CN116981448A - 包括工程化的植物来源的细胞外囊泡的组合物及其用作疫苗的用途 - Google Patents

包括工程化的植物来源的细胞外囊泡的组合物及其用作疫苗的用途 Download PDF

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CN116981448A
CN116981448A CN202280014285.7A CN202280014285A CN116981448A CN 116981448 A CN116981448 A CN 116981448A CN 202280014285 A CN202280014285 A CN 202280014285A CN 116981448 A CN116981448 A CN 116981448A
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乔瓦尼·卡穆西
基亚拉·加伊
玛格丽塔·阿尔巴·卡洛塔·普马托
弗朗切斯科·朱塞佩·德·罗萨
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Abstract

本发明提供了一种用作疫苗的包括非免疫调节的工程化的植物来源的细胞外囊泡(EV)的组合物，所述囊泡装载有编码蛋白抗原的外源核酸分子。进一步提供了一种制备所述组合物的方法，所述方法利用一种或多种聚阳离子物质和一个或多个糖分子。

Description

包括工程化的植物来源的细胞外囊泡的组合物及其用作疫苗 的用途
技术领域
本发明涉及包括植物来源的细胞外囊泡的组合物，以用作疫苗和/或用于预防应用。更具体地，本发明涉及一种包括装载有外源核酸分子的非免疫调节的工程化的植物来源的细胞外囊泡(EV)的组合物。
背景技术
疫苗接种是预防和控制传染性和非传染性疾病的最有效的公共健康干预措施之一。存在不同类型的疫苗，包括减毒活疫苗、灭活疫苗、亚单位或重组或结合疫苗、类毒素疫苗以及基于核酸的那些疫苗。
基于核酸的疫苗包括病毒载体、质粒DNA和mRNA。它们已成为传统疫苗方法的有潜力的替代品，因为它们诱导广泛的保护性免疫反应的能力，并且它们通过快速且灵活的生产工艺生产的潜力。在基于核酸的疫苗中，RNA疫苗与其他疫苗类型相比具有能提供潜在优势的几种属性。事实上，RNA疫苗的特征是不存在真核生物污染物。与DNA疫苗相反，RNA疫苗不需要到达细胞核就可以发挥作用并且它们更安全，因为质粒DNA疫苗可以整合到免疫宿主的基因组中。在该上下文中，mRNA分子诱导针对编码抗原的免疫反应。
该机制已被证明用于包括报告基因、病毒抗原、肿瘤抗原和过敏原的多种报告基因(reporter gene)。然而，mRNA的不稳定性、高度先天免疫原性和低效的体内递送限制了RNA疫苗的临床应用。这些疫苗所面临的主要挑战是细胞内递送。由于其对降解酶的敏感性，mRNA在体内的生理条件下高度不稳定。
到目前为止，在临床应用中，mRNA分子通过与阳离子聚合物络合或者使用合成脂质纳米颗粒(LNP)(也称为脂质体、阳离子纳米粒、EV模拟纳米囊泡或基于多肽的囊泡)来运载以提高其功效。LNP可以保护mRNA免受酶的影响，从而促进了更高的稳定性，延长了RNA的循环寿命和体内递送。LNP颗粒是通过将mRNA分子与不同的合成脂质或聚合物混合来产生的。然而，LNP代表一种低效的递送系统。
事实上，LNP可以聚集在非预期组织(unintended tissue)中，从而限制了其对感兴趣的靶组织的作用，并且LNP的特征是通过网状内皮系统或单核吞噬细胞系统进行快速清除(Koppers-Lalic D.,et al.“Virus-modified exosomes for targeted RNAdelivery；a new approach in nanomedicine”.Adv Drug Deliv Rev.2013Mar；65(3):348-56)。此外，LNP可在体内诱导促炎症反应和细胞凋亡。
此外，LNP的细胞摄入是通过内吞作用介导的，这可能会激活细胞的自噬-溶酶体途径。越来越多的证据表明，纳米颗粒的内吞作用会产生自噬体，并且随后它们与溶酶体的融合，导致其内容物消化。
近年来，已经尝试通过使用细胞外囊泡(EV)来克服LNP的限制。事实上，EV是由细胞自然分泌的，并且与合成纳米材料(如LNP)相比更安全。EV可以利用其天然作用机制，并且克服了组装颗粒的一些限制，包括免疫原性、毒性、外源颗粒的施用、有限的细胞摄入和颗粒的化学组装。EV摄入的途径与LNP不同，并且不太可能引发自噬-溶酶体途径，因为它们会在没有溶酶体捕获的情况下将其内容物释放到细胞质中。此外，由于它们的尺寸小，EV可以逃避快速吞噬作用，并在循环中稳定携带和递送核酸，从而通过血管内皮到靶细胞。
而且，与LNP相比，EV在生物相容性、循环过程中的低清除、低毒性、低安全问题和高特异性方面，显示出了几种优势(Sancho-Albero M，等人(2020)“使用外泌体作为载体进行高级疗法(Sancho-Albero M,et al(2020)“Use of exosomes as vectors to carryadvanced therapies”.RSC Adv 10,23975-23987)。EV的天然来源使其膜具有高且固有的生物相容性，并且在受体细胞中有效吸收。此外，植物来源的EV可抵抗胃环境，并且在口服施用后到达肠道。已对包裹核酸分子的EV的多个临床应用进行了研究，包括RNA干扰、用于神经退行性疾病的基于RNA的基因治疗、癌症、含有miRNA和siRNA分子的癌症疫苗。
US20200069594公开了植物来源的细胞外囊泡的用途，该植物来源的细胞外囊泡包括用于递送包括编码和非编码核酸分子的治疗剂的阳离子聚合物。
本领域众所周知，EV能够保护并且递送核酸分子。对于疫苗制剂，EV的有益活性主要基于其对先天性和适应性免疫系统细胞的促进作用，例如，如在Jesus S.等人对HBV疫苗制剂进行的研究中所示的((Jesus S.,et al“Exosomes as adjuvants for therecombinant hepatitis B antigen:First report”.Eur J Pharm Biopharm.2018Dec；133:1-11)。
WO2020191361公开了EV用作疫苗来诱导细胞免疫反应并且治疗和/或预防一系列医学疾病的用途。
WO2020050808描述了植物来源的外泌体作为佐剂在疫苗应用中的用途，以及这些囊泡在激活或抑制免疫系统细胞中的免疫调节特性。
从各种植物中分离的细胞外囊泡已显示出通过减少肠道炎症来对免疫系统的细胞发挥调节活性。植物来源包括姜黄素(Ohno M.,et al,“Nanoparticle curcuminameliorates experimental colitis via modulation of gut microbiota andinduction of regulatory T cells”PLoS One.(2017)Oct 6；12(10):e0185999)、姜(Zhang M.,et al,(2016)“Edible ginger-derived nanoparticles:Anovel therapeuticapproach for the prevention and treatment of inflammatory bowel disease andcolitis-associated cancer”,Biomaterials 101:321-40)、橙子(Berger E.,et al,“Useof Nanovesicles from Orange Juice to Reverse Diet-Induced Gut Modificationsin Diet-Induced Obese Mice”(2020)Mol Ther Methods Clin Dev.18:880-892)、葡萄、葡萄柚和胡萝卜(Ju S,et al.“Grape exosome-like nanoparticles induce intestinalstem cells and protect mice from DSS-induced colitis”(2013)Mol Ther.21(7):1345-57)。此外，来自蓝莓的EV已显示出可减少用TNF-α刺激的内皮细胞中的促炎基因的基因表达，并且保护内皮细胞免受TNF诱导的细胞毒性和氧化应激(De Robertis M等人，“蓝莓来源的类外泌体纳米颗粒对抗TNF-α诱导的EA.hy926细胞基因表达变化的反应(DeRobertis M,et al.“Blueberry-Derived Exosome-Like Nanoparticles Counter theResponse to TNF-α-Induced Change on Gene Expression in EA.hy926 Cells”(2020)Biomolecules 10(5):742)。
尽管EV具有有益的作用，但这些囊泡的免疫调节特性可能是疫苗应用中的重大限制。事实上，免疫系统的非特异性激活或抑制对疫苗可能是有害的。具有免疫抑制活性的EV的使用可以通过抑制免疫细胞反应来显著降低疫苗功效。此外，对EV的免疫反应的发展可能会导致疫苗清除的加速。相反地，通过免疫刺激EV来促进免疫系统可以是有害的并且会导致受试者的免疫系统的有害激活和/或过度反应。对囊泡的免疫反应可以限制疫苗的重复应用。此外，囊泡的先天免疫感应可以与抗原表达的抑制有关，并且可能会对免疫反应产生负面影响(Pardi N,et al“mRNA vaccines-a new era in vaccinology”(2018)Nat RevDrug Discov.17(4):261-279)。总之，基于这些囊泡对免疫系统所显示出的影响，上述证据突出了在疫苗制剂中使用EV的显著缺点。
为了克服现有技术的限制和缺点，本发明提供了一种用作疫苗的包括非免疫调节的工程化的植物来源的细胞外囊泡(EV)的组合物，以及一种制备如所附独立权利要求所限定的所述组合物的方法。从属权利要求确定了所要求保护的组合物和方法的进一步优势特征。所附权利要求的主题构成了本说明书不可分割的部分。
发明内容
本发明涉及一种包括非免疫调节的工程化的植物来源的细胞外囊泡(EV)的组合物，其中所述细胞外囊泡(EV)由包括外脂质层和内脂质层的脂质双层膜界定，
其中所述EV在内部装载有编码至少一种蛋白抗原的外源核酸分子；
其中所述EV具有20至500nm的范围内，优选200至300nm的范围内的直径；
其中所述EV的脂质双层膜上的膜电位在+5至-5mV的范围内；
其中在用作疫苗的组合物中≤44％的EV在脂质双层膜的外层中包含磷脂酰丝氨酸。
如本文所用，术语“细胞外囊泡(EV)”是指由几乎所有活细胞释放的异质颗粒群体，其由磷脂双层界定或包裹并且携带脂质、蛋白、核酸和来源于它们所来源的细胞的其他分子。这些囊泡主要包括通过质膜出芽释放的微囊泡，以及来源于内体室的外泌体。细胞外囊泡称为“颗粒”、“微粒”、“纳米囊泡”、“微囊泡”和“外泌体”。EV的固有细胞靶向特性取决于其脂质组成和蛋白含量，以及其在循环中的内在稳定性，这使得这些囊泡有资格作为用于治疗剂递送的载体。
在本说明书中，术语“免疫调节”是指通过增强(免疫刺激)或减少(免疫抑制)免疫反应来使免疫系统功能发生改变的过程。因此，本文所用的表达“非免疫调节的EV”是指对免疫系统没有任何促进或免疫抑制作用的细胞外囊泡。
如本文所用，术语“工程化的EV”是指已通过将外源核酸分子装载到囊泡的供体细胞来体外修饰以表达异源组分的细胞外囊泡。因此，旨在工程化的EV是一种非天然存在的囊泡。
在本说明书的上下文中的表达“在内部装载”是指通过例如转染、转化或转导在细胞外囊泡(例如植物来源的EV)中引入核酸分子。
本说明书中所用的术语“外源核酸分子”是指一种不是本发明的EV的天然物本身的部分的异源核酸分子。表达“异源的”是指核酸分子来源于动物或其他植物种类而不是根据本发明的细胞外囊泡，或者来源于不同的供体细胞、不同的条件，或者来源于经基因修饰的供体细胞。
本文所用的术语“抗原蛋白”是指能够引发免疫反应的蛋白分子。
根据本发明，装载在植物来源的EV中的外源核酸分子优选选自由以下组成的组：DNA、cDNA、信使RNA(mRNA)、前体mRNA、长链RNA、编码RNA、单链RNA、双链RNA、线性RNA、RNA寡核苷酸、自复制RNA(复制子RNA)、逆转录病毒RNA、病毒RNA(vRNA)。
在本发明的优选的实施方式中，外源核酸分子是信使RNA(mRNA)分子。在本发明的上下文中，外源mRNA分子可以包括一个或多个修饰，诸如，例如5’帽结构、5’UTR、开放阅读框、3’UTR和多腺苷酸尾(polyA tail)。
根据本发明，组合物中的EV可以装载有单核酸分子或者两个或更多个核酸分子的组合。
在本发明的一个优选的实施方式中，EV中所装载的外源核酸分子的含量在20至200ng/109EV的范围内，优选30至100ng/109EV，更优选40至60ng/109EV。
将外源核酸分子装载到根据本发明的EV中可通过本领域已知的多种不同的技术来完成，包括例如电穿孔、超声处理、脂质体介导、微量注射(microinjection)、共孵育、透析和冻融循环。
本发明利用具有在20至500nm、优选100至400nm、更优选200至300nm范围内的直径的细胞外囊泡。
根据本发明，组合物中的EV的脂质双层膜上的膜电位的值在+5至-5mV、优选+2至-4mV、更优选0至-3的范围内。
在本发明的另一个更优选的实施方式中，EV的膜电位的值为-2mV。
在根据本发明的组合物中，组合物中小于或等于(≤)44％的总EV的EV量在脂质双层膜的外层中包括磷脂酰丝氨酸。
优选地，在脂质双层膜的外层中在包括磷脂酰丝氨酸的组合物中的EV量包含在总EV的25％至44％，更优选地总EV的35％至44％，甚至更优选地总EV的40％至44％的范围内。
本发明使用的植物来源的EV优选地来源于选自由以下组成的组的一种或多种植物：柑橘属，包括柠檬和橙子；猕猴桃属，包括奇异果；葫芦属，包括西葫芦；芸苔属，包括卷心菜和羽衣甘蓝；石榴属，包括石榴；越橘属，包括蓝莓，以及芹属，包括芹菜。
本发明的范围既包括含有来源于单个植物种类的EV的组合物又包括含有来源于多个植物种类的EV的组合物。应理解，植物来源的EV可以以其天然形式或通过化学修饰来使用。
优选地，根据本发明的组合物中的植物来源的EV可从果汁、植物的部分或植物细胞的培养基中纯化得到。植物细胞和部分可来源于叶子、果肉、芽或嫩枝(sprout)。
合适的EV纯化技术包括但不限于，超速离心、过滤和切向流过滤。选择用于纯化植物来源的EV的最合适的方法属于本领域普通技术人员的知识和技能范围。
在本发明的一个实施方式中，本发明组合物中EV的总蛋白含量在100至200ng/1010EV、更优选120至160ng/1010EV的范围内。
在另一个实施方式中，本发明组合物中EV的总RNA含量在20至200ng/109EV、更优选30至100ng/109EV、甚至更优选40至60ng/109EV的范围内。
表达“总蛋白含量”既包括内源蛋白物(EV的内部和膜含量)又包括本发明中使用的EV中所装载的蛋白。
在本说明书的上下文中，表达“总RNA含量”既包括内源RNA物又包括根据本发明的EV中所装载的外源RNA。
如下文实验部分进一步所示，本发明人惊奇地发现，具有如上文限定的结构和功能特征的工程化的植物来源的EV没有表现出任何免疫调节活性，即它们缺乏影响免疫系统的细胞的任何能力，既不促进也不降低这些细胞的激活和功效。
与天然的植物来源的EV不同，根据本发明的非天然存在的EV有利地能够将抗原分子递送至靶细胞，但不会对免疫系统的细胞本身产生任何影响。因此，根据本发明的EV的使用能够克服与基于EV的疫苗制剂有关的安全问题，并且避免免疫系统的有害激活或抑制，从而增强疫苗的功效。
此外，根据本发明的EV被证明能有效地将核酸试剂装载和运载至受体细胞中，并且保护它们免受环境降解。具体地，对胃环境的高抗性允许口服施用根据本发明的组合物。
施用后，所装载的EV与抗原呈递细胞(APC)(包括巨噬细胞和树突状细胞)的相互作用允许核酸分子转移至抗原呈递细胞中。在靶细胞中，表达包含DNA和mRNA分子的核酸分子，从而导致蛋白抗原翻译。然后，将抗原呈递在APC的表面上，诱导直接针对肿瘤细胞或病原体的免疫细胞的特异性激活，从而允许高效的免疫保护。
由于如上文所述的非天然存在的EV的有利特征，本发明的组合物特别适合用作疫苗。
在本发明的上下文中，本发明的组合物可以用作疫苗以用于治疗现有疾病或预防性地预防该疾病的发生。
由包裹至本发明的EV中的外源核酸分子编码的示例性蛋白抗原包括但不限于，细菌、病毒、真菌、原生动物和肿瘤抗原、其哺乳动物同源物，以及来自其兽医或工业上感兴趣的动物的同源物。
因此，本发明的组合物特别地用于治疗或预防传染性疾病或癌症疾病。
示例性的癌症疾病包括但不限于，膀胱癌、宫颈癌、肾细胞癌、睾丸癌、结直肠癌、肺癌、头颈癌、卵巢、淋巴瘤、肝癌、胶质母细胞瘤、黑色素瘤、骨髓瘤、白血病、胰腺癌。
例如但不限于，传染性疾病可以是病毒性疾病、细菌性疾病、真菌性疾病或原生动物性疾病，诸如，例如COVID-19疾病、流感、HPV感染、HIV感染、鼻病毒感染、肝炎、黄病毒感染、脑炎、脑膜炎、胃肠炎、霍乱、白喉、衣原体、结核病、伤寒、性传播感染(STI)、疟疾、真菌病、弓形虫病。
在本发明的一个实施方式中，由装载至EV中的外源核酸分子编码的至少一种抗原是选自由以下组成的组的肿瘤抗原：人激肽释放酶相关肽酶3(也称为前列腺特异性抗原(PSA))、人前列腺干细胞抗原(PSCA)、人膀胱特异性膜抗原(PSMA)、人金属还原酶(前列腺的六跨膜上皮抗原1(STEAP1))、人受体酪氨酸-蛋白激酶erbB-2(也称为酪氨酸激酶型细胞表面受体HER2)、人细胞表面相关粘蛋白1蛋白(MUC1)(也称为乳腺癌相关抗原DF3)、人酪氨酸酶相关蛋白2(TRP-2)、人丝氨酸/苏氨酸蛋白激酶B-raf(也称为原癌基因B-raf)、人肥大细胞/干细胞生长因子受体Kit(也称为原癌基因c-Kit)、人GTP酶NRas(也称为转化蛋白N-Ras)、人黑色素瘤相关抗原1、人黑色素瘤相关抗原1蛋白、人NY-ESO-1蛋白，及它们的任何组合。
在本发明的另一个实施方式中，至少一种蛋白抗原是来自选自由以下组成的组的细菌的细菌抗原：金黄色葡萄球菌、结核分枝杆菌、沙眼衣原体、化脓性链球菌、肺炎链球菌、伯氏疏螺旋体、马约氏尼疏螺旋体(例如，莱姆病)、克雷伯氏菌属、铜绿假单胞菌、肠球菌属、变形杆菌属(例如寻常菌(vulgaris)、奇异菌(mirabilis)、彭氏菌(penneri))、淋病奈瑟菌、肠杆菌属、放线菌属、凝固酶阴性葡萄球菌(CoNS)、支原体属、艰难梭菌、炭疽杆菌、霍乱弧菌、肉毒杆菌、破伤风梭菌、沙门氏菌属、梅毒螺旋体、疟原虫属，以及它们的任何组合。
在本发明的又一个实施方式中，至少一种蛋白抗原是来自选自由以下组成的组的真菌的真菌抗原：芽生菌、格特隐球菌、新型隐球菌、镰孢菌、曲霉、念珠菌、白色念珠菌、耳念珠菌、隐球菌、组织胞浆菌、芽生菌、球孢子菌、毛霉菌、卡氏肺囊虫、皮癣菌、孢子丝菌，以及它们的任何组合。
在又一个实施方式中，至少一种蛋白抗原是来自选自由以下组成的组的原生动物的原生动物抗原：疟原虫(例如，间日疟原虫和恶性疟原虫)、肠贾第虫(Giardiaintestinalis)、鲑六鞭毛虫、组织滴虫(Histomonas meleagridis)、胎三毛滴虫(Trichomonas foetus)、脆弱双核阿米巴(Dientamoeba fragilis)、阴道毛滴虫、利什曼原虫、克氏锥虫、布氏罗得西亚锥虫(Trypanosoma brucei rhodensiense)、布氏甘比亚锥虫(Trypanosoma brucei gambiense)、疟原虫寄生虫、痢疾阿米巴(Entamoebahistolytica)、内格里阿米巴(Naeglaria)、棘阿米巴(Acanthomoeba)、卵菌纲(Peronosporomycetes)、致病疫霉(Phytophthora infestans)、兰氏贾第鞭毛虫(Giardialamblia)、十二指肠贾第虫、弓形虫、大肠绒毛虫(Balantidium Coli)、牛泰勒虫(Theileria parva)、环状泰勒虫(Theileria parva)、棕色耳螨(Phipicephalusappendiculatus)、莫氏原藻(Prototheca moriformis)，以及它们的任何组合。
优选地，原生动物抗原选自由以下组成的组：弓形虫的致密颗粒蛋白6(GRA6)、棒状蛋白2A(ROP2A)、棒状蛋白18(ROP18)、表面抗原1(SAG1)、表面抗原2A(SAG2A)、顶膜抗原1(AMA1)，以及它们的任何组合。
在又一个实施方式中，至少一种蛋白抗原是来自选自由以下组成的组的病毒的病毒抗原：人乳突病毒(HPV)、人免疫缺陷病毒HIV(例如，HIV-1、HIV-2)、A型肝炎病毒、B型肝炎病毒(HBV)、C型肝炎病毒、D型肝炎病毒、E型肝炎病毒、疱疹病毒(人λ疱疹病毒4型(爱泼斯坦-巴尔(EB)病毒)、人单纯疱疹病毒2型(HSV2)、人疱疹病毒8型、流感病毒(例如，A型流感病毒、B型流感病毒)、巨细胞病毒、克里米亚-刚果出血热正内罗病毒(Crimean-Congohemorrhagic fever orthonairovirus)、冠状病毒、人多瘤病毒2型、BK病毒、严重急性呼吸道症候群冠状病毒(SARS-CoV、SARS-Cov-2、COVID-19)、中东呼吸综合征相关冠状病毒(MERS-CoV)、诺如病毒、丝状病毒(奎瓦病毒(Cueva)、马尔堡病毒(Marburg)和埃博拉病毒(Ebola))、基孔肯雅热病毒(Chikungunya virus)、人α疱疹病毒3型(HHV-3)或水痘带状疱疹病毒(VZV)、风疹病毒、Merkel细胞多瘤病毒(MCV)、布尼亚病毒(例如，汉塔病毒)、沙粒病毒(例如，淋巴细胞性脉络丛脑膜炎病毒(LCMV，lymphocytic choriomeningitismammarenavirus)和拉萨病毒)、黄病毒(登革热病毒、寨卡病毒、日本脑炎病毒、西尼罗河病毒、蜱虫脑炎病毒(TBEV)和黄热病毒)、鼻病毒、人副流感病毒(HPIV)、肠病毒(例如，脊髓灰质炎病毒)、呼吸道合胞病毒(RSV)、腮腺炎病毒、克沙奇病毒(Coxsackievirus)、麻疹病毒、星状病毒(例如，肠胃炎病毒)、弹状病毒(例如，狂犬病病毒)、腺病毒、腺相关病毒(AAV)、癌基因病毒(oncogenic virus)(包括人乳突病毒、B型肝炎病毒、C型肝炎病毒、爱泼斯坦-巴尔病毒、卡波西肉瘤相关的疱疹病毒、人嗜T淋巴细胞病毒和Merkel细胞多瘤病毒)，以及它们的任何组合。
根据本发明优选的实施方式，病毒抗原选自由以下组成的组：刺突蛋白，也称为严重急性呼吸道症候群冠状病毒2型或SARS-COV-2或COVID-19的表面糖蛋白；N蛋白，也称为严重急性呼吸道症候群冠状病毒2型或SARS-COV-2或COVID-19的核鞘蛋白；M蛋白，也称为严重急呼吸道症候群冠状病毒2型或SARS-COV-2或COVID-19的膜糖蛋白；A型流感病毒H5N1的血凝素(HA)蛋白；A型流感病毒H3N2的血凝素(HA)蛋白；A型流感病毒H1N1的血凝素(HA)蛋白；A型流感病毒H7N9的血凝素(HA)蛋白；A型流感病毒H1N1的血凝素(HA)蛋白；A型流感病毒H2N2的血凝素(HA)蛋白；B型流感病毒的血凝素(HA)蛋白；A型流感病毒H5N1的神经氨酸酶(NA)蛋白；A型流感病毒H1N1的神经氨酸酶(NA)蛋白；A型流感毒H3N2的神经氨酸酶(NA)蛋白；A型流感病毒H7N9的神经氨酸酶(NA)蛋白；A型流感病毒H9N2的神经氨酸酶(NA)蛋白；A型流感病毒H2N2的神经氨酸酶(NA)蛋白；A型流感病毒H1N1的神经氨酸酶(NA)蛋白；B型流感病毒的神经氨酸酶(NA)蛋白；人免疫缺陷病毒(HIV1)的包膜蛋白；人免疫缺陷病毒(HIV2)的包膜蛋白；人乳突病毒(HPV)的主要衣壳蛋白L1；人乳突病毒(HPV)的次要衣壳蛋白L2；狂犬病毒的糖蛋白；人巨细胞病毒的糖蛋白；C型肝炎病毒的包膜糖蛋白E1E2；呼吸道合胞病毒(RSV)的融合蛋白(F)；扎伊尔(Zaire)埃博拉病毒的刺突糖蛋白；寨卡病毒的蛋白prM；寨卡病毒的丝氨酸蛋白酶NS3；寨卡病毒的丝氨酸蛋白酶亚基NS2B；寨卡病毒的包膜蛋白E；寨卡病毒的衣壳蛋白C；SARS-CoV-2刺突(S)RBD蛋白，以及它们的任何组合。
在示例性的实施方式中，如上文所限定的所编码的至少一种蛋白抗原包括氨基酸序列、基本上由或由氨基酸序列组成，该氨基酸序列选自由SEQ ID NO：1-13、15、16、18和20-49组成的组。
在本发明的另一个实施方式中，装载在EV中的外源核酸分子是包括核苷酸序列或由核苷酸序列组成的mRNA分子，该核苷酸序列选自由SEQ ID NO：14、17、19和50组成的组。更具体地，SEQ ID NO：14、17、19和50分别对应于编码SARS-COV-2 S蛋白、N蛋白、M蛋白和刺突(S)RBD蛋白的mRNA序列。
根据本发明，设想组合物可以包括装载有单个外源核酸分子的工程化的植物来源的EV，或者替代地，组合物可以包括装载有不同外源核酸分子的工程化的植物来源的EV的组合。
应理解，本发明范围内的蛋白抗原可以包括一个或多个修饰，以提高抗原免疫原性和/或稳定性。示例性的修饰包括翻译后修饰。
根据本发明的组合物可以单独使用，也可以与其他疫苗组合使用。
在一个实施方式中，根据本发明的组合物进一步包括一种或多种聚阳离子物质，所述一种或多种聚阳离子物质通过静电相互作用与EV的脂质双层膜的外脂质层相关联。
优选地，一种或多种聚阳离子物质选自由以下组成的组：阳离子蛋白，包括鱼精蛋白、降钙素肽、菌丝霉素、乳铁蛋白、鱼精蛋白样蛋白(如精胺或亚精胺)、核仁蛋白、组蛋白、细胞穿透肽(CPP)；阳离子肽，包括富含组氨酸的肽、富含精氨酸的肽、富含赖氨酸的肽和富含阳离子精氨酸的肽(CARP)；多肽，包括聚精氨酸、聚赖氨酸、聚组氨酸、富含组氨酸的肽、富含精氨酸的肽、富含赖氨酸的肽；多糖，包括壳聚糖、糖胺聚糖(如多硫酸化糖胺聚糖(PSGAG))、阳离子葡聚糖；甘油、聚乙二醇(PEG)。
优选的聚阳离子物质是鱼精蛋白。
优选地，组合物中一种或多种聚阳离子物质的含量在0.001至2μg/1010EV，更优选0.05至1μg/1010EV，甚至更优选0.1至0.4μg/1010EV的范围内。
根据本发明，一种或多种聚阳离子物质可以单独使用，也可以组合使用。应理解，可以以其天然形式或采用化学修饰来使用聚阳离子物质。这种成分可以单独使用，也可以组合使用。
在根据本发明的另一个实施方式中，本发明的组合物中的EV额外地装载有一个或多个糖分子，所述一个或多个糖分子通过静电相互作用和氢键与装载至EV中的外源核酸分子相关联。
优选地，一个或多个糖分子选自由以下组成的组：二糖，包括海藻糖、麦芽糖、乳糖、蔗糖、纤维二糖、壳二糖、曲二糖、黑曲霉糖、异麦芽糖、β,β-海藻糖、α,β-海藻糖、槐二糖、昆布二糖、龙胆二糖、海藻酮糖、松二糖、麦芽酮糖、白菌二糖、异麦芽酮糖、龙胆二酮糖(gentiobiulose)、甘露二糖、蜜二糖、车前二糖、芦丁糖、芦丁酮糖、木二糖；糖醇，包括阿拉伯糖醇、赤藓糖醇、甘油、氢化葡萄糖浆(HSH)、异麦芽酮糖醇(isomalt)、乳糖醇、麦芽糖醇、甘露醇、山梨醇、木糖醇；多糖，包括淀粉、糖原、半乳多糖、菊粉、阿拉伯糖基木聚糖、纤维素、甲壳素和果胶。
优选的糖分子是海藻糖。海藻糖是一种非还原性二糖，通常用作稳定蛋白和核酸的细胞保护剂。此外，海藻糖可以分解RNA的二级结构。
优选地，根据本发明的EV中的一个或多个糖分子的含量在0.1至10mg/1010EV，更优选0.5至5mg/1010EV，甚至更优选1至2mg/1010EV的范围内。
在另一个实施方式中，根据本发明的EV中的一个或多个糖分子的含量在所装载的外源核酸的0.1至20mg/μg，优选地在所装载的外源核酸的1至10mg/μg，更优选地在所装载的外源核酸的2至6mg/μg的范围内。
应理解，糖分子可以以其天然形式或采用化学修饰来使用。这种组分可以单独使用，也可以组合使用。
使用前，可以将本发明的组合物中非天然存在的EV冻干并且用水重悬。替代地，本发明的组合物中使用的非天然存在的EV可以新鲜制备或在4℃、-20℃或-80℃下存储。
根据本发明的组合物可以配制成几种可施用的形式，包括粉剂、颗粒剂、片剂、胶囊剂、混悬剂、乳剂、糖浆剂、气雾剂、丸剂、糖衣片剂、胶囊剂、液剂、凝胶剂、糖浆剂、浆液和混悬剂。
根据常规的方法，本发明的组合物可任选地含有适合的赋形剂、防腐剂、溶剂或稀释剂。
示例性的赋形剂包括但不限于，糖，包括蔗糖、D-甘露糖、D-果糖、葡萄糖、无水乳糖、D-海藻糖、D-山梨醇；蛋白，包括人血清白蛋白、水解酪蛋白、MRC-5细胞蛋白、水解明胶、CRM197载体蛋白、来自植物、酵母、细菌、鸡蛋的蛋白；必需和非必需氨基酸，如天冬酰胺、苯丙氨酸、精氨酸、组氨酸；钠，包括氯化钠、碳酸氢钠、碳酸钠、硼酸钠、苯甲酸钠、牛磺脱氧胆酸钠、脱氧胆酸钠、磷酸二氢钠、磷酸氢二钠、偏亚硫酸氢钠；钾，包括磷酸钾、波拉克林钾(polacrilin potassium)、磷酸二氢钾和磷酸氢二钾、氯化钾；硬脂酸镁、氯化钙、磷酸钙、硅酸钙、谷氨酸、纤维素、微晶纤维素、乙酸邻苯二甲酸纤维素、铝、氢氧化铝、磷酸铝、无定形羟基磷酸硫酸铝、硫酸铝钾、柠檬酸、柠檬酸铁铵、蓖麻油、新霉素、链霉素、氨基糖苷、卡那霉素、庆大霉素、金霉素、两性霉素B、plasdone C、乙醇、丙酮、苄索氯铵、甲醛、甘油、抗坏血酸、氨丁三醇(trometamol)、尿素、戊二醛、2-苯氧基乙醇、聚山梨醇酯80(吐温80)、多粘菌素B、硫氰酸铵、氨基丁三醇(tromethamine)、宿主细胞DNA核酸酶(host cell DNAbenzonase)、福尔马林、磷酸盐缓冲盐溶液、聚山梨醇酯20、脱氧胆酸盐、十二水合二碱盐(dibasic dodecahydrate)、一碱脱水化物(monobasic dehydrate)、福尔马林、多粘菌素B、β-丙内酯、氢化可的松、角鲨烯、山梨醇三油酸酯、钡、十六烷基三甲基溴化铵(CTAB)、辛苯聚醇-10(octoxynol-10)(TRITON X-100)、α-生育酚琥珀酸氢酯、十六烷基三甲基溴化铵和β-丙内酯、硫柳汞、乙二胺四乙酸(EDTA)、苯酚、β-丙内酯、DMEM、HEPES、聚二甲基硅氧烷、维生素、二油酰基磷脂酰胆碱(DOPC)、3-O-脱酰基-4’单磷酰脂质A(MPL)、脂质、胆固醇、泛醇、树胶(包括瓜尔胶)、硼酸和硼酸盐(包括四硼酸钠)、甘油、尿囊素、三乙醇胺、海藻酸盐；pluronic、泊洛沙姆(包括P188、P331)；PEG，包括PEG8000；二醇(包括乙二醇、丙二醇)和甘油；胞磷胆碱(胞苷-5-二磷酸胆碱；CDP胆碱)、胆固醇。
适用于在本发明的组合物中的防腐剂的说明性的非限制性的实例包括对羟基苯甲酸酯(paraben，尼泊金酯)，包括对羟基苯甲酸乙酯、对羟基苯甲酸甲酯、对羟基苯甲酸丙酯，甲醛供体，包括DMDM乙内酰脲、咪唑烷基脲，以及戊二醛、苯酚衍生物、苯甲酸、苯甲醇。
在本发明中使用的适合的溶剂或稀释剂可以选自纯化水、乙醇和苄醇。
根据本发明，考虑可以将佐剂添加至组合物中用作疫苗。
适用于在本发明的免疫原性组合物中的佐剂的说明性的非限制性实例是矿物质组合物，包括铝盐(如氢氧化铝、磷酸铝钾、AS04等)、钙盐、氢氧化物(例如，氧氢氧化物)、磷酸盐(例如，羟基磷酸盐、正磷酸盐)、硫酸盐；乳剂，包括水包油和油包水乳液，如弗氏佐剂(Freund’s adjuvant)、完全弗氏佐剂和不完全弗氏助剂、MF59、AF03、AS03、AS02、吡喃葡萄糖苷脂质佐剂(GLA-SE)、吡喃葡萄糖基脂质佐剂(GLA)；细菌或微生物衍生物，包括肠细菌脂多糖(LPS)、单磷酰脂质A(MPL)、3-O-脱酰化MPL(3dMPL)、脂质A、来自大肠杆菌的脂质A(如OM-174)的非毒性衍生物。OM-174；免疫刺激性寡核苷酸，包括含有CpG基序的核苷酸序列、细菌双链RNA、含有回文或多(dG)序列的寡核苷酸、ADP-核糖基化毒素和解毒衍生物、RC529；环状GMP-AMP佐剂、STING激动剂、CAF01、免疫刺激性复合物(ISCOM)、ISCOMATRIX、AS01；聚氧乙烯醚和聚氧乙烯酯制剂、聚合物颗粒(如聚(丙交酯-乙交酯)(PLG)微粒、聚磷腈(PCPP))、皂苷制剂(如来源于牙买加菝葜(Smilax ornata)(沙士)(sarsaprilla)、圆锥石头花(Gypsophilla paniculata)(新娘面)(brides veil)和肥皂草(Saponariaofficinalis)(皂根)的皂苷)、纯化制剂(如QS7、QS17、QS18、QS21、QH-A、QH-B和QH-C)；人免疫调节剂，包括细胞因子，诸如白细胞介素(例如IL-1、IL-2、IL-4、IL-5、IL-6、IL-7、IL-12等)、干扰素(例如干扰素-γ)、巨噬细胞集落刺激因子和肿瘤坏死因子；生物粘合剂和粘膜粘合剂，包括酯化透明质酸微球或粘膜粘合剂，如聚(丙烯酸)、聚乙烯醇、聚乙烯吡咯烷酮、多糖和羧甲基纤维素的交联衍生物、壳聚糖及其衍生物；胞壁酰肽，包括N-乙酰基-胞壁酰-L-苏氨酰-D-异谷氨酰胺(thr-MDP)、N-乙酰基-去甲胞壁酰基-L-丙氨酰-D-异谷氨酰胺(nor-MDP)和N-乙酰基胞壁酰基-L-丙氨酰-D-异谷氨酰胺酰-L-丙氨酸-2-(1’-2’-二棕榈酰基-sn-甘油-3-羟基磷酰氧基)-乙胺MTP-PE)；咪唑并喹诺酮化合物，包括咪喹莫特(Imiquamod)及其同源物；病毒体和病毒样颗粒(VLP)。
本发明的组合物可以通过各种途径施用，包括口服、鼻内、肠胃外，包括皮下、腹膜内、静脉内、皮内、肌内、脾内和结肠内。
优选地，本发明的药物组合物是以适用于口服、鼻内或肠胃外施用的形式。
施用剂量、应用的数目和频次可以根据各种因素来确定，诸如治疗或预防的疾病以及患者的特征，并且可以由本领域的普通技术人员利用他/她的一般知识来确定。
此外，根据本发明的组合物可以冻干并且是稳定的而不需要冷链存储。
在本发明的范围内还有一种制备具有如上文所限定的特征的组合物的方法。
根据本发明，方法包括以下步骤：
(i)将植物来源的细胞外囊泡(EV)的悬浮液与一种或多种聚阳离子物质接触并混合以得到第一混合物；
(ii)将核酸分子的配制品与一个或多个糖分子接触并混合以得到第二混合物，所述核酸分子编码至少一种蛋白抗原；
(iii)将所述第一混合物与所述第二混合物混合以得到第三混合物；以及
(iv)将预定体积的水添加至所述第三混合物，其中水的预定体积与第三混合物的体积之比包含在5:1至15:1内，优选地在8:1至12:1内。
水的预定体积与第三混合物的体积的优选比例是10:1。
任选地，根据本发明的方法可进一步包括浓缩步骤(iv)中获得的组合物。浓缩技术是众所周知的并且包括例如过滤、超速离心、切向流过滤、色谱法和沉淀。本领域技术人员将清楚用于浓缩组合物的技术，并且可以使用任何这种合适的方法。
在本发明的方法的一个实施方式中，在步骤(i)中，混合进一步包括将第一混合物在30至40℃范围内(优选在37℃)的温度下孵育30分钟至2小时范围内(优选1小时)的时间的步骤。
在本发明的方法的另一个实施方式中，在步骤(ii)中，混合进一步包括将第二混合物在0至25℃范围内(优选在20℃)的温度下孵育5至30分钟范围内(优选10分钟)的时间的步骤。
在本发明的方法的又一个实施方式中，在步骤(iii)中，混合进一步包括将第三混合物在30至40℃范围内(优选在37℃)的温度下孵育1至5小时范围内(优选3小时)的时间的步骤。
在本发明的方法的另一个实施方式中，步骤(iv)进一步包括孵育步骤，该孵育步骤在0至10℃范围内(优选在4℃)的温度下进行5至24小时范围内(优选12小时)的时间。
用于根据本发明的方法的合适的聚阳离子物质和糖分子是参照组合物如上文所述的。
不希望受任何理论约束，本发明人认为聚阳离子物质可以改变植物来源的EV的脂质双层膜的电荷，并且允许核酸分子吸附在这种膜的外表面。此外，本发明人认为糖对核酸分子可具有保护作用以允许将这些分子高效引入至植物来源的EV中。
优选地，第一混合物中植物来源的EV的浓度包含在所述第一混合物的总体积的5x1010至1012EV/ml，更优选所述第一混合物的总体积的1x1011至5x1011EV/ml的范围内。
根据本发明的方法的第一混合物可以进一步包含盐，优选NaCl，更优选以所述第一混合物的总体积的0.9％(w/v)的浓度的NaCl。
在本发明的方法的一个实施方式中，一种或多种聚阳离子物质以包含在所述第一混合物的总体积的0.1至2μg/ml、优选所述第一混合物的总体积的0.1至1μg/ml、更优选所述第一混合物的总体积的0.4至0.6μg/ml范围内的浓度存在于第一混合物中。
在本发明的另一个实施方式中，核酸分子以包含在所述第二混合物的总体积的0.1至10μg/ml、优选所述第二混合物的总体积的0.1至1μg/ml、更优选所述第二混合物的总体积的0.1至0.5μg/ml范围内的浓度存在于第二混合物中。
在根据本发明的又一个实施方式中，一个或多个糖分子以包含在所述第二混合物的总体积的1至20％(w/v)、优选所述第二混合物的总体积的1至10％(w/v)、更优选所述第二混合物的总体积的1至5％(w/v)范围内的浓度存在于第二混合物中。
根据本发明的方法，步骤(i)中将包括植物来源的EV的悬浮液与聚阳离子物质的混合和/或步骤(ii)中将核酸分子的配制品与一个或多个糖分子的混合可以通过涡旋进行，优选至少30秒的时间段。
根据本发明，设想方法可以包括进一步的操作以改善将核酸分子装载到植物来源的EV中，包括但不限于，电穿孔、超声处理、转染、孵育、细胞挤压(cell extrusion)、皂苷介导的渗透和冻融。
本发明的另一个方面是一种用作疫苗的组合物，包括非免疫调节的工程化的植物来源的细胞外囊泡(EV)，可通过如上文所限定的方法获得。
附图说明
以下实验部分单纯地通过说明的方式提供并且不旨在限制所附权利要求中所限定的本发明的范围。在以下实验部分中参考附图，其中：
图1示出了与天然的植物来源的EV相比，实验例1中本发明的工程化的植物来源的EV的表征。来自奇异果的天然的EV(A)和本发明的工程化的EV(B)的Nanosight分析的代表性图像在尺寸上表现出显著差异。通过Nanosight分析的天然的植物来源的EV和本发明的工程化的植物来源的EV的n＝3个制剂的平均直径的统计分析(C)。E1＝来自卷心菜EV的工程化的EV，E2＝来自蓝莓EV的工程化的EV。p:****<0.001。
图2示出了实验例1中在EV中测量的脂质双层膜上的膜电位(Z电位)的值。在天然的EV(天然EV)和来自西葫芦(E1)和蓝莓(E2)的本发明的工程化的EV中以毫伏(mV)为单位测量膜电位。将对工程化的植物来源的EV所测量的膜电位与对天然的植物来源的EV所测定的值进行比较，计算出统计学意义。p:***<0.005。每个数据集进行N＝3次实验。数据以平均值±标准偏差(SD)显示。
图3A示出了实验例1中天然的植物来源的EV和本发明的工程化的植物来源的EV的蛋白含量，以1010个EV中的纳克(ng)蛋白表示。对天然的植物来源的EV(天然EV)以及来自石榴(E1)和奇异果(E2)的工程化的植物来源的EV进行测量。将工程化的植物来源的EV的蛋白含量与天然的植物来源的EV所测量的值进行比较，计算出统计学意义。p:**<0.01。每个数据集进行N＝3次实验。图3B示出了实验例1中本发明的组合物中的EV在脂质双层膜的外层中含有磷酯酰丝氨酸的百分比。在包括天然的植物来源的EV(天然EV)的组合物以及在包括来自卷心菜(E1)和蓝莓(E2)的工程化的植物来源的EV的组合物中分析囊泡膜的外层中的磷酯酰丝氨酸的存在。在每个样品中，使用细胞荧光测定法(FACS)测量磷脂酰丝氨酸含量，以膜联蛋白V(Annexin V)染色，并且以荧光信号的百分比表示。将含有磷脂酰丝氨酸的工程化的植物来源的EV的百分比与天然的植物来源的EV的百分比进行比较，计算出统计学意义。p:**<0.01。每个数据集进行N＝3次实验。数据以平均值±标准偏差(SD)显示。
图4示出了实验例1中对本发明的工程化的植物来源的EV的免疫调节测定的结果。将PBMC细胞与来自石榴的工程化的植物来源的EV(剂量为50,000个颗粒/细胞)一起孵育48小时，并通过BrdU掺入测量细胞增殖。(A)直方图示出了未处理的PBMC(CTR)和用本发明的EV刺激的PBMC的吸光度(平均值±SD)。吸光度与细胞增殖直接成比例。与对照(CTR)相比，用本发明的EV刺激的PBMC的增殖率未发生变化并且不具有统计学意义。然后，淋巴细胞用LPS(剂量为100ng/ml)激活并用本发明的EV(剂量为50,000个颗粒/细胞)刺激48小时，并通过BrdU掺入测量增殖。(B)直方图示出了未刺激的PBMC(CTR)、用LPS处理的PBMC(CTR+)、用LPS和来自石榴的本发明的EV处理的PBMC(E1)以及用LPS和来自奇异果的本发明的EV处理的PBMC(E2)的吸光度(平均值±SD)。吸光度直接与细胞增殖成比例。与未处理的细胞相比，LPS显著激活了PBMC增殖，而与用LPS处理的PBMC相比，用LPS和本发明的EV刺激的PBMC的增殖率未改变并且不具有统计学意义。PBMC增殖还可通过使用荧光染料CFSE来测量。PBMC用本发明的EV(剂量为50,000个囊泡/细胞)刺激24小时，然后通过流式细胞术(C、D)分析增殖。直方图(C)示出了未处理的PBMC(CTR)和用来自卷心菜(E1)、芹菜(E2)和西葫芦(E3)的本发明的EV刺激的PBMC的荧光FITC强度(平均值±SD)。与CTR相比，用本发明不同的EV样品刺激的PBMC的增殖率未发生改变并且不具有统计学意义。事实上，CTR、E1、E2和E3的流式细胞术分析的直方图完全重叠(D)。p:*<0.05；ns>0.05。
