CN115867658A - 冠状病毒疫苗 - Google Patents
冠状病毒疫苗 Download PDFInfo
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- CN115867658A CN115867658A CN202180028581.8A CN202180028581A CN115867658A CN 115867658 A CN115867658 A CN 115867658A CN 202180028581 A CN202180028581 A CN 202180028581A CN 115867658 A CN115867658 A CN 115867658A
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Abstract
本文提供了分离的多核苷酸，其编码甲病毒非结构蛋白nsp1、nsp2、nsp3和nsp4以及包含与信号序列和/或跨膜结构域融合的冠状病毒蛋白的多肽。该冠状病毒蛋白可以是冠状病毒刺突(S)蛋白的S1亚基的受体结合结构域。该多核苷酸诸如RNA可用于作为针对冠状病毒感染、特别是COVID‑19感染的疫苗。
Description
技术领域
本公开内容总体上涉及疫苗领域，并涉及用于针对病毒感染进行治疗和/或免疫的方法和组合物。具体而言，本公开内容涉及用于冠状病毒诸如SARS-CoV-2(COVID-19)的疫苗。
背景技术
冠状病毒是大的病毒科，其经常引起轻度至中度上呼吸道疾病，如普通感冒。但是，在过去的二十年中，三种新的冠状病毒已经从动物储主中出现，引起严重和广泛的疾病和死亡。
存在数百种冠状病毒，其中大部分在诸如猪、骆驼、蝙蝠和猫等动物中传播。有时那些病毒会跳至人类(被称为溢出事件)并可以引起疾病。在已知的七种使人患病的冠状病毒中，四种仅引起轻度至中度疾病。三种可以引起更严重的、甚至致死的疾病。SARS冠状病毒(SARS-CoV)引起严重急性呼吸综合症(SARS)。中东呼吸综合征(MERS)是由MERS冠状病毒(MERS-CoV)引起的。MERS从骆驼中的动物储主传播，于2012年9月被鉴定，并继续引起零星和局部暴发。在本世纪出现的第三种新型冠状病毒被称为SARS-CoV-2，其引起COVID-19。
大多数感染了COVID-19病毒的人会经历轻度至中度呼吸疾病，且无需特殊治疗即可康复。老年人以及具有基础医学问题(如心血管疾病、糖尿病、慢性呼吸系统疾病和癌症)的人更可能发生严重疾病。目前，没有用于COVID-19的特异性疫苗或治疗(https://www.who.int/health-topics/coronavirus#tab＝tab_1)。
因为疫苗是给大量健康人使用，所以疫苗通常具有比给已经生病的人施用的药物更高的安全性门槛。对于SARS-CoV-2(COVID-19)疫苗，研究人员的主要安全性担忧是避免被称为疾病增强的现象，其中确实被感染的接种疫苗的人比从未接种疫苗的人发生更严重的疾病形式。在2004年报道的实验性SARS疫苗的研究中，接种疫苗的雪貂在被病毒感染后在其肝脏中发生破坏性炎症。
抗体依赖性增强(ADE)是一种机制，作为一种替代策略，一些病毒(诸如登革热病毒、猫冠状病毒和HIV)通过该机制可以感染展示Fc受体的宿主细胞并从而利用抗病毒体液免疫应答。ADE是否发生在SARS-冠状病毒感染中存在争议，但已经报道，针对SARS-冠状病毒的刺突蛋白的抗体可能引起ADE效应。
得克萨斯州休斯顿贝勒医学院(Baylor College of Medicine)的疫苗科学家Peter Hotez认为，在将试验转移至人类之前，潜在的疫苗应该首先在动物中进行试验以排除疾病增强。他说，他理解将SARS-CoV-2(COVID-19)疫苗迅速推向人类试验的原因，但补充说，由于疫苗可能加剧疾病，“我不确定这是你想做的疫苗”。(非专利文献1)
因此，本领域需要可以避免ADE效应的更安全的冠状病毒疫苗。
引文列表
专利文献
[PL1]WO 2019/124441
非专利文献
[NPL 1]Nature 579,481(2020)。
发明内容
本公开内容涉及能够诱导针对冠状病毒的保护同时使ADE的可能性最小化的新型抗原活性蛋白/多肽。本文公开的蛋白/多肽包括与信号序列和/或跨膜结构域融合的冠状病毒结构蛋白。所述冠状病毒结构蛋白可以是刺突(S)蛋白、核衣壳(N)蛋白、膜(M)蛋白、小包膜蛋白(E)或其组合。具体实例可以包括刺突蛋白的S1和/或S2亚基，并且尤其是S1亚基的受体结合结构域。
在另一个方面，本公开内容涉及编码上面讨论的能够诱导针对冠状病毒的保护的新型抗原活性蛋白/多肽的新型多核苷酸。
在另一个方面，本公开内容涉及可以表达上面讨论的抗原活性蛋白/多肽的新型甲病毒复制子。该甲病毒复制子包括多核苷酸，诸如编码甲病毒非结构蛋白nsp1、nsp2、nsp3和nsp4的RNA以及编码上面讨论的抗原活性蛋白/多肽的作为目标基因的多核苷酸。
在仍然另一个方面，本公开内容涉及包含上面讨论的多肽或多核苷酸的疫苗。特别地，本公开内容提供了一种疫苗，其包含编码甲病毒非结构蛋白nsp1、nsp2、nsp3和nsp4的多核苷酸以及包含与信号序列和/或跨膜结构域融合的冠状病毒结构蛋白的多肽。该疫苗可以被用于预防和/或治疗受试者免于冠状病毒感染。
在仍然另一个方面，本公开内容涉及用于免疫、预防或治疗受试者免于冠状病毒感染的方法，所述方法包含给需要其的受试者施用有效量的上面讨论的多肽或多肽。
在仍然另一个方面，本公开内容涉及上面讨论的多肽或多核苷酸用于药物制造的用途。
附图说明
[图1]甲病毒复制子的构建体。
[图2]在实施例3中产生的包括SS1-RBD-GS-IgG4-CH3(接头)-GS-TM1的构建体C的全长VEEV TC-83载体。
[图3]用在实施例3中制备的甲病毒复制子质粒载体转染的HEK293T细胞中的RBD表达。
[图4-1]在用在实施例4中制备的甲病毒复制子颗粒免疫的第14天和第35天，小鼠血清中针对COVID-19刺突蛋白的抗体滴度。
[图4-2]在用在实施例4中制备的甲病毒复制子颗粒免疫的第14天和第35天，小鼠血清中针对COVID-19刺突蛋白的抗体滴度。
[图5-1]在用在实施例4中制备的甲病毒复制子颗粒免疫的第14天和第35天，小鼠血清中针对COVID-19RBD的抗体滴度。
[图5-2]在用在实施例4中制备的甲病毒复制子颗粒免疫的第14天和第35天，小鼠血清中针对COVID-19RBD的抗体滴度。
[图6]在用在实施例4中制备的甲病毒复制子颗粒免疫的第7天，小鼠血清中针对COVID-19刺突S1抗原的抗体滴度。
[图7]在用在实施例8中制备的甲病毒自扩增RNA免疫的第14、28和46天，在小鼠血清中针对COVID-19S1蛋白的抗体滴度。
具体实施方式
如本文中使用的，“冠状病毒”意在指属于冠状病毒科的单链有义RNA病毒。示例性的冠状病毒科病毒包括但不限于SARS-Cov、MERS-Cov和SARS-CoV-2(COVID-19)。SARS-CoV-2(COVID-19)可以包括已知和未知的突变体。已知的突变体可以包括COVID-19E484K_N501Y_K417T突变体(巴西株突变体)、E484K_N501Y_K417N突变体(南非突变体)和E484K突变体。冠状病毒基因组编码众多非结构蛋白和包括刺突(S)蛋白、核衣壳(N)蛋白、膜(M)蛋白和小包膜(E)蛋白在内的四种主要结构蛋白。刺突(S)蛋白是大包膜糖蛋白，由S1和S2亚基组成。“受体结合结构域(RBD)”位于S1亚基中。本文使用的结构蛋白的优选实例是冠状病毒RBD。COVID-19及其突变体的刺突蛋白、刺突蛋白的S1和S2亚基以及RBD已被鉴定和公开(Wrapp等人,Science 10.1126/science.abb2507(2020)、Z.Wang等人,(预印本)bioRxiv2021.01.15.426911；doi:https://doi.org/10.1101/2021.01.15.426911)。
本文使用的“冠状病毒结构蛋白”可以是天然存在的病毒结构蛋白或其修饰蛋白。修饰的蛋白可以是天然存在的病毒结构蛋白的片段。在一个实施方案中，修饰的蛋白与天然存在的病毒结构蛋白或其片段具有至少70％、75％、80％、85％、90％、95％或98％的氨基酸序列同一性。在一个实施方案中，修饰的蛋白是突变体，其中基于天然存在的病毒包膜蛋白或其片段，至多10％的氨基酸被删除、置换和/或添加。
如本文中使用的，“跨膜结构域(TM)”是衍生自天然或合成来源的蛋白。在来源是天然的情况下，在一些方面，该结构域衍生自任何膜结合蛋白或跨膜蛋白。在一个方面，所述膜结合蛋白或跨膜蛋白是与COVID-19异源的蛋白。所述膜结合蛋白或跨膜蛋白的实例可以包括T细胞受体的α、β或ζ链、CD28、CD3ε、CD45、CD4、CD5、CDS、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD137、CD154；toll样受体(TLR)，诸如人类中的TLR1-TLR10和小鼠中的TLR1-TLR9、TLR11-TLR13；白介素(IL)受体，诸如IL-1-28受体、RANTES受体(CCR1、CCR3、CCR5)、MIP-1受体、PF4受体、M-CSF受体和NAP-2受体(属于GPCR趋化因子受体)；血凝素(HA)。在另一个方面，所述膜结合蛋白或跨膜蛋白是衍生自COVID-19的蛋白，诸如COVID-19刺突蛋白。
跨膜蛋白的实例还可以包括以下：5-脂氧合酶活化蛋白、ABC转运蛋白、ACBP、淀粉样蛋白β(A4)、Bcl-2抑制剂、BNIP、CAAX蛋白酶、细胞色素P450、E-NPP、EPHA1、EPHA2、EPHA3、EPHA4、脂肪酸去饱和酶、γ分泌酶、葡萄糖转运蛋白、血型糖蛋白、GPCR、HER2/ErbB2、HER3/ErbB3、HER4/ErbB4、HSD-11β、低氧诱导的蛋白、免疫球蛋白、胰岛素受体、整联蛋白、离子通道、MAPEG、MFS、MinK家族、MPP、肽酶AD、肽酶家族M48、肽酶MA、锯齿状蛋白(ProteinJagged)、受体-型激酶、SNARE复合物、硫酸酯酶、TNF受体、跨膜蛋白14、转运蛋白、TROBP、VEGF受体、醛脱氢酶、氨和脲转运蛋白、FMN-连接的氧化还原酶、含有亮氨酸富集重复(LRR)的跨膜蛋白、白三烯C4合酶、溶酶体相关的膜糖蛋白、主要内在蛋白(MIP)/FNT超家族、微粒体前列腺素E合酶、N-(脱氧)核糖基转移酶样膜蛋白、中性/碱性神经酰胺酶、寡糖基转移酶、五聚体配体门控的离子通道、视紫质样受体和泵、单螺旋ATP酶调节剂、角鲨烯/八氢番茄红素合酶、硬脂酰基-辅酶A去饱和酶1、Stannin(SNN)膜蛋白、T-细胞表面糖蛋白CD3ζ链、三角四肽(Tetratricopeptide)重复(TPR)α-螺旋重复蛋白、具有结合NAD(P)的Rossmann折叠结构域的跨膜蛋白。
此外，附着至脂质双层的单型/外周蛋白或其它整合蛋白和肽也可以被用作跨膜蛋白。实例可以包括α/β-水解酶、膜联蛋白、Bet V1样蛋白、含有C1结构域的蛋白、含有C2结构域的蛋白、辅酶A依赖性的酰基转移酶、含有CRAL-TRIO结构域的蛋白、DNA酶I样蛋白、纤维蛋白原、FYVE/PHD锌指蛋白、半乳糖结合结构域样蛋白、糖脂转移蛋白、免疫球蛋白样超家族(E Set)蛋白、脂质运载蛋白(Lipocalin)、脂氧合酶、PGBD超家族、PH结构域样蛋白、磷脂酰肌醇3-/4-激酶、PLC样磷酸二酯酶、磷酸酪氨酸蛋白磷酸酶II、含有P-环的三磷酸核苷水解酶、蛋白激酶超家族、含有PX结构域的蛋白、皂化蛋白(Saposin)、突触核蛋白和桶状转录因子(Transcriptional factor tubby)。
在本公开内容中使用的表述“跨膜结构域”至少包括膜结合蛋白或跨膜蛋白的跨膜区域。此外，该跨膜结构域还可以包括膜结合蛋白或跨膜蛋白的近膜结构域(JMD)和/或胞质尾。
或者，在一些实施方案中，该跨膜结构域是合成的。在一些方面，该合成的跨膜结构域主要包含疏水残基，诸如亮氨酸和缬氨酸。在一些方面，苯丙氨酸、色氨酸和缬氨酸的三联体将出现在合成跨膜结构域的每一端。
优选的跨膜结构域可以是衍生自流感病毒血凝素(HA)、CD80、Toll样受体4(TLR4)或COVID-19刺突蛋白的那些。具体实例可以包括：由流感病毒血凝素的柔性近膜区域或柔性接头、跨膜结构域和胞质尾组成的蛋白“HA(柔性-TM-Cyt)”；由人CD80的跨膜结构域和胞质尾组成的蛋白；由跨膜结构域(TM)和Toll/白介素-1受体结构域(TIR)组成的蛋白；以及由COVID-19刺突(S)的近膜结构域(JMD)和TM组成的蛋白。
如本文中使用的，“信号序列”(有时被称作信号肽、靶向信号、定位信号、定位序列、转运肽、前导序列或前导肽)是多核苷酸或多肽，取决于上下文。信号序列的长度为约9-200个核苷酸或3-70个氨基酸，其任选地掺入在编码区或蛋白的5'或N-端处。一些信号序列从蛋白切割下，例如在蛋白被运输到期望的部位以后被信号肽酶切割。
在一些实施方案中，可以采用IL-2、尤其是人IL-2的信号序列。在另一个实施方案中，可以使用COVID-19刺突蛋白的信号序列。
冠状病毒结构蛋白、跨膜结构域和/或信号序列可以直接地或间接地融合。在一个实施方案中，一个或两个接头可以插进它们之间。
冠状病毒结构蛋白、跨膜结构域和/或信号序列也可以被截短和被短接头替换。在一些实施方案中，冠状病毒结构蛋白、跨膜结构域和/或信号序列包括一个或多个肽接头。
短接头的一个实例由2-25个氨基酸(例如2、3、4、5或6个氨基酸)组成。通常，其长度为2-15个氨基酸，诸如SG、GS、SGG、GGS SGSG和TRGGS。在某些情况下，所述接头可以仅由一个氨基酸组成，所述氨基酸诸如甘氨酸(G)、丝氨酸(S)和半胱氨酸(C)。
当冠状病毒结构蛋白通过化学交联剂化学缀合至跨膜结构域和/或信号序列时，所述交联剂的实例包括但不限于SMPH、磺基-MBS、磺基-EMCS、磺基-GMBS、磺基-SIAB、磺基-SMPB、磺基-SMCC、SVSB和SIA。也可以采用在市场上可获得的化学交联剂。
IgG-衍生的物质也可以被用作接头。IgG-衍生的物质的实例包括IgG1至IgG4，其包含(i)全(铰链-CH2CH3)、(ii)半(铰链-CH3)和(iii)短(仅12氨基酸铰链)。优选实例是IgG4-CH3。
如本文中使用的，“甲病毒”意在指属于病毒的披膜病毒科的含有RNA的病毒。示例性的披膜病毒科病毒包括但不限于东方马脑炎病毒(EEEV)、委内瑞拉马脑炎病毒(VEEV)、沼泽地病毒(Everglades Virus)、穆坎布病毒(Mucambo Virus)、皮舒纳病毒(PixunaVirus)、西方马脑炎病毒(WEEV)、辛德毕斯病毒(Sindbis Virus)、塞姆利基森林病毒(Semliki Forest Virus)、米德尔堡病毒、基孔肯雅病毒(CHIKV)、阿尼昂尼昂病毒、罗斯河病毒、巴马森林病毒、格塔病毒(Getah Virus)、鹭山病毒、贝巴鲁病毒、马亚罗病毒、乌纳病毒(Una Virus)、奥拉病毒(Aura Virus)、怀塔罗瓦病毒(Whataroa Virus)、巴班肯病毒(Babanki Virus)、孜拉加奇病毒(Kyzylagach Virus)、高地J病毒、摩根堡病毒(FortMorgan Virus)、恩杜茂病毒(Ndumu Virus)、博吉河病毒(Buggy CreekVirus)和奥克尔布病毒(Ockelbo virus)。
“甲病毒结构蛋白”意指与天然存在的病毒衣壳或包膜蛋白具有至少约80％的氨基酸序列同一性的多肽或其片段。在一个实施方案中，所述甲病毒结构蛋白与东方马脑炎病毒(EEEV)、委内瑞拉马脑炎病毒(VEEV)、沼泽地病毒、穆坎布病毒、皮舒纳病毒、西方马脑炎病毒(WEEV)、辛德毕斯病毒、塞姆利基森林病毒、米德尔堡病毒、基孔肯雅病毒(CHIKV)、阿尼昂尼昂病毒、罗斯河病毒、巴马森林病毒、格塔病毒、鹭山病毒、贝巴鲁病毒、马亚罗病毒、乌纳病毒、奥拉病毒、怀塔罗瓦病毒、巴班肯病毒、孜拉加奇病毒、高地J病毒、摩根堡病毒、恩杜茂病毒或博吉河病毒具有至少约85％、90％、95％或更大的氨基酸序列同一性。甲病毒结构蛋白的野生型氨基酸序列可以得自GenBank。
在具体实施方案中，所述甲病毒是CHIKV，例如CHIKV株37997或LR2006 OPY-1。在其它实施方案中，所述甲病毒是VEEV，例如VEEV株TC-83。
“甲病毒复制子”意指可以在靶细胞中指导其自身体内扩增的RNA分子。该复制子编码催化RNA扩增的聚合酶(nspl、nsp2、nsp3、nsp4)，并含有复制所需的顺式RNA序列，该序列被编码的聚合酶识别和利用。甲病毒复制子通常含有以下有序元件：5'UTR、编码甲病毒非结构蛋白(nspl、nsp2、nsp3、nsp4)的序列、3'UTR和多聚A信号。甲病毒复制子还含有一个或多个指导目标基因表达的病毒亚基因组启动子。那些序列可以具有在现有技术中教导的一种或多种突变。
由本公开内容提供的甲病毒复制子可以具有在图1中所示的构建体。
在本公开内容中，“包含(comprises)”、“包含(comprising)”、“含有”和“具有”等可以具有在美国专利法中赋予它们的含义，并且可以意指“包括(includes)”、“包括(including)”等；“基本上由...组成(consisting essentially of)”或“基本上由...组成(consists essentially)”同样具有在美国专利法中所赋予的含义，且该术语是开放式的，允许存在超过所述的对象，只要所述对象的基本或新颖特征不被超过所述对象的存在改变，但现有技术实施方案除外。
“片段”意指多肽或核酸分子的部分。该部分优选地含有参考核酸分子或多肽的整个长度的至少10％、20％、30％、40％、50％、60％、70％、80％或90％。片段可以含有10、20、30、40、50、60、70、80、90或100、200、300、400、500、600、700、800、900或1000个核苷酸或氨基酸。
“参考”意指标准或对照条件。
“参考序列”是用作序列对比基础的规定序列。参考序列可以是指定序列的子集或全部；例如全长cDNA或基因序列的区段，或完整的cDNA或基因序列。对于多肽，参考多肽序列的长度通常将为至少约16个氨基酸，优选地至少约20个氨基酸，更优选地至少约25个氨基酸，和甚至更优选约35个氨基酸、约50个氨基酸或约100个氨基酸。对于核酸，参考核酸序列的长度通常将为至少约50个核苷酸，优选地至少约60个核苷酸，更优选地至少约75个核苷酸，且甚至更优选地约100个核苷酸或约300个核苷酸或在其附近或其之间的任何整数。
通常使用序列分析软件(例如威斯康辛大学生物技术中心(University ofWisconsin Biotechnology Center)，1710University Avenue,Madison,Wis.53705的遗传学计算机组的序列分析软件包(Sequence Analysis Software Package of the GeneticsComputer Group)、BLAST、BESTFIT、GAP或PILEUP/PRETTYBOX程序)来测量序列同一性。此类软件通过对各种置换、缺失和/或其它修饰分配同源性程度来匹配相同或相似的序列。保守置换通常包括在以下组内的置换：甘氨酸、丙氨酸；缬氨酸、异亮氨酸、亮氨酸；天冬氨酸、谷氨酸、天冬酰胺、谷氨酰胺；丝氨酸、苏氨酸；赖氨酸、精氨酸；以及苯丙氨酸、酪氨酸。在确定同一性程度的示例性方案中，可以使用BLAST程序，其中e<"3>和e<"100>之间的概率评分指明密切相关的序列。
“有效量”意指相对于未经治疗的患者，改善疾病症状所需的药剂的量。用于实践本发明以预防或治疗疾病的活性化合物的有效量根据施用方式，受试者的年龄、体重和一般健康而变化。最终，主治医师或兽医将决定适当的量和剂量方案。此种量被称为“有效”量。
通过全身施用可以获得令人满意的效果，所述全身施用例如肌肉内施用、皮下施用或静脉内施用1-4次，以每次103-1010感染单位(IU)或0.01-500μg的量，优选每次105-1010IU或0.1-100μg，例如每次107-109IU或1-50μg。可以优选地将所述复制子配制在适合以常规方式施用的疫苗组合物中。
“受试者”意指哺乳动物，包括但不限于人或非人哺乳动物，诸如牛、马、犬、羊或猫。
如本文中使用的，术语“治疗(treat)”、“治疗(treating)”、“治疗(treatment)”等是指减轻或改善与其相关的障碍和/或症状。应当理解，尽管没有排除，但是治疗障碍或病症并不需要完全消除与其相关的障碍、病症或症状。
如本文中使用的，术语“预防(prevent)”、“预防(preventing)”、“预防(prevention)”、“预防性治疗”等是指降低在受试者中发生障碍或病症的可能性，所述受试者不具有障碍或病症，但是处于发生障碍或病症的风险中或易于发生障碍或病症。
除非特别说明或者从上下文显而易见，否则如本文中使用的术语“或”被理解为包含性的。
除非特别说明或者从上下文显而易见，否则如本文中使用的术语“一个(a)”、“一种(an)”和“该(the)”被理解为单数或复数的。
本领域将承认，在说明书和权利要求书中描述的多核苷酸序列将在代表性DNA序列中列举“T”，但是当该序列代表RNA时，则“T”将代替“U”。
本文提供的任何疫苗组合物或方法可以与本文提供的任何其它疫苗组合物和方法中的一种或多种组合。
术语“载体”是指核酸序列可以通过其在生物体、细胞或细胞组分之间传播和/或转移的方式。载体包括质粒、病毒、噬菌体、原病毒、噬菌粒、转座子、人工染色体等，它们自主复制或者可以整合到宿主细胞的染色体中。载体也可以是裸RNA多核苷酸、裸DNA多核苷酸、在同一链内由DNA和RNA组成的多核苷酸、聚赖氨酸缀合的DNA或RNA、肽缀合的DNA或RNA、脂质体缀合的DNA等，其不能自主复制。在许多但非全部的常见实施方案中，本发明的载体为质粒或杆粒。
通常，待表达的核酸分子“可操作地连接”至启动子和/或增强子，并且受到启动子和/或增强子的转录调节控制。
转染的方法和表达载体的选择将取决于所选的宿主系统。转染方法描述于例如Ausubel等人(上文)；表达载体可以选自例如在Cloning Vectors:A Laboratory Manual(P.H.Pouwels等人,1985,Supp.1987)中提供的那些。在本段落中引用的参考文献通过参考并入本文。
存在多种表达系统用于产生本发明的构建体。可用于产生所述构建体的表达载体包括而不限于染色体载体、附加体载体和病毒衍生的载体，例如衍生自细菌质粒、噬菌体、转座子、酵母附加体、插入元件、酵母染色体元件、病毒的载体以及衍生自其组合的载体，所述病毒诸如甲病毒(例如切昆贡亚病毒(CHIKV)和委内瑞拉马脑炎病毒(VEEV))、杆状病毒、诸如SV40等乳多泡病毒、痘苗病毒、腺病毒、禽痘病毒(fowl pox virus)、假狂犬病病毒和逆转录病毒。
本文使用的构建体和/或载体包含编码非结构蛋白nsp1、nsp2、nsp3和nsp4的甲病毒多核苷酸和编码包含与如上面讨论的信号序列和/或跨膜结构域融合的冠状病毒结构蛋白的多肽的目标基因。所述构建体或载体的具体实例为图1所示的。
载体可以是例如噬菌体、质粒、病毒载体或逆转录病毒载体。包含核苷酸的构建体和/或载体应当可操作地连接到适当的启动子，诸如CMV启动子、噬菌体λPL启动子、大肠杆菌lac、phoA和tac启动子、SV40早期和晚期启动子，并且逆转录病毒LTR的启动子为非限制性实例。取决于宿主细胞和/或期望的表达速率，其它合适的启动子将是熟练的技术人员已知的。表达构建体将进一步含有用于转录起始、终止的位点，以及在转录的区域中用于翻译的核糖体结合位点。由构建体表达的转录本的编码部分将优选包括在起始处的翻译起始密码子和适当位于待翻译多肽末端处的终止密码子。
载体将优选地包括至少一种选择标记。此类标记包括二氢叶酸还原酶，用于真核细胞培养的G418或新霉素抗性以及用于在大肠杆菌和其它细菌中培养的四环素、卡那霉素或氨苄青霉素抗性基因。在载体中优选的是病毒载体，诸如杆状病毒、痘病毒(例如痘苗病毒、禽痘病毒(avipox virus)、金丝雀痘病毒、禽痘病毒(fowlpox virus)、浣熊痘病毒、猪痘病毒等)、腺病毒(例如犬腺病毒)、疱疹病毒和逆转录病毒。可以与本发明一起使用的其它载体包含用于细菌中的载体，其包含pQE70、pQE60和pQE-9、pBluescript载体、Phagescript载体、pNH8A、pNH16a、pNH18A、pNH46A、ptrc99a、pKK223-3、pKK233-3、pDR540、pRIT5。优选的真核载体为pFastBacl pWINEO、pSV2CAT、pOG44、pXTl和pSG、pSVK3、pBPV、pMSG和pSVL。其它合适载体对于熟练的技术人员而言将是显而易见的。
可被制备并用于转染的重组构建体可以将病毒蛋白(包括本文描述的那些)表达到真核细胞和/或原核细胞中。因此，在一个实施方案中，本公开内容提供了处于允许形成甲病毒复制子颗粒的条件下的宿主细胞，其包含含有编码甲病毒结构蛋白(包括衣壳、E3、E2、6K和El或其部分)的核酸的一种或多种载体以及包含编码甲病毒nsp1、nsp2、nsp3和nsp4的核酸和至少一个冠状病毒目标基因的载体。
在一个实施方案中，所述载体是重组杆状病毒。在另一个实施方案中，将所述重组杆状病毒转染到昆虫细胞中。在优选的实施方案中，所述细胞是昆虫细胞。在另一个实施方案中，所述昆虫细胞是Sf9细胞。
一种用于多肽生产的特定细菌表达系统是大肠杆菌pET表达系统(Novagen,Inc.,Madison,Wis)。根据该表达系统，将编码多肽的DNA以被设计为允许表达的取向插入到pET载体中。由于编码此种多肽的基因是在T7调节信号的控制下，所以多肽的表达通过在宿主细胞中诱导T7 RNA聚合酶的表达来实现。这通常使用响应于IPTG诱导而表达T7RNA聚合酶的宿主菌株来实现。一旦产生，然后便根据本领域已知的标准方法分离重组多肽，所述方法例如本文描述的那些。
根据所选择的载体和宿主细胞，通过在藉此表达重组蛋白并产生甲病毒复制子的条件下培养被载体转染的宿主细胞来生产构建体，并且形成了含有与甲病毒结构蛋白的颗粒一起包装的甲病毒复制子的构建体。在一个实施方案中，本发明包含产生构建体的方法，其涉及将包含编码甲病毒非结构蛋白nsp1、nsp2、nsp3和nsp4的多核苷酸和至少一个编码包含与信号序列和/或跨膜结构域融合的冠状病毒结构蛋白的多肽的目标基因以及至少一种各自编码至少一种甲病毒结构蛋白的载体共转染到合适的宿主细胞中，并在允许构建体形成的条件下表达所述甲病毒结构蛋白。在另一个实施方案中，真核细胞选自酵母、昆虫、两栖动物、禽类或哺乳动物细胞。适当的生长条件的选择在本领域的技术或本领域普通技术人员的技术范围内。
