CN114717201A - 适应vero细胞的肠道病毒D68、肠道病毒D68疫苗及其用途 - Google Patents

适应vero细胞的肠道病毒D68、肠道病毒D68疫苗及其用途 Download PDF

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CN114717201A
CN114717201A CN202210399314.8A CN202210399314A CN114717201A CN 114717201 A CN114717201 A CN 114717201A CN 202210399314 A CN202210399314 A CN 202210399314A CN 114717201 A CN114717201 A CN 114717201A
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黃立民
廖怡珍
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Abstract

本发明提供了适应vero细胞的肠道病毒D68、肠道病毒D68疫苗及其用途，其中包含适应vero细胞的肠道病毒D68的疫苗，在适应之前在基本上不含血清的培养基中培养所述vero细胞。本发明还公开了在受试者中诱导针对肠道病毒D68感染的免疫应答的方法，所述方法包括向所述受试者给药有效量的所述疫苗。

Description

适应vero细胞的肠道病毒D68、肠道病毒D68疫苗及其用途
技术领域
本发明涉及肠道病毒技术领域，具体而言，涉及适应vero细胞的肠道病毒D68、肠道病毒D68疫苗及其用途。
背景技术
肠道病毒D68(EV-D68)是小核糖核酸病毒科肠道病毒属的单链正义RNA病毒。EV-D68于1962年于加利福尼亚首次分离自四例肺炎及细支气管炎的住院儿科患者，并且其直到2000年代早期为止仍为罕见报导的感染(M.Vogt等人，Current Understanding ofHumoral Immunity to Enterovirus D68,JPIDS 2018:7(增刊2)s49-s53)。此后，EV-D68已经因在全球爆发而被认为是日益重要的病原体。越来越多的证据表明，它可引起急性弛缓性脊髓炎(AFM)，一种脊髓灰质炎样神经肌肉无力综合征)的爆发，与EV-D68呼吸系统疾病爆发同时发生。
EV-D68的治疗主要是支持性护理，因为没有许可的疫苗或治疗药物来预防或治疗EV-D68感染或AFM疾病。
Vero细胞系是监管机构(例如世界卫生组织)最广泛认可的用于生产人用病毒疫苗的连续细胞系之一。然而，W.Wei等人(ICAM-5/Telencephalin Is a Functional EntryReceptor for Enterovirus D68,Cell Host Microbe.2016年11月9日；20(5):631-641)和J Sun等人(Current Understanding of Human Enterovirus D68,Viruses 2019,11(6),490)进行的研究报告，vero细胞不允许EV-D68感染，除非ICAM5在EV-D68不允许的vero细胞中外源性表达。动物血清(诸如牛血清白蛋白)用于病毒培养和疫苗生产的用途具有许多缺点，包括暴露于不利因素，诸如内毒素、支原体、病毒污染物或FBS中的朊病毒蛋白(G.Gstranthuler等人，A plea to reduce or replace fetal bovine serum in cellculture media,cytotechnology.2013年10月；65(5):791-793)并且可能引起疫苗超敏反应，尤其是对牛肉或牛乳过敏的超敏反应(R.de Silva等人，Sensitization to bovineserum albumin as a possible cause of allergic reactions to vaccines,Vaccine，2017年3月13日；35(11):1494-1500)。
对EV-D68感染进行安全和有效的预防远未满足需求。本发明提供EV-D68疫苗以满足这些和其它需要。
发明内容
在一个实施方案中，提供了适应vero细胞的肠道病毒D68(EV-D68)，其与SEQ IDNO:2或SEQ ID NO:3具有95％-100％同一性的氨基酸序列。
在另一实施方案中，提供了适应vero细胞的肠道病毒D68(EV-D68)，所述EV-D68具有SEQ ID NO:1的氨基酸序列且具有至少一个氨基酸取代：
(a)氨基酸残基730被丝氨酸取代(G730S)；
(b)氨基酸残基1028被丙氨酸取代(T1028A)；
(c)氨基酸残基1171被丙氨酸取代(E1171A)；
(d)氨基酸残基1178被赖氨酸取代(E1178K)；
(e)氨基酸残基1365被丝氨酸取代(N1365S)；
(f)氨基酸残基1822被苏氨酸取代(I1822T)；以及
(g)氨基酸残基2179被甘氨酸取代(S2179G)。
本发明还公开了包含本文公开的适应vero细胞的EV-D68的疫苗和包含本文公开的疫苗的药物组合物。
本发明涉及生产本文所述的适应vero细胞的EV-D68的方法。
本发明还涉及通过给药有效量的本文所述疫苗在受试者中诱导针对EV-D68感染的免疫应答的方法。
在本专利中使用的术语“发明”、“本发明(this invention)”和“本发明(thepresent invention)”意图广泛地指本专利和以下专利权利要求的所有主题。包含这些术语的陈述不应被理解为限制本文所述的主题或限制以下专利权利要求的含义或范围。本专利所覆盖的本发明的实施方案由所附权利要求书而非本发明内容来限定。本发明内容是对本发明各方面的高度概括，并介绍了在以下具体实施方式部分中将进一步描述的一些概念。本发明内容并非意图在标识所要求保护的主题的关键或必要特征，也并非意图单独用于确定所要求保护的主题的范围。通过参考整个说明书的适当部分以及每个权利要求，应该理解本主题。
具体实施方式
下面对本申请实施例中的技术方案进行清楚、详尽地描述。
本申请实施例中，术语“一个”和“一种”(“a”和“an”)是指一个或多于一个(即，至少一个)该冠词的语法对象。
术语“受试者”可指怀疑患有EV-D68感染或处于患EV-D68感染风险中的脊椎动物。受试者包括温血动物，诸如哺乳动物，例如灵长类，更优选是人。非人灵长类动物也是受试者。术语受试者包括驯养的动物(诸如猫、狗等)、家畜(例如：牛、马、猪、绵羊、山羊等)和实验室动物(例如：小鼠、兔、大鼠、沙鼠、豚鼠等)。
本申请实施例中，“有效量”指足以引发针对EV-D68感染的免疫应答或中和抗体的疫苗或药物组合物的剂量。术语“有效量”和“治疗有效量”可互换使用。
本申请实施例中，“基本上不含”是指培养基或组合物含有少于5％、4％、3％、2％、1％或0.5％的特定物质，例如动物血清。在一些实施方案中，培养基或组合物不含特定物质。
本申请实施例中，所有数字可以理解为由“约”修饰。
本申请实施例中，术语“约”意指包括±10％的变化。
本发明提供了包含本文所述的适应vero细胞的EV-D68的疫苗。
本发明还提供了包含一种或多种适应vero细胞的EV-D68抗原或其免疫原性片段的疫苗。如本文所用，表达“适应vero细胞的EV-D68抗原”指能够刺激针对EV-D68的中和抗体的任何抗原。病毒抗原可包括外壳/衣壳蛋白或其片段、抗原决定簇和/或其它EV-D68蛋白。
在一个实施方案中，适应vero细胞的EV-D68与SEQ ID NO:2具有95、96、97、98、99或100％同一性的氨基酸序列。
在另一实施方案中，适应vero细胞的EV-D68与SEQ ID NO:3具有95、96、97、98、99或100％同一性的氨基酸序列。
在另一实施方案中，适应vero细胞的EV-D68具有SEQ ID NO:1的氨基酸序列和至少一个氨基酸取代，其选自由以下组成的组：
(a)氨基酸残基730被丝氨酸取代(G730S)；
(b)氨基酸残基1028被丙氨酸取代(T1028A)；
(c)氨基酸残基1171被丙氨酸取代(E1171A)；
(d)氨基酸残基1178被赖氨酸取代(E1178K)；
(e)氨基酸残基1365被丝氨酸取代(N1365S)；
(f)氨基酸残基1822被苏氨酸取代(I1822T)；以及
(g)氨基酸残基2179被甘氨酸取代(S2179G)。
