CN116113638A - 包含SARS-CoV-2受体结合结构域的融合蛋白 - Google Patents
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Abstract
一种融合蛋白，包含SARS‑CoV‑2刺突蛋白的SARS‑CoV‑2受体结合结构域(RBD)或其片段和N‑末端信号肽，以及多组氨酸标签、接头、寡聚化标签、刺突蛋白中RBD外的区域、辣根过氧化物酶结合结构域或蛋白酶裂解位点中的至少一种。
Description
技术领域
本申请涉及COVID-19诊断或治疗的医学领域，并且特别是涉及包含严重急性呼吸综合征冠状病毒2(SARS-CoV-2)受体结合结构域(RBD)或其片段的融合蛋白。所述融合蛋白可用于开发能够筛选抑制病毒刺突(S)蛋白与血管紧张素转化酶2(ACE2)结合的试剂的测定。
背景
SARS-CoV-2是一种来自冠状病毒科(Coronaviridae)的包膜RNA病毒(Gorbalenya,A.E等人,2020,Nature Microbiology,5(4):p.536-544)，具有若干种结构组分，包括刺突(S)、包膜(E)、膜(M)和核衣壳(N)蛋白(Lu,R.等人,2020,Lancet 395(10224):p.565-574)。S蛋白由两个亚基(S1和S2)组成，它们在病毒膜上形成三聚体；S1包含负责与ACE2宿主细胞受体结合的RBD(Hoffmann,M.等人,2020,Cell,181(2):p.271-280.e8)，而S2使宿主和病毒膜之间的融合成为可能(Lan,J.,等人,2020,Nature,581(7807):215-220；Wrapp,D.,等人,2020,Science,367(6483):p.1260-1263)。
SARS-CoV-2已经造成了广泛的COVID-19大流行，感染了全球数百万人，并且夺去了数十万人的生命。目前，主要和最准确的诊断方法是通过鼻咽拭子的PCR测试(Peng等人,2020,J Med Virol.24；10.1002/jmv.25936)；然而，迫切需要开发能够不论临床症状如何而识别所有受感染的个体的可靠、高度灵敏和特异性的抗体测试。这一信息将对于建立社区监测和实施遏制病毒传播的政策至关重要。
美国食品药品监督管理局(FDA)已经批准了市场上多种免疫测定测试的紧急使用授权(EUA)，但这些测定都没有得到充分验证。由于缺乏有效的免疫测定(是理解风险、流行病学因素、发病机理和死亡率的关键)，本发明人开发了包含RBD分子设计的融合蛋白，旨在作为SARS-CoV-2免疫测定中的试剂。
刺突RBD代表用于检测旨在鉴定当前和过去的感染的抗SARS-CoV-2IgG的有前途的抗原；并且因为RBD在其他SARS-CoV和致病性人类冠状病毒之间保守性很差，所以它显示出识别总抗SARS-CoV-2Ig和IgM的增强能力(Premkumar,L.等人,2020,ScienceImmunology,(10):p1126-1140)。由于RBD蛋白的小尺寸而导致的测定灵敏度较低的担忧可以通过RBD和N蛋白的分子融合来克服。本发明的目的是改进测定特异性(RBD截短物和RBD突变物)和灵敏度(RBD-N融合物，RBD-多聚化结构域；RBD-辣根过氧化物酶(HRP))。
本发明的发明人开发了RBD融合蛋白和分子设计，其有助于鉴定将用作治疗剂或用于治疗剂开发的超免疫人类血清。针对RBD开发的大部分抗体显示出中和特性，其基本原理是这些mAb破坏了S蛋白和hACE2蛋白之间的相互作用，阻止了病毒的进入。截至2020年6月29日，FDA尚未批准康复期血浆疗法，但在调查研究和临床试验中，建议在人类被动免疫研究中使用至少1:160的滴度。因为RBD引发了对具有抗病毒活性的抗体的开发，所以这些蛋白对于开发鉴定针对SARS-CoV-2的中和抗体的抑制性测定将是必不可少的。
本发明描述了一种用于产生RBD融合蛋白的新的物质组合物。本发明体现了用于产生RBD融合蛋白的方法以及编码RBD的核酸分子、其表达载体和宿主细胞。本发明还包括RBD截短物、多聚化结构域和与N蛋白的融合物。这种新的物质组合物还体现了通过分子动力学模拟和亲和力成熟鉴定出的突变，这些突变被描述为表达的增强因素或与ACE2的亲和力的增强因素。所描述的分子设计可用作抗体滴度、抑制剂/中和筛选测定、疫苗开发中的关键试剂，或用作引发产生具有抗病毒活性的治疗性抗体的试剂。这些融合蛋白也可以与HRP融合，用于实现SARS-CoV-2检测和定量。
本发明人开发了包含IgG1、IgG2aFc和p53二聚化和四聚化结构域的并非显而易见的RBD分子设计，目标是增加测定亲合力和灵敏度；同时也生产高质量、良好表征和可再现的材料。此外，设计了RBD与N蛋白融合在一起的实施方案，以及RBD和HRP融合在一起的实施方案，目标是提高感染急性期期间的测定灵敏度，因为N蛋白在感染早期被检测到。
所述分子被抗SARS-CoV-2S/S1/RBD多克隆兔抗体特异性识别，并且可用作免疫测定平台中捕获抗SARS-CoV-2总IgG或IgM抗体的单独实体。当开发完整测定时，这些分子可以固定在固体支持物中，诸如微量滴定板、膜、珠、多肽芯片或层析柱。所展示的设计的子集经过实验测试，具有与其他商业对应物相似或更好的性能(以对hACE2的亲和力来衡量)。
最后，由于RBD特异性抗体的强抗病毒活性，本文描述的RBD蛋白可用作疫苗候选物以引发广泛有效的抗SARS-CoV-2抗体(Robbiani,D.等人,2020,Nature,doi:https://doi.org/10.1101/2020.05.13.092619；Huo,J.等人,2020,Cell Host&Microbe,(28):p1-10)。
概述
在第一方面，本发明涉及一种融合蛋白，包含SARS-CoV-2刺突蛋白的SARS-CoV-2受体结合结构域(RBD)或其片段和N-末端信号肽，以及多组氨酸标签、接头、寡聚化标签、刺突蛋白中RBD外的区域、辣根过氧化物酶结合结构域或蛋白酶裂解位点中的至少一种。
在一种实施方案中，所述N-末端信号肽选自SARS-CoV-2刺突内源性信号肽或组织纤溶酶原激活物(tPa)信号肽。在一种实施方案中，所述N-末端信号肽具有选自SEQ ID NO:1和SEQ ID NO:2的氨基酸序列。
在一种实施方案中，所述多组氨酸标签由8个或10个组氨酸残基组成。在一种实施方案中，所述多组氨酸标签具有选自SEQ ID NO:7和SEQ ID NO:8的氨基酸序列。
在一种实施方案中，所述寡聚化标签选自鼠IgG1-Fc(仅CH2、CH3)、鼠IgG1-Fc二聚化结构域、鼠IgG-2a-Fc(仅CH2、CH3)、鼠IgG-2a-Fc二聚化结构域、p53四聚化结构域、SARS-CoV-2核衣壳N-末端结构域和SARS-CoV-2核衣壳C-末端结构域。在一种实施方案中，所述寡聚化标签具有选自SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14和SEQ ID NO:15的氨基酸序列。
在一种实施方案中，所述接头是柔性接头。在一种实施方案中，所述接头具有选自SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6的氨基酸序列。
在一种实施方案中，链霉亲和素结合肽标签具有或包含SEQ ID NO:17的氨基酸序列。在一种实施方案中，所述辣根过氧化物酶结合结构域具有选自SEQ ID NO:18的氨基酸序列。
在一种实施方案中，所述蛋白酶裂解位点选自烟草蚀刻病毒裂解位点(TEV)。在一种实施方案中，所述蛋白酶裂解位点具有选自SEQ ID NO:19的氨基酸序列。
在一种实施方案中，SARS-CoV-2刺突蛋白的所述受体结合结构域(RBD)或其片段具有与SEQ ID NO:20具有至少约90％或至少95％序列同一性的氨基酸序列。
在一种实施方案中，所述融合蛋白具有与SEQ ID NO:21、SEQ ID NO:22、SEQ IDNO:23、SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:28、SEQ IDNO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:34、SEQ IDNO:35、SEQ ID NO:36、SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:39、SEQ ID NO:40、SEQ IDNO:41、SEQ ID NO:42、SEQ ID NO:43、SEQ ID NO:44、SEQ ID NO:45、SEQ ID NO:46、SEQ IDNO:47、SEQ ID NO:48、SEQ ID NO:49、SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:52、SEQ IDNO:53、SEQ ID NO:54、SEQ ID NO:55、SEQ ID NO:56或SEQ ID NO:57具有至少90％或至少95％序列同一性的氨基酸序列。
在一种实施方案中，所述SARS-CoV-2RBD蛋白包含以下一个或更多个位置的突变：G404、A475、T478、N481、G485、F490、Q493、G496、Q498、N501或V503。
在另外的方面，本发明涉及包含上述融合蛋白的细胞。
在另外的方面，本发明涉及包含编码融合蛋白的核苷酸序列、可操作地连接到该核苷酸序列的启动子和选择性标志物的核酸。
在另一方面，本发明涉及包含上述核酸的细胞。
最后，本发明涉及包含上述融合蛋白和固体支持物的组合物，其中融合蛋白与固体支持物共价或非共价结合。
序列表
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<120> 包含SARS-CoV-2受体结合结构域的融合蛋白
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Ser Leu Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp
20 25 30
Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His
35 40 45
Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg
50 55 60
Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys
65 70 75 80
Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu
85 90 95
Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr
100 105 110
Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu
115 120 125
Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp
130 135 140
Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val
145 150 155 160
Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu
165 170 175
Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His
180 185 190
Glu Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro
195 200 205
Gly Lys
210
<210> 12
<211> 232
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 鼠IgG2a-Fc二聚化结构域
<400> 12
Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala
1 5 10 15
Pro Asn Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile
20 25 30
Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val Thr Cys Val Val
35 40 45
Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val
50 55 60
Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr His Arg Glu Asp
65 70 75 80
Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro Ile Gln His Gln
85 90 95
Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp
100 105 110
Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val
115 120 125
Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr
130 135 140
Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu
145 150 155 160
Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr
165 170 175
Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr
180 185 190
Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr
195 200 205
Ser Cys Ser Val Val His Glu Gly Leu His Asn His His Thr Thr Lys
210 215 220
Ser Phe Ser Arg Thr Pro Gly Lys
225 230
<210> 13
<211> 40
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> p53四聚化结构域
<400> 13
Lys Pro Leu Asp Gly Glu Tyr Phe Thr Leu Gln Ile Arg Gly Arg Glu
1 5 10 15
Arg Phe Glu Met Phe Arg Glu Leu Asn Glu Ala Leu Glu Leu Lys Asp
20 25 30
Ala Gln Ala Gly Lys Glu Pro Gly
35 40
<210> 14
<211> 124
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> SARS-CoV2核衣壳N-末端结构域
<400> 14
Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Asp Leu Lys
1 5 10 15
Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro Asp
20 25 30
Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly Gly
35 40 45
Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr Leu
50 55 60
Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn Lys Asp Gly
65 70 75 80
Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp His
85 90 95
Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val Leu Gln Leu
100 105 110
Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala
115 120
<210> 15
<211> 101
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> SARS-CoV2核衣壳C-末端结构域
<400> 15
Ala Glu Ala Ser Lys Lys Asn Val Thr Gln Ala Phe Gly Arg Arg Gly
1 5 10 15
Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp Gln Glu Leu Ile Arg Gln
20 25 30
Gly Thr Asp Tyr Lys His Trp Pro Gln Ile Ala Gln Phe Ala Pro Ser
35 40 45
Ala Ser Ala Phe Phe Gly Met Ser Arg Ile Gly Met Glu Val Thr Pro
50 55 60
Ser Gly Thr Trp Leu Thr Tyr Thr Gly Ala Ile Lys Leu Asp Asp Lys
65 70 75 80
Asp Pro Asn Phe Lys Asp Gln Val Ile Leu Leu Asn Lys His Ile Asp
85 90 95
Ala Tyr Lys Thr Phe
100
<210> 16
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 鼠IgG1-Fc铰链结构域
<400> 16
Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr
1 5 10
<210> 17
<211> 38
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> 链霉亲和素结合肽标签
<400> 17
Met Asp Glu Lys Thr Thr Gly Trp Arg Gly Gly His Val Val Glu Gly
1 5 10 15
Leu Ala Gly Glu Leu Glu Gln Leu Arg Ala Arg Leu Glu His His Pro
20 25 30
Gln Gly Gln Arg Glu Pro
35
<210> 18
<211> 308
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> HRP酶
<400> 18
Gln Leu Thr Pro Thr Phe Tyr Asp Asn Ser Cys Pro Asn Val Ser Asn
1 5 10 15
Ile Val Arg Asp Thr Ile Val Asn Glu Leu Arg Ser Asp Pro Arg Ile
20 25 30
Ala Ala Ser Ile Leu Arg Leu His Phe His Asp Cys Phe Val Asn Gly
35 40 45
Cys Asp Ala Ser Ile Leu Leu Asp Asn Thr Thr Ser Phe Arg Thr Glu
50 55 60
Lys Asp Ala Phe Gly Asn Ala Asn Ser Ala Arg Gly Phe Pro Val Ile
65 70 75 80
Asp Arg Met Lys Ala Ala Val Glu Ser Ala Cys Pro Arg Thr Val Ser
85 90 95
Cys Ala Asp Leu Leu Thr Ile Ala Ala Gln Gln Ser Val Thr Leu Ala
100 105 110
Gly Gly Pro Ser Trp Arg Val Pro Leu Gly Arg Arg Asp Ser Leu Gln
115 120 125
Ala Phe Leu Asp Leu Ala Asn Ala Asn Leu Pro Ala Pro Phe Phe Thr
130 135 140
Leu Pro Gln Leu Lys Asp Ser Phe Arg Asn Val Gly Leu Asn Arg Ser
145 150 155 160
Ser Asp Leu Val Ala Leu Ser Gly Gly His Thr Phe Gly Lys Asn Gln
165 170 175
Cys Arg Phe Ile Met Asp Arg Leu Tyr Asn Phe Ser Asn Thr Gly Leu
180 185 190
Pro Asp Pro Thr Leu Asn Thr Thr Tyr Leu Gln Thr Leu Arg Gly Leu
195 200 205
Cys Pro Leu Asn Gly Asn Leu Ser Ala Leu Val Asp Phe Asp Leu Arg
210 215 220
Thr Pro Thr Ile Phe Asp Asn Lys Tyr Tyr Val Asn Leu Glu Glu Gln
225 230 235 240
Lys Gly Leu Ile Gln Ser Asp Gln Glu Leu Phe Ser Ser Pro Asn Ala
245 250 255
Thr Asp Thr Ile Pro Leu Val Arg Ser Phe Ala Asn Ser Thr Gln Thr
260 265 270
Phe Phe Asn Ala Phe Val Glu Ala Met Asp Arg Met Gly Asn Ile Thr
275 280 285
Pro Leu Thr Gly Thr Gln Gly Gln Ile Arg Leu Asn Cys Arg Val Val
290 295 300
Asn Ser Asn Ser
305
<210> 19
<211> 6
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> TEV裂解位点
<400> 19
Glu Asn Leu Tyr Phe Gln
1 5
<210> 20
<211> 223
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> RBD
<400> 20
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
20 25 30
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
35 40 45
Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
50 55 60
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
65 70 75 80
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
85 90 95
Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
100 105 110
Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly
115 120 125
Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
130 135 140
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr
145 150 155 160
Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser
165 170 175
Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val
180 185 190
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
195 200 205
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe
210 215 220
<210> 21
<211> 255
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> pxENB14-RBD
<400> 21
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln His His
1 5 10 15
His His His His His His Gly Gly Gly Ser Glu Asn Leu Tyr Phe Gln
20 25 30
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
35 40 45
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
50 55 60
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
65 70 75 80
Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
85 90 95