培养产生本发明的甲病毒复制子颗粒的细胞的方法包括但不限于分批式、分批补料式、连续式和灌注式细胞培养技术。在一个实施方案中，在细胞于其中繁殖并表达蛋白(例如重组蛋白)以供纯化和分离的生物反应器或发酵室中培养用编码甲病毒复制子的载体和包含编码衣壳的多肽的载体以及包含编码包膜蛋白的多核苷酸的载体(诸如衍生自CHIKV或VEEV的那些)共转染的细胞。细胞培养通常在无菌的、受控制的温度和气氛条件下进行。生物反应器是用于培养细胞的室，其中诸如温度、气氛、搅拌和/或pH等环境条件可被监测。在一个实施方案中，所述生物反应器是不锈钢室。在另一个实施方案中，所述生物反应器是预灭菌的塑料袋(例如Cellbag.RTM.,Wave Biotech,Bridgewater,N.J.，引用的文件的内容通过参考并入本文)。在其它实施方案中，所述预灭菌的塑料袋为约10L至1000L袋。
在另一个实施方案中，可以通过本领域已知的常规程序从模板DNA序列产生RNA分子，诸如甲病毒复制子。体外转录(IVT)方法允许RNA分子的模板指导合成。IVT方法允许合成大量的RNA转录本。通常，IVT利用包含目标序列上游的启动子序列的DNA模板。该启动子序列最常见地具有细菌噬菌体来源，诸如T7、T3或SP6启动子序列，但许多其它启动子序列是可耐受的，包括从头设计的那些。DNA模板的转录通常最好通过使用对应于特定噬菌体启动子序列的RNA聚合酶来实现。示例性的RNA聚合酶包括但不限于T7RNA聚合酶、T3 RNA聚合酶或SP6 RNA聚合酶等。IVT通常在dsDNA处开始，但可以在单链上进行。用于体外转录的试剂盒诸如T7转录试剂盒(RiboMaxTM Express Large Scale RNAproduction System,Promega(WIUSA))。
如本文中使用的，术语“药学上可接受的载体”意指适合施用给人或其它脊椎动物的一种或多种相容的固体或液体填充剂、稀释剂或包封物质，包括任意的和所有的水性溶剂(例如水、醇/水溶液、盐水溶液、胃肠外媒介物诸如氯化钠和林格氏右旋糖)、非水性溶剂(例如丙二醇、聚乙二醇、植物油和可注射的有机酯诸如油酸乙酯)、分散介质、包衣剂、表面活性剂、抗氧化剂、防腐剂(例如抗细菌剂或抗真菌剂、抗氧化剂、螯合剂和惰性气体)、等渗剂、吸收延迟剂、盐、药物、药物稳定剂、凝胶、粘合剂、赋形剂、崩解剂、润滑剂、甜味剂、矫味剂、染料、流体和营养补充剂、此类材料及其组合，如本领域普通技术人员已知的。根据众所周知的参数调整疫苗组合物中各种组分的pH和准确浓度。
包封物质是指多核苷酸或载体包装在其中的递送媒介物，诸如复制子颗粒(例如在美国专利公开号2019-0185822中描述的甲病毒复制子颗粒，该文件的内容通过参考并入)和脂质递送系统(例如脂质体)。
在一些实施方案中，本公开内容的疫苗组合物或制剂包含脂质递送系统，例如脂质体、脂质复合物、脂质纳米颗粒或其任意组合。可以使用一种或多种脂质体、脂质复合物或脂质纳米颗粒来配制本文所述的多核苷酸，诸如α病毒复制子。脂质体、脂质复合物或脂质纳米颗粒可以被用于改善多核苷酸指导的蛋白生产的效力，因为这些制剂可以增加多核苷酸对细胞的转染；和/或增加编码的蛋白的翻译。脂质体、脂质复合物或脂质纳米颗粒也可以被用于增加多核苷酸的稳定性。
脂质体是人工制备的囊泡，其可以主要由脂质双层组成并可以被用作用于施用药物制剂的递送媒介物。脂质体可以具有不同的大小。多层囊泡(MLV)可以具有数百纳米的直径，并且可以含有一系列被狭窄水性隔室隔开的同心双层。小单室囊泡(SUV)可以具有小于50nm的直径，而大单层囊泡(LUV)可以具有50至500nm之间的直径。脂质体设计可以包括但不限于调理素或配体以改善脂质体与不健康组织的附着或者活化诸如但不限于胞吞作用的事件。脂质体可以含有低或高pH值以便改善药物制剂的递送。
脂质体的形成可能取决于所包埋的药物制剂和脂质体成分，脂质囊泡在其中分散的介质的性质，包埋物质的有效浓度及其潜在毒性，在囊泡的的应用和/或递送过程中涉及的任何额外过程，对于预期应用而言囊泡的最佳尺寸、多分散性和贮存期限，以及安全且有效的脂质体产品的批次间再现性和放大生产等。
在一些实施方案中，本文所述的诸如甲病毒复制子等多核苷酸可以被脂质体包封和/或它可以被包含在随后可以被脂质体包封的水性核心中。
在一些实施方案中，本文所述的诸如甲病毒复制子等多核苷酸可以被配制在阳离子水包油乳剂中，其中乳剂颗粒包含油核心和阳离子脂质，其可以与将分子锚定至乳剂颗粒的多核苷酸相互作用。在某些实施方案中，本文所述的多核苷酸可以被配制在油包水乳剂中，所述乳剂包含其中分散有亲水相的连续疏水相。
在一些实施方案中，本文所述的诸如甲病毒复制子等多核苷酸可以被配制在脂质-聚阳离子复合物中。作为非限制性实例，所述聚阳离子可以包括阳离子肽或多肽，诸如但不限于聚赖氨酸、聚鸟氨酸和/或聚精氨酸以及阳离子肽。
在一些实施方案中，本文所述的诸如甲病毒复制子等多核苷酸可以被配制在脂质纳米颗粒(LNP)中。
脂质纳米颗粒制剂通常包含一种或多种脂质。在一些实施方案中，所述脂质是阳离子脂质或可电离脂质。在一些实施方案中，脂质纳米颗粒制剂进一步包含其它组分，包括磷脂、结构脂质、季胺化合物和能够减少颗粒聚集的分子例如PEG或PEG修饰的脂质。在一些实施方案中，脂质组合物中阳离子脂质和可电离脂质的量在从约0.01mol％至约99mol％的范围内。
LNP含有pH敏感的可电离的阳离子脂质，其吸引阴离子核酸以形成自组装纳米颗粒的核心，以确保高度封装。在生理pH下，LNP是中性的，消除用永久性阳离子分子所看到的毒性机制。
这些相同的pH敏感的脂质负责响应核内体的酸性环境，并触发核内体的破坏和核酸向细胞中的释放。
这种基于复制子的疫苗技术是用于疫苗接种的独特的平台技术，因为RNA可以自我扩增以产生疫苗抗原并递送到细胞器官中。此外，这种基于复制子的疫苗技术克服了通常与基于DNA的疫苗相关的挑战，诸如基因组整合的风险或施用(例如电穿孔)所需的高剂量和装置，并预计基于自我复制系统用最小剂量获得比mRNA技术更高的免疫原性。
根据本发明，新型抗原活性蛋白/多肽也可用于生产用于诊断和保护免于冠状病毒的抗体，同时使ADE的可能性最小化。本文公开的蛋白/多肽包括编码与信号序列和/或与跨膜结构域(TMD)序列融合的冠状病毒RBD的最小序列，预期最大化免疫原性和最小化ADE。
将参考以下实施例来详细描述本发明，然而所述实施例并不预期限制本申请的范围。
实施例1
编码下面显示的构建体1-8的每个基因由Integrated DNA Technologies,Inc.(https://www.idtdna.com/pages)合成。
1.与hIL-2信号序列和HA(柔性结构域-跨膜(TM)-胞质尾(Cyt))融合的COVID-19-RBD序列的构建体
[化学式1]
构建体1
MYRMQLLSCIALSLALVTNSVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSGVKLESMGIYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI(SEQ ID NO:1)；
hIL-2信号序列：MYRMQLLSCIALSLALVTNS(SEQ ID NO:2)；
COVID19-RBD：VRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKS(SEQID NO:3)；
HA(柔性-TM-Cyt)：GVKLESMGIYQILAIYSTVASSLVLLVSLGAISFWMC SNGSLQCRICI(SEQID NO:4)；
hIL-2：人白介素-2；
HA(柔性-TM-Cyt)：流感病毒的多肽。
2.与hIL-2信号序列和HA(柔性-TM-Cyt)融合的COVID-19-RBD序列的构建体
[化学式2]
构建体2
MYRMQLLSCIALSLALVTNSCVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSCGVKLESMGIYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI(SEQ ID NO:5)；
在示意图中的“C”：接头(半胱氨酸)；
在氨基酸序列中的接头标有下划线。
3.具有接头的与hIL-2信号序列和HA(柔性-TM-Cyt)融合的COVID-19-RBD序列的构建体
[化学式3]
构建体3
MYRMQLLSCIALSLALVTNSVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSGSGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGSGVKLESMGIYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI(SEQ ID NO:6)；
在示意图中的“GS”：接头(GGATCC)；
在氨基酸序列中接头标有下划线；
人IgG4 CH3：
GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:7)。
4.具有接头的与hIL-2信号序列和HA(柔性-TM-Cyt)融合的COVID-19-RBD序列的构建体
[化学式4]
构建体4
MYRMQLLSCIALSLALVTNSCVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSCGSGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGSGVKLESMGIYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI(SEQ ID NO:8)；
在示意图中的“C”、“CGS”和“GS”：接头；
在氨基酸序列中接头标有下划线。
5.与hIL-2信号序列和人TLR4(TM-Toll/白介素-1受体结构域(TIR))融合的COVID-19-RBD序列的构建体
[化学式5]
构建体5
MYRMQLLSCIALSLALVTNSVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSGSKTIIGVSVLSVLVVSVVAVLVYKFYFHLMLLAGCIKYGRGENIYDAFVIYSSQDEDWVRNELVKNLEEGVPPFQLCLHYRDFIPGVAIAANIIHEGFHKSRKVIVVVSQHFIQSRWCIFEYEIAQTWQFLSSRAGIIFIVLQKVEKTLLRQQVELYRLLSRNTYLEWEDSVLGRHIFWRRLRKALLDGKSWNPEGTVGTGCNWQEATSI(SEQ ID NO:9)；
人TLR4(TM-TIR)：
KTIIGVSVLSVLVVSVVAVLVYKFYFHLMLLAGCIKYGRGENIYDAFVIYSSQDEDWVRNELVKNLEEGVPPFQLCLHYRDFIPGVAIAANIIHEGFHKSRKVIVVVSQHFIQSRWCIFEYEIAQTWQFLSSRAGIIFIVLQKVEKTLLRQQVELYRLLSRNTYLEWEDSVLGRHIFWRRLRKALLDGKSWNPEGTVGTGCNWQEATSI(SEQ ID NO:10)。
6.与hIL-2信号序列和人TLR4(TM-TIR)融合的COVID-19-RBD基因序列的构建体[化学式6]
构建体6
MYRMQLLSCIALSLALVTNSCVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSCGSKTIIGVSVLSVLVVSVVAVLVYKFYFHLMLLAGCIKYGRGENIYDAFVIYSSQDEDWVRNELVKNLEEGVPPFQLCLHYRDFIPGVAIAANIIHEGFHKSRKVIVVVSQHFIQSRWCIFEYEIAQTWQFLSSRAGIIFIVLQKVEKTLLRQQVELYRLLSRNTYLEWEDSVLGRHIFWRRLRKALLDGKSWNPEGTVGTGCNWQEATSI(SEQ ID NO:11)；
在示意图中的“C”和“CGS”：接头；
在氨基酸序列中接头标有下划线。
7.具有接头的与hIL-2信号序列和人TLR4(TM-TIR)融合的COVID-19-R0BD序列的构建体
[化学式7]
构建体7
MYRMQLLSCIALSLALVTNSVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSGSGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGSKTIIGVSVLSVLVVSVVAVLVYKFYFHLMLLAGCIKYGRGENIYDAFVIYSSQDEDWVRNELVKNLEEGVPPFQLCLHYRDFIPGVAIAANIIHEGFHKSRKVIVVVSQHFIQSRWCIFEYEIAQTWQFLSSRAGIIFIVLQKVEKTLLRQQVELYRLLSRNTYLEWEDSVLGRHIFWRRLRKALLDGKSWNPEGTVGTGCNWQEATSI(SEQ ID NO:12)；
在示意图中的“GS”：接头；
在氨基酸序列中接头标有下划线。
8.具有接头的与hIL-2信号序列和人TLR4(TM-TIR)融合的COVID19 RBD序列的构建体
[化学式8]
构建体8
MYRMQLLSCIALSLALVTNSCVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSCGSGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKGSKTIIGVSVLSVLVVSVVAVLVYKFYFHLMLLAGCIKYGRGENIYDAFVIYSSQDEDWVRNELVKNLEEGVPPFQLCLHYRDFIPGVAIAANIIHEGFHKSRKVIVVVSQHFIQSRWCIFEYEIAQTWQFLSSRAGIIFIVLQKVEKTLLRQQVELYRLLSRNTYLEWEDSVLGRHIFWRRLRKALLDGKSWNPEGTVGTGCNWQEATSI(SEQ ID NO:13)；
在示意图中的“C、“CGS”和“GS”：接头；
在氨基酸序列中接头标有下划线。
关于hIL-2信号序列、COVID19-RBD、人IgG4 CH3和HA(柔性-TM-Cyt)的信息也可得自美国国家生物技术信息中心(National Center for Biotechnology Information)网站和UniProt网站。
hIL-2信号序列
https://www.ncbi.nlm.nih.gov/protein/P60568
UniProtKB/Swiss-Prot：P60568.1；
COVID19-RBD
https://www.ncbi.nlm.nih.gov/nuccore/NC_045512
NCBI参考序列：NC_045512.2；
HA(柔性-TM-Cyt)
https://www.ncbi.nlm.nih.gov/protein/P03452
UniProtKB/Swiss-Prot：P03452.2；
人IgG4
https://www.uniprot.org/uniprot/P01861
UniProtKB：P01861。
实施例2
编码下面显示的构建体A-O的每个基因都由Integrated DNA Technologies,Inc.(https://www.idtdna.com/pages)合成。
[化学式9]
构建体A：SS1-RBD1(330-521)(SEQ ID NO:37)
构建体B：SS2-RBD1(330-521)-TM2(SEQ ID NO:38)
构建体C：SS1-RBD1(330-521)-GS-接头-GS-TM1(SEQ ID NO:39)
构建体D：SS1-RBD1(330-521)-GS-接头-GS-TM2(SEQ ID NO:40)
构建体E：SS1-RBD2(330-530)-GS-接头-GS-TM1(SEQ ID NO:41)
构建体F：SS1-RBD2(330-530)-GS-接头-GS-TM2(SEQ ID NO:42)
构建体G：SS2-RBD1(330-521)-GS-接头-GS-TM1(SEQ ID NO:43)
构建体H：SS2-RBD1(330-521)-GS-接头-GS-TM2(SEQ ID NO:44)
构建体I：SS2-RBD2(330-530)-GS-接头-GS-TM1(SEQ ID NO:45)
构建体J：SS2-RBD2(330-530)-GS-接头-GS-TM2(SEQ ID NO:46)
构建体K：SS3-RBD3(327-531)-TM2(SEQ ID NO:47)
构建体L：SS1-RBD2(330-521)-TM2(SEQ ID NO:48)
构建体M：SS1-RBD3(327-531)-C-TM3(SEQ ID NO:49)
构建体N：SS3-RBD3(327-531)-TM3(SEQ ID NO:50)
构建体O：SS3-RBD1(330-521)-TM3(SEQ ID NO:51)。
信号序列
SS1：COVID-19信号序列(1-15)：MFVFLVLLPLVSSQC(SEQ ID NO:14)
SS2：hIL-2信号序列：MYRMQLLSCIALSLALVTNS(SEQ ID NO:15)
SS3：COVID-19信号序列(1-13)：MFVFLVLLPLVSS(SEQ ID NO:16)。
COVID-19-RBD
RBD1：COVID-19RBD氨基酸位置330和521
RBD2：COVID-19RBD氨基酸位置330和530
RBD3：COVID-19RBD氨基酸位置327和531。
RBD1：
PNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAP(SEQ ID NO:17)。
RBD2：
PNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKS(SEQ ID NO:18)。
RBD3：
VRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKST(SEQ ID NO:19)。
接头
人IgG4-CH3
GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO:20)。
跨膜-细胞质(TM-Cyt)
TM1：人CD80*(TM-Cyt)：
LLPSWAIT LISVNGIFVI CCLTYCFAPR CRERRRNERLRRESVRPV(SEQ ID NO:21)
*CD80：https://www.ncbi.nlm.nih.gov/protein/NP_005182；
TM2：HA(柔性-TM-Cyt)：
GVKLESMGIYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI(SEQ ID NO:22)；
TM3：COVID19(JMD-TM)：
GKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT(SEQ ID NO:23)。
刺突蛋白的近膜结构域(JMD)是靠近跨膜结构域的富含芳族氨基酸的区域，该区域在所有冠状病毒中都是高度保守的(Howard等人,JOURNAL OF VIROLOGY,2008年3月，第2883-2894页，第82卷，第6期，该参考文献的内容通过参考并入本文)。
JMD：GKYEQYIKWPWYIWL(SEQ ID NO:24)；
TM：GFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT(SEQ ID NO:25)。
实施例3
复制子载体的制备。在图1中显示了甲病毒复制子的示意构建体。
将实施例1和2中制备的每个目标基因DNA都克隆到处于亚基因组(SG)启动子控制下的VEEV复制子载体中。通过在AscI和SbfI限制位点插入来创建编码每个片段的VEEV复制子质粒，以获得全长VEEV TC-83复制子构建体。在图2中显示了全长VEEV TC-83质粒载体的一个实例。
SG启动子、5'UTR、3'UTR和多聚A尾的核苷酸序列如下。使用那些DNA序列作为模板来得到RNA序列。
SG启动子：cctgaatggactacgacatagtctagtccgccaag(SEQ ID NO:26)
5'UTR：ataggcggcgcatgagagaagcccagaccaattacctacccaaa(SEQ ID NO:27)
3'UTR：gcgatcgcatacagcagcaattggcaagctgcttacatagaactcgcggcgattggcatgccgccttaaaatttttattttatttttcttttcttttccgaatcggattttgtttttaatatttc(SEQ ID NO:28)
多聚A尾：aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa(SEQID NO:29)。
VEEVTC-83复制子nsP1-4氨基酸序列如下。
MEKVHVDIEEDSPFLRALQRSFPQFEVEAKQVTDNDHANARAFSHLASKLIETEVDPSDTILDIGSAPARRMYSKHKYHCICPMRCAEDPDRLYKYATKLKKNCKEITDKELDKKMKELAAVMSDPDLETETMCLHDDESCRYEGQVAVYQDVYAVDGPTSLYHQANKGVRVAYWIGFDTTPFMFKNLAGAYPSYSTNWADETVLTARNIGLCSSDVMERSRRGMSILRKKYLKPSNNVLFSVGSTIYHEKRDLLRSWHLPSVFHLRGKQNYTCRCETIVSCDGYVVKRIAISPGLYGKPSGYAATMHREGFLCCKVTDTLNGERVSFPVCTYVPATLCDQMTGILATDVSADDAQKLLVGLNQRIVVNGRTQRNTNTMKNYLLPVVAQAFARWAKEYKEDQEDERPLGLRDRQLVMGCCWAFRRHKITSIYKRPDTQTIIKVNSDFHSFVLPRIGSNTLEIGLRTRIRKMLEEHKEPSPLITAEDIQEAKCAADEAKEVREAEELRAALPPLAADFEEPTLEADVDLMLQEAGAGSVETPRGLIKVTSYAGEDKIGSYAVLSPQAVLKSEKLSCIHPLAEQVIVITHSGRKGRYAVEPYHGKVVVPEGHAIPVQDFQALSESATIVYNEREFVNRYLHHIATHGGALNTDEEYYKTVKPSEHDGEYLYDIDRKQCVKKELVTGLGLTGELVDPPFHEFAYESLRTRPAAPYQVPTIGVYGVPGSGKSGIIKSAVTKKDLVVSAKKENCAEIIRDVKKMKGLDVNARTVDSVLLNGCKHPVETLYIDEAFACHAGTLRALIAIIRPKKAVLCGDPKQCGFFNMMCLKVHFNHEICTQVFHKSISRRCTKSVTSVVSTLFYDKRMRTTNPKETKIVIDTTGSTKPKQDDLILTCFRGWVKQLQIDYKGNEIMTAAASQGLTRKGVYAVRYKVNENPLYAPTSEHVNVLLTRTEDRIVWKTLAGDPWIKILTAKYPGNFTATIEEWQAEHDAIMRHILERPDPTDVFQNKANVCWAKALVPVLKTAGIDMTTEQWNTVDYFETDKAHSAEIVLNQLCVRFFGLDLDSGLFSAPTVPLSIRNNHWDNSPSPNMYGLNKEVVRQLSRRYPQLPRAVATGRVYDMNTGTLRNYDPRINLVPVNRRLPHALVLHHNEHPQSDFSSFVSKLKGRTVLVVGEKLSVPGKKVDWLSDQPEATFRARLDLGIPGDVPKYDIVFINVRTPYKYHHYQQCEDHAIKLSMLTKKACLHLNPGGTCVSIGYGYADRASESIIGAIARQFKFSRVCKPKSSHEETEVLFVFIGYDRKARTHNPYKLSSTLTNIYTGSRLHEAGCAPSYHVVRGDIATATEGVIINAANSKGQPGG GVCGALYKKFPESFDLQPIEVGKARLVKGAAKHIIHAVGPNFNKVSEVEGDKQLAEAYESIAKIVNDNNYKSVAIP LLSTGIFSGNKDRLTQSLNHLLTALDTTDADVAIYCRDKKWEMTLKEAVARREAVEEICISDDSSVTEPDAELVRV HPKSSLAGRKGYSTSDGKTFSYLEGTKFHQAAKDIAEINAMWPVATEANEQVCMYILGESMSSIRSKCPVEESEAS TPPSTLPCLCIHAMTPERVQRLKASRPEQITVCSSFPLPKYRITGVQKIQCSQPILFSPKVPAYIHPRKYLVETPP VEETPESPAENQSTEGTPEQPALVNVDATRTRMPEPIIIEEEEEDSISLLSDGPTHQVLQVEADIHGSPSVSSSSW SIPHASDFDVDSLSILDTLDGASVTSGAVSAETNSYFARSMEFRARPVPAPRTVFRNPPHPAPRTRTPPLAHSRAS SRTSLVSTPPGVNRVITREELEALTPSRAPSRSASRTSLVSNPPGVNRVITREEFEAFVAQQQXRFDAGAYIFSSDTGQGHLQQKSVRQTVLSEVVLERTELEISYAPRLDQEKEELLRKKLQLNPTPANRSRYQSRRVENMKAITARRILQGLGHYLKAEGKVECYRTLHPVPLYSSSVNRAFSSPKVAVEACNAMLKENFPTVASYCIIPEYDAYLDMVDGASCCLDTASFCPAKLRSFPKKHSYLEPTIRSAVPSAIQNTLQNVLAAATKRNCNVTQMRELPVLDSAAFNVECFKKYACNNEYWETFKENPIRLTEENVVNYITKLKGPKAAALFAKTHNLNMLQDIPMDRFVMDLKRDVKVTPGTKHTEERPKVQVIQAADPLATADLCGIHRELVRRLNAVLLPNIHTLFDMSAEDFDAIIAEHFQPGDCVLETDIASFDKSEDDAMALTALMILEDLGVDAELLTLIEAAFGEISSIHLPTKTKFKFGAMMKSGMFLTLFVNTVINIVIASRVLRERLTGSPCAAFIGDDNIVKGVKSDKLMADRCATWLNMEVKIIDAVVGEKAPYFCGGFILCDSVTGTACRVADPLKRLFKLGKPLAVDDEHDDDRRRALHEESTRWNRVGILPELCKAVESRYETVGTSIIVMAMTTLASSVKSFSYLRGAPITLYG(SEQ IDNO:30)；
与nsp3对应的氨基酸序列标有下划线。
在该实施例中，将对应于SEQ ID NO:30中1330-1886的nsp3的氨基酸序列替换为以下所示的序列。标有下划线的序列不同于SEQ ID NO:30。