图5示出了实验例1中EV的总RNA含量。在天然的植物来源的EV以及来自芹菜(E1)、石榴(E2)和奇异果(E3)的工程化的植物来源的EV中测量总RNA含量。总RNA含量通过在RNA提取后对每个样品中所含RNA进行绝对量化来测量，并以针对囊泡数量(ng/109个囊泡)归一化的RNA量(ng)表示。将本发明的每个EV样品与天然的植物来源的EV的总RNA含量值进行比较，计算出统计学意义。p:*<0.05，***<0.005，****<0.001。每个数据集进行N＝3次实验。数据以平均值±标准偏差(SD)显示。
图6示出了实验例2中装载在本发明的工程化的植物来源的EV中的外源核酸分子的量化。对于该测定，使用编码SARS-CoV-2核壳(N)蛋白的mRNA分子，并且在天然的植物来源的EV(天然EV)以及来自奇异果(E1和E3)和芹菜(E2和E4)的工程化的植物来源的EV中通过qRT-PCR测量所装载的核酸分子。使用两种不同剂量的mRNA：样品E1和E2为0.1μg/ml并且样品E3和E4为1μg/ml。根据所述的方法，以RQ值表示所装载的mRNA的量。将本发明的每个EV样品与天然的植物来源的EV中所装载的核酸的量进行比较，计算出统计学意义。p:****<0.001。每个数据集进行N＝3次实验。数据以平均值±标准偏差(SD)显示。
图7示出了实验例3中装载在本发明的工程化的植物来源的EV中的核酸分子对降解环境的抗性。对于该实验，使用mRNA分子并且通过qRT-PCR测定测量。图表表示了与起始材料相比在降解测定后仍存在的mRNA分子的百分比，并且示出了本发明的EV中保留了总共100％的mRNA。(A)在用RNA酶处理后测量对酶降解的抗性，而(B)在用类胃溶液处理后评估对胃肠道环境的抗性。在所有实验中，使用裸mRNA作为对照。对来自石榴(7A)和奇异果(7B)的工程化的植物来源的EV进行实验。将本发明的EV与裸mRNA中保留的核酸的百分比进行比较，计算出统计学意义。p:****<0.001。每个数据集进行N＝3次实验。数据以平均值±标准偏差(SD)显示。
图8示出了实验例4中装载在本发明的工程化的植物来源的EV中的核酸分子对存储的抗性。对于这些实验，来源于芹菜(E1)和奇异果(E2)的EV装载有编码SARS-CoV-2核壳(N)蛋白的mRNA分子。在冻干并在+4℃下存储7天后，通过qRT-PCR测定测量到EV的所保留的mRNA量，并以相对起始量的百分比表示。每个数据集进行N＝3次实验。数据以平均值±标准偏差(SD)显示。
图9示出了实验例5中装载至本发明的工程化的植物来源的EV中的核酸分子向受体细胞的转移。将装载有编码SARS-CoV-2核壳(N)蛋白的mRNA分子的本发明的EV与巨噬细胞一起孵育。24小时后，使用分子分析(qRT-PCR)在受体细胞中测量mRNA量，归一化为作为管家基因(housekeeping)的GAPDH并且以RQ值表示，如在方法部分中所述的。RQ值归一化为对照(未处理的细胞，NT)，并且RQ值为1是指在样品中检测不到mRNA。用天然的植物来源的EV(天然EV)、工程化的植物来源的EV(E1、E2、E3)、与mRNA(EV+mRNA)孵育而没有核酸装载的植物来源的EV、裸mRNA处理巨噬细胞。以50,000个颗粒/细胞的剂量处理受体细胞。用来自卷心菜(E1)、石榴(E2)和奇异果(E3)的EV进行实验。将每个样品与作为对照(NT)的未处理的细胞的mRNA的RQ值进行比较，计算出统计学意义。p:***<0.005，****<0.001。每个数据集进行N＝3次实验。数据以平均值±标准偏差(SD)显示。
图10示出了实验例5中受体细胞中由本发明的工程化的植物来源的EV携带的核酸分子的功能。对于这些实验，本发明的装载有编码绿色荧光蛋白(GFP)的mRNA分子的EV与作为受体细胞的(A)内皮细胞和(B)巨噬细胞一起孵育。共孵育24小时后，使用细胞荧光分析(FACS)，以荧光信号检测由外源mRNA编码的蛋白在受体细胞中的表达。以50,000个颗粒/细胞的剂量用天然的植物来源的EV(天然EV)、本发明的工程化的植物来源的EV或裸mRNA处理受体细胞。用来自西葫芦(图10A)和羽衣甘蓝(图10B)的植物来源的EV进行实验。将每个样品与作为对照(NT)的未处理的细胞的信号强度百分比进行比较，计算出统计学意义。p:***<0.005，****<0.001。每个数据集进行N＝3次实验。数据以平均值±标准偏差(SD)显示。
图11示出了实验例5中用由本发明的工程化的植物来源的EV携带的核酸分子处理的靶受体细胞中的蛋白表达。对于这些实验，将本发明的装载有编码SARS-CoV-2刺突糖蛋白(S1)RBD蛋白(SEQ ID NO.50)、SARS-CoV-2刺突糖蛋白全蛋白(SEQ ID NO.14)、或SARS-CoV-2核壳蛋白(SEQ ID NO.17)的mRNA分子的EV与作为受体细胞的内皮细胞一起孵育。共孵育24小时后，使用荧光标记的二级抗体检测由外源mRNA编码的蛋白在受体细胞中的表达，并且用细胞荧光分析(FACS)测量荧光信号。以1.2x1010个颗粒的剂量用天然的植物来源的EV(天然EV)或本发明的工程化的植物来源的EV处理受体细胞。用源于橙子的EV进行实验。将每个样品与作为对照(NT)的未处理的细胞的信号强度百分比进行比较，计算出统计学意义。p:****<0.001。每个数据集进行N＝3次实验。数据以平均值±标准偏差(SD)显示。
图12示出了实验例6中本发明的用核酸工程化的植物来源的EV和非天然的EV能够在掺入巨噬细胞后激活淋巴细胞。对于这些实验，本发明的装载有编码SARS-CoV-2刺突糖蛋白(S1)RBD蛋白(SEQ ID NO.50)、SARS-CoV-2刺突糖蛋白全蛋白(SEQ ID NO.14)、或SARS-CoV-2核壳蛋白(SEQ ID NO.17)的mRNA分子的EV与作为受体细胞的APC细胞(巨噬细胞)一起孵育。在EV掺入APC后，添加PBMC细胞，并且用工程化的EV进行的处理每5天重复两次。最后，通过细胞荧光分析(FACS)来分析淋巴细胞。评估通过CD4+的表达鉴定的淋巴细胞的激活。淋巴细胞激活以淋巴细胞增殖(A)的增加以及淋巴细胞CD4+的淋巴细胞激活标记CD25+(B)和HLADR+(C)的表达的增加来衡量。以1.2x1010个颗粒的剂量用天然的植物来源的EV(天然EV)或本发明的工程化的植物来源的EV处理细胞。用源于橙子的EV进行实验。将每个样品与作为对照(NT)的未处理的细胞或用天然EV处理的细胞的信号强度的百分比进行比较，计算出统计学意义。阳性对照通过用珠状人T激活剂CD3/CD28(CTR+)和纯化蛋白(SARS-CoV-2刺突糖蛋白RBD蛋白(S蛋白)或SARS-CoV-2核壳蛋白(N蛋白))进行处理来表示。p:*<0.05，**<0.01，***<0.005，****<0.001。每个数据集进行N＝3次实验。数据以平均值±标准偏差(SD)显示。
图13示出了实验例7中本发明的用核酸工程化的植物来源的EV和非天然的EV能够诱导小鼠中的特异性免疫反应。图表示出了通过疫苗接种诱导的对SARS-CoV-2刺突糖蛋白(S1)RBD蛋白具有特异性的IgA免疫球蛋白的吸光度测量。对于该实验，在第0天和第21天将小鼠免疫化，并在第35天处死小鼠后分析血清。用天然的EV(天然EV)或者用编码SARS-CoV-2刺突糖蛋白RBD(S1)的mRNA分子工程化的EV处理小鼠。经肌内或口服途径施用处理。使用用SARS-CoV-2刺突糖蛋白RBD(S1)包被的ELISA板通过ELISA评估对IgA诱导的体液免疫反应。对每个施用途径的天然的和工程化的EV的信号强度进行比较，计算出统计学意义。p:*<0.05,**<0.01每个数据集N＝3个动物。数据以平均值±标准偏差(SD)显示。
具体实施方式
材料与方法
细胞外囊泡分离
从新鲜果汁(猕猴桃、石榴、蓝莓、橙子、柠檬)或新鲜植物提取物(西葫芦、卷心菜、羽衣甘蓝、芹菜)中分离细胞外囊泡。使用递减级别的孔依次过滤果汁或提取物以去除纤维。然后用差速超速离心(differential ultracentrifugation)或切向流过滤纯化EV。对于差速超速离心，将果汁在1,500g离心30分钟以去除碎屑和其他污染物。然后，在10,000g进行超速离心接着在100,000g进行超速离心，在4℃进行1小时(Beck-man Coulter OptimaL-90K)来纯化EV。用添加有1％ DMSO的磷酸盐缓冲溶液重悬最终丸粒，并且用0.22微米过滤器过滤以进行灭菌。
细胞外囊泡被使用或长时间在-80℃下存储。对于切向流过滤，首先通过用深层过滤片盘Supracap 50(Pall)过滤以去除纤维和碎屑来净化果汁。然后，使用切向流过滤盒TFF-Omega(Pall-Cadence)通过浓缩和渗滤来纯化过滤的果汁。最后，切向流过滤产生的保留物(retentate)通过用0.2nm过滤器过滤来灭菌。
纳米颗粒跟踪分析(NTA)
使用装配有405nm激光和NTA 3.1分析软件的NanoSight LM10系统(Malvern)进行纳米颗粒跟踪分析(NTA)以界定EV尺寸和轮廓(profile)。用相机记录在受到激光光源照射的样品中存在的EV的布朗运动并通过NTA用Stokes-Einstein方程将其转换为尺寸和浓度参数。所有采集的相机水平均为16，并且对于每个样品，记录了三个持续时间30秒的视频。简言之，在1ml无囊泡盐水溶液(Fresenius Kabi)中以1:2000稀释纯化的EV。优化NTA采集后设置并使其在所有样品中保持不变，然后分析每个视频以测量EV的平均值、模式和浓度。
本发明的EV的生产
通过如下文所述的顺序步骤生产本发明的工程化的植物来源的EV。简言之，将植物来源的EV与阳离子肽混合，并在37℃下进行反应1小时。将核酸分子的配制品与糖混合，并在20℃下进行反应10分钟。然后，将这两种溶液混合，并在37℃下进行反应3小时。然后，将水添加至反应中，并将样品在4℃下放置12小时。为了从剩余的游离核酸分子中纯化工程化的植物来源的EV，在100,000g通过超速离心在4℃下2小时来洗涤样品(Beckman CoulterOptima L-90K,Fullerton,CA,USA)，并将样品重悬在盐水溶液中。
EV膜电位的测量
通过使用Zeta粒度仪纳米仪器(Malvern Instruments,Malvern,UK)进行分析。在25℃下，在过滤(截止值＝200nm)的盐水溶液中分析所有样品。在距囊泡x距离处产生Zeta电位(滑动平面)，这表明了分散体中相邻的带类似电荷的囊泡之间的静电排斥程度。
蛋白提取与量化
使用补充有蛋白酶和磷酸酶抑制剂的混合物(Sigma-Aldrich,St.Louis,Missouri,USA)的RIPA缓冲液(150nM NaCl，20nM Tris-HCl，0.1％十二烷基硫酸钠，1％脱氧胆酸盐，1％ Triton X-100，pH 7.8)从EV样品中提取蛋白。根据制造商方案，通过BCA蛋白测定试剂盒(Thermo Fisher Scientific,Waltham,Massachusetts,USA)量化蛋白含量。简言之，将10μl样品分配至96孔板的孔中，并使用用牛血清白蛋白(BSA)建立的线性标准曲线确定总蛋白浓度。
磷脂酰丝氨酸分析
对于磷脂酰丝氨酸分析，EV样品用膜联蛋白V FITC和FITC同种型(MiltenyiBiotec,Germany)染色30分钟，并在采集前用盐水溶液稀释。使用带有CytExpert软件的CytoFLEX流式细胞仪(Beckman Coulter)通过细胞荧光分析表征样品，并使用FITC同种型作为背景来测量每个样品的信号阳性(signal positivity)百分比。
RNA提取与量化
根据制造商方案，使用miRNeasy迷你试剂盒(Qiagen,Hilden,Germany)从EV和细胞中分离总RNA，并将其重悬于水中。使用分光光度计(mySPEC,VWR,Radnor,PA,USA)量化样品的RNA浓度。
使用qRT-PCR进行mRNA检测
使用高容量cDNA逆转录试剂盒(Applied Biosystems)从RNA样品中获得cDNA。将5纳克cDNA添加至SYBR GREEN PCR Master Mix(Applied Biosystems)中，并在96孔QuantStudio12K Flex实时PCR(qRT-PCR)系统(Thermo Fisher Scientific,Waltham,MA,USA)上运行。使用GAPDH作为细胞样品中的管家基因。所有样品中mRNA表达的倍数变化(Rq)计算为相对于对照样品的2-ΔΔCt。
细胞培养
通过对人真皮微血管内皮细胞的猴病毒40进行永生化获得人微血管内皮细胞(HMEC)。在补充有bullet试剂盒(EBM,Lonza,Basel,Switzerland)和1ml Mycozap CL(Lonza)的内皮基础培养基中培养HMEC。通过ATCC获得巨噬细胞MV-4-11细胞系(CRL9591TM)，并在补充有10％胎牛血清(ATCC,USA)的柯夫改良杜氏培养基(Iscove'sModified Dulbecco's Medium)中进行培养。外周血单核细胞(PBMC)分离如下：用PBS以1:1稀释来自健康志愿者供体的全血，然后将30ml全血轻轻分层在50ml离心管的15ml中性粒细胞分离液(Histopaque)(Sigma-Aldrich)中。在400g下离心管30分钟。将含有PBMC的白色浑浊层收集在50ml离心管中，用40ml PBS稀释并在300g下离心5分钟以洗涤两次。对丸粒化的细胞计数，并使用台盼蓝染色来估计存活率。在24孔板中，在含有10％胎牛血清的RPMI中培养细胞。
受体细胞中的核酸掺入
为了评估装载至本发明的工程化的植物来源的EV中的eGFP mRNA进入细胞的摄入，将这些囊泡与HMEC细胞和巨噬细胞一起孵育。将总共50,000个受体细胞/孔铺板在24孔板中，并用50,000个囊泡/受体细胞进行刺激。24小时后，彻底洗涤细胞，用胰蛋白酶分离，并使用具有CytExpert软件的CytoFLEX流式细胞仪(Beckman Coulter Optima L-90K,Fullerton,CA,USA)通过FACS测量翻译的GFP蛋白的荧光。
体外核酸降解测定
为了测试对装载至本发明的EV中的核酸分子的酶降解的抗性，本发明人进行了RNA酶测定。简言之，在37℃下，使用0.4mg/mL的浓度，用RNA酶A(ThermoFisherScientific)处理样品30分钟。如制造商方案所述的，使用RNA酶抑制剂(Thermo FisherScientific)终止反应，并使用10mL聚碳酸酯管(SW 90Ti转子,Beckman Coulter OptimaL-90K超速离心机)在100,000g下通过超速离心洗涤样品，在4℃下进行2小时。最终，将EV丸粒样品重悬在盐水缓冲溶液中并进行分子分析。
为了测试对装载至本发明的EV中的核酸分子的胃消化的抗性，本发明人进行了胃消化测定。简言之，制备含有18.5％w/v HCl(pH 2.0)、24mg/mL胆汁提取物、胃蛋白酶溶液(在0.1N HCl中80mg/mL，pH 2.0；Sigma)和4mg/mL胰蛋白酶(Sigma)(在0.1N NaHCO3中)的类胃溶液。，水溶液中1μL每个EV样品的量与1.34μL类胃溶液以慢速旋转一起孵育，在37℃下进行60分钟。用1N NaHCO3将类胃溶液的pH值调节至6.5，并且称为肠道溶液。然后，在肠道溶液中额外孵育EV样品60分钟。通过如上文描述的分子分析评估装载至本发明的EV中的核酸分子的稳定性。对于所有抗性实验，以裸RNA用作对照。
样品冻干
使用仪器Heto lyolab 3000(Thermo Fisher Scientific)将样品冻干3小时，并在4℃下保持7天。在存储时间点后，使用分子分析来分析EV样品的核酸含量。因此，在冻干和存储前，将所测量的含量与起始量进行比较。
免疫细胞激活测定
为了通过流式细胞术分析评估PBMC增殖，根据制造商的说明书，用来自CellTrace细胞增殖试剂盒(Invitrogen,ThermoFisher Scientific)的CFSE染料对PBMC染色。然后以50,000个细胞/孔的密度将PBMC铺板在48孔板中。为了评估本发明的工程化的植物来源的EV是否会影响PBMC增殖，用以50,000个颗粒/孔的剂量的这些囊泡刺激PBMC。使用未刺激的PBMC作为对照。24小时孵育后，收集PBMC，并通过装配有CytoExpert软件的CytoFLEX流式细胞仪(Beckman Coulter)测量荧光。CFSE染料被检测为FITC荧光。
为了通过溴脱氧尿苷(BrdU)掺入测定分析PBMC增殖，以20,000个细胞/孔的密度将PBMC铺板在96孔板中，并将10μl BrdU标记溶液(BrdU colorimetric assay,Roche)添加至每个孔中。为了评估本发明的工程化的植物来源的EV是否会影响PBMC增殖，用以50,000个颗粒/孔的密度的这些囊泡刺激PBMC。使用未刺激的PBMC作为对照。而且，为了评估本发明的EV是否会降低激活的PBMC增殖，用以50,000个颗粒/细胞的剂量的这些囊泡和用以100ng/ml的浓度的LPS(来自E.Coli,Sigma-Aldrich)刺激PBMC。使用未刺激的PBMC作为阴性对照。48小时孵育后分析刺激的作用。根据制造商的说明书进行测定。通过ELISA酶标仪在420nm处测量吸光度，参考波长在490nm处。计算每种条件下的平均吸光度。吸光度与增殖率直接成比例。
蛋白表达的检测
为了评估蛋白在靶细胞中的表达，用1.2x1010EV刺激内皮细胞。所测定的细胞样品包括未处理的细胞(NT)、用植物来源的天然EV处理的细胞和用编码SARS-CoV-2刺突糖蛋白(S1)RBD蛋白(SEQ ID NO.50)、SARS-CoV-2刺突糖蛋白全蛋白(SEQ ID NO.14)或SARS-CoV-2核壳蛋白(SEQ ID NO.17)的mRNA分子工程化的本发明的植物来源的EV处理的细胞。24小时后，按照制造商的说明书(Inside Stain Kit,Miltenyi Biotec)，细胞被彻底洗涤、用胰蛋白酶分离，并且固定和渗透。然后，在室温下，用特异性抗体对细胞染色30分钟以检测蛋白表达(针对SARS-CoV-2刺突糖蛋白和核壳蛋白的抗体，Invitrogen)。洗涤后，在室温下添加荧光第二抗体1小时(Alexa Fluor Plus 594或488,Invitrogen,ThermoFisherScientific)。洗涤后，将细胞重悬在适当的缓冲液中，并使用具有CytExpert软件的CytoFLEX流式细胞仪(Beckman Coulter Optima L-90K,Fullerton,CA,USA)通过FACS获得该细胞。
淋巴细胞激活测定
为了评估本发明的工程化的EV在掺入APC后诱导淋巴细胞激活的能力，以20,000个细胞/孔将巨噬细胞铺板在24孔板中，并用1.2x1010EV进行刺激。所测定的细胞样品包括未处理的细胞(NT)、用植物来源的天然EV处理的细胞以及用编码SARS-CoV-2刺突糖蛋白(S1)RBD蛋白(SEQ ID NO.50)、SARS-CoV-2刺突糖蛋白全蛋白(SEQ ID NO.14)或SARS-CoV-2核壳蛋白(SEQ ID NO.17)的mRNA分子工程化的本发明的植物来源的EV处理的细胞。掺入后，以200,000个细胞/孔的浓度添加PBMC，并在5天后用EV重复进行处理。在初始处理10天后，收获细胞，并使用合适的同种型(Miltenyi Biotec)用CD4、CD25和HLA DR的荧光抗体在室温下对细胞染色30分钟。洗涤后，获得细胞。
对于增殖分析，根据制造商的说明书，预先用来自CellTrace细胞增殖试剂盒(Invitrogen,ThermoFisher Scientific)的CSFE染料对采用的PBMC染色。
最后，使用具有CytExpert软件的CytoFLEX流式细胞仪(Beckman Coulter OptimaL-90K,Fullerton,CA,USA)通过FACS分析样品。
小鼠疫苗接种
6-10周龄的雌性BALB/cAnNCrl小鼠在第0天和第21天以相当于30μg mRNA的本发明的工程化的植物来源的EV的剂量接受2次免疫接种，并在第35天处死。使用口服(使用灌服)和肌内(右腿)途径，用植物来源的天然EV或用编码SARS-CoV-2刺突糖蛋白RBD(S1)的mRNA分子工程化的本发明的植物来源的EV处理小鼠。处死后，收集血液以分离血清，用于抗体检测。
抗体测量
通过ELISA测定血清的SARS-CoV-2特异性IgA抗体的滴度。简言之，MaxiSorpELISA板(Nunc)在每孔100μl 50mM碳酸钠/碳酸氢钠pH 9.6缓冲液中涂有1μg/ml SARS-CoV-2刺突蛋白(Thermofisher Scientific)，在4℃下过夜。涂覆的板用200μl的1X PBS洗涤3次，并在每孔1X PBS中用200μl的3％ BSA饱和。用1X PBS洗涤板三次，并在3％ BSA中以及与100倍小鼠血清稀释液孵育2小时。随后用每孔200μl的1X PBS洗涤3次，以及与每孔100μl的以1:10,000稀释的二级驴抗鼠IgA-HRP共轭抗体(secondary donkey anti-mouse IgAHRP conjugated antibody)一起孵育。与二级抗体一起孵育后，用每孔200μl的1X PBS洗涤板5次，并用每孔100μl的TMB(Thermofisher Scientific)显影30分钟。通过添加每孔100μl的终止液(Thermofisher Scientific)来终止反应。使用酶标仪读取450nm处的吸光度。
统计分析
用软件Graph Pad 8(demo版)进行数据分析。结果以平均值±标准偏差(SD)表示。使用单因素方差分析(ANOVA)来证实组间的统计学差异，同时使用学生t检验在两个样品之间进行比较。我们使用p<0.05作为最低显著性水平。
结果/实施例
实施例1
为了研究本发明方法的可行性，本发明人工程化了不同的植物来源的EV，并根据传统上在物理特性方面用于表征EV的许多特征对其进行了表征，如颗粒尺寸和表面电荷(Théry C.,et al,(2018)“Minimal information for studies of extracellularvesicles 2018(MISEV2018):a position statement of the International Societyfor Extracellular Vesicles and update of the MISEV2014 guidelines”；Journal ofExtracellular Vesicles,7:1,1535750,DOI:10.1080/20013078.2018.1535750)。图1示出了来自不同植物(包括奇异果、卷心菜和蓝莓)的本发明的工程化的植物来源的EV的尺寸。用来自其他植物来源(如石榴、羽衣甘蓝、芹菜、西葫芦、橙子和柠檬)的EV也获得了相似的结果。颗粒尺寸是EV的基本参数。所获得的数据表明与天然的植物来源的EV相比，本发明的EV具有更大的平均直径(图1A、B、C)。EV尺寸的测量由本发明人利用纳米颗粒跟踪分析(NTA)技术(测量EV尺寸的最常用方法，其利用光散射和布朗运动的特性来获得样品在液体悬浮液中的纳米颗粒尺寸分布)进行。特别地，NTA经光散射通过跟踪颗粒运动进行以评估在由激光束照射的样品室中在布朗运动下运动的颗粒的均方位移。颗粒的跟踪能够计算扩散常数，其可用于斯托克斯-爱因斯坦方程(Stokes-Einstein equation)中计算流体动力学直径。斯托克斯-爱因斯坦方程还考虑了悬浮液的温度和粘度。由本发明人进行的分析的结果表明，与天然的植物来源的EV相比，本发明的EV具有更大的尺寸，因为天然囊泡具有100至150nm范围内的直径，平均直径为134±6nm，而本发明的EV的直径在200和250nm之间的范围内，平均直径为220nm。尺寸分布表明本发明的EV具有20至500nm范围内的直径，优选200至300nm的范围。
为了表征本发明的工程化的植物来源的EV的膜特性，测量囊泡的膜电位。事实上，Zeta电位是测量EV的表面电位的流行方法，并且它被用作表面电荷和胶体稳定性的指标。EV的表面电荷取决于在其表面表达的分子的性质，并且它影响在分散系统(如人体)中的EV相互作用，从而限定了其在生物过程中的活性。例如，已知表面电荷会影响与颗粒相关的不同的生物过程，如细胞摄入和细胞毒性。Zeta电位是颗粒之间静电或电荷排斥/吸引大小的量度，并且它可以在通过施加电场并测量得到的颗粒速度(电泳光散射)所确定的悬浮液中的电泳迁移率来测量(Midekessa G,et al.Zeta Potential of ExtracellularVesicles:Toward Understanding the Attributes that Determine ColloidalStability.ACS Omega.2020Jun 30；5(27):16701-16710.doi:10.1021/acsomega.0c01582.PMID:32685837；PMCID:PMC7364712.)如图2所示，已知植物来源的EV具有负表面电荷，并且膜电位(Z电位)在-10和-15毫伏之间的范围，平均值为-13毫伏。相反地，本发明的工程化的植物来源的EV采用具有包含在0和-3毫伏之间，平均值为-2毫伏的膜电位的不同的膜。对来自西葫芦(E1)和蓝莓(E2)的工程化的植物来源的EV进行上述实验，而且用来源于其他植物来源(如奇异果、卷心菜、羽衣甘蓝、石榴、柠檬、橙子和芹菜)的EV获得了相似的结果。
为了进一步表征本发明的EV，测量这些囊泡和天然的植物来源的EV的蛋白含量(图3A)。由此获得的数据表明，本发明的EV具有在120至160ng/1010EV范围内的较高的蛋白含量，而天然的植物来源的EV具有在50至100ng/1010EV范围内的蛋白含量。本发明人对来自石榴(E1)和奇异果(E2)的工程化的植物来源的EV进行了实验，而且用来自其他植物来源(如西葫芦、卷心菜、羽衣甘蓝、蓝莓、柠檬、橙子和芹菜)的EV获得了相似的结果。
此外，本发明人进行了专门的实验以更好地表征本发明的EV的膜。如本领域所知，在天然的EV中，磷脂酰丝氨酸(PS)主要位于质膜的外表面。不希望受任何理论约束，本发明人认为，在由于渗透应力引起的膜重排后，PS失去其在磷脂双层中的不对称分布，并在本发明的EV中易位到膜的内侧。通过荧光标记的膜联蛋白V检测囊泡的细胞外膜中的磷脂酰丝氨酸。事实上，已知膜联蛋白V与囊泡膜上的PS特异性结合。膜联蛋白V的荧光信号数量反映了EV膜外表面上的PS含量(Montoro-García S,et al.“An innovative flow cytometricapproach for small-size platelet microparticles:influence of calcium”.ThrombHaemost.2012 Aug；108(2):373-83)。本发明人获得的结果表明，在本发明的组合物中≤44％百分比的EV在膜的外层中具有磷脂酰丝氨酸(图3B)，该百分比的范围为40至44％，平均值为43％。不同的是，在包含天然的植物来源的EV的组合物中，在膜外层中具有磷脂酰丝氨酸的这些囊泡的百分比的范围为55至48％，平均值为49％。
对来自卷心菜(E1)和蓝莓(E2)的工程化的植物来源的EV进行上文所述的实验，而且用来自其他植物来源(如奇异果、柠檬、橙子、西葫芦、羽衣甘蓝、石榴和芹菜)的EV获得了相似的结果。
作为进一步评估，本发明人进行了专门的实验，目的是评估本发明的工程化的植物来源的EV的免疫调节活性。简言之，用本发明的EV刺激PBMC(即来自人血的淋巴细胞、单核细胞和其他免疫细胞的混合群体)，并且测量细胞增殖率。
如图4A所示，用来自石榴的本发明的EV刺激48小时的PBMC与未处理的PBMC的增殖率相同，表明这些囊泡既不促进PBMC增殖也不发挥免疫刺激作用。此外，为了验证本发明的EV是否具有免疫抑制作用，本发明人用LPS(已知可诱导炎症反应并促进淋巴细胞增殖)处理PBMC，然后用本发明的工程化的植物来源的EV刺激细胞。如图4B所示，由LPS激活的PBMC的增殖率不受本发明的EV(来自石榴(E1)和奇异果(E2)的)的影响。
这些结果证实了根据本发明的EV既不具有免疫刺激作用也不具有免疫抑制作用。
此外，还用荧光染料(CFSE)对PBMC染色，该荧光染料允许通过流式细胞术来检测PBMC增殖。如图4C和4D所示，用来自卷心菜(E1)、芹菜(E2)和西葫芦(E3)的工程化的植物来源的EV刺激的PBMC与未刺激的PBMC的增殖率相同。用来自其他植物(如蓝莓、柠檬、橙子和羽衣甘蓝)的工程化的EV获得了相似的结果。
总之，上述结果表明，本发明的工程化的植物来源的EV不能刺激或抑制免疫细胞激活和增殖，并且证实了这些囊泡的非免疫调节特性，与囊泡的植物来源无关。
最后，测量本发明的EV和天然的植物来源的EV的总RNA含量(图5)。所获得的数据表明，与天然的囊泡相比，本发明的EV具有更高的RNA含量，范围为30至100ng/109EV，平均值为50ng/109EV。天然的植物来源的EV具有范围为5至15ng/109EV的RNA含量，平均值为10ng/109EV。对来自芹菜(E1)、石榴(E2)和奇异果(E3)的工程化的EV进行实验，而且用来自其他植物来源(如西葫芦、卷心菜、羽衣甘蓝、柠檬、橙子和蓝莓)的EV也获得了相似的结果。
总之，所有这些数据表明，本发明的工程化的植物来源的EV与天然的植物来源的EV显著不同。特别地，在外源核酸装载后，与天然的囊泡相比，植物来源的EV结构和功能发生了令人惊讶的独特变化，从而导致更大的平均直径、更高的表面电荷、更低的磷脂酰丝氨酸含量和对免疫系统细胞的免疫调节作用的丧失。
实施例2
为了证明根据本发明的EV适于作为核酸递送载体，通过在内部装载mRNA分子来生产工程化的EV，并通过qRT-PCR分析测量所装载的mRNA的量(图6)。所获得的结果表明，根据本发明的EV可以装载有增加剂量的核酸分子。事实上，使用两种不同剂量的mRNA从奇异果(E1和E3)和芹菜(E2和E4)中生产工程化的EV(E1和E2为0.1μg/ml，并且E3和E4为1μg/ml)。核酸剂量的增加可以检测为囊泡中mRNA量的增加(分别地，E3和E4与E1和E2)。用来自其他植物来源(如西葫芦、卷心菜、羽衣甘蓝、柠檬、橙子、石榴和蓝莓)的EV也获得了相似的结果。总之，这些数据表明，本发明的EV包裹了所装载的核酸分子，并且它们的量可以增加。
实施例3
本发明人进行了专门的实验来评估本发明的EV保护装载的核酸分子免受降解的能力(图7)。特别地，这些研究表明，本发明的工程化的植物来源的EV能够保护所装载的核酸分子免受用降解酶(RNA酶)的处理。简言之，在用RNA酶处理EV后，qRT-PCR分析显示，大约80％的装载的mRNA仍存在于本发明的囊泡中，而用作对照的裸mRNA几乎完全被降解(图7A)。
进一步的实验表明，根据本发明的EV也能够保护所装载的核酸分子，因为在用模拟胃肠道环境的类胃溶液处理囊泡后，约90％所装载的mRNA仍然存在于EV中，而裸mRNA几乎完全被降解(图7B)。对来自石榴(6A)和奇异果(6B)的工程化的植物来源的EV进行上述实验，而且用来自其他植物来源(如西葫芦、卷心菜、羽衣甘蓝、蓝莓、柠檬、橙子和芹菜)的EV也获得了相似的结果。
总之，这些数据表明，根据本发明的EV保护所包裹的外源核酸免受降解条件的影响。此外，保护免受胃肠道环境影响支持了根据本发明的用于使用的组合物的口服施用。
实施例4
根据本发明的工程化的植物来源的EV可以被高效地冻干和存储。特别地，在EV冻干并在+4℃下存储7天后，与起始条件相比，囊泡中所装载的mRNA含量没有降低(图8)。对来自芹菜(E1)和奇异果(E2)的工程化的植物来源的EV进行实验，而且用来自其他植物来源(如西葫芦、卷心菜、羽衣甘蓝、蓝莓、柠檬、橙子和石榴)的EV也获得了相似的结果。
这些数据表明，根据本发明的EV可以很容易地被冻干，并高效地在+4℃或室温下存储，而不需要使用典型适于核酸试剂存储的非常低的温度，如-80℃。
实施例5
本发明人进一步表明，本发明的工程化的植物来源的EV适用于将所装载的核酸递送至受体细胞中(图9)。在这些实验中，使用巨噬细胞作为示例性受体细胞，并且通过qRT-PCR分析测量这些细胞中的mRNA分子的转移。特别地，上述实验表明，相对于未处理的细胞(未处理，NT)，来源于自不同类型植物(E1、E2、E3)的本发明的EV能够将mRNA分子转移至巨噬细胞中，而在天然的植物来源的EV(天然EV)、与mRNA共孵育的植物来源的EV(无核酸装载)(EV+mRNA)和裸mRNA中未检测到mRNA转移。对来自卷心菜(E1)、石榴(E2)和奇异果(E3)的工程化的植物来源的EV进行实验，而且用来自其他植物来源(如西葫芦、芹菜、羽衣甘蓝、柠檬、橙子和蓝莓)的EV也获得了相似的结果。
为了表明递送至受体细胞的核酸保持了其功能活性，对含有编码GFP蛋白的mRNA分子的本发明的EV进行测定。在掺入到受体细胞后，如果mRNA具有功能性，则被翻译成GFP蛋白，并且在细胞中可检测到功能蛋白的荧光。
由本发明人进行的实验表明，本发明的EV携带的mRNA具有功能性，并且可以以荧光信号在内皮细胞(图10A)和巨噬细胞(图10B)中检测，而使用天然的植物来源的(图10A中的天然EV)或裸mRNA(图10B中的裸mRNA)则没有功能性mRNA的转移。对来自西葫芦(图10A)和羽衣甘蓝(图10B)的工程化的植物来源的EV进行实验，而且用来自其他植物来源(如芹菜、卷心菜、奇异果、蓝莓、柠檬、橙子和石榴)的EV获得了相似的结果。
此外，本发明人表明，本发明的工程化的植物来源的EV能够将翻译成蛋白抗原的功能性mRNA转移到受体细胞中，并以正确折叠的抗原表示(图11)。在实验过程中，测定本发明的EV，其含有编码病毒蛋白抗原的不同序列的mRNA分子：SEQ ID NO.50，SARS-CoV-2刺突糖蛋白(S1)RBD蛋白；SEQ ID No.14，SARS-CoV-2刺突糖蛋白全蛋白；SEQ ID NO.17，SARS-CoV-2核壳蛋白。在掺入内皮细胞后，如果mRNA具有功能性，则被翻译成可被特异性抗体检测的蛋白。图11示出了本发明的工程化的植物来源的EV能够将mRNA转移至靶细胞中，该靶细胞将其翻译为特异性蛋白抗原，而在未处理的细胞(NT)或用天然的植物来源的未工程化的EV处理的细胞中未检测到蛋白抗原。
总之，这些数据表明，根据本发明的EV可以高效地将外源核酸分子递送至不同类型的受体细胞中，包括作为巨噬细胞的抗原呈递细胞(APC)，同时保留核酸功能及其翻译成蛋白的能力。因此，正确折叠的表达的蛋白可以作为抗原促进APC的免疫。此外，如上所述进行的实验表明，本发明的EV适于与编码不同蛋白(如病毒抗原(SARS-CoV-2核壳(N)蛋白和SARS-CoV-2刺突糖蛋白))或其他蛋白(如GFP)的核酸分子一起使用。
实施例6
本发明人进一步表明，本发明的工程化的植物来源的EV可以将核酸递送至表达和呈递抗原的APC细胞，然后刺激特异性免疫激活(图12)。在这些实验中，使用巨噬细胞作为示例性APC受体细胞，并在与PBMC(即来自人类血液的淋巴细胞、单核细胞和其他免疫细胞的混合群体)一起孵育之前用本发明的工程化的EV进行刺激。对于该实验，测定本发明的EV，其含有编码病毒蛋白抗原的不同序列的mRNA分子，作为示例：序列1，SARS-CoV-2刺突糖蛋白(S1)RBD蛋白；序列2，SARS-CoV-2刺突糖蛋白全蛋白；序列3，ARS-CoV-2核壳蛋白。10天后，使用FACS分析测量淋巴细胞的激活，即淋巴细胞(细胞CD4+)增殖和激活标记物CD25和HLA DR的表达的增加。为了检测淋巴细胞增殖，还用荧光染料(CFSE)对PBMC染色，该荧光染料允许通过流式细胞术检测PBMC增殖。如图12A所示，与阴性对照、未处理的细胞(NT)和用天然的EV处理的细胞相比，用工程化的植物来源的EV刺激的淋巴细胞CD4+的增殖率增加。如所预期的，阳性对照刺激了淋巴细胞增殖：人T激活剂CD3/CD28(CTR+)和纯化蛋白(SARS-CoV-2刺突糖蛋白RBD蛋白(S蛋白)或SARS-CoV-2核壳蛋白(N蛋白)。淋巴细胞增殖率的增加表明了它们被激活。
此外，用本发明的工程化的植物来源的EV刺激APC增加了淋巴细胞中激活标记物CD25(图12B)和HLA DR(图12C)的表达。
用本发明的植物来源的EV的刺激诱导了CD4+淋巴细胞两种激活标记物的表达增加，表明免疫细胞被激活。将刺激与阴性对照、未处理的细胞(NT)和用天然的植物来源的EV处理的细胞进行比较。如所预期的，阳性对照刺激了淋巴细胞增殖：人T激活剂CD3/CD28(CTR+)和纯化蛋白(SARS-CoV-2刺突糖蛋白RBD蛋白(S蛋白)或SARS-CoV-2核壳蛋白(N蛋白)。
总体而言，上述结果表明，本发明的工程化的植物来源的EV在掺入APC(如巨噬细胞)后激活了免疫反应，并且它们可以用编码不同蛋白抗原的不同核酸分子进行测定。
总之，所有这些数据表明，本发明的工程化的植物来源的EV能够将功能性mRNA转移至APC，进而被翻译成正确折叠的蛋白抗原，并且可以特异性地激活免疫反应。值得注意的是，只有本发明的工程化的植物来源的EV而非天然的植物来源的EV，才能诱导淋巴细胞激活。
实施例7
为了表明根据本发明的作为核酸递送载体的EV适合用作疫苗，在小鼠模型上进行了体内实验。
特别地，对小鼠进行两次免疫(两次治疗之间间隔三周)，并在最后一次剂量后两周测量血清中特异性抗的存在。使用肌内或口服施用途径，用天然的植物来源的EV(天然EV)或用编码病毒蛋白抗原(例如，SARS-CoV-2刺突糖蛋白RBD(S1))的mRNA分子工程化的本发明的植物来源的EV(工程化的EV)处理小鼠。
图13示出了小鼠血清中对SARS-CoV-2刺突糖蛋白RBD(S1)具有特异性的IgA抗体的测量。在口服和肌内施用后，与用天然的植物来源的EV进行的疫苗接种相比，用本发明的工程化的植物来源的EV进行的疫苗接种诱导了特异性抗体的产生。口服施用后检测的抗体阳性反应表明了保护根据本发明的用于使用的组合物免受胃肠道环境的影响。
总之，所有这些数据表明，本发明的工程化的植物来源的EV适合用作疫苗，因为它可以装载有核酸，其被转移至APC并且被翻译成正确折叠的抗原，激活免疫反应并在体内产生特异性抗体。免疫反应的激活对抗原具有特异性，因为本发明的EV本身既不发挥免疫刺激作用也不发挥免疫抑制作用。此外，本发明的工程化的植物来源的EV可以有效地保护核酸免受降解，从而允许使用不同的途径来施用疫苗。
序列表
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Leu Cys Tyr Gln Asp Thr Ile Leu Trp Lys Asp Ile Phe His Lys Asn
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His Pro Cys Ser Pro Met Cys Lys Gly Ser Arg Cys Trp Gly Glu Ser
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Ser Glu Asp Cys Gln Ser Leu Thr Arg Thr Val Cys Ala Gly Gly Cys
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Ala Arg Cys Lys Gly Pro Leu Pro Thr Asp Cys Cys His Glu Gln Cys
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Ala Ala Gly Cys Thr Gly Pro Lys His Ser Asp Cys Leu Ala Cys Leu
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His Phe Asn His Ser Gly Ile Cys Glu Leu His Cys Pro Ala Leu Val
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Thr Tyr Asn Thr Asp Thr Phe Glu Ser Met Pro Asn Pro Glu Gly Arg
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Tyr Thr Phe Gly Ala Ser Cys Val Thr Ala Cys Pro Tyr Asn Tyr Leu
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Ser Thr Asp Val Gly Ser Cys Thr Leu Val Cys Pro Leu His Asn Gln
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Glu Val Thr Ala Glu Asp Gly Thr Gln Arg Cys Glu Lys Cys Ser Lys
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Pro Cys Ala Arg Val Cys Tyr Gly Leu Gly Met Glu His Leu Arg Glu
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Val Arg Ala Val Thr Ser Ala Asn Ile Gln Glu Phe Ala Gly Cys Lys
