根据α病毒家族中委内瑞拉马脑脊髓炎病毒(TC83VenezuelanEquine Encephalitis Virus，VEEV)的基因组进行设计的，其包含了可编码α病毒自复制组件的基因，但是缺乏编码用于制造具有传染性的α病毒颗粒的结构蛋白，并且构建到的质粒中做为模板来扩增，将设计好的序列通过合成直接获得，基因序列为序列表中SEQ ID NO.2所示
步骤三，重组质粒JCXH-102的制备方法如下：
将上游携带ApaI、下游携带NotI的Spike基因插入TC-83复制载体的ApaI和NotⅠ酶切位点之间，得到重组质粒JCXH-102(见图1)，基因序列为序列表中SEQ ID NO.3所示；
步骤四，JCXH-102质粒用BspQI酶切线性化；
步骤五，线性化后JCXH-102质粒第一步使用T7 RNA聚合酶开始的体外转录反应(IVT)，第二步使用Turbo DNase酶对模板DNA进行降解和使用加帽酶(Vaccinia cappingenzyme)将7-甲基化鸟苷酸帽结构(7-methylguanylate cap structure，称为Cap 0)加至转录的mRNA的5’端，基因序列为序列表中SEQ ID NO.4所示。
步骤六，mRNA与脂类(其组成以物质的量计包括2％的1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000(DMG-PEG2000)、48％的胆固醇、10％的二硬脂酰基磷脂酰胆碱(DSPC)以及40％的阳离子脂类(DLinDMA))通过Nanoassemblr混合器进行快速的混合，从而引起脂类的沉淀及在电荷作用mRNA包裹进入LNP内。mRNA-LNP复合物随后通过浓缩及换液至制剂溶液中。
本发明采用的试材皆为普通市售品，皆可于市场购得。需要说明的是：mRNA-Spike体外转录mRNA产物，且本申请说明书已经详细描述了体外转录及脂质体包裹的全部过程，任何公司或个人，按照说明书上的步骤均能制备的体外转录脂质体包裹的mRNA疫苗。下面结合实施例，进一步阐述本发明：
实施例1.疫苗mRNA-Spike的制备；
1.Spike基因片段合成
从NCBI上查询Spike(SARS-CoV-2，Wuhan seafoodmarket pneumonia virusisolate Wuhan-Hu-1株)蛋白基因，然后根据人密码子进行相应优化(SEQ ID NO:1所示)，并在上游加入Apa I酶切位点(GGGCCC)和启动子(subgenomic promoter，序列为ctataactctctacggctaacctgaatggactacgacatagtctagtccgccaag)，下游加入Not I酶切位点(GCGGCCGC)和保护碱基(taaggcgcgcccaccca)，最后通过合成直接获得DNA序列(以公司提供克隆质粒pUC57-Spike的形式获得)。
2.构建TC-83自复制载体
自复制mRNA是根据α病毒家族中委内瑞拉马脑脊髓炎病毒(TC83 VenezuelanEquine Encephalitis Virus，VEEV)的基因组进行设计的，其包含了可编码α病毒自复制组件的基因，但是缺乏编码用于制造具有传染性的α病毒颗粒的结构蛋白，并且构建到的质粒中做为模板来扩增，将设计好的序列通过合成直接获得，序列如SEQ ID NO:2所示。
3.重组质粒JCXH-102的制备方法如下，
pUC57-Spike和TC-83自复制载体用ApaⅠ和NotⅠ双酶切，酶切体系20μL：pUC57-Spike或TC-83自复制载体＜1μg，ApaⅠ1μL，NotⅠ1μL，10×CutSmartBuffer 2μL，ddH2O将体系补足20μL。25℃水浴1h酶切质粒，然后37℃水浴1h酶切质粒，载体片段需加0.5μLCIP，37℃水浴30min去磷酸化。将酶切混合物与6×Loading Buffer混合，电泳(1％琼脂糖，94V)并胶回收相应长度的片段(pUC57-Spike回收片段Spike基因片段，长度为3897bp；TC-83自复制载体回收片段，长度约9.5Kb)，以30μL Elution Buffer洗脱。
分别取Spike基因片段与TC-83自复制载体片段按照连接体系：载体50ng，插入片段摩尔数：载体片段摩尔数＝5:1，T4 Ligase 1μL，10×Ligase Buffer5μL，ddH2O将体系补足10μl，22℃连接1h。将连接产物按体积1:10与大肠杆菌DH5α的感受态细胞轻柔混匀，冰浴30min，42℃热击45s，冰浴3min，加入预热的SOB肉汤500μL，混匀后于37℃，180rpm培养1h，涂布于LK平板(LB-Kan平板：含50μg/mL Kan的LB平板)，37℃培养16-20h。
PCR筛选：以煮沸法提取的细菌质粒为模板，扩增目的条带Spike，上游引物F：5’-TATGGCCATGACTACTCTAGCTA-3’，下游引物R：5’-GGGAAACGCCTGGTATCTTT-3’，反应循环条件为：94℃3min→(94℃1min，47℃30s，72℃3min)×30个循环→72℃10min→4℃；电泳观察PCR结果(电泳条件：1％琼脂糖凝胶；90V，上样量：5μlPCR产物)，预期结果：Spike片段约4300bp。
酶切验证：PCR筛选验证的阳性菌提取质粒，按照上述酶切体系以ApaⅠ和NotⅠ进行酶切，将酶切混合物与6×Loading Buffer混合进行电泳(1％琼脂糖，94V)。
测序验证：提取PCR及酶切验证符合预期的质粒送测序公司测序。将测序验证后完全正确的携带阳性质粒的大肠杆菌保存于-80℃。
4.JCXH-102质粒用BspQI酶切线性化及回收方法如下，
JCXH-102质粒10μg，BspQI 1μL，10×NEBuffer 3.15μL，ddH2O将体系补足50μL，50℃水浴1h酶切质粒。将酶切混合物与6×Loading Buffer混合，电泳(1％琼脂糖，94V)并胶回收相应长度的片段(JCXH-102质粒用，长度约13Kb)，以30μL Elution Buffer洗脱。
5.线性化后JCXH-102质粒使用T7 RNA聚合酶开始的体外转录反应(IVT)，TurboDNase酶对模板DNA进行降解，和加帽酶(Vaccinia capping enzyme)将7-甲基化鸟苷酸帽结构(7-methylguanylate cap structure，称为Cap 0)加至转录的mRNA的5’端，具体方法如下。
10×Reaction Buffer 2μL，NTP每种0.5mM，线性化JCXH-102 1μg，T7 RNAPolymerase 2μL，补水至20μL，30℃反应1小时后加入，1μL TURBOTMDNase，10×CappingBuffer4μL，GTP(10mM)2μL，SAM(2mM)2μL，Vaccinia Capping Enzyme 2μL，总体积补水至总体积40μL，30℃反应1小时。后补水至200μL再加入7.5M氯化锂120μL混匀后，-20℃静置30分钟后，14000g 4℃离心30分钟，弃上清后用70％酒精清洗沉淀，14000g 4℃离心5分钟，弃上清后风干5分钟，溶解在40μL水内。用分光光度计定量后，取400ng和10μLNorthernMax-GlySample Loading Dye混合，50℃孵育30分钟后，用NorthernMax-Gly Gel Prep/Running缓冲液电泳(1％琼脂糖，70V)。电泳图片见图2。
6.mRNA-Spike与脂类包裹步骤如下。
摩尔比8:1N:P(N为DLinDMA，P为RNA)，mRNA与脂类(摩尔组成包括2％的1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000(DMG-PEG2000)、48％的胆固醇、10％的二硬脂酰基磷脂酰胆碱(DSPC)以及40％的阳离子脂类(DLinDMA)，溶解在酒精内。Geall,Andrew J.,et al.“Nonviral Delivery of Self-Amplifying RNAVaccines.”Proceedings of the National Academy of Sciences of the UnitedStates of America,vol.109,no.36,2012,pp.14604–14609.)通过Nanoassemblr混合器进行快速的混合，从而引起脂类的沉淀及在电荷作用mRNA包裹进入LNP内。mRNA-LNP复合物随后通过浓缩及换液至制剂溶液中。
7.Western blot检测mRNA-Spike在细胞中的表达情况。将购于上海细胞中心的BHK-21细胞传代培养，待细胞数量足够，胰酶消化至细胞培养6孔板每孔，铺板37℃CO2培养箱过夜。第二天将脂质体包裹的mRNA-Spike转染到铺好BHK-21细胞中，培养72h后，将细胞裂解收集蛋白样品。具体方法：
1)细胞培养及铺板：将复苏好的BHK-21细胞接种在75cm2的培养瓶中，培养基为DMEM高糖培养基+5％的双抗+10％胎牛血清，待细胞在瓶底的汇集度达到80％以上，用胰酶将细胞消化下来，并计数。在6孔细胞培养板中铺入适宜数量的细胞，37℃CO2培养箱过夜。
2)脂质体mRNA转染BHK-21细胞：吸干铺好的6孔板中的培养基，用PBS缓冲液清洗一遍，将脂质体包裹的mRNA与1mLOpti-MEM培养基混合，加至清洗好的6孔板中，37℃CO2培养箱6h后补充1mL含有20％胎牛血清的DMEM高糖培养基。37℃CO2培养箱，培养72h。
3)蛋白样品的处理：在72小时后的BHK-21细胞中加入200μL细胞裂解液及1％的PMSF，冰上放置5分钟，离心机14000g离心5分钟，将上清转移至新的离心管，加入5×SDS 95℃金属浴12分钟。放在-20℃备用。
4)Western blot检测：
电泳：聚丙稀酰胺分离胶浓度为6％，蛋白上样量20μL，积层胶电泳条件150V，10min，分离胶电泳条件为200V，30min。
电转：使用硝酸纤维素膜膜，在100V电压下转膜1小时。
封闭：使用1×PBST配制5％BSA作为封闭液，4℃封闭过夜。
一抗孵育：使用封闭液将SARA-Cov-2(2019-nCov)spike抗体以1：500进行稀释，在室温下孵育2小时。用1×PBST洗3次，每次5分钟。
二抗孵育：使用封闭液稀释二抗，稀释比为1：10000，在室温下孵育1小时。用1×PBST洗3次，每次5分钟。
显影：显影液A液：B液＝1：1，在成像系统中进行显影。结果见图片3。
8.mRNA-Spike免疫小鼠诱导的抗Spike抗体滴度检测
6～8周龄的BALB/c雌鼠适应性喂养3天后，3只为对照组注射PBS，5只为0.5μg组，分别肌肉免疫未经过密码子优化后mRNA-Spike的疫苗，注射0.5μg剂量。35天后，再加强注射0.5μg剂量。70天后，取血检测针对spike抗体滴度。
或者6～8周龄的BALB/c雌鼠适应性喂养3天后，2只为对照组注射PBS，4只为0.2μg组，2只为0.5μg组，5只为1μg组，分别肌肉免疫经过密码子优化后mRNA-Spike的疫苗，注射0.2μg，0.5μg和1μg剂量。35天后，再加强注射0.2μg，0.5μg和1μg剂量。70天后，取血检测针对spike抗体滴度。
抗体滴度检测方法为ELISA具体操作如下，96孔酶标板每孔包被50ng Spike RBD结构域蛋白室温过夜，第二天用PBST清洗3遍，用5％牛奶封闭，37度1小时后，PBST清洗3遍，加入对应稀释比例的小鼠血清，37℃1小时后，PBST清洗3遍，加入抗小鼠IGg重链轻链HRP，37℃1小时后，PBST清洗3遍，最后加入显色液显色。如图4A所示；为未经过密码子的mRNA-Spike疫苗免疫小鼠后，血清中抗spike抗体滴度，如图4B所示；为经过密码子优化后mRNA-Spike疫苗免疫小鼠后，血清中抗spike抗体滴度。对比可知经密码子优化后的mRNA-Spike疫苗，免疫小鼠后产生的抗体滴度更高，效果更好。
以上仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 成都欧林生物科技股份有限公司
嘉晨西海（杭州）生物技术有限公司
<120> 编码SARS-CoV-2病毒刺突蛋白的核酸及其的应用
<130> MP2024794
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3822
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atgttcgtgt tcctcgtgct gctgcccctg gtgtctagcc agtgcgtgaa cctgaccacc 60
agaacccagc tgcctccagc ctacaccaac agcttcacca ggggagtgta ctaccccgac 120
aaggtgttcc ggagcagcgt gctgcatagc acccaggacc tgttcctgcc cttcttcagc 180
aacgtgactt ggttccacgc catccacgtc agcggaacca acggcaccaa gcgctttgac 240
aacccagtgc tgcccttcaa cgacggagtg tacttcgcca gcaccgagaa gtccaacatc 300
atccggggtt ggatcttcgg caccacactg gacagcaaga cccagagcct gctgatcgtg 360
aacaacgcca ccaacgtcgt gatcaaggtc tgcgagttcc agttttgcaa cgaccccttc 420
ctgggcgtgt actaccacaa gaacaacaag tcttggatgg agagcgagtt ccgggtgtac 480
agcagcgcca acaattgcac cttcgagtac gtgtcccagc ccttcctgat ggacctggaa 540
ggcaagcagg gcaacttcaa gaacctgcgg gagttcgtgt tcaagaacat cgacggctac 600
ttcaagatct acagcaagca cacccccatc aacctcgtga gagacctgcc acagggcttc 660
agcgctctgg aacctctggt ggatctgcca atcggcatca acatcacccg gttccagacc 720
ctgctggctc tgcacagaag ctacctgacc ccaggcgatt cttctagcgg ctggacagca 780
ggagccgccg cttattacgt gggctatctg cagccccgga ccttcctgct gaagtacaac 840
gagaacggca ccatcaccga cgcagtggat tgcgctctgg atcctctgag cgagaccaag 900