表1显示在基本上不含血清的培养基中适应vero细胞的EV-D68的氨基酸取代
Figure BDA0003598982120000041
Figure BDA0003598982120000051
术语“取代”可以指用另一种氨基酸置换未修饰或野生型氨基酸序列中特定位置的氨基酸。例如，取代S2179G是指第2179位的丝氨酸被甘氨酸替代。
在一个实施方案中，在基本上不含血清的培养基中进行EV-D68在vero细胞中的适应。与在血清培养基中具有适应vero细胞的EV-D68的疫苗相比，在基本上不含血清的培养基中包含适应vero细胞的EV-D68的疫苗导致更好的免疫原性和更少的副作用。
在一个实施方案中，所述疫苗包含本文公开的无活性适应vero细胞的EV-D68。在另一个实施方案中，所述疫苗包含本文公开的活减毒的适应vero细胞的EV-D68。术语“减毒的”在本领域中具有其一般倾向，特别是倾向于毒性较小的病毒。
疫苗可进一步包含佐剂，与在不存在试剂的情况下给药抗原相比增强抗原特异性免疫应答产生的试剂。佐剂的非限制性实例包括铝、皂苷、ASO4、MF59、AS01b CpG1018(参见疾病控制和预防中心：佐剂和疫苗)。
制备疫苗的方法包括灭活或减毒EV-D68或其抗原，诸如通过加热、化学方法(例如：福尔马林)或物理方法或其组合，并分别或平行混合佐剂或其它添加剂；混合药学上可接受的溶媒、辅料或载体。
在儿科疫苗中，也可以包括其它相容的抗原，例如已知有效抗脑膜炎、链球菌感染或中耳炎的抗原。
本发明还提供了包含本文所述疫苗和药学上可接受的溶媒、辅料或载体的药物组合物。合适的溶媒是例如水、盐水、葡萄糖、甘油、乙醇等及其组合。合适的辅料是，例如，润湿剂、乳化剂或pH缓冲剂。药学上可接受的载体可含有生理上可接受的化合物，所述化合物用于例如稳定或增加或降低本发明的药物组合物的吸收或清除速率。生理学上可接受的化合物可以包括，例如，碳水化合物，诸如葡萄糖、蔗糖或葡聚糖，抗氧化剂，诸如抗坏血酸或谷胱甘肽，螯合剂、低分子量蛋白质、去污剂、脂质体载体，或其它稳定剂和/或缓冲剂。其它生理上可接受的化合物包括润湿剂、乳化剂、分散剂或防腐剂。辅料可以是非离子型表面活性剂、聚乙烯吡咯烷酮、人血清白蛋白、氢氧化铝、具有麻醉作用的试剂和各种未修饰的和衍生的环糊精。在一个实施方案中，非离子型表面活性剂可包括聚山梨酯20、聚山梨酯40、聚山梨酯60和聚山梨酯80。聚乙烯吡咯烷酮可以优选为Plasdone C15，一种药用级聚乙烯吡咯烷酮。包含这种辅料或载体的药物组合物通过公知的常规方法配制。
本发明的疫苗或药物组合物可被配制用于以下给药途径：肌内、皮内、口服、皮肤或通过皮下给药。只要可以诱导令人满意的免疫原性，也可采用其它给药方式。
本发明的疫苗或药物组合物可以制备成可注射的液体溶液或混悬液剂型，或制备为适于在注射前溶解或混悬在液体溶媒中的固体剂型。
本发明的另一方面涉及诱导针对EV-D68的免疫应答的方法，包括将有效量的本文所述疫苗或药物组合物给药于有需要的受试者。免疫应答包括但不限于IgM、IgG、IgA、IgD或IgE产生。由疫苗产生的抗体(诸如抗EV-D68抗体)固有地抑制EV-D68和/或中和EV-D68。
在本发明的另一方面，提供疫苗用于预防、改善或治疗EV-D68感染和/或其并发症。
EV-D68在每个疫苗剂量中的量选择为在受试者中诱导免疫保护应答而无明显不良副作用的量。特定疫苗的最佳量可以通过标准研究确定，包括观察受试者中的抗体滴度和其它应答。初次接种过程可包括1-3剂疫苗，以提供免疫保护应答的最佳时间间隔给予。疫苗可以间隔(例如：每两年)通过加强剂来补充，被设计为维持令人满意的保护性免疫水平。
疫苗可以以特定时间间隔以立即常用的方式给药，加上或不加上一个或多个加强剂量，以实现几个月至几年之间的长期免疫保护作用。给药频率可以根据多种因素中的任一因素变化，例如症状的严重性、所需的免疫保护程度、疫苗是用于预防性目的还是治疗性目的等。在一个示例性实施方案中，根据本发明的疫苗或药物组合物每10年一次、每5年一次、每4年一次、每3年一次、每两年一次、每年一次、每6个月一次、每月一次、每月两次、每月三次、每隔一周(qow)、每周一次(QW)给药。该疫苗也可与EV-D68感染的其它支持性疗法同时或相继给药。
用于在基本上不含血清的培养基中在vero细胞中适应EV-D68的方法
本发明还涉及产生本文所述的适应vero细胞的EV-D68的方法，所述方法包含：
(a)用EV-D68感染vero细胞以繁殖EV-D68；
(b)在基本上不含血清的培养基中培养步骤(a)中的vero细胞；以及
(c)收获步骤(b)中的EV-D68以用于疫苗中。
具体而言，在基本上不含血清的培养基或不含血清的培养基中进行此类方法。基本上不含血清的培养基的非限制性实例包括OptiPROTMSFM。
仅出于说明性目的提供以下用于实施本发明的具体方面的实施例，并非意图以任何方式限制本发明的范围。
实施例1：EV-D68复制
第1步：EV-D68的分离和培养
EV-D68分离自台湾大学医院的患者，RD细胞获自ATCC、USA并在37℃下维持在含有10％胎牛血清(FBS)的达尔伯克改良Eagle培养基(DMEM)中。EV-D68感染的RD细胞在含有2％FBS的培养基中于33℃下在5％CO2中生长。3次传代后收集细胞上清液，其中培养的EV-D68(D68-P3)的基因组被扩增，并用SEQ ID NO:1的氨基酸序列测序。
第2步：EV-D68在血清饥饿的vero细胞中的适应
进行步骤1中EV-D68在血清饥饿的vero细胞系上的适应以及适应vero细胞的病毒的表征。
首先将vero细胞(可从台湾生物资源保存及研究中心商购)在含5％胎牛血清(FBS)的最低必需培养基(MEM)中继代培养，然后在无血清培养基(最低必需培养基)中继代培养24小时，形成血清饥饿的vero细胞。
在步骤1中，用EV-D68(D68-P3)以10的感染复数(MOI)感染血清饥饿的Vero细胞。随后通过在含有2％FBS的最低必需培养基(MEM)中于33℃下用病毒连续感染血清饥饿的Vero细胞系进行EV-D68在血清饥饿Vero细胞系上的3次传代。具体而言，收获第1代(P1)病毒/上清液并用于下一代。以这种方式，在血清饥饿的vero细胞系上对病毒进行三次连续传代，在该细胞系中观察到特征性细胞病变效应(CPE)。空斑测定后获得单空斑纯化的克隆，纯化的EV-D68(V#4)具有SEQ ID NO:2的氨基酸序列(含EV-D68-P3病毒株的三个氨基酸取代，即2C-E1171A、2C-E1178K和3D-S2179G)。
第3步：EV-D68在无血清培养基中在血清饥饿的Vero细胞中的适应
在步骤2中，用纯化的EV-D68(V#4)以0.025的感染复数(MOI)感染血清饥饿的vero细胞。随后通过在无血清培养基OptiPRO-SFM(可从Gibco，USA商购)中于33℃下用EV-D68病毒连续感染vero细胞进行EV-D68在血清饥饿的vero细胞系中适应的12次传代。当观察到90％的细胞病变效应(CPE)时收获无细胞上清液并储存在-80℃下。
扩增并测序第8代EV-D68和第12代EV-D68(OP-V#4)的基因组，其均具有SEQ IDNO:3的氨基酸序列(具有D68-P3病毒株的7个氨基酸取代，即E1171A、E1178、S2179G、G730S、T1028A、N1365S和I1822T)。
实施例2：EV-D68疫苗的制备
当观察到90％的细胞病变效应(CPE)时，以3dpi从实施例1的无细胞上清培养物中收集EV-D68(OP-V#4)。随后使用Amicon Ultra 100kDa离心过滤器浓缩和纯化EV-D68(OP-V#4)，然后使用0.02％甲醛在37℃下灭活长达10天(下文称为灭活的EV-D68)。将灭活的EV-D68(5μg)与氢氧化铝(佐剂)混合，形成EV-D68疫苗的实施方案。
实施例3：EV-D68疫苗的免疫原性
EV-D68疫苗的免疫原性使用6周龄雌性BALB/c小鼠评价。三只小鼠，每只在第0周、第2周和第4周通过肌内注射给予实施例2的EV-D68疫苗。每次免疫接种两周(第2周、第4周和第6周)后收集血清样品，并加热(56℃，30min)以灭活补体。使用微中和试验测量抗EV-D68的血清中和抗体滴度。表2显示了三只小鼠中的中和抗体滴度，表3显示了log2转化的中和抗体滴度。
表2、小鼠中的中和抗体滴度
Figure BDA0003598982120000091