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
100 105 110
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
115 120 125
Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
130 135 140
Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly
145 150 155 160
Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
165 170 175
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr
180 185 190
Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser
195 200 205
Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val
210 215 220
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
225 230 235 240
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe
245 250 255
<210> 22
<211> 249
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> pxENB17-RBD
<400> 22
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Arg Val
1 5 10 15
Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys
20 25 30
Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala
35 40 45
Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu
50 55 60
Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro
65 70 75 80
Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe
85 90 95
Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly
100 105 110
Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys
115 120 125
Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn
130 135 140
Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe
145 150 155 160
Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys
165 170 175
Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly
180 185 190
Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val
195 200 205
Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys
210 215 220
Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Gly Gly Gly
225 230 235 240
Ser His His His His His His His His
245
<210> 23
<211> 264
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> pxENB15-RBD
<400> 23
Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly
1 5 10 15
Ala Val Phe Val Ser Pro Ser His His His His His His His His Gly
20 25 30
Gly Gly Ser Glu Asn Leu Tyr Phe Gln Arg Val Gln Pro Thr Glu Ser
35 40 45
Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val
50 55 60
Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg
65 70 75 80
Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser
85 90 95
Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp
100 105 110
Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp
115 120 125
Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr
130 135 140
Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn
145 150 155 160
Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr
165 170 175
Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser
180 185 190
Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly
195 200 205
Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn
210 215 220
Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu
225 230 235 240
Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu
245 250 255
Val Lys Asn Lys Cys Val Asn Phe
260
<210> 24
<211> 258
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> pxENB18-RBD
<400> 24
Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly
1 5 10 15
Ala Val Phe Val Ser Pro Ser Arg Val Gln Pro Thr Glu Ser Ile Val
20 25 30
Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn
35 40 45
Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser
50 55 60
Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser
65 70 75 80
Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys
85 90 95
Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val
100 105 110
Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr
115 120 125
Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn
130 135 140
Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu
145 150 155 160
Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu
165 170 175
Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn
180 185 190
Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val
195 200 205
Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His
210 215 220
Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys
225 230 235 240
Asn Lys Cys Val Asn Phe Gly Gly Gly Ser His His His His His His
245 250 255
His His
<210> 25
<211> 473
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> pxEBNCP21-RBD
<400> 25
Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly
1 5 10 15
Ala Val Phe Val Ser Pro Ser Arg Val Gln Pro Thr Glu Ser Ile Val
20 25 30
Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn
35 40 45
Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser
50 55 60
Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser
65 70 75 80
Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys
85 90 95
Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val
100 105 110
Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr
115 120 125
Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn
130 135 140
Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu
145 150 155 160
Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu
165 170 175
Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn
180 185 190
Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val
195 200 205
Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His
210 215 220
Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys
225 230 235 240
Asn Lys Cys Val Asn Phe Val Pro Arg Asp Cys Gly Cys Lys Pro Cys
245 250 255
Ile Cys Thr Val Pro Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys
260 265 270
Pro Lys Asp Val Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val
275 280 285
Val Val Asp Ile Ser Lys Asp Asp Pro Glu Val Gln Phe Ser Trp Phe
290 295 300
Val Asp Asp Val Glu Val His Thr Ala Gln Thr Gln Pro Arg Glu Glu
305 310 315 320
Gln Phe Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Met His
325 330 335
Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys Cys Arg Val Asn Ser Ala
340 345 350
Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg
355 360 365
Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met
370 375 380
Ala Lys Asp Lys Val Ser Leu Thr Cys Met Ile Thr Asp Phe Phe Pro
385 390 395 400
Glu Asp Ile Thr Val Glu Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn
405 410 415
Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr Asp Gly Ser Tyr Phe Val
420 425 430
Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr
435 440 445
Phe Thr Cys Ser Val Leu His Glu Gly Leu His Asn His His Thr Glu
450 455 460
Lys Ser Leu Ser His Ser Pro Gly Ile
465 470
<210> 26
<211> 478
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> pxEBNCP22-RBD
<400> 26
Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly
1 5 10 15
Ala Val Phe Val Ser Pro Ser Arg Val Gln Pro Thr Glu Ser Ile Val
20 25 30
Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn
35 40 45
Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser
50 55 60
Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser
65 70 75 80
Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys
85 90 95
Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val
100 105 110
Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr
115 120 125
Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn
130 135 140
Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu
145 150 155 160
Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu
165 170 175
Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn
180 185 190
Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val
195 200 205
Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His
210 215 220
Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys
225 230 235 240
Asn Lys Cys Val Asn Phe Pro Arg Gly Pro Thr Ile Lys Pro Cys Pro
245 250 255
Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly Pro Ser Val Phe
260 265 270
Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro
275 280 285
Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val
290 295 300
Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr
305 310 315 320
Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala
325 330 335
Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys
340 345 350
Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser
355 360 365
Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro
370 375 380
Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val
385 390 395 400
Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly
405 410 415
Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp
420 425 430
Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp
435 440 445
Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His
450 455 460
Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys
465 470 475
<210> 27
<211> 464
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> pxEBNCP23-RBD
<400> 27
Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly
1 5 10 15
Ala Val Phe Val Ser Pro Ser Arg Val Gln Pro Thr Glu Ser Ile Val
20 25 30
Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn
35 40 45
Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser
50 55 60
Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser
65 70 75 80
Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys
85 90 95
Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val
100 105 110
Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr
115 120 125
Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn
130 135 140
Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu
145 150 155 160
Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu
165 170 175
Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn
180 185 190
Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val
195 200 205
Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His
210 215 220
Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys
225 230 235 240
Asn Lys Cys Val Asn Phe Gly Gly Gly Pro Val Pro Glu Val Ser Ser
245 250 255
Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu
260 265 270
Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro
275 280 285
Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala
290 295 300
Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val
305 310 315 320
Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe
325 330 335
Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr
340 345 350
Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile
355 360 365
Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys
370 375 380
Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp
385 390 395 400
Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp
405 410 415
Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser
420 425 430
Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly
435 440 445
Leu His Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Ile
450 455 460
<210> 28
<211> 460
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> pxEBNCP24-RBD
<400> 28
Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly
1 5 10 15
Ala Val Phe Val Ser Pro Ser Arg Val Gln Pro Thr Glu Ser Ile Val
20 25 30
Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn
35 40 45
Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser
50 55 60
Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser
65 70 75 80
Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys
85 90 95
Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val