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Lys Ile Phe Gly Ser Leu Ala Phe Leu Pro Glu Ser Phe Asp Gly Asp
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Pro Ala Ser Asn Thr Ala Pro Leu Gln Pro Glu Gln Leu Gln Val Phe
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Glu Thr Leu Glu Glu Ile Thr Gly Tyr Leu Tyr Ile Ser Ala Trp Pro
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Asp Ser Leu Pro Asp Leu Ser Val Phe Gln Asn Leu Gln Val Ile Arg
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Gly Arg Ile Leu His Asn Gly Ala Tyr Ser Leu Thr Leu Gln Gly Leu
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Gly Ile Ser Trp Leu Gly Leu Arg Ser Leu Arg Glu Leu Gly Ser Gly
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Leu Ala Leu Ile His His Asn Thr His Leu Cys Phe Val His Thr Val
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Pro Trp Asp Gln Leu Phe Arg Asn Pro His Gln Ala Leu Leu His Thr
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Ala Asn Arg Pro Glu Asp Glu Cys Val Gly Glu Gly Leu Ala Cys His
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Gln Leu Cys Ala Arg Gly His Cys Trp Gly Pro Gly Pro Thr Gln Cys
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Val Asn Cys Ser Gln Phe Leu Arg Gly Gln Glu Cys Val Glu Glu Cys
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Arg Val Leu Gln Gly Leu Pro Arg Glu Tyr Val Asn Ala Arg His Cys
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Leu Pro Cys His Pro Glu Cys Gln Pro Gln Asn Gly Ser Val Thr Cys
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Phe Gly Pro Glu Ala Asp Gln Cys Val Ala Cys Ala His Tyr Lys Asp
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Pro Pro Phe Cys Val Ala Arg Cys Pro Ser Gly Val Lys Pro Asp Leu
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Ser Tyr Met Pro Ile Trp Lys Phe Pro Asp Glu Glu Gly Ala Cys Gln
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Pro Cys Pro Ile Asn Cys Thr His Ser Cys Val Asp Leu Asp Asp Lys
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Gly Cys Pro Ala Glu Gln Arg Ala Ser Pro Leu Thr Ser Ile Ile Ser
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Ala Val Val Gly Ile Leu Leu Val Val Val Leu Gly Val Val Phe Gly
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Ile Leu Ile Lys Arg Arg Gln Gln Lys Ile Arg Lys Tyr Thr Met Arg
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Arg Leu Leu Gln Glu Thr Glu Leu Val Glu Pro Leu Thr Pro Ser Gly
690 695 700
Ala Met Pro Asn Gln Ala Gln Met Arg Ile Leu Lys Glu Thr Glu Leu
705 710 715 720
Arg Lys Val Lys Val Leu Gly Ser Gly Ala Phe Gly Thr Val Tyr Lys
725 730 735
Gly Ile Trp Ile Pro Asp Gly Glu Asn Val Lys Ile Pro Val Ala Ile
740 745 750
Lys Val Leu Arg Glu Asn Thr Ser Pro Lys Ala Asn Lys Glu Ile Leu
755 760 765
Asp Glu Ala Tyr Val Met Ala Gly Val Gly Ser Pro Tyr Val Ser Arg
770 775 780
Leu Leu Gly Ile Cys Leu Thr Ser Thr Val Gln Leu Val Thr Gln Leu
785 790 795 800
Met Pro Tyr Gly Cys Leu Leu Asp His Val Arg Glu Asn Arg Gly Arg
805 810 815
Leu Gly Ser Gln Asp Leu Leu Asn Trp Cys Met Gln Ile Ala Lys Gly
820 825 830
Met Ser Tyr Leu Glu Asp Val Arg Leu Val His Arg Asp Leu Ala Ala
835 840 845
Arg Asn Val Leu Val Lys Ser Pro Asn His Val Lys Ile Thr Asp Phe
850 855 860
Gly Leu Ala Arg Leu Leu Asp Ile Asp Glu Thr Glu Tyr His Ala Asp
865 870 875 880
Gly Gly Lys Val Pro Ile Lys Trp Met Ala Leu Glu Ser Ile Leu Arg
885 890 895
Arg Arg Phe Thr His Gln Ser Asp Val Trp Ser Tyr Gly Val Thr Val
900 905 910
Trp Glu Leu Met Thr Phe Gly Ala Lys Pro Tyr Asp Gly Ile Pro Ala
915 920 925
Arg Glu Ile Pro Asp Leu Leu Glu Lys Gly Glu Arg Leu Pro Gln Pro
930 935 940
Pro Ile Cys Thr Ile Asp Val Tyr Met Ile Met Val Lys Cys Trp Met
945 950 955 960
Ile Asp Ser Glu Cys Arg Pro Arg Phe Arg Glu Leu Val Ser Glu Phe
965 970 975
Ser Arg Met Ala Arg Asp Pro Gln Arg Phe Val Val Ile Gln Asn Glu
980 985 990
Asp Leu Gly Pro Ala Ser Pro Leu Asp Ser Thr Phe Tyr Arg Ser Leu
995 1000 1005
Leu Glu Asp Asp Asp Met Gly Asp Leu Val Asp Ala Glu Glu Tyr Leu
1010 1015 1020
Val Pro Gln Gln Gly Phe Phe Cys Pro Asp Pro Ala Pro Gly Ala Gly
1025 1030 1035 1040
Gly Met Val His His Arg His Arg Ser Ser Ser Thr Arg Ser Gly Gly
1045 1050 1055
Gly Asp Leu Thr Leu Gly Leu Glu Pro Ser Glu Glu Glu Ala Pro Arg
1060 1065 1070
Ser Pro Leu Ala Pro Ser Glu Gly Ala Gly Ser Asp Val Phe Asp Gly
1075 1080 1085
Asp Leu Gly Met Gly Ala Ala Lys Gly Leu Gln Ser Leu Pro Thr His
1090 1095 1100
Asp Pro Ser Pro Leu Gln Arg Tyr Ser Glu Asp Pro Thr Val Pro Leu
1105 1110 1115 1120
Pro Ser Glu Thr Asp Gly Tyr Val Ala Pro Leu Thr Cys Ser Pro Gln
1125 1130 1135
Pro Glu Tyr Val Asn Gln Pro Asp Val Arg Pro Gln Pro Pro Ser Pro
1140 1145 1150
Arg Glu Gly Pro Leu Pro Ala Ala Arg Pro Ala Gly Ala Thr Leu Glu
1155 1160 1165
Arg Pro Lys Thr Leu Ser Pro Gly Lys Asn Gly Val Val Lys Asp Val
1170 1175 1180
Phe Ala Phe Gly Gly Ala Val Glu Asn Pro Glu Tyr Leu Thr Pro Gln
1185 1190 1195 1200
Gly Gly Ala Ala Pro Gln Pro His Pro Pro Pro Ala Phe Ser Pro Ala
1205 1210 1215
Phe Asp Asn Leu Tyr Tyr Trp Asp Gln Asp Pro Pro Glu Arg Gly Ala
1220 1225 1230
Pro Pro Ser Thr Phe Lys Gly Thr Pro Thr Ala Glu Asn Pro Glu Tyr
1235 1240 1245
Leu Gly Leu Asp Val Pro Val
1250 1255
<210> 6
<211> 1255
<212> PRT
<213> 智人
<220>
<223> 粘蛋白1，细胞表面相关蛋白（MUC1）
<400> 6
Met Thr Pro Gly Thr Gln Ser Pro Phe Phe Leu Leu Leu Leu Leu Thr
1 5 10 15
Val Leu Thr Val Val Thr Gly Ser Gly His Ala Ser Ser Thr Pro Gly
20 25 30
Gly Glu Lys Glu Thr Ser Ala Thr Gln Arg Ser Ser Val Pro Ser Ser
35 40 45
Thr Glu Lys Asn Ala Val Ser Met Thr Ser Ser Val Leu Ser Ser His
50 55 60
Ser Pro Gly Ser Gly Ser Ser Thr Thr Gln Gly Gln Asp Val Thr Leu
65 70 75 80
Ala Pro Ala Thr Glu Pro Ala Ser Gly Ser Ala Ala Thr Trp Gly Gln
85 90 95
Asp Val Thr Ser Val Pro Val Thr Arg Pro Ala Leu Gly Ser Thr Thr
100 105 110
Pro Pro Ala His Asp Val Thr Ser Ala Pro Asp Asn Lys Pro Ala Pro
115 120 125
Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr
130 135 140
Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser
145 150 155 160
Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His
165 170 175
Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala
180 185 190
Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro
195 200 205
Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr
210 215 220
Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser
225 230 235 240
Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His
245 250 255
Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala
260 265 270
Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro
275 280 285
Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr
290 295 300
Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser
305 310 315 320
Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His
325 330 335
Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala
340 345 350
Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro
355 360 365
Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr
370 375 380
Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser
385 390 395 400
Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His
405 410 415
Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala
420 425 430
Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro
435 440 445
Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr
450 455 460
Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser
465 470 475 480
Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His
485 490 495
Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala
500 505 510
Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro
515 520 525
Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr
530 535 540
Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser
545 550 555 560
Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His
565 570 575
Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala
580 585 590
Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro
595 600 605
Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr
610 615 620
Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser
625 630 635 640
Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His
645 650 655
Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala
660 665 670
Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro
675 680 685
Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr
690 695 700
Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser
705 710 715 720
Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His
725 730 735
Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala
740 745 750
Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro
755 760 765
Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr
770 775 780
Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser
785 790 795 800
Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His
805 810 815
Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala
820 825 830
Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro
835 840 845
Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr
850 855 860
Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser
865 870 875 880
Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His
885 890 895
Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala
900 905 910
Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro
915 920 925
Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Asn
930 935 940
Arg Pro Ala Leu Gly Ser Thr Ala Pro Pro Val His Asn Val Thr Ser
945 950 955 960
Ala Ser Gly Ser Ala Ser Gly Ser Ala Ser Thr Leu Val His Asn Gly
965 970 975
Thr Ser Ala Arg Ala Thr Thr Thr Pro Ala Ser Lys Ser Thr Pro Phe
980 985 990
Ser Ile Pro Ser His His Ser Asp Thr Pro Thr Thr Leu Ala Ser His
995 1000 1005
Ser Thr Lys Thr Asp Ala Ser Ser Thr His His Ser Ser Val Pro Pro
1010 1015 1020
Leu Thr Ser Ser Asn His Ser Thr Ser Pro Gln Leu Ser Thr Gly Val
1025 1030 1035 1040
Ser Phe Phe Phe Leu Ser Phe His Ile Ser Asn Leu Gln Phe Asn Ser
1045 1050 1055
Ser Leu Glu Asp Pro Ser Thr Asp Tyr Tyr Gln Glu Leu Gln Arg Asp
1060 1065 1070
Ile Ser Glu Met Phe Leu Gln Ile Tyr Lys Gln Gly Gly Phe Leu Gly
1075 1080 1085
Leu Ser Asn Ile Lys Phe Arg Pro Gly Ser Val Val Val Gln Leu Thr
1090 1095 1100
Leu Ala Phe Arg Glu Gly Thr Ile Asn Val His Asp Val Glu Thr Gln
1105 1110 1115 1120
Phe Asn Gln Tyr Lys Thr Glu Ala Ala Ser Arg Tyr Asn Leu Thr Ile
1125 1130 1135
Ser Asp Val Ser Val Ser Asp Val Pro Phe Pro Phe Ser Ala Gln Ser
1140 1145 1150
Gly Ala Gly Val Pro Gly Trp Gly Ile Ala Leu Leu Val Leu Val Cys
1155 1160 1165
Val Leu Val Ala Leu Ala Ile Val Tyr Leu Ile Ala Leu Ala Val Cys
1170 1175 1180
Gln Cys Arg Arg Lys Asn Tyr Gly Gln Leu Asp Ile Phe Pro Ala Arg
1185 1190 1195 1200
Asp Thr Tyr His Pro Met Ser Glu Tyr Pro Thr Tyr His Thr His Gly
1205 1210 1215
Arg Tyr Val Pro Pro Ser Ser Thr Asp Arg Ser Pro Tyr Glu Lys Val
1220 1225 1230
Ser Ala Gly Asn Gly Gly Ser Ser Leu Ser Tyr Thr Asn Pro Ala Val
1235 1240 1245
Ala Ala Thr Ser Ala Asn Leu
1250 1255
<210> 7
<211> 519
<212> PRT
<213> 智人
<220>
<223> 酪氨酸酶相关蛋白2 （TRP-2）
<400> 7
Met Ser Pro Leu Trp Trp Gly Phe Leu Leu Ser Cys Leu Gly Cys Lys
1 5 10 15
Ile Leu Pro Gly Ala Gln Gly Gln Phe Pro Arg Val Cys Met Thr Val
20 25 30
Asp Ser Leu Val Asn Lys Glu Cys Cys Pro Arg Leu Gly Ala Glu Ser
35 40 45
Ala Asn Val Cys Gly Ser Gln Gln Gly Arg Gly Gln Cys Thr Glu Val
50 55 60
Arg Ala Asp Thr Arg Pro Trp Ser Gly Pro Tyr Ile Leu Arg Asn Gln
65 70 75 80
Asp Asp Arg Glu Leu Trp Pro Arg Lys Phe Phe His Arg Thr Cys Lys
85 90 95
Cys Thr Gly Asn Phe Ala Gly Tyr Asn Cys Gly Asp Cys Lys Phe Gly
100 105 110
Trp Thr Gly Pro Asn Cys Glu Arg Lys Lys Pro Pro Val Ile Arg Gln
115 120 125
Asn Ile His Ser Leu Ser Pro Gln Glu Arg Glu Gln Phe Leu Gly Ala
130 135 140
Leu Asp Leu Ala Lys Lys Arg Val His Pro Asp Tyr Val Ile Thr Thr
145 150 155 160
Gln His Trp Leu Gly Leu Leu Gly Pro Asn Gly Thr Gln Pro Gln Phe
165 170 175
Ala Asn Cys Ser Val Tyr Asp Phe Phe Val Trp Leu His Tyr Tyr Ser
180 185 190
Val Arg Asp Thr Leu Leu Gly Pro Gly Arg Pro Tyr Arg Ala Ile Asp
195 200 205
Phe Ser His Gln Gly Pro Ala Phe Val Thr Trp His Arg Tyr His Leu
210 215 220
Leu Cys Leu Glu Arg Asp Leu Gln Arg Leu Ile Gly Asn Glu Ser Phe
225 230 235 240
Ala Leu Pro Tyr Trp Asn Phe Ala Thr Gly Arg Asn Glu Cys Asp Val
245 250 255
Cys Thr Asp Gln Leu Phe Gly Ala Ala Arg Pro Asp Asp Pro Thr Leu
260 265 270
Ile Ser Arg Asn Ser Arg Phe Ser Ser Trp Glu Thr Val Cys Asp Ser
275 280 285
Leu Asp Asp Tyr Asn His Leu Val Thr Leu Cys Asn Gly Thr Tyr Glu
290 295 300
Gly Leu Leu Arg Arg Asn Gln Met Gly Arg Asn Ser Met Lys Leu Pro
305 310 315 320
Thr Leu Lys Asp Ile Arg Asp Cys Leu Ser Leu Gln Lys Phe Asp Asn
325 330 335
Pro Pro Phe Phe Gln Asn Ser Thr Phe Ser Phe Arg Asn Ala Leu Glu
340 345 350
Gly Phe Asp Lys Ala Asp Gly Thr Leu Asp Ser Gln Val Met Ser Leu
355 360 365
His Asn Leu Val His Ser Phe Leu Asn Gly Thr Asn Ala Leu Pro His
370 375 380
Ser Ala Ala Asn Asp Pro Ile Phe Val Val Leu His Ser Phe Thr Asp
385 390 395 400
Ala Ile Phe Asp Glu Trp Met Lys Arg Phe Asn Pro Pro Ala Asp Ala
405 410 415
Trp Pro Gln Glu Leu Ala Pro Ile Gly His Asn Arg Met Tyr Asn Met
420 425 430
Val Pro Phe Phe Pro Pro Val Thr Asn Glu Glu Leu Phe Leu Thr Ser
435 440 445
Asp Gln Leu Gly Tyr Ser Tyr Ala Ile Asp Leu Pro Val Ser Val Glu
450 455 460
Glu Thr Pro Gly Trp Pro Thr Thr Leu Leu Val Val Met Gly Thr Leu
465 470 475 480
Val Ala Leu Val Gly Leu Phe Val Leu Leu Ala Phe Leu Gln Tyr Arg
485 490 495
Arg Leu Arg Lys Gly Tyr Thr Pro Leu Met Glu Thr His Leu Ser Ser
500 505 510
Lys Arg Tyr Thr Glu Glu Ala
515
<210> 8
<211> 766
<212> PRT
<213> 智人
<220>
<223> 丝氨酸/苏氨酸-蛋白激酶B-raf
<400> 8
Met Ala Ala Leu Ser Gly Gly Gly Gly Gly Gly Ala Glu Pro Gly Gln
1 5 10 15
Ala Leu Phe Asn Gly Asp Met Glu Pro Glu Ala Gly Ala Gly Ala Gly
20 25 30
Ala Ala Ala Ser Ser Ala Ala Asp Pro Ala Ile Pro Glu Glu Val Trp
35 40 45
Asn Ile Lys Gln Met Ile Lys Leu Thr Gln Glu His Ile Glu Ala Leu
50 55 60
Leu Asp Lys Phe Gly Gly Glu His Asn Pro Pro Ser Ile Tyr Leu Glu
65 70 75 80
Ala Tyr Glu Glu Tyr Thr Ser Lys Leu Asp Ala Leu Gln Gln Arg Glu
85 90 95
Gln Gln Leu Leu Glu Ser Leu Gly Asn Gly Thr Asp Phe Ser Val Ser
100 105 110
Ser Ser Ala Ser Met Asp Thr Val Thr Ser Ser Ser Ser Ser Ser Leu
115 120 125
Ser Val Leu Pro Ser Ser Leu Ser Val Phe Gln Asn Pro Thr Asp Val
130 135 140
Ala Arg Ser Asn Pro Lys Ser Pro Gln Lys Pro Ile Val Arg Val Phe
145 150 155 160
Leu Pro Asn Lys Gln Arg Thr Val Val Pro Ala Arg Cys Gly Val Thr
165 170 175
Val Arg Asp Ser Leu Lys Lys Ala Leu Met Met Arg Gly Leu Ile Pro
180 185 190
Glu Cys Cys Ala Val Tyr Arg Ile Gln Asp Gly Glu Lys Lys Pro Ile
195 200 205
Gly Trp Asp Thr Asp Ile Ser Trp Leu Thr Gly Glu Glu Leu His Val
210 215 220
Glu Val Leu Glu Asn Val Pro Leu Thr Thr His Asn Phe Val Arg Lys
225 230 235 240
Thr Phe Phe Thr Leu Ala Phe Cys Asp Phe Cys Arg Lys Leu Leu Phe
245 250 255
Gln Gly Phe Arg Cys Gln Thr Cys Gly Tyr Lys Phe His Gln Arg Cys
260 265 270
Ser Thr Glu Val Pro Leu Met Cys Val Asn Tyr Asp Gln Leu Asp Leu
275 280 285
Leu Phe Val Ser Lys Phe Phe Glu His His Pro Ile Pro Gln Glu Glu
290 295 300
Ala Ser Leu Ala Glu Thr Ala Leu Thr Ser Gly Ser Ser Pro Ser Ala
305 310 315 320
Pro Ala Ser Asp Ser Ile Gly Pro Gln Ile Leu Thr Ser Pro Ser Pro
325 330 335
Ser Lys Ser Ile Pro Ile Pro Gln Pro Phe Arg Pro Ala Asp Glu Asp
340 345 350
His Arg Asn Gln Phe Gly Gln Arg Asp Arg Ser Ser Ser Ala Pro Asn
355 360 365
Val His Ile Asn Thr Ile Glu Pro Val Asn Ile Asp Asp Leu Ile Arg
370 375 380
Asp Gln Gly Phe Arg Gly Asp Gly Gly Ser Thr Thr Gly Leu Ser Ala
385 390 395 400
Thr Pro Pro Ala Ser Leu Pro Gly Ser Leu Thr Asn Val Lys Ala Leu
405 410 415
Gln Lys Ser Pro Gly Pro Gln Arg Glu Arg Lys Ser Ser Ser Ser Ser
420 425 430
Glu Asp Arg Asn Arg Met Lys Thr Leu Gly Arg Arg Asp Ser Ser Asp
435 440 445
Asp Trp Glu Ile Pro Asp Gly Gln Ile Thr Val Gly Gln Arg Ile Gly
450 455 460
Ser Gly Ser Phe Gly Thr Val Tyr Lys Gly Lys Trp His Gly Asp Val
465 470 475 480
Ala Val Lys Met Leu Asn Val Thr Ala Pro Thr Pro Gln Gln Leu Gln
485 490 495
Ala Phe Lys Asn Glu Val Gly Val Leu Arg Lys Thr Arg His Val Asn
500 505 510
Ile Leu Leu Phe Met Gly Tyr Ser Thr Lys Pro Gln Leu Ala Ile Val
515 520 525
Thr Gln Trp Cys Glu Gly Ser Ser Leu Tyr His His Leu His Ile Ile
530 535 540
Glu Thr Lys Phe Glu Met Ile Lys Leu Ile Asp Ile Ala Arg Gln Thr
545 550 555 560
Ala Gln Gly Met Asp Tyr Leu His Ala Lys Ser Ile Ile His Arg Asp
565 570 575
Leu Lys Ser Asn Asn Ile Phe Leu His Glu Asp Leu Thr Val Lys Ile
580 585 590
Gly Asp Phe Gly Leu Ala Thr Val Lys Ser Arg Trp Ser Gly Ser His
595 600 605
Gln Phe Glu Gln Leu Ser Gly Ser Ile Leu Trp Met Ala Pro Glu Val
610 615 620
Ile Arg Met Gln Asp Lys Asn Pro Tyr Ser Phe Gln Ser Asp Val Tyr
625 630 635 640
Ala Phe Gly Ile Val Leu Tyr Glu Leu Met Thr Gly Gln Leu Pro Tyr
645 650 655
Ser Asn Ile Asn Asn Arg Asp Gln Ile Ile Phe Met Val Gly Arg Gly
660 665 670
Tyr Leu Ser Pro Asp Leu Ser Lys Val Arg Ser Asn Cys Pro Lys Ala
675 680 685
Met Lys Arg Leu Met Ala Glu Cys Leu Lys Lys Lys Arg Asp Glu Arg
690 695 700
Pro Leu Phe Pro Gln Ile Leu Ala Ser Ile Glu Leu Leu Ala Arg Ser
705 710 715 720
Leu Pro Lys Ile His Arg Ser Ala Ser Glu Pro Ser Leu Asn Arg Ala
725 730 735
Gly Phe Gln Thr Glu Asp Phe Ser Leu Tyr Ala Cys Ala Ser Pro Lys
740 745 750
Thr Pro Ile Gln Ala Gly Gly Tyr Gly Ala Phe Pro Val His
755 760 765
<210> 9
<211> 976
<212> PRT
<213> 智人
<220>
<223> 肥大细胞/干细胞生长因子受体KIT
<400> 9
Met Arg Gly Ala Arg Gly Ala Trp Asp Phe Leu Cys Val Leu Leu Leu
1 5 10 15
Leu Leu Arg Val Gln Thr Gly Ser Ser Gln Pro Ser Val Ser Pro Gly
20 25 30
Glu Pro Ser Pro Pro Ser Ile His Pro Gly Lys Ser Asp Leu Ile Val
35 40 45
Arg Val Gly Asp Glu Ile Arg Leu Leu Cys Thr Asp Pro Gly Phe Val
50 55 60
Lys Trp Thr Phe Glu Ile Leu Asp Glu Thr Asn Glu Asn Lys Gln Asn
65 70 75 80
Glu Trp Ile Thr Glu Lys Ala Glu Ala Thr Asn Thr Gly Lys Tyr Thr
85 90 95
Cys Thr Asn Lys His Gly Leu Ser Asn Ser Ile Tyr Val Phe Val Arg
100 105 110
Asp Pro Ala Lys Leu Phe Leu Val Asp Arg Ser Leu Tyr Gly Lys Glu
115 120 125