表3、小鼠中的中和抗体滴度(log2)
Figure BDA0003598982120000092
表2和3的结果显示本发明的EV-D68疫苗诱导强的EV-D68特异性免疫应答。
虽然本发明公开披露如上，但本发明公开的保护范围并非仅限于此。本领域技术人员在不脱离本发明公开的精神和范围的前提下，可进行各种变更与修改，这些变更与修改均将落入本发明的保护范围。
序列表
<110> 景均股份有限公司
<120> 适应 vero细胞的肠道病毒 D68、肠道病毒 D68疫苗及其用途
<150> US 63/176,274
<151> 2021-04-17
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2068
<212> PRT
<213> 肠道病毒 D68(肠道病毒 D68 )
<400> 1
Met Gly Ala Gln Val Thr Arg Gln Gln Thr Gly Thr His Glu Asn Ala
1 5 10 15
Asn Val Ala Thr Asn Gly Ser His Ile Thr Tyr Asn Gln Ile Asn Phe
20 25 30
Tyr Lys Asp Ser Tyr Ala Ala Ser Ala Ser Lys Gln Asp Phe Ser Gln
35 40 45
Asp Pro Ser Lys Phe Thr Glu Pro Val Val Glu Gly Leu Lys Ala Gly
50 55 60
Ala Pro Val Leu Lys Ser Pro Ser Ala Glu Ala Cys Gly Tyr Ser Asp
65 70 75 80
Arg Val Leu Gln Leu Lys Leu Gly Asn Ser Ala Ile Val Thr Gln Glu
85 90 95
Ala Ala Asn Tyr Cys Cys Ala Tyr Gly Lys Pro Thr Gln Pro Glu Thr
100 105 110
Ala Thr Asp Arg Phe Tyr Thr Leu Arg Ser Val Lys Trp Glu Ala Thr
115 120 125
Ser Thr Gly Trp Trp Trp Lys Leu Pro Asp Ala Leu Asn Asn Ile Gly
130 135 140
Met Phe Gly Gln Asn Val Gln His His Tyr Leu Tyr Arg Ser Gly Phe
145 150 155 160
Leu Ile His Val Gln Cys Asn Ala Thr Lys Phe His Gln Gly Ala Leu
165 170 175
Leu Val Val Ala Ile Pro Glu His Gln Arg Gly Ala His Asn Thr Thr
180 185 190
Thr Ser Pro Gly Phe Asp Asp Ile Met Lys Gly Glu Ala Gly Gly Thr
195 200 205
Phe Asn His Pro Tyr Val Leu Asp Asp Gly Thr Ser Leu Ala Cys Ala
210 215 220
Thr Ile Phe Pro His Gln Trp Ile Asn Leu Arg Thr Asn Asn Ser Ala
225 230 235 240
Thr Ile Val Leu Pro Trp Met Asn Ala Ala Pro Met Asp Phe Pro Leu
245 250 255
Arg His Asn Gln Trp Thr Leu Ala Ile Ile Pro Val Val Pro Leu Gly
260 265 270
Thr Arg Thr Met Ser Ser Met Val Pro Ile Thr Val Ser Thr Gln Gly
275 280 285
Val Pro Thr Tyr Leu Leu Pro Gly Ser Gly Gln Phe Leu Thr Thr Asp
290 295 300
Asp His Ser Ser Ala Pro Val Leu Pro Cys Phe Asn Pro Thr Pro Glu
305 310 315 320
Met His Ile Pro Gly Gln Val Arg Asn Met Leu Glu Val Val Gln Val
325 330 335
Glu Ser Met Met Glu Ile Asn Asn Thr Glu Ser Ala Val Gly Met Glu
340 345 350
Arg Leu Lys Val Asp Ile Ser Ala Leu Thr Asp Val Asp Gln Leu Leu
355 360 365
Phe Asn Ile Pro Leu Asp Ile Gln Leu Asp Gly Pro Leu Arg Asn Thr
370 375 380
Leu Val Gly Asn Ile Ser Arg Tyr Tyr Thr His Trp Ser Gly Ser Leu
385 390 395 400
Glu Met Thr Phe Met Phe Cys Gly Ser Phe Met Ala Thr Gly Lys Leu
405 410 415
Ile Leu Cys Tyr Thr Pro Pro Gly Gly Ser Cys Pro Thr Thr Arg Glu
420 425 430
Thr Ala Met Leu Gly Thr His Ile Val Trp Asp Phe Gly Leu Gln Ser
435 440 445
Ser Ile Thr Leu Ile Ile Pro Trp Ile Ser Gly Ser His Tyr Arg Met
450 455 460
Phe Cys Phe Met Gln Thr Asn Leu Ile Val Pro Ser Glu Ser Ser Asp
465 470 475 480
Thr Cys Ser Leu Ile Gly Phe Ile Ala Ala Lys Asp Asp Phe Ser Leu
485 490 495
Arg Leu Met Arg Asp Ser Pro Asp Ile Gly Gln Leu Asp His Leu His
500 505 510
Gly Ala Glu Ala Ala Tyr Gln Ile Glu Ser Ile Ile Lys Thr Ala Thr
515 520 525
Asp Thr Val Lys Ser Glu Ile Asn Ala Glu Leu Gly Val Val Pro Ser
530 535 540
Leu Asn Ala Val Glu Thr Gly Ala Thr Ser Asn Thr Glu Pro Glu Glu
545 550 555 560
Ala Ile Gln Thr Arg Thr Val Ile Asn Gln His Gly Val Ser Glu Thr
565 570 575
Leu Val Glu Asn Phe Leu Gly Arg Ala Ala Leu Val Ser Lys Arg Ser
580 585 590
Phe Lys Tyr Lys Asp His Thr Ser Ser Ala Ala Arg Ala His Lys Asn
595 600 605
Phe Phe Lys Trp Thr Ile Asn Thr Arg Ser Phe Val Gln Leu Arg Arg
610 615 620
Lys Leu Glu Leu Phe Thr Tyr Leu Arg Phe Asp Ala Glu Ile Thr Ile
625 630 635 640
Leu Thr Thr Val Ala Val Asn Ser Ser Ser Asn Asn Thr Tyr Val Gly
645 650 655
Leu Pro Asp Leu Thr Leu Gln Ala Met Phe Val Pro Thr Gly Ala Leu
660 665 670
Thr Pro Glu Lys Gln Asp Ser Phe His Trp Gln Ser Gly Ser Asn Ala