100 105 110
Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr
115 120 125
Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn
130 135 140
Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu
145 150 155 160
Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu
165 170 175
Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn
180 185 190
Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val
195 200 205
Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His
210 215 220
Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys
225 230 235 240
Asn Lys Cys Val Asn Phe Gly Gly Gly Pro Pro Ser Val Phe Ile Phe
245 250 255
Pro Pro Lys Ile Lys Asp Val Leu Met Ile Ser Leu Ser Pro Ile Val
260 265 270
Thr Cys Val Val Val Asp Val Ser Glu Asp Asp Pro Asp Val Gln Ile
275 280 285
Ser Trp Phe Val Asn Asn Val Glu Val His Thr Ala Gln Thr Gln Thr
290 295 300
His Arg Glu Asp Tyr Asn Ser Thr Leu Arg Val Val Ser Ala Leu Pro
305 310 315 320
Ile Gln His Gln Asp Trp Met Ser Gly Lys Glu Phe Lys Cys Lys Val
325 330 335
Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Pro
340 345 350
Lys Gly Ser Val Arg Ala Pro Gln Val Tyr Val Leu Pro Pro Pro Glu
355 360 365
Glu Glu Met Thr Lys Lys Gln Val Thr Leu Thr Cys Met Val Thr Asp
370 375 380
Phe Met Pro Glu Asp Ile Tyr Val Glu Trp Thr Asn Asn Gly Lys Thr
385 390 395 400
Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val Leu Asp Ser Asp Gly Ser
405 410 415
Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu Lys Lys Asn Trp Val Glu
420 425 430
Arg Asn Ser Tyr Ser Cys Ser Val Val His Glu Gly Leu His Asn His
435 440 445
His Thr Thr Lys Ser Phe Ser Arg Thr Pro Gly Lys
450 455 460
<210> 29
<211> 298
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> pxEBNCP25-RBD
<400> 29
Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly
1 5 10 15
Ala Val Phe Val Ser Pro Ser Arg Val Gln Pro Thr Glu Ser Ile Val
20 25 30
Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn
35 40 45
Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser
50 55 60
Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser
65 70 75 80
Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys
85 90 95
Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val
100 105 110
Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr
115 120 125
Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn
130 135 140
Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu
145 150 155 160
Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu
165 170 175
Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn
180 185 190
Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val
195 200 205
Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His
210 215 220
Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys
225 230 235 240
Asn Lys Cys Val Asn Phe Gly Gly Gly Pro Lys Pro Leu Asp Gly Glu
245 250 255
Tyr Phe Thr Leu Gln Ile Arg Gly Arg Glu Arg Phe Glu Met Phe Arg
260 265 270
Glu Leu Asn Glu Ala Leu Glu Leu Lys Asp Ala Gln Ala Gly Lys Glu
275 280 285
Pro Gly His His His His His His His His
290 295
<210> 30
<211> 285
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> pxEBNCP29-RBD
<400> 30
Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly
1 5 10 15
Ala Val Phe Val Ser Pro Ser Val Glu Lys Gly Ile Tyr Gln Thr Ser
20 25 30
Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile
35 40 45
Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala
50 55 60
Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp
65 70 75 80
Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr
85 90 95
Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr
100 105 110
Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro
115 120 125
Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp
130 135 140
Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys
145 150 155 160
Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn
165 170 175
Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly
180 185 190
Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu
195 200 205
Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr
210 215 220
Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val
225 230 235 240
Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn
245 250 255
Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn
260 265 270
Lys Gly Gly Gly Ser His His His His His His His His
275 280 285
<210> 31
<211> 222
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> pxEBNCP30-RBD
<400> 31
Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly
1 5 10 15
Ala Val Phe Val Ser Pro Ser Ile Thr Asn Leu Cys Pro Phe Gly Glu
20 25 30
Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys
35 40 45
Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala
50 55 60
Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn
65 70 75 80
Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly
85 90 95
Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp
100 105 110
Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp
115 120 125
Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu
130 135 140
Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile
145 150 155 160
Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu
165 170 175
Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr
180 185 190
Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu
195 200 205
Leu Leu Gly Gly Gly Ser His His His His His His His His
210 215 220
<210> 32
<211> 261
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> pxEBNCP31-RBD
<400> 32
Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly
1 5 10 15
Ala Val Phe Val Ser Pro Ser Ile Thr Asn Leu Cys Pro Phe Gly Glu
20 25 30
Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys
35 40 45
Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala
50 55 60
Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn
65 70 75 80
Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly
85 90 95
Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp
100 105 110
Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp
115 120 125
Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu
130 135 140
Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile
145 150 155 160
Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu
165 170 175
Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr
180 185 190
Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu
195 200 205
Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn
210 215 220
Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn Gly Leu Thr Gly
225 230 235 240
Thr Gly Val Leu Thr Glu Ser Asn Lys Gly Gly Gly Ser His His His
245 250 255
His His His His His
260
<210> 33
<211> 389
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> pxENBEP32-NucRBD
<400> 33
Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly
1 5 10 15
Ala Val Phe Val Ser Pro Ser Ala Ser Trp Phe Thr Ala Leu Thr Gln
20 25 30
His Gly Lys Glu Asp Leu Lys Phe Pro Arg Gly Gln Gly Val Pro Ile
35 40 45
Asn Thr Asn Ser Ser Pro Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala
50 55 60
Thr Arg Arg Ile Arg Gly Gly Asp Gly Lys Met Lys Asp Leu Ser Pro
65 70 75 80
Arg Trp Tyr Phe Tyr Tyr Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro
85 90 95
Tyr Gly Ala Asn Lys Asp Gly Ile Ile Trp Val Ala Thr Glu Gly Ala
100 105 110
Leu Asn Thr Pro Lys Asp His Ile Gly Thr Arg Asn Pro Ala Asn Asn
115 120 125
Ala Ala Ile Val Leu Gln Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly
130 135 140
Phe Tyr Ala Gly Gly Ser Gly Gly Arg Val Gln Pro Thr Glu Ser Ile
145 150 155 160
Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe
165 170 175
Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile
180 185 190
Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe
195 200 205
Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu
210 215 220
Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu
225 230 235 240
Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn
245 250 255
Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser
260 265 270
Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg
275 280 285
Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr
290 295 300
Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe
305 310 315 320
Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly
325 330 335
Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu
340 345 350
His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val
355 360 365
Lys Asn Lys Cys Val Asn Phe Gly Gly Gly Ser His His His His His
370 375 380
His His His His His
385
<210> 34
<211> 389
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> pxENBEP33-RBDNuc
<400> 34
Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly
1 5 10 15
Ala Val Phe Val Ser Pro Ser Arg Val Gln Pro Thr Glu Ser Ile Val
20 25 30
Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn
35 40 45
Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser
50 55 60
Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser
65 70 75 80
Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys
85 90 95
Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val
100 105 110
Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr
115 120 125
Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn
130 135 140
Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu
145 150 155 160
Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu
165 170 175
Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn
180 185 190
Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val
195 200 205
Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His
210 215 220
Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys
225 230 235 240
Asn Lys Cys Val Asn Phe Gly Gly Ser Gly Gly Ala Ser Trp Phe Thr
245 250 255
Ala Leu Thr Gln His Gly Lys Glu Asp Leu Lys Phe Pro Arg Gly Gln
260 265 270
Gly Val Pro Ile Asn Thr Asn Ser Ser Pro Asp Asp Gln Ile Gly Tyr
275 280 285
Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly Gly Asp Gly Lys Met Lys
290 295 300
Asp Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr Leu Gly Thr Gly Pro Glu
305 310 315 320
Ala Gly Leu Pro Tyr Gly Ala Asn Lys Asp Gly Ile Ile Trp Val Ala
325 330 335
Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp His Ile Gly Thr Arg Asn
340 345 350
Pro Ala Asn Asn Ala Ala Ile Val Leu Gln Leu Pro Gln Gly Thr Thr
355 360 365
Leu Pro Lys Gly Phe Tyr Ala Gly Gly Gly Ser His His His His His
370 375 380
His His His His His
385
<210> 35
<211> 498
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> pxENBEP34-NucRBD
<400> 35
Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly
1 5 10 15
Ala Val Phe Val Ser Pro Ser Ala Ser Trp Phe Thr Ala Leu Thr Gln
20 25 30
His Gly Lys Glu Asp Leu Lys Phe Pro Arg Gly Gln Gly Val Pro Ile
35 40 45
Asn Thr Asn Ser Ser Pro Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala
50 55 60
Thr Arg Arg Ile Arg Gly Gly Asp Gly Lys Met Lys Asp Leu Ser Pro
65 70 75 80
Arg Trp Tyr Phe Tyr Tyr Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro
85 90 95
Tyr Gly Ala Asn Lys Asp Gly Ile Ile Trp Val Ala Thr Glu Gly Ala
100 105 110
Leu Asn Thr Pro Lys Asp His Ile Gly Thr Arg Asn Pro Ala Asn Asn
115 120 125
Ala Ala Ile Val Leu Gln Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly
130 135 140
Phe Tyr Ala Gly Gly Ser Gly Gly Gly Gly Ser Ala Glu Ala Ser Lys
145 150 155 160
Lys Asn Val Thr Gln Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln
165 170 175
Gly Asn Phe Gly Asp Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys
180 185 190
His Trp Pro Gln Ile Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe
195 200 205
Gly Met Ser Arg Ile Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu
210 215 220
Thr Tyr Thr Gly Ala Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys
225 230 235 240
Asp Gln Val Ile Leu Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe
245 250 255
Gly Gly Ser Gly Gly Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe
260 265 270
Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr
275 280 285
Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys
290 295 300
Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe
305 310 315 320
Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr
325 330 335
Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln
340 345 350
Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu
355 360 365
Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu
370 375 380
Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg
385 390 395 400
Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr
405 410 415
Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr
420 425 430
Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr
435 440 445
Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro
450 455 460
Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys
465 470 475 480
Cys Val Asn Phe Gly Gly Gly Ser His His His His His His His His
485 490 495
His His
<210> 36
<211> 498
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> pxENBEP35-RBDNuc
<400> 36
Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly
1 5 10 15
Ala Val Phe Val Ser Pro Ser Arg Val Gln Pro Thr Glu Ser Ile Val
20 25 30
Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn
35 40 45
Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser
50 55 60
Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser
65 70 75 80
Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys
85 90 95
Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val
100 105 110
Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr
115 120 125
Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn
130 135 140
Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu
145 150 155 160
Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu
165 170 175
Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn
180 185 190
Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val
195 200 205
Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His
210 215 220
Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys
225 230 235 240
Asn Lys Cys Val Asn Phe Gly Gly Ser Gly Gly Ala Ser Trp Phe Thr
245 250 255
Ala Leu Thr Gln His Gly Lys Glu Asp Leu Lys Phe Pro Arg Gly Gln
260 265 270
Gly Val Pro Ile Asn Thr Asn Ser Ser Pro Asp Asp Gln Ile Gly Tyr
275 280 285
Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly Gly Asp Gly Lys Met Lys
290 295 300
Asp Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr Leu Gly Thr Gly Pro Glu
305 310 315 320
Ala Gly Leu Pro Tyr Gly Ala Asn Lys Asp Gly Ile Ile Trp Val Ala
325 330 335
Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp His Ile Gly Thr Arg Asn
340 345 350
Pro Ala Asn Asn Ala Ala Ile Val Leu Gln Leu Pro Gln Gly Thr Thr
355 360 365
Leu Pro Lys Gly Phe Tyr Ala Gly Gly Ser Gly Gly Gly Gly Ser Ala
370 375 380
Glu Ala Ser Lys Lys Asn Val Thr Gln Ala Phe Gly Arg Arg Gly Pro
385 390 395 400
Glu Gln Thr Gln Gly Asn Phe Gly Asp Gln Glu Leu Ile Arg Gln Gly
405 410 415
Thr Asp Tyr Lys His Trp Pro Gln Ile Ala Gln Phe Ala Pro Ser Ala
420 425 430
Ser Ala Phe Phe Gly Met Ser Arg Ile Gly Met Glu Val Thr Pro Ser
435 440 445
Gly Thr Trp Leu Thr Tyr Thr Gly Ala Ile Lys Leu Asp Asp Lys Asp
450 455 460
Pro Asn Phe Lys Asp Gln Val Ile Leu Leu Asn Lys His Ile Asp Ala
465 470 475 480
Tyr Lys Thr Phe Gly Gly Gly Ser His His His His His His His His
485 490 495
His His
<210> 37
<211> 597
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> pxEBNCP26-RBD
<400> 37
Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly
1 5 10 15
Ala Val Phe Val Ser Pro Ser Val Glu Lys Gly Ile Tyr Gln Thr Ser
20 25 30
Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile
35 40 45
Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala
50 55 60
Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp
65 70 75 80
Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr
85 90 95
Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr
100 105 110
Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro
115 120 125
Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp
130 135 140
Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys
145 150 155 160
Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn
165 170 175
Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly
180 185 190
Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu
195 200 205
Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr
210 215 220
Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val
225 230 235 240
Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn
245 250 255
Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn
260 265 270
Lys Gly Gly Gly Ser Gln Leu Thr Pro Thr Phe Tyr Asp Asn Ser Cys
275 280 285
Pro Asn Val Ser Asn Ile Val Arg Asp Thr Ile Val Asn Glu Leu Arg
290 295 300
Ser Asp Pro Arg Ile Ala Ala Ser Ile Leu Arg Leu His Phe His Asp
305 310 315 320
Cys Phe Val Asn Gly Cys Asp Ala Ser Ile Leu Leu Asp Asn Thr Thr
325 330 335
Ser Phe Arg Thr Glu Lys Asp Ala Phe Gly Asn Ala Asn Ser Ala Arg
340 345 350
Gly Phe Pro Val Ile Asp Arg Met Lys Ala Ala Val Glu Ser Ala Cys
355 360 365
Pro Arg Thr Val Ser Cys Ala Asp Leu Leu Thr Ile Ala Ala Gln Gln
370 375 380
Ser Val Thr Leu Ala Gly Gly Pro Ser Trp Arg Val Pro Leu Gly Arg
385 390 395 400
Arg Asp Ser Leu Gln Ala Phe Leu Asp Leu Ala Asn Ala Asn Leu Pro
405 410 415
Ala Pro Phe Phe Thr Leu Pro Gln Leu Lys Asp Ser Phe Arg Asn Val
420 425 430
Gly Leu Asn Arg Ser Ser Asp Leu Val Ala Leu Ser Gly Gly His Thr
435 440 445
Phe Gly Lys Asn Gln Cys Arg Phe Ile Met Asp Arg Leu Tyr Asn Phe
450 455 460
Ser Asn Thr Gly Leu Pro Asp Pro Thr Leu Asn Thr Thr Tyr Leu Gln
465 470 475 480
Thr Leu Arg Gly Leu Cys Pro Leu Asn Gly Asn Leu Ser Ala Leu Val
485 490 495
Asp Phe Asp Leu Arg Thr Pro Thr Ile Phe Asp Asn Lys Tyr Tyr Val
500 505 510
Asn Leu Glu Glu Gln Lys Gly Leu Ile Gln Ser Asp Gln Glu Leu Phe
515 520 525
Ser Ser Pro Asn Ala Thr Asp Thr Ile Pro Leu Val Arg Ser Phe Ala
530 535 540
Asn Ser Thr Gln Thr Phe Phe Asn Ala Phe Val Glu Ala Met Asp Arg
545 550 555 560
Met Gly Asn Ile Thr Pro Leu Thr Gly Thr Gln Gly Gln Ile Arg Leu
565 570 575
Asn Cys Arg Val Val Asn Ser Asn Ser Gly Gly Gly Ser His His His
580 585 590
His His His His His
595
<210> 38
<211> 597
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> pxEBNCP27-RBD
<400> 38
Met Asp Ala Met Lys Arg Gly Leu Cys Cys Val Leu Leu Leu Cys Gly
1 5 10 15
Ala Val Phe Val Ser Pro Ser His His His His His His His His Gly
20 25 30
Gly Gly Ser Gln Leu Thr Pro Thr Phe Tyr Asp Asn Ser Cys Pro Asn
35 40 45
Val Ser Asn Ile Val Arg Asp Thr Ile Val Asn Glu Leu Arg Ser Asp
50 55 60
Pro Arg Ile Ala Ala Ser Ile Leu Arg Leu His Phe His Asp Cys Phe
65 70 75 80
Val Asn Gly Cys Asp Ala Ser Ile Leu Leu Asp Asn Thr Thr Ser Phe
85 90 95
Arg Thr Glu Lys Asp Ala Phe Gly Asn Ala Asn Ser Ala Arg Gly Phe
100 105 110
Pro Val Ile Asp Arg Met Lys Ala Ala Val Glu Ser Ala Cys Pro Arg
115 120 125
Thr Val Ser Cys Ala Asp Leu Leu Thr Ile Ala Ala Gln Gln Ser Val
130 135 140
Thr Leu Ala Gly Gly Pro Ser Trp Arg Val Pro Leu Gly Arg Arg Asp
145 150 155 160
Ser Leu Gln Ala Phe Leu Asp Leu Ala Asn Ala Asn Leu Pro Ala Pro
165 170 175
Phe Phe Thr Leu Pro Gln Leu Lys Asp Ser Phe Arg Asn Val Gly Leu
180 185 190
Asn Arg Ser Ser Asp Leu Val Ala Leu Ser Gly Gly His Thr Phe Gly
195 200 205
Lys Asn Gln Cys Arg Phe Ile Met Asp Arg Leu Tyr Asn Phe Ser Asn
210 215 220
Thr Gly Leu Pro Asp Pro Thr Leu Asn Thr Thr Tyr Leu Gln Thr Leu
225 230 235 240
Arg Gly Leu Cys Pro Leu Asn Gly Asn Leu Ser Ala Leu Val Asp Phe
245 250 255
Asp Leu Arg Thr Pro Thr Ile Phe Asp Asn Lys Tyr Tyr Val Asn Leu
260 265 270
Glu Glu Gln Lys Gly Leu Ile Gln Ser Asp Gln Glu Leu Phe Ser Ser
275 280 285
Pro Asn Ala Thr Asp Thr Ile Pro Leu Val Arg Ser Phe Ala Asn Ser
290 295 300
Thr Gln Thr Phe Phe Asn Ala Phe Val Glu Ala Met Asp Arg Met Gly
305 310 315 320
Asn Ile Thr Pro Leu Thr Gly Thr Gln Gly Gln Ile Arg Leu Asn Cys
325 330 335
Arg Val Val Asn Ser Asn Ser Gly Gly Gly Ser Val Glu Lys Gly Ile
340 345 350
Tyr Gln Thr Ser Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg
355 360 365
Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala
370 375 380
Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn
385 390 395 400
Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr
405 410 415
Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe
420 425 430
Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg
435 440 445
Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys
450 455 460
Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn
465 470 475 480
Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe
485 490 495
Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile
500 505 510
Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys
515 520 525
Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly
530 535 540
Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala
545 550 555 560
Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn
565 570 575
Lys Cys Val Asn Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu
580 585 590
Thr Glu Ser Asn Lys
595
<210> 39
<211> 300
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> pxENB36-H8RBDgpp53
<400> 39
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys His
1 5 10 15
His His His His His His His Gly Gly Gly Ser Glu Asn Leu Tyr Phe
20 25 30
Gln Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr
35 40 45
Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser
50 55 60
Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr
65 70 75 80
Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly
85 90 95
Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala
100 105 110
Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly
115 120 125
Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe
130 135 140
Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val
145 150 155 160
Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu
165 170 175
Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser
180 185 190
Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln
195 200 205
Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg
210 215 220
Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys
225 230 235 240
Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe
245 250 255
Gly Gly Gly Pro Lys Pro Leu Asp Gly Glu Tyr Phe Thr Leu Gln Ile
260 265 270
Arg Gly Arg Glu Arg Phe Glu Met Phe Arg Glu Leu Asn Glu Ala Leu
275 280 285
Glu Leu Lys Asp Ala Gln Ala Gly Lys Glu Pro Gly
290 295 300
<210> 40
<211> 300
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> pxENB37-H8RBDgsp53
<400> 40
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys His
1 5 10 15
His His His His His His His Gly Gly Gly Ser Glu Asn Leu Tyr Phe
20 25 30
Gln Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr
35 40 45
Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser
50 55 60
Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr
65 70 75 80
Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly
85 90 95
Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala
100 105 110
Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly
115 120 125
Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe
130 135 140
Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val
145 150 155 160
Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu
165 170 175
Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser
180 185 190
Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln
195 200 205
Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg
210 215 220
Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys
225 230 235 240
Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe
245 250 255
Gly Gly Gly Ser Lys Pro Leu Asp Gly Glu Tyr Phe Thr Leu Gln Ile
260 265 270
Arg Gly Arg Glu Arg Phe Glu Met Phe Arg Glu Leu Asn Glu Ala Leu
275 280 285
Glu Leu Lys Asp Ala Gln Ala Gly Lys Glu Pro Gly
290 295 300
<210> 41
<211> 290
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> pxENB38-RBDgpp53H8
<400> 41
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Arg
1 5 10 15
Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu
20 25 30
Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr
35 40 45
Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val
50 55 60
Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser
65 70 75 80
Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser
85 90 95
Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr
100 105 110
Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly
115 120 125
Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly
130 135 140
Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro
145 150 155 160
Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro
165 170 175
Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr
180 185 190
Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val
195 200 205
Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro
210 215 220
Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Gly Gly
225 230 235 240
Gly Pro Lys Pro Leu Asp Gly Glu Tyr Phe Thr Leu Gln Ile Arg Gly
245 250 255
Arg Glu Arg Phe Glu Met Phe Arg Glu Leu Asn Glu Ala Leu Glu Leu
260 265 270
Lys Asp Ala Gln Ala Gly Lys Glu Pro Gly His His His His His His
275 280 285
His His
290
<210> 42
<211> 290
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> pxENB39-RBDgsp53H8
<400> 42
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Arg
1 5 10 15
Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu
20 25 30
Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr
35 40 45
Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val
50 55 60
Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser
65 70 75 80
Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser
85 90 95
Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr
100 105 110
Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly
115 120 125
Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly
130 135 140
Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro
145 150 155 160
Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro
165 170 175
Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr
180 185 190
Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val
195 200 205
Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro
210 215 220
Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Gly Gly
225 230 235 240
Gly Ser Lys Pro Leu Asp Gly Glu Tyr Phe Thr Leu Gln Ile Arg Gly
245 250 255
Arg Glu Arg Phe Glu Met Phe Arg Glu Leu Asn Glu Ala Leu Glu Leu
260 265 270
Lys Asp Ala Gln Ala Gly Lys Glu Pro Gly His His His His His His
275 280 285
His His
290
<210> 43
<211> 483
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> pxENB40-H8RBDFc
<400> 43
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys His
1 5 10 15
His His His His His His His Gly Gly Gly Ser Glu Asn Leu Tyr Phe
20 25 30
Gln Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr
35 40 45
Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser
50 55 60
Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr
65 70 75 80
Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly
85 90 95
Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala
100 105 110
Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly
115 120 125
Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe
130 135 140
Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val
145 150 155 160
Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu
165 170 175
Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser
180 185 190
Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln
195 200 205
Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg
210 215 220
Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys
225 230 235 240
Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe
245 250 255
Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu
260 265 270
Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr
275 280 285
Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser Lys
290 295 300
Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val
305 310 315 320
His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe
325 330 335
Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly
340 345 350
Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile
355 360 365
Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val
370 375 380
Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser
385 390 395 400
Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu
405 410 415
Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro
420 425 430
Ile Met Asn Thr Asn Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val
435 440 445
Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu
450 455 460
His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His Ser
465 470 475 480
Pro Gly Lys
<210> 44
<211> 525
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> pxENB41-H8RBDFcSBP
<400> 44
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys His
1 5 10 15
His His His His His His His Gly Gly Gly Ser Glu Asn Leu Tyr Phe
20 25 30
Gln Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr
35 40 45
Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser
50 55 60
Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr
65 70 75 80
Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly
85 90 95
Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala
100 105 110
Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly
115 120 125
Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe
130 135 140
Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val
145 150 155 160
Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu
165 170 175
Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser
180 185 190
Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln
195 200 205
Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg
210 215 220
Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys
225 230 235 240
Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe
245 250 255
Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu
260 265 270
Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr
275 280 285
Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser Lys
290 295 300
Asp Asp Pro Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val
305 310 315 320
His Thr Ala Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe
325 330 335
Arg Ser Val Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly
340 345 350
Lys Glu Phe Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile
355 360 365
Glu Lys Thr Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val
370 375 380
Tyr Thr Ile Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser
385 390 395 400
Leu Thr Cys Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu
405 410 415
Trp Gln Trp Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro
420 425 430
Ile Met Asn Thr Asn Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val
435 440 445
Gln Lys Ser Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu
450 455 460
His Glu Gly Leu His Asn His His Thr Glu Lys Ser Leu Ser His Ser
465 470 475 480
Pro Gly Lys Gly Gly Gly Ser Met Asp Glu Lys Thr Thr Gly Trp Arg
485 490 495
Gly Gly His Val Val Glu Gly Leu Ala Gly Glu Leu Glu Gln Leu Arg
500 505 510
Ala Arg Leu Glu His His Pro Gln Gly Gln Arg Glu Pro
515 520 525
<210> 45
<211> 269
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> pxENB42-H8RBDFcHG
<400> 45
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys His
1 5 10 15
His His His His His His His Gly Gly Gly Ser Glu Asn Leu Tyr Phe
20 25 30
Gln Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr
35 40 45
Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser
50 55 60
Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr
65 70 75 80
Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly
85 90 95
Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala
100 105 110
Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly
115 120 125
Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe
130 135 140
Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val
145 150 155 160
Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu
165 170 175
Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser
180 185 190
Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln
195 200 205
Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg
210 215 220
Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys
225 230 235 240
Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe
245 250 255
Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr
260 265
<210> 46
<211> 311
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> pxENB43-RBDFcHGSBP
<400> 46
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys His
1 5 10 15
His His His His His His His Gly Gly Gly Ser Glu Asn Leu Tyr Phe
20 25 30
Gln Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr
35 40 45
Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser
50 55 60
Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr
65 70 75 80
Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly
85 90 95
Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala
100 105 110
Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly
115 120 125
Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe
130 135 140
Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val
145 150 155 160
Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu
165 170 175
Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser
180 185 190
Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln
195 200 205
Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg
210 215 220
Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys
225 230 235 240
Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe
245 250 255
Val Pro Arg Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Gly Gly Gly
260 265 270
Ser Met Asp Glu Lys Thr Thr Gly Trp Arg Gly Gly His Val Val Glu
275 280 285
Gly Leu Ala Gly Glu Leu Glu Gln Leu Arg Ala Arg Leu Glu His His
290 295 300
Pro Gln Gly Gln Arg Glu Pro
305 310
<210> 47
<211> 479
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> pxENB44-H8RBDRBD