APSYHVVRGDIATATEGVIINAANSKGQPGGGVCGALYKKFPESFDLQPIEVGKARLVKGAAKHIIHAVGPNFNKVSEVEGDKQLAEAYESIAKIVNDNNYKSVAIPLLSTGIFSGNKDRLTQSLNHLLTALDTTDADVAIYCRDKKWEMTLKEAVARREAVEEICISDDSSVTEPDAELVRVHPKSSLAGRKGYSTSDGKTFSYLEGTKFHQAAKDIAEINAMWPVATEANEQVCMYILGKSMSSIRSKCPVEESEASTPPSTLPCLCIHAMTPERVQRLKASRPEQITVCSSFPLPKYRITGVQKIQCSQPILFSPKVPAYIHPRKYLVETPPVDETPEPSAENQSTEGTPEQPPLITEDETRTRTPEPIIIEEEEEDSISLLSDGPTHQVLQVEADIHGPPSVSSSSWSIPHASDFDVDSLSILDTLEGASVTSGATSAETNSYFAKSMEFLARPVPAPRTVFRNPPHPAPRTRTPSLAPSRACSRTSLVSTPPGVNRVITREELEALTPSRTPSRSVSRTSLVSNPPGVNRVITREEFEAFVAQQQXRFDAGA(SEQ ID NO:31)。
实施例4
甲病毒复制子颗粒的制备
将10μg在实施例3中制备的全长复制子质粒、1μg VEEV Env表达质粒和1μg VEEV衣壳NLS突变体(或1μg VEEV衣壳表达质粒)转染进HEK293T细胞中。在转染后48-96小时收获上清液。通过使用离子交换柱纯化复制子颗粒。用纯化的颗粒制品的稀释液感染HET293T或Vero细胞以确定感染滴度。将纯化的复制子颗粒用于生产用于诊断和疫苗接种的抗原。
实施例5
在HEK293T细胞中表达RBD
用Fectopro转染试剂(Polyplus)和2ug的每种复制子质粒转染293T细胞(6.25x105个细胞/孔)。使用与Covid-19-RBD反应的抗血清作为一级抗体，并使用与辣根过氧化物酶连接的山羊抗兔免疫球蛋白作为二级抗体，在转染后72h直接对没有纯化颗粒的上清液(左)或对细胞裂解物级分(右)进行蛋白质印迹法。此外，使用与Covid-19-RBD反应的抗血清作为一级抗体，并使用与辣根过氧化物酶连接的山羊抗兔免疫球蛋白作为二级抗体，在转染后72h进行蛋白质印迹法，以证实细胞裂解物级分中来自构建体K和构建体O的抗原表达。
结果显示在图3中。
缩写意指如下：
ssRBD：构建体A；
hIL-2ssRBD-TM：构建体1(小鼠HA)；
ssRBD-接头-TM：构建体C(TM1：小鼠CD80，接头：小鼠IgG4-CH3)。
小鼠:CD80(TM-Cyt)
TLVLFGAGFGAVITVVVIVVIIKCFCKHRSCFRRNEASRETNNSLTFGPEEALAEQTVFL(SEQ IDNO:32)；
小鼠IgG4-CH3
GRPKAPQVYTIPPPKEQMAKDKVSLTCMITNFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK(SEQ ID NO:33)。
在细胞中确认所有试验颗粒的表达。如预期，没有跨膜序列的ssRBD被分泌到细胞培养物的培养基中，而与跨膜序列相连的每个RBD构建体都保留在细胞中且没有分泌到培养基中。
实施例6
免疫原性
使用在实施例4中制备的以下复制子颗粒疫苗。
VRep ssRBD：VEEV颗粒包装构建体A；
VRep sshIL2 RBD HA：VEEV颗粒包装构建体1；
VRep ssRBD mIgG4 CD80：VEEV复制子颗粒包装构建体C(TM1：小鼠CD80，接头：小鼠IgG4-CH3)；
对照：没有目标基因的VEEV复制子；
DNA疫苗：表达COVID-19的刺突蛋白的DNA。
用指明的复制子颗粒(RBD或RBD-接头-TM，107IU/剂)、对照复制子颗粒或DNA疫苗(20ug/剂)肌肉内免疫小鼠3次(第0天、第7天和第14天)，并在第14天(第2次免疫后7天)和第35天(第3次免疫后21天)收集血清。在针对COVID-19-刺突S1或RBD抗原的ELISA测定中检查这些血清。符号显示每组五只小鼠的平均值，且误差条显示均值标准差。通过Prism软件计算曲线拟合。这些结果显示在图4(4-1和4-2)和图5(5-1和5-2)中。
图4和图5显示，在用复制子疫苗免疫后，实现了显著更高的针对重组冠状病毒RBD和刺突S1的抗体滴度。相反，用DNA疫苗诱导了非常低的抗体滴度。
实施例7
免疫原性
使用在实施例4中制备的以下复制子颗粒疫苗。
CD80：VEEV颗粒包装构建体C；
HANo.1：VEEV颗粒包装构建体K；
对照：PBS。
用指明的复制子疫苗(CD80或HA No.1，105IU/剂)或PBS肌肉内免疫小鼠，并在免疫后7天收集血清。这些血清接受针对COVID-19-刺突S1抗原的ELISA分析。符号显示每组五只小鼠的平均值，且误差条显示均值标准差。这些结果显示在图6中。
图6显示，在用复制子疫苗免疫后，实现了显著更高的针对重组冠状病毒刺突S1的抗体滴度。
实施例8
包封在脂质纳米颗粒(LNP)中的自扩增RNA(saRNA)的制备
使用在实施例3中制备的包含编码构建体C的DNA序列的载体。将DNA线性化并用作模板。根据由T7转录试剂盒(RiboMaxTM Express大规模RNA生产系统,Promega,(WI USA))提供的方案进行T7体外转录。将线性DNA模板与T7酶和rNTP混合以合成RNA。使用苗痘加帽酶对纯化的RNA产物加帽以产生自扩增RNA。
将得到的saRNA包封在脂质纳米颗粒中以产生saRNA(RBD-CD80TM)颗粒。将没有RNA的脂质纳米颗粒用作对照。
实施例9
免疫
使用在实施例8中制备的用LNP配制的saRNA。
免疫量：
0.3ug saRNA(RBD-CD80TM)；
1ug saRNA(RBD-CD80TM)；
10ug saRNA(RBD-CD80TM)；
无RNA(对照)。
使用指明量的用LNP(Genvoy)配制的saRNA或无RNA(对照)。在第0天和第28天通过肌肉内注射使用用LNP配制的saRNA免疫4-6周龄小鼠(每组n＝5)。
在第14、28和46天将小鼠放血。通过ELISA测量来自免疫小鼠的血清中针对S1蛋白的抗体滴度。用S1蛋白(Sinobio 100ng/ml)包被平板。结果显示在图6中。
实施例10
COVID-19RBD可以衍生自COVID-19突变体。包含COVID-19信号序列、衍生自COVID-19突变体的COVID-19RBD和人HA(柔性-TM-Cyt)的构建体的以下氨基酸序列也被用于生成甲病毒复制子，如在图1中所示。在该实施例中，目标基因被替换为编码以下氨基酸序列的多核苷酸。以与实施例8相同的方式产生包封在脂质纳米颗粒(LNP)中的自扩增RNA(saRNA)。
衍生自COVID-19E484K_N501Y_K417T突变体(巴西株突变体)的构建体MFVFLVLLPLVSSVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGTIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTGVKLESMGIYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI(SEQ ID NO:34)。
衍生自COVID-19E484K_N501Y_K417N突变体(南非突变体)的构建体MFVFLVLLPLVSSVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGFNCYFPLQSYGFQPTYGVGYQPYRVVVLSFELLHAPATVCGPKKSTGVKLESMGIYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI(SEQ ID NO:35)。
衍生自COVID-19E484K突变体的构建体
MFVFLVLLPLVSSVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTGVKLESMGIYQILAIYSTVASSLVLLVSLGAISFWMCSNGSLQCRICI(SEQ ID NO 36)。
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245 250 255
Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile
260 265 270
Cys Ile
<210> 6
<211> 383
<212> PRT
<213> 人工序列
<220>
<223> 包含hIL-2信号序列、COVID-19-RBD、人IgG4CH3和HA(柔性-TM-Cyt)的融合蛋白
<400> 6
Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu
1 5 10 15
Val Thr Asn Ser Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe
20 25 30
Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn
35 40 45
Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn
50 55 60
Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys
65 70 75 80
Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile
85 90 95
Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile
100 105 110
Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile
115 120 125
Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn
130 135 140
Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg
145 150 155 160
Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly
165 170 175
Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln
180 185 190
Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser
195 200 205
Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser
210 215 220
Gly Ser Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
225 230 235 240
Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
245 250 255
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
260 265 270
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
275 280 285
Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln
290 295 300
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
305 310 315 320
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Gly Ser Gly
325 330 335
Val Lys Leu Glu Ser Met Gly Ile Tyr Gln Ile Leu Ala Ile Tyr Ser
340 345 350
Thr Val Ala Ser Ser Leu Val Leu Leu Val Ser Leu Gly Ala Ile Ser
355 360 365
Phe Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile
370 375 380
<210> 7
<211> 107
<212> PRT
<213> 人工序列
<220>
<223> 人IgG4 CH3 (接头)
<400> 7
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu
1 5 10 15
Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
20 25 30
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
35 40 45
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
50 55 60
Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly
65 70 75 80
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
85 90 95
Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
100 105
<210> 8
<211> 385
<212> PRT
<213> 人工序列
<220>
<223> 包含hIL-2信号序列、COVID-19-RBD、人IgG4CH3和HA (柔性-TM-Cyt)的融合蛋白
<400> 8
Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu
1 5 10 15
Val Thr Asn Ser Cys Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro
20 25 30
Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp
35 40 45
Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr
50 55 60
Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr
65 70 75 80
Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val
85 90 95
Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys
100 105 110
Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val
115 120 125
Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr
130 135 140
Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu
145 150 155 160
Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn
165 170 175
Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe
180 185 190
Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu
195 200 205
Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys
210 215 220
Ser Cys Gly Ser Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
225 230 235 240
Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
245 250 255
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
260 265 270
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
275 280 285
Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg
290 295 300
Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
305 310 315 320
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Gly
325 330 335
Ser Gly Val Lys Leu Glu Ser Met Gly Ile Tyr Gln Ile Leu Ala Ile
340 345 350
Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Leu Val Ser Leu Gly Ala
355 360 365
Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys
370 375 380
Ile
385
<210> 9
<211> 435
<212> PRT
<213> 人工序列
<220>
<223> 包含hIL-2信号序列、COVID-19-RBD和人TLR4(TM-TIR)的融合蛋白
<400> 9
Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu
1 5 10 15
Val Thr Asn Ser Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe
20 25 30
Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn
35 40 45
Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn
50 55 60
Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys
65 70 75 80
Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile
85 90 95
Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile
100 105 110
Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile
115 120 125
Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn
130 135 140
Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg
145 150 155 160
Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly
165 170 175
Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln
180 185 190
Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser
195 200 205
Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser
210 215 220
Gly Ser Lys Thr Ile Ile Gly Val Ser Val Leu Ser Val Leu Val Val
225 230 235 240
Ser Val Val Ala Val Leu Val Tyr Lys Phe Tyr Phe His Leu Met Leu
245 250 255
Leu Ala Gly Cys Ile Lys Tyr Gly Arg Gly Glu Asn Ile Tyr Asp Ala
260 265 270
Phe Val Ile Tyr Ser Ser Gln Asp Glu Asp Trp Val Arg Asn Glu Leu
275 280 285
Val Lys Asn Leu Glu Glu Gly Val Pro Pro Phe Gln Leu Cys Leu His
290 295 300
Tyr Arg Asp Phe Ile Pro Gly Val Ala Ile Ala Ala Asn Ile Ile His
305 310 315 320
Glu Gly Phe His Lys Ser Arg Lys Val Ile Val Val Val Ser Gln His
325 330 335
Phe Ile Gln Ser Arg Trp Cys Ile Phe Glu Tyr Glu Ile Ala Gln Thr
340 345 350
Trp Gln Phe Leu Ser Ser Arg Ala Gly Ile Ile Phe Ile Val Leu Gln
355 360 365
Lys Val Glu Lys Thr Leu Leu Arg Gln Gln Val Glu Leu Tyr Arg Leu
370 375 380
Leu Ser Arg Asn Thr Tyr Leu Glu Trp Glu Asp Ser Val Leu Gly Arg
385 390 395 400
His Ile Phe Trp Arg Arg Leu Arg Lys Ala Leu Leu Asp Gly Lys Ser
405 410 415
Trp Asn Pro Glu Gly Thr Val Gly Thr Gly Cys Asn Trp Gln Glu Ala
420 425 430
Thr Ser Ile
435
<210> 10
<211> 209
<212> PRT
<213> 人工序列
<220>
<223> 人TLR4 (TM-Toll/白介素-1受体结构域)
<400> 10
Lys Thr Ile Ile Gly Val Ser Val Leu Ser Val Leu Val Val Ser Val
1 5 10 15
Val Ala Val Leu Val Tyr Lys Phe Tyr Phe His Leu Met Leu Leu Ala
20 25 30
Gly Cys Ile Lys Tyr Gly Arg Gly Glu Asn Ile Tyr Asp Ala Phe Val
35 40 45
Ile Tyr Ser Ser Gln Asp Glu Asp Trp Val Arg Asn Glu Leu Val Lys
50 55 60
Asn Leu Glu Glu Gly Val Pro Pro Phe Gln Leu Cys Leu His Tyr Arg
65 70 75 80
Asp Phe Ile Pro Gly Val Ala Ile Ala Ala Asn Ile Ile His Glu Gly
85 90 95
Phe His Lys Ser Arg Lys Val Ile Val Val Val Ser Gln His Phe Ile
100 105 110
Gln Ser Arg Trp Cys Ile Phe Glu Tyr Glu Ile Ala Gln Thr Trp Gln
115 120 125
Phe Leu Ser Ser Arg Ala Gly Ile Ile Phe Ile Val Leu Gln Lys Val
130 135 140
Glu Lys Thr Leu Leu Arg Gln Gln Val Glu Leu Tyr Arg Leu Leu Ser
145 150 155 160
Arg Asn Thr Tyr Leu Glu Trp Glu Asp Ser Val Leu Gly Arg His Ile
165 170 175
Phe Trp Arg Arg Leu Arg Lys Ala Leu Leu Asp Gly Lys Ser Trp Asn
180 185 190
Pro Glu Gly Thr Val Gly Thr Gly Cys Asn Trp Gln Glu Ala Thr Ser
195 200 205
Ile
<210> 11
<211> 437
<212> PRT
<213> 人工序列
<220>
<223> 包含hIL-2信号序列、COVID-19-RBD和人TLR4 (TM-TIR)的融合蛋白
<400> 11
Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu
1 5 10 15
Val Thr Asn Ser Cys Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro
20 25 30
Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp
35 40 45
Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr
50 55 60
Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr
65 70 75 80
Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val
85 90 95
Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys
100 105 110
Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val