Asp Asn Asp Thr Leu Val Arg Cys Pro Leu Thr Asp Pro Glu Val Thr
130 135 140
Asn Tyr Ser Leu Lys Gly Cys Gln Gly Lys Pro Leu Pro Lys Asp Leu
145 150 155 160
Arg Phe Ile Pro Asp Pro Lys Ala Gly Ile Met Ile Lys Ser Val Lys
165 170 175
Arg Ala Tyr His Arg Leu Cys Leu His Cys Ser Val Asp Gln Glu Gly
180 185 190
Lys Ser Val Leu Ser Glu Lys Phe Ile Leu Lys Val Arg Pro Ala Phe
195 200 205
Lys Ala Val Pro Val Val Ser Val Ser Lys Ala Ser Tyr Leu Leu Arg
210 215 220
Glu Gly Glu Glu Phe Thr Val Thr Cys Thr Ile Lys Asp Val Ser Ser
225 230 235 240
Ser Val Tyr Ser Thr Trp Lys Arg Glu Asn Ser Gln Thr Lys Leu Gln
245 250 255
Glu Lys Tyr Asn Ser Trp His His Gly Asp Phe Asn Tyr Glu Arg Gln
260 265 270
Ala Thr Leu Thr Ile Ser Ser Ala Arg Val Asn Asp Ser Gly Val Phe
275 280 285
Met Cys Tyr Ala Asn Asn Thr Phe Gly Ser Ala Asn Val Thr Thr Thr
290 295 300
Leu Glu Val Val Asp Lys Gly Phe Ile Asn Ile Phe Pro Met Ile Asn
305 310 315 320
Thr Thr Val Phe Val Asn Asp Gly Glu Asn Val Asp Leu Ile Val Glu
325 330 335
Tyr Glu Ala Phe Pro Lys Pro Glu His Gln Gln Trp Ile Tyr Met Asn
340 345 350
Arg Thr Phe Thr Asp Lys Trp Glu Asp Tyr Pro Lys Ser Glu Asn Glu
355 360 365
Ser Asn Ile Arg Tyr Val Ser Glu Leu His Leu Thr Arg Leu Lys Gly
370 375 380
Thr Glu Gly Gly Thr Tyr Thr Phe Leu Val Ser Asn Ser Asp Val Asn
385 390 395 400
Ala Ala Ile Ala Phe Asn Val Tyr Val Asn Thr Lys Pro Glu Ile Leu
405 410 415
Thr Tyr Asp Arg Leu Val Asn Gly Met Leu Gln Cys Val Ala Ala Gly
420 425 430
Phe Pro Glu Pro Thr Ile Asp Trp Tyr Phe Cys Pro Gly Thr Glu Gln
435 440 445
Arg Cys Ser Ala Ser Val Leu Pro Val Asp Val Gln Thr Leu Asn Ser
450 455 460
Ser Gly Pro Pro Phe Gly Lys Leu Val Val Gln Ser Ser Ile Asp Ser
465 470 475 480
Ser Ala Phe Lys His Asn Gly Thr Val Glu Cys Lys Ala Tyr Asn Asp
485 490 495
Val Gly Lys Thr Ser Ala Tyr Phe Asn Phe Ala Phe Lys Gly Asn Asn
500 505 510
Lys Glu Gln Ile His Pro His Thr Leu Phe Thr Pro Leu Leu Ile Gly
515 520 525
Phe Val Ile Val Ala Gly Met Met Cys Ile Ile Val Met Ile Leu Thr
530 535 540
Tyr Lys Tyr Leu Gln Lys Pro Met Tyr Glu Val Gln Trp Lys Val Val
545 550 555 560
Glu Glu Ile Asn Gly Asn Asn Tyr Val Tyr Ile Asp Pro Thr Gln Leu
565 570 575
Pro Tyr Asp His Lys Trp Glu Phe Pro Arg Asn Arg Leu Ser Phe Gly
580 585 590
Lys Thr Leu Gly Ala Gly Ala Phe Gly Lys Val Val Glu Ala Thr Ala
595 600 605
Tyr Gly Leu Ile Lys Ser Asp Ala Ala Met Thr Val Ala Val Lys Met
610 615 620
Leu Lys Pro Ser Ala His Leu Thr Glu Arg Glu Ala Leu Met Ser Glu
625 630 635 640
Leu Lys Val Leu Ser Tyr Leu Gly Asn His Met Asn Ile Val Asn Leu
645 650 655
Leu Gly Ala Cys Thr Ile Gly Gly Pro Thr Leu Val Ile Thr Glu Tyr
660 665 670
Cys Cys Tyr Gly Asp Leu Leu Asn Phe Leu Arg Arg Lys Arg Asp Ser
675 680 685
Phe Ile Cys Ser Lys Gln Glu Asp His Ala Glu Ala Ala Leu Tyr Lys
690 695 700
Asn Leu Leu His Ser Lys Glu Ser Ser Cys Ser Asp Ser Thr Asn Glu
705 710 715 720
Tyr Met Asp Met Lys Pro Gly Val Ser Tyr Val Val Pro Thr Lys Ala
725 730 735
Asp Lys Arg Arg Ser Val Arg Ile Gly Ser Tyr Ile Glu Arg Asp Val
740 745 750
Thr Pro Ala Ile Met Glu Asp Asp Glu Leu Ala Leu Asp Leu Glu Asp
755 760 765
Leu Leu Ser Phe Ser Tyr Gln Val Ala Lys Gly Met Ala Phe Leu Ala
770 775 780
Ser Lys Asn Cys Ile His Arg Asp Leu Ala Ala Arg Asn Ile Leu Leu
785 790 795 800
Thr His Gly Arg Ile Thr Lys Ile Cys Asp Phe Gly Leu Ala Arg Asp
805 810 815
Ile Lys Asn Asp Ser Asn Tyr Val Val Lys Gly Asn Ala Arg Leu Pro
820 825 830
Val Lys Trp Met Ala Pro Glu Ser Ile Phe Asn Cys Val Tyr Thr Phe
835 840 845
Glu Ser Asp Val Trp Ser Tyr Gly Ile Phe Leu Trp Glu Leu Phe Ser
850 855 860
Leu Gly Ser Ser Pro Tyr Pro Gly Met Pro Val Asp Ser Lys Phe Tyr
865 870 875 880
Lys Met Ile Lys Glu Gly Phe Arg Met Leu Ser Pro Glu His Ala Pro
885 890 895
Ala Glu Met Tyr Asp Ile Met Lys Thr Cys Trp Asp Ala Asp Pro Leu
900 905 910
Lys Arg Pro Thr Phe Lys Gln Ile Val Gln Leu Ile Glu Lys Gln Ile
915 920 925
Ser Glu Ser Thr Asn His Ile Tyr Ser Asn Leu Ala Asn Cys Ser Pro
930 935 940
Asn Arg Gln Lys Pro Val Val Asp His Ser Val Arg Ile Asn Ser Val
945 950 955 960
Gly Ser Thr Ala Ser Ser Ser Gln Pro Leu Leu Val His Asp Asp Val
965 970 975
<210> 10
<211> 189
<212> PRT
<213> 智人
<220>
<223> GTP酶 NRas
<400> 10
Met Thr Glu Tyr Lys Leu Val Val Val Gly Ala Gly Gly Val Gly Lys
1 5 10 15
Ser Ala Leu Thr Ile Gln Leu Ile Gln Asn His Phe Val Asp Glu Tyr
20 25 30
Asp Pro Thr Ile Glu Asp Ser Tyr Arg Lys Gln Val Val Ile Asp Gly
35 40 45
Glu Thr Cys Leu Leu Asp Ile Leu Asp Thr Ala Gly Gln Glu Glu Tyr
50 55 60
Ser Ala Met Arg Asp Gln Tyr Met Arg Thr Gly Glu Gly Phe Leu Cys
65 70 75 80
Val Phe Ala Ile Asn Asn Ser Lys Ser Phe Ala Asp Ile Asn Leu Tyr
85 90 95
Arg Glu Gln Ile Lys Arg Val Lys Asp Ser Asp Asp Val Pro Met Val
100 105 110
Leu Val Gly Asn Lys Cys Asp Leu Pro Thr Arg Thr Val Asp Thr Lys
115 120 125
Gln Ala His Glu Leu Ala Lys Ser Tyr Gly Ile Pro Phe Ile Glu Thr
130 135 140
Ser Ala Lys Thr Arg Gln Gly Val Glu Asp Ala Phe Tyr Thr Leu Val
145 150 155 160
Arg Glu Ile Arg Gln Tyr Arg Met Lys Lys Leu Asn Ser Ser Asp Asp
165 170 175
Gly Thr Gln Gly Cys Met Gly Leu Pro Cys Val Val Met
180 185
<210> 11
<211> 309
<212> PRT
<213> 智人
<220>
<223> 黑色素瘤相关抗原1
<400> 11
Met Ser Leu Glu Gln Arg Ser Leu His Cys Lys Pro Glu Glu Ala Leu
1 5 10 15
Glu Ala Gln Gln Glu Ala Leu Gly Leu Val Cys Val Gln Ala Ala Thr
20 25 30
Ser Ser Ser Ser Pro Leu Val Leu Gly Thr Leu Glu Glu Val Pro Thr
35 40 45
Ala Gly Ser Thr Asp Pro Pro Gln Ser Pro Gln Gly Ala Ser Ala Phe
50 55 60
Pro Thr Thr Ile Asn Phe Thr Arg Gln Arg Gln Pro Ser Glu Gly Ser
65 70 75 80
Ser Ser Arg Glu Glu Glu Gly Pro Ser Thr Ser Cys Ile Leu Glu Ser
85 90 95
Leu Phe Arg Ala Val Ile Thr Lys Lys Val Ala Asp Leu Val Gly Phe
100 105 110
Leu Leu Leu Lys Tyr Arg Ala Arg Glu Pro Val Thr Lys Ala Glu Met
115 120 125
Leu Glu Ser Val Ile Lys Asn Tyr Lys His Cys Phe Pro Glu Ile Phe
130 135 140
Gly Lys Ala Ser Glu Ser Leu Gln Leu Val Phe Gly Ile Asp Val Lys
145 150 155 160
Glu Ala Asp Pro Thr Gly His Ser Tyr Val Leu Val Thr Cys Leu Gly
165 170 175
Leu Ser Tyr Asp Gly Leu Leu Gly Asp Asn Gln Ile Met Pro Lys Thr
180 185 190
Gly Phe Leu Ile Ile Val Leu Val Met Ile Ala Met Glu Gly Gly His
195 200 205
Ala Pro Glu Glu Glu Ile Trp Glu Glu Leu Ser Val Met Glu Val Tyr
210 215 220
Asp Gly Arg Glu His Ser Ala Tyr Gly Glu Pro Arg Lys Leu Leu Thr
225 230 235 240
Gln Asp Leu Val Gln Glu Lys Tyr Leu Glu Tyr Arg Gln Val Pro Asp
245 250 255
Ser Asp Pro Ala Arg Tyr Glu Phe Leu Trp Gly Pro Arg Ala Leu Ala
260 265 270
Glu Thr Ser Tyr Val Lys Val Leu Glu Tyr Val Ile Lys Val Ser Ala
275 280 285
Arg Val Arg Phe Phe Phe Pro Ser Leu Arg Glu Ala Ala Leu Arg Glu
290 295 300
Glu Glu Glu Gly Val
305
<210> 12
<211> 314
<212> PRT
<213> 智人
<220>
<223> 黑色素瘤相关抗原1
<400> 12
Met Pro Leu Glu Gln Arg Ser Gln His Cys Lys Pro Glu Glu Gly Leu
1 5 10 15
Glu Ala Arg Gly Glu Ala Leu Gly Leu Val Gly Ala Gln Ala Pro Ala
20 25 30
Thr Glu Glu Gln Glu Ala Ala Ser Ser Ser Ser Thr Leu Val Glu Val
35 40 45
Thr Leu Gly Glu Val Pro Ala Ala Glu Ser Pro Asp Pro Pro Gln Ser
50 55 60
Pro Gln Gly Ala Ser Ser Leu Pro Thr Thr Met Asn Tyr Pro Leu Trp
65 70 75 80
Ser Gln Ser Tyr Glu Asp Ser Ser Asn Gln Glu Glu Glu Gly Pro Ser
85 90 95
Thr Phe Pro Asp Leu Glu Ser Glu Phe Gln Ala Ala Leu Ser Arg Lys
100 105 110
Val Ala Glu Leu Val His Phe Leu Leu Leu Lys Tyr Arg Ala Arg Glu
115 120 125
Pro Val Thr Lys Ala Glu Met Leu Gly Ser Val Val Gly Asn Trp Gln
130 135 140
Tyr Phe Phe Pro Val Ile Phe Ser Lys Ala Ser Ser Ser Leu Gln Leu
145 150 155 160
Val Phe Gly Ile Glu Leu Met Glu Val Asp Pro Ile Gly His Leu Tyr
165 170 175
Ile Phe Ala Thr Cys Leu Gly Leu Ser Tyr Asp Gly Leu Leu Gly Asp
180 185 190
Asn Gln Ile Met Pro Lys Ala Gly Leu Leu Ile Ile Val Leu Ala Ile
195 200 205
Ile Ala Arg Glu Gly Asp Cys Ala Pro Glu Glu Lys Ile Trp Glu Glu
210 215 220
Leu Ser Val Leu Glu Val Phe Glu Gly Arg Glu Asp Ser Ile Leu Gly
225 230 235 240
Asp Pro Lys Lys Leu Leu Thr Gln His Phe Val Gln Glu Asn Tyr Leu
245 250 255
Glu Tyr Arg Gln Val Pro Gly Ser Asp Pro Ala Cys Tyr Glu Phe Leu
260 265 270
Trp Gly Pro Arg Ala Leu Val Glu Thr Ser Tyr Val Lys Val Leu His
275 280 285
His Met Val Lys Ile Ser Gly Gly Pro His Ile Ser Tyr Pro Pro Leu
290 295 300
His Glu Trp Val Leu Arg Glu Gly Glu Glu
305 310
<210> 13
<211> 180
<212> PRT
<213> 智人
<220>
<223> NY-ESO-1蛋白
<400> 13
Met Gln Ala Glu Gly Arg Gly Thr Gly Gly Ser Thr Gly Asp Ala Asp
1 5 10 15
Gly Pro Gly Gly Pro Gly Ile Pro Asp Gly Pro Gly Gly Asn Ala Gly
20 25 30
Gly Pro Gly Glu Ala Gly Ala Thr Gly Gly Arg Gly Pro Arg Gly Ala
35 40 45
Gly Ala Ala Arg Ala Ser Gly Pro Gly Gly Gly Ala Pro Arg Gly Pro
50 55 60
His Gly Gly Ala Ala Ser Gly Leu Asn Gly Cys Cys Arg Cys Gly Ala
65 70 75 80
Arg Gly Pro Glu Ser Arg Leu Leu Glu Phe Tyr Leu Ala Met Pro Phe
85 90 95
Ala Thr Pro Met Glu Ala Glu Leu Ala Arg Arg Ser Leu Ala Gln Asp
100 105 110
Ala Pro Pro Leu Pro Val Pro Gly Val Leu Leu Lys Glu Phe Thr Val
115 120 125
Ser Gly Asn Ile Leu Thr Ile Arg Leu Thr Ala Ala Asp His Arg Gln
130 135 140
Leu Gln Leu Ser Ile Ser Ser Cys Leu Gln Gln Leu Ser Leu Leu Met
145 150 155 160
Trp Ile Thr Gln Cys Phe Leu Pro Val Phe Leu Ala Gln Pro Pro Ser
165 170 175
Gly Gln Arg Arg
180
<210> 14
<211> 3822
<212> RNA
<213> SARS冠状病毒
<220>
<223> 编码SARS-CoV-2刺突（S）蛋白的mRNA
<400> 14
atgtttgttt ttcttgtttt attgccacta gtctctagtc agtgtgttaa tcttacaacc 60
agaactcaat taccccctgc atacactaat tctttcacac gtggtgttta ttaccctgac 120
aaagttttca gatcctcagt tttacattca actcaggact tgttcttacc tttcttttcc 180
aatgttactt ggttccatgc tatacatgtc tctgggacca atggtactaa gaggtttgat 240
aaccctgtcc taccatttaa tgatggtgtt tattttgctt ccactgagaa gtctaacata 300
ataagaggct ggatttttgg tactacttta gattcgaaga cccagtccct acttattgtt 360
aataacgcta ctaatgttgt tattaaagtc tgtgaatttc aattttgtaa tgatccattt 420
ttgggtgttt attaccacaa aaacaacaaa agttggatgg aaagtgagtt cagagtttat 480
tctagtgcga ataattgcac ttttgaatat gtctctcagc cttttcttat ggaccttgaa 540
ggaaaacagg gtaatttcaa aaatcttagg gaatttgtgt ttaagaatat tgatggttat 600
tttaaaatat attctaagca cacgcctatt aatttagtgc gtgatctccc tcagggtttt 660
tcggctttag aaccattggt agatttgcca ataggtatta acatcactag gtttcaaact 720
ttacttgctt tacatagaag ttatttgact cctggtgatt cttcttcagg ttggacagct 780
ggtgctgcag cttattatgt gggttatctt caacctagga cttttctatt aaaatataat 840
gaaaatggaa ccattacaga tgctgtagac tgtgcacttg accctctctc agaaacaaag 900
tgtacgttga aatccttcac tgtagaaaaa ggaatctatc aaacttctaa ctttagagtc 960
caaccaacag aatctattgt tagatttcct aatattacaa acttgtgccc ttttggtgaa 1020
gtttttaacg ccaccagatt tgcatctgtt tatgcttgga acaggaagag aatcagcaac 1080
tgtgttgctg attattctgt cctatataat tccgcatcat tttccacttt taagtgttat 1140
ggagtgtctc ctactaaatt aaatgatctc tgctttacta atgtctatgc agattcattt 1200
gtaattagag gtgatgaagt cagacaaatc gctccagggc aaactggaaa gattgctgat 1260
tataattata aattaccaga tgattttaca ggctgcgtta tagcttggaa ttctaacaat 1320
cttgattcta aggttggtgg taattataat tacctgtata gattgtttag gaagtctaat 1380
ctcaaacctt ttgagagaga tatttcaact gaaatctatc aggccggtag cacaccttgt 1440
aatggtgttg aaggttttaa ttgttacttt cctttacaat catatggttt ccaacccact 1500
aatggtgttg gttaccaacc atacagagta gtagtacttt cttttgaact tctacatgca 1560
ccagcaactg tttgtggacc taaaaagtct actaatttgg ttaaaaacaa atgtgtcaat 1620
ttcaacttca atggtttaac aggcacaggt gttcttactg agtctaacaa aaagtttctg 1680
cctttccaac aatttggcag agacattgct gacactactg atgctgtccg tgatccacag 1740
acacttgaga ttcttgacat tacaccatgt tcttttggtg gtgtcagtgt tataacacca 1800
ggaacaaata cttctaacca ggttgctgtt ctttatcagg atgttaactg cacagaagtc 1860
cctgttgcta ttcatgcaga tcaacttact cctacttggc gtgtttattc tacaggttct 1920
aatgtttttc aaacacgtgc aggctgttta ataggggctg aacatgtcaa caactcatat 1980
gagtgtgaca tacccattgg tgcaggtata tgcgctagtt atcagactca gactaattct 2040
cctcggcggg cacgtagtgt agctagtcaa tccatcattg cctacactat gtcacttggt 2100
gcagaaaatt cagttgctta ctctaataac tctattgcca tacccacaaa ttttactatt 2160
agtgttacca cagaaattct accagtgtct atgaccaaga catcagtaga ttgtacaatg 2220
tacatttgtg gtgattcaac tgaatgcagc aatcttttgt tgcaatatgg cagtttttgt 2280
acacaattaa accgtgcttt aactggaata gctgttgaac aagacaaaaa cacccaagaa 2340
gtttttgcac aagtcaaaca aatttacaaa acaccaccaa ttaaagattt tggtggtttt 2400
aatttttcac aaatattacc agatccatca aaaccaagca agaggtcatt tattgaagat 2460
ctacttttca acaaagtgac acttgcagat gctggcttca tcaaacaata tggtgattgc 2520
cttggtgata ttgctgctag agacctcatt tgtgcacaaa agtttaacgg ccttactgtt 2580
ttgccacctt tgctcacaga tgaaatgatt gctcaataca cttctgcact gttagcgggt 2640
acaatcactt ctggttggac ctttggtgca ggtgctgcat tacaaatacc atttgctatg 2700
caaatggctt ataggtttaa tggtattgga gttacacaga atgttctcta tgagaaccaa 2760
aaattgattg ccaaccaatt taatagtgct attggcaaaa ttcaagactc actttcttcc 2820
acagcaagtg cacttggaaa acttcaagat gtggtcaacc aaaatgcaca agctttaaac 2880
acgcttgtta aacaacttag ctccaatttt ggtgcaattt caagtgtttt aaatgatatc 2940
ctttcacgtc ttgacaaagt tgaggctgaa gtgcaaattg ataggttgat cacaggcaga 3000
cttcaaagtt tgcagacata tgtgactcaa caattaatta gagctgcaga aatcagagct 3060
tctgctaatc ttgctgctac taaaatgtca gagtgtgtac ttggacaatc aaaaagagtt 3120
gatttttgtg gaaagggcta tcatcttatg tccttccctc agtcagcacc tcatggtgta 3180
gtcttcttgc atgtgactta tgtccctgca caagaaaaga acttcacaac tgctcctgcc 3240
atttgtcatg atggaaaagc acactttcct cgtgaaggtg tctttgtttc aaatggcaca 3300
cactggtttg taacacaaag gaatttttat gaaccacaaa tcattactac agacaacaca 3360
tttgtgtctg gtaactgtga tgttgtaata ggaattgtca acaacacagt ttatgatcct 3420
ttgcaacctg aattagactc attcaaggag gagttagata aatattttaa gaatcataca 3480
tcaccagatg ttgatttagg tgacatctct ggcattaatg cttcagttgt aaacattcaa 3540
aaagaaattg accgcctcaa tgaggttgcc aagaatttaa atgaatctct catcgatctc 3600
caagaacttg gaaagtatga gcagtatata aaatggccat ggtacatttg gctaggtttt 3660
atagctggct tgattgccat agtaatggtg acaattatgc tttgctgtat gaccagttgc 3720
tgtagttgtc tcaagggctg ttgttcttgt ggatcctgct gcaaatttga tgaagacgac 3780
tctgagccag tgctcaaagg agtcaaatta cattacacat aa 3822
<210> 15
<211> 254
<212> PRT
<213> SARS冠状病毒
<220>
<223> SARS-CoV-2刺突(S)蛋白
<400> 15
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 Thr Asn Leu Val Lys Asn Lys
195 200 205
Cys Val Asn Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr
210 215 220
Glu Ser Asn Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile
225 230 235 240
Ala Asp Thr Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu
245 250
<210> 16
<211> 609
<212> PRT
<213> SARS冠状病毒
<220>
<223> SARS-CoV-2刺突（S）蛋白
<400> 16
Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala Ser Tyr Gln Thr Gln
1 5 10 15
Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala Ser Gln Ser Ile Ile
20 25 30
Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser Val Ala Tyr Ser Asn
35 40 45
Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile Ser Val Thr Thr Glu
50 55 60
Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val Asp Cys Thr Met Tyr
65 70 75 80
Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu Leu Leu Gln Tyr Gly
85 90 95
Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr Gly Ile Ala Val Glu
100 105 110
Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln Val Lys Gln Ile Tyr
115 120 125
Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe Asn Phe Ser Gln Ile
130 135 140
Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser Phe Ile Glu Asp Leu
145 150 155 160
Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly Phe Ile Lys Gln Tyr
165 170 175
Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp Leu Ile Cys Ala Gln
180 185 190
Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr Asp Glu Met
195 200 205
Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly Thr Ile Thr Ser Gly
210 215 220
Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe Ala Met Gln
225 230 235 240
Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn Val Leu Tyr
245 250 255
Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn Ser Ala Ile Gly Lys
260 265 270
Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala Leu Gly Lys Leu Gln
275 280 285
Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu Val Lys Gln
290 295 300
Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val Leu Asn Asp Ile Leu
305 310 315 320
Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln Ile Asp Arg Leu Ile
325 330 335
Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln Gln Leu Ile
340 345 350
Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala Thr Lys Met
355 360 365
Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val Asp Phe Cys Gly Lys
370 375 380
Gly Tyr His Leu Met Ser Phe Pro Gln Ser Ala Pro His Gly Val Val
385 390 395 400
Phe Leu His Val Thr Tyr Val Pro Ala Gln Glu Lys Asn Phe Thr Thr
405 410 415
Ala Pro Ala Ile Cys His Asp Gly Lys Ala His Phe Pro Arg Glu Gly
420 425 430
Val Phe Val Ser Asn Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe
435 440 445
Tyr Glu Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn
450 455 460
Cys Asp Val Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu
465 470 475 480
Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys
485 490 495
Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn
500 505 510
Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val
515 520 525
Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly Lys
530 535 540
Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu Gly Phe Ile
545 550 555 560
Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met Leu Cys Cys Met
565 570 575
Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys Ser Cys Gly Ser Cys
580 585 590
Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro Val Leu Lys Gly Val Lys
595 600 605
Leu
<210> 17
<211> 1260
<212> RNA
<213> SARS冠状病毒
<220>
<223>编码SARS-CoV-2核壳（N）蛋白的mRNA
<400> 17
atgtctgata atggacccca aaatcagcga aatgcacccc gcattacgtt tggtggaccc 60
tcagattcaa ctggcagtaa ccagaatgga gaacgcagtg gggcgcgatc aaaacaacgt 120
cggccccaag gtttacccaa taatactgcg tcttggttca ccgctctcac tcaacatggc 180
aaggaagacc ttaaattccc tcgaggacaa ggcgttccaa ttaacaccaa tagcagtcca 240
gatgaccaaa ttggctacta ccgaagagct accagacgaa ttcgtggtgg tgacggtaaa 300
atgaaagatc tcagtccaag atggtatttc tactacctag gaactgggcc agaagctgga 360
cttccctatg gtgctaacaa agacggcatc atatgggttg caactgaggg agccttgaat 420
acaccaaaag atcacattgg cacccgcaat cctgctaaca atgctgcaat cgtgctacaa 480
cttcctcaag gaacaacatt gccaaaaggc ttctacgcag aagggagcag aggcggcagt 540