<400> 47
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys His
1 5 10 15
His His His His His His His Gly Gly Gly Ser Glu Asn Leu Tyr Phe
20 25 30
Gln Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr
35 40 45
Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser
50 55 60
Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr
65 70 75 80
Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly
85 90 95
Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala
100 105 110
Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly
115 120 125
Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe
130 135 140
Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val
145 150 155 160
Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu
165 170 175
Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser
180 185 190
Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln
195 200 205
Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg
210 215 220
Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys
225 230 235 240
Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe
245 250 255
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
260 265 270
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
275 280 285
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
290 295 300
Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
305 310 315 320
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
325 330 335
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
340 345 350
Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
355 360 365
Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly
370 375 380
Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
385 390 395 400
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr
405 410 415
Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser
420 425 430
Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val
435 440 445
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
450 455 460
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe
465 470 475
<210> 48
<211> 473
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> pxENB46-RBDRBDH8
<400> 48
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Arg
1 5 10 15
Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu
20 25 30
Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr
35 40 45
Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val
50 55 60
Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser
65 70 75 80
Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser
85 90 95
Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr
100 105 110
Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly
115 120 125
Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly
130 135 140
Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro
145 150 155 160
Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro
165 170 175
Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr
180 185 190
Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val
195 200 205
Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro
210 215 220
Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Arg Val
225 230 235 240
Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys
245 250 255
Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala
260 265 270
Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu
275 280 285
Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro
290 295 300
Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe
305 310 315 320
Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly
325 330 335
Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys
340 345 350
Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn
355 360 365
Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe
370 375 380
Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys
385 390 395 400
Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly
405 410 415
Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val
420 425 430
Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys
435 440 445
Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Gly Gly Gly
450 455 460
Ser His His His His His His His His
465 470
<210> 49
<211> 298
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> pxENB48-H8RBDSBP
<400> 49
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys His
1 5 10 15
His His His His His His His Gly Gly Gly Ser Glu Asn Leu Tyr Phe
20 25 30
Gln Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr
35 40 45
Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser
50 55 60
Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr
65 70 75 80
Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly
85 90 95
Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala
100 105 110
Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly
115 120 125
Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe
130 135 140
Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val
145 150 155 160
Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu
165 170 175
Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser
180 185 190
Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln
195 200 205
Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg
210 215 220
Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys
225 230 235 240
Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe
245 250 255
Gly Gly Gly Ser Met Asp Glu Lys Thr Thr Gly Trp Arg Gly Gly His
260 265 270
Val Val Glu Gly Leu Ala Gly Glu Leu Glu Gln Leu Arg Ala Arg Leu
275 280 285
Glu His His Pro Gln Gly Gln Arg Glu Pro
290 295
<210> 50
<211> 255
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> pxENB14-RBD-B.1.1.7
<400> 50
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln His His
1 5 10 15
His His His His His His Gly Gly Gly Ser Glu Asn Leu Tyr Phe Gln
20 25 30
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
35 40 45
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
50 55 60
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
65 70 75 80
Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
85 90 95
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
100 105 110
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
115 120 125
Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
130 135 140
Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly
145 150 155 160
Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
165 170 175
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr
180 185 190
Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser
195 200 205
Tyr Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val
210 215 220
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
225 230 235 240
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe
245 250 255
<210> 51
<211> 255
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> pxENB14-RBD-B.1.351
<400> 51
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln His His
1 5 10 15
His His His His His His Gly Gly Gly Ser Glu Asn Leu Tyr Phe Gln
20 25 30
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
35 40 45
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
50 55 60
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
65 70 75 80
Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
85 90 95
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
100 105 110
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
115 120 125
Thr Gly Asn Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
130 135 140
Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly
145 150 155 160
Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
165 170 175
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr
180 185 190
Pro Cys Asn Gly Val Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser
195 200 205
Tyr Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val
210 215 220
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
225 230 235 240
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe
245 250 255
<210> 52
<211> 255
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> pxENB14-RBD-B.1.617.2
<400> 52
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln His His
1 5 10 15
His His His His His His Gly Gly Gly Ser Glu Asn Leu Tyr Phe Gln
20 25 30
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
35 40 45
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
50 55 60
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
65 70 75 80
Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
85 90 95
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
100 105 110
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
115 120 125
Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
130 135 140
Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly
145 150 155 160
Gly Asn Tyr Asn Tyr Arg Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
165 170 175
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Lys
180 185 190
Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser
195 200 205
Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val
210 215 220
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
225 230 235 240
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe
245 250 255
<210> 53
<211> 255
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> pxENB14-RBD-B.1.427
<400> 53
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln His His
1 5 10 15
His His His His His His Gly Gly Gly Ser Glu Asn Leu Tyr Phe Gln
20 25 30
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
35 40 45
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
50 55 60
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
65 70 75 80
Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
85 90 95
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
100 105 110
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
115 120 125
Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
130 135 140
Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly
145 150 155 160
Gly Asn Tyr Asn Tyr Arg Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
165 170 175
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr
180 185 190
Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser
195 200 205
Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val
210 215 220
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
225 230 235 240
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe
245 250 255
<210> 54
<211> 255
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> pxENB14-RBD-P.1
<400> 54
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln His His
1 5 10 15
His His His His His His Gly Gly Gly Ser Glu Asn Leu Tyr Phe Gln
20 25 30
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
35 40 45
Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val
50 55 60
Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser
65 70 75 80
Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val
85 90 95
Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp
100 105 110
Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln
115 120 125
Thr Gly Thr Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr
130 135 140
Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly
145 150 155 160
Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys
165 170 175
Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr
180 185 190