115 120 125
Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr
130 135 140
Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu
145 150 155 160
Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn
165 170 175
Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe
180 185 190
Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu
195 200 205
Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys
210 215 220
Ser Cys Gly Ser Lys Thr Ile Ile Gly Val Ser Val Leu Ser Val Leu
225 230 235 240
Val Val Ser Val Val Ala Val Leu Val Tyr Lys Phe Tyr Phe His Leu
245 250 255
Met Leu Leu Ala Gly Cys Ile Lys Tyr Gly Arg Gly Glu Asn Ile Tyr
260 265 270
Asp Ala Phe Val Ile Tyr Ser Ser Gln Asp Glu Asp Trp Val Arg Asn
275 280 285
Glu Leu Val Lys Asn Leu Glu Glu Gly Val Pro Pro Phe Gln Leu Cys
290 295 300
Leu His Tyr Arg Asp Phe Ile Pro Gly Val Ala Ile Ala Ala Asn Ile
305 310 315 320
Ile His Glu Gly Phe His Lys Ser Arg Lys Val Ile Val Val Val Ser
325 330 335
Gln His Phe Ile Gln Ser Arg Trp Cys Ile Phe Glu Tyr Glu Ile Ala
340 345 350
Gln Thr Trp Gln Phe Leu Ser Ser Arg Ala Gly Ile Ile Phe Ile Val
355 360 365
Leu Gln Lys Val Glu Lys Thr Leu Leu Arg Gln Gln Val Glu Leu Tyr
370 375 380
Arg Leu Leu Ser Arg Asn Thr Tyr Leu Glu Trp Glu Asp Ser Val Leu
385 390 395 400
Gly Arg His Ile Phe Trp Arg Arg Leu Arg Lys Ala Leu Leu Asp Gly
405 410 415
Lys Ser Trp Asn Pro Glu Gly Thr Val Gly Thr Gly Cys Asn Trp Gln
420 425 430
Glu Ala Thr Ser Ile
435
<210> 12
<211> 544
<212> PRT
<213> 人工序列
<220>
<223> 包含hIL-2信号序列、COVID-19-RBD、人IgG4CH3和人TLR4(TM-TIR)的融合蛋白
<400> 12
Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu
1 5 10 15
Val Thr Asn Ser Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe
20 25 30
Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn
35 40 45
Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn
50 55 60
Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys
65 70 75 80
Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile
85 90 95
Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile
100 105 110
Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile
115 120 125
Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn
130 135 140
Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg
145 150 155 160
Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly
165 170 175
Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln
180 185 190
Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser
195 200 205
Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser
210 215 220
Gly Ser Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
225 230 235 240
Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
245 250 255
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
260 265 270
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
275 280 285
Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln
290 295 300
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
305 310 315 320
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Gly Ser Lys
325 330 335
Thr Ile Ile Gly Val Ser Val Leu Ser Val Leu Val Val Ser Val Val
340 345 350
Ala Val Leu Val Tyr Lys Phe Tyr Phe His Leu Met Leu Leu Ala Gly
355 360 365
Cys Ile Lys Tyr Gly Arg Gly Glu Asn Ile Tyr Asp Ala Phe Val Ile
370 375 380
Tyr Ser Ser Gln Asp Glu Asp Trp Val Arg Asn Glu Leu Val Lys Asn
385 390 395 400
Leu Glu Glu Gly Val Pro Pro Phe Gln Leu Cys Leu His Tyr Arg Asp
405 410 415
Phe Ile Pro Gly Val Ala Ile Ala Ala Asn Ile Ile His Glu Gly Phe
420 425 430
His Lys Ser Arg Lys Val Ile Val Val Val Ser Gln His Phe Ile Gln
435 440 445
Ser Arg Trp Cys Ile Phe Glu Tyr Glu Ile Ala Gln Thr Trp Gln Phe
450 455 460
Leu Ser Ser Arg Ala Gly Ile Ile Phe Ile Val Leu Gln Lys Val Glu
465 470 475 480
Lys Thr Leu Leu Arg Gln Gln Val Glu Leu Tyr Arg Leu Leu Ser Arg
485 490 495
Asn Thr Tyr Leu Glu Trp Glu Asp Ser Val Leu Gly Arg His Ile Phe
500 505 510
Trp Arg Arg Leu Arg Lys Ala Leu Leu Asp Gly Lys Ser Trp Asn Pro
515 520 525
Glu Gly Thr Val Gly Thr Gly Cys Asn Trp Gln Glu Ala Thr Ser Ile
530 535 540
<210> 13
<211> 546
<212> PRT
<213> 人工序列
<220>
<223> 包含hIL-2信号序列、COVID19-RBD、人IgG4CH3和人TLR4(TM-TIR)的融合蛋白
<400> 13
Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu
1 5 10 15
Val Thr Asn Ser Cys Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro
20 25 30
Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp
35 40 45
Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr
50 55 60
Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr
65 70 75 80
Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val
85 90 95
Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys
100 105 110
Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val
115 120 125
Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr
130 135 140
Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu
145 150 155 160
Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn
165 170 175
Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe
180 185 190
Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu
195 200 205
Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys
210 215 220
Ser Cys Gly Ser Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
225 230 235 240
Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
245 250 255
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
260 265 270
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
275 280 285
Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg
290 295 300
Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
305 310 315 320
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Gly
325 330 335
Ser Lys Thr Ile Ile Gly Val Ser Val Leu Ser Val Leu Val Val Ser
340 345 350
Val Val Ala Val Leu Val Tyr Lys Phe Tyr Phe His Leu Met Leu Leu
355 360 365
Ala Gly Cys Ile Lys Tyr Gly Arg Gly Glu Asn Ile Tyr Asp Ala Phe
370 375 380
Val Ile Tyr Ser Ser Gln Asp Glu Asp Trp Val Arg Asn Glu Leu Val
385 390 395 400
Lys Asn Leu Glu Glu Gly Val Pro Pro Phe Gln Leu Cys Leu His Tyr
405 410 415
Arg Asp Phe Ile Pro Gly Val Ala Ile Ala Ala Asn Ile Ile His Glu
420 425 430
Gly Phe His Lys Ser Arg Lys Val Ile Val Val Val Ser Gln His Phe
435 440 445
Ile Gln Ser Arg Trp Cys Ile Phe Glu Tyr Glu Ile Ala Gln Thr Trp
450 455 460
Gln Phe Leu Ser Ser Arg Ala Gly Ile Ile Phe Ile Val Leu Gln Lys
465 470 475 480
Val Glu Lys Thr Leu Leu Arg Gln Gln Val Glu Leu Tyr Arg Leu Leu
485 490 495
Ser Arg Asn Thr Tyr Leu Glu Trp Glu Asp Ser Val Leu Gly Arg His
500 505 510
Ile Phe Trp Arg Arg Leu Arg Lys Ala Leu Leu Asp Gly Lys Ser Trp
515 520 525
Asn Pro Glu Gly Thr Val Gly Thr Gly Cys Asn Trp Gln Glu Ala Thr
530 535 540
Ser Ile
545
<210> 14
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> COVID-19信号序列(1-15)
<400> 14
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys
1 5 10 15
<210> 15
<211> 20
<212> PRT
<213> 人工序列
<220>
<223> 人IL-2信号序列
<400> 15
Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu
1 5 10 15
Val Thr Asn Ser
20
<210> 16
<211> 13
<212> PRT
<213> 人工序列
<220>
<223> COVID-19信号序列(1-13)
<400> 16
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser
1 5 10
<210> 17
<211> 192
<212> PRT
<213> 人工序列
<220>
<223> COVID-19 RBD (氨基酸330-521)
<400> 17
Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr
1 5 10 15
Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys
20 25 30
Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe
35 40 45
Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr
50 55 60
Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln
65 70 75 80
Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu
85 90 95
Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu
100 105 110
Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg
115 120 125
Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr
130 135 140
Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr
145 150 155 160
Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr
165 170 175
Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro
180 185 190
<210> 18
<211> 201
<212> PRT
<213> 人工序列
<220>
<223> COVID-19 RBD (氨基酸330-530)
<400> 18
Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr
1 5 10 15
Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys
20 25 30
Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe
35 40 45
Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr
50 55 60
Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln
65 70 75 80
Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu
85 90 95
Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu
100 105 110
Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg
115 120 125
Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr
130 135 140
Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr
145 150 155 160
Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr
165 170 175
Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro
180 185 190
Ala Thr Val Cys Gly Pro Lys Lys Ser
195 200
<210> 19
<211> 205
<212> PRT
<213> 人工序列
<220>
<223> COVID-19 RBD (氨基酸327-531)
<400> 19
Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe
1 5 10 15
Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile
20 25 30
Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe
35 40 45
Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu
50 55 60
Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu
65 70 75 80
Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn
85 90 95
Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser
100 105 110
Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg
115 120 125
Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr
130 135 140
Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe
145 150 155 160
Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly
165 170 175
Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu
180 185 190
His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr
195 200 205
<210> 20
<211> 107
<212> PRT
<213> 人工序列
<220>
<223> 人IgG4-CH3 (接头)
<400> 20
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu
1 5 10 15
Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
20 25 30
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
35 40 45
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
50 55 60
Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly
65 70 75 80
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
85 90 95
Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
100 105
<210> 21
<211> 46
<212> PRT
<213> 人工序列
<220>
<223> 人CD80跨膜-细胞质
<400> 21
Leu Leu Pro Ser Trp Ala Ile Thr Leu Ile Ser Val Asn Gly Ile Phe
1 5 10 15
Val Ile Cys Cys Leu Thr Tyr Cys Phe Ala Pro Arg Cys Arg Glu Arg
20 25 30
Arg Arg Asn Glu Arg Leu Arg Arg Glu Ser Val Arg Pro Val
35 40 45
<210> 22
<211> 48
<212> PRT
<213> 人工序列
<220>
<223> 流感HA (柔性-TM-Cyt)
<400> 22
Gly Val Lys Leu Glu Ser Met Gly Ile Tyr Gln Ile Leu Ala Ile Tyr
1 5 10 15
Ser Thr Val Ala Ser Ser Leu Val Leu Leu Val Ser Leu Gly Ala Ile
20 25 30
Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile
35 40 45
<210> 23
<211> 70
<212> PRT
<213> COVID-19近膜结构域和跨膜结构域
<400> 23
Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu Gly
1 5 10 15
Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met Leu Cys
20 25 30
Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys Ser Cys Gly
35 40 45
Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro Val Leu Lys Gly
50 55 60
Val Lys Leu His Tyr Thr
65 70
<210> 24
<211> 15
<212> PRT
<213> 人工序列
<220>
<223> COVID-19刺突蛋白的近膜结构域
<400> 24
Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu
1 5 10 15
<210> 25
<211> 55
<212> PRT
<213> 人工序列
<220>
<223> COVID-19刺突蛋白的跨膜结构域
<400> 25
Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met Leu
1 5 10 15
Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys Ser Cys