tgcaccctga agtccttcac cgtggagaag ggcatctacc agaccagcaa cttccgggtg 960
cagcctacag agagcatcgt gcggttccct aacatcacca acctctgccc cttcggcgag 1020
gtgttcaacg ccaccagatt cgccagcgtc tacgcctgga accggaagcg gatcagcaat 1080
tgcgtggccg actacagcgt gctgtacaac agcgccagct tcagcacctt caagtgctac 1140
ggcgtgtccc ctaccaagct gaacgatctg tgcttcacca acgtgtacgc cgacagcttc 1200
gtgatcaggg gcgacgaagt gcgacagatc gccccaggac agacaggaaa gatcgccgat 1260
tacaactaca agctgcccga cgacttcacc ggttgcgtga tcgcttggaa cagcaacaac 1320
ctggacagca aggtcggcgg caactacaac tacctgtacc ggctgttccg gaagtccaac 1380
ctgaagccct tcgagcggga catcagcaca gagatctacc aggccggcag cacaccttgt 1440
aacggcgtgg agggcttcaa ctgctacttc cctctgcaga gctacggctt ccagcctaca 1500
aacggcgtgg gataccagcc ttacagggtg gtggtgctga gcttcgaact gctgcacgct 1560
ccagccaccg tctgcggacc taaaaagagc accaacctgg tgaagaacaa gtgcgtgaat 1620
ttcaacttca acggcctgac cggaaccggc gtgctgaccg agagcaacaa gaagttcctg 1680
cccttccagc agttcggcag agacatcgca gacaccacag acgccgtgag agatcctcag 1740
accctggaga tcctggacat caccccttgc agctttggcg gagtgtccgt gatcacccca 1800
ggaaccaaca ccagcaatca ggtggccgtg ctgtaccagg acgtgaattg caccgaggtg 1860
ccagtggcca ttcacgcaga tcagctgacc cctacctgga gagtgtacag caccggaagc 1920
aacgtgttcc agaccagggc aggttgcctg atcggagccg agcacgtgaa caacagctac 1980
gagtgcgata tccctatcgg cgccggcatt tgcgcctctt accagaccca gaccaacagc 2040
cctaggagag ccagaagcgt ggcctctcag agcatcatcg cctacaccat gagcctggga 2100
gccgagaaca gcgtggccta cagcaacaac agcatcgcca tccccaccaa cttcaccatc 2160
agcgtgacca ccgagattct gcccgtgtct atgaccaaga ccagcgtcga ctgcaccatg 2220
tacatctgcg gcgacagcac cgagtgctct aacctgctgc tgcagtacgg ctccttttgc 2280
acccagctga acagggctct gacaggcatc gcagtggagc aggacaagaa cacccaggag 2340
gtcttcgccc aggtcaagca gatctacaag acccccccta tcaaggactt cggcggcttt 2400
aacttcagcc agatcctgcc cgatcccagc aagcctagca agcggagctt catcgaggac 2460
ctgctgttca acaaggtcac cctggccgac gcaggcttca tcaagcagta cggcgattgc 2520
ctgggcgaca ttgccgccag agacctgatt tgcgcccaga agttcaacgg cctgacagtg 2580
ctgcctcctc tgctgaccga cgagatgatc gcccagtata caagcgctct gctggcagga 2640
accatcacca gcggttggac ctttggagcc ggagccgctc tgcagatccc ttttgccatg 2700
cagatggcct accgcttcaa cggaatcggc gtgacccaga acgtgctcta cgagaaccag 2760
aagctgatcg ccaaccagtt caacagcgcc atcggcaaga tccaggacag cctgagcagc 2820
acagctagcg ctctgggcaa gctgcaggat gtggtgaacc agaacgccca ggctctgaac 2880
accctggtga agcagctgag cagcaacttc ggagccatca gcagcgtgct gaacgacatc 2940
ctgagcaggc tggacaaggt ggaagccgaa gtgcagatcg acaggctgat caccggcaga 3000
ctgcagtctc tgcagaccta cgtgacccag cagctgatca gagccgccga gatcagagct 3060
agcgctaatc tggccgccac caagatgagc gagtgcgtgc tgggacagag caagagagtg 3120
gacttctgcg gcaagggcta ccacctgatg agctttcctc agagcgctcc tcacggagtg 3180
gtgtttctgc acgtgaccta cgtgccagcc caggagaaga acttcaccac agccccagcc 3240
atttgccacg acggaaaggc ccacttcccc agagaaggcg tgttcgtgtc caacggcacc 3300
cattggttcg tgacccagcg gaacttctac gagccccaga tcatcaccac cgacaacacc 3360
ttcgtgagcg gcaattgcga cgtggtcatc ggcatcgtga acaacaccgt gtacgacccc 3420
ctgcagccag agctggactc cttcaaggag gagctggaca agtacttcaa gaaccacacc 3480
agcccagacg tggacctggg agatatcagc ggcatcaacg ccagcgtggt gaacatccag 3540
aaggagatcg acaggctgaa cgaggtggcc aagaacctga acgagagcct gatcgacctg 3600
caggagctgg gcaagtacga gcagtacatc aagtggcctt ggtacatttg gctgggcttc 3660
atcgccggcc tgatcgcaat cgtcatggtg accatcatgc tctgttgcat gaccagttgc 3720
tgcagctgcc tgaagggttg ctgcagttgc gggagttgct gcaagttcga cgaggacgac 3780
agcgagccag tgctgaaagg cgtgaagctg cactacacat ga 3822
<210> 2
<211> 9636
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atgggcggcg catgagagaa gcccagacca attacctacc caaaatggag aaagttcacg 60
ttgacatcga ggaagacagc ccattcctca gagctttgca gcggagcttc ccgcagtttg 120
aggtagaagc caagcaggtc actgataatg accatgctaa tgccagagcg ttttcgcatc 180
tggcttcaaa actgatcgaa acggaggtgg acccatccga cacgatcctt gacattggaa 240
gtgcgcccgc ccgcagaatg tattctaagc acaagtatca ttgtatctgt ccgatgagat 300
gtgcggaaga tccggacaga ttgtataagt atgcaactaa gctgaagaaa aactgtaagg 360
aaataactga taaggaattg gacaagaaaa tgaaggagct cgccgccgtc atgagcgacc 420
ctgacctgga aactgagact atgtgcctcc acgacgacga gtcgtgtcgc tacgaagggc 480
aagtcgctgt ttaccaggat gtatacgcgg ttgacggacc gacaagtctc tatcaccaag 540
ccaataaggg agttagagtc gcctactgga taggctttga caccacccct tttatgttta 600
agaacttggc tggagcatat ccatcatact ctaccaactg ggccgacgaa accgtgttaa 660
cggctcgtaa cataggccta tgcagctctg acgttatgga gcggtcacgt agagggatgt 720
ccattcttag aaagaagtat ttgaaaccat ccaacaatgt tctattctct gttggctcga 780
ccatctacca cgagaagagg gacttactga ggagctggca cctgccgtct gtatttcact 840
tacgtggcaa gcaaaattac acatgtcggt gtgagactat agttagttgc gacgggtacg 900
tcgttaaaag aatagctatc agtccaggcc tgtatgggaa gccttcaggc tatgctgcta 960
cgatgcaccg cgagggattc ttgtgctgca aagtgacaga cacattgaac ggggagaggg 1020
tctcttttcc cgtgtgcacg tatgtgccag ctacattgtg tgaccaaatg actggcatac 1080
tggcaacaga tgtcagtgcg gacgacgcgc aaaaactgct ggttgggctc aaccagcgta 1140
tagtcgtcaa cggtcgcacc cagagaaaca ccaataccat gaaaaattac cttttgcccg 1200
tagtggccca ggcatttgct aggtgggcaa aggaatataa ggaagatcaa gaagatgaaa 1260
ggccactagg actacgagat agacagttag tcatggggtg ttgttgggct tttagaaggc 1320
acaagataac atctatttat aagcgcccgg atacccaaac catcatcaaa gtgaacagcg 1380
atttccactc attcgtgctg cccaggatag gcagtaacac attggagatc gggctgagaa 1440
caagaatcag gaaaatgtta gaggagcaca aggagccgtc acctctcatt accgccgagg 1500
acgtacaaga agctaagtgc gcagccgatg aggctaagga ggtgcgtgaa gccgaggagt 1560
tgcgcgcagc tctaccacct ttggcagctg atgttgagga gcccactctg gaagccgatg 1620
tcgacttgat gttacaagag gctggggccg gctcagtgga gacacctcgt ggcttgataa 1680
aggttaccag ctacgatggc gaggacaaga tcggctctta cgctgtgctt tctccgcagg 1740
ctgtactcaa gagtgaaaaa ttatcttgca tccaccctct cgctgaacaa gtcatagtga 1800
taacacactc tggccgaaaa gggcgttatg ccgtggaacc ataccatggt aaagtagtgg 1860
tgccagaggg acatgcaata cccgtccagg actttcaagc tctgagtgaa agtgccacca 1920
ttgtgtacaa cgaacgtgag ttcgtaaaca ggtacctgca ccatattgcc acacatggag 1980
gagcgctgaa cactgatgaa gaatattaca aaactgtcaa gcccagcgag cacgacggcg 2040
aatacctgta cgacatcgac aggaaacagt gcgtcaagaa agaactagtc actgggctag 2100
ggctcacagg cgagctggtg gatcctccct tccatgaatt cgcctacgag agtctgagaa 2160
cacgaccagc cgctccttac caagtaccaa ccataggggt gtatggcgtg ccaggatcag 2220
gcaagtctgg catcattaaa agcgcagtca ccaaaaaaga tctagtggtg agcgccaaga 2280
aagaaaactg tgcagaaatt ataagggacg tcaagaaaat gaaagggctg gacgtcaatg 2340
ccagaactgt ggactcagtg ctcttgaatg gatgcaaaca ccccgtagag accctgtata 2400
ttgacgaagc ttttgcttgt catgcaggta ctctcagagc gctcatagcc attataagac 2460