caagcctctt ctcgttcctc atcacgtagt cgcaacagtt caagaaattc aactccaggc 600
agcagtaggg gaacttctcc tgctagaatg gctggcaatg gcggtgatgc tgctcttgct 660
ttgctgctgc ttgacagatt gaaccagctt gagagcaaaa tgtctggtaa aggccaacaa 720
caacaaggcc aaactgtcac taagaaatct gctgctgagg cttctaagaa gcctcggcaa 780
aaacgtactg ccactaaagc atacaatgta acacaagctt tcggcagacg tggtccagaa 840
caaacccaag gaaattttgg ggaccaggaa ctaatcagac aaggaactga ttacaaacat 900
tggccgcaaa ttgcacaatt tgcccccagc gcttcagcgt tcttcggaat gtcgcgcatt 960
ggcatggaag tcacaccttc gggaacgtgg ttgacctaca caggtgccat caaattggat 1020
gacaaagatc caaatttcaa agatcaagtc attttgctga ataagcatat tgacgcatac 1080
aaaacattcc caccaacaga gcctaaaaag gacaaaaaga agaaggctga tgaaactcaa 1140
gccttaccgc agagacagaa gaaacagcaa actgtgactc ttcttcctgc tgcagatttg 1200
gatgatttct ccaaacaatt gcaacaatcc atgagcagtg ctgactcaac tcaggcctaa 1260
<210> 18
<211> 355
<212> PRT
<213> SARS冠状病毒
<220>
<223> SARS-CoV-2核壳（N）蛋白
<400> 18
Arg Ile Thr Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn
1 5 10 15
Gly Glu Arg Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu
20 25 30
Pro Asn Asn Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys
35 40 45
Glu Asp Leu Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn
50 55 60
Ser Ser Pro Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg
65 70 75 80
Ile Arg Gly Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr
85 90 95
Phe Tyr Tyr Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala
100 105 110
Asn Lys Asp Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr
115 120 125
Pro Lys Asp His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile
130 135 140
Val Leu Gln Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala
145 150 155 160
Glu Gly Ser Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg
165 170 175
Ser Arg Asn Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Thr
180 185 190
Ser Pro Ala Arg Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu
195 200 205
Leu Leu Leu Asp Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys
210 215 220
Gly Gln Gln Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu
225 230 235 240
Ala Ser Lys Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn
245 250 255
Val Thr Gln Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn
260 265 270
Phe Gly Asp Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp
275 280 285
Pro Gln Ile Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met
290 295 300
Ser Arg Ile Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr
305 310 315 320
Thr Gly Ala Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln
325 330 335
Val Ile Leu Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro
340 345 350
Thr Glu Pro
355
<210> 19
<211> 669
<212> RNA
<213> SARS冠状病毒
<220>
<223> 编码SARS-CoV-2膜（M）蛋白的mRNA
<400> 19
atggcagatt ccaacggtac tattaccgtt gaagagctta aaaagctcct tgaacaatgg 60
aacctagtaa taggtttcct attccttaca tggatttgtc ttctacaatt tgcctatgcc 120
aacaggaata ggtttttgta tataattaag ttaattttcc tctggctgtt atggccagta 180
actttagctt gttttgtgct tgctgctgtt tacagaataa attggatcac cggtggaatt 240
gctatcgcaa tggcttgtct tgtaggcttg atgtggctca gctacttcat tgcttctttc 300
agactgtttg cgcgtacgcg ttccatgtgg tcattcaatc cagaaactaa cattcttctc 360
aacgtgccac tccatggcac tattctgacc agaccgcttc tagaaagtga actcgtaatc 420
ggagctgtga tccttcgtgg acatcttcgt attgctggac accatctagg acgctgtgac 480
atcaaggacc tgcctaaaga aatcactgtt gctacatcac gaacgctttc ttattacaaa 540
ttgggagctt cgcagcgtgt agcaggtgac tcaggttttg ctgcatacag tcgctacagg 600
attggcaact ataaattaaa cacagaccat tccagtagca gtgacaatat tgctttgctt 660
gtacagtaa 669
<210> 20
<211> 218
<212> PRT
<213> SARS冠状病毒
<220>
<223> SARS-CoV-2膜（M）蛋白
<400> 20
Ser Asn Gly Thr Ile Thr Val Glu Glu Leu Lys Lys Leu Leu Glu Gln
1 5 10 15
Trp Asn Leu Val Ile Gly Phe Leu Phe Leu Thr Trp Ile Cys Leu Leu
20 25 30
Gln Phe Ala Tyr Ala Asn Arg Asn Arg Phe Leu Tyr Ile Ile Lys Leu
35 40 45
Ile Phe Leu Trp Leu Leu Trp Pro Val Thr Leu Ala Cys Phe Val Leu
50 55 60
Ala Ala Val Tyr Arg Ile Asn Trp Ile Thr Gly Gly Ile Ala Ile Ala
65 70 75 80
Met Ala Cys Leu Val Gly Leu Met Trp Leu Ser Tyr Phe Ile Ala Ser
85 90 95
Phe Arg Leu Phe Ala Arg Thr Arg Ser Met Trp Ser Phe Asn Pro Glu
100 105 110
Thr Asn Ile Leu Leu Asn Val Pro Leu His Gly Thr Ile Leu Thr Arg
115 120 125
Pro Leu Leu Glu Ser Glu Leu Val Ile Gly Ala Val Ile Leu Arg Gly
130 135 140
His Leu Arg Ile Ala Gly His His Leu Gly Arg Cys Asp Ile Lys Asp
145 150 155 160
Leu Pro Lys Glu Ile Thr Val Ala Thr Ser Arg Thr Leu Ser Tyr Tyr
165 170 175
Lys Leu Gly Ala Ser Gln Arg Val Ala Gly Asp Ser Gly Phe Ala Ala
180 185 190
Tyr Ser Arg Tyr Arg Ile Gly Asn Tyr Lys Leu Asn Thr Asp His Ser
195 200 205
Ser Ser Ser Asp Asn Ile Ala Leu Leu Val
210 215
<210> 21
<211> 195
<212> PRT
<213> A型流感病毒
<220>
<223> A型流感病毒H5N1的血凝素（HA）蛋白
<400> 21
Asn Asp Gln Gly Ser Gly Tyr Ala Ala Asp Lys Glu Ser Thr Gln Lys
1 5 10 15
Ala Phe Asn Gly Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met
20 25 30
Asn Thr Gln Phe Glu Ala Val Gly Lys Glu Phe Ser Asn Leu Glu Lys
35 40 45
Arg Leu Glu Asn Leu Asn Lys Lys Met Glu Asp Gly Phe Leu Asp Val
50 55 60
Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu Met Glu Asn Glu Arg Thr
65 70 75 80
Leu Asp Phe His Asp Ser Asn Val Lys Asn Leu Tyr Asp Lys Val Arg
85 90 95
Met Gln Leu Arg Asp Asn Val Lys Glu Leu Gly Asn Gly Cys Phe Glu
100 105 110
Phe Tyr His Lys Cys Asp Asn Glu Cys Met Asp Ser Val Lys Asn Gly
115 120 125
Thr Tyr Asp Tyr Pro Lys Tyr Glu Glu Glu Ser Lys Leu Asn Arg Asn
130 135 140
Glu Ile Lys Gly Val Lys Leu Ser Ser Met Gly Val Tyr Gln Ile Leu
145 150 155 160
Ala Ile Tyr Ala Thr Val Ala Gly Ser Leu Ser Leu Ala Ile Met Met
165 170 175
Ala Gly Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg
180 185 190
Ile Cys Ile
195
<210> 22
<211> 538
<212> PRT
<213> A型流感病毒
<220>
<223> A型流感病毒H3N2的血凝素（HA）蛋白
<400> 22
Leu Cys Leu Gly His His Ala Val Pro Asn Gly Thr Ile Val Lys Thr
1 5 10 15
Ile Thr Asn Asp Gln Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gln
20 25 30
Ser Ser Ser Thr Gly Gly Ile Cys Asp Ser Pro His Gln Ile Leu Asp
35 40 45
Gly Glu Asn Cys Thr Leu Ile Asp Ala Leu Leu Gly Asp Pro Gln Cys
50 55 60
Asp Gly Phe Gln Asn Lys Lys Trp Asp Leu Phe Val Glu Arg Ser Lys
65 70 75 80
Ala Tyr Ser Asn Cys Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser Leu
85 90 95
Arg Ser Leu Val Ala Ser Ser Gly Thr Leu Glu Phe Asn Asn Glu Ser
100 105 110
Phe Asn Trp Thr Gly Val Thr Gln Asn Gly Thr Ser Ser Ala Cys Lys
115 120 125
Arg Arg Ser Asn Asn Ser Phe Phe Ser Arg Leu Asn Trp Leu Thr His
130 135 140
Leu Lys Phe Lys Tyr Pro Ala Leu Asn Val Thr Met Pro Asn Asn Glu
145 150 155 160
Lys Phe Asp Lys Leu Tyr Ile Trp Gly Val His His Pro Gly Thr Asp
165 170 175
Asn Asp Gln Ile Ser Leu Tyr Ala Gln Ala Ser Gly Arg Ile Thr Val
180 185 190
Ser Thr Lys Arg Ser Gln Gln Thr Val Ile Pro Ser Ile Gly Ser Arg
195 200 205
Pro Arg Ile Arg Asp Val Pro Ser Arg Ile Ser Ile Tyr Trp Thr Ile
210 215 220
Val Lys Pro Gly Asp Ile Leu Leu Ile Asn Ser Thr Gly Asn Leu Ile
225 230 235 240
Ala Pro Arg Gly Tyr Phe Lys Ile Arg Ser Gly Lys Ser Ser Ile Met
245 250 255
Arg Ser Asp Ala Pro Ile Gly Lys Cys Asn Ser Glu Cys Ile Thr Pro
260 265 270
Asn Gly Ser Ile Pro Asn Asp Lys Pro Phe Gln Asn Val Asn Arg Ile
275 280 285
Thr Tyr Gly Ala Cys Pro Arg Tyr Val Lys Gln Asn Thr Leu Lys Leu
290 295 300
Ala Thr Gly Met Arg Asn Val Pro Glu Lys Gln Thr Arg Gly Ile Phe
305 310 315 320
Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly Met Val Asp
325 330 335
Gly Trp Tyr Gly Phe Arg His Gln Asn Ser Glu Gly Thr Gly Gln Ala
340 345 350
Ala Asp Leu Lys Ser Thr Gln Ala Ala Ile Asn Gln Ile Asn Gly Lys
355 360 365
Leu Asn Arg Leu Ile Gly Lys Thr Asn Glu Lys Phe His Gln Ile Glu
370 375 380
Lys Glu Phe Ser Glu Val Glu Gly Arg Ile Gln Asp Leu Glu Lys Tyr
385 390 395 400
Val Glu Asp Thr Lys Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu
405 410 415
Val Ala Leu Glu Asn Gln His Thr Ile Asp Leu Thr Asp Ser Glu Met
420 425 430
Asn Lys Leu Phe Glu Arg Thr Lys Lys Gln Leu Arg Glu Asn Ala Glu
435 440 445
Asp Met Gly Asn Gly Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala
450 455 460
Cys Ile Gly Ser Ile Arg Asn Gly Thr Tyr Asp His Asp Val Tyr Arg
465 470 475 480
Asp Glu Ala Leu Asn Asn Arg Phe Gln Ile Lys Gly Val Glu Leu Lys
485 490 495
Ser Gly Tyr Lys Asp Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cys
500 505 510
Phe Leu Leu Cys Val Ala Leu Leu Gly Phe Ile Met Trp Ala Cys Gln
515 520 525
Lys Gly Asn Ile Arg Cys Asn Ile Cys Ile
530 535
<210> 23
<211> 538
<212> PRT
<213> A型流感病毒
<220>
<223> A型流感病毒H1N1的血凝素（HA）蛋白
<400> 23
Leu Cys Leu Gly His His Ala Val Pro Asn Gly Thr Ile Val Lys Thr
1 5 10 15
Ile Thr Asn Asp Gln Ile Glu Val Thr Asn Ala Thr Glu Leu Val Gln
20 25 30
Ser Ser Ser Thr Gly Gly Ile Cys Asp Ser Pro His Gln Ile Leu Asp
35 40 45
Gly Glu Asn Cys Thr Leu Ile Asp Ala Leu Leu Gly Asp Pro Gln Cys
50 55 60
Asp Gly Phe Gln Asn Lys Lys Trp Asp Leu Phe Val Glu Arg Ser Lys
65 70 75 80
Ala Tyr Ser Asn Cys Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser Leu
85 90 95
Arg Ser Leu Val Ala Ser Ser Gly Thr Leu Glu Phe Asn Asn Glu Ser
100 105 110
Phe Asn Trp Thr Gly Val Thr Gln Asn Gly Thr Ser Ser Ala Cys Lys
115 120 125
Arg Arg Ser Asn Asn Ser Phe Phe Ser Arg Leu Asn Trp Leu Thr His
130 135 140
Leu Lys Phe Lys Tyr Pro Ala Leu Asn Val Thr Met Pro Asn Asn Glu
145 150 155 160
Lys Phe Asp Lys Leu Tyr Ile Trp Gly Val His His Pro Gly Thr Asp
165 170 175
Asn Asp Gln Ile Ser Leu Tyr Ala Gln Ala Ser Gly Arg Ile Thr Val
180 185 190
Ser Thr Lys Arg Ser Gln Gln Thr Val Ile Pro Ser Ile Gly Ser Arg
195 200 205
Pro Arg Ile Arg Asp Val Pro Ser Arg Ile Ser Ile Tyr Trp Thr Ile
210 215 220
Val Lys Pro Gly Asp Ile Leu Leu Ile Asn Ser Thr Gly Asn Leu Ile
225 230 235 240
Ala Pro Arg Gly Tyr Phe Lys Ile Arg Ser Gly Lys Ser Ser Ile Met
245 250 255
Arg Ser Asp Ala Pro Ile Gly Lys Cys Asn Ser Glu Cys Ile Thr Pro
260 265 270
Asn Gly Ser Ile Pro Asn Asp Lys Pro Phe Gln Asn Val Asn Arg Ile
275 280 285
Thr Tyr Gly Ala Cys Pro Arg Tyr Val Lys Gln Asn Thr Leu Lys Leu
290 295 300
Ala Thr Gly Met Arg Asn Val Pro Glu Lys Gln Thr Arg Gly Ile Phe
305 310 315 320
Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly Met Val Asp
325 330 335
Gly Trp Tyr Gly Phe Arg His Gln Asn Ser Glu Gly Thr Gly Gln Ala
340 345 350
Ala Asp Leu Lys Ser Thr Gln Ala Ala Ile Asn Gln Ile Asn Gly Lys
355 360 365
Leu Asn Arg Leu Ile Gly Lys Thr Asn Glu Lys Phe His Gln Ile Glu
370 375 380
Lys Glu Phe Ser Glu Val Glu Gly Arg Ile Gln Asp Leu Glu Lys Tyr
385 390 395 400
Val Glu Asp Thr Lys Ile Asp Leu Trp Ser Tyr Asn Ala Glu Leu Leu
405 410 415
Val Ala Leu Glu Asn Gln His Thr Ile Asp Leu Thr Asp Ser Glu Met
420 425 430
Asn Lys Leu Phe Glu Arg Thr Lys Lys Gln Leu Arg Glu Asn Ala Glu
435 440 445
Asp Met Gly Asn Gly Cys Phe Lys Ile Tyr His Lys Cys Asp Asn Ala
450 455 460
Cys Ile Gly Ser Ile Arg Asn Gly Thr Tyr Asp His Asp Val Tyr Arg
465 470 475 480
Asp Glu Ala Leu Asn Asn Arg Phe Gln Ile Lys Gly Val Glu Leu Lys
485 490 495
Ser Gly Tyr Lys Asp Trp Ile Leu Trp Ile Ser Phe Ala Ile Ser Cys
500 505 510
Phe Leu Leu Cys Val Ala Leu Leu Gly Phe Ile Met Trp Ala Cys Gln
515 520 525
Lys Gly Asn Ile Arg Cys Asn Ile Cys Ile
530 535
<210> 24
<211> 540
<212> PRT
<213> A型流感病毒
<220>
<223> A型流感病毒H7N9的血凝素（HA）蛋白
<400> 24
Ile Cys Leu Gly His His Ala Val Ser Asn Gly Thr Lys Val Asn Thr
1 5 10 15
Leu Thr Glu Arg Gly Val Glu Val Val Asn Ala Thr Glu Thr Val Glu
20 25 30
Arg Thr Asn Ile Pro Arg Ile Cys Ser Lys Gly Lys Arg Thr Val Asp
35 40 45
Leu Gly Gln Cys Gly Leu Leu Gly Thr Ile Thr Gly Pro Pro Gln Cys
50 55 60
Asp Gln Phe Leu Glu Phe Ser Ala Asp Leu Ile Ile Glu Arg Arg Glu
65 70 75 80
Gly Ser Asp Val Cys Tyr Pro Gly Lys Phe Val Asn Glu Glu Ala Leu
85 90 95
Arg Gln Ile Leu Arg Glu Ser Gly Gly Ile Asp Lys Glu Ala Met Gly
100 105 110
Phe Thr Tyr Ser Gly Ile Arg Thr Asn Gly Ala Thr Ser Ala Cys Arg
115 120 125
Arg Ser Gly Ser Ser Phe Tyr Ala Glu Met Lys Trp Leu Leu Ser Asn
130 135 140
Thr Asp Asn Ala Ala Phe Pro Gln Met Thr Lys Ser Tyr Lys Asn Thr
145 150 155 160
Arg Lys Ser Pro Ala Leu Ile Val Trp Gly Ile His His Ser Val Ser
165 170 175
Thr Ala Glu Gln Thr Lys Leu Tyr Gly Ser Gly Asn Lys Leu Val Thr
180 185 190
Val Gly Ser Ser Asn Tyr Gln Gln Ser Phe Val Pro Ser Pro Gly Ala
195 200 205
Arg Pro Gln Val Asn Gly Leu Ser Gly Arg Ile Asp Phe His Trp Leu
210 215 220
Met Leu Asn Pro Asn Asp Thr Val Thr Phe Ser Phe Asn Gly Ala Phe
225 230 235 240
Ile Ala Pro Asp Arg Ala Ser Phe Leu Arg Gly Lys Ser Met Gly Ile
245 250 255
Gln Ser Gly Val Gln Val Asp Ala Asn Cys Glu Gly Asp Cys Tyr His
260 265 270
Ser Gly Gly Thr Ile Ile Ser Asn Leu Pro Phe Gln Asn Ile Asp Ser
275 280 285
Arg Ala Val Gly Lys Cys Pro Arg Tyr Val Lys Gln Arg Ser Leu Leu
290 295 300
Leu Ala Thr Gly Met Lys Asn Val Pro Glu Ile Pro Lys Gly Arg Gly
305 310 315 320
Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly Leu
325 330 335
Ile Asp Gly Trp Tyr Gly Phe Arg His Gln Asn Ala Gln Gly Glu Gly
340 345 350
Thr Ala Ala Asp Tyr Lys Ser Thr Gln Ser Ala Ile Asp Gln Ile Thr
355 360 365
Gly Lys Leu Asn Arg Leu Ile Glu Lys Thr Asn Gln Gln Phe Glu Leu
370 375 380
Ile Asp Asn Glu Phe Asn Glu Val Glu Lys Gln Ile Gly Asn Val Ile
385 390 395 400
Asn Trp Thr Arg Asp Ser Ile Thr Glu Val Trp Ser Tyr Asn Ala Glu
405 410 415
Leu Leu Val Ala Met Glu Asn Gln His Thr Ile Asp Leu Ala Asp Ser
420 425 430
Glu Met Asp Lys Leu Tyr Glu Arg Val Lys Arg Gln Leu Arg Glu Asn
435 440 445
Ala Glu Glu Asp Gly Thr Gly Cys Phe Glu Ile Phe His Lys Cys Asp
450 455 460
Asp Asp Cys Met Ala Ser Ile Arg Asn Asn Thr Tyr Asp His Ser Lys
465 470 475 480
Tyr Arg Glu Glu Ala Met Gln Asn Arg Ile Gln Ile Asp Pro Val Lys
485 490 495
Leu Ser Ser Gly Tyr Lys Asp Val Ile Leu Trp Phe Ser Phe Gly Ala
500 505 510
Ser Cys Phe Ile Leu Leu Ala Ile Val Met Gly Leu Val Phe Ile Cys
515 520 525
Val Lys Asn Gly Asn Met Arg Cys Thr Ile Cys Ile
530 535 540
<210> 25
<211> 540
<212> PRT
<213> A型流感病毒
<220>
<223> A型流感病毒H1N1的血凝素（HA）蛋白
<400> 25
Ile Cys Ile Gly His Gln Ser Thr Asn Ser Thr Glu Thr Val Asp Thr
1 5 10 15
Leu Thr Glu Thr Asn Val Pro Val Thr His Ala Lys Glu Leu Leu His
20 25 30
Thr Glu His Asn Gly Met Leu Cys Ala Thr Ser Leu Gly His Pro Leu
35 40 45
Ile Leu Asp Thr Cys Thr Ile Glu Gly Leu Val Tyr Gly Asn Pro Ser
50 55 60
Cys Asp Leu Leu Leu Gly Gly Arg Glu Trp Ser Tyr Ile Val Glu Arg
65 70 75 80
Ser Ser Ala Val Asn Gly Thr Cys Tyr Pro Gly Asn Val Glu Asn Leu
85 90 95
Glu Glu Leu Arg Thr Leu Phe Ser Ser Ala Ser Ser Tyr Gln Arg Ile
100 105 110
Gln Ile Phe Pro Asp Thr Thr Trp Asn Val Thr Tyr Thr Gly Thr Ser
115 120 125
Arg Ala Cys Ser Gly Ser Phe Tyr Arg Ser Met Arg Trp Leu Thr Gln
130 135 140
Lys Ser Gly Phe Tyr Pro Val Gln Asp Ala Gln Tyr Thr Asn Asn Arg
145 150 155 160
Gly Lys Ser Ile Leu Phe Val Trp Gly Ile His His Pro Pro Thr Tyr
165 170 175
Thr Glu Gln Thr Asn Leu Tyr Ile Arg Asn Asp Thr Thr Thr Ser Val
180 185 190
Thr Thr Glu Asp Leu Asn Arg Thr Phe Lys Pro Val Ile Gly Pro Arg
195 200 205
Pro Leu Val Asn Gly Leu Gln Gly Arg Ile Asp Tyr Tyr Trp Ser Val
210 215 220
Leu Lys Pro Gly Gln Thr Leu Arg Val Arg Ser Asn Gly Asn Leu Ile
225 230 235 240
Ala Pro Trp Tyr Gly His Val Leu Ser Gly Gly Ser His Gly Arg Ile
245 250 255
Leu Lys Thr Asp Leu Lys Gly Gly Asn Cys Val Val Gln Cys Gln Thr
260 265 270
Glu Lys Gly Gly Leu Asn Ser Thr Leu Pro Phe His Asn Ile Ser Lys
275 280 285
Tyr Ala Phe Gly Thr Cys Pro Lys Tyr Val Arg Val Asn Ser Leu Lys
290 295 300
Leu Ala Val Gly Leu Arg Asn Val Pro Ala Arg Ser Ser Arg Gly Leu
305 310 315 320
Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly Trp Pro Gly Leu Val
325 330 335
Ala Gly Trp Tyr Gly Phe Gln His Ser Asn Asp Gln Gly Val Gly Met
340 345 350
Ala Ala Asp Arg Asp Ser Thr Gln Lys Ala Ile Asp Lys Ile Thr Ser
355 360 365
Lys Val Asn Asn Ile Val Asp Lys Met Asn Lys Gln Tyr Glu Ile Ile
370 375 380
Asp His Glu Phe Ser Glu Val Glu Thr Arg Leu Asn Met Ile Asn Asn
385 390 395 400
Lys Ile Asp Asp Gln Ile Gln Asp Val Trp Ala Tyr Asn Ala Glu Leu
405 410 415
Leu Val Leu Leu Glu Asn Gln Lys Thr Leu Asp Glu His Asp Ala Asn
420 425 430
Val Asn Asn Leu Tyr Asn Lys Val Lys Arg Ala Leu Gly Ser Asn Ala
435 440 445
Met Glu Asp Gly Lys Gly Cys Phe Glu Leu Tyr His Lys Cys Asp Asp
450 455 460
Gln Cys Met Glu Thr Ile Arg Asn Gly Thr Tyr Asn Arg Arg Lys Tyr
465 470 475 480
Arg Glu Glu Ser Arg Leu Glu Arg Gln Lys Ile Glu Gly Val Lys Leu
485 490 495
Glu Ser Glu Gly Thr Tyr Lys Ile Leu Thr Ile Tyr Ser Thr Val Ala
500 505 510
Ser Ser Leu Val Leu Ala Met Gly Phe Ala Ala Phe Leu Phe Trp Ala
515 520 525
Met Ser Asn Gly Ser Cys Arg Cys Asn Ile Cys Ile
530 535 540
<210> 26
<211> 545
<212> PRT
<213> A型流感病毒
<220>
<223> A型流感病毒H2N2的血凝素（HA）蛋白
<400> 26
Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Lys Val Asp Thr
1 5 10 15
Ile Leu Glu Arg Asn Val Thr Val Thr His Ala Lys Asp Ile Leu Glu
20 25 30
Lys Thr His Asn Gly Lys Leu Cys Lys Leu Asn Gly Ile Pro Pro Leu
35 40 45
Glu Leu Gly Asp Cys Ser Ile Ala Gly Trp Leu Leu Gly Asn Pro Glu
50 55 60
Cys Asp Arg Leu Leu Ser Val Pro Glu Trp Ser Tyr Ile Met Glu Lys
65 70 75 80
Glu Asn Pro Arg Tyr Ser Leu Cys Tyr Pro Gly Ser Phe Asn Asp Tyr
85 90 95
Glu Glu Leu Lys His Leu Leu Ser Ser Val Lys His Phe Glu Lys Val
100 105 110
Lys Ile Leu Pro Lys Asp Arg Trp Thr Gln His Thr Thr Thr Gly Gly
115 120 125
Ser Trp Ala Cys Ala Val Ser Gly Lys Pro Ser Phe Phe Arg Asn Met
130 135 140
Val Trp Leu Thr Arg Lys Gly Ser Asn Tyr Pro Val Ala Lys Gly Ser
145 150 155 160
Tyr Asn Asn Thr Ser Gly Glu Gln Met Leu Ile Ile Trp Gly Val His
165 170 175
His Pro Asn Asp Glu Ala Glu Gln Arg Ala Leu Tyr Gln Asn Val Gly
180 185 190
Thr Tyr Val Ser Val Ala Thr Ser Thr Leu Tyr Lys Arg Ser Ile Pro
195 200 205
Glu Ile Ala Ala Arg Pro Lys Val Asn Gly Leu Gly Arg Arg Met Glu
210 215 220
Phe Ser Trp Thr Leu Leu Asp Met Trp Asp Thr Ile Asn Phe Glu Ser
225 230 235 240
Thr Gly Asn Leu Val Ala Pro Glu Tyr Gly Phe Lys Ile Ser Lys Arg
245 250 255
Gly Ser Ser Gly Ile Met Lys Thr Glu Gly Thr Leu Glu Asn Cys Glu
260 265 270
Thr Lys Cys Gln Thr Pro Leu Gly Ala Ile Asn Thr Thr Leu Pro Phe
275 280 285
His Asn Val His Pro Leu Thr Ile Gly Glu Cys Pro Lys Tyr Val Lys
290 295 300
