CN111393532B - 新型冠状病毒优势表位融合蛋白、诊断试剂及应用 - Google Patents

新型冠状病毒优势表位融合蛋白、诊断试剂及应用 Download PDF

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CN111393532B
CN111393532B CN202010119472.4A CN202010119472A CN111393532B CN 111393532 B CN111393532 B CN 111393532B CN 202010119472 A CN202010119472 A CN 202010119472A CN 111393532 B CN111393532 B CN 111393532B
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周建平
周裕军
许秀丽
张望
吴鸣月
王艳新
常缘荣
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BEIJING DEAOPING BIOTECHNOLOGY CO.,LTD.
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Abstract

本发明涉及新型冠状病毒优势表位融合蛋白、诊断试剂及应用，属于病毒诊断试剂。本发明所述新型冠状病毒优势表位融合蛋白包括通过连接臂连接的N蛋白优势表位肽和S蛋白优势表位肽、E蛋白优势表位肽，所述N蛋白优势表位肽包括优势表位肽N1、优势表位肽N2、优势表位肽N3和优势表位肽N4中的一个或两个以上。本发明所述新型冠状病毒优势表位融合蛋白特异性更高，灵敏度更高(阳性检出率更高)，生物相容性、溶解度高，稳定性好，更有利于制备相应的检测试剂；基于本发明所述融合蛋白制备的检测试剂，灵敏度高，特异性好，可实现新型冠状病毒的早期筛查。