ctaaaaaggc agtgctctgc ggggatccca aacagtgcgg tttttttaac atgatgtgcc 2520
tgaaagtgca ttttaaccac gagatttgca cacaagtctt ccacaaaagc atctctcgcc 2580
gttgcactaa atctgtgact tcggtcgtct caaccttgtt ttacgacaaa aaaatgagaa 2640
cgacgaatcc gaaagagact aagattgtga ttgacactac cggcagtacc aaacctaagc 2700
aggacgatct cattctcact tgtttcagag ggtgggtgaa gcagttgcaa atagattaca 2760
aaggcaacga aataatgacg gcagctgcct ctcaagggct gacccgtaaa ggtgtgtatg 2820
ccgttcggta caaggtgaat gaaaatcctc tgtacgcacc cacctcagaa catgtgaacg 2880
tcctactgac ccgcacggag gaccgcatcg tgtggaaaac actagccggc gacccatgga 2940
taaaaacact gactgccaag taccctggga atttcactgc cacgatagag gagtggcaag 3000
cagagcatga tgccatcatg aggcacatct tggagagacc ggaccctacc gacgtcttcc 3060
agaataaggc aaacgtgtgt tgggccaagg ctttagtgcc ggtgctgaag accgctggca 3120
tagacatgac cactgaacaa tggaacactg tggattattt tgaaacggac aaagctcact 3180
cagcagagat agtattgaac caactatgcg tgaggttctt tggactcgat ctggactccg 3240
gtctattttc tgcacccact gttccgttat ccattaggaa taatcactgg gataactccc 3300
cgtcgcctaa catgtacggg ctgaataaag aagtggtccg tcagctctct cgcaggtacc 3360
cacaactgcc tcgggcagtt gccactggaa gagtctatga catgaacact ggtacactgc 3420
gcaattatga tccgcgcata aacctagtac ctgtaaacag aagactgcct catgctttag 3480
tcctccacca taatgaacac ccacagagtg acttttcttc attcgtcagc aaattgaagg 3540
gcagaactgt cctggtggtc ggggaaaagt tgtccgtccc aggcaaaatg gttgactggt 3600
tgtcagaccg gcctgaggct accttcagag ctcggctgga tttaggcatc ccaggtgatg 3660
tgcccaaata tgacataata tttgttaatg tgaggacccc atataaatac catcactatc 3720
agcagtgtga agaccatgcc attaagctta gcatgttgac caagaaagct tgtctgcatc 3780
tgaatcccgg cggaacctgt gtcagcatag gttatggtta cgctgacagg gccagcgaaa 3840
gcatcattgg tgctatagcg cggctgttca agttttcccg ggtatgcaaa ccgaaatcct 3900
cacttgaaga gacggaagtt ctgtttgtat tcattgggta cgatcgcaag gcccgtacgc 3960
acaatcctta caagctttca tcaaccttga ccaacattta tacaggttcc agactccacg 4020
aagccggatg tgcaccctca tatcatgtgg tgcgagggga tattgccacg gccaccgaag 4080
gagtgattat aaatgctgct aacagcaaag gacaacctgg cggaggggtg tgcggagcgc 4140
tgtataagaa attcccggaa agcttcgatt tacagccgat cgaagtagga aaagcgcgac 4200
tggtcaaagg tgcagctaaa catatcattc atgccgtagg accaaacttc aacaaagttt 4260
cggaggttga aggtgacaaa cagttggcag aggcttatga gtccatcgct aagattgtca 4320
acgataacaa ttacaagtca gtagcgattc cactgttgtc caccggcatc ttttccggga 4380
acaaagatcg actaacccaa tcattgaacc atttgctgac agctttagac accactgatg 4440
cagatgtagc catatactgc agggacaaga aatgggaaat gactctcaag gaagcagtgg 4500
ctaggagaga agcagtggag gagatatgca tatccgacga ctcttcagtg acagaacctg 4560
atgcagagct ggtgagggtg catccgaaga gttctttggc tggaaggaag ggctacagca 4620
caagcgatgg caaaactttc tcatatttgg aagggaccaa gtttcaccag gcggccaagg 4680
atatagcaga aattaatgcc atgtggcccg ttgcaacgga ggccaatgag caggtatgca 4740
tgtatatcct cggagaaagc atgagcagta ttaggtcgaa atgccccgtc gaagagtcgg 4800
aagcctccac accacctagc acgctgcctt gcttgtgcat ccatgccatg actccagaaa 4860
gagtacagcg cctaaaagcc tcacgtccag aacaaattac tgtgtgctca tcctttccat 4920
tgccgaagta tagaatcact ggtgtgcaga agatccaatg ctcccagcct atattgttct 4980
caccgaaagt gcctgcgtat attcatccaa ggaagtatct cgtggaaaca ccaccggtag 5040
acgagactcc ggagccatcg gcagagaacc aatccacaga ggggacacct gaacaaccac 5100
cacttataac cgaggatgag accaggacta gaacgcctga gccgatcatc atcgaagagg 5160
aagaagagga tagcataagt ttgctgtcag atggcccgac ccaccaggtg ctgcaagtcg 5220
aggcagacat tcacgggccg ccctctgtat ctagctcatc ctggtccatt cctcatgcat 5280
ccgactttga tgtggacagt ttatccatac ttgacaccct ggagggagct agcgtgacca 5340
gcggggcaac gtcagccgag actaactctt acttcgcaaa gagtatggag tttctggcgc 5400
gaccggtgcc tgcgcctcga acagtattca ggaaccctcc acatcccgct ccgcgcacaa 5460
gaacaccgtc acttgcaccc agcagggcct gctcgagaac cagcctagtt tccaccccgc 5520
caggcgtgaa tagggtgatc actagagagg agctcgaggc gcttaccccg tcacgcactc 5580
ctagcaggtc ggtctcgaga accagcctgg tctccaaccc gccaggcgta aatagggtga 5640
ttacaagaga ggagtttgag gcgttcgtag cacaacaaca atgacggttt gatgcgggtg 5700
catacatctt ttcctccgac accggtcaag ggcatttaca acaaaaatca gtaaggcaaa 5760
cggtgctatc cgaagtggtg ttggagagga ccgaattgga gatttcgtat gccccgcgcc 5820
tcgaccaaga aaaagaagaa ttactacgca agaaattaca gttaaatccc acacctgcta 5880
acagaagcag ataccagtcc aggaaggtgg agaacatgaa agccataaca gctagacgta 5940
ttctgcaagg cctagggcat tatttgaagg cagaaggaaa agtggagtgc taccgaaccc 6000
tgcatcctgt tcctttgtat tcatctagtg tgaaccgtgc cttttcaagc cccaaggtcg 6060
cagtggaagc ctgtaacgcc atgttgaaag agaactttcc gactgtggct tcttactgta 6120
ttattccaga gtacgatgcc tatttggaca tggttgacgg agcttcatgc tgcttagaca 6180
ctgccagttt ttgccctgca aagctgcgca gctttccaaa gaaacactcc tatttggaac 6240
ccacaatacg atcggcagtg ccttcagcga tccagaacac gctccagaac gtcctggcag 6300
ctgccacaaa aagaaattgc aatgtcacgc aaatgagaga attgcccgta ttggattcgg 6360
cggcctttaa tgtggaatgc ttcaagaaat atgcgtgtaa taatgaatat tgggaaacgt 6420
ttaaagaaaa ccccatcagg cttactgaag aaaacgtggt aaattacatt accaaattaa 6480
aaggaccaaa agctgctgct ctttttgcga agacacataa tttgaatatg ttgcaggaca 6540
taccaatgga caggtttgta atggacttaa agagagacgt gaaagtgact ccaggaacaa 6600
aacatactga agaacggccc aaggtacagg tgatccaggc tgccgatccg ctagcaacag 6660
cgtatctgtg cggaatccac cgagagctgg ttaggagatt aaatgcggtc ctgcttccga 6720
acattcatac actgtttgat atgtcggctg aagactttga cgctattata gccgagcact 6780
tccagcctgg ggattgtgtt ctggaaactg acatcgcgtc gtttgataaa agtgaggacg 6840
acgccatggc tctgaccgcg ttaatgattc tggaagactt aggtgtggac gcagagctgt 6900
tgacgctgat tgaggcggct ttcggcgaaa tttcatcaat acatttgccc actaaaacta 6960
aatttaaatt cggagccatg atgaaatctg gaatgttcct cacactgttt gtgaacacag 7020
tcattaacat tgtaatcgca agcagagtgt tgagagaacg gctaaccgga tcaccatgtg 7080
cagcattcat tggagatgac aatatcgtga aaggagtcaa atcggacaaa ttaatggcag 7140
acaggtgcgc cacctggttg aatatggaag tcaagattat agatgctgtg gtgggcgaga 7200
aagcgcctta tttctgtgga gggtttattt tgtgtgactc cgtgaccggc acagcgtgcc 7260
gtgtggcaga ccccctaaaa aggctgttta agcttggcaa acctctggca gcagacgatg 7320
aacatgatga tgacaggaga agggcattgc atgaagagtc aacacgctgg aaccgagtgg 7380
gtattctttc agagctgtgc aaggcagtag aatcaaggta tgaaaccgta ggaacttcca 7440
tcatagttat ggccatgact actctagcta gcagtgttaa atcattcagc tacctgagag 7500
gggcccctat aactctctac ggctaacctg aatggactac gacatagtct agtccgccaa 7560
gtaaggcgcg cccacccagc ggccgcatac agcagcaatt ggcaagctgc ttacatagaa 7620
ctcgcggcga ttggcatgcc gccttaaaat ttttatttta tttttctttt cttttccgaa 7680
tcggattttg tttttaatat ttcaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 7740
aaaagaagag ctagggataa cagggtaatt gagcaaaagg ccagcaaaag gccaggaacc 7800
gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca 7860
aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt 7920
ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc 7980
tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc 8040
tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc 8100
ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact 8160
tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg 8220
ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta 8280
tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca 8340
aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa 8400
aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg 8460
aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc 8520
ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg 8580
acagttagaa aaactcatcg agcatcaaat gaaactgcaa tttattcata tcaggattat 8640
caataccata tttttgaaaa agccgtttct gtaatgaagg agaaaactca ccgaggcagt 8700
tccataggat ggcaagatcc tggtatcggt ctgcgattcc gactcgtcca acatcaatac 8760
aacctattaa tttcccctcg tcaaaaataa ggttatcaag tgagaaatca ccatgagtga 8820
cgactgaatc cggtgagaat ggcaaaagtt tatgcatttc tttccagact tgttcaacag 8880
gccagccatt acgctcgtca tcaaaatcac tcgcatcaac caaaccgtta ttcattcgtg 8940
attgcgcctg agcgagacga aatacgcgat cgctgttaaa aggacaatta caaacaggaa 9000
tcgaatgcaa ccggcgcagg aacactgcca gcgcatcaac aatattttca cctgaatcag 9060
gatattcttc taatacctgg aatgctgttt tcccagggat cgcagtggtg agtaaccatg 9120
catcatcagg agtacggata aaatgcttga tggtcggaag aggcataaat tccgtcagcc 9180
agtttagtct gaccatctca tctgtaacat cattggcaac gctacctttg ccatgtttca 9240
gaaacaactc tggcgcatcg ggcttcccat acaatcgata gattgtcgca cctgattgcc 9300
cgacattatc gcgagcccat ttatacccat ataaatcagc atccatgttg gaatttaatc 9360
gcggcctaga gcaagacgtt tcccgttgaa tatggctcat actcttcctt tttcaatatt 9420
attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga 9480
aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag 9540
aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc 9600
tagggataac agggtaatta atacgactca ctatag 9636
<210> 3
<211> 13458
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atgggcggcg catgagagaa gcccagacca attacctacc caaaatggag aaagttcacg 60
ttgacatcga ggaagacagc ccattcctca gagctttgca gcggagcttc ccgcagtttg 120
aggtagaagc caagcaggtc actgataatg accatgctaa tgccagagcg ttttcgcatc 180
tggcttcaaa actgatcgaa acggaggtgg acccatccga cacgatcctt gacattggaa 240
gtgcgcccgc ccgcagaatg tattctaagc acaagtatca ttgtatctgt ccgatgagat 300
gtgcggaaga tccggacaga ttgtataagt atgcaactaa gctgaagaaa aactgtaagg 360
aaataactga taaggaattg gacaagaaaa tgaaggagct cgccgccgtc atgagcgacc 420
ctgacctgga aactgagact atgtgcctcc acgacgacga gtcgtgtcgc tacgaagggc 480
aagtcgctgt ttaccaggat gtatacgcgg ttgacggacc gacaagtctc tatcaccaag 540
ccaataaggg agttagagtc gcctactgga taggctttga caccacccct tttatgttta 600
agaacttggc tggagcatat ccatcatact ctaccaactg ggccgacgaa accgtgttaa 660
cggctcgtaa cataggccta tgcagctctg acgttatgga gcggtcacgt agagggatgt 720
ccattcttag aaagaagtat ttgaaaccat ccaacaatgt tctattctct gttggctcga 780
ccatctacca cgagaagagg gacttactga ggagctggca cctgccgtct gtatttcact 840
tacgtggcaa gcaaaattac acatgtcggt gtgagactat agttagttgc gacgggtacg 900
tcgttaaaag aatagctatc agtccaggcc tgtatgggaa gccttcaggc tatgctgcta 960
cgatgcaccg cgagggattc ttgtgctgca aagtgacaga cacattgaac ggggagaggg 1020
tctcttttcc cgtgtgcacg tatgtgccag ctacattgtg tgaccaaatg actggcatac 1080
tggcaacaga tgtcagtgcg gacgacgcgc aaaaactgct ggttgggctc aaccagcgta 1140
tagtcgtcaa cggtcgcacc cagagaaaca ccaataccat gaaaaattac cttttgcccg 1200
tagtggccca ggcatttgct aggtgggcaa aggaatataa ggaagatcaa gaagatgaaa 1260
ggccactagg actacgagat agacagttag tcatggggtg ttgttgggct tttagaaggc 1320
acaagataac atctatttat aagcgcccgg atacccaaac catcatcaaa gtgaacagcg 1380
atttccactc attcgtgctg cccaggatag gcagtaacac attggagatc gggctgagaa 1440
caagaatcag gaaaatgtta gaggagcaca aggagccgtc acctctcatt accgccgagg 1500
acgtacaaga agctaagtgc gcagccgatg aggctaagga ggtgcgtgaa gccgaggagt 1560
tgcgcgcagc tctaccacct ttggcagctg atgttgagga gcccactctg gaagccgatg 1620
tcgacttgat gttacaagag gctggggccg gctcagtgga gacacctcgt ggcttgataa 1680
aggttaccag ctacgatggc gaggacaaga tcggctctta cgctgtgctt tctccgcagg 1740
ctgtactcaa gagtgaaaaa ttatcttgca tccaccctct cgctgaacaa gtcatagtga 1800
taacacactc tggccgaaaa gggcgttatg ccgtggaacc ataccatggt aaagtagtgg 1860
tgccagaggg acatgcaata cccgtccagg actttcaagc tctgagtgaa agtgccacca 1920
ttgtgtacaa cgaacgtgag ttcgtaaaca ggtacctgca ccatattgcc acacatggag 1980
gagcgctgaa cactgatgaa gaatattaca aaactgtcaa gcccagcgag cacgacggcg 2040
aatacctgta cgacatcgac aggaaacagt gcgtcaagaa agaactagtc actgggctag 2100
ggctcacagg cgagctggtg gatcctccct tccatgaatt cgcctacgag agtctgagaa 2160
cacgaccagc cgctccttac caagtaccaa ccataggggt gtatggcgtg ccaggatcag 2220
gcaagtctgg catcattaaa agcgcagtca ccaaaaaaga tctagtggtg agcgccaaga 2280
aagaaaactg tgcagaaatt ataagggacg tcaagaaaat gaaagggctg gacgtcaatg 2340
ccagaactgt ggactcagtg ctcttgaatg gatgcaaaca ccccgtagag accctgtata 2400
ttgacgaagc ttttgcttgt catgcaggta ctctcagagc gctcatagcc attataagac 2460
ctaaaaaggc agtgctctgc ggggatccca aacagtgcgg tttttttaac atgatgtgcc 2520
tgaaagtgca ttttaaccac gagatttgca cacaagtctt ccacaaaagc atctctcgcc 2580
gttgcactaa atctgtgact tcggtcgtct caaccttgtt ttacgacaaa aaaatgagaa 2640
cgacgaatcc gaaagagact aagattgtga ttgacactac cggcagtacc aaacctaagc 2700
aggacgatct cattctcact tgtttcagag ggtgggtgaa gcagttgcaa atagattaca 2760
aaggcaacga aataatgacg gcagctgcct ctcaagggct gacccgtaaa ggtgtgtatg 2820
ccgttcggta caaggtgaat gaaaatcctc tgtacgcacc cacctcagaa catgtgaacg 2880
tcctactgac ccgcacggag gaccgcatcg tgtggaaaac actagccggc gacccatgga 2940
taaaaacact gactgccaag taccctggga atttcactgc cacgatagag gagtggcaag 3000
cagagcatga tgccatcatg aggcacatct tggagagacc ggaccctacc gacgtcttcc 3060
agaataaggc aaacgtgtgt tgggccaagg ctttagtgcc ggtgctgaag accgctggca 3120
tagacatgac cactgaacaa tggaacactg tggattattt tgaaacggac aaagctcact 3180
cagcagagat agtattgaac caactatgcg tgaggttctt tggactcgat ctggactccg 3240
gtctattttc tgcacccact gttccgttat ccattaggaa taatcactgg gataactccc 3300
cgtcgcctaa catgtacggg ctgaataaag aagtggtccg tcagctctct cgcaggtacc 3360
cacaactgcc tcgggcagtt gccactggaa gagtctatga catgaacact ggtacactgc 3420
gcaattatga tccgcgcata aacctagtac ctgtaaacag aagactgcct catgctttag 3480
tcctccacca taatgaacac ccacagagtg acttttcttc attcgtcagc aaattgaagg 3540
gcagaactgt cctggtggtc ggggaaaagt tgtccgtccc aggcaaaatg gttgactggt 3600
tgtcagaccg gcctgaggct accttcagag ctcggctgga tttaggcatc ccaggtgatg 3660
tgcccaaata tgacataata tttgttaatg tgaggacccc atataaatac catcactatc 3720