20 25 30
Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro Val Leu Lys
35 40 45
Gly Val Lys Leu His Tyr Thr
50 55
<210> 26
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> SG启动子
<400> 26
cctgaatgga ctacgacata gtctagtccg ccaag 35
<210> 27
<211> 44
<212> DNA
<213> 人工序列
<220>
<223> 5' UTR
<400> 27
ataggcggcg catgagagaa gcccagacca attacctacc caaa 44
<210> 28
<211> 125
<212> DNA
<213> 人工序列
<220>
<223> 3' UTR
<400> 28
gcgatcgcat acagcagcaa ttggcaagct gcttacatag aactcgcggc gattggcatg 60
ccgccttaaa atttttattt tatttttctt ttcttttccg aatcggattt tgtttttaat 120
atttc 125
<210> 29
<211> 55
<212> DNA
<213> 人工序列
<220>
<223> 多聚A尾
<400> 29
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaa 55
<210> 30
<211> 2493
<212> PRT
<213> 人工序列
<220>
<223> VEEV TC-83复制子nsP1-4氨基酸序列
<220>
<221> 杂项特征
<222> (1880)..(1880)
<223> Xaa可以是任何天然存在的氨基酸
<400> 30
Met Glu Lys Val His Val Asp Ile Glu Glu Asp Ser Pro Phe Leu Arg
1 5 10 15
Ala Leu Gln Arg Ser Phe Pro Gln Phe Glu Val Glu Ala Lys Gln Val
20 25 30
Thr Asp Asn Asp His Ala Asn Ala Arg Ala Phe Ser His Leu Ala Ser
35 40 45
Lys Leu Ile Glu Thr Glu Val Asp Pro Ser Asp Thr Ile Leu Asp Ile
50 55 60
Gly Ser Ala Pro Ala Arg Arg Met Tyr Ser Lys His Lys Tyr His Cys
65 70 75 80
Ile Cys Pro Met Arg Cys Ala Glu Asp Pro Asp Arg Leu Tyr Lys Tyr
85 90 95
Ala Thr Lys Leu Lys Lys Asn Cys Lys Glu Ile Thr Asp Lys Glu Leu
100 105 110
Asp Lys Lys Met Lys Glu Leu Ala Ala Val Met Ser Asp Pro Asp Leu
115 120 125
Glu Thr Glu Thr Met Cys Leu His Asp Asp Glu Ser Cys Arg Tyr Glu
130 135 140
Gly Gln Val Ala Val Tyr Gln Asp Val Tyr Ala Val Asp Gly Pro Thr
145 150 155 160
Ser Leu Tyr His Gln Ala Asn Lys Gly Val Arg Val Ala Tyr Trp Ile
165 170 175
Gly Phe Asp Thr Thr Pro Phe Met Phe Lys Asn Leu Ala Gly Ala Tyr
180 185 190
Pro Ser Tyr Ser Thr Asn Trp Ala Asp Glu Thr Val Leu Thr Ala Arg
195 200 205
Asn Ile Gly Leu Cys Ser Ser Asp Val Met Glu Arg Ser Arg Arg Gly
210 215 220
Met Ser Ile Leu Arg Lys Lys Tyr Leu Lys Pro Ser Asn Asn Val Leu
225 230 235 240
Phe Ser Val Gly Ser Thr Ile Tyr His Glu Lys Arg Asp Leu Leu Arg
245 250 255
Ser Trp His Leu Pro Ser Val Phe His Leu Arg Gly Lys Gln Asn Tyr
260 265 270
Thr Cys Arg Cys Glu Thr Ile Val Ser Cys Asp Gly Tyr Val Val Lys
275 280 285
Arg Ile Ala Ile Ser Pro Gly Leu Tyr Gly Lys Pro Ser Gly Tyr Ala
290 295 300
Ala Thr Met His Arg Glu Gly Phe Leu Cys Cys Lys Val Thr Asp Thr
305 310 315 320
Leu Asn Gly Glu Arg Val Ser Phe Pro Val Cys Thr Tyr Val Pro Ala
325 330 335
Thr Leu Cys Asp Gln Met Thr Gly Ile Leu Ala Thr Asp Val Ser Ala
340 345 350
Asp Asp Ala Gln Lys Leu Leu Val Gly Leu Asn Gln Arg Ile Val Val
355 360 365
Asn Gly Arg Thr Gln Arg Asn Thr Asn Thr Met Lys Asn Tyr Leu Leu
370 375 380
Pro Val Val Ala Gln Ala Phe Ala Arg Trp Ala Lys Glu Tyr Lys Glu
385 390 395 400
Asp Gln Glu Asp Glu Arg Pro Leu Gly Leu Arg Asp Arg Gln Leu Val
405 410 415
Met Gly Cys Cys Trp Ala Phe Arg Arg His Lys Ile Thr Ser Ile Tyr
420 425 430
Lys Arg Pro Asp Thr Gln Thr Ile Ile Lys Val Asn Ser Asp Phe His
435 440 445
Ser Phe Val Leu Pro Arg Ile Gly Ser Asn Thr Leu Glu Ile Gly Leu
450 455 460
Arg Thr Arg Ile Arg Lys Met Leu Glu Glu His Lys Glu Pro Ser Pro
465 470 475 480
Leu Ile Thr Ala Glu Asp Ile Gln Glu Ala Lys Cys Ala Ala Asp Glu
485 490 495
Ala Lys Glu Val Arg Glu Ala Glu Glu Leu Arg Ala Ala Leu Pro Pro
500 505 510
Leu Ala Ala Asp Phe Glu Glu Pro Thr Leu Glu Ala Asp Val Asp Leu
515 520 525
Met Leu Gln Glu Ala Gly Ala Gly Ser Val Glu Thr Pro Arg Gly Leu
530 535 540
Ile Lys Val Thr Ser Tyr Ala Gly Glu Asp Lys Ile Gly Ser Tyr Ala
545 550 555 560
Val Leu Ser Pro Gln Ala Val Leu Lys Ser Glu Lys Leu Ser Cys Ile
565 570 575
His Pro Leu Ala Glu Gln Val Ile Val Ile Thr His Ser Gly Arg Lys
580 585 590
Gly Arg Tyr Ala Val Glu Pro Tyr His Gly Lys Val Val Val Pro Glu
595 600 605
Gly His Ala Ile Pro Val Gln Asp Phe Gln Ala Leu Ser Glu Ser Ala
610 615 620
Thr Ile Val Tyr Asn Glu Arg Glu Phe Val Asn Arg Tyr Leu His His
625 630 635 640
Ile Ala Thr His Gly Gly Ala Leu Asn Thr Asp Glu Glu Tyr Tyr Lys
645 650 655
Thr Val Lys Pro Ser Glu His Asp Gly Glu Tyr Leu Tyr Asp Ile Asp
660 665 670
Arg Lys Gln Cys Val Lys Lys Glu Leu Val Thr Gly Leu Gly Leu Thr
675 680 685
Gly Glu Leu Val Asp Pro Pro Phe His Glu Phe Ala Tyr Glu Ser Leu
690 695 700
Arg Thr Arg Pro Ala Ala Pro Tyr Gln Val Pro Thr Ile Gly Val Tyr
705 710 715 720
Gly Val Pro Gly Ser Gly Lys Ser Gly Ile Ile Lys Ser Ala Val Thr
725 730 735
Lys Lys Asp Leu Val Val Ser Ala Lys Lys Glu Asn Cys Ala Glu Ile
740 745 750
Ile Arg Asp Val Lys Lys Met Lys Gly Leu Asp Val Asn Ala Arg Thr
755 760 765
Val Asp Ser Val Leu Leu Asn Gly Cys Lys His Pro Val Glu Thr Leu
770 775 780
Tyr Ile Asp Glu Ala Phe Ala Cys His Ala Gly Thr Leu Arg Ala Leu
785 790 795 800
Ile Ala Ile Ile Arg Pro Lys Lys Ala Val Leu Cys Gly Asp Pro Lys
805 810 815
Gln Cys Gly Phe Phe Asn Met Met Cys Leu Lys Val His Phe Asn His
820 825 830
Glu Ile Cys Thr Gln Val Phe His Lys Ser Ile Ser Arg Arg Cys Thr
835 840 845
Lys Ser Val Thr Ser Val Val Ser Thr Leu Phe Tyr Asp Lys Arg Met
850 855 860
Arg Thr Thr Asn Pro Lys Glu Thr Lys Ile Val Ile Asp Thr Thr Gly
865 870 875 880
Ser Thr Lys Pro Lys Gln Asp Asp Leu Ile Leu Thr Cys Phe Arg Gly
885 890 895
Trp Val Lys Gln Leu Gln Ile Asp Tyr Lys Gly Asn Glu Ile Met Thr
900 905 910
Ala Ala Ala Ser Gln Gly Leu Thr Arg Lys Gly Val Tyr Ala Val Arg
915 920 925
Tyr Lys Val Asn Glu Asn Pro Leu Tyr Ala Pro Thr Ser Glu His Val
930 935 940
Asn Val Leu Leu Thr Arg Thr Glu Asp Arg Ile Val Trp Lys Thr Leu
945 950 955 960
Ala Gly Asp Pro Trp Ile Lys Ile Leu Thr Ala Lys Tyr Pro Gly Asn
965 970 975
Phe Thr Ala Thr Ile Glu Glu Trp Gln Ala Glu His Asp Ala Ile Met
980 985 990
Arg His Ile Leu Glu Arg Pro Asp Pro Thr Asp Val Phe Gln Asn Lys
995 1000 1005
Ala Asn Val Cys Trp Ala Lys Ala Leu Val Pro Val Leu Lys Thr
1010 1015 1020
Ala Gly Ile Asp Met Thr Thr Glu Gln Trp Asn Thr Val Asp Tyr
1025 1030 1035
Phe Glu Thr Asp Lys Ala His Ser Ala Glu Ile Val Leu Asn Gln
1040 1045 1050
Leu Cys Val Arg Phe Phe Gly Leu Asp Leu Asp Ser Gly Leu Phe
1055 1060 1065
Ser Ala Pro Thr Val Pro Leu Ser Ile Arg Asn Asn His Trp Asp
1070 1075 1080
Asn Ser Pro Ser Pro Asn Met Tyr Gly Leu Asn Lys Glu Val Val
1085 1090 1095
Arg Gln Leu Ser Arg Arg Tyr Pro Gln Leu Pro Arg Ala Val Ala
1100 1105 1110
Thr Gly Arg Val Tyr Asp Met Asn Thr Gly Thr Leu Arg Asn Tyr
1115 1120 1125
Asp Pro Arg Ile Asn Leu Val Pro Val Asn Arg Arg Leu Pro His
1130 1135 1140
Ala Leu Val Leu His His Asn Glu His Pro Gln Ser Asp Phe Ser
1145 1150 1155
Ser Phe Val Ser Lys Leu Lys Gly Arg Thr Val Leu Val Val Gly
1160 1165 1170
Glu Lys Leu Ser Val Pro Gly Lys Lys Val Asp Trp Leu Ser Asp
1175 1180 1185
Gln Pro Glu Ala Thr Phe Arg Ala Arg Leu Asp Leu Gly Ile Pro
1190 1195 1200
Gly Asp Val Pro Lys Tyr Asp Ile Val Phe Ile Asn Val Arg Thr
1205 1210 1215
Pro Tyr Lys Tyr His His Tyr Gln Gln Cys Glu Asp His Ala Ile
1220 1225 1230
Lys Leu Ser Met Leu Thr Lys Lys Ala Cys Leu His Leu Asn Pro
1235 1240 1245
Gly Gly Thr Cys Val Ser Ile Gly Tyr Gly Tyr Ala Asp Arg Ala
1250 1255 1260
Ser Glu Ser Ile Ile Gly Ala Ile Ala Arg Gln Phe Lys Phe Ser
1265 1270 1275
Arg Val Cys Lys Pro Lys Ser Ser His Glu Glu Thr Glu Val Leu
1280 1285 1290
Phe Val Phe Ile Gly Tyr Asp Arg Lys Ala Arg Thr His Asn Pro
1295 1300 1305
Tyr Lys Leu Ser Ser Thr Leu Thr Asn Ile Tyr Thr Gly Ser Arg
1310 1315 1320
Leu His Glu Ala Gly Cys Ala Pro Ser Tyr His Val Val Arg Gly
1325 1330 1335
Asp Ile Ala Thr Ala Thr Glu Gly Val Ile Ile Asn Ala Ala Asn
1340 1345 1350
Ser Lys Gly Gln Pro Gly Gly Gly Val Cys Gly Ala Leu Tyr Lys
1355 1360 1365
Lys Phe Pro Glu Ser Phe Asp Leu Gln Pro Ile Glu Val Gly Lys
1370 1375 1380
Ala Arg Leu Val Lys Gly Ala Ala Lys His Ile Ile His Ala Val
1385 1390 1395
Gly Pro Asn Phe Asn Lys Val Ser Glu Val Glu Gly Asp Lys Gln
1400 1405 1410
Leu Ala Glu Ala Tyr Glu Ser Ile Ala Lys Ile Val Asn Asp Asn
1415 1420 1425
Asn Tyr Lys Ser Val Ala Ile Pro Leu Leu Ser Thr Gly Ile Phe
1430 1435 1440
Ser Gly Asn Lys Asp Arg Leu Thr Gln Ser Leu Asn His Leu Leu
1445 1450 1455
Thr Ala Leu Asp Thr Thr Asp Ala Asp Val Ala Ile Tyr Cys Arg
1460 1465 1470
Asp Lys Lys Trp Glu Met Thr Leu Lys Glu Ala Val Ala Arg Arg
1475 1480 1485
Glu Ala Val Glu Glu Ile Cys Ile Ser Asp Asp Ser Ser Val Thr
1490 1495 1500
Glu Pro Asp Ala Glu Leu Val Arg Val His Pro Lys Ser Ser Leu
1505 1510 1515
Ala Gly Arg Lys Gly Tyr Ser Thr Ser Asp Gly Lys Thr Phe Ser
1520 1525 1530
Tyr Leu Glu Gly Thr Lys Phe His Gln Ala Ala Lys Asp Ile Ala
1535 1540 1545
Glu Ile Asn Ala Met Trp Pro Val Ala Thr Glu Ala Asn Glu Gln
1550 1555 1560
Val Cys Met Tyr Ile Leu Gly Glu Ser Met Ser Ser Ile Arg Ser
1565 1570 1575
Lys Cys Pro Val Glu Glu Ser Glu Ala Ser Thr Pro Pro Ser Thr
1580 1585 1590
Leu Pro Cys Leu Cys Ile His Ala Met Thr Pro Glu Arg Val Gln
1595 1600 1605
Arg Leu Lys Ala Ser Arg Pro Glu Gln Ile Thr Val Cys Ser Ser
1610 1615 1620
Phe Pro Leu Pro Lys Tyr Arg Ile Thr Gly Val Gln Lys Ile Gln
1625 1630 1635
Cys Ser Gln Pro Ile Leu Phe Ser Pro Lys Val Pro Ala Tyr Ile
1640 1645 1650
His Pro Arg Lys Tyr Leu Val Glu Thr Pro Pro Val Glu Glu Thr
1655 1660 1665
Pro Glu Ser Pro Ala Glu Asn Gln Ser Thr Glu Gly Thr Pro Glu
1670 1675 1680
Gln Pro Ala Leu Val Asn Val Asp Ala Thr Arg Thr Arg Met Pro
1685 1690 1695
Glu Pro Ile Ile Ile Glu Glu Glu Glu Glu Asp Ser Ile Ser Leu
1700 1705 1710
Leu Ser Asp Gly Pro Thr His Gln Val Leu Gln Val Glu Ala Asp
1715 1720 1725
Ile His Gly Ser Pro Ser Val Ser Ser Ser Ser Trp Ser Ile Pro
1730 1735 1740
His Ala Ser Asp Phe Asp Val Asp Ser Leu Ser Ile Leu Asp Thr
1745 1750 1755
Leu Asp Gly Ala Ser Val Thr Ser Gly Ala Val Ser Ala Glu Thr
1760 1765 1770
Asn Ser Tyr Phe Ala Arg Ser Met Glu Phe Arg Ala Arg Pro Val
1775 1780 1785
Pro Ala Pro Arg Thr Val Phe Arg Asn Pro Pro His Pro Ala Pro
1790 1795 1800
Arg Thr Arg Thr Pro Pro Leu Ala His Ser Arg Ala Ser Ser Arg
1805 1810 1815
Thr Ser Leu Val Ser Thr Pro Pro Gly Val Asn Arg Val Ile Thr
1820 1825 1830
Arg Glu Glu Leu Glu Ala Leu Thr Pro Ser Arg Ala Pro Ser Arg
1835 1840 1845
Ser Ala Ser Arg Thr Ser Leu Val Ser Asn Pro Pro Gly Val Asn
1850 1855 1860
Arg Val Ile Thr Arg Glu Glu Phe Glu Ala Phe Val Ala Gln Gln
1865 1870 1875
Gln Xaa Arg Phe Asp Ala Gly Ala Tyr Ile Phe Ser Ser Asp Thr
1880 1885 1890
Gly Gln Gly His Leu Gln Gln Lys Ser Val Arg Gln Thr Val Leu
1895 1900 1905
Ser Glu Val Val Leu Glu Arg Thr Glu Leu Glu Ile Ser Tyr Ala
1910 1915 1920
Pro Arg Leu Asp Gln Glu Lys Glu Glu Leu Leu Arg Lys Lys Leu
1925 1930 1935
Gln Leu Asn Pro Thr Pro Ala Asn Arg Ser Arg Tyr Gln Ser Arg
1940 1945 1950
Arg Val Glu Asn Met Lys Ala Ile Thr Ala Arg Arg Ile Leu Gln
1955 1960 1965
Gly Leu Gly His Tyr Leu Lys Ala Glu Gly Lys Val Glu Cys Tyr
1970 1975 1980
Arg Thr Leu His Pro Val Pro Leu Tyr Ser Ser Ser Val Asn Arg
1985 1990 1995
Ala Phe Ser Ser Pro Lys Val Ala Val Glu Ala Cys Asn Ala Met
2000 2005 2010
Leu Lys Glu Asn Phe Pro Thr Val Ala Ser Tyr Cys Ile Ile Pro
2015 2020 2025
Glu Tyr Asp Ala Tyr Leu Asp Met Val Asp Gly Ala Ser Cys Cys
2030 2035 2040
Leu Asp Thr Ala Ser Phe Cys Pro Ala Lys Leu Arg Ser Phe Pro
2045 2050 2055
Lys Lys His Ser Tyr Leu Glu Pro Thr Ile Arg Ser Ala Val Pro
2060 2065 2070
Ser Ala Ile Gln Asn Thr Leu Gln Asn Val Leu Ala Ala Ala Thr
2075 2080 2085
Lys Arg Asn Cys Asn Val Thr Gln Met Arg Glu Leu Pro Val Leu
2090 2095 2100
Asp Ser Ala Ala Phe Asn Val Glu Cys Phe Lys Lys Tyr Ala Cys
2105 2110 2115
Asn Asn Glu Tyr Trp Glu Thr Phe Lys Glu Asn Pro Ile Arg Leu
2120 2125 2130
Thr Glu Glu Asn Val Val Asn Tyr Ile Thr Lys Leu Lys Gly Pro
2135 2140 2145
Lys Ala Ala Ala Leu Phe Ala Lys Thr His Asn Leu Asn Met Leu
2150 2155 2160
Gln Asp Ile Pro Met Asp Arg Phe Val Met Asp Leu Lys Arg Asp
2165 2170 2175
Val Lys Val Thr Pro Gly Thr Lys His Thr Glu Glu Arg Pro Lys
2180 2185 2190