Description

新型冠状病毒优势表位融合蛋白、诊断试剂及应用
技术领域
本发明涉及病毒诊断试剂技术领域，具体涉及新型冠状病毒优势表位融合蛋白、诊断试剂及应用。
背景技术
冠状病毒是一个大型病毒家族，已知可引起感冒、中东呼吸综合征（MERS）和严重急性呼吸综合征（SARS）等较严重疾病。新型冠状病毒是以前从未在人体中发现的冠状病毒新毒株，2019-nCoV是人类分离的第七类冠状病毒。相关疾病监测，发现多起病毒性肺炎病例，均诊断为病毒性肺炎/肺部感染。
新型冠状病毒感染人体后一般有3~14天（有个别案例报道最长24天）的潜伏期，潜伏期内可无临床症状，不易发现。目前的证据表明，潜伏期感染者也有传染性，增大了阻断病毒传播的难度。新型冠状病毒感染临床症状主要表现为发热、咳嗽、乏力等非特异症状，与普通感冒难以区分，其确诊依赖实验室诊断技术。
目前新型冠状病毒感染诊断主要依赖基于聚合酶链式反应（PCR）或核酸测序检测病毒特异性基因序列来进行。但是核酸诊断需要有特殊的实验室（要求有4个在物理空间上完全相互独立的区域）和数十种配套设备以及专业操作人员（需认证）才能完成，且检测过程操作复杂，需要数小时才能完成；极易产生气溶胶污染，造成假阳性，也存在因为取样不合适造成漏检的情况，因此核酸检测不适合现场大规模开展。
免疫学诊断主要是通过抗原抗体免疫反应检测人体感染病毒以后产生的特异性抗体来诊断。免疫学诊断方法具有操作简单、检测速度快、无需特殊场地及（复杂）仪器设备，且敏感性高等优点，适用于现场大规模开展新型冠状病毒感染筛查及诊断工作。
为实现快速，特异性高，灵敏度高的免疫学诊断手段，诊断原料尤为重要，主要体现在抗原稳定性和特异性检出率方面。病毒在感染人体后，释放大量的棘突蛋白（Spikeprotein，S蛋白）和核蛋白（Nucleoprotein，N蛋白）刺激机体产生免疫反应，产生针对这两种蛋白的抗体，单一采用N蛋白或S蛋白包被生产诊断试剂会造成捕获不完全，存在漏检灵敏度不高的情况。采用两种蛋白混合包被会使生产工艺复杂，批间控制难度大。
发明内容
本发明的目的在于提供新型冠状病毒优势表位融合蛋白、诊断试剂及应用。本发明所述新型冠状病毒优势表位融合蛋白去除了非表位相关的氨基酸序列，减少了融合蛋白同检测样本的非特异性反应，相应制备的检测试剂特异性更高；同时表位丰富度高，暴露更好，相应制备的检测试剂灵敏度更高（阳性检出率更高）；通过亲水及柔性的连接臂连接多个抗原表位，有效的改善了融合蛋白的生物相容性，溶解度高，稳定性好，更有利于制备相应的检测试剂；基于上述的多种优势表位融合蛋白制备的检测试剂，灵敏度高，特异性好，可实现新型冠状病毒的早期筛查。
本发明提供了新型冠状病毒优势表位融合蛋白，所述优势表位融合蛋白包括通过连接臂连接的N蛋白优势表位肽、S蛋白优势表位肽和E蛋白优势表位肽；
所述N蛋白优势表位肽包括优势表位肽N1、优势表位肽N2、优势表位肽N3和优势表位肽N4中的一个或两个以上，两个以上优势表位肽之间通过连接臂连接；所述优势表位肽N1的氨基酸序列如SEQ ID NO.1所示，所述优势表位肽N2的氨基酸序列如SEQ ID NO.2所示，所述优势表位肽N3的氨基酸序列如SEQ ID NO.3所示，所述优势表位肽N4的氨基酸序列如SEQ ID NO.4所示；
所述S蛋白优势表位肽的氨基酸序列如SEQ ID NO.5所示；
所述E蛋白优势表位肽的氨基酸序列如SEQ ID NO.6所示；
所述连接臂氨基酸序列如SEQ ID NO.7所示。
优选的是，所述N蛋白优势表位肽由优势表位肽N1和优势表位肽N4通过连接臂连接得到。
优选的是，所述优势表位融合蛋白的氨基酸序列如SEQ ID NO.8所示。
优选的是，所述N蛋白优势表位肽由优势表位肽N1、优势表位肽N2和优势表位肽N4通过连接臂连接得到。
优选的是，所述优势表位融合蛋白的氨基酸序列如SEQ ID NO.9所示。
优选的是，所述N蛋白优势表位肽由优势表位肽N1、优势表位肽N2、优势表位肽N3和优势表位肽N4通过连接臂连接得到。
优选的是，所述优势表位融合蛋白的氨基酸序列如SEQ ID NO.10所示。
本发明还提供了上述技术方案所述优势表位融合蛋白在制备新型冠状病毒诊断用试剂中的应用。
本发明还提供了一种新型冠状病毒诊断试剂，所述诊断试剂包括上述技术方案所述新型冠状病毒优势表位融合蛋白。
本发明还提供了一种双抗夹心体外诊断试剂，所述双抗夹心体外诊断试剂包括上述技术方案所述新型冠状病毒优势表位融合蛋白。
本发明提供了新型冠状病毒优势表位融合蛋白。本发明提供的新型冠状病毒优势表位融合蛋白（含不同融合表位的重组蛋白），能够提高试剂生产工艺可控性、灵敏度高、特异性好，可获得高稳定性抗原，免疫产生高亲和度抗体。