agcagtgtga agaccatgcc attaagctta gcatgttgac caagaaagct tgtctgcatc 3780
tgaatcccgg cggaacctgt gtcagcatag gttatggtta cgctgacagg gccagcgaaa 3840
gcatcattgg tgctatagcg cggctgttca agttttcccg ggtatgcaaa ccgaaatcct 3900
cacttgaaga gacggaagtt ctgtttgtat tcattgggta cgatcgcaag gcccgtacgc 3960
acaatcctta caagctttca tcaaccttga ccaacattta tacaggttcc agactccacg 4020
aagccggatg tgcaccctca tatcatgtgg tgcgagggga tattgccacg gccaccgaag 4080
gagtgattat aaatgctgct aacagcaaag gacaacctgg cggaggggtg tgcggagcgc 4140
tgtataagaa attcccggaa agcttcgatt tacagccgat cgaagtagga aaagcgcgac 4200
tggtcaaagg tgcagctaaa catatcattc atgccgtagg accaaacttc aacaaagttt 4260
cggaggttga aggtgacaaa cagttggcag aggcttatga gtccatcgct aagattgtca 4320
acgataacaa ttacaagtca gtagcgattc cactgttgtc caccggcatc ttttccggga 4380
acaaagatcg actaacccaa tcattgaacc atttgctgac agctttagac accactgatg 4440
cagatgtagc catatactgc agggacaaga aatgggaaat gactctcaag gaagcagtgg 4500
ctaggagaga agcagtggag gagatatgca tatccgacga ctcttcagtg acagaacctg 4560
atgcagagct ggtgagggtg catccgaaga gttctttggc tggaaggaag ggctacagca 4620
caagcgatgg caaaactttc tcatatttgg aagggaccaa gtttcaccag gcggccaagg 4680
atatagcaga aattaatgcc atgtggcccg ttgcaacgga ggccaatgag caggtatgca 4740
tgtatatcct cggagaaagc atgagcagta ttaggtcgaa atgccccgtc gaagagtcgg 4800
aagcctccac accacctagc acgctgcctt gcttgtgcat ccatgccatg actccagaaa 4860
gagtacagcg cctaaaagcc tcacgtccag aacaaattac tgtgtgctca tcctttccat 4920
tgccgaagta tagaatcact ggtgtgcaga agatccaatg ctcccagcct atattgttct 4980
caccgaaagt gcctgcgtat attcatccaa ggaagtatct cgtggaaaca ccaccggtag 5040
acgagactcc ggagccatcg gcagagaacc aatccacaga ggggacacct gaacaaccac 5100
cacttataac cgaggatgag accaggacta gaacgcctga gccgatcatc atcgaagagg 5160
aagaagagga tagcataagt ttgctgtcag atggcccgac ccaccaggtg ctgcaagtcg 5220
aggcagacat tcacgggccg ccctctgtat ctagctcatc ctggtccatt cctcatgcat 5280
ccgactttga tgtggacagt ttatccatac ttgacaccct ggagggagct agcgtgacca 5340
gcggggcaac gtcagccgag actaactctt acttcgcaaa gagtatggag tttctggcgc 5400
gaccggtgcc tgcgcctcga acagtattca ggaaccctcc acatcccgct ccgcgcacaa 5460
gaacaccgtc acttgcaccc agcagggcct gctcgagaac cagcctagtt tccaccccgc 5520
caggcgtgaa tagggtgatc actagagagg agctcgaggc gcttaccccg tcacgcactc 5580
ctagcaggtc ggtctcgaga accagcctgg tctccaaccc gccaggcgta aatagggtga 5640
ttacaagaga ggagtttgag gcgttcgtag cacaacaaca atgacggttt gatgcgggtg 5700
catacatctt ttcctccgac accggtcaag ggcatttaca acaaaaatca gtaaggcaaa 5760
cggtgctatc cgaagtggtg ttggagagga ccgaattgga gatttcgtat gccccgcgcc 5820
tcgaccaaga aaaagaagaa ttactacgca agaaattaca gttaaatccc acacctgcta 5880
acagaagcag ataccagtcc aggaaggtgg agaacatgaa agccataaca gctagacgta 5940
ttctgcaagg cctagggcat tatttgaagg cagaaggaaa agtggagtgc taccgaaccc 6000
tgcatcctgt tcctttgtat tcatctagtg tgaaccgtgc cttttcaagc cccaaggtcg 6060
cagtggaagc ctgtaacgcc atgttgaaag agaactttcc gactgtggct tcttactgta 6120
ttattccaga gtacgatgcc tatttggaca tggttgacgg agcttcatgc tgcttagaca 6180
ctgccagttt ttgccctgca aagctgcgca gctttccaaa gaaacactcc tatttggaac 6240
ccacaatacg atcggcagtg ccttcagcga tccagaacac gctccagaac gtcctggcag 6300
ctgccacaaa aagaaattgc aatgtcacgc aaatgagaga attgcccgta ttggattcgg 6360
cggcctttaa tgtggaatgc ttcaagaaat atgcgtgtaa taatgaatat tgggaaacgt 6420
ttaaagaaaa ccccatcagg cttactgaag aaaacgtggt aaattacatt accaaattaa 6480
aaggaccaaa agctgctgct ctttttgcga agacacataa tttgaatatg ttgcaggaca 6540
taccaatgga caggtttgta atggacttaa agagagacgt gaaagtgact ccaggaacaa 6600
aacatactga agaacggccc aaggtacagg tgatccaggc tgccgatccg ctagcaacag 6660
cgtatctgtg cggaatccac cgagagctgg ttaggagatt aaatgcggtc ctgcttccga 6720
acattcatac actgtttgat atgtcggctg aagactttga cgctattata gccgagcact 6780
tccagcctgg ggattgtgtt ctggaaactg acatcgcgtc gtttgataaa agtgaggacg 6840
acgccatggc tctgaccgcg ttaatgattc tggaagactt aggtgtggac gcagagctgt 6900
tgacgctgat tgaggcggct ttcggcgaaa tttcatcaat acatttgccc actaaaacta 6960
aatttaaatt cggagccatg atgaaatctg gaatgttcct cacactgttt gtgaacacag 7020
tcattaacat tgtaatcgca agcagagtgt tgagagaacg gctaaccgga tcaccatgtg 7080
cagcattcat tggagatgac aatatcgtga aaggagtcaa atcggacaaa ttaatggcag 7140
acaggtgcgc cacctggttg aatatggaag tcaagattat agatgctgtg gtgggcgaga 7200
aagcgcctta tttctgtgga gggtttattt tgtgtgactc cgtgaccggc acagcgtgcc 7260
gtgtggcaga ccccctaaaa aggctgttta agcttggcaa acctctggca gcagacgatg 7320
aacatgatga tgacaggaga agggcattgc atgaagagtc aacacgctgg aaccgagtgg 7380
gtattctttc agagctgtgc aaggcagtag aatcaaggta tgaaaccgta ggaacttcca 7440
tcatagttat ggccatgact actctagcta gcagtgttaa atcattcagc tacctgagag 7500
gggcccctat aactctctac ggctaacctg aatggactac gacatagtct agtccgccaa 7560
gatgttcgtg ttcctcgtgc tgctgcccct ggtgtctagc cagtgcgtga acctgaccac 7620
cagaacccag ctgcctccag cctacaccaa cagcttcacc aggggagtgt actaccccga 7680
caaggtgttc cggagcagcg tgctgcatag cacccaggac ctgttcctgc ccttcttcag 7740
caacgtgact tggttccacg ccatccacgt cagcggaacc aacggcacca agcgctttga 7800
caacccagtg ctgcccttca acgacggagt gtacttcgcc agcaccgaga agtccaacat 7860
catccggggt tggatcttcg gcaccacact ggacagcaag acccagagcc tgctgatcgt 7920
gaacaacgcc accaacgtcg tgatcaaggt ctgcgagttc cagttttgca acgacccctt 7980
cctgggcgtg tactaccaca agaacaacaa gtcttggatg gagagcgagt tccgggtgta 8040
cagcagcgcc aacaattgca ccttcgagta cgtgtcccag cccttcctga tggacctgga 8100
aggcaagcag ggcaacttca agaacctgcg ggagttcgtg ttcaagaaca tcgacggcta 8160
cttcaagatc tacagcaagc acacccccat caacctcgtg agagacctgc cacagggctt 8220
cagcgctctg gaacctctgg tggatctgcc aatcggcatc aacatcaccc ggttccagac 8280
cctgctggct ctgcacagaa gctacctgac cccaggcgat tcttctagcg gctggacagc 8340
aggagccgcc gcttattacg tgggctatct gcagccccgg accttcctgc tgaagtacaa 8400
cgagaacggc accatcaccg acgcagtgga ttgcgctctg gatcctctga gcgagaccaa 8460
gtgcaccctg aagtccttca ccgtggagaa gggcatctac cagaccagca acttccgggt 8520
gcagcctaca gagagcatcg tgcggttccc taacatcacc aacctctgcc ccttcggcga 8580
ggtgttcaac gccaccagat tcgccagcgt ctacgcctgg aaccggaagc ggatcagcaa 8640
ttgcgtggcc gactacagcg tgctgtacaa cagcgccagc ttcagcacct tcaagtgcta 8700
cggcgtgtcc cctaccaagc tgaacgatct gtgcttcacc aacgtgtacg ccgacagctt 8760
cgtgatcagg ggcgacgaag tgcgacagat cgccccagga cagacaggaa agatcgccga 8820
ttacaactac aagctgcccg acgacttcac cggttgcgtg atcgcttgga acagcaacaa 8880
cctggacagc aaggtcggcg gcaactacaa ctacctgtac cggctgttcc ggaagtccaa 8940
cctgaagccc ttcgagcggg acatcagcac agagatctac caggccggca gcacaccttg 9000
taacggcgtg gagggcttca actgctactt ccctctgcag agctacggct tccagcctac 9060
aaacggcgtg ggataccagc cttacagggt ggtggtgctg agcttcgaac tgctgcacgc 9120