675 680 685
Ser Val Phe Phe Lys Ile Ser Asp Pro Pro Ala Arg Met Thr Ile Pro
690 695 700
Phe Met Cys Ile Asn Ser Ala Tyr Ser Val Phe Tyr Asp Gly Phe Ala
705 710 715 720
Gly Phe Glu Lys Thr Gly Leu Tyr Gly Ile Asn Pro Ala Asp Thr Ile
725 730 735
Gly Asn Leu Cys Ile Arg Ile Val Asn Glu His Gln Pro Val Gly Phe
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Thr Val Thr Val Arg Val Tyr Met Lys Pro Lys His Ile Lys Ala Trp
755 760 765
Ala Pro Arg Pro Pro Arg Thr Leu Pro Tyr Met Ser Ile Ala Asn Ala
770 775 780
Asn Tyr Lys Gly Lys Glu Arg Ala Pro Asn Ala Leu Asn Ala Ile Ile
785 790 795 800
Gly Asn Arg Asp Ser Val Lys Thr Met Pro His Asn Ile Val Thr Thr
805 810 815
Gly Pro Gly Phe Gly Gly Val Phe Val Arg Gln Ser Ala Ile Tyr Val
820 825 830
Asp Trp Gln Ser Asp Ile Leu Val Thr Pro Ile Ala Ala His Gly Arg
835 840 845
His Gln Ile Ala Arg Cys Lys Cys Asn Thr Gly Val Tyr Tyr Cys Arg
850 855 860
His Lys Asp Arg Ser Tyr Pro Ile Cys Phe Glu Gly Pro Arg Ile Gln
865 870 875 880
Trp Ile Glu Gln Asn Glu Tyr Tyr Pro Ala Arg Tyr Gln Thr Asn Val
885 890 895
Leu Leu Ala Val Gly Pro Ala Glu Ala Gly Asp Cys Gly Gly Leu Leu
900 905 910
Val Cys Pro His Gly Val Ile Gly Leu Leu Thr Ala Gly Gly Gly Gly
915 920 925
Ile Val Ala Phe Thr Asp Ile Arg Asn Leu Leu Trp Leu Asp Thr Asp
930 935 940
Val Met Glu Gln Gly Ile Thr Asp Tyr Ile Gln Asn Leu Gly Asn Ala
945 950 955 960
Phe Gly Ala Gly Phe Thr Glu Thr Ile Ser Asn Lys Ala Lys Glu Val
965 970 975
Gln Asp Met Leu Ile Gly Glu Ser Ser Leu Leu Glu Lys Leu Leu Lys
980 985 990
Ala Leu Ile Lys Ile Ile Ser Ala Leu Val Ile Val Ile Leu Gly Cys
995 1000 1005
His Asp Ser Pro Trp Ser Tyr Leu Lys Gln Lys Val Cys Ser Tyr Leu
1010 1015 1020
Gly Ile Pro Tyr Val Pro Arg Gln Ser Glu Ser Trp Leu Lys Lys Phe
1025 1030 1035 1040
Thr Glu Ala Cys Asn Ala Leu Arg Gly Leu Asp Trp Leu Ser Gln Lys
1045 1050 1055
Ile Asp Lys Phe Ile Asn Trp Leu Lys Thr Lys Ile Leu Pro Glu Ala
1060 1065 1070
Arg Glu Lys Tyr Glu Phe Val Gln Arg Leu Lys Gln Leu Pro Val Ile
1075 1080 1085
Glu Asn Gln Val Ser Thr Ile Glu His Ser Cys Pro Thr Thr Glu Gln
1090 1095 1100
Gln Gln Ala Leu Phe Asn Asn Val Gln Tyr Tyr Ser His Tyr Cys Arg
1105 1110 1115 1120
Lys Tyr Ala Pro Leu Tyr Ala Val Glu Ala Lys Arg Val Met Ala Leu
1125 1130 1135
Glu Lys Lys Ile Asn Asn Tyr Ile Gln Phe Lys Ser Lys Ser Arg Ile
1140 1145 1150
Glu Pro Val Cys Leu Ile Ile His Gly Ser Pro Gly Thr Gly Lys Ser
1155 1160 1165
Val Ala Ser Asn Leu Ile Ala Arg Ala Ile Thr Glu Lys Leu Gly Gly
1170 1175 1180
Asp Ile Tyr Ser Leu Pro Pro Asp Pro Lys Tyr Phe Asp Gly Tyr Lys
1185 1190 1195 1200
Gln Gln Thr Val Val Leu Met Asp Asp Leu Met Gln Asn Pro Asp Gly
1205 1210 1215
Asn Asp Ile Ser Met Phe Cys Gln Met Val Ser Thr Val Asp Phe Ile
1220 1225 1230
Pro Pro Met Ala Ser Leu Glu Glu Lys Gly Thr Leu Tyr Thr Ser Pro
1235 1240 1245
Phe Leu Ile Ala Thr Thr Asn Ala Gly Ser Ile His Ala Pro Thr Val
1250 1255 1260
Ser Asp Ser Lys Ala Leu Ser Arg Arg Phe Lys Phe Asp Val Asp Ile
1265 1270 1275 1280
Glu Val Thr Asp Ser Tyr Lys Asp Ser Asn Lys Leu Asp Met Ser Arg
1285 1290 1295
Ala Val Glu Met Cys Lys Pro Asp Ser Cys Ala Pro Ala Asn Tyr Lys
1300 1305 1310
Arg Cys Cys Pro Leu Ile Cys Gly Lys Ala Ile Gln Phe Arg Asp Arg
1315 1320 1325
Arg Thr Asn Ala Arg Ser Thr Ile Asp Met Leu Val Thr Asp Ile Ile
1330 1335 1340
Lys Glu Tyr Arg Thr Arg Asn Ser Thr Gln Asp Lys Leu Glu Ala Leu
1345 1350 1355 1360
Phe Gln Gly Pro Pro Pro Asp Ala Ile Asn Asp Leu Leu Arg Ser Val
1365 1370 1375
Asp Ser Gln Glu Val Arg Asp Tyr Cys Gln Lys Lys Gly Trp Ile Val
1380 1385 1390
Ile His Pro Ser Asn Glu Leu Leu Val Glu Lys His Ile Ser Arg Ala
1395 1400 1405