Ser Glu Lys Leu Val Leu Ala Thr Gly Leu Arg Asn Val Pro Gln Ile
305 310 315 320
Glu Ser Arg Gly Leu Phe Gly Ala Ile Ala Gly Phe Ile Glu Gly Gly
325 330 335
Trp Gln Gly Met Val Asp Gly Trp Tyr Gly Tyr His His Ser Asn Asp
340 345 350
Gln Gly Ser Gly Tyr Ala Ala Asp Lys Glu Ser Thr Gln Lys Ala Phe
355 360 365
Asn Gly Ile Thr Asn Lys Val Asn Ser Val Ile Glu Lys Met Asn Thr
370 375 380
Gln Phe Glu Ala Val Gly Lys Glu Phe Ser Asn Leu Glu Lys Arg Leu
385 390 395 400
Glu Asn Leu Asn Lys Lys Met Glu Asp Gly Phe Leu Asp Val Trp Thr
405 410 415
Tyr Asn Ala Glu Leu Leu Val Leu Met Glu Asn Glu Arg Thr Leu Asp
420 425 430
Phe His Asp Ser Asn Val Lys Asn Leu Tyr Asp Lys Val Arg Met Gln
435 440 445
Leu Arg Asp Asn Val Lys Glu Leu Gly Asn Gly Cys Phe Glu Phe Tyr
450 455 460
His Lys Cys Asp Asn Glu Cys Met Asp Ser Val Lys Asn Gly Thr Tyr
465 470 475 480
Asp Tyr Pro Lys Tyr Glu Glu Glu Ser Lys Leu Asn Arg Asn Glu Ile
485 490 495
Lys Gly Val Lys Leu Ser Ser Met Gly Val Tyr Gln Ile Leu Ala Ile
500 505 510
Tyr Ala Thr Val Ala Gly Ser Leu Ser Leu Ala Ile Met Met Ala Gly
515 520 525
Ile Ser Phe Trp Met Cys Ser Asn Gly Ser Leu Gln Cys Arg Ile Cys
530 535 540
Ile
545
<210> 27
<211> 567
<212> PRT
<213> B型流感病毒
<220>
<223> 血凝素（HA）蛋白
<400> 27
Ile Cys Thr Gly Ile Thr Ser Ser Asn Ser Pro His Val Val Lys Thr
1 5 10 15
Ala Thr Gln Gly Glu Val Asn Val Thr Gly Val Ile Pro Leu Thr Thr
20 25 30
Thr Pro Thr Lys Ser His Phe Ala Asn Leu Lys Gly Thr Gln Thr Arg
35 40 45
Gly Lys Leu Cys Pro Asn Cys Phe Asn Cys Thr Asp Leu Asp Val Ala
50 55 60
Leu Gly Arg Pro Lys Cys Met Gly Asn Thr Pro Ser Ala Lys Val Ser
65 70 75 80
Ile Leu His Glu Val Lys Pro Ala Thr Ser Gly Cys Phe Pro Ile Met
85 90 95
His Asp Arg Thr Lys Ile Arg Gln Leu Pro Asn Leu Leu Arg Gly Tyr
100 105 110
Glu Asn Ile Arg Leu Ser Thr Ser Asn Val Ile Asn Thr Glu Thr Ala
115 120 125
Pro Gly Gly Pro Tyr Lys Val Gly Thr Ser Gly Ser Cys Pro Asn Val
130 135 140
Ala Asn Gly Asn Gly Phe Phe Asn Thr Met Ala Trp Val Ile Pro Lys
145 150 155 160
Asp Asn Asn Lys Thr Ala Ile Asn Pro Val Thr Val Glu Val Pro Tyr
165 170 175
Ile Cys Ser Glu Gly Glu Asp Gln Ile Thr Val Trp Gly Phe His Ser
180 185 190
Asp Asp Lys Thr Gln Met Glu Arg Leu Tyr Gly Asp Ser Asn Pro Gln
195 200 205
Lys Phe Thr Ser Ser Ala Asn Gly Val Thr Thr His Tyr Val Ser Gln
210 215 220
Ile Gly Gly Phe Pro Asn Gln Thr Glu Asp Glu Gly Leu Lys Gln Ser
225 230 235 240
Gly Arg Ile Val Val Asp Tyr Met Val Gln Lys Pro Gly Lys Thr Gly
245 250 255
Thr Ile Val Tyr Gln Arg Gly Ile Leu Leu Pro Gln Lys Val Trp Cys
260 265 270
Ala Ser Gly Arg Ser Lys Val Ile Lys Gly Ser Leu Pro Leu Ile Gly
275 280 285
Glu Ala Asp Cys Leu His Glu Lys Tyr Gly Gly Leu Asn Lys Ser Lys
290 295 300
Pro Tyr Tyr Thr Gly Glu His Ala Lys Ala Ile Gly Asn Cys Pro Ile
305 310 315 320
Trp Val Lys Thr Pro Leu Lys Leu Ala Asn Gly Thr Lys Tyr Arg Pro
325 330 335
Pro Ala Lys Leu Leu Lys Glu Arg Gly Phe Phe Gly Ala Ile Ala Gly
340 345 350
Phe Leu Glu Gly Gly Trp Glu Gly Met Ile Ala Gly Trp His Gly Tyr
355 360 365
Thr Ser His Gly Ala His Gly Val Ala Val Ala Ala Asp Leu Lys Ser
370 375 380
Thr Gln Glu Ala Ile Asn Lys Ile Thr Lys Asn Leu Asn Tyr Leu Ser
385 390 395 400
Glu Leu Glu Val Lys Asn Leu Gln Arg Leu Ser Gly Ala Met Asn Glu
405 410 415
Leu His Asp Glu Ile Leu Glu Leu Asp Glu Lys Val Asp Asp Leu Arg
420 425 430
Ala Asp Thr Ile Ser Ser Gln Ile Glu Leu Ala Val Leu Leu Ser Asn
435 440 445
Glu Gly Ile Ile Asn Ser Glu Asp Glu His Leu Leu Ala Leu Glu Arg
450 455 460
Lys Leu Lys Lys Met Leu Gly Pro Ser Ala Val Glu Ile Gly Asn Gly
465 470 475 480
Cys Phe Glu Thr Lys His Lys Cys Asn Gln Thr Cys Leu Asp Arg Ile
485 490 495
Ala Ala Gly Thr Phe Asn Ala Gly Asp Phe Ser Leu Pro Thr Phe Asp
500 505 510
Ser Leu Asn Ile Thr Ala Ala Ser Leu Asn Asp Asp Gly Leu Asp Asn
515 520 525
His Thr Ile Leu Leu Tyr Tyr Ser Thr Ala Ala Ser Ser Leu Ala Val
530 535 540
Thr Leu Met Ile Ala Ile Phe Ile Val Tyr Met Val Ser Arg Asp Asn
545 550 555 560
Val Ser Cys Ser Ile Cys Leu
565
<210> 28
<211> 381
<212> PRT
<213> A型流感病毒
<220>
<223> A型流感病毒H5N1的神经氨酸酶（NA）蛋白
<400> 28
Leu Ala Gly Asn Ser Ser Leu Cys Pro Ile Ser Gly Trp Ala Val His
1 5 10 15
Ser Lys Asp Asn Gly Ile Arg Ile Gly Ser Lys Gly Asp Val Phe Val
20 25 30
Ile Arg Glu Pro Phe Ile Ser Cys Ser His Leu Glu Cys Arg Thr Phe
35 40 45
Phe Leu Thr Gln Gly Ala Leu Leu Asn Asp Lys His Ser Asn Gly Thr
50 55 60
Val Lys Asp Arg Ser Pro His Arg Thr Leu Met Ser Cys Pro Val Gly
65 70 75 80
Glu Ala Pro Ser Pro Tyr Asn Ser Arg Phe Glu Ser Val Ala Trp Ser
85 90 95
Ala Ser Ala Cys His Asp Gly Thr Ser Trp Leu Thr Ile Gly Ile Ser
100 105 110
Gly Pro Asp Asn Gly Ala Val Ala Val Leu Lys Tyr Asn Gly Ile Ile
115 120 125
Thr Asp Thr Ile Lys Ser Trp Arg Asn Asn Ile Leu Arg Thr Gln Glu
130 135 140
Ser Glu Cys Ala Cys Val Asn Gly Ser Cys Phe Thr Val Met Thr Asp
145 150 155 160
Gly Pro Ser Asn Gly Gln Ala Ser Tyr Lys Ile Phe Lys Met Glu Lys
165 170 175
Gly Lys Val Val Lys Ser Val Glu Leu Asn Ala Pro Asn Tyr His Tyr
180 185 190
Glu Glu Cys Ser Cys Tyr Pro Asp Ala Gly Glu Ile Thr Cys Val Cys
195 200 205
Arg Asp Asn Trp His Gly Ser Asn Arg Pro Trp Val Ser Phe Asn Gln
210 215 220
Asn Leu Glu Tyr Gln Ile Gly Tyr Ile Cys Ser Gly Val Phe Gly Asp
225 230 235 240
Asn Pro Arg Pro Asn Asp Gly Thr Gly Ser Cys Gly Pro Val Ser Pro
245 250 255
Asn Gly Ala Tyr Gly Val Lys Gly Phe Ser Phe Lys Tyr Gly Asn Gly
260 265 270
Val Trp Ile Gly Arg Thr Lys Ser Thr Asn Ser Arg Ser Gly Phe Glu
275 280 285
Met Ile Trp Asp Pro Asn Gly Trp Thr Gly Thr Asp Ser Ser Phe Ser
290 295 300
Val Lys Gln Asp Ile Val Ala Ile Thr Asp Trp Ser Gly Tyr Ser Gly
305 310 315 320
Ser Phe Val Gln His Pro Glu Leu Thr Gly Leu Asp Cys Ile Arg Pro
325 330 335
Cys Phe Trp Val Glu Leu Ile Arg Gly Arg Pro Lys Glu Ser Thr Ile
340 345 350
Trp Thr Ser Gly Ser Ser Ile Ser Phe Cys Gly Val Asn Ser Asp Thr
355 360 365
Val Gly Trp Ser Trp Pro Asp Asp Ala Glu Leu Pro Phe
370 375 380
<210> 29
<211> 383
<212> PRT
<213> A型流感病毒
<220>
<223> A型流感病毒H1N1的神经氨酸酶（NA）蛋白
<400> 29
Val Ile Leu Thr Gly Asn Ser Ser Leu Cys Pro Ile Arg Gly Trp Ala
1 5 10 15
Ile Tyr Ser Lys Asp Asn Ser Ile Arg Ile Gly Ser Lys Gly Asp Val
20 25 30
Phe Val Ile Arg Glu Pro Phe Ile Ser Cys Ser His Leu Glu Cys Arg
35 40 45
Thr Phe Phe Leu Thr Gln Gly Ala Leu Leu Asn Asp Arg His Ser Asn
50 55 60
Gly Thr Val Lys Asp Arg Ser Pro Tyr Arg Ala Leu Met Ser Cys Pro
65 70 75 80
Val Gly Glu Ala Pro Ser Pro Tyr Asn Ser Arg Phe Glu Ser Val Ala
85 90 95
Trp Ser Ala Ser Ala Cys His Asp Gly Met Gly Trp Leu Thr Ile Gly
100 105 110
Ile Ser Gly Pro Asp Asn Gly Ala Val Ala Val Leu Lys Tyr Asn Gly
115 120 125
Ile Ile Thr Glu Thr Ile Lys Ser Trp Arg Lys Lys Ile Leu Arg Thr
130 135 140
Gln Glu Ser Glu Cys Ala Cys Val Asn Gly Ser Cys Phe Thr Ile Met
145 150 155 160
Thr Asp Gly Pro Ser Asp Gly Leu Ala Ser Tyr Lys Ile Phe Lys Ile
165 170 175
Glu Lys Gly Lys Val Thr Lys Ser Ile Glu Leu Asn Ala Pro Asn Ser
180 185 190
His Tyr Glu Glu Cys Ser Cys Tyr Pro Asp Thr Gly Lys Val Met Cys
195 200 205
Val Cys Arg Asp Asn Trp His Gly Ser Asn Arg Pro Trp Val Ser Phe
210 215 220
Asp Gln Asn Leu Asp Tyr Gln Ile Gly Tyr Ile Cys Ser Gly Val Phe
225 230 235 240
Gly Asp Asn Pro Arg Pro Lys Asp Gly Thr Gly Ser Cys Gly Pro Val
245 250 255
Tyr Val Asp Gly Ala Asn Gly Val Lys Gly Phe Ser Tyr Arg Tyr Gly
260 265 270
Asn Gly Val Trp Ile Gly Arg Thr Lys Ser His Ser Ser Arg His Gly
275 280 285
Phe Glu Met Ile Trp Asp Pro Asn Gly Trp Thr Glu Thr Asp Ser Lys
290 295 300
Phe Ser Val Arg Gln Asp Val Val Ala Met Thr Asp Trp Ser Gly Tyr
305 310 315 320
Ser Gly Ser Phe Val Gln His Pro Glu Leu Thr Gly Leu Asp Cys Ile
325 330 335
Arg Pro Cys Phe Trp Val Glu Leu Ile Arg Gly Arg Pro Lys Glu Lys
340 345 350
Thr Ile Trp Thr Ser Ala Ser Ser Ile Ser Phe Cys Gly Val Asn Ser
355 360 365
Asp Thr Val Asp Trp Ser Trp Pro Asp Gly Ala Glu Leu Pro Phe
370 375 380
<210> 30
<211> 384
<212> PRT
<213> A型流感病毒
<220>
<223> A型流感病毒H3N2的神经氨酸酶（NA）蛋白
<400> 30
Glu Tyr Arg Asn Trp Ser Lys Pro Gln Cys Asp Ile Thr Gly Phe Ala
1 5 10 15
Pro Phe Ser Lys Asp Asn Ser Ile Arg Leu Ser Ala Gly Gly Asp Ile
20 25 30
Trp Val Thr Arg Glu Pro Tyr Val Ser Cys Asp Pro Asp Lys Cys Tyr
35 40 45
Gln Phe Ala Leu Gly Gln Gly Thr Thr Leu Asn Asn Val His Ser Asn
50 55 60
Asp Thr Val His Asp Arg Thr Pro Tyr Arg Thr Leu Leu Met Asn Glu
65 70 75 80
Leu Gly Val Pro Phe His Leu Gly Thr Lys Gln Val Cys Ile Ala Trp
85 90 95
Ser Ser Ser Ser Cys His Asp Gly Lys Ala Trp Leu His Val Cys Val
100 105 110
Thr Gly Asp Asp Lys Asn Ala Thr Ala Ser Phe Ile Tyr Asn Gly Arg
115 120 125
Leu Val Asp Ser Ile Val Ser Trp Ser Lys Lys Ile Leu Arg Thr Gln
130 135 140
Glu Ser Glu Cys Val Cys Ile Asn Gly Thr Cys Thr Val Val Met Thr
145 150 155 160
Asp Gly Ser Ala Ser Gly Lys Ala Asp Thr Lys Ile Leu Phe Ile Glu
165 170 175
Glu Gly Lys Ile Ile His Thr Ser Thr Leu Ser Gly Ser Ala Gln His
180 185 190
Val Glu Glu Cys Ser Cys Tyr Pro Arg Tyr Pro Gly Val Arg Cys Val
195 200 205
Cys Arg Asp Asn Trp Lys Gly Ser Asn Arg Pro Ile Val Asp Ile Asn
210 215 220
Ile Lys Asp Tyr Ser Ile Val Ser Ser Tyr Val Cys Ser Gly Leu Val
225 230 235 240
Gly Asp Thr Pro Arg Lys Asn Asp Ser Ser Ser Ser Ser His Cys Leu
245 250 255
Asp Pro Asn Asn Glu Glu Gly Gly His Gly Val Lys Gly Trp Ala Phe
260 265 270
Asp Asp Gly Asn Asp Val Trp Met Gly Arg Thr Ile Ser Glu Lys Leu
275 280 285
Arg Ser Gly Tyr Glu Thr Phe Lys Val Ile Glu Gly Trp Ser Lys Pro
290 295 300
Asn Ser Lys Leu Gln Ile Asn Arg Gln Val Ile Val Asp Arg Gly Asn
305 310 315 320
Arg Ser Gly Tyr Ser Gly Ile Phe Ser Val Glu Gly Lys Ser Cys Ile
325 330 335
Asn Arg Cys Phe Tyr Val Glu Leu Ile Arg Gly Arg Lys Glu Glu Thr
340 345 350
Glu Val Leu Trp Thr Ser Asn Ser Ile Val Val Phe Cys Gly Thr Ser
355 360 365
Gly Thr Tyr Gly Thr Gly Ser Trp Pro Asp Gly Ala Asp Ile Asn Leu
370 375 380
<210> 31
<211> 384
<212> PRT
<213> A型流感病毒
<220>
<223> A型流感病毒H7N9的神经氨酸酶（NA）蛋白
<400> 31
Phe Asn Asn Leu Thr Lys Gly Leu Cys Thr Ile Asn Ser Trp His Ile
1 5 10 15
Tyr Gly Lys Asp Asn Ala Val Arg Ile Gly Glu Ser Ser Asp Val Leu
20 25 30
Val Thr Arg Glu Pro Tyr Val Ser Cys Asp Pro Asp Glu Cys Arg Phe
35 40 45
Tyr Ala Leu Ser Gln Gly Thr Thr Ile Arg Gly Lys His Ser Asn Gly
50 55 60
Thr Ile His Asp Arg Ser Gln Tyr Arg Ala Leu Ile Ser Trp Pro Leu
65 70 75 80
Ser Ser Pro Pro Thr Val Tyr Asn Ser Arg Val Glu Cys Ile Gly Trp
85 90 95
Ser Ser Thr Ser Cys His Asp Gly Lys Ser Arg Met Ser Ile Cys Ile
100 105 110
Ser Gly Pro Asn Asn Asn Ala Ser Ala Val Val Trp Tyr Asn Arg Arg
115 120 125
Pro Val Ala Glu Ile Asn Thr Trp Ala Arg Asn Ile Leu Arg Thr Gln
130 135 140
Glu Ser Glu Cys Val Cys His Asn Gly Val Cys Pro Val Val Phe Thr
145 150 155 160
Asp Gly Ser Ala Thr Gly Pro Ala Asp Thr Arg Ile Tyr Tyr Phe Lys
165 170 175
Glu Gly Lys Ile Leu Lys Trp Glu Ser Leu Thr Gly Thr Ala Lys His
180 185 190
Ile Glu Glu Cys Ser Cys Tyr Gly Glu Arg Thr Gly Ile Thr Cys Thr
195 200 205
Cys Arg Asp Asn Trp Gln Gly Ser Asn Arg Pro Val Ile Gln Ile Asp
210 215 220
Pro Val Ala Met Thr His Thr Ser Gln Tyr Ile Cys Ser Pro Val Leu
225 230 235 240
Thr Asp Asn Pro Arg Pro Asn Asp Pro Asn Ile Gly Lys Cys Asn Asp
245 250 255
Pro Tyr Pro Gly Asn Asn Asn Asn Gly Val Lys Gly Phe Ser Tyr Leu
260 265 270
Asp Gly Ala Asn Thr Trp Leu Gly Arg Thr Ile Ser Thr Ala Ser Arg
275 280 285
Ser Gly Tyr Glu Met Leu Lys Val Pro Asn Ala Leu Thr Asp Asp Arg
290 295 300
Ser Lys Pro Ile Gln Gly Gln Thr Ile Val Leu Asn Ala Asp Trp Ser
305 310 315 320
Gly Tyr Ser Gly Ser Phe Met Asp Tyr Trp Ala Glu Gly Asp Cys Tyr
325 330 335
Arg Ala Cys Phe Tyr Val Glu Leu Ile Arg Gly Arg Pro Lys Glu Asp
340 345 350
Lys Val Trp Trp Thr Ser Asn Ser Ile Val Ser Met Cys Ser Ser Thr
355 360 365
Glu Phe Leu Gly Gln Trp Asn Trp Pro Asp Gly Ala Lys Ile Glu Tyr
370 375 380
<210> 32
<211> 384
<212> PRT
<213> A型流感病毒
<220>
<223> A型流感病毒H9N2的神经氨酸酶（NA）蛋白
<400> 32
Glu Tyr Lys Asn Trp Ser Lys Pro Gln Cys Gln Ile Thr Gly Phe Ala
1 5 10 15
Pro Phe Ser Lys Asp Asn Ser Ile Arg Leu Ser Ala Gly Gly Asp Ile
20 25 30
Trp Val Thr Arg Glu Pro Tyr Val Ser Cys Gly Leu Gly Lys Cys Tyr
35 40 45
Gln Phe Ala Leu Gly Gln Gly Thr Thr Leu Asn Asn Lys His Ser Asn
50 55 60
Gly Thr Ile His Asp Arg Ser Pro His Arg Thr Leu Leu Met Asn Glu
65 70 75 80
Leu Gly Val Pro Phe His Leu Gly Thr Lys Gln Val Cys Ile Ala Trp
85 90 95
Ser Ser Ser Ser Cys His Asp Gly Lys Ala Trp Leu His Val Cys Val
100 105 110
Thr Gly Asp Asp Arg Asn Ala Thr Ala Ser Ile Ile Tyr Asp Gly Met
115 120 125
Leu Thr Asp Ser Ile Gly Ser Trp Ser Lys Asn Ile Leu Arg Thr Gln
130 135 140
Glu Ser Glu Cys Val Cys Ile Asn Gly Thr Cys Thr Val Val Met Thr
145 150 155 160
Asp Gly Ser Ala Ser Gly Arg Ala Asp Thr Lys Ile Leu Phe Ile Arg
165 170 175
Glu Gly Lys Ile Val His Ile Gly Pro Leu Ser Gly Ser Ala Gln His
180 185 190
Val Glu Glu Cys Ser Cys Tyr Pro Arg Tyr Pro Glu Val Arg Cys Val
195 200 205
Cys Arg Asp Asn Trp Lys Gly Ser Asn Arg Pro Val Leu Tyr Ile Asn
210 215 220
Val Ala Asp Tyr Ser Val Asp Ser Ser Tyr Val Cys Ser Gly Leu Val
225 230 235 240
Gly Asp Thr Pro Arg Asn Asp Asp Ser Ser Ser Ser Ser Asn Cys Arg
245 250 255
Asp Pro Asn Asn Glu Arg Gly Gly Pro Gly Val Lys Gly Trp Ala Phe
260 265 270
Asp Asn Gly Asn Asp Val Trp Met Gly Arg Thr Ile Lys Lys Asp Ser
275 280 285
Arg Ser Gly Tyr Glu Thr Phe Arg Val Val Gly Gly Trp Thr Thr Ala
290 295 300
Asn Ser Lys Ser Gln Ile Asn Arg Gln Val Ile Val Asp Ser Asp Asn
305 310 315 320
Trp Ser Gly Tyr Ser Gly Ile Phe Ser Val Glu Gly Lys Thr Cys Ile
325 330 335
Asn Arg Cys Phe Tyr Val Glu Leu Ile Arg Gly Arg Pro Gln Glu Thr
340 345 350
Arg Val Trp Trp Thr Ser Asn Ser Ile Ile Val Phe Cys Gly Thr Ser
355 360 365
Gly Thr Tyr Gly Thr Gly Ser Trp Pro Asp Gly Ala Asn Ile Asn Phe
370 375 380
<210> 33
<211> 384
<212> PRT
<213> A型流感病毒
<220>
<223> A型流感病毒H2N2的神经氨酸酶（NA）蛋白
<400> 33
Glu Tyr Arg Asn Trp Ser Lys Pro Gln Cys Gln Ile Thr Gly Phe Ala
1 5 10 15
Pro Phe Ser Lys Asp Asn Ser Ile Arg Leu Ser Ala Gly Gly Asp Ile
20 25 30
Trp Val Thr Arg Glu Pro Tyr Val Ser Cys Asp Pro Gly Lys Cys Tyr
35 40 45
Gln Phe Ala Leu Gly Gln Gly Thr Thr Leu Asp Asn Lys His Ser Asn
50 55 60
Asp Thr Ile His Asp Arg Ile Pro His Arg Thr Leu Leu Met Asn Glu
65 70 75 80
Leu Gly Val Pro Phe His Leu Gly Thr Arg Gln Val Cys Val Ala Trp
85 90 95
Ser Ser Ser Ser Cys His Asp Gly Lys Ala Trp Leu His Val Cys Val
100 105 110
Thr Gly Asp Asp Lys Asn Ala Thr Ala Ser Phe Ile Tyr Asp Gly Arg
115 120 125
Leu Met Asp Ser Ile Gly Ser Trp Ser Gln Asn Ile Leu Arg Thr Gln
130 135 140
Glu Ser Glu Cys Val Cys Ile Asn Gly Thr Cys Thr Val Val Met Thr
145 150 155 160
Asp Gly Ser Ala Ser Gly Arg Ala Asp Thr Arg Ile Leu Phe Ile Glu
165 170 175
Glu Gly Lys Ile Val His Ile Ser Pro Leu Ser Gly Ser Ala Gln His
180 185 190
Val Glu Glu Cys Ser Cys Tyr Pro Arg Tyr Pro Asp Val Arg Cys Ile
195 200 205
Cys Arg Asp Asn Trp Lys Gly Ser Asn Arg Pro Val Ile Asp Ile Asn
210 215 220
Met Glu Asp Tyr Ser Ile Asp Ser Ser Tyr Val Cys Ser Gly Leu Val
225 230 235 240
Gly Asp Thr Pro Arg Asn Asp Asp Arg Ser Ser Asn Ser Asn Cys Arg
245 250 255
Asn Pro Asn Asn Glu Arg Gly Asn Pro Gly Val Lys Gly Trp Ala Phe
260 265 270
Asp Asn Gly Asp Asp Val Trp Met Gly Arg Thr Ile Ser Lys Asp Leu
275 280 285
Arg Ser Gly Tyr Glu Thr Phe Lys Val Ile Gly Gly Trp Ser Thr Pro
290 295 300
Asn Ser Lys Ser Gln Ile Asn Arg Gln Val Ile Val Asp Ser Asn Asn
305 310 315 320
Trp Ser Gly Tyr Ser Gly Ile Phe Ser Val Glu Gly Lys Arg Cys Ile
325 330 335
Asn Arg Cys Phe Tyr Val Glu Leu Ile Arg Gly Arg Gln Gln Glu Thr
340 345 350
Arg Val Trp Trp Thr Ser Asn Ser Ile Val Val Phe Cys Gly Thr Ser
355 360 365
Gly Thr Tyr Gly Thr Gly Ser Trp Pro Asp Gly Ala Asn Ile Asn Phe
370 375 380
<210> 34
<211> 381
<212> PRT
<213> A型流感病毒
<220>
<223> A型流感病毒H1N1的神经氨酸酶（NA）蛋白
<400> 34
Leu Ala Gly Asn Ser Ser Leu Cys Pro Val Ser Gly Trp Ala Ile Tyr
1 5 10 15
Ser Lys Asp Asn Ser Val Arg Ile Gly Ser Lys Gly Asp Val Phe Val
20 25 30
Ile Arg Glu Pro Phe Ile Ser Cys Ser Pro Leu Glu Cys Arg Thr Phe
35 40 45
Phe Leu Thr Gln Gly Ala Leu Leu Asn Asp Lys His Ser Asn Gly Thr
50 55 60
Ile Lys Asp Arg Ser Pro Tyr Arg Thr Leu Met Ser Cys Pro Ile Gly
65 70 75 80
Glu Val Pro Ser Pro Tyr Asn Ser Arg Phe Glu Ser Val Ala Trp Ser
85 90 95
Ala Ser Ala Cys His Asp Gly Ile Asn Trp Leu Thr Ile Gly Ile Ser
100 105 110
Gly Pro Asp Asn Gly Ala Val Ala Val Leu Lys Tyr Asn Gly Ile Ile
115 120 125
Thr Asp Thr Ile Lys Ser Trp Arg Asn Asn Ile Leu Arg Thr Gln Glu
130 135 140
Ser Glu Cys Ala Cys Val Asn Gly Ser Cys Phe Thr Val Met Thr Asp
145 150 155 160
Gly Pro Ser Asn Gly Gln Ala Ser Tyr Lys Ile Phe Arg Ile Glu Lys
165 170 175
Gly Lys Ile Val Lys Ser Val Glu Met Asn Ala Pro Asn Tyr His Tyr
180 185 190
Glu Glu Cys Ser Cys Tyr Pro Asp Ser Ser Glu Ile Thr Cys Val Cys
195 200 205
Arg Asp Asn Trp His Gly Ser Asn Arg Pro Trp Val Ser Phe Asn Gln
210 215 220
Asn Leu Glu Tyr Gln Ile Gly Tyr Ile Cys Ser Gly Ile Phe Gly Asp
225 230 235 240
Asn Pro Arg Pro Asn Asp Lys Thr Gly Ser Cys Gly Pro Val Ser Ser
245 250 255
Asn Gly Ala Asn Gly Val Lys Gly Phe Ser Phe Lys Tyr Gly Asn Gly
260 265 270
Val Trp Ile Gly Arg Thr Lys Ser Ile Ser Ser Arg Asn Gly Phe Glu
275 280 285
Met Ile Trp Asp Pro Asn Gly Trp Thr Gly Thr Asp Asn Asn Phe Ser
290 295 300
Ile Lys Gln Asp Ile Val Gly Ile Asn Glu Trp Ser Gly Tyr Ser Gly
305 310 315 320
Ser Phe Val Gln His Pro Glu Leu Thr Gly Leu Asp Cys Ile Arg Pro
325 330 335
Cys Phe Trp Val Glu Leu Ile Arg Gly Arg Pro Lys Glu Asn Thr Ile
340 345 350
Trp Thr Ser Gly Ser Ser Ile Ser Phe Cys Gly Val Asn Ser Asp Thr
355 360 365
Val Gly Trp Ser Trp Pro Asp Gly Ala Glu Leu Pro Phe
370 375 380
<210> 35
<211> 466
<212> PRT
<213> B型流感病毒
<220>
<223> 神经氨酸酶（NA）蛋白
<400> 35
Met Leu Pro Ser Thr Val Gln Thr Leu Thr Leu Leu Leu Thr Ser Gly
1 5 10 15
Gly Val Leu Leu Ser Leu Tyr Val Ser Ala Ser Leu Ser Tyr Leu Leu
20 25 30
Tyr Ser Asp Val Leu Leu Lys Phe Ser Ser Thr Lys Thr Thr Ala Pro
35 40 45
Thr Met Ser Leu Glu Cys Thr Asn Ala Ser Asn Ala Gln Thr Val Asn
50 55 60
His Ser Ala Thr Lys Glu Met Thr Phe Pro Pro Pro Glu Pro Glu Trp
65 70 75 80
Thr Tyr Pro Arg Leu Ser Cys Gln Gly Ser Thr Phe Gln Lys Ala Leu
85 90 95
Leu Ile Ser Pro His Arg Phe Gly Glu Ile Lys Gly Asn Ser Ala Pro
100 105 110
Leu Ile Ile Arg Glu Pro Phe Val Ala Cys Gly Pro Lys Glu Cys Arg
115 120 125