tccagccacc gtctgcggac ctaaaaagag caccaacctg gtgaagaaca agtgcgtgaa 9180
tttcaacttc aacggcctga ccggaaccgg cgtgctgacc gagagcaaca agaagttcct 9240
gcccttccag cagttcggca gagacatcgc agacaccaca gacgccgtga gagatcctca 9300
gaccctggag atcctggaca tcaccccttg cagctttggc ggagtgtccg tgatcacccc 9360
aggaaccaac accagcaatc aggtggccgt gctgtaccag gacgtgaatt gcaccgaggt 9420
gccagtggcc attcacgcag atcagctgac ccctacctgg agagtgtaca gcaccggaag 9480
caacgtgttc cagaccaggg caggttgcct gatcggagcc gagcacgtga acaacagcta 9540
cgagtgcgat atccctatcg gcgccggcat ttgcgcctct taccagaccc agaccaacag 9600
ccctaggaga gccagaagcg tggcctctca gagcatcatc gcctacacca tgagcctggg 9660
agccgagaac agcgtggcct acagcaacaa cagcatcgcc atccccacca acttcaccat 9720
cagcgtgacc accgagattc tgcccgtgtc tatgaccaag accagcgtcg actgcaccat 9780
gtacatctgc ggcgacagca ccgagtgctc taacctgctg ctgcagtacg gctccttttg 9840
cacccagctg aacagggctc tgacaggcat cgcagtggag caggacaaga acacccagga 9900
ggtcttcgcc caggtcaagc agatctacaa gaccccccct atcaaggact tcggcggctt 9960
taacttcagc cagatcctgc ccgatcccag caagcctagc aagcggagct tcatcgagga 10020
cctgctgttc aacaaggtca ccctggccga cgcaggcttc atcaagcagt acggcgattg 10080
cctgggcgac attgccgcca gagacctgat ttgcgcccag aagttcaacg gcctgacagt 10140
gctgcctcct ctgctgaccg acgagatgat cgcccagtat acaagcgctc tgctggcagg 10200
aaccatcacc agcggttgga cctttggagc cggagccgct ctgcagatcc cttttgccat 10260
gcagatggcc taccgcttca acggaatcgg cgtgacccag aacgtgctct acgagaacca 10320
gaagctgatc gccaaccagt tcaacagcgc catcggcaag atccaggaca gcctgagcag 10380
cacagctagc gctctgggca agctgcagga tgtggtgaac cagaacgccc aggctctgaa 10440
caccctggtg aagcagctga gcagcaactt cggagccatc agcagcgtgc tgaacgacat 10500
cctgagcagg ctggacaagg tggaagccga agtgcagatc gacaggctga tcaccggcag 10560
actgcagtct ctgcagacct acgtgaccca gcagctgatc agagccgccg agatcagagc 10620
tagcgctaat ctggccgcca ccaagatgag cgagtgcgtg ctgggacaga gcaagagagt 10680
ggacttctgc ggcaagggct accacctgat gagctttcct cagagcgctc ctcacggagt 10740
ggtgtttctg cacgtgacct acgtgccagc ccaggagaag aacttcacca cagccccagc 10800
catttgccac gacggaaagg cccacttccc cagagaaggc gtgttcgtgt ccaacggcac 10860
ccattggttc gtgacccagc ggaacttcta cgagccccag atcatcacca ccgacaacac 10920
cttcgtgagc ggcaattgcg acgtggtcat cggcatcgtg aacaacaccg tgtacgaccc 10980
cctgcagcca gagctggact ccttcaagga ggagctggac aagtacttca agaaccacac 11040
cagcccagac gtggacctgg gagatatcag cggcatcaac gccagcgtgg tgaacatcca 11100
gaaggagatc gacaggctga acgaggtggc caagaacctg aacgagagcc tgatcgacct 11160
gcaggagctg ggcaagtacg agcagtacat caagtggcct tggtacattt ggctgggctt 11220
catcgccggc ctgatcgcaa tcgtcatggt gaccatcatg ctctgttgca tgaccagttg 11280
ctgcagctgc ctgaagggtt gctgcagttg cgggagttgc tgcaagttcg acgaggacga 11340
cagcgagcca gtgctgaaag gcgtgaagct gcactacaca tgataaggcg cgcccaccca 11400
gcggccgcat acagcagcaa ttggcaagct gcttacatag aactcgcggc gattggcatg 11460
ccgccttaaa atttttattt tatttttctt ttcttttccg aatcggattt tgtttttaat 11520
atttcaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaagaag agctagggat 11580
aacagggtaa ttgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc 11640
tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc 11700
agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc 11760
tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt 11820
cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg 11880
ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat 11940
ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag 12000
ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt 12060
ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc 12120
cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta 12180
gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 12240
atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga 12300
ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa 12360
gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttag aaaaactcat 12420
cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa 12480
aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat 12540
cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct 12600
cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga 12660
atggcaaaag tttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt 12720
catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac 12780
gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca 12840
ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct 12900
ggaatgctgt tttcccaggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga 12960
taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct 13020
catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat 13080
cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc 13140
atttataccc atataaatca gcatccatgt tggaatttaa tcgcggccta gagcaagacg 13200
tttcccgttg aatatggctc atactcttcc tttttcaata ttattgaagc atttatcagg 13260
gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg 13320
ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga 13380
cattaaccta taaaaatagg cgtatcacga ggccctttcg tctagggata acagggtaat 13440
taatacgact cactatag 13458
<210> 4
<211> 11566
<212> RNA
<213> 人工序列(Artificial Sequence)
<400> 4
gaugggcggc gcaugagaga agcccagacc aauuaccuac ccaaaaugga gaaaguucac 60
guugacaucg aggaagacag cccauuccuc agagcuuugc agcggagcuu cccgcaguuu 120
gagguagaag ccaagcaggu cacugauaau gaccaugcua augccagagc guuuucgcau 180
cuggcuucaa aacugaucga aacggaggug gacccauccg acacgauccu ugacauugga 240
agugcgcccg cccgcagaau guauucuaag cacaaguauc auuguaucug uccgaugaga 300
ugugcggaag auccggacag auuguauaag uaugcaacua agcugaagaa aaacuguaag 360
gaaauaacug auaaggaauu ggacaagaaa augaaggagc ucgccgccgu caugagcgac 420
ccugaccugg aaacugagac uaugugccuc cacgacgacg agucgugucg cuacgaaggg 480
caagucgcug uuuaccagga uguauacgcg guugacggac cgacaagucu cuaucaccaa 540
gccaauaagg gaguuagagu cgccuacugg auaggcuuug acaccacccc uuuuauguuu 600
aagaacuugg cuggagcaua uccaucauac ucuaccaacu gggccgacga aaccguguua 660
acggcucgua acauaggccu augcagcucu gacguuaugg agcggucacg uagagggaug 720
uccauucuua gaaagaagua uuugaaacca uccaacaaug uucuauucuc uguuggcucg 780
accaucuacc acgagaagag ggacuuacug aggagcuggc accugccguc uguauuucac 840
uuacguggca agcaaaauua cacaugucgg ugugagacua uaguuaguug cgacggguac 900
gucguuaaaa gaauagcuau caguccaggc cuguauggga agccuucagg cuaugcugcu 960
acgaugcacc gcgagggauu cuugugcugc aaagugacag acacauugaa cggggagagg 1020
gucucuuuuc ccgugugcac guaugugcca gcuacauugu gugaccaaau gacuggcaua 1080
cuggcaacag augucagugc ggacgacgcg caaaaacugc ugguugggcu caaccagcgu 1140
auagucguca acggucgcac ccagagaaac accaauacca ugaaaaauua ccuuuugccc 1200