Phe Ile Thr Leu Gln Ala Ile Ala Thr Phe Val Ser Ile Ala Gly Val
1410 1415 1420
Val Tyr Val Ile Tyr Lys Leu Phe Ala Gly Ile Gln Gly Pro Tyr Thr
1425 1430 1435 1440
Gly Ile Pro Asn Pro Lys Pro Lys Val Pro Ser Leu Arg Thr Ala Lys
1445 1450 1455
Val Gln Gly Pro Gly Phe Asp Phe Ala Gln Ala Ile Met Lys Lys Asn
1460 1465 1470
Thr Val Ile Ala Arg Thr Glu Lys Gly Glu Phe Thr Met Leu Gly Val
1475 1480 1485
Tyr Asp Arg Val Ala Val Ile Pro Thr His Ala Ser Val Gly Glu Thr
1490 1495 1500
Ile Tyr Ile Asp Asp Val Glu Thr Arg Val Leu Asp Ala Cys Ala Leu
1505 1510 1515 1520
Arg Asp Leu Thr Asp Thr Asn Leu Glu Ile Thr Ile Val Lys Leu Asp
1525 1530 1535
Arg Asn Gln Lys Phe Arg Asp Ile Arg Ser Val His Thr Ser Lys Phe
1540 1545 1550
Pro Asn Met Tyr Ile Pro Val Gly Gln Val Thr Asn Tyr Gly Phe Leu
1555 1560 1565
Asn Leu Gly Gly Thr Pro Thr His Arg Ile Leu Met Tyr Asn Phe Pro
1570 1575 1580
Thr Arg Ala Gly Gln Cys Gly Gly Val Val Thr Thr Thr Gly Lys Val
1585 1590 1595 1600
Ile Gly Ile His Val Gly Gly Asn Gly Ala Gln Gly Phe Ala Ala Met
1605 1610 1615
Leu Leu His Ser Tyr Phe Thr Asp Thr Gln Gly Glu Ile Val Ser Ser
1620 1625 1630
Glu Lys Ser Gly Val Cys Ile Asn Ala Pro Ala Lys Thr Lys Leu Gln
1635 1640 1645
Pro Ser Val Phe His Gln Val Phe Glu Gly Ser Lys Glu Pro Ala Val
1650 1655 1660
Leu Asn Pro Lys Asp Pro Arg Leu Lys Thr Asp Phe Glu Glu Ala Ile
1665 1670 1675 1680
Phe Ser Lys Tyr Thr Gly Asn Lys Ile Met Leu Met Asp Glu Tyr Met
1685 1690 1695
Glu Glu Ala Val Asp His Tyr Val Gly Cys Leu Glu Pro Leu Asp Ile
1700 1705 1710
Ser Val Asp Pro Ile Pro Leu Glu Ser Ala Met Tyr Gly Met Asp Gly
1715 1720 1725
Leu Glu Ala Leu Asp Leu Thr Thr Ser Ala Gly Phe Pro Tyr Leu Leu
1730 1735 1740
Gln Gly Lys Lys Lys Arg Asp Ile Phe Asn Arg His Thr Arg Asp Thr
1745 1750 1755 1760
Ser Glu Met Thr Lys Met Leu Glu Lys Tyr Gly Val Asp Leu Pro Phe
1765 1770 1775
Val Thr Phe Val Lys Asp Glu Leu Arg Ser Arg Glu Lys Val Glu Lys
1780 1785 1790
Gly Lys Ser Arg Leu Ile Glu Ala Ser Ser Leu Asn Asp Ser Val Ala
1795 1800 1805
Met Arg Val Ala Phe Gly Asn Leu Tyr Ala Thr Phe His Asn Asn Pro
1810 1815 1820
Gly Thr Ala Thr Gly Ser Ala Val Gly Cys Asp Pro Asp Ile Phe Trp
1825 1830 1835 1840
Ser Lys Ile Pro Ile Leu Leu Asp Gly Glu Ile Phe Ala Phe Asp Tyr
1845 1850 1855
Thr Gly Tyr Asp Ala Ser Leu Ser Pro Val Trp Phe Ala Cys Leu Lys
1860 1865 1870
Lys Val Leu Val Lys Leu Gly Tyr Thr His Gln Thr Ser Phe Ile Asp
1875 1880 1885
Tyr Leu Cys His Ser Val His Leu Tyr Lys Asp Arg Lys Tyr Ile Val
1890 1895 1900
Asn Met Ile Asn Asn Ile Ile Ile Arg Thr Leu Leu Ile Arg Val Tyr
1905 1910 1915 1920
Lys Gly Ile Asp Leu Asp Gln Phe Lys Met Ile Ala Tyr Gly Asp Asp
1925 1930 1935
Val Ile Ala Ser Tyr Pro His Lys Ile Asp Pro Gly Leu Leu Ala Glu
1940 1945 1950
Ala Gly Lys His Tyr Gly Leu Val Met Thr Pro Ala Asp Lys Gly Thr
1955 1960 1965
Ser Phe Val Asp Thr Asn Trp Glu Asn Val Thr Phe Leu Lys Arg Tyr
1970 1975 1980
Phe Arg Ala Asp Asp Gln Tyr Pro Phe Leu Ile His Pro Val Met Pro
1985 1990 1995 2000
Met Lys Glu Ile His Glu Ser Ile Arg Trp Thr Lys Asp Pro Arg Asn
2005 2010 2015
Thr Gln Asp His Val Arg Ser Leu Cys Tyr Leu Ala Trp His Asn Gly
2020 2025 2030
Glu Glu Ala Tyr Asn Glu Phe Cys Arg Lys Ile Arg Ser Val Pro Val
2035 2040 2045
Gly Arg Ala Leu Thr Leu Pro Ala Tyr Ser Ser Leu Arg Arg Lys Trp
2050 2055 2060
Leu Asp Ser Phe
2065
<210> 2
<211> 1965
<212> PRT
<213> 肠道病毒 D68(肠道病毒 D68 )
<400> 2
Met Gly Ala Gln Val Thr Arg Gln Gln Thr Gly Thr His Glu Asn Ala
1 5 10 15
Asn Val Ala Thr Asn Gly Ser His Ile Thr Tyr Asn Gln Ile Asn Phe
20 25 30
Tyr Lys Asp Ser Tyr Ala Ala Ser Ala Ser Lys Gln Asp Phe Ser Gln
35 40 45
Asp Pro Ser Lys Phe Thr Glu Pro Val Val Glu Gly Leu Lys Ala Gly