Pro Cys Asn Gly Val Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser
195 200 205
Tyr Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val
210 215 220
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
225 230 235 240
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe
245 250 255
<210> 55
<211> 473
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> pxENB46-RBD2-B.1.1.7
<400> 55
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Arg
1 5 10 15
Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu
20 25 30
Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr
35 40 45
Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val
50 55 60
Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser
65 70 75 80
Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser
85 90 95
Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr
100 105 110
Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly
115 120 125
Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly
130 135 140
Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro
145 150 155 160
Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro
165 170 175
Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr
180 185 190
Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val Val
195 200 205
Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro
210 215 220
Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Arg Val
225 230 235 240
Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys
245 250 255
Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala
260 265 270
Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu
275 280 285
Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro
290 295 300
Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe
305 310 315 320
Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly
325 330 335
Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys
340 345 350
Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn
355 360 365
Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe
370 375 380
Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys
385 390 395 400
Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly
405 410 415
Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val
420 425 430
Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys
435 440 445
Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Gly Gly Gly
450 455 460
Ser His His His His His His His His
465 470
<210> 56
<211> 473
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> pxENB46-RBD2-B.1.351
<400> 56
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Arg
1 5 10 15
Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu
20 25 30
Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr
35 40 45
Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val
50 55 60
Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser
65 70 75 80
Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser
85 90 95
Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr
100 105 110
Gly Asn Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly
115 120 125
Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly
130 135 140
Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro
145 150 155 160
Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro
165 170 175
Cys Asn Gly Val Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr
180 185 190
Gly Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val Val
195 200 205
Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro
210 215 220
Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Arg Val
225 230 235 240
Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys
245 250 255
Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala
260 265 270
Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu
275 280 285
Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro
290 295 300
Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe
305 310 315 320
Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly
325 330 335
Asn Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys
340 345 350
Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn
355 360 365
Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe
370 375 380
Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys
385 390 395 400
Asn Gly Val Lys Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly
405 410 415
Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val
420 425 430
Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys
435 440 445
Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Gly Gly Gly
450 455 460
Ser His His His His His His His His
465 470
<210> 57
<211> 473
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> pxENB46-RBD2-B.1.617.2
<400> 57
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Arg
1 5 10 15
Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu
20 25 30
Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr
35 40 45
Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val
50 55 60
Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser
65 70 75 80
Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser
85 90 95
Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr
100 105 110
Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly
115 120 125
Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly
130 135 140
Asn Tyr Asn Tyr Arg Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro
145 150 155 160
Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Lys Pro
165 170 175
Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr
180 185 190
Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val
195 200 205
Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro
210 215 220
Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Arg Val
225 230 235 240
Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys
245 250 255
Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala
260 265 270
Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu
275 280 285
Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro
290 295 300
Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe
305 310 315 320
Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly
325 330 335
Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys
340 345 350
Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn
355 360 365
Tyr Asn Tyr Arg Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe
370 375 380
Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Lys Pro Cys
385 390 395 400
Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly
405 410 415
Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val
420 425 430
Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys
435 440 445
Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Gly Gly Gly
450 455 460
Ser His His His His His His His His
465 470
附图简述
图1示出SARS-CoV-2融合蛋白的表达和纯化。A)示出pxENB14-RBD构建体(上图)和pxENB17-RBD构建体(下图)的特征的示意图。B)在第3天收获的在Expi293细胞中产生的pxENB14-RBD和pxENB17-RBD的平均产量，和C)使用抗His标签小鼠单克隆抗体对在第3天收获的Expi293上清液的蛋白印迹分析。在还原条件下处理样品。D)使用镍亲和层析纯化RBD蛋白。E)显示pxENB14-RBD和pxENB17-RBD纯化物的表观分子量和纯度的SDS-PAGE。F)和G)最终纯化样品的SDS-PAGE，还原(R)和非还原(NR)，在8％-16％ TGX免染色凝胶上运行。M：蛋白梯(Precision Plus未染色蛋白标准品)。H)和I)使用1:1000稀释的S1兔多克隆抗体(Sino Biological)进行的蛋白印迹分析。
图2是与RBD对接的ACE2的Cryo-EM结构。结构是从PDB结构6M1710检索的。ACE2(绿色)。RBD(青色)。
图3是说明人类ACE2受体-RBD相互作用的生物层干涉测量感测图。A)固定化hACE2与SARS-CoV-2RBD的结合曲线：B)pxENB14-His-TEV-RBD；C)pxENB17-RBD和D)从商业来源产生的RBD。根据分析物浓度，数据以不同的颜色线显示，并且将数据针对1:1结合模型最佳拟合，如红线所示。
图4是来自表达所描绘的每种构建体的Expi293细胞的上清液的SDS-PAGE。在存在DTT的情况下，所有样品被还原。样品在8％-16％ TGX免染色凝胶上运行。M：蛋白梯(Precision Plus未染色蛋白标准品)。使用1:1000的抗His mAb进行的蛋白印迹分析；SP：上清液；PL：沉淀物(pellet)。箭头标出蛋白条带。
图5是说明人类ACE2受体-多聚化(multimeric)RBD蛋白相互作用的生物层干涉测量感测图。
图6是说明人类ACE2受体-pxENB14突变体的生物层干涉测量感测图。
图7是说明人类ACE2受体-pxENB46突变体的生物层干涉测量感测图。
详细描述
除非另外定义，否则本文使用的所有技术术语和科学术语具有与所描述的方法和组合物所属领域的普通技术人员通常理解的相同的含义。如本文使用的，以下术语和短语具有归于它们的含义，除非另有说明。
除非上下文另有明确指示，否则术语“一(a)”、“一(an)”和“该(the)”包括复数指代物。
贯穿本说明书，除非上下文另外要求，否则词语“包含(comprise)”或变化形式诸如“包含(comprises)”和“包含(comprising)”将被理解为暗示包括所陈述的要素或整数，或者要素或整数的组，但不排除任何其他的要素或整数，或者要素或整数的组。
除非另有定义，否则本文使用的所有技术和科学术语具有与本发明所属领域的普通技术人员通常理解的相同含义。下文描述了示例性方法和材料，尽管与本文描述的方法和材料相似或等效的方法和材料也可以被使用，并且对本领域技术人员来说将是明显的。本文提及的所有出版物及其他参考文献均通过引用以其整体并入。在冲突的情况下，应以本说明书(包括定义)为准。材料、方法和实例仅为说明性的而并非意图限制。
除非另有明确说明，否则本说明书中的每种实施方案经必要修改后将适用于每一种其他的实施方案。
除非另外指示，否则以下术语应被理解为具有以下含义：
如本文使用的，术语“核酸”是指包括DNA或RNA的任何材料。核酸可以合成地制备或由活细胞制备。
如本文使用的，术语“蛋白”是指由一个或更多个氨基酸残基的链组成的大的生物分子或大分子。许多蛋白是催化生化反应的酶，并且对代谢至关重要。蛋白还具有结构功能或机械功能，诸如肌肉中的肌动蛋白和肌球蛋白以及细胞骨架中的蛋白，它们形成了维持细胞形状的支架系统。其他蛋白在细胞信号传导、免疫应答、细胞粘附和细胞周期中是重要的。然而，蛋白可以是完全人工的或重组的，即，不是天然存在于生物系统中的。
如本文使用的，术语“多肽”是指天然存在和非天然存在的蛋白，以及其片段、突变体、衍生物和类似物。多肽可以是单体的或聚合的。多肽可以包含许多不同的结构域(肽)，结构域(肽)中的每一个具有一种或更多种不同的活性。
如本文使用的，术语“重组体(recombinant)”是指这样的生物分子，例如基因或蛋白，所述生物分子(1)已从其天然存在的环境中取出，(2)不与自然界中发现该基因的多核苷酸的全部或部分缔合，(3)与自然界中不与其连接的多核苷酸可操作地连接，或(4)在自然界中不存在。术语“重组体”可以用于指克隆的DNA分离物、化学合成的多核苷酸类似物、或由异源系统生物合成的多核苷酸类似物，以及由这样的核酸编码的蛋白和/或mRNA。
如本文使用的，术语“融合蛋白”是指包含两个或更多个不共存于天然存在的蛋白中的氨基酸序列的蛋白。融合蛋白可以包含两个或更多个来自相同或不同生物体的氨基酸序列。融合蛋白的两个或更多个氨基酸序列通常同框(in frame)，它们之间没有终止密码子，并且通常作为融合蛋白的一部分从mRNA翻译。
术语“融合蛋白”和术语“重组体”在本文可以互换使用。
如本文使用的，术语“抗原”是指与相应的抗体特异性地结合的生物分子。来自不同库(repertoire)的抗体借助于其可变区相互作用结合特定的抗原结构。
本文中使用的术语“抗体”或“免疫球蛋白”具有相同的含义，并且将在本发明中等同使用。本文中使用的术语“抗体”是指免疫球蛋白分子和免疫球蛋白分子的免疫活性部分，即含有特异性结合抗原的抗原结合位点的分子。因此，术语抗体不仅涵盖完整抗体分子，还涵盖抗体片段或衍生物。
本文中使用的术语“结合亲和力”是指抗原的表位和抗体的抗原结合位点之间的相互作用强度。
如本文所用，“启动子”是由DNA依赖性RNA聚合酶(“转录酶”)识别为结合核酸并在特定位点开始RNA转录的信号的特定核酸序列。
术语“修饰的序列”和“修饰的基因”在本文可互换使用以指包括对天然存在的核酸序列的缺失、插入或中断的序列。在一些优选的实施方案中，修饰的序列的表达产物是截短的蛋白(例如，如果修饰是序列的缺失或中断)。在一些特别优选的实施方案中，截短的蛋白保留生物活性。在替代实施方案中，修饰的序列的表达产物是延长的蛋白(例如，包含向核酸序列中的插入的修饰)。在一些实施方案中，插入导致截短的蛋白(例如，当插入导致终止密码子的形成时)。因此，插入可以导致截短的蛋白或延长的蛋白作为表达产物。
如本文使用的，术语“突变序列”和“突变基因”可互换使用，并指在宿主细胞的野生型序列中具有至少一个密码子的改变的序列。突变序列的表达产物是相对于野生型具有改变的氨基酸序列的蛋白。表达产物可以具有改变的功能性能力(例如，增强的结合亲和力)。
本文中使用的术语“区域”或“片段”是指氨基酸序列的一部分，其中所述部分小于整个氨基酸序列。在一些实施方案中，指SARS-CoV-2的受体结合结构域(RBD)的一部分，其与RBD的氨基酸序列的序列同一性为至少约90％。在一些实施方案中，指SARS-CoV-2的刺突蛋白的RBD外的部分，其与刺突蛋白的RBD外的氨基酸序列的序列同一性为至少约90％。
术语“受体结合结构域”或“RBD”是指SARS-CoV-2S中与人类和蝙蝠血管紧张素转化酶2(ACE2)受体强烈结合的蛋白。
术语“刺突蛋白”、“S蛋白”或“S”是指一种大的I型跨膜蛋白，其范围为禽类感染性支气管炎病毒(IBV)的1,160个氨基酸和多达猫冠状病毒(FCoV)的1,400个氨基酸。此外，这种蛋白是高度糖基化的，因为它含有21至35个N-糖基化位点。刺突蛋白在病毒粒子表面组装成三聚体，形成独特的“冠”，或冠状外观。所有CoV刺突蛋白的胞外结构域共有以下两个结构域的相同的组织：负责受体结合的名为S1的N-末端结构域，和负责融合的C-末端S2结构域。CoV的多样性反映在可变刺突蛋白(S蛋白)中，这些蛋白演化成在其受体相互作用和对病毒-细胞膜融合的各种环境触发因素的响应方面不同的形式。据报道，2019-nCoV可以通过与人类ACE2受体的相互作用感染人类呼吸道上皮细胞。事实上，重组刺突蛋白可以与重组ACE2蛋白结合。
术语“血管紧张素转化酶2”或“ACE2”是指附着在肺、动脉、心脏、肾脏和肠道细胞的细胞膜的酶。ACE2通过催化血管紧张素II(一种血管收缩肽)水解成血管紧张素(1-7)(一种血管扩张剂)来降低血压。ACE2通过减少血管紧张素II的量和增加Ang(1-7)来对抗相关的血管紧张素转化酶(ACE)的活性，使得ACE2成为用于治疗心血管疾病的有前途的药物靶。ACE2也用作包括HCoV-NL63、SARS-CoV和SARS-CoV-2的一些冠状病毒进入细胞的进入点。这种酶的人类形式通常被称为hACE2。
术语“辣根过氧化物酶”或“HRP”在生物化学应用中被广泛使用。它是一种具有许多同种型的金属酶，其中研究最多的类型是C。它催化过氧化氢对各种有机底物的氧化。
如本文使用的，术语“N-末端信号肽”是存在于大多数新合成的通向分泌途径的蛋白的N-末端的短肽(通常长10-30个氨基酸)。这些蛋白包括那些驻留于某些细胞器(内质网、高尔基体或内体)内、从细胞分泌或插入大多数细胞膜的蛋白。尽管大多数I型膜结合蛋白具有信号肽，但大多数II型和多次跨膜结合蛋白通过其第一跨膜结构域靶向分泌途径，该第一跨膜结构域在生物化学上类似于信号序列，只是它不被裂解。它们是一种靶向肽。
如本文使用的，术语“纯化标签”或“亲和标签”是指用于纯化蛋白的多肽，其简化了纯化并使得能够使用标准方案。在本发明中，纯化标签是具有4个、6个、7个、8个、9个、10个、11个或12个组氨酸残基的多组氨酸标签。优选地，组氨酸标签具有8个或10个组氨酸残基。
如本文使用的，术语“接头”是指包含1-10个氨基酸，优选地3-6个氨基酸的多肽。接头的氨基酸可选自由以下组成的组：亮氨酸(Leu，L)、异亮氨酸(Ile，I)、丙氨酸(Ala，A)、甘氨酸(Gly，G)、缬氨酸(Val，V)、脯氨酸(Pro，P)、赖氨酸(Lys，K)、精氨酸(Arg，R)、丝氨酸(Ser，S)、天冬酰胺(Asn，N)和谷氨酰胺(Gln，Q)、色氨酸(Trp，W)、甲硫氨酸(Met，M)、天冬氨酸(Asp，D)、半胱氨酸(Cys，C)、谷氨酸(Glu，E)、组氨酸(His，H)、苯丙氨酸(Phe，F)、苏氨酸(The，T)和酪氨酸(Tyr，Y)。在一些优选的实施方案中，接头是柔性接头，其可以由通常包括至少一个甘氨酸和至少一个丝氨酸的连续氨基酸序列组成。示例性柔性接头包括SEQ IDNO:3(GGGS)、SEQ ID NO:4(GGGP)、SEQ ID NO:5(GGSGG)或SEQ ID NO:6(GGSGGGGS)中列出的氨基酸序列，尽管接头的准确氨基酸序列不是特别限制性的。如本文使用的，术语“寡聚化标签”是指用于增加测定亲合力和灵敏度的多肽。在本发明中，寡聚化标签选自鼠IgG1-Fc(仅CH2、CH3)、鼠IgG1-Fc二聚化结构域、鼠IgG-2a-Fc(仅CH2、CH3)、鼠IgG-2a-Fc二聚化结构域、p53四聚化结构域、SARS-CoV-2核衣壳N-末端结构域和SARS-CoV-2核衣壳C-末端结构域。