guaguggccc aggcauuugc uaggugggca aaggaauaua aggaagauca agaagaugaa 1260
aggccacuag gacuacgaga uagacaguua gucauggggu guuguugggc uuuuagaagg 1320
cacaagauaa caucuauuua uaagcgcccg gauacccaaa ccaucaucaa agugaacagc 1380
gauuuccacu cauucgugcu gcccaggaua ggcaguaaca cauuggagau cgggcugaga 1440
acaagaauca ggaaaauguu agaggagcac aaggagccgu caccucucau uaccgccgag 1500
gacguacaag aagcuaagug cgcagccgau gaggcuaagg aggugcguga agccgaggag 1560
uugcgcgcag cucuaccacc uuuggcagcu gauguugagg agcccacucu ggaagccgau 1620
gucgacuuga uguuacaaga ggcuggggcc ggcucagugg agacaccucg uggcuugaua 1680
aagguuacca gcuacgaugg cgaggacaag aucggcucuu acgcugugcu uucuccgcag 1740
gcuguacuca agagugaaaa auuaucuugc auccacccuc ucgcugaaca agucauagug 1800
auaacacacu cuggccgaaa agggcguuau gccguggaac cauaccaugg uaaaguagug 1860
gugccagagg gacaugcaau acccguccag gacuuucaag cucugaguga aagugccacc 1920
auuguguaca acgaacguga guucguaaac agguaccugc accauauugc cacacaugga 1980
ggagcgcuga acacugauga agaauauuac aaaacuguca agcccagcga gcacgacggc 2040
gaauaccugu acgacaucga caggaaacag ugcgucaaga aagaacuagu cacugggcua 2100
gggcucacag gcgagcuggu ggauccuccc uuccaugaau ucgccuacga gagucugaga 2160
acacgaccag ccgcuccuua ccaaguacca accauagggg uguauggcgu gccaggauca 2220
ggcaagucug gcaucauuaa aagcgcaguc accaaaaaag aucuaguggu gagcgccaag 2280
aaagaaaacu gugcagaaau uauaagggac gucaagaaaa ugaaagggcu ggacgucaau 2340
gccagaacug uggacucagu gcucuugaau ggaugcaaac accccguaga gacccuguau 2400
auugacgaag cuuuugcuug ucaugcaggu acucucagag cgcucauagc cauuauaaga 2460
ccuaaaaagg cagugcucug cggggauccc aaacagugcg guuuuuuuaa caugaugugc 2520
cugaaagugc auuuuaacca cgagauuugc acacaagucu uccacaaaag caucucucgc 2580
cguugcacua aaucugugac uucggucguc ucaaccuugu uuuacgacaa aaaaaugaga 2640
acgacgaauc cgaaagagac uaagauugug auugacacua ccggcaguac caaaccuaag 2700
caggacgauc ucauucucac uuguuucaga ggguggguga agcaguugca aauagauuac 2760
aaaggcaacg aaauaaugac ggcagcugcc ucucaagggc ugacccguaa agguguguau 2820
gccguucggu acaaggugaa ugaaaauccu cuguacgcac ccaccucaga acaugugaac 2880
guccuacuga cccgcacgga ggaccgcauc guguggaaaa cacuagccgg cgacccaugg 2940
auaaaaacac ugacugccaa guacccuggg aauuucacug ccacgauaga ggaguggcaa 3000
gcagagcaug augccaucau gaggcacauc uuggagagac cggacccuac cgacgucuuc 3060
cagaauaagg caaacgugug uugggccaag gcuuuagugc cggugcugaa gaccgcuggc 3120
auagacauga ccacugaaca auggaacacu guggauuauu uugaaacgga caaagcucac 3180
ucagcagaga uaguauugaa ccaacuaugc gugagguucu uuggacucga ucuggacucc 3240
ggucuauuuu cugcacccac uguuccguua uccauuagga auaaucacug ggauaacucc 3300
ccgucgccua acauguacgg gcugaauaaa gaaguggucc gucagcucuc ucgcagguac 3360
ccacaacugc cucgggcagu ugccacugga agagucuaug acaugaacac ugguacacug 3420
cgcaauuaug auccgcgcau aaaccuagua ccuguaaaca gaagacugcc ucaugcuuua 3480
guccuccacc auaaugaaca cccacagagu gacuuuucuu cauucgucag caaauugaag 3540
ggcagaacug uccugguggu cggggaaaag uuguccgucc caggcaaaau gguugacugg 3600
uugucagacc ggccugaggc uaccuucaga gcucggcugg auuuaggcau cccaggugau 3660
gugcccaaau augacauaau auuuguuaau gugaggaccc cauauaaaua ccaucacuau 3720
cagcagugug aagaccaugc cauuaagcuu agcauguuga ccaagaaagc uugucugcau 3780
cugaaucccg gcggaaccug ugucagcaua gguuaugguu acgcugacag ggccagcgaa 3840
agcaucauug gugcuauagc gcggcuguuc aaguuuuccc ggguaugcaa accgaaaucc 3900
ucacuugaag agacggaagu ucuguuugua uucauugggu acgaucgcaa ggcccguacg 3960
cacaauccuu acaagcuuuc aucaaccuug accaacauuu auacagguuc cagacuccac 4020
gaagccggau gugcacccuc auaucaugug gugcgagggg auauugccac ggccaccgaa 4080
ggagugauua uaaaugcugc uaacagcaaa ggacaaccug gcggaggggu gugcggagcg 4140
cuguauaaga aauucccgga aagcuucgau uuacagccga ucgaaguagg aaaagcgcga 4200
cuggucaaag gugcagcuaa acauaucauu caugccguag gaccaaacuu caacaaaguu 4260
ucggagguug aaggugacaa acaguuggca gaggcuuaug aguccaucgc uaagauuguc 4320
aacgauaaca auuacaaguc aguagcgauu ccacuguugu ccaccggcau cuuuuccggg 4380
aacaaagauc gacuaaccca aucauugaac cauuugcuga cagcuuuaga caccacugau 4440
gcagauguag ccauauacug cagggacaag aaaugggaaa ugacucucaa ggaagcagug 4500
gcuaggagag aagcagugga ggagauaugc auauccgacg acucuucagu gacagaaccu 4560
gaugcagagc uggugagggu gcauccgaag aguucuuugg cuggaaggaa gggcuacagc 4620
acaagcgaug gcaaaacuuu cucauauuug gaagggacca aguuucacca ggcggccaag 4680
gauauagcag aaauuaaugc cauguggccc guugcaacgg aggccaauga gcagguaugc 4740
auguauaucc ucggagaaag caugagcagu auuaggucga aaugccccgu cgaagagucg 4800
gaagccucca caccaccuag cacgcugccu ugcuugugca uccaugccau gacuccagaa 4860
agaguacagc gccuaaaagc cucacgucca gaacaaauua cugugugcuc auccuuucca 4920
uugccgaagu auagaaucac uggugugcag aagauccaau gcucccagcc uauauuguuc 4980
ucaccgaaag ugccugcgua uauucaucca aggaaguauc ucguggaaac accaccggua 5040
gacgagacuc cggagccauc ggcagagaac caauccacag aggggacacc ugaacaacca 5100
ccacuuauaa ccgaggauga gaccaggacu agaacgccug agccgaucau caucgaagag 5160
gaagaagagg auagcauaag uuugcuguca gauggcccga cccaccaggu gcugcaaguc 5220
gaggcagaca uucacgggcc gcccucugua ucuagcucau ccugguccau uccucaugca 5280
uccgacuuug auguggacag uuuauccaua cuugacaccc uggagggagc uagcgugacc 5340
agcggggcaa cgucagccga gacuaacucu uacuucgcaa agaguaugga guuucuggcg 5400
cgaccggugc cugcgccucg aacaguauuc aggaacccuc cacaucccgc uccgcgcaca 5460
agaacaccgu cacuugcacc cagcagggcc ugcucgagaa ccagccuagu uuccaccccg 5520
ccaggcguga auagggugau cacuagagag gagcucgagg cgcuuacccc gucacgcacu 5580
ccuagcaggu cggucucgag aaccagccug gucuccaacc cgccaggcgu aaauagggug 5640
auuacaagag aggaguuuga ggcguucgua gcacaacaac aaugacgguu ugaugcgggu 5700
gcauacaucu uuuccuccga caccggucaa gggcauuuac aacaaaaauc aguaaggcaa 5760
acggugcuau ccgaaguggu guuggagagg accgaauugg agauuucgua ugccccgcgc 5820
cucgaccaag aaaaagaaga auuacuacgc aagaaauuac aguuaaaucc cacaccugcu 5880
aacagaagca gauaccaguc caggaaggug gagaacauga aagccauaac agcuagacgu 5940
auucugcaag gccuagggca uuauuugaag gcagaaggaa aaguggagug cuaccgaacc 6000
cugcauccug uuccuuugua uucaucuagu gugaaccgug ccuuuucaag ccccaagguc 6060
gcaguggaag ccuguaacgc cauguugaaa gagaacuuuc cgacuguggc uucuuacugu 6120
auuauuccag aguacgaugc cuauuuggac augguugacg gagcuucaug cugcuuagac 6180
acugccaguu uuugcccugc aaagcugcgc agcuuuccaa agaaacacuc cuauuuggaa 6240
cccacaauac gaucggcagu gccuucagcg auccagaaca cgcuccagaa cguccuggca 6300
gcugccacaa aaagaaauug caaugucacg caaaugagag aauugcccgu auuggauucg 6360
gcggccuuua auguggaaug cuucaagaaa uaugcgugua auaaugaaua uugggaaacg 6420
uuuaaagaaa accccaucag gcuuacugaa gaaaacgugg uaaauuacau uaccaaauua 6480
aaaggaccaa aagcugcugc ucuuuuugcg aagacacaua auuugaauau guugcaggac 6540
auaccaaugg acagguuugu aauggacuua aagagagacg ugaaagugac uccaggaaca 6600