50 55 60
Ala Pro Val Leu Lys Ser Pro Ser Ala Glu Ala Cys Gly Tyr Ser Asp
65 70 75 80
Arg Val Leu Gln Leu Lys Leu Gly Asn Ser Ala Ile Val Thr Gln Glu
85 90 95
Ala Ala Asn Tyr Cys Cys Ala Tyr Gly Glu Trp Pro Asn Tyr Leu Pro
100 105 110
Asp His Glu Ala Val Ala Ile Asp Lys Pro Thr Gln Pro Glu Thr Ala
115 120 125
Thr Asp Arg Phe Tyr Thr Leu Arg Ser Val Lys Trp Glu Ala Thr Ser
130 135 140
Thr Gly Trp Trp Trp Lys Leu Pro Asp Ala Leu Asn Asn Ile Gly Met
145 150 155 160
Phe Gly Gln Asn Val Cys Asn Ala Thr Lys Phe His Gln Gly Ala Leu
165 170 175
Leu Val Val Ala Ile Pro Glu His Gln Arg Gly Ala His Asn Thr Thr
180 185 190
Thr Ser Pro Gly Phe Asp Asp Ile Met Lys Gly Glu Ala Gly Gly Thr
195 200 205
Phe Asn His Pro Tyr Val Leu Asp Asp Gly Thr Ser Leu Ala Cys Ala
210 215 220
Thr Ile Phe Pro His Gln Trp Ile Asn Leu Arg Thr Asn Asn Ser Ala
225 230 235 240
Thr Ile Val Leu Pro Trp Met Asn Ala Ala Pro Met Asp Phe Pro Leu
245 250 255
Arg His Asn Gln Trp Thr Leu Ala Ile Ile Pro Val Val Pro Leu Gly
260 265 270
Thr Arg Thr Met Ser Ser Met Val Pro Ile Thr Val Ser Ile Ala Pro
275 280 285
Met Cys Cys Glu Phe Asn Gly Leu Arg His Ala Ile Thr Gln Gly Val
290 295 300
Pro Thr Tyr Leu Leu Pro Gly Ser Gly Gln Phe Leu Thr Thr Asp Asp
305 310 315 320
His Ser Ser Ala Pro Val Leu Pro Cys Phe Asn Pro Thr Pro Glu Met
325 330 335
His Ile Pro Gly Gln Val Arg Asn Met Glu Ser Ala Val Gly Met Glu
340 345 350
Arg Leu Lys Val Asp Ile Ser Ala Leu Thr Asp Val Asp Gln Leu Leu
355 360 365
Phe Asn Ile Pro Leu Asp Ile Gln Leu Asp Gly Pro Leu Arg Asn Thr
370 375 380
Leu Val Gly Asn Ile Ser Arg Tyr Tyr Thr His Trp Ser Gly Ser Leu
385 390 395 400
Glu Met Thr Phe Met Phe Cys Gly Ser Phe Met Ala Thr Gly Lys Leu
405 410 415
Ile Leu Cys Tyr Thr Pro Pro Gly Gly Ser Cys Pro Thr Thr Arg Glu
420 425 430
Thr Ala Met Leu Gly Thr His Ile Val Trp Asp Phe Gly Leu Gln Ser
435 440 445
Ser Ile Thr Leu Ile Ile Pro Trp Ile Ser Gly Ser His Tyr Arg Met
450 455 460
Phe Asn Asn Asp Ala Lys Ser Thr Asn Ala Asn Val Gly Tyr Val Thr
465 470 475 480
Cys Phe Met Gln Thr Asn Leu Ile Val Pro Ser Glu Ser Ser Asp Thr
485 490 495
Cys Ser Leu Ile Gly Phe Ile Ala Ala Lys Asp Asp Phe Ser Leu Arg
500 505 510
Leu Met Arg Asp Ser Pro Asp Ile Gly Gln Leu Asp His Leu His Gly
515 520 525
Ala Glu Ala Ala Tyr Gln Ile Glu Ser Ile Ile Lys Thr Ala Thr Asp
530 535 540
Thr Val Lys Ser Glu Ile Asn Ala Glu Leu Gly Val Val Pro Ser Leu
545 550 555 560
Asn Ala Val Glu Thr Gly Ala Thr Ser Asn Thr Glu Pro Glu Glu Ala
565 570 575
Ile Gln Thr Arg Thr Val Ile Asn Gln His Gly Val Ser Glu Thr Leu
580 585 590
Val Glu Asn Phe Leu Gly Arg Ala Ala Leu Val Ser Lys Arg Ser Phe
595 600 605
Lys Tyr Lys Asp His Thr Ser Ser Ala Ala Arg Ala His Lys Asn Phe
610 615 620
Phe Lys Trp Thr Ile Asn Thr Arg Ser Phe Val Gln Leu Arg Arg Lys
625 630 635 640
Leu Glu Leu Phe Thr Tyr Leu Arg Phe Asp Ala Glu Ile Thr Ile Leu
645 650 655
Thr Thr Val Ala Val Asn Ser Ser Ser Asn Asn Thr Tyr Val Gly Leu
660 665 670
Pro Asp Leu Thr Leu Gln Ala Met Phe Val Pro Thr Gly Ala Leu Thr
675 680 685
Pro Glu Lys Gln Asp Ser Phe His Trp Gln Ser Gly Ser Asn Ala Ser
690 695 700
Val Met Cys Ile Asn Ser Ala Tyr Ser Val Phe Tyr Asp Gly Phe Ala
705 710 715 720
Gly Phe Glu Lys Thr Gly Leu Tyr Gly Ile Asn Pro Ala Asp Thr Ile
725 730 735
Gly Asn Leu Cys Ile Arg Ile Val Asn Glu His Gln Pro Val Gly Phe
740 745 750
Thr Val Thr Val Arg Val Tyr Met Lys Pro Lys His Ile Lys Ala Trp
755 760 765
Ala Pro Arg Pro Pro Arg Thr Leu Pro Tyr Met Ser Ile Ala Asn Ala
770 775 780
Asn Tyr Lys Gly Lys Glu Arg Ala Pro Asn Ala Leu Asn Ala Ile Ile
785 790 795 800
Gly Asn Arg Asp Ser Val Lys Thr Met Pro His Asn Ile Val Thr Thr
805 810 815