His Phe Ala Leu Thr His Tyr Ala Ala Gln Pro Gly Gly Tyr Tyr Asn
130 135 140
Gly Thr Arg Lys Asp Arg Asn Lys Leu Arg His Leu Val Ser Val Lys
145 150 155 160
Leu Gly Lys Ile Pro Thr Val Glu Asn Ser Ile Phe His Met Ala Ala
165 170 175
Trp Ser Gly Ser Ala Cys His Asp Gly Arg Glu Trp Thr Tyr Ile Gly
180 185 190
Val Asp Gly Pro Asp Asn Asp Ala Leu Val Lys Ile Lys Tyr Gly Glu
195 200 205
Ala Tyr Thr Asp Thr Tyr His Ser Tyr Ala His Asn Ile Leu Arg Thr
210 215 220
Gln Glu Ser Ala Cys Asn Cys Ile Gly Gly Asp Cys Tyr Leu Met Ile
225 230 235 240
Thr Asp Gly Ser Ala Ser Gly Ile Ser Lys Cys Arg Phe Leu Lys Ile
245 250 255
Arg Glu Gly Arg Ile Ile Lys Glu Ile Leu Pro Thr Gly Arg Val Glu
260 265 270
His Thr Glu Glu Cys Thr Cys Gly Phe Ala Ser Asn Lys Thr Ile Glu
275 280 285
Cys Ala Cys Arg Asp Asn Ser Tyr Thr Ala Lys Arg Pro Phe Val Lys
290 295 300
Leu Asn Val Glu Thr Asp Thr Ala Glu Ile Arg Leu Met Cys Thr Lys
305 310 315 320
Thr Tyr Leu Asp Thr Pro Arg Pro Asp Asp Gly Ser Ile Ala Gly Pro
325 330 335
Cys Glu Ser Asn Gly Asp Lys Trp Leu Gly Gly Ile Lys Gly Gly Phe
340 345 350
Val His Gln Arg Met Ala Ser Lys Ile Gly Arg Trp Tyr Ser Arg Thr
355 360 365
Met Ser Lys Thr Asn Arg Met Gly Met Glu Leu Tyr Val Lys Tyr Asp
370 375 380
Gly Asp Pro Trp Thr Asp Ser Asp Ala Leu Thr Leu Ser Gly Val Met
385 390 395 400
Val Ser Ile Glu Glu Pro Gly Trp Tyr Ser Phe Gly Phe Glu Ile Lys
405 410 415
Asp Lys Lys Cys Asp Val Pro Cys Ile Gly Ile Glu Met Val His Asp
420 425 430
Gly Gly Lys Asp Thr Trp His Ser Ala Ala Thr Ala Ile Tyr Cys Leu
435 440 445
Met Gly Ser Gly Gln Leu Leu Trp Asp Thr Val Thr Gly Val Asp Met
450 455 460
Ala Leu
465
<210> 36
<211> 842
<212> PRT
<213> 人免疫缺陷病毒1
<220>
<223> 包膜蛋白
<400> 36
Met Arg Val Arg Gly Met Gln Arg Asn Trp Gln His Leu Gly Lys Trp
1 5 10 15
Gly Leu Leu Phe Leu Gly Met Leu Ile Ile Cys Lys Ala Ala Asp Asp
20 25 30
Leu Trp Val Thr Ile Tyr Tyr Gly Val Pro Val Trp Lys Glu Ala Asn
35 40 45
Thr Thr Leu Phe Cys Ala Ser Asp Ala Lys Ser Tyr Glu Lys Glu Val
50 55 60
His Asn Val Trp Ala Thr His Ala Cys Val Pro Thr Asp Pro Asn Pro
65 70 75 80
Gln Glu Val Ala Leu Asn Val Thr Glu Asn Phe Asn Met Trp Glu Asn
85 90 95
Asp Met Val Glu Gln Met His Lys Asp Ile Ile Ser Leu Trp Asp Gln
100 105 110
Ser Leu Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Thr Leu Asn
115 120 125
Cys Thr Asn Ala Thr Thr Thr Asn Asp Thr Leu Ser Asp Gln Ser Ser
130 135 140
Thr Leu Lys Glu Glu Pro Gly Ala Ile Gln Asn Cys Ser Phe Asn Met
145 150 155 160
Thr Thr Glu Val Glu Asp Lys Lys Gln Lys Val His Ala Leu Phe Tyr
165 170 175
Arg Leu Asp Ile Glu Pro Ile Ser Asn Asn Asn Ser Arg Glu Glu Tyr
180 185 190
Arg Leu Ile Thr Cys Asn Thr Ser Thr Ile Thr Gln Ala Cys Pro Lys
195 200 205
Val Ser Trp Asp Pro Ile Pro Ile His Tyr Cys Ala Pro Ala Gly Tyr
210 215 220
Ala Ile Leu Lys Cys Lys Asp Lys Arg Phe Asn Gly Thr Gly Pro Cys
225 230 235 240
Arg Asn Val Ser Thr Val Gln Cys Thr His Gly Ile Arg Pro Val Val
245 250 255
Ser Thr Gln Leu Leu Leu Asn Gly Ser Leu Ser Glu Gly Gly Ile Ile
260 265 270
Ile Arg Ser Gln Asn Leu Ser Asp Asn Ala Lys Thr Ile Ile Val His
275 280 285
Leu Asn Glu Ser Val Gln Ile Asn Cys Thr Arg Pro Asn Asn Asn Thr
290 295 300
Arg Lys Ser Ile Arg Ile Gly Pro Gly Gln Ser Phe Tyr Ala Thr Gly
305 310 315 320
Glu Ile Ile Gly Asp Ile Arg Lys Ala His Cys Asn Ile Ser Gly Glu
325 330 335
Gln Trp Asn Lys Thr Leu Asp Arg Val Lys Ala Glu Leu Lys Leu His
340 345 350
Phe Asn Lys Thr Ile Gln Phe Asn Ser Ser Ser Gly Gly Asp Leu Glu
355 360 365
Ile Thr Met His Ser Phe Asn Cys Arg Gly Glu Phe Phe Tyr Cys Asn
370 375 380
Thr Ser Leu Leu Phe Asn Asn Thr Val Pro Asn Asn Gly Thr Ile Thr
385 390 395 400
Leu Pro Cys Arg Ile Lys Gln Phe Val Asn Met Trp Gln Glu Val Gly
405 410 415
Arg Ala Met Tyr Ala Ala Pro Ile Ala Gly Asn Ile Thr Cys Asn Ser
420 425 430
Asn Ile Thr Gly Leu Leu Leu Thr Arg Asp Gly Gly Gln Ser Asn Asn
435 440 445
Ser Asp Ser Glu Thr Phe Arg Pro Gly Gly Gly Asp Met Lys Asp Asn
450 455 460
Trp Arg Ser Glu Leu Tyr Lys Tyr Lys Val Val Glu Ile Glu Pro Leu
465 470 475 480
Gly Val Ala Pro Thr Arg Pro Lys Arg Pro Val Val Arg Arg Glu Arg
485 490 495
Arg Ala Val Ala Ile Gly Ala Val Phe Leu Gly Phe Leu Ser Ala Ala
500 505 510
Gly Ser Thr Met Gly Ala Ala Ser Leu Thr Leu Thr Val Gln Ala Arg
515 520 525
Gln Leu Leu Ser Gly Ile Val Gln Gln Gln Asn Asn Leu Leu Gln Ala
530 535 540
Ile Glu Ala Gln Gln His Met Leu Gln Leu Thr Val Trp Gly Ile Lys
545 550 555 560
Gln Leu Gln Ala Arg Val Leu Ala Val Glu Arg Tyr Leu Lys Asp Gln
565 570 575
Gln Leu Leu Gly Leu Trp Gly Cys Ser Gly Lys Leu Ile Cys Thr Thr
580 585 590
Asn Val Pro Trp Asn Ser Ser Trp Ser Asn Lys Ser Gln Asp Glu Ile
595 600 605
Trp Asn Asn Met Thr Trp Met Gln Trp Glu Lys Glu Ile Ser Asn Tyr
610 615 620
Ser Lys Thr Ile Tyr Met Leu Ile Glu Lys Ser Gln Ser Gln Gln Glu
625 630 635 640
Arg Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Asp Ser Leu Trp
645 650 655
Ser Trp Phe Asp Ile Thr Asn Trp Leu Trp Tyr Ile Lys Ile Phe Ile
660 665 670
Met Ile Val Gly Gly Leu Ile Gly Leu Arg Ile Val Phe Ala Val Leu
675 680 685
Ser Ile Val Asn Arg Val Arg Lys Gly Tyr Ser Pro Leu Ser Leu Gln
690 695 700
Thr Leu Ile Pro Ala Pro Thr Glu Pro Asp Arg Pro Glu Gly Ile Glu
705 710 715 720
Glu Gly Gly Gly Glu Gln Gly Lys Asp Arg Ser Val Arg Leu Val Asn
725 730 735
Gly Phe Leu Ala Leu Val Trp Asp Asp Leu Arg Asn Leu Cys Leu Phe
740 745 750
Ser Tyr Arg His Leu Arg Asp Phe Ile Leu Ile Ala Ala Arg Ile Val
755 760 765
Asp Arg Gly Leu Arg Arg Gly Trp Glu Ala Leu Lys Tyr Leu Gly Asn
770 775 780
Ile Ile Gln Tyr Trp Ser Gln Glu Leu Lys Asn Ser Ala Ile Ser Leu
785 790 795 800
Phe Asn Thr Thr Ala Ile Val Val Ala Glu Gly Thr Asp Arg Val Ile
805 810 815
Glu Ala Leu Gln Arg Ala Val Arg Ala Val Leu Asn Ile Pro Arg Arg
820 825 830
Ile Arg Gln Arg Val Glu Arg Ala Leu Ile
835 840
<210> 37
<211> 850
<212> PRT
<213> 人免疫缺陷病毒2
<220>
<223> 包膜蛋白
<400> 37
Met Thr Ser Glu Lys Thr Gln Leu Leu Ile Ala Ile Leu Leu Ala Ser
1 5 10 15
Thr Cys Leu Leu Tyr Cys Lys Gln Tyr Val Thr Val Phe Tyr Gly Val
20 25 30
Pro Ala Trp Arg Asn Ala Ser Ile Pro Leu Phe Cys Ala Thr Lys Asn
35 40 45
Arg Asp Thr Trp Gly Thr Ile Gln Cys Leu Pro Asp Asn Asp Asp Tyr
50 55 60
Gln Glu Ile Ala Leu Asn Val Thr Glu Ala Phe Asp Ala Trp Asn Asn
65 70 75 80
Thr Val Thr Glu Gln Ala Ile Asp Asp Val Lys Arg Leu Phe Glu Thr
85 90 95
Ser Ile Lys Pro Cys Val Lys Leu Thr Pro Leu Cys Val Ala Met Asn
100 105 110
Cys Thr Asn Val Thr Ser Thr Ala Asn Thr Thr Ile Thr Ile Ser Ser
115 120 125
Thr Asn Met Ile Val Asn Asp Ser Ser Pro Cys Ala Ser Asn Asp Ser
130 135 140
Cys Pro Gly Met Gly Glu Glu Glu Met Val Gln Cys Asn Phe Ser Met
145 150 155 160
Thr Gly Leu Gln Arg Asp Lys Val Lys Gln Tyr Asn Glu Thr Trp Tyr
165 170 175
Ser Arg Asp Val Val Cys Asp Gln Gly Glu Asn Asp Thr Arg Arg Cys
180 185 190
Tyr Met Asn His Cys Asn Thr Ser Val Ile Thr Glu Ser Cys Asp Lys
195 200 205
His Tyr Trp Asp Asp Met Arg Phe Arg Tyr Cys Ala Pro Pro Gly Tyr
210 215 220
Ala Leu Leu Arg Cys Asn Asp Thr Asn Tyr Ser Gly Phe Met Pro Asn
225 230 235 240
Cys Ser Lys Val Val Ala Ala Thr Cys Thr Arg Met Met Glu Thr Gln
245 250 255
Thr Ser Thr Trp Phe Gly Phe Asn Gly Thr Arg Ala Glu Asn Arg Thr
260 265 270
Tyr Ile Tyr Trp His Ser Lys Ser Asn Arg Thr Ile Ile Ser Leu Asn
275 280 285
Lys Ser Phe Asn Leu Ser Leu His Cys Lys Arg Pro Gly Asn Lys Thr
290 295 300
Val Val Pro Ile Thr Leu Met Ser Gly Leu Val Phe His Ser Gln Pro
305 310 315 320
Ile Asn Thr Arg Pro Arg Gln Ala Trp Cys Trp Phe Glu Gly Glu Trp
325 330 335
Lys Glu Ala Met Gln Glu Val Lys Arg Ala Ile Ala Lys His Pro Arg
340 345 350
Tyr Thr Gly Thr Asn Asp Thr Gly Lys Ile Asn Leu Thr Ala Pro Gly
355 360 365
Arg Gly Ser Asp Pro Glu Val Ser Tyr Met Trp Thr Asn Cys Arg Gly
370 375 380
Glu Phe Leu Tyr Cys Asn Met Thr Trp Phe Leu Asn Trp Val Glu Asn
385 390 395 400
Arg Thr Gly Ile Gln Arg Asn Tyr Ala Pro Cys His Ile Arg Gln Ile
405 410 415
Ile Asn Thr Trp His Lys Val Gly Lys Asn Val Tyr Leu Pro Pro Arg
420 425 430
Glu Gly Glu Leu Val Cys Asn Ser Thr Val Thr Ser Leu Ile Ala Asp
435 440 445
Ile Asp Trp Ile Asp Lys Asn Gly Thr Asn Ile Thr Phe Ser Ala Glu
450 455 460
Val Ser Glu Leu Tyr Arg Leu Glu Leu Gly Asp Tyr Lys Leu Val Glu
465 470 475 480
Ile Thr Pro Ile Gly Phe Ala Pro Thr Thr Glu Lys Arg Tyr Ser Pro
485 490 495
Asn His Gly Arg Pro Lys Arg Gly Val Phe Val Leu Gly Phe Leu Gly
500 505 510
Phe Leu Ala Thr Ala Gly Ser Ala Met Gly Ala Ala Ser Leu Thr Leu
515 520 525
Ser Ala Gln Ser Arg Thr Leu Leu Ala Gly Ile Val Gln Gln Gln Gln
530 535 540
Gln Leu Leu Asp Val Val Lys Arg Gln Gln Glu Met Leu Arg Leu Thr
545 550 555 560
Val Trp Gly Thr Lys Asn Leu Gln Ala Arg Val Thr Ala Ile Glu Lys
565 570 575
Tyr Leu Gly Asp Gln Ala Arg Leu Asn Ser Trp Gly Cys Ala Phe Arg
580 585 590
Gln Val Cys His Thr Thr Val Pro Trp Val Asn Asp Thr Leu Met Pro
595 600 605
Asp Trp Lys Asn Met Thr Trp Gln Lys Trp Glu Glu Gln Ile Arg Tyr
610 615 620
Leu Glu Ala Asn Ile Ser Asp Ser Leu Glu Gln Ala Gln Ile Gln Gln
625 630 635 640
Glu Lys Asn Thr Tyr Glu Leu Gln Lys Leu Asn Ser Trp Asp Val Phe
645 650 655
Thr Asn Trp Leu Asp Leu Thr Ser Trp Ile Lys Tyr Ile Gln Tyr Gly
660 665 670
Val Tyr Ile Val Val Gly Ile Ile Ala Leu Arg Ile Val Ile Tyr Val
675 680 685
Val Gln Met Leu Ser Arg Leu Arg Lys Gly Tyr Arg Pro Val Phe Ser
690 695 700
Ser Pro Pro Gly Tyr Ile Gln Gln Ile His Ile His Lys Gly Gln Glu
705 710 715 720
Gln Pro Thr Arg Glu Gly Thr Glu Glu Asp Val Gly Asp Asn Gly Gly
725 730 735
Asp Arg Ser Trp Pro Trp Pro Ile Ala Tyr Leu His Phe Leu Ile Arg
740 745 750
Leu Leu Ile Arg Leu Leu Ile Thr Leu Tyr Asn Ser Cys Arg Asp Leu
755 760 765
Leu Ser Arg Ile Phe Gln Thr Leu Gln Pro Ile Leu Arg Asn Leu Arg
770 775 780
Asp Trp Leu Arg Ile Lys Thr Ala Leu Leu Gln Tyr Gly Cys Glu Trp
785 790 795 800
Ile Gln Glu Ala Phe Gln Ala Ala Ala Arg Thr Thr Gly Glu Thr Leu
805 810 815
Ala Gly Ala Cys Arg Gly Leu Trp Arg Thr Leu Gly Arg Ile Gly Arg
820 825 830
Gly Ile Phe Ala Val Pro Arg Arg Ile Arg Gln Gly Ala Glu Ile Ala
835 840 845
Leu Leu
850
<210> 38
<211> 424
<212> PRT
<213> 人乳突病毒
<220>
<223> 主要衣壳L1蛋白
<400> 38
Ala Val Val Ser Thr Asp Glu Tyr Val Ala Arg Thr Asn Ile Tyr Tyr
1 5 10 15
His Ala Gly Thr Ser Arg Leu Leu Ala Val Gly His Pro Tyr Phe Pro
20 25 30
Ile Lys Lys Pro Asn Asn Asn Lys Ile Leu Val Pro Lys Val Ser Gly
35 40 45
Leu Gln Tyr Arg Val Phe Arg Ile His Leu Pro Asp Pro Asn Lys Phe
50 55 60
Gly Phe Pro Asp Thr Ser Phe Tyr Asn Pro Asp Thr Gln Arg Leu Val
65 70 75 80
Trp Ala Cys Val Gly Val Glu Val Gly Arg Gly Gln Pro Leu Gly Val
85 90 95
Gly Ile Ser Gly His Pro Leu Leu Asn Lys Leu Asp Asp Thr Glu Asn
100 105 110
Ala Ser Ala Tyr Ala Ala Asn Ala Gly Val Asp Asn Arg Glu Cys Ile
115 120 125
Ser Met Asp Tyr Lys Gln Thr Gln Leu Cys Leu Ile Gly Cys Lys Pro
130 135 140
Pro Ile Gly Glu His Trp Gly Lys Gly Ser Pro Cys Thr Gln Val Ala
145 150 155 160
Val Gln Pro Gly Asp Cys Pro Pro Leu Glu Leu Ile Asn Thr Val Ile
165 170 175
Gln Asp Gly Asp Met Val Asp Thr Gly Phe Gly Ala Met Asp Phe Thr
180 185 190
Thr Leu Gln Ala Asn Lys Ser Glu Val Pro Leu Asp Ile Cys Thr Ser
195 200 205
Ile Cys Lys Tyr Pro Asp Tyr Ile Lys Met Val Ser Glu Pro Tyr Gly
210 215 220
Asp Ser Leu Phe Phe Tyr Leu Arg Arg Glu Gln Met Phe Val Arg His
225 230 235 240
Leu Phe Asn Arg Ala Gly Thr Val Gly Glu Asn Val Pro Asp Asp Leu
245 250 255
Tyr Ile Lys Gly Ser Gly Ser Thr Ala Asn Leu Ala Ser Ser Asn Tyr
260 265 270
Phe Pro Thr Pro Ser Gly Ser Met Val Thr Ser Asp Ala Gln Ile Phe
275 280 285
Asn Lys Pro Tyr Trp Leu Gln Arg Ala Gln Gly His Asn Asn Gly Ile
290 295 300
Cys Trp Gly Asn Gln Leu Phe Val Thr Val Val Asp Thr Thr Arg Ser
305 310 315 320
Thr Asn Met Ser Leu Cys Ala Ala Ile Ser Thr Ser Glu Thr Thr Tyr
325 330 335
Lys Asn Thr Asn Phe Lys Glu Tyr Leu Arg His Gly Glu Glu Tyr Asp
340 345 350
Leu Gln Phe Ile Phe Gln Leu Cys Lys Ile Thr Leu Thr Ala Asp Val
355 360 365
Met Thr Tyr Ile His Ser Met Asn Ser Thr Ile Leu Glu Asp Trp Asn
370 375 380
Gly Gly Ser Gly Gly Glu Asp Pro Leu Lys Lys Tyr Thr Phe Trp Glu
385 390 395 400
Val Asn Leu Lys Glu Lys Phe Ser Ala Asp Leu Asp Gln Phe Pro Leu
405 410 415
Gly Arg Lys Phe Leu Leu Gln Leu
420
<210> 39
<211> 523
<212> PRT
<213> 人乳突病毒
<220>
<223> 次要衣壳L2蛋白
<400> 39
Met Ala Arg Ala Arg Arg Thr Lys Arg Asp Ser Val Thr Asn Ile Tyr
1 5 10 15
Arg Thr Cys Lys Ala Ala Gly Thr Cys Pro Pro Asp Val Val Asn Lys
20 25 30
Val Glu Gln Thr Thr Val Ala Asp Gln Ile Leu Lys Tyr Gly Ser Thr
35 40 45
Gly Val Phe Phe Gly Gly Leu Gly Ile Gly Thr Gly Arg Gly Thr Gly
50 55 60
Gly Ser Thr Gly Tyr Gly Pro Leu Gly Glu Gly Thr Ser Val Arg Val
65 70 75 80
Gly Asn Thr Pro Thr Val Ile Arg Pro Ala Leu Val Pro Glu Ala Ile
85 90 95
Gly Pro Ser Glu Leu Ile Pro Ile Asp Ser Val Asn Pro Ile Asp Pro
100 105 110
Ser Ala Ser Ser Ile Ile Pro Leu Thr Glu Ser Thr Gly Pro Asp Leu
115 120 125
Leu Pro Gly Glu Ile Glu Thr Ile Ala Glu Val His Pro Ala Pro Asp
130 135 140
Ile Pro Thr Val Asp Thr Pro Val Val Thr Gly Gly Arg Asn Ser Asn
145 150 155 160
Ala Val Leu Glu Val Ala Asp Pro Ser Pro Pro Thr Arg Asn Arg Val
165 170 175
Ser Arg Thr Gln Tyr Asn Asn Pro Ala Phe Gln Ile Ile Ser Glu Thr
180 185 190
Thr Pro Ser Ala Gly Glu Thr Ser Leu Ser Asp Gln Ile Val Val Gln
195 200 205
Ser Phe Asp Gly Gly Gln Tyr Ile Gly Gly Asn Pro Pro Pro Arg Ser
210 215 220
Val Val Glu Ile Glu Leu Gln Glu Ile Pro Ser Gln Tyr Ser Phe Glu
225 230 235 240
Ile Glu Glu Pro Thr Pro Pro Arg Gln Thr Ser Thr Pro Val Arg Gln
245 250 255
Ala Gln Gln Met Ala Ser Ala Leu Arg Arg Ala Leu Tyr Asn Arg Arg
260 265 270
Phe Thr Gln Gln Val Gln Val Glu Asp Pro Met Phe Tyr Ser Arg Pro
275 280 285
Ser Arg Leu Val Arg Phe Gln Phe Asp Asn Pro Val Phe Glu Glu Glu
290 295 300
Val Thr Gln Val Phe Glu Arg Asp Leu Glu Thr Ile Glu Glu Pro Pro
305 310 315 320
Asp Arg Gln Phe Leu Asp Val Gln Lys Leu Gly Arg Pro Thr Tyr Ala
325 330 335
Glu Thr Pro Ala Gly Tyr Ile Arg Val Ser Arg Leu Gly Lys Arg Ala
340 345 350
Thr Ile Arg Thr Arg Ser Gly Thr Thr Ile Gly Gly Gln Val His Phe
355 360 365
Phe Arg Asp Ile Ser Ser Ile Asp Thr Gln Pro Ser Ile Glu Leu Gln
370 375 380
Val Leu Gly Glu His Ser Gly Asp Ala Thr Ile Val Gln Gly Pro Val
385 390 395 400
Glu Ser Thr Phe Val Asn Ile Asp Leu Glu Glu Leu Pro Asn Leu Glu
405 410 415
Glu Asn Val His Leu Glu Ser Asp Asp Ile Leu Ile Asp Glu Ala Ile
420 425 430
Glu Asp Phe Ser Gly Ala Gln Leu Val Phe Gly Asn Ser Arg Arg Ser
435 440 445
Asn Thr Val Thr Leu Pro Arg Phe Glu Thr Val Arg Glu Thr Ser Leu
450 455 460
Tyr Thr Val Asp Leu Asp Gly Phe His Val Ser Tyr Pro Glu Ser Arg
465 470 475 480
Ala Tyr Pro Glu Val Ile Pro Thr Glu Pro Asp Asn Thr Pro Thr Val
485 490 495
Ile Ile His Thr Glu Asp Phe Ser Gly Asp Tyr Tyr Leu His Pro Ser
500 505 510
Leu Lys Trp Lys Lys Arg Lys Arg Ala Tyr Leu
515 520
<210> 40
<211> 237
<212> PRT
<213> 狂犬病毒
<220>
<223> 糖蛋白
<400> 40
Met Ala Pro Gln Val Leu Pro Phe Val Leu Leu Leu Ala Leu Ser Met
1 5 10 15
Cys Leu Gly Lys Phe Pro Ile Tyr Thr Ile Pro Asp Lys Leu Gly Pro
20 25 30
Trp Ser Pro Ile Asp Ile His His Leu Ser Cys Pro Asn Asn Leu Val
35 40 45
Val Glu Asp Glu Gly Cys Thr Asn Leu Ser Gly Phe Ser Tyr Met Glu
50 55 60
Leu Lys Val Gly Tyr Ile Ser Ala Ile Lys Val Asn Gly Phe Thr Cys
65 70 75 80
Thr Gly Val Val Thr Glu Ala Glu Thr Tyr Thr Asn Phe Val Gly Tyr
85 90 95
Val Thr Thr Thr Phe Lys Arg Lys His Phe Arg Pro Thr Pro Asp Ala
100 105 110
Cys Arg Ala Ala Tyr Asn Trp Lys Met Ala Gly Asp Pro Arg Tyr Glu
115 120 125
Glu Ser Leu His Asn Pro Tyr Pro Asp Tyr His Trp Leu Arg Thr Val
130 135 140
Lys Thr Thr Lys Glu Ser Leu Val Ile Ile Ser Pro Ser Val Ala Asp
145 150 155 160
Leu Asp Pro Tyr Asp Lys Ser Leu His Ser Arg Val Phe Pro Ser Gly
165 170 175
Lys Cys Ser Gly Ile Thr Ile Ser Ser Thr Tyr Cys Ser Thr Asn His
180 185 190
Asp Tyr Thr Ile Trp Met Pro Glu Asn Pro Arg Leu Gly Thr Ser Cys
195 200 205
Asp Ile Phe Thr Asn Ser Arg Gly Lys Arg Ala Ser Lys Gly Gly Lys
210 215 220
Thr Cys Gly Phe Val Asp Glu Arg Gly Leu Tyr Lys Ser
225 230 235
<210> 41
<211> 368
<212> PRT
<213> 巨细胞病毒
<220>
<223> 糖蛋白
<400> 41
Met Met Thr Met Trp Cys Leu Thr Leu Phe Val Leu Trp Met Leu Arg
1 5 10 15
Val Val Gly Met His Val Leu Arg Tyr Gly Tyr Thr Gly Ile Phe Asp
20 25 30
Asp Thr Ser His Met Thr Leu Thr Val Val Gly Ile Phe Asp Gly Gln
35 40 45
His Phe Phe Thr Tyr His Val Asn Ser Ser Asp Lys Ala Ser Ser Arg
50 55 60
Ala Asn Gly Thr Ile Ser Trp Met Ala Asn Val Ser Ala Ala Tyr Pro
65 70 75 80
Thr Tyr Leu Asp Gly Glu Arg Ala Lys Gly Asp Leu Ile Phe Asn Gln
85 90 95
Thr Glu Gln Asn Leu Leu Glu Leu Glu Ile Ala Leu Gly Tyr Arg Ser
100 105 110
Gln Ser Val Leu Thr Trp Thr His Glu Cys Asn Thr Thr Glu Asn Gly
115 120 125
Ser Phe Val Ala Gly Tyr Glu Gly Phe Gly Trp Asp Gly Glu Thr Leu
130 135 140
Met Glu Leu Lys Asp Asn Leu Thr Leu Trp Thr Gly Pro Asn Tyr Glu
145 150 155 160
Ile Ser Trp Leu Lys Gln Asn Lys Thr Tyr Ile Asp Gly Lys Ile Lys
165 170 175
Asn Ile Ser Glu Gly Asp Thr Thr Ile Gln Arg Asn Tyr Leu Lys Gly
180 185 190
Asn Cys Thr Gln Trp Ser Val Ile Tyr Ser Gly Phe Gln Pro Pro Val
195 200 205
Thr His Pro Val Val Lys Gly Gly Val Arg Asn Gln Asn Asp Asn Arg
210 215 220
Ala Glu Ala Phe Cys Thr Ser Tyr Gly Phe Phe Pro Gly Glu Ile Asn
225 230 235 240
Ile Thr Phe Ile His Tyr Gly Asp Lys Val Pro Glu Asp Ser Glu Pro
245 250 255
Gln Cys Asn Pro Leu Leu Pro Thr Leu Asp Gly Thr Phe His Gln Gly
260 265 270
Cys Tyr Val Ala Ile Phe Cys Asn Gln Asn Tyr Thr Cys Arg Val Thr
275 280 285
His Gly Asn Trp Thr Val Glu Ile Pro Ile Ser Val Thr Ser Pro Asp
290 295 300
Asp Ser Ser Ser Gly Glu Val Pro Asp His Pro Thr Ala Asn Lys Arg
305 310 315 320
Tyr Asn Thr Met Thr Ile Ser Ser Val Leu Leu Ala Leu Leu Leu Cys
325 330 335
Ala Leu Leu Phe Ala Phe Leu His Tyr Phe Thr Thr Leu Lys Gln Tyr
340 345 350
Leu Arg Asn Leu Ala Phe Ala Trp Arg Tyr Arg Lys Val Arg Ser Ser
355 360 365
<210> 42
<211> 621
<212> PRT
<213> C型肝炎病毒
<220>
<223> 包膜糖蛋白E1E2
<400> 42
Ile Arg Leu Asp Gly Val Asn Tyr Ala Thr Gly Asn Leu Pro Gly Cys
1 5 10 15
Ser Phe Ser Ile Phe Leu Leu Ala Leu Leu Ser Cys Leu Thr Ile Pro
20 25 30
Ala Ser Ala Tyr Glu Val Arg Asn Val Ser Gly Val Tyr His Val Thr
35 40 45
Asn Asp Cys Ser Asn Ser Ser Ile Val Tyr Glu Ala Ala Asp Met Ile
50 55 60
Met His Thr Pro Gly Cys Val Pro Cys Val Arg Glu Asn Asn Phe Ser
65 70 75 80