aaacauacug aagaacggcc caagguacag gugauccagg cugccgaucc gcuagcaaca 6660
gcguaucugu gcggaaucca ccgagagcug guuaggagau uaaaugcggu ccugcuuccg 6720
aacauucaua cacuguuuga uaugucggcu gaagacuuug acgcuauuau agccgagcac 6780
uuccagccug gggauugugu ucuggaaacu gacaucgcgu cguuugauaa aagugaggac 6840
gacgccaugg cucugaccgc guuaaugauu cuggaagacu uaggugugga cgcagagcug 6900
uugacgcuga uugaggcggc uuucggcgaa auuucaucaa uacauuugcc cacuaaaacu 6960
aaauuuaaau ucggagccau gaugaaaucu ggaauguucc ucacacuguu ugugaacaca 7020
gucauuaaca uuguaaucgc aagcagagug uugagagaac ggcuaaccgg aucaccaugu 7080
gcagcauuca uuggagauga caauaucgug aaaggaguca aaucggacaa auuaauggca 7140
gacaggugcg ccaccugguu gaauauggaa gucaagauua uagaugcugu ggugggcgag 7200
aaagcgccuu auuucugugg aggguuuauu uugugugacu ccgugaccgg cacagcgugc 7260
cguguggcag acccccuaaa aaggcuguuu aagcuuggca aaccucuggc agcagacgau 7320
gaacaugaug augacaggag aagggcauug caugaagagu caacacgcug gaaccgagug 7380
gguauucuuu cagagcugug caaggcagua gaaucaaggu augaaaccgu aggaacuucc 7440
aucauaguua uggccaugac uacucuagcu agcaguguua aaucauucag cuaccugaga 7500
ggggccccua uaacucucua cggcuaaccu gaauggacua cgacauaguc uaguccgcca 7560
agauguucgu guuccucgug cugcugcccc uggugucuag ccagugcgug aaccugacca 7620
ccagaaccca gcugccucca gccuacacca acagcuucac caggggagug uacuaccccg 7680
acaagguguu ccggagcagc gugcugcaua gcacccagga ccuguuccug cccuucuuca 7740
gcaacgugac uugguuccac gccauccacg ucagcggaac caacggcacc aagcgcuuug 7800
acaacccagu gcugcccuuc aacgacggag uguacuucgc cagcaccgag aaguccaaca 7860
ucauccgggg uuggaucuuc ggcaccacac uggacagcaa gacccagagc cugcugaucg 7920
ugaacaacgc caccaacguc gugaucaagg ucugcgaguu ccaguuuugc aacgaccccu 7980
uccugggcgu guacuaccac aagaacaaca agucuuggau ggagagcgag uuccgggugu 8040
acagcagcgc caacaauugc accuucgagu acguguccca gcccuuccug auggaccugg 8100
aaggcaagca gggcaacuuc aagaaccugc gggaguucgu guucaagaac aucgacggcu 8160
acuucaagau cuacagcaag cacaccccca ucaaccucgu gagagaccug ccacagggcu 8220
ucagcgcucu ggaaccucug guggaucugc caaucggcau caacaucacc cgguuccaga 8280
cccugcuggc ucugcacaga agcuaccuga ccccaggcga uucuucuagc ggcuggacag 8340
caggagccgc cgcuuauuac gugggcuauc ugcagccccg gaccuuccug cugaaguaca 8400
acgagaacgg caccaucacc gacgcagugg auugcgcucu ggauccucug agcgagacca 8460
agugcacccu gaaguccuuc accguggaga agggcaucua ccagaccagc aacuuccggg 8520
ugcagccuac agagagcauc gugcgguucc cuaacaucac caaccucugc cccuucggcg 8580
agguguucaa cgccaccaga uucgccagcg ucuacgccug gaaccggaag cggaucagca 8640
auugcguggc cgacuacagc gugcuguaca acagcgccag cuucagcacc uucaagugcu 8700
acggcguguc cccuaccaag cugaacgauc ugugcuucac caacguguac gccgacagcu 8760
ucgugaucag gggcgacgaa gugcgacaga ucgccccagg acagacagga aagaucgccg 8820
auuacaacua caagcugccc gacgacuuca ccgguugcgu gaucgcuugg aacagcaaca 8880
accuggacag caaggucggc ggcaacuaca acuaccugua ccggcuguuc cggaagucca 8940
accugaagcc cuucgagcgg gacaucagca cagagaucua ccaggccggc agcacaccuu 9000
guaacggcgu ggagggcuuc aacugcuacu ucccucugca gagcuacggc uuccagccua 9060
caaacggcgu gggauaccag ccuuacaggg ugguggugcu gagcuucgaa cugcugcacg 9120
cuccagccac cgucugcgga ccuaaaaaga gcaccaaccu ggugaagaac aagugcguga 9180
auuucaacuu caacggccug accggaaccg gcgugcugac cgagagcaac aagaaguucc 9240
ugcccuucca gcaguucggc agagacaucg cagacaccac agacgccgug agagauccuc 9300
agacccugga gauccuggac aucaccccuu gcagcuuugg cggagugucc gugaucaccc 9360
caggaaccaa caccagcaau cagguggccg ugcuguacca ggacgugaau ugcaccgagg 9420
ugccaguggc cauucacgca gaucagcuga ccccuaccug gagaguguac agcaccggaa 9480
gcaacguguu ccagaccagg gcagguugcc ugaucggagc cgagcacgug aacaacagcu 9540
acgagugcga uaucccuauc ggcgccggca uuugcgccuc uuaccagacc cagaccaaca 9600
gcccuaggag agccagaagc guggccucuc agagcaucau cgccuacacc augagccugg 9660
gagccgagaa cagcguggcc uacagcaaca acagcaucgc cauccccacc aacuucacca 9720
ucagcgugac caccgagauu cugcccgugu cuaugaccaa gaccagcguc gacugcacca 9780
uguacaucug cggcgacagc accgagugcu cuaaccugcu gcugcaguac ggcuccuuuu 9840
gcacccagcu gaacagggcu cugacaggca ucgcagugga gcaggacaag aacacccagg 9900
aggucuucgc ccaggucaag cagaucuaca agaccccccc uaucaaggac uucggcggcu 9960
uuaacuucag ccagauccug cccgauccca gcaagccuag caagcggagc uucaucgagg 10020
accugcuguu caacaagguc acccuggccg acgcaggcuu caucaagcag uacggcgauu 10080
gccugggcga cauugccgcc agagaccuga uuugcgccca gaaguucaac ggccugacag 10140
ugcugccucc ucugcugacc gacgagauga ucgcccagua uacaagcgcu cugcuggcag 10200
gaaccaucac cagcgguugg accuuuggag ccggagccgc ucugcagauc ccuuuugcca 10260
ugcagauggc cuaccgcuuc aacggaaucg gcgugaccca gaacgugcuc uacgagaacc 10320
agaagcugau cgccaaccag uucaacagcg ccaucggcaa gauccaggac agccugagca 10380
gcacagcuag cgcucugggc aagcugcagg auguggugaa ccagaacgcc caggcucuga 10440
acacccuggu gaagcagcug agcagcaacu ucggagccau cagcagcgug cugaacgaca 10500
uccugagcag gcuggacaag guggaagccg aagugcagau cgacaggcug aucaccggca 10560
gacugcaguc ucugcagacc uacgugaccc agcagcugau cagagccgcc gagaucagag 10620
cuagcgcuaa ucuggccgcc accaagauga gcgagugcgu gcugggacag agcaagagag 10680
uggacuucug cggcaagggc uaccaccuga ugagcuuucc ucagagcgcu ccucacggag 10740
ugguguuucu gcacgugacc uacgugccag cccaggagaa gaacuucacc acagccccag 10800
ccauuugcca cgacggaaag gcccacuucc ccagagaagg cguguucgug uccaacggca 10860
cccauugguu cgugacccag cggaacuucu acgagcccca gaucaucacc accgacaaca 10920
ccuucgugag cggcaauugc gacgugguca ucggcaucgu gaacaacacc guguacgacc 10980
cccugcagcc agagcuggac uccuucaagg aggagcugga caaguacuuc aagaaccaca 11040
ccagcccaga cguggaccug ggagauauca gcggcaucaa cgccagcgug gugaacaucc 11100
agaaggagau cgacaggcug aacgaggugg ccaagaaccu gaacgagagc cugaucgacc 11160
ugcaggagcu gggcaaguac gagcaguaca ucaaguggcc uugguacauu uggcugggcu 11220
ucaucgccgg ccugaucgca aucgucaugg ugaccaucau gcucuguugc augaccaguu 11280
gcugcagcug ccugaagggu ugcugcaguu gcgggaguug cugcaaguuc gacgaggacg 11340
acagcgagcc agugcugaaa ggcgugaagc ugcacuacac augauaaggc gcgcccaccc 11400
agcggccgca uacagcagca auuggcaagc ugcuuacaua gaacucgcgg cgauuggcau 11460
gccgccuuaa aauuuuuauu uuauuuuucu uuucuuuucc gaaucggauu uuguuuuuaa 11520
uauuucaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa 11566

Claims (8)

1. 编码SARS-CoV-2病毒刺突蛋白的核酸，其核苷酸序列如SEQ ID NO:1所示。
2.线性化mRNA，其核酸序列如SEQ ID NO:4所示。
3.权利要求1所述的核酸或权利要求2所述的线性化mRNA，在制备新型冠状病毒mRNA疫苗中的应用。
4.mRNA-LNP复合物，其制备原料包括权利要求2所述的线性化mRNA和脂类。
5.根据权利要求4所述的复合物，其特征在于，所述脂类包括：DMG-PEG2000、胆固醇、二硬脂酰基磷脂酰胆碱和DLinDMA；其中DMG-PEG2000、胆固醇、二硬脂酰基磷脂酰胆碱和DLinDMA的摩尔比为2:48:10:40。
6.权利要求4或5所述复合物的制备方法，其特征在于，将权利要求2所述的线性化mRNA和脂类经快速混合，制成脂质体。
7.一种新型冠状病毒疫苗，其特征在于，包括权利要求4或5所述的复合物。
8.根据权利要求7所述的疫苗，其特征在于，还包括疫苗中可接受的辅料；其中权利要求4或5所述的复合物的浓度为 30mg/mL。
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