Gly Pro Gly Phe Gly Gly Val Phe Val Gly Ser Phe Lys Ile Ile Asn
820 825 830
Tyr His Leu Ala Thr Thr Glu Glu Arg Gln Ser Ala Ile Tyr Val Asp
835 840 845
Trp Gln Ser Asp Ile Leu Val Thr Pro Ile Ala Ala His Gly Arg His
850 855 860
Gln Ile Ala Arg Cys Lys Cys Asn Thr Gly Val Tyr Tyr Cys Arg His
865 870 875 880
Lys Asp Arg Ser Tyr Glu Tyr Tyr Pro Ala Arg Tyr Gln Thr Asn Val
885 890 895
Leu Leu Ala Val Gly Pro Ala Glu Ala Gly Asp Cys Gly Gly Leu Leu
900 905 910
Val Cys Pro His Gly Val Ile Gly Leu Leu Thr Ala Gly Gly Gly Gly
915 920 925
Ile Val Ala Phe Thr Asp Ile Arg Asn Leu Leu Trp Leu Asp Thr Asp
930 935 940
Val Met Glu Gln Gly Ile Thr Asp Tyr Ile Gln Asn Leu Gly Asn Ala
945 950 955 960
Phe Gly Ala Gly Phe Thr Glu Thr Ile Ser Asn Lys Ala Lys Glu Val
965 970 975
Gln Asp Met Leu Ile Gly Glu Ser Ser Leu Leu Glu Lys Leu Leu Lys
980 985 990
Ala Leu Ile Lys Ile Ile Ser Ala Leu Val Ile Val Ile Arg Asn Ser
995 1000 1005
Glu Asp Leu Val Thr Val Thr Ala Thr Leu Ala Leu Leu Gly Cys His
1010 1015 1020
Asp Ser Pro Trp Ser Tyr Leu Lys Gln Lys Val Cys Ser Tyr Leu Gly
1025 1030 1035 1040
Ile Pro Tyr Val Pro Arg Gln Ser Glu Ser Trp Leu Lys Lys Phe Thr
1045 1050 1055
Glu Ala Cys Asn Ala Leu Arg Gly Leu Asp Trp Leu Ser Gln Lys Ile
1060 1065 1070
Asp Lys Phe Ile Asn Trp Leu Lys Thr Lys Ile Leu Pro Glu Ala Arg
1075 1080 1085
Glu Lys Tyr Glu Phe Val Gln Arg Leu Lys Gln Leu Pro Val Ile Glu
1090 1095 1100
Asn Gln Val Ser Thr Ile Ala His Ser Cys Pro Thr Thr Lys Gln Gln
1105 1110 1115 1120
Gln Ala Leu Phe Asn Asn Val Gln Tyr Tyr Ser His Tyr Cys Arg Lys
1125 1130 1135
Tyr Ala Pro Leu Tyr Ala Val Glu Ala Lys Arg Val Met Ala Leu Glu
1140 1145 1150
Lys Lys Ile Asn Asn Tyr Ile Gln Phe Lys Ser Lys Ser Arg Ile Glu
1155 1160 1165
Pro Val Cys Leu Ile Ile His Gly Ser Pro Gly Thr Gly Lys Ser Val
1170 1175 1180
Ala Ser Asn Leu Ile Ala Arg Ala Ile Thr Glu Lys Leu Gly Gly Asp
1185 1190 1195 1200
Ile Tyr Ser Leu Pro Pro Asp Pro Lys Tyr Phe Asp Gly Tyr Lys Gln
1205 1210 1215
Gln Thr Val Val Leu Met Asp Asp Leu Met Gln Asn Pro Asp Gly Asn
1220 1225 1230
Asp Ile Ser Met Phe Cys Gln Met Val Ser Thr Val Asp Thr Ser Pro
1235 1240 1245
Phe Leu Ile Ala Thr Thr Asn Ala Gly Ser Ile His Ala Pro Thr Val
1250 1255 1260
Ser Asp Ser Lys Ala Leu Ser Arg Arg Phe Lys Phe Asp Val Asp Ile
1265 1270 1275 1280
Glu Val Thr Asp Ser Tyr Lys Asp Ser Asn Lys Leu Asp Met Ser Arg
1285 1290 1295
Ala Val Glu Met Cys Lys Pro Asp Ser Cys Ala Pro Ala Asn Tyr Lys
1300 1305 1310
Arg Cys Cys Pro Leu Ile Cys Gly Lys Ala Ile Gln Phe Arg Asp Arg
1315 1320 1325
Arg Thr Asn Ala Arg Ser Thr Ile Asp Met Leu Val Thr Asp Ile Ile
1330 1335 1340
Lys Glu Tyr Arg Thr Arg Asn Ser Thr Gln Asp Lys Leu Glu Ala Leu
1345 1350 1355 1360
Phe Gln Gly Pro Pro Gln Phe Lys Glu Ile Lys Ile Ser Val Thr Pro
1365 1370 1375
Asp Thr Pro Ala Pro Asp Ala Ile Asn Asp Leu Leu Arg Ser Val Asp
1380 1385 1390
Ser Gln Glu Val Arg Asp Tyr Cys Gln Lys Lys Gly Trp Ile Val Ile
1395 1400 1405
His Pro Ser Asn Glu Leu Leu Val Glu Lys His Ile Ser Arg Ala Phe
1410 1415 1420
Ile Tyr Val Ile Tyr Lys Leu Phe Ala Gly Ile Gln Gly Pro Tyr Thr
1425 1430 1435 1440
Gly Ile Pro Asn Pro Lys Pro Lys Val Pro Ser Leu Arg Thr Ala Lys
1445 1450 1455
Val Gln Gly Pro Gly Phe Asp Phe Ala Gln Ala Ile Met Lys Lys Asn
1460 1465 1470
Thr Val Ile Ala Arg Thr Glu Lys Gly Glu Phe Thr Met Leu Gly Val
1475 1480 1485
Tyr Asp Arg Val Ala Val Ile Pro Thr His Ala Ser Val Gly Glu Thr
1490 1495 1500
Ile Tyr Ile Asp Asp Val Glu Thr Arg Val Leu Asp Ala Cys Ala Leu
1505 1510 1515 1520
Arg Asp Leu Thr Asp Thr Asn Leu Glu Ile Thr Ile Val Lys Leu Asp
1525 1530 1535
Arg Asn Gln Lys Phe Arg Asp Ile Arg His Phe Leu Pro Arg Tyr Glu