Val Gln Val Ile Gln Ala Ala Asp Pro Leu Ala Thr Ala Asp Leu
2195 2200 2205
Cys Gly Ile His Arg Glu Leu Val Arg Arg Leu Asn Ala Val Leu
2210 2215 2220
Leu Pro Asn Ile His Thr Leu Phe Asp Met Ser Ala Glu Asp Phe
2225 2230 2235
Asp Ala Ile Ile Ala Glu His Phe Gln Pro Gly Asp Cys Val Leu
2240 2245 2250
Glu Thr Asp Ile Ala Ser Phe Asp Lys Ser Glu Asp Asp Ala Met
2255 2260 2265
Ala Leu Thr Ala Leu Met Ile Leu Glu Asp Leu Gly Val Asp Ala
2270 2275 2280
Glu Leu Leu Thr Leu Ile Glu Ala Ala Phe Gly Glu Ile Ser Ser
2285 2290 2295
Ile His Leu Pro Thr Lys Thr Lys Phe Lys Phe Gly Ala Met Met
2300 2305 2310
Lys Ser Gly Met Phe Leu Thr Leu Phe Val Asn Thr Val Ile Asn
2315 2320 2325
Ile Val Ile Ala Ser Arg Val Leu Arg Glu Arg Leu Thr Gly Ser
2330 2335 2340
Pro Cys Ala Ala Phe Ile Gly Asp Asp Asn Ile Val Lys Gly Val
2345 2350 2355
Lys Ser Asp Lys Leu Met Ala Asp Arg Cys Ala Thr Trp Leu Asn
2360 2365 2370
Met Glu Val Lys Ile Ile Asp Ala Val Val Gly Glu Lys Ala Pro
2375 2380 2385
Tyr Phe Cys Gly Gly Phe Ile Leu Cys Asp Ser Val Thr Gly Thr
2390 2395 2400
Ala Cys Arg Val Ala Asp Pro Leu Lys Arg Leu Phe Lys Leu Gly
2405 2410 2415
Lys Pro Leu Ala Val Asp Asp Glu His Asp Asp Asp Arg Arg Arg
2420 2425 2430
Ala Leu His Glu Glu Ser Thr Arg Trp Asn Arg Val Gly Ile Leu
2435 2440 2445
Pro Glu Leu Cys Lys Ala Val Glu Ser Arg Tyr Glu Thr Val Gly
2450 2455 2460
Thr Ser Ile Ile Val Met Ala Met Thr Thr Leu Ala Ser Ser Val
2465 2470 2475
Lys Ser Phe Ser Tyr Leu Arg Gly Ala Pro Ile Thr Leu Tyr Gly
2480 2485 2490
<210> 31
<211> 557
<212> PRT
<213> 人工序列
<220>
<223> VEEV TC-83 nsp3 E242K
<220>
<221> 杂项特征
<222> (551)..(551)
<223> Xaa可以是任何天然存在的氨基酸
<400> 31
Ala Pro Ser Tyr His Val Val Arg Gly Asp Ile Ala Thr Ala Thr Glu
1 5 10 15
Gly Val Ile Ile Asn Ala Ala Asn Ser Lys Gly Gln Pro Gly Gly Gly
20 25 30
Val Cys Gly Ala Leu Tyr Lys Lys Phe Pro Glu Ser Phe Asp Leu Gln
35 40 45
Pro Ile Glu Val Gly Lys Ala Arg Leu Val Lys Gly Ala Ala Lys His
50 55 60
Ile Ile His Ala Val Gly Pro Asn Phe Asn Lys Val Ser Glu Val Glu
65 70 75 80
Gly Asp Lys Gln Leu Ala Glu Ala Tyr Glu Ser Ile Ala Lys Ile Val
85 90 95
Asn Asp Asn Asn Tyr Lys Ser Val Ala Ile Pro Leu Leu Ser Thr Gly
100 105 110
Ile Phe Ser Gly Asn Lys Asp Arg Leu Thr Gln Ser Leu Asn His Leu
115 120 125
Leu Thr Ala Leu Asp Thr Thr Asp Ala Asp Val Ala Ile Tyr Cys Arg
130 135 140
Asp Lys Lys Trp Glu Met Thr Leu Lys Glu Ala Val Ala Arg Arg Glu
145 150 155 160
Ala Val Glu Glu Ile Cys Ile Ser Asp Asp Ser Ser Val Thr Glu Pro
165 170 175
Asp Ala Glu Leu Val Arg Val His Pro Lys Ser Ser Leu Ala Gly Arg
180 185 190
Lys Gly Tyr Ser Thr Ser Asp Gly Lys Thr Phe Ser Tyr Leu Glu Gly
195 200 205
Thr Lys Phe His Gln Ala Ala Lys Asp Ile Ala Glu Ile Asn Ala Met
210 215 220
Trp Pro Val Ala Thr Glu Ala Asn Glu Gln Val Cys Met Tyr Ile Leu
225 230 235 240
Gly Lys Ser Met Ser Ser Ile Arg Ser Lys Cys Pro Val Glu Glu Ser
245 250 255
Glu Ala Ser Thr Pro Pro Ser Thr Leu Pro Cys Leu Cys Ile His Ala
260 265 270
Met Thr Pro Glu Arg Val Gln Arg Leu Lys Ala Ser Arg Pro Glu Gln
275 280 285
Ile Thr Val Cys Ser Ser Phe Pro Leu Pro Lys Tyr Arg Ile Thr Gly
290 295 300
Val Gln Lys Ile Gln Cys Ser Gln Pro Ile Leu Phe Ser Pro Lys Val
305 310 315 320
Pro Ala Tyr Ile His Pro Arg Lys Tyr Leu Val Glu Thr Pro Pro Val
325 330 335
Asp Glu Thr Pro Glu Pro Ser Ala Glu Asn Gln Ser Thr Glu Gly Thr
340 345 350
Pro Glu Gln Pro Pro Leu Ile Thr Glu Asp Glu Thr Arg Thr Arg Thr
355 360 365
Pro Glu Pro Ile Ile Ile Glu Glu Glu Glu Glu Asp Ser Ile Ser Leu
370 375 380
Leu Ser Asp Gly Pro Thr His Gln Val Leu Gln Val Glu Ala Asp Ile
385 390 395 400
His Gly Pro Pro Ser Val Ser Ser Ser Ser Trp Ser Ile Pro His Ala
405 410 415
Ser Asp Phe Asp Val Asp Ser Leu Ser Ile Leu Asp Thr Leu Glu Gly
420 425 430
Ala Ser Val Thr Ser Gly Ala Thr Ser Ala Glu Thr Asn Ser Tyr Phe
435 440 445
Ala Lys Ser Met Glu Phe Leu Ala Arg Pro Val Pro Ala Pro Arg Thr
450 455 460
Val Phe Arg Asn Pro Pro His Pro Ala Pro Arg Thr Arg Thr Pro Ser
465 470 475 480
Leu Ala Pro Ser Arg Ala Cys Ser Arg Thr Ser Leu Val Ser Thr Pro
485 490 495
Pro Gly Val Asn Arg Val Ile Thr Arg Glu Glu Leu Glu Ala Leu Thr
500 505 510
Pro Ser Arg Thr Pro Ser Arg Ser Val Ser Arg Thr Ser Leu Val Ser
515 520 525
Asn Pro Pro Gly Val Asn Arg Val Ile Thr Arg Glu Glu Phe Glu Ala
530 535 540
Phe Val Ala Gln Gln Gln Xaa Arg Phe Asp Ala Gly Ala
545 550 555
<210> 32
<211> 60
<212> PRT
<213> 人工序列
<220>
<223> 小鼠CD80 (TM-Cyt)
<400> 32
Thr Leu Val Leu Phe Gly Ala Gly Phe Gly Ala Val Ile Thr Val Val
1 5 10 15
Val Ile Val Val Ile Ile Lys Cys Phe Cys Lys His Arg Ser Cys Phe
20 25 30
Arg Arg Asn Glu Ala Ser Arg Glu Thr Asn Asn Ser Leu Thr Phe Gly
35 40 45
Pro Glu Glu Ala Leu Ala Glu Gln Thr Val Phe Leu
50 55 60
<210> 33
<211> 107
<212> PRT
<213> 人工序列
<220>
<223> 小鼠IgG4-CH3
<400> 33
Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu
1 5 10 15
Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asn Phe
20 25 30
Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala
35 40 45
Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr
50 55 60
Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly
65 70 75 80
Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His
85 90 95
Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys
100 105
<210> 34
<211> 266
<212> PRT
<213> 人工序列
<220>
<223> 衍生自COVID-19 E484K_N501Y_K417T突变体(巴西株突变体)的构建体
<400> 34
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Val Arg Phe
1 5 10 15
Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr
20 25 30
Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys
35 40 45
Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe
50 55 60
Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr
65 70 75 80
Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln
85 90 95
Ile Ala Pro Gly Gln Thr Gly Thr Ile Ala Asp Tyr Asn Tyr Lys Leu
100 105 110
Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu
115 120 125
Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg
130 135 140
Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr
145 150 155 160
Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Lys Gly Phe Asn Cys Tyr
165 170 175
Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr
180 185 190
Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro
195 200 205
Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Gly Val Lys Leu Glu Ser
210 215 220
Met Gly Ile Tyr Gln Ile Leu Ala Ile Tyr Ser Thr Val Ala Ser Ser
225 230 235 240
Leu Val Leu Leu Val Ser Leu Gly Ala Ile Ser Phe Trp Met Cys Ser
245 250 255
Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile
260 265
<210> 35
<211> 266
<212> PRT
<213> 人工序列
<220>
<223> 衍生自COVID-19 E484K_N501Y_K417N突变体(南非突变体)的构建体
<400> 35
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Val Arg Phe
1 5 10 15
Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr
20 25 30
Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys
35 40 45
Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe
50 55 60
Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr
65 70 75 80
Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln
85 90 95
Ile Ala Pro Gly Gln Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys Leu
100 105 110
Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu
115 120 125
Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg
130 135 140
Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr
145 150 155 160
Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Lys Gly Phe Asn Cys Tyr
165 170 175
Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr
180 185 190
Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro
195 200 205
Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Gly Val Lys Leu Glu Ser
210 215 220
Met Gly Ile Tyr Gln Ile Leu Ala Ile Tyr Ser Thr Val Ala Ser Ser
225 230 235 240
Leu Val Leu Leu Val Ser Leu Gly Ala Ile Ser Phe Trp Met Cys Ser
245 250 255
Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile
260 265
<210> 36
<211> 266
<212> PRT
<213> 人工序列
<220>
<223> 衍生自COVID-19 E484K突变体的构建体
<400> 36
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Val Arg Phe
1 5 10 15
Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr
20 25 30
Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys
35 40 45
Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe
50 55 60
Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr
65 70 75 80
Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln
85 90 95
Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu
100 105 110
Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu
115 120 125
Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg
130 135 140
Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr
145 150 155 160
Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Lys Gly Phe Asn Cys Tyr
165 170 175
Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr
180 185 190
Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro
195 200 205
Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Gly Val Lys Leu Glu Ser
210 215 220
Met Gly Ile Tyr Gln Ile Leu Ala Ile Tyr Ser Thr Val Ala Ser Ser
225 230 235 240
Leu Val Leu Leu Val Ser Leu Gly Ala Ile Ser Phe Trp Met Cys Ser
245 250 255
Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile
260 265
<210> 37
<211> 207
<212> PRT
<213> 人工序列
<220>
<223> 包含COVID-19信号序列(1-15)和COVID-19 RBD(330-521)的融合蛋白
<400> 37
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Pro
1 5 10 15
Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg
20 25 30
Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val
35 40 45
Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys
50 55 60
Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn
65 70 75 80
Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile
85 90 95
Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro
100 105 110
Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp
115 120 125
Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys
130 135 140
Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln
145 150 155 160
Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe
165 170 175
Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln
180 185 190
Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro
195 200 205
<210> 38
<211> 260
<212> PRT
<213> 人工序列
<220>
<223> 包含hIL-2信号序列、COVID-19 RBD(330-521)和HA(柔性-TM-Cyt)的融合蛋白
<400> 38
Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu
1 5 10 15
Val Thr Asn Ser Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val
20 25 30
Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg
35 40 45
Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser
50 55 60
Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp
65 70 75 80
Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp
85 90 95
Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr
100 105 110
Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn
115 120 125
Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr
130 135 140
Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser
145 150 155 160
Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly
165 170 175
Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn
180 185 190
Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu
195 200 205
Leu His Ala Pro Gly Val Lys Leu Glu Ser Met Gly Ile Tyr Gln Ile
210 215 220
Leu Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Leu Val Ser
225 230 235 240
Leu Gly Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln Cys
245 250 255
Arg Ile Cys Ile
260
<210> 39
<211> 364
<212> PRT
<213> 人工序列
<220>
<223> 包含COVID-19信号序列(1-15)、COVID-19 RBD(330-521)、人IgG4CH3和人CD80(TM-Cyt)的融合蛋白
<400> 39
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Pro
1 5 10 15
Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg
20 25 30
Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val
35 40 45
Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys
50 55 60
Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn
65 70 75 80
Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile
85 90 95
Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro
100 105 110
Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp
115 120 125
Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys
130 135 140
Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln
145 150 155 160
Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe
165 170 175
Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln
180 185 190
Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Gly
195 200 205
Ser Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
210 215 220
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
225 230 235 240
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
245 250 255
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
260 265 270
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
275 280 285
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
290 295 300
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Gly Ser Leu Leu
305 310 315 320
Pro Ser Trp Ala Ile Thr Leu Ile Ser Val Asn Gly Ile Phe Val Ile
325 330 335
Cys Cys Leu Thr Tyr Cys Phe Ala Pro Arg Cys Arg Glu Arg Arg Arg
340 345 350
Asn Glu Arg Leu Arg Arg Glu Ser Val Arg Pro Val
355 360
<210> 40
<211> 366
<212> PRT
<213> 人工序列
<220>
<223> 包含COVID-19信号序列(1-15)、COVID-19 RBD(330-521)、人IgG4CH3和HA(柔性-TM-Cyt)的融合蛋白
<400> 40
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Pro
1 5 10 15
Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg
20 25 30
Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val
35 40 45
Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys
50 55 60
Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn
65 70 75 80
Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile
85 90 95
Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro
100 105 110
Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp
115 120 125
Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys
130 135 140
Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln
145 150 155 160
Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe
165 170 175
Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln
180 185 190
Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Gly
195 200 205
Ser Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln
210 215 220
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
225 230 235 240
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
245 250 255
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
260 265 270
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu
275 280 285
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
290 295 300
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Gly Ser Gly Val
305 310 315 320
Lys Leu Glu Ser Met Gly Ile Tyr Gln Ile Leu Ala Ile Tyr Ser Thr
325 330 335
Val Ala Ser Ser Leu Val Leu Leu Val Ser Leu Gly Ala Ile Ser Phe
340 345 350
Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile
355 360 365
<210> 41
<211> 373
<212> PRT
<213> 人工序列
<220>
<223> 包含COVID-19信号序列(1-15)、COVID-19 RBD(330-530)、人IgG4CH3和人CD80(TM-Cyt)的融合蛋白
<400> 41
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Pro
1 5 10 15
Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg
20 25 30
Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val
35 40 45
Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys
50 55 60
Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn
65 70 75 80
Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile
85 90 95
Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro
100 105 110
Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp
115 120 125
Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys
130 135 140
Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln
145 150 155 160
Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe
165 170 175
Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln
180 185 190
Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala
195 200 205
Thr Val Cys Gly Pro Lys Lys Ser Gly Ser Gly Gln Pro Arg Glu Pro
210 215 220
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
225 230 235 240
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
245 250 255
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
260 265 270
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
275 280 285
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
290 295 300
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
305 310 315 320
Leu Ser Leu Gly Lys Gly Ser Leu Leu Pro Ser Trp Ala Ile Thr Leu
325 330 335
Ile Ser Val Asn Gly Ile Phe Val Ile Cys Cys Leu Thr Tyr Cys Phe
340 345 350
Ala Pro Arg Cys Arg Glu Arg Arg Arg Asn Glu Arg Leu Arg Arg Glu
355 360 365
Ser Val Arg Pro Val
370
<210> 42
<211> 375
<212> PRT
<213> 人工序列
<220>
<223> 包含COVID-19信号序列(1-15)、COVID-19 RBD(330-530)、人IgG4CH3和HA(柔性-TM-Cyt)的融合蛋白
<400> 42
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Pro
1 5 10 15
Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg
20 25 30
Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val
35 40 45
Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys
50 55 60
Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn
65 70 75 80
Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile
85 90 95
Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro
100 105 110
Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp
115 120 125
Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys
130 135 140
Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln
145 150 155 160
Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe
165 170 175
Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln
180 185 190
Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala
195 200 205
Thr Val Cys Gly Pro Lys Lys Ser Gly Ser Gly Gln Pro Arg Glu Pro
210 215 220
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
225 230 235 240
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
245 250 255
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
260 265 270
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
275 280 285
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
290 295 300
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
305 310 315 320
Leu Ser Leu Gly Lys Gly Ser Gly Val Lys Leu Glu Ser Met Gly Ile
325 330 335
Tyr Gln Ile Leu Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu
340 345 350
Leu Val Ser Leu Gly Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser
355 360 365
Leu Gln Cys Arg Ile Cys Ile
370 375
<210> 43
<211> 369
<212> PRT
<213> 人工序列
<220>
<223> 包含hIL-2信号序列、COVID-19 RBD(330-521)、人IgG4CH3和人CD80 (TM-Cyt)的融合蛋白
<400> 43
Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu
1 5 10 15
Val Thr Asn Ser Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val
20 25 30
Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg
35 40 45
Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser
50 55 60
Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp
65 70 75 80
Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp
85 90 95
Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr
100 105 110
Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn
115 120 125
Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr
130 135 140
Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser
145 150 155 160
Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly
165 170 175
Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn
180 185 190
Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu
195 200 205
Leu His Ala Pro Gly Ser Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
210 215 220
Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
225 230 235 240
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
245 250 255
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
260 265 270
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys
275 280 285
Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu
290 295 300
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
305 310 315 320
Lys Gly Ser Leu Leu Pro Ser Trp Ala Ile Thr Leu Ile Ser Val Asn
325 330 335
Gly Ile Phe Val Ile Cys Cys Leu Thr Tyr Cys Phe Ala Pro Arg Cys
340 345 350
Arg Glu Arg Arg Arg Asn Glu Arg Leu Arg Arg Glu Ser Val Arg Pro
355 360 365
Val
<210> 44
<211> 371
<212> PRT
<213> 人工序列
<220>
<223> 包含hIL-2信号序列、COVID-19 RBD(330-521)、人IgG4CH3和HA(柔性-TM-Cyt)的融合蛋白
<400> 44
Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu
1 5 10 15
Val Thr Asn Ser Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val
20 25 30
Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg
35 40 45
Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser
50 55 60
Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp
65 70 75 80
Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp
85 90 95
Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr
100 105 110
Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn
115 120 125
Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr
130 135 140
Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser
145 150 155 160
Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly
165 170 175
Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn
180 185 190
Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu
195 200 205
Leu His Ala Pro Gly Ser Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
210 215 220
Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
225 230 235 240
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
245 250 255
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
260 265 270
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys
275 280 285
Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu
290 295 300
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
305 310 315 320
Lys Gly Ser Gly Val Lys Leu Glu Ser Met Gly Ile Tyr Gln Ile Leu
325 330 335
Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu Val Leu Leu Val Ser Leu
340 345 350
Gly Ala Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg
355 360 365
Ile Cys Ile
370
<210> 45
<211> 378
<212> PRT
<213> 人工序列
<220>
<223> 包含hIL-2信号序列、COVID-19 RBD(330-530)、人IgG4CH3和人CD80 (TM-Cyt)的融合蛋白
<400> 45
Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu
1 5 10 15
Val Thr Asn Ser Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val
20 25 30
Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg
35 40 45
Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser
50 55 60
Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp
65 70 75 80
Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp
85 90 95
Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr
100 105 110
Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn
115 120 125
Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr
130 135 140
Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser
145 150 155 160
Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly
165 170 175
Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn
180 185 190
Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu
195 200 205
Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Gly Ser Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu
225 230 235 240
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Gly Ser Leu Leu Pro Ser
325 330 335
Trp Ala Ile Thr Leu Ile Ser Val Asn Gly Ile Phe Val Ile Cys Cys
340 345 350
Leu Thr Tyr Cys Phe Ala Pro Arg Cys Arg Glu Arg Arg Arg Asn Glu
355 360 365
Arg Leu Arg Arg Glu Ser Val Arg Pro Val
370 375
<210> 46
<211> 380
<212> PRT
<213> 人工序列
<220>