Arg Cys Trp Val Ala Leu Thr Pro Thr Leu Ala Ala Arg Asn Ala Ser
85 90 95
Val Pro Thr Thr Thr Ile Arg Arg His Val Asp Leu Leu Val Gly Ala
100 105 110
Ala Ala Phe Cys Ser Ala Met Tyr Val Gly Asp Leu Cys Gly Ser Val
115 120 125
Phe Leu Val Ser Gln Leu Phe Thr Phe Ser Pro Arg Gln His Glu Thr
130 135 140
Val Gln Asp Cys Asn Cys Ser Ile Tyr Pro Gly His Val Ser Gly His
145 150 155 160
Arg Met Ala Trp Asp Met Met Met Asn Trp Ser Pro Thr Ala Ala Ile
165 170 175
Val Val Ser Gln Leu Leu Arg Ile Pro Gln Ala Val Val Asp Met Val
180 185 190
Ala Gly Ala His Trp Gly Val Leu Ala Gly Leu Ala Tyr Tyr Thr Met
195 200 205
Ile Gly Asn Trp Ala Lys Ala Leu Ile Val Met Leu Leu Phe Ala Gly
210 215 220
Val Asp Gly His Thr His Val Thr Gly Gly Ser Ala Ser Arg Ala Thr
225 230 235 240
Arg Gly Leu Thr Ala Leu Phe Asp Phe Gly Ala Ser Gln Asn Leu Gln
245 250 255
Leu Ile Lys Thr Asn Gly Ser Trp His Ile Asn Arg Thr Ser Leu Asn
260 265 270
Cys Asn Asp Ser Leu Gln Thr Gly Phe Ile Ala Ala Leu Phe Tyr Thr
275 280 285
His Arg Phe Asn Ser Ser Gly Cys Pro Glu Arg Met Ala Ser Cys Arg
290 295 300
Pro Ile Asp Trp Phe Ala Gln Gly Trp Gly Pro Ile Thr His Asp Lys
305 310 315 320
Gly Gly Ser Ser Asp Gln Arg Pro Tyr Cys Trp His Tyr Ala Pro Arg
325 330 335
Pro Cys Gly Ile Val Pro Ala Ala Gln Val Cys Gly Pro Val Tyr Cys
340 345 350
Phe Thr Pro Ser Pro Val Val Val Gly Thr Thr Asp Arg Phe Gly Val
355 360 365
Pro Thr Tyr Ser Trp Gly Glu Asn Glu Thr Asp Val Leu Leu Leu Asn
370 375 380
Asn Thr Arg Pro Pro Gln Gly Asn Trp Phe Gly Cys Thr Trp Met Asn
385 390 395 400
Ser Thr Gly Phe Thr Lys Thr Cys Gly Gly Pro Pro Cys Asn Ile Gly
405 410 415
Gly Val Gly Asn Asn Thr Leu Ile Cys Pro Thr Asp Cys Phe Arg Lys
420 425 430
His Pro Glu Ala Thr Tyr Thr Lys Cys Gly Ser Gly Pro Trp Leu Thr
435 440 445
Pro Arg Cys Ile Val Asp Tyr Pro Tyr Arg Leu Trp His Tyr Pro Cys
450 455 460
Thr Val Asn Phe Thr Ile Phe Lys Val Arg Met Tyr Val Gly Gly Val
465 470 475 480
Glu His Arg Leu Asn Ala Ala Cys Asn Trp Thr Arg Gly Glu Arg Cys
485 490 495
Asn Leu Glu Asp Arg Asp Arg Ser Glu Leu Ser Pro Leu Leu Leu Ser
500 505 510
Thr Thr Glu Trp Gln Val Leu Pro Cys Ser Phe Thr Thr Leu Pro Ala
515 520 525
Leu Ser Thr Gly Leu Ile His Leu His Gln Asn Ile Val Asp Val Gln
530 535 540
Tyr Leu Tyr Gly Ile Gly Ser Val Val Val Ser Phe Val Ile Lys Trp
545 550 555 560
Glu Tyr Val Val Leu Leu Phe Leu Leu Leu Ala Asp Ala Arg Val Cys
565 570 575
Ala Cys Leu Trp Met Met Leu Leu Ile Ala Gln Ala Glu Ala Ala Leu
580 585 590
Glu Asn Leu Val Val Leu Asn Ala Ala Ser Val Ala Gly Ala His Ser
595 600 605
Ile Leu Ser Phe Leu Val Phe Phe Cys Glu Ala Glu Phe
610 615 620
<210> 43
<211> 492
<212> PRT
<213> 呼吸道合胞病毒
<220>
<223> 融合（F）蛋白
<400> 43
Thr Glu Glu Phe Tyr Gln Thr Thr Cys Ser Ala Val Ser Lys Gly Tyr
1 5 10 15
Leu Ser Ala Leu Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu
20 25 30
Leu Ser Asn Ile Lys Glu Asn Lys Cys Asn Gly Thr Asp Ala Lys Val
35 40 45
Lys Leu Ile Lys Gln Glu Leu Asp Lys Tyr Lys Ser Ala Val Thr Glu
50 55 60
Leu Gln Leu Leu Met Gln Ser Thr Pro Ala Thr Asn Asn Arg Ala Arg
65 70 75 80
Arg Glu Leu Pro Arg Phe Met Asn Tyr Thr Leu Asn Asn Thr Lys Asn
85 90 95
Thr Asn Val Thr Leu Ser Lys Lys Arg Lys Arg Arg Phe Leu Gly Phe
100 105 110
Leu Leu Gly Val Gly Ser Ala Ile Ala Ser Gly Ile Ala Val Ser Lys
115 120 125
Val Leu His Leu Glu Gly Glu Val Asn Lys Ile Lys Ser Ala Leu Leu
130 135 140
Ser Thr Asn Lys Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu
145 150 155 160
Thr Ser Lys Val Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu
165 170 175
Pro Ile Val Asn Lys Gln Ser Cys Ser Ile Ser Asn Ile Glu Thr Val
180 185 190
Ile Glu Phe Gln Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr Arg Glu
195 200 205
Phe Ser Val Asn Ala Gly Val Thr Thr Pro Val Ser Thr Tyr Met Leu
210 215 220
Thr Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn
225 230 235 240
Asp Gln Lys Lys Leu Met Ser Asn Asn Val Gln Ile Val Arg Gln Gln
245 250 255
Ser Tyr Ser Ile Met Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val
260 265 270
Val Gln Leu Pro Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu
275 280 285
His Thr Ser Pro Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile
290 295 300
Cys Leu Thr Arg Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala Gly Ser
305 310 315 320
Val Ser Phe Phe Pro Leu Ala Glu Thr Cys Lys Val Gln Ser Asn Arg
325 330 335
Val Phe Cys Asp Thr Met Asn Ser Leu Thr Leu Pro Ser Glu Val Asn
340 345 350
Leu Cys Asn Ile Asp Ile Phe Asn Pro Lys Tyr Asp Cys Lys Ile Met
355 360 365
Thr Ser Lys Thr Asp Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala
370 375 380
Ile Val Ser Cys Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn
385 390 395 400
Arg Gly Ile Ile Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn
405 410 415
Lys Gly Val Asp Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn
420 425 430
Lys Gln Glu Gly Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile Ile Asn
435 440 445
Phe Tyr Asp Pro Leu Val Phe Pro Ser Asp Glu Phe Asp Ala Ser Ile
450 455 460
Ser Gln Val Asn Glu Lys Ile Asn Gln Ser Leu Ala Phe Ile Arg Lys
465 470 475 480
Ser Asp Glu Leu Leu His Asn Val Asn Ala Gly Lys
485 490
<210> 44
<211> 334
<212> PRT
<213> 扎伊尔埃博拉病毒
<220>
<223> 刺突糖蛋白
<400> 44
Lys Arg Thr Ser Phe Phe Leu Trp Val Ile Ile Leu Phe Gln Arg Thr
1 5 10 15
Phe Ser Ile Pro Leu Gly Val Ile His Asn Ser Thr Leu Gln Val Ser
20 25 30
Asp Val Asp Lys Leu Val Cys Arg Asp Lys Leu Ser Ser Thr Asn Gln
35 40 45
Leu Arg Ser Val Gly Leu Asn Leu Glu Gly Asn Gly Val Ala Thr Asp
50 55 60
Val Pro Ser Ala Thr Lys Arg Trp Gly Phe Arg Ser Gly Val Pro Pro
65 70 75 80
Lys Val Val Asn Tyr Glu Ala Gly Glu Trp Ala Glu Asn Cys Tyr Asn
85 90 95
Leu Glu Ile Lys Lys Pro Asp Gly Ser Glu Cys Leu Pro Ala Ala Pro
100 105 110
Asp Gly Ile Arg Gly Phe Pro Arg Cys Arg Tyr Val His Lys Val Ser
115 120 125
Gly Thr Gly Pro Cys Ala Gly Asp Phe Ala Phe His Lys Glu Gly Ala
130 135 140
Phe Phe Leu Tyr Asp Arg Leu Ala Ser Thr Val Ile Tyr Arg Gly Thr
145 150 155 160
Thr Phe Ala Glu Gly Val Val Ala Phe Leu Ile Leu Pro Gln Ala Lys
165 170 175
Lys Asp Phe Phe Ser Ser His Pro Leu Arg Glu Pro Val Asn Ala Thr
180 185 190
Glu Asp Pro Ser Ser Gly Tyr Tyr Ser Thr Thr Ile Arg Tyr Gln Ala
195 200 205
Thr Gly Phe Gly Thr Asn Glu Thr Glu Tyr Leu Phe Glu Val Asp Asn
210 215 220
Leu Thr Tyr Val Gln Leu Glu Ser Arg Phe Thr Pro Gln Phe Leu Leu
225 230 235 240
Gln Leu Asn Glu Thr Ile Tyr Thr Ser Gly Lys Arg Ser Asn Thr Thr
245 250 255
Gly Lys Leu Ile Trp Lys Val Asn Pro Glu Ile Asp Thr Thr Ile Gly
260 265 270
Glu Trp Ala Phe Trp Glu Thr Lys Lys Asn Leu Thr Arg Lys Ile Arg
275 280 285
Ser Glu Glu Leu Ser Phe Thr Val Val Ser Asn Gly Ala Lys Asn Ile
290 295 300
Ser Gly Gln Ser Pro Ala Arg Thr Ser Ser Asp Pro Gly Thr Asn Thr
305 310 315 320
Thr Thr Glu Asp His Lys Ile Met Ala Ser Glu Asn Ser Ser
325 330
<210> 45
<211> 168
<212> PRT
<213> 寨卡病毒
<220>
<223> prM蛋白
<400> 45
Ala Glu Ile Thr Arg Arg Gly Ser Ala Tyr Tyr Met Tyr Leu Asp Arg
1 5 10 15
Ser Asp Ala Gly Lys Ala Ile Ser Phe Ala Thr Thr Leu Gly Val Asn
20 25 30
Lys Cys His Val Gln Ile Met Asp Leu Gly His Met Cys Asp Ala Thr
35 40 45
Met Ser Tyr Glu Cys Pro Met Leu Asp Glu Gly Val Glu Pro Asp Asp
50 55 60
Val Asp Cys Trp Cys Asn Thr Thr Ser Thr Trp Val Val Tyr Gly Thr
65 70 75 80
Cys His His Lys Lys Gly Glu Ala Arg Arg Ser Arg Arg Ala Val Thr
85 90 95
Leu Pro Ser His Ser Thr Arg Lys Leu Gln Thr Arg Ser Gln Thr Trp
100 105 110
Leu Glu Ser Arg Glu Tyr Thr Lys His Leu Ile Lys Val Glu Asn Trp
115 120 125
Ile Phe Arg Asn Pro Gly Phe Ala Leu Val Ala Val Ala Ile Ala Trp
130 135 140
Leu Leu Gly Ser Ser Thr Ser Gln Lys Val Ile Tyr Leu Val Met Ile
145 150 155 160
Leu Leu Ile Ala Pro Ala Tyr Ser
165
<210> 46
<211> 617
<212> PRT
<213> 寨卡病毒
<220>
<223> 丝氨酸蛋白酶NS3
<400> 46
Ser Gly Ala Leu Trp Asp Val Pro Ala Pro Lys Glu Val Lys Lys Gly
1 5 10 15
Glu Thr Thr Asp Gly Val Tyr Arg Val Met Thr Arg Arg Leu Leu Gly
20 25 30
Ser Thr Gln Val Gly Val Gly Val Met Gln Glu Gly Val Phe His Thr
35 40 45
Met Trp His Val Thr Lys Gly Ala Ala Leu Arg Ser Gly Glu Gly Arg
50 55 60
Leu Asp Pro Tyr Trp Gly Asp Val Lys Gln Asp Leu Val Ser Tyr Cys
65 70 75 80
Gly Pro Trp Lys Leu Asp Ala Ala Trp Asp Gly Leu Ser Glu Val Gln
85 90 95
Leu Leu Ala Val Pro Pro Gly Glu Arg Ala Arg Asn Ile Gln Thr Leu
100 105 110
Pro Gly Ile Phe Lys Thr Lys Asp Gly Asp Ile Gly Ala Val Ala Leu
115 120 125
Asp Tyr Pro Ala Gly Thr Ser Gly Ser Pro Ile Leu Asp Lys Cys Gly
130 135 140
Arg Val Ile Gly Leu Tyr Gly Asn Gly Val Val Ile Lys Asn Gly Ser
145 150 155 160
Tyr Val Ser Ala Ile Thr Gln Gly Lys Arg Glu Glu Glu Thr Pro Val
165 170 175
Glu Cys Phe Glu Pro Ser Met Leu Lys Lys Lys Gln Leu Thr Val Leu
180 185 190
Asp Leu His Pro Gly Ala Gly Lys Thr Arg Arg Val Leu Pro Glu Ile
195 200 205
Val Arg Glu Ala Ile Lys Lys Arg Leu Arg Thr Val Ile Leu Ala Pro
210 215 220
Thr Arg Val Val Ala Ala Glu Met Glu Glu Ala Leu Arg Gly Leu Pro
225 230 235 240
Val Arg Tyr Met Thr Thr Ala Val Asn Val Thr His Ser Gly Thr Glu
245 250 255
Ile Val Asp Leu Met Cys His Ala Thr Phe Thr Ser Arg Leu Leu Gln
260 265 270
Pro Ile Arg Val Pro Asn Tyr Asn Leu Asn Ile Met Asp Glu Ala His
275 280 285
Phe Thr Asp Pro Ser Ser Ile Ala Ala Arg Gly Tyr Ile Ser Thr Arg
290 295 300
Val Glu Met Gly Glu Ala Ala Ala Ile Phe Met Thr Ala Thr Pro Pro
305 310 315 320
Gly Thr Arg Asp Ala Phe Pro Asp Ser Asn Ser Pro Ile Met Asp Thr
325 330 335
Glu Val Glu Val Pro Glu Arg Ala Trp Ser Ser Gly Phe Asp Trp Val
340 345 350
Thr Asp His Ser Gly Lys Thr Val Trp Phe Val Pro Ser Val Arg Asn
355 360 365
Gly Asn Glu Ile Ala Ala Cys Leu Thr Lys Ala Gly Lys Arg Val Ile
370 375 380
Gln Leu Ser Arg Lys Thr Phe Glu Thr Glu Phe Gln Lys Thr Lys Asn
385 390 395 400
Gln Glu Trp Asp Phe Val Ile Thr Thr Asp Ile Ser Glu Met Gly Ala
405 410 415
Asn Phe Lys Ala Asp Arg Val Ile Asp Ser Arg Arg Cys Leu Lys Pro
420 425 430
Val Ile Leu Asp Gly Glu Arg Val Ile Leu Ala Gly Pro Met Pro Val
435 440 445
Thr His Ala Ser Ala Ala Gln Arg Arg Gly Arg Ile Gly Arg Asn Pro
450 455 460
Asn Lys Pro Gly Asp Glu Tyr Met Tyr Gly Gly Gly Cys Ala Glu Thr
465 470 475 480
Asp Glu Gly His Ala His Trp Leu Glu Ala Arg Met Leu Leu Asp Asn
485 490 495
Ile Tyr Leu Gln Asp Gly Leu Ile Ala Ser Leu Tyr Arg Pro Glu Ala
500 505 510
Asp Lys Val Ala Ala Ile Glu Gly Glu Phe Lys Leu Arg Thr Glu Gln
515 520 525
Arg Lys Thr Phe Val Glu Leu Met Lys Arg Gly Asp Leu Pro Val Trp
530 535 540
Leu Ala Tyr Gln Val Ala Ser Ala Gly Ile Thr Tyr Thr Asp Arg Arg
545 550 555 560
Trp Cys Phe Asp Gly Thr Thr Asn Asn Thr Ile Met Glu Asp Ser Val
565 570 575
Pro Ala Glu Val Trp Thr Lys Tyr Gly Glu Lys Arg Val Leu Lys Pro
580 585 590
Arg Trp Met Asp Ala Arg Val Cys Ser Asp His Ala Ala Leu Lys Ser
595 600 605
Phe Lys Glu Phe Ala Ala Gly Lys Arg
610 615
<210> 47
<211> 130
<212> PRT
<213> 寨卡病毒
<220>
<223> 丝氨酸蛋白酶亚基NS2B
<400> 47
Ser Trp Pro Pro Ser Glu Val Leu Thr Ala Val Gly Leu Ile Cys Ala
1 5 10 15
Leu Ala Gly Gly Phe Ala Lys Ala Asp Ile Glu Met Ala Gly Pro Met
20 25 30
Ala Ala Val Gly Leu Leu Ile Val Ser Tyr Val Val Ser Gly Lys Ser
35 40 45
Val Asp Met Tyr Ile Glu Arg Ala Gly Asp Ile Thr Trp Glu Lys Asp
50 55 60
Ala Glu Val Thr Gly Asn Ser Pro Arg Leu Asp Val Ala Leu Asp Glu
65 70 75 80
Ser Gly Asp Phe Ser Leu Val Glu Glu Asp Gly Pro Pro Met Arg Glu
85 90 95
Ile Ile Leu Lys Val Val Leu Met Ala Ile Cys Gly Met Asn Pro Ile
100 105 110
Ala Ile Pro Phe Ala Ala Gly Ala Trp Tyr Val Tyr Val Lys Thr Gly
115 120 125
Lys Arg
130
<210> 48
<211> 500
<212> PRT
<213> 寨卡病毒
<220>
<223> 包膜蛋白E
<400> 48
Ile Arg Cys Ile Gly Val Ser Asn Arg Asp Phe Val Glu Gly Met Ser
1 5 10 15
Gly Gly Thr Trp Val Asp Val Val Leu Glu His Gly Gly Cys Val Thr
20 25 30
Val Met Ala Gln Asp Lys Pro Thr Val Asp Ile Glu Leu Val Thr Thr
35 40 45
Thr Val Ser Asn Met Ala Glu Val Arg Ser Tyr Cys Tyr Glu Ala Ser
50 55 60
Ile Ser Asp Met Ala Ser Asp Ser Arg Cys Pro Thr Gln Gly Glu Ala
65 70 75 80
Tyr Leu Asp Lys Gln Ser Asp Thr Gln Tyr Val Cys Lys Arg Thr Leu
85 90 95
Val Asp Arg Gly Trp Gly Asn Gly Cys Gly Leu Phe Gly Lys Gly Ser
100 105 110
Leu Val Thr Cys Ala Lys Phe Thr Cys Ser Lys Lys Met Thr Gly Lys
115 120 125
Ser Ile Gln Pro Glu Asn Leu Glu Tyr Arg Ile Met Leu Ser Val His
130 135 140
Gly Ser Gln His Ser Gly Met Ile Gly Tyr Glu Thr Asp Glu Asp Arg
145 150 155 160
Ala Lys Val Glu Val Thr Pro Asn Ser Pro Arg Ala Glu Ala Thr Leu
165 170 175
Gly Gly Phe Gly Ser Leu Gly Leu Asp Cys Glu Pro Arg Thr Gly Leu
180 185 190
Asp Phe Ser Asp Leu Tyr Tyr Leu Thr Met Asn Asn Lys His Trp Leu
195 200 205
Val His Lys Glu Trp Phe His Asp Ile Pro Leu Pro Trp His Ala Gly
210 215 220
Ala Asp Thr Gly Thr Pro His Trp Asn Asn Lys Glu Ala Leu Val Glu
225 230 235 240
Phe Lys Asp Ala His Ala Lys Arg Gln Thr Val Val Val Leu Gly Ser
245 250 255
Gln Glu Gly Ala Val His Thr Ala Leu Ala Gly Ala Leu Glu Ala Glu
260 265 270
Met Asp Gly Ala Lys Gly Arg Leu Phe Ser Gly His Leu Lys Cys Arg
275 280 285
Leu Lys Met Asp Lys Leu Arg Leu Lys Gly Val Ser Tyr Ser Leu Cys
290 295 300
Thr Ala Ala Phe Thr Phe Thr Lys Val Pro Ala Glu Thr Leu His Gly
305 310 315 320
Thr Val Thr Val Glu Val Gln Tyr Ala Gly Thr Asp Gly Pro Cys Lys
325 330 335
Ile Pro Val Gln Met Ala Val Asp Met Gln Thr Leu Thr Pro Val Gly
340 345 350
Arg Leu Ile Thr Ala Asn Pro Val Ile Thr Glu Ser Thr Glu Asn Ser
355 360 365
Lys Met Met Leu Glu Leu Asp Pro Pro Phe Gly Asp Ser Tyr Ile Val
370 375 380
Ile Gly Val Gly Asp Lys Lys Ile Thr His His Trp His Arg Ser Gly
385 390 395 400
Ser Thr Ile Gly Lys Ala Phe Glu Ala Thr Val Arg Gly Ala Lys Arg
405 410 415
Met Ala Val Leu Gly Asp Thr Ala Trp Asp Phe Gly Ser Val Gly Gly
420 425 430
Val Phe Asn Ser Leu Gly Lys Gly Ile His Gln Ile Phe Gly Ala Ala
435 440 445
Phe Lys Ser Leu Phe Gly Gly Met Ser Trp Phe Ser Gln Ile Leu Ile
450 455 460
Gly Thr Leu Leu Val Trp Leu Gly Leu Asn Thr Lys Asn Gly Ser Ile
465 470 475 480
Ser Leu Thr Cys Leu Ala Leu Gly Gly Val Met Ile Phe Leu Ser Thr
485 490 495
Ala Val Ser Ala
500
<210> 49
<211> 104
<212> PRT
<213> 寨卡病毒
<220>
<223> 衣壳蛋白C
<400> 49
Met Lys Asn Pro Lys Glu Glu Ile Arg Arg Ile Arg Ile Val Asn Met
1 5 10 15
Leu Lys Arg Gly Val Ala Arg Val Asn Pro Leu Gly Gly Leu Lys Arg
20 25 30
Leu Pro Ala Gly Leu Leu Leu Gly His Gly Pro Ile Arg Met Val Leu
35 40 45
Ala Ile Leu Ala Phe Leu Arg Phe Thr Ala Ile Lys Pro Ser Leu Gly
50 55 60
Leu Ile Asn Arg Trp Gly Ser Val Gly Lys Lys Glu Ala Met Glu Ile
65 70 75 80
Ile Lys Lys Phe Lys Lys Asp Leu Ala Ala Met Leu Arg Ile Ile Asn
85 90 95
Ala Arg Lys Glu Arg Lys Arg Arg
100
<210> 50
<211> 669
<212> RNA
<213> SARS冠状病毒
<220>
<223> 编码SARS-CoV-2刺突（S）RBD蛋白的mRNA
<400> 50
agagtccaac caacagaatc tattgttaga tttcctaata ttacaaactt gtgccctttt 60
ggtgaagttt ttaacgccac cagatttgca tctgtttatg cttggaacag gaagagaatc 120
agcaactgtg ttgctgatta ttctgtccta tataattccg catcattttc cacttttaag 180
tgttatggag tgtctcctac taaattaaat gatctctgct ttactaatgt ctatgcagat 240
tcatttgtaa ttagaggtga tgaagtcaga caaatcgctc cagggcaaac tggaaagatt 300
gctgattata attataaatt accagatgat tttacaggct gcgttatagc ttggaattct 360
aacaatcttg attctaaggt tggtggtaat tataattacc tgtatagatt gtttaggaag 420
tctaatctca aaccttttga gagagatatt tcaactgaaa tctatcaggc cggtagcaca 480
ccttgtaatg gtgttgaagg ttttaattgt tactttcctt tacaatcata tggtttccaa 540
cccactaatg gtgttggtta ccaaccatac agagtagtag tactttcttt tgaacttcta 600
catgcaccag caactgtttg tggacctaaa aagtctacta atttggttaa aaacaaatgt 660
gtcaatttc 669

Claims (16)

1.一种用作疫苗的组合物，包括非免疫调节的工程化的植物来源的细胞外囊泡(EV)，其中所述细胞外囊泡(EV)由包括外脂质层和内脂质层的脂质双层膜界定，
其中所述EV在内部装载有编码至少一种蛋白抗原的外源核酸分子；其中所述EV具有在20至500nm、优选200至300nm的范围内的直径；
其中所述EV的脂质双层膜上的膜电位在+5至-5mV的范围内；
其中所述组合物中≤44％的EV在所述脂质双层膜的外层中包括磷脂酰丝氨酸。
2.根据权利要求1所述的用于使用的组合物，其中装载的外源核酸分子选自由以下组成的组：DNA、cDNA、信使RNA(mRNA)、前体mRNA、长链RNA、编码RNA、单链RNA、双链RNA、线性RNA、RNA寡核苷酸、自复制RNA(复制子RNA)、逆转录病毒RNA、病毒RNA(vRNA)。
3.根据权利要求2所述的用于使用的组合物，其中所述装载的外源核酸分子是包括选自由SEQ ID NO.14、17、19和50组成的组的核苷酸序列的mRNA分子。
4.根据权利要求1至3中任一项所述的用于使用的组合物，其中编码的至少一种蛋白抗原选自由以下组成的组：肿瘤抗原、病毒抗原、细菌抗原、真菌抗原和原生动物抗原。
5.根据权利要求4所述的用于使用的组合物，其中所述编码的至少一种蛋白抗原选自由以下组成的组：前列腺特异性抗原(PSA)；前列腺干细胞抗原(PSCA)；前列腺特异性膜抗原(PSMA)；(前列腺的六跨膜上皮抗原1)(STEAP1)；受体酪氨酸-蛋白激酶erbB-2；细胞表面相关粘蛋白1蛋白(MUC1)；酪氨酸酶相关蛋白2(TRP-2)；原癌基因B-Raf；原癌基因c-Kit；GTP酶NRas；黑色素瘤相关抗原1；黑色素瘤相关抗原1蛋白；NY-ESO-1蛋白；SARS-COV-2的刺突蛋白；SARS-COV-2的N蛋白；SARS-COV-2的M蛋白；A型流感病毒的血凝素蛋白；B型流感病毒的血凝素蛋白；A型流感病毒的神经氨酸酶蛋白；B型流感病毒的神经氨酸酶蛋白；HIV1的包膜蛋白；包膜蛋白HIV2；HPV的主要衣壳蛋白L1；HPV的次要衣壳蛋白L2；狂犬病毒的糖蛋白；人巨细胞病毒的糖蛋白；C型肝炎病毒的包膜糖蛋白E1E2；RSV的融合蛋白；扎伊尔埃博拉病毒的刺突糖蛋白；寨卡病毒的蛋白prM；寨卡病毒的丝氨酸蛋白酶NS3；寨卡病毒的丝氨酸蛋白酶亚基NS2B；寨卡病毒的包膜蛋白E；寨卡病毒的衣壳蛋白C；刚地弓形虫蛋白，包括致密颗粒蛋白6、棒状体蛋白2A、棒状体蛋白18、表面抗原1、表面抗原2A；刚地弓形虫顶端膜抗原1；SARS-CoV-2刺突(S)RBD蛋白；以及它们的任何组合。
6.根据权利要求5所述的用于使用的组合物，其中所述编码的至少一种蛋白抗原包括选自由SEQ ID NO.:1-13、15、16、18和20-49组成的组的氨基酸序列。
7.根据权利要求1至6中任一项所述的用于使用的组合物，其中在所述EV中装载的外源核酸分子的含量在20至200ng/109EV、优选30至100ng/109EV的范围内。
8.根据权利要求1至7中任一项所述的用于使用的组合物，其中所述EV来源于选自由以下组成的组的一种或多种植物：柑橘属，包括柠檬和橙子；猕猴桃属，包括奇异果；葫芦属，包括西葫芦；芸苔属，包括卷心菜和羽衣甘蓝；石榴属，包括石榴；越橘属，包括蓝莓，以及芹属，包括芹菜。
9.根据权利要求1至8中任一项所述的用于使用的组合物，进一步包括一种或多种聚阳离子物质，所述一种或多种聚阳离子物质通过静电相互作用与所述EV的脂质双层膜的外脂质层相关联。
10.根据权利要求9所述的用于使用的组合物，其中所述一种或多种聚阳离子物质选自由以下组成的组：阳离子蛋白，包括鱼精蛋白；阳离子肽；多肽；多糖；甘油；聚乙二醇(PEG)；以及它们的任何组合，并且所述一种或多种聚阳离子物质优选以0.001至2μg/1010EV范围内的量存在于所述组合物中。
11.根据权利要求1至10中任一项所述的用于使用的组合物，其中所述EV额外地装载有一个或多个糖分子，所述一个或多个糖分子通过静电相互作用和氢键与装载到所述EV中的所述外源核酸分子相关联。
12.根据权利要求11所述的用于使用的组合物，其中所述一个或多个糖分子选自由以下组成的组：二糖、糖醇、多糖以及它们的任何组合，并且在所述EV中所述一个或多个糖分子的含量优选在0.1至10mg/1010EV的范围内。
13.根据权利要求1至12中任一项所述的用于使用的组合物，所述组合物为适用于口服、鼻内或肠胃外施用的形式。
14.一种用于制备根据权利要求1至12中任一项所述的组合物的方法，包括以下步骤：
(i)将植物来源的细胞外囊泡(EV)的悬浮液与一种或多种聚阳离子物质接触并混合以得到第一混合物；
(ii)将核酸分子的配制品与一个或多个糖分子接触并混合以得到第二混合物，所述核酸分子编码至少一种蛋白抗原；
(iii)将所述第一混合物与所述第二混合物混合以得到第三混合物；以及
(iv)将预定体积的水添加至所述第三混合物，其中水的所述预定体积与所述第三混合物的体积的比例包含在5:1至15:1内。
15.根据权利要求14所述的方法，进一步包括将步骤(iv)中得到的组合物优选通过过滤浓缩。
16.根据权利要求14或15所述的方法，其中所述一种或多种聚阳离子物质选自由以下组成的组：阳离子蛋白，包括鱼精蛋白、降钙素肽、菌丝霉素、乳铁蛋白、鱼精蛋白样蛋白，如精胺或亚精胺、核仁蛋白、组蛋白、细胞穿透肽(CPP)；阳离子肽，包括富含组氨酸的肽、富含精氨酸的肽、富含赖氨酸的肽和富含阳离子精氨酸的肽(CARP)；多肽，包括聚精氨酸、聚赖氨酸、聚组氨酸、富含组氨酸的肽、富含精氨酸的肽、富含赖氨酸的肽；多糖，包括壳聚糖、糖胺聚糖，如多硫酸化糖胺聚糖(PSGAG)、阳离子葡聚糖；甘油、聚乙二醇(PEG)，以及它们的任何组合，和/或其中所述一个或多个糖分子选自由以下组成的组：二糖，包括海藻糖、麦芽糖、乳糖、蔗糖、纤维二糖、壳二糖、曲二糖、黑曲霉糖、异麦芽糖、β,β-海藻糖、α,β-海藻糖、槐二糖、昆布二糖、龙胆二糖、海藻酮糖、松二糖、麦芽酮糖、白菌二糖、异麦芽酮糖、龙胆二酮糖、甘露二糖、蜜二糖、车前二糖、芦丁糖、芦丁酮糖、木二糖；糖醇，包括阿拉伯糖醇、赤藓糖醇、甘油、氢化葡萄糖浆(HSH)、异麦芽酮糖醇、乳糖醇、麦芽糖醇、甘露醇、山梨醇、木糖醇；多糖，包括淀粉、糖原、半乳多糖、菊粉、阿拉伯糖基木聚糖、纤维素、甲壳素和果胶，以及它们的任何组合。
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