1540 1545 1550
Asp Asp Tyr Asn Asp Ala Val Leu Ser Val His Thr Ser Lys Phe Pro
1555 1560 1565
Asn Met Tyr Ile Pro Val Gly Gln Val Thr Asn Tyr Gly Phe Leu Asn
1570 1575 1580
Leu Gly Gly Thr Pro Thr His Arg Ile Leu Met Tyr Asn Phe Pro Thr
1585 1590 1595 1600
Arg Ala Gly Gln Cys Gly Gly Val Val Thr Thr Thr Gly Lys Val Ile
1605 1610 1615
Gly Ile His Val Gly Gly Asn Gly Ala Gln Gly Phe Ala Ala Met Leu
1620 1625 1630
Leu His Ser Tyr Phe Thr Asp Thr Gln Gly Glu Ile Val Ser Ser Glu
1635 1640 1645
Lys Ser Gly Val Cys Ile Asn Ala Pro Ala Lys Thr Lys Leu Gln Pro
1650 1655 1660
Ser Val Phe His Gln Val Phe Glu Gly Ser Lys Glu Pro Ala Val Leu
1665 1670 1675 1680
Asn Pro Lys Asp Pro Arg Leu Lys Thr Asp Phe Glu Glu Ala Ile Phe
1685 1690 1695
Ser Lys Tyr Thr Gly Asn Lys Ile Met Leu Met Asp Glu Tyr Met Glu
1700 1705 1710
Glu Ala Val Asp His Tyr Val Gly Cys Leu Glu Pro Leu Asp Ile Ser
1715 1720 1725
Val Asp Pro Ile Pro Leu Glu Ser Ala Met Tyr Gly Met Asp Gly Leu
1730 1735 1740
Glu Ala Leu Asp Leu Thr Thr Ser Ala Gly Phe Pro Tyr Leu Leu Gln
1745 1750 1755 1760
Gly Lys Lys Lys Arg Asp Ile Phe Asn Arg His Thr Arg Asp Thr Ser
1765 1770 1775
Glu Met Thr Lys Met Leu Glu Lys Tyr Ser Arg Glu Lys Val Glu Lys
1780 1785 1790
Gly Lys Ser Arg Leu Ile Glu Ala Ser Ser Leu Asn Asp Ser Val Ala
1795 1800 1805
Met Arg Val Ala Phe Gly Asn Leu Tyr Ala Thr Phe His Asn Asn Pro
1810 1815 1820
Gly Thr Ala Thr Gly Ser Ala Val Gly Cys Asp Pro Asp Ile Phe Trp
1825 1830 1835 1840
Ser Lys Ile Pro Ile Leu Leu Asp Gly Glu Ile Phe Ala Phe Asp Tyr
1845 1850 1855
Thr Gly Tyr Asp Ala Ser Leu Ser Pro Val Trp Phe Ala Cys Leu Lys
1860 1865 1870
Lys Val Leu Val Lys Leu Gly Tyr Thr His Gln Thr Ser Phe Ile Asp
1875 1880 1885
Tyr Leu Cys His Ser Val His Leu Tyr Lys Asp Arg Lys Tyr Ile Val
1890 1895 1900
Asn Gly Gly Met Pro Ser Gly Ser Ser Gly Thr Ser Ile Phe Asn Thr
1905 1910 1915 1920
Met Ile Asn Asn Ile Ile Ile Arg Thr Leu Leu Ile Arg Val Tyr Lys
1925 1930 1935
Gly Ile Asp Leu Asp Gln Phe Lys Met Ile Ala Tyr Gly Asp Asp Val
1940 1945 1950
Ile Ala Ser Tyr Pro His Lys Ile Asp Pro Gly Leu Leu
1955 1960 1965

Claims (17)

1.一种适应vero细胞的肠道病毒D68，其特征在于，所述肠道病毒D68与SEQ ID NO:2或SEQ ID NO:3具有95％-100％同一性的氨基酸序列。
2.如权利要求1所述的适应vero细胞的肠道病毒D68，其特征在于，所述肠道病毒D68具有SEQ ID NO:2的氨基酸序列。
3.如权利要求1所述的适应vero细胞的肠道病毒D68，其特征在于，所述肠道病毒D68具有SEQ ID NO:3的氨基酸序列。
4.一种适应vero细胞的肠道病毒D68，其特征在于，所述肠道病毒D68具有SEQ ID NO:1的氨基酸序列和至少一个氨基酸取代，所述氨基酸取代选自由以下组成的组：
(a)氨基酸残基730被丝氨酸取代；
(b)氨基酸残基1028被丙氨酸取代；
(c)氨基酸残基1171被丙氨酸取代；
(d)氨基酸残基1178被赖氨酸取代；
(e)氨基酸残基1365被丝氨酸取代；
(f)氨基酸残基1822被苏氨酸取代；以及
(g)氨基酸残基2179被甘氨酸取代。
5.一种疫苗，其特征在于，包含如权利要求1-4中任一项所述的适应vero细胞的肠道病毒D68。
6.如权利要求5所述的疫苗，其特征在于，所述适应vero细胞的肠道病毒D68是无活性的。
7.如权利要求5所述的疫苗，其特征在于，所述适应vero细胞的肠道病毒D68具有SEQID NO:3的氨基酸序列。
8.如权利要求5所述的疫苗，其特征在于，所述适应vero细胞的肠道病毒D68具有SEQID NO:1的氨基酸序列和至少一个氨基酸取代，所述氨基酸取代选自由以下组成的组：
(a)氨基酸残基730被丝氨酸取代；
(b)氨基酸残基1028被丙氨酸取代；
(c)氨基酸残基1171被丙氨酸取代；
(d)氨基酸残基1178被赖氨酸取代；
(e)氨基酸残基1365被丝氨酸取代；
(f)氨基酸残基1822被苏氨酸取代；以及
(g)氨基酸残基2179被甘氨酸取代。
9.如权利要求5所述的疫苗，其特征在于，还包含佐剂。
10.如权利要求5所述的疫苗，其特征在于，所述疫苗包含所述适应vero细胞的肠道病毒D68的一种或多种抗原或其免疫原性片段。
11.一种药物组合物，其特征在于，包含如权利要求5-10中任一项所述的疫苗和药学上可接受的溶媒、辅料或载体。
12.一种在受试者中诱导针对肠道病毒D68感染的免疫应答的方法，其特征在于，所述方法包括向所述受试者给药有效量的如权利要求5-10中任一项所述的疫苗。
13.如权利要求12所述的方法，其特征在于，受试者在18岁以下。
14.如权利要求12所述的方法，其特征在于，所述疫苗通过肌内途径给药。
15.一种生产用于疫苗的适应vero细胞的肠道病毒D68的方法，其特征在于，所述方法包括：
(a)用肠道病毒D68感染vero细胞以繁殖肠道病毒D68；
(b)在基本上不含血清的培养基中培养步骤(a)中的vero细胞；以及
(c)收获步骤(b)中的肠道病毒D68以用于疫苗中。
16.如权利要求15所述的方法，其特征在于，所述基本上不含血清的培养基是OptiPROTM-SFM或VP-SFM。
17.如权利要求15所述的方法，其特征在于，还包括：在基本上不含血清的培养基中培养vero细胞之前，在含血清的培养基中培养vero细胞。
CN202210399314.8A 2021-04-17 2022-04-15 适应vero细胞的肠道病毒D68、肠道病毒D68疫苗及其用途 Pending CN114717201A (zh)

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