<223> 包含hIL-2信号序列、COVID-19 RBD(330-530)、人IgG4CH3和HA(柔性-TM-Cyt)的融合蛋白
<400> 46
Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu
1 5 10 15
Val Thr Asn Ser Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val
20 25 30
Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg
35 40 45
Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser
50 55 60
Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp
65 70 75 80
Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp
85 90 95
Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr
100 105 110
Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn
115 120 125
Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr
130 135 140
Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser
145 150 155 160
Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly
165 170 175
Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn
180 185 190
Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu
195 200 205
Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Gly Ser Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu
225 230 235 240
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Gly Ser Gly Val Lys Leu
325 330 335
Glu Ser Met Gly Ile Tyr Gln Ile Leu Ala Ile Tyr Ser Thr Val Ala
340 345 350
Ser Ser Leu Val Leu Leu Val Ser Leu Gly Ala Ile Ser Phe Trp Met
355 360 365
Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile
370 375 380
<210> 47
<211> 266
<212> PRT
<213> 人工序列
<220>
<223> 包含COVID-19信号序列(1-13)、COVID-19 RBD(327-531)和HA(柔性-TM-Cyt)的融合蛋白
<400> 47
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Val Arg Phe
1 5 10 15
Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr
20 25 30
Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys
35 40 45
Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe
50 55 60
Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr
65 70 75 80
Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln
85 90 95
Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu
100 105 110
Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu
115 120 125
Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg
130 135 140
Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr
145 150 155 160
Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr
165 170 175
Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr
180 185 190
Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro
195 200 205
Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Gly Val Lys Leu Glu Ser
210 215 220
Met Gly Ile Tyr Gln Ile Leu Ala Ile Tyr Ser Thr Val Ala Ser Ser
225 230 235 240
Leu Val Leu Leu Val Ser Leu Gly Ala Ile Ser Phe Trp Met Cys Ser
245 250 255
Asn Gly Ser Leu Gln Cys Arg Ile Cys Ile
260 265
<210> 48
<211> 264
<212> PRT
<213> 人工序列
<220>
<223> 包含COVID-19信号序列(1-15)、COVID-19 RBD(330-521)和HA(柔性-TM-Cyt)的融合蛋白
<400> 48
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Pro
1 5 10 15
Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg
20 25 30
Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val
35 40 45
Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys
50 55 60
Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn
65 70 75 80
Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile
85 90 95
Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro
100 105 110
Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp
115 120 125
Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys
130 135 140
Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln
145 150 155 160
Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe
165 170 175
Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln
180 185 190
Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala
195 200 205
Thr Val Cys Gly Pro Lys Lys Ser Gly Val Lys Leu Glu Ser Met Gly
210 215 220
Ile Tyr Gln Ile Leu Ala Ile Tyr Ser Thr Val Ala Ser Ser Leu Val
225 230 235 240
Leu Leu Val Ser Leu Gly Ala Ile Ser Phe Trp Met Cys Ser Asn Gly
245 250 255
Ser Leu Gln Cys Arg Ile Cys Ile
260
<210> 49
<211> 291
<212> PRT
<213> 人工序列
<220>
<223> 包含COVID-19信号序列(1-15)、COVID-19 RBD(327-531)和COVID19 (JMD-TM)的融合蛋白
<400> 49
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val
1 5 10 15
Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn
20 25 30
Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser
35 40 45
Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser
50 55 60
Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys
65 70 75 80
Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val
85 90 95
Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr
100 105 110
Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn
115 120 125
Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu
130 135 140
Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu
145 150 155 160
Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn
165 170 175
Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val
180 185 190
Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His
195 200 205
Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Cys Gly Lys Tyr
210 215 220
Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu Gly Phe Ile Ala
225 230 235 240
Gly Leu Ile Ala Ile Val Met Val Thr Ile Met Leu Cys Cys Met Thr
245 250 255
Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys Ser Cys Gly Ser Cys Cys
260 265 270
Lys Phe Asp Glu Asp Asp Ser Glu Pro Val Leu Lys Gly Val Lys Leu
275 280 285
His Tyr Thr
290
<210> 50
<211> 288
<212> PRT
<213> 人工序列
<220>
<223> 包含COVID-19信号序列(1-13)、COVID-19 RBD(327-531)和COVID19 (JMD-TM)的融合蛋白
<400> 50
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Val Arg Phe
1 5 10 15
Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr
20 25 30
Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys
35 40 45
Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe
50 55 60
Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr
65 70 75 80
Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln
85 90 95
Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu
100 105 110
Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu
115 120 125
Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg
130 135 140
Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr
145 150 155 160
Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr
165 170 175
Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr
180 185 190
Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro
195 200 205
Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Gly Lys Tyr Glu Gln Tyr
210 215 220
Ile Lys Trp Pro Trp Tyr Ile Trp Leu Gly Phe Ile Ala Gly Leu Ile
225 230 235 240
Ala Ile Val Met Val Thr Ile Met Leu Cys Cys Met Thr Ser Cys Cys
245 250 255
Ser Cys Leu Lys Gly Cys Cys Ser Cys Gly Ser Cys Cys Lys Phe Asp
260 265 270
Glu Asp Asp Ser Glu Pro Val Leu Lys Gly Val Lys Leu His Tyr Thr
275 280 285
<210> 51
<211> 275
<212> PRT
<213> 人工序列
<220>
<223> 包含COVID-19信号序列(1-13)、COVID-19 RBD(330-521)和COVID19 (JMD-TM)的融合蛋白
<400> 51
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Pro Asn Ile
1 5 10 15
Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala
20 25 30
Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp
35 40 45
Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr
50 55 60
Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr
65 70 75 80
Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro
85 90 95
Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp
100 105 110
Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys
115 120 125
Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn
130 135 140
Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly
145 150 155 160
Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu
165 170 175
Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr
180 185 190
Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Gly Lys Tyr
195 200 205
Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu Gly Phe Ile Ala
210 215 220
Gly Leu Ile Ala Ile Val Met Val Thr Ile Met Leu Cys Cys Met Thr
225 230 235 240
Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys Ser Cys Gly Ser Cys Cys
245 250 255
Lys Phe Asp Glu Asp Asp Ser Glu Pro Val Leu Lys Gly Val Lys Leu
260 265 270
His Tyr Thr
275
Claims (30)
1.分离的多核苷酸，其编码甲病毒非结构蛋白nsp1、nsp2、nsp3和nsp4以及包含与信号序列和/或跨膜结构域融合的冠状病毒蛋白的多肽。
2.权利要求1的多核苷酸，其中所述冠状病毒蛋白是刺突(S)蛋白、核衣壳(N)蛋白、膜(M)蛋白、小包膜(E)蛋白或其组合。
3.权利要求1的多核苷酸，其中所述冠状病毒蛋白是刺突(S)蛋白。
4.权利要求1的多核苷酸，其中所述冠状病毒蛋白是刺突(S)蛋白的S1和/或S2亚基。
5.权利要求4的多核苷酸，其中所述冠状病毒蛋白是刺突(S)蛋白中的S1亚基。
6.权利要求5的多核苷酸，其中所述冠状病毒蛋白是所述S1亚基的受体结合结构域。
7.权利要求1-6中任一项的多核苷酸，其中所述跨膜结构域衍生自流感血凝素(HA)、CD80或TLR4。
8.权利要求1-6中任一项的多核苷酸，其中所述跨膜结构域是衍生自冠状病毒结构蛋白的经修饰的跨膜结构域。
9.权利要求8的多核苷酸，所述经修饰的跨膜结构域包含COVID-19刺突(S)的近膜结构域和跨膜结构域。
10.权利要求1-9中任一项的多核苷酸，其中冠状病毒蛋白融合至信号序列和跨膜结构域。
11.权利要求1-10中任一项的多核苷酸，其中所述信号序列衍生自人IL-2。
12.权利要求1-10中任一项的多核苷酸，其中所述信号序列衍生自COVID-19刺突蛋白。
13.权利要求1-12中任一项的多核苷酸，其中所述跨膜结构域和/或信号序列通过接头融合至所述冠状病毒蛋白。
14.权利要求13的多核苷酸，其中所述接头是IgG4CH3和/或短接头。
15.权利要求1-14中任一项的多核苷酸，其中所述冠状病毒是COVID-19。
16.权利要求15的多核苷酸，其中所述多肽编码选自SEQ ID NO 1、5、6、8、9、11、12、13、34、35和36的氨基酸序列。
17.权利要求15的多核苷酸，其中所述多肽编码选自SEQ ID NO 37-51的氨基酸序列。
18.权利要求1-17中任一项的多核苷酸，其中所述多核苷酸是RNA。
19.权利要求1-17中任一项的多核苷酸，其中所述多核苷酸是DNA。
20.包含权利要求1-19中任一项的多核苷酸的载体。
21.权利要求20的载体，所述载体包含启动子，5' UTR，编码甲病毒非结构蛋白nsp1、nsp2、nsp3和nsp4的多核苷酸，SG启动子，编码包含与信号序列和/或跨膜结构域融合的冠状病毒蛋白的多肽的目标基因，3' UTR和多聚A尾。
22.包含权利要求1-21中任一项的多核苷酸或载体以及药学上可接受的载体的疫苗组合物。
23.权利要求22的疫苗组合物，其中所述药学上可接受的载体是递送媒介物。
24.权利要求23的疫苗组合物，其中所述递送媒介物是由一种或多种甲病毒结构蛋白组成的颗粒或脂质递送系统。
25.权利要求1-21中任一项的多核苷酸或载体用于药物制造的用途。
26.权利要求25的用途，其中所述药物用于在受试者中诱导免疫调节。
27.权利要求25的用途，其中所述药物用于治疗或预防受试者免于由冠状病毒感染引起的病症。
28.在受试者中免疫调节的方法，其包含给需要其的受试者施用免疫学上有效量的权利要求22-24中任一项的疫苗组合物。
29.在受试者中针对冠状病毒的病毒感染进行治疗、预防和/或免疫的方法，其包含给需要其的受试者施用有效量的权利要求22-24中任一项的疫苗组合物。
30.包含与信号序列和/或异源跨膜结构域融合的冠状病毒结构蛋白的多肽。
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| CN112029781B (zh) * | 2020-08-14 | 2023-01-03 | 中山大学 | 一种新型冠状病毒SARS-CoV-2的安全型复制子系统及其应用 |
| WO2023113016A1 (en) * | 2021-12-17 | 2023-06-22 | Vlp Therapeutics Japan, Inc. | Efficient vaccine |
| US20230366001A1 (en) * | 2022-05-12 | 2023-11-16 | SunVax mRNA Therapeutics Inc. | Synthetic self-amplifying mrna molecules with secretion antigen and immunomodulator |
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| US8795682B2 (en) * | 2007-05-02 | 2014-08-05 | Emory University | Virus-like particles comprising chimeric human immunodeficiency virus (HIV)/mouse mammary tumor virus (MMTV) envelopes |
| CN104293740B (zh) * | 2013-07-18 | 2018-03-09 | 特菲(天津)生物医药科技有限公司 | 表面展示sars双价抗原的重组杆状病毒及其制备方法和应用 |
| CN106928372B (zh) * | 2016-12-30 | 2019-09-13 | 北京大学深圳研究生院 | 乙肝重组抗原及其表达基因、构建方法、病毒样颗粒及其制备方法、应用与疫苗 |
| BR112020012190A8 (pt) | 2017-12-20 | 2023-04-11 | Vlp Therapeutics Llc | Partícula de replicon de alfavírus |
| RU2733832C1 (ru) * | 2020-07-28 | 2020-10-07 | Федеральное бюджетное учреждение науки "Государственный научный центр вирусологии и биотехнологии "Вектор" Федеральной службы по надзору в сфере защиты прав потребителей и благополучия человека (ФБУН ГНЦ ВБ "Вектор" Роспотребнадзора) | Искусственный ген Stbl_RBD_TrM_SC2, кодирующий бицистронную структуру, образованную последовательностями рецепторсвязывающего домена гликопротеина S коронавируса SARS-CoV-2, трансмембранного региона, P2A-пептида и гликопротеина G VSV, рекомбинантная плазмида pStem-rVSV-Stbl_RBD_TrM_SC2, обеспечивающая экспрессию искусственного гена, и рекомбинантный штамм вируса везикулярного стоматита rVSV-Stbl_RBD_TrM_SC2, используемый для создания вакцины против коронавируса SARS-CoV-2 |
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| US20210322541A1 (en) | 2021-10-21 |
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| JP2023523580A (ja) | 2023-06-06 |
| EP4135847A1 (en) | 2023-02-22 |
| EP4135847A4 (en) | 2024-05-15 |
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