如本文使用的，术语“刺突蛋白中RBD外的区域”是指包含不是RBD蛋白一部分的SARS-CoV-2的1-30个氨基酸的多肽。
如本文使用的，术语“辣根过氧化物酶结合结构域”是指用于缀合物(已经遗传或化学连接的分子)中以确定分子靶的存在的酶。
如本文使用的，术语“烟草蚀刻病毒裂解位点”或“TEV”是指存在于来自融合蛋白的标签中的高度位点特异性半胱氨酸蛋白酶。裂解的最适温度为30℃；它也可以在低至4℃的温度使用。建议通过改变重组病毒TEV蛋白酶的量、反应时间或孵育温度来优化每种融合蛋白的裂解。可以用Ni2+亲和树脂去除。这种酶的最佳识别位点是序列Glu-Asn-Leu-Tyr-Phe-Gln-(Gly/Ser)[ENLYFQ(G/S)]，并且裂解发生在Gln和Gly/Ser残基之间。最常用的序列是ENLYFQG。蛋白酶用于从融合蛋白裂解亲和标签。
如本文使用的术语“诊断(diagnostic)”或“诊断(diagnosed)”意指鉴定病理状况的存在或性质或易受疾病影响的患者。诊断方法的灵敏度和特异性不同。诊断测定的“灵敏度”是患病个体被测试为阳性的百分比(“真阳性”的百分比)。未被测定检测出的患病个体是“假阴性”。未患病并且在测定中被测试为阴性的受试者称为“真阴性”。诊断测定的“特异性”是1减去假阳性率，其中“假阳性”率被定义为无疾病、测试为阳性的那些的比例。尽管特定的诊断方法可能不提供状况的确定性诊断，但是如果该方法提供了有助于诊断的有用指示，它就合格了。
如本文使用的，术语“生物层干涉测量(BLI)”是用于测量生物分子相互作用的无标记技术。这是一种光学分析技术，分析从以下两个表面反射的白光的干涉模式：生物传感器尖端上固定的蛋白质层和内部参考层。结合到生物传感器尖端的分子数量的任何变化导致可以实时测量的干涉模式的位移。
I.融合蛋白
本发明涉及一种融合蛋白，包含SARS-CoV-2刺突蛋白的SARS-CoV-2受体结合结构域(RBD)或其片段和N-末端信号肽，以及多组氨酸标签、接头、寡聚化标签、刺突蛋白中RBD外的区域、辣根过氧化物酶结合结构域或蛋白酶裂解位点中的至少一种。
SARS-CoV-2全长刺突(FLS，GenBank MN908947.3)包含两个结构域，即S1和S2，它们负责结合步骤。S1含有RBD，它直接与ACE2的肽酶结构域(PD)结合，而S2负责膜融合。当S1与宿主受体ACE2结合时，S2上的另一个裂解位点暴露出来，并被宿主蛋白酶裂解，这一过程对病毒感染至关重要。SARS-CoV-2的S蛋白也可能利用ACE2感染宿主。
本发明的融合蛋白可以通过本领域技术人员熟知的方法获得。例如，所述融合蛋白可以在细菌、酵母、真菌或哺乳动物细胞中重组获得。在一种实施方案中，本发明的融合蛋白在原核细胞诸如大肠杆菌(Escherichia coli)中产生，但可以使用其他原核细胞。在另一种实施方案中，本发明的融合蛋白在人类胚胎肾(HEK)细胞或中国仓鼠卵巢(CHO)细胞中产生，但可以使用其他真核细胞。
本发明的融合蛋白可以通过本领域技术人员熟知的方法从细胞纯化。所述方法包括但不限于过滤、缀合、亲和层析、离子交换层析、疏水相互作用层析和尺寸排阻层析。
关于本发明的融合蛋白中包含的信号肽，这些信号肽可以导致重组产生期间蛋白表达和/或分泌的改进。此外，包含不同的信号肽可以改变翻译后修饰(PTM)和潜在的蛋白功能。因此，本发明的融合蛋白能否产生或具有功能性并不是明显的。在一种实施方案中，所述N-末端信号肽选自刺突内源性信号肽和组织纤溶酶原激活物(tPa)。所述N-末端信号肽具有选自SEQ ID NO:1和SEQ ID NO:2的氨基酸序列。
如前所述，多组氨酸标签的使用简化了纯化，并使得能够在融合蛋白的产生中使用标准方案。例如，已知组氨酸(His)标签(也称为多组氨酸或polyHis)可用于例如通过固定化金属亲和层析(IMAC)的纯化。多组氨酸标签的其他用途也是本领域技术人员熟知的，并且因此本发明的多组氨酸标签不限于纯化功能。在本发明中，所述多组氨酸标签可以具有6个、8个或10个组氨酸残基。评价在蛋白N末端和C末端二者的标签对产生蛋白以及蛋白的功能和聚集状态的影响是重要的。标签将具有的位置的影响并不是明显的。此外，标签在纯化或任何测定开发中的效用是未知的。TEV裂解位点的包含通过N-末端加标签完成。如果选择N-末端加标签的构建体，将有可能产生无标签的形式。此外，TEV标签的混杂性(promiscuity)被用来支持无瘢痕(scar-free)蛋白的可能产生。优选地，所述多组氨酸标签具有选自SEQ ID NO:7和SEQ ID NO:8的氨基酸序列。
在另一种实施方案中，寡聚化标签或结构域被包含在本发明的融合蛋白中，其选自鼠IgG1-Fc(仅CH2、CH3)、鼠IgG1-Fc二聚化结构域、鼠IgG-2a-Fc(仅CH2、CH3)、鼠IgG-2a-Fc二聚化结构域、p53四聚化结构域、SARS-CoV-2核衣壳N-末端结构域和SARS-CoV-2核衣壳C-末端结构域。所述寡聚化标签具有选自SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11、SEQID NO:12、SEQ ID NO:13、SEQ ID NO:14和SEQ ID NO:15的氨基酸序列。RBD分子设计包含IgG1、IgG2aFc和p53二聚化和四聚化结构域，目的是提高测定亲合力和灵敏度。
接头也可以存在于本发明的融合蛋白中。在一种实施方案中，所述接头可以是柔性接头。当将不同蛋白的结构域融合在一起时，柔性接头被包括在内。这些接头大多是甘氨酸和丝氨酸的组合，而在一些情况下，添加脯氨酸以使蛋白弯折。这些柔性接头可以有助于提高对这些结构域组装的耐受性，并且通常是甘氨酸和丝氨酸的组合。然而，对于本领域技术人员来说，包含所选接头是否会产生功能性融合蛋白并不是明显的。在一种实施方案中，所述接头是柔性接头以增加柔性。所述接头具有选自SEQ ID NO:3、SEQ ID NO:4、SEQ IDNO:5和SEQ ID NO:6的氨基酸序列。
使用链霉亲和素结合结构域(SBP)(SEQ ID NO:17)来支持平板包被或荧光团或HRP标签缀合中的测定开发，以用于读出。目标是避免标记对于蛋白与hACE2受体或抗体相互作用关键的残基。辣根过氧化物酶(HRP)结合结构域是指缀合物(已经遗传或化学连接的分子)中用以确定分子靶的存在的酶。在一些实施方案中，所述辣根过氧化物酶结合结构域具有选自SEQ ID NO:18的氨基酸序列。
在一些实施方案中，所述蛋白酶裂解位点是烟草蚀刻病毒裂解位点(TEV)。所述蛋白酶裂解位点具有选自SEQ ID NO:19的氨基酸序列。
在一些实施方案中，SARS-CoV-2刺突蛋白的所述受体结合结构域(RBD)或其片段具有与SEQ ID NO:20具有至少90％或至少95％序列同一性的氨基酸序列。
本发明还涵盖特定RBD形式的高亲和力RBD突变，以覆盖出现的增强与hACE2结合的SARS-CoV-2突变。这些新的蛋白设计中的一些包含在自然界中出现的SARS-CoV-2突变(Pango谱系变体：B1.1.7、B.1.351、B1.617.2、B.1.427和P.1)。此外，使用来自Schrodinger的分子动力学模拟和亲和力成熟软件(Bio luminate)来预测RBD一级序列中将赋予与hACE2更高的亲和力的AA突变。在这些突变中，我们通过计算机模拟(in silico)和根据文献中所描述的发现了突变V367F和G502D(其增加了RBD的表达)和N501F、N501T和Q498Y。
II.示例性融合蛋白
在一些实施方案中，所述融合蛋白具有与SEQ ID NO:21、SEQ ID NO:22、SEQ IDNO:23、SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26、SEQ ID NO:27、SEQ ID NO:28、SEQ IDNO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:34、SEQ IDNO:35、SEQ ID NO:36、SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:39、SEQ ID NO:40、SEQ IDNO:41、SEQ ID NO:42、SEQ ID NO:43、SEQ ID NO:44、SEQ ID NO:45、SEQ ID NO:46、SEQ IDNO:47、SEQ ID NO:48、SEQ ID NO:49、SEQ ID NO:50、SEQ ID NO:51、SEQ ID NO:52、SEQ IDNO:53、SEQ ID NO:54、SEQ ID NO:55、SEQ ID NO:56或SEQ ID NO:57具有至少90％或至少95％序列同一性的氨基酸序列。
本发明人还设计了RBD与N蛋白融合在一起的实施方案，以及RBD和HRP融合在一起的实施方案，目标是提高感染急性期期间的测定灵敏度，因为N蛋白在感染早期被检测到。
在一些实施方案中，本发明还体现了增强与人类ACE2结合的高亲和力RBD突变。本发明人使用来自Schrodinger的分子动力学模拟和亲和力成熟软件(Bio luminate)来预测RBD一级序列中将赋予与hACE2更高的亲和力的AA突变。这些突变中有V367F和G502D(其增加了RBD的表达)和N501F、N501T和Q498Y。在一些实施方案中，所述SARS-CoV-2RBD蛋白包含以下一个或更多个位置的突变：G404、A475、T478、N481、G485、F490、Q493、G496、Q498、N501或V503。
III.核酸、克隆细胞和表达细胞
本发明还涉及包含编码本文描述的融合蛋白的核苷酸序列的核酸。核酸可以是DNA或RNA。包含编码本文描述的融合蛋白的核苷酸序列的DNA通常包含与该核苷酸序列可操作地连接的启动子。启动子优选地能够在感兴趣的表达细胞中驱动核苷酸序列的组成型或诱导型表达。所述核酸还可以包含可用于选择包含感兴趣的所述核酸的细胞的选择性标志物。有用的选择性标志物是技术人员熟知的。核酸的精确核苷酸序列不受特别限制，只要核苷酸序列编码本文描述的融合蛋白。密码子可以选择为，例如，匹配感兴趣的表达细胞(例如，哺乳动物细胞，诸如人类细胞)的密码子偏好和/或为了克隆期间的方便。DNA可以是质粒，例如，质粒可以包含复制起点(例如，用于质粒在原核细胞中的复制)。
在本文描述的一种实施方案中，本发明涉及包含编码融合蛋白的核苷酸序列、可操作地连接到该核苷酸序列的启动子和选择性标志物的核酸。
本发明的各方面还涉及包含核酸的细胞，该核酸包含编码如本文描述的融合蛋白的核苷酸序列。细胞可以是表达细胞或克隆细胞。核酸通常在大肠杆菌(E.coli)中克隆，尽管可以使用其他克隆细胞。
如果细胞是表达细胞，则所述核酸任选地是染色体的核酸，即，其中核苷酸序列被整合到染色体中，尽管核酸可以例如作为染色体外DNA或载体(诸如质粒、粘粒、噬菌体等)存在于表达细胞中。载体的形式不应被视为限制性的。
在本文描述的一种实施方案中，细胞通常是表达细胞。表达细胞的性质不特别限制。可以使用的表达细胞是原核细胞，诸如大肠杆菌和芽孢杆菌属的种(Bacillus spp.)以及真核细胞诸如酵母细胞(例如酿酒酵母(S.cerevisiae)、粟酒裂殖酵母(S.pombe)、巴斯德毕赤酵母(P.pastoris)、乳酸克鲁维酵母(K lactis)、多形汉逊酵母(H polymorpha))、昆虫细胞(例如Sf9)、真菌、植物细胞或哺乳动物细胞。哺乳动物表达细胞可以允许融合蛋白的有利折叠、翻译后修饰和/或分泌，尽管其他真核细胞或原核细胞也可以用作表达细胞。示例性表达细胞包括TunaCHO、ExpiCHO、Expi293、BHK、NS0、Sp2/0、COS、C127、HEK、HT-1080、PER.C6、HeLa和Jurkat细胞。细胞还可以针对载体的整合(更优选地针对质粒DNA的整合)进行选择。
本发明的融合蛋白可以通过包含编码融合蛋白的核苷酸序列的核酸向哺乳动物细胞中的适当转染策略来产生。技术人员知悉可用于将核酸转染到所选择的细胞系中的不同技术(脂质体转染、电穿孔等)。因此，哺乳动物细胞系和转染策略的选择不应被认为是限制性的。细胞系可以进一步针对质粒DNA的整合进行选择。
本发明的各方面还涉及包含本文所述融合蛋白的细胞。
IV.与测定相关的组合物和方法
本发明的各方面涉及包含如本文描述的融合蛋白的组合物。在一些实施方案中，组合物可以包含药学上可接受的载体和/或药学上可接受的赋形剂。组合物可以是例如疫苗。
本发明的各种实施方案涉及治疗或预防人类患者SARS-CoV-2感染的方法，包括向患者施用包含本文所述融合蛋白的组合物。本文中使用的术语“预防(preventing)”是指预防(prophylaxis)，其包括向患者施用组合物以减少患者相对于未接受组合物的其他类似患者感染SARS-CoV-2的可能性。术语预防还包括向一组患者施用组合物，以相对于未接受组合物的其他类似的患者组减少该组中感染SARS-CoV-2的患者数量。
本发明的各种实施方案涉及治疗或预防人类患者SARS-CoV-2感染的方法，包括向患者施用根据本文所述实施方案的疫苗。
患者可能感染了SARS-CoV-2，患者可能已经暴露于SARS-CoV-2，或者患者可能呈现暴露于SARS-CoV-2和/或感染SARS-CoV-2的风险增加。
在本文描述的一种实施方案中，组合物包含本发明的融合蛋白和固体支持物。
在其他实施方案中，组合物包含本发明的融合蛋白和固体支持物，其中融合蛋白与固体支持物共价或非共价结合。如本文使用的术语“非共价结合”是指特异性结合，诸如在抗体和其抗原之间、配体和其受体之间、或者酶和其底物之间的特异性结合，例如由链霉亲和素结合蛋白和链霉亲和素之间或抗体和其抗原之间的相互作用例示。
在其他实施方案中，组合物包含本发明的融合蛋白和固体支持物，其中融合蛋白与固体支持物直接或间接结合。如本文使用的术语“直接”结合是指分子与固体支持物的直接缀合，例如，将融合蛋白的半胱氨酸硫醇结合至金表面的金-硫醇相互作用。如本文使用的术语“间接”结合包括融合蛋白与直接结合至固体支持物的另一分子的特异性结合，例如，融合蛋白可以结合直接结合至固体支持物的抗体，从而将融合蛋白间接结合至固体支持物。术语“间接”结合与融合蛋白和固体支持物之间的分子数目无关，只要(a)分子的菊花链(daisy chain)之间的每个相互作用是特异性或共价的相互作用，以及(b)菊花链的末端分子直接结合至固体支持物。
固体支持物可以包括颗粒、珠、膜、表面、多肽芯片、微量滴定板或层析柱的固相。
组合物可以包含多于一个珠或颗粒，其中所述多于一个珠或颗粒中的每个珠或颗粒与至少一种如本文描述的融合蛋白直接或间接结合。组合物可以包含多于一个珠或颗粒，其中所述多于一个珠或颗粒中的每个珠或颗粒与至少一种如本文描述的融合蛋白共价或非共价结合。
实施方案的各方面涉及用于检测样品中针对本发明的融合蛋白的抗体和/或其片段的存在的试剂盒，所述试剂盒包含本文所述的融合蛋白和固体支持物或组合物。
本文描述的组合物和试剂盒可以用于测定或用于在测定进行期间产生的组合物。本发明的各方面涉及包括如本文描述的组合物的诊断医学装置。
本发明的各方面涉及用于检测抗SARS-CoV-2抗体的测定。
测定可以是用于测量本发明的融合蛋白与样品中的抗RBD、片段抗RBD和/或片段抗RBD的相对结合亲和力(例如，相对于一个或更多个对照样品或标准)的测定。测定可以是用于测量本发明的融合蛋白与任何抗RBD的相对结合亲和力(例如，相对于一个或更多个对照样品或标准)的测定。
测定通常以允许测量(诸如通过透射比浊法、散射比浊法、UV/Vis/IR光谱法(例如，吸收、发射)、荧光或磷光光谱法、或表面等离子体共振)，或者有助于将直接或间接结合固体支持物的组分与不直接或间接结合固体支持物的组分分离，或者两者的固体支持物为特征。例如，测定可以包括包含颗粒或珠和/或有助于直接或间接结合颗粒或珠的组分的机械分离的组合物。
可以包括本发明的融合蛋白或组合物的其他示例性测定包括但不限于ELISA、侧向流、单分子计数(SMC)、粘弹性测试诸如Sonoclot、凝胶技术、荧光测定和使用这些技术中的任何一种的其他护理点测试。
本发明的融合蛋白将通过以下非限制性实例进一步说明。
实施例
实施例1：本发明的pxENB14-RBD和pxENB17-RBD蛋白的表达和纯化
RBD蛋白在Expi293细胞中产生，并从上清液亲和纯化。亲和纯化根据包括咪唑洗涤和洗脱的IMAC标准方案进行。在旋转浓缩和缓冲液交换后，通过SDS-PAGE蛋白印迹在还原和非还原条件下对蛋白进行功能评价。图1示出了两种分子设计的实验数据，通过SDS-PAGE表征的最终纯化样品。
通过SDS-PAGE蛋白印迹评价pxENB14-RBD和pxENB17-RBD蛋白揭示了RBD单体、二聚体和四聚体的存在。这一数据得到了SECMALS的证实。在蛋白印迹上，这两种蛋白都被兔多克隆抗体识别，展示了生物活性。使用N-和O、D-、糖基化和还原条件进行完整质量分析(表1)。pxENB14-RBD和pxENB17-RBD都显示了伪MW移位，表明存在通过完整的质谱分析(intact mass spectrometry analysis)未鉴定的PTM。
表1：通过完整质谱法测量的最终分子量
#tables0#
实施例2：RBD-hACE2相互作用的评价
SARS-CoV-2大流行RBD序列的多样性仍然很低。然而，已经观察到一个突变子集，其中10种特定的突变体表现为处于世界范围内传播的高度的正选择压力下。根据一些研究，在中国武汉、深圳、香港地区和法国出现了三种RBD突变体，并且当与原型Wuhan-Hu-1毒株相比时，这些突变体对ACE2受体表现出更高的亲和力。两种突变(F342L、R408I)显示出与原始Wuhan毒株相似的ACE2亲和力，但鉴定出四种突变(N354D、D364Y、V367F、W436R)(Ou,J.等人.2020,bioRxiv,doi:https://doi.org/10.1101/2020.03.15.991844)。
鉴于出现的RBD突变，通过残基扫描和与人类ACE2受体复合的SARS-CoV-2受体结合结构域结构的亲和力成熟进行蛋白建模。这些研究使用Schrodinger的BioLuminate软件进行，并且聚焦于RBD-ACE2相互作用(图2)。
实施例3：受体结合结构域突变的评价
这项研究的目的是鉴定新的和潜在的出现的可能导致与ACE2更强的结合的突变。来自该研究的结果总结在表2中。这些突变可以单独使用或组合使用，并且突变的数量对于本发明中提出的任何设计都不是限制性的。
为了发现增强与人类ACE2结合的高亲和力RBD突变，本发明人使用来自Schrodinger的分子动力学模拟和亲和力成熟软件(Bio luminate)来预测RBD一级序列中将赋予对hACE2的更高亲和力的AA突变。这些突变中有V367F和G502D(其增加了RBD的表达)和N501F、N501T和Q498Y。
表2：通过残基扫描和亲和力成熟鉴定出的RBD突变体。
#tables1#
实施例4：本发明的pxENB14-RBD和pxENB17-RBD蛋白的功能的证实
通过BLI评价了pxENB14-RBD和pxENB17-RBD的功能。简而言之，将生物素化hACE2固定在链霉亲和素生物传感器的表面，并与浓度范围为12.5nM至0.38nM的RBD蛋白一起孵育(图3)。基于KD值，pxENB14-RBD和pxENB17-RBD显示出比来自商业来源的RBD更好的亲和力；表明RBD蛋白更有效。
本发明人评价了Expi293中RBD截短物和融合物子集的表达。RBD截短物和多聚化形式在Expi293细胞中产生(图4)。通过SDS-PAGE和蛋白印迹在还原条件下进行表达评价。所有构建体均被表达并且蛋白被分泌至细胞培养上清液。
此外，多聚化RBD蛋白在范围为25nM至0.38nM的蛋白浓度孵育，并通过结合固定在链霉亲和素生物传感器表面的生物素化hACE2进行测试，类似于图3中所描述的。除RBD41外，所有测试的蛋白都显示出比pxENB14更紧密的与rhACE2的结合，如通过解离速率(koff)值观察到的，参见图5。
图5中固定化hACE2与SARS-CoV-2多聚化RBD蛋白的结合曲线显示，多聚化结构域的添加增加了亲和力，并且与pxENB14RBD相比对koff具有积极影响，RBD41除外。所有蛋白均显示解离速率(koff)低于pxENB14RBD，表明与rhACE2的结合更紧密。根据分析物浓度，数据以不同的颜色线显示，并且将数据针对1:1结合模型进行最佳拟合，如红线所示。
通过BLI基于pxENB14RBD(图6)和pxENB46RBD(图7)评价了RBD突变蛋白的功能。
图6示出了固定化hACE2与描述了当前SARS-CoV-2变体的SARS-CoV-2pxENB14RBD突变体(Pango谱系)的结合曲线。
突变体pxENBRBD14-B1.617(SEQ ID NO:52)对rhACE2受体显示出特别高的亲和力，如观察到亲和常数从17nM增加到76.1nM所示。除了pxENB-RBD14 B1.1.7(SEQ ID NO:50)之外，所有RBD突变体都显示出比pxENB14RBD更高的解离速率，表明这些突变体与rhACE2的结合比原始蛋白更强。
图7示出了固定化hACE2与描述了当前SARS-CoV-2变体的SARS-CoV-2pxENB46RBD突变体(Pango谱系)的结合曲线。
序列
Claims (20)
1.一种融合蛋白，包含SARS-CoV-2刺突蛋白的SARS-CoV-2受体结合结构域(RBD)或其片段和N-末端信号肽，以及多组氨酸标签、接头、寡聚化标签、刺突蛋白中RBD外的区域、辣根过氧化物酶结合结构域或蛋白酶裂解位点中的至少一种。
2.根据权利要求1所述的融合蛋白，其中所述N-末端信号肽选自刺突内源性信号肽、组织纤溶酶原激活物(tPa)。
3.根据权利要求1或2所述的融合蛋白，其中所述N-末端信号肽具有选自SEQ ID NO:1和SEQ ID NO:2的氨基酸序列。
4.根据前述权利要求中任一项所述的融合蛋白，其中所述多组氨酸标签由8个或10个组氨酸残基组成。
5.根据权利要求4所述的融合蛋白，其中所述多组氨酸标签具有选自SEQ ID NO:7和SEQ ID NO:8的氨基酸序列。
6.根据前述权利要求中任一项所述的融合蛋白，其中所述寡聚化标签选自鼠IgG1-Fc(仅CH2、CH3)、鼠IgG1-Fc二聚化结构域、鼠IgG-2a-Fc(仅CH2、CH3)、鼠IgG-2a-Fc二聚化结构域、p53四聚化结构域、SARS-CoV-2核衣壳N-末端结构域和SARS-CoV-2核衣壳C-末端结构域。
7.根据权利要求6所述的融合蛋白，其中所述寡聚化标签具有选自SEQ ID NO:9、SEQID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ IDNO:13、SEQ ID NO:14和SEQ ID NO:15的氨基酸序列。
8.根据前述权利要求中任一项所述的融合蛋白，其中所述接头是柔性接头。
9.根据权利要求8所述的融合蛋白，其中所述接头具有选自SEQ IDNO:3、SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6的氨基酸序列。
10.根据前述权利要求中任一项所述的融合蛋白，其中所述链霉亲和素结合肽标签是SEQ ID NO:17或包含SEQ ID NO:17。
11.根据前述权利要求中任一项所述的融合蛋白，其中所述辣根过氧化物酶结合结构域具有选自SEQ ID NO:18的氨基酸序列。
12.根据前述权利要求中任一项所述的融合蛋白，其中所述蛋白酶裂解位点是烟草蚀刻病毒裂解位点(TEV)。
13.根据权利要求12所述的融合蛋白，其中所述蛋白酶裂解位点具有选自SEQ ID NO:19的氨基酸序列。
14.根据前述权利要求中任一项所述的融合蛋白，其中所述SARS-CoV-2刺突蛋白的所述受体结合结构域(RBD)或其片段具有与SEQ ID NO:20具有至少90％序列同一性的氨基酸序列。
15.根据前述权利要求中任一项所述的融合蛋白，其中所述融合蛋白具有与SEQ IDNO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26、SEQ IDNO:27、SEQ ID NO:28、SEQ IDNO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32、SEQ IDNO:33、SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:37、SEQ IDNO:38、SEQ IDNO:39、SEQ ID NO:40、SEQ ID NO:41、SEQ ID NO:42、SEQ ID NO:43、SEQ ID NO:44、SEQ IDNO:45、SEQ ID NO:46、SEQ IDNO:47、SEQ ID NO:48、SEQ ID NO:49、SEQ ID NO:50、SEQ IDNO:51、SEQ ID NO:52、SEQ ID NO:53、SEQ ID NO:54、SEQ ID NO:55、SEQ IDNO:56或SEQ IDNO:57具有至少90％序列同一性的氨基酸序列。
16.根据前述权利要求中任一项所述的融合蛋白，其中所述SARS-CoV-2RBD蛋白包含以下一个或更多个位置的突变：G404、A475、T478、N481、G485、F490、Q493、G496、Q498、N501或V503。
17.一种细胞，包含根据前述权利要求中任一项所述的融合蛋白。
18.一种核酸，包含编码根据权利要求1至16中任一项所述的融合蛋白的核苷酸序列、可操作地连接至所述核苷酸序列的启动子和选择性标志物。
19.一种细胞，包含权利要求18所述的核酸。
20.一种组合物，包含权利要求1至16中任一项所述的融合蛋白和固体支持物，其中所述融合蛋白与所述固体支持物共价或非共价结合。
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