本发明所述新型冠状病毒优势表位融合蛋白是基于2019-nCoV病毒N、S蛋白和E蛋白的多种优势表位融合蛋白，相比于常规的N蛋白、S蛋白、或者N+S蛋白，本发明融合蛋白去除了非表位相关的氨基酸序列，减少了融合蛋白同检测样本的非特异性反应，相应制备的检测试剂特异性更高；同时表位丰富度高，暴露更好，相应制备的检测试剂灵敏度更高（阳性检出率更高）；通过亲水及柔性的连接臂连接多个抗原表位，有效的改善了融合蛋白的生物相容性，溶解度高，稳定性好，更有利于制备相应的检测试剂。因此，基于本发明新型冠状病毒优势表位融合蛋白制备的检测试剂，灵敏度高，特异性好，可实现新型冠状病毒的早期筛查。
对比单独的N蛋白或者S蛋白作为捕获/检测抗原，本发明的新型冠状病毒优势表位融合蛋白具备更丰富的表位，能够检测到样本中更多的病毒抗体，可以更早期的检出感染人群；同时融合蛋白生物稳定性好，溶解度相比于全长的蛋白更好，制备的检测试剂性能更好。
对比混合的N蛋白和S蛋白作为捕获/检测抗原，本发明的新型冠状病毒优势表位融合蛋白通过特殊的亲水柔性连接臂把N蛋白和S蛋白的优势表位串联在一起，使用更简单（不需要摸索N蛋白和S蛋白混合的比例），捕获密度更高（如包被在NC膜上时，同样大小的空间，融合蛋白可以包被更多数量的表位）从而灵敏度提高，生物相容性更好，有利于试剂性能提高（低等电点和亲水性的柔性连接臂，有效降低原N蛋白的高等电点，并提高水溶性，避免蛋白聚集）。另外，试剂生产工艺更为简单，易于控制批间差。
对比新冠病毒裂解液作为捕获/检测抗原，本发明的新型冠状病毒优势表位融合蛋白特异性高（新冠病毒裂解液存在多种其他病毒类似表位，造成交叉反应高），生产批间差小（病毒裂解液批次之间差异较大，控制难度大，均一性差），生物安全性高（病毒裂解液即使灭活也存在潜在的生物危害性）。
具体实施方式
本发明提供了新型冠状病毒优势表位融合蛋白，所述优势表位融合蛋白包括通过连接臂连接的N蛋白优势表位肽、S蛋白优势表位肽和E蛋白优势表位肽；
所述N蛋白优势表位肽包括优势表位肽N1（N蛋白21-43）、优势表位肽N2（N蛋白65-86）、优势表位肽N3（N蛋白248-271）和优势表位肽N4（N蛋白348-406）中的一个或两个以上，两个以上优势表位肽之间通过连接臂连接；所述优势表位肽N1的氨基酸序列如SEQ IDNO.1所示（SDSTGSNQNGERSGARSKQRRPQ），所述优势表位肽N2的氨基酸序列如SEQ ID NO.2所示（KFPRGQGVPINTNSSPDDQIGY），所述优势表位肽N3的氨基酸序列如SEQ ID NO.3所示（KKSAAEASKKPRQKRTATKAYNVT），所述优势表位肽N4的氨基酸序列如SEQ ID NO.4所示（DQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAADLDDFSKQ）；
所述S蛋白优势表位肽（S蛋白452-611）的氨基酸序列如SEQ ID NO.5所示（DISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHA）；
所述E蛋白优势表位肽（E蛋白30-75）的氨基酸序列如SEQ ID NO.6所示（TLAILTALRLCAYCCNIVNVSLVKPSFYVYSRVKNLNSSRVPDLLV）；
所述连接臂氨基酸序列如SEQ ID NO.7所示（GGGS）。
在本发明中，所述N蛋白优势表位肽由优势表位肽N1和优势表位肽N4通过连接臂连接得到。本发明所述新型冠状病毒优势表位融合蛋白，通过linker连接降低等电点，能够提高亲和力，溶解度，有利于体外诊断试剂的开发。
在本发明中，所述优势表位融合蛋白的氨基酸序列如SEQ ID NO.8所示。
在本发明中，所述N蛋白优势表位肽由优势表位肽N1、优势表位肽N2和优势表位肽N4通过连接臂连接得到。
在本发明中，所述优势表位融合蛋白的氨基酸序列如SEQ ID NO.9所示。
在本发明中，所述N蛋白优势表位肽由优势表位肽N1、优势表位肽N2、优势表位肽N3和优势表位肽N4通过连接臂连接得到。
在本发明中，所述优势表位融合蛋白的氨基酸序列如SEQ ID NO.10所示。
本发明具体实施例还提供了当所述N蛋白优势表位肽为优势表位肽N4的优势表位融合蛋白。所述优势表位融合蛋白的氨基酸序列如SEQ ID NO.11所示。
本发明对上述技术方案所述优势表位融合蛋白的合成方法没有特殊限定，采用本领域技术人员熟知的常规融合蛋白表达方法即可。如委托专业基因合成公司进行基因合成；将合成好的基因转入载体，通过真核系统和原核系统进行表达；收集表达后的产物，采用亲和层析方法进行纯化，获得多抗原表位的融合蛋白。本发明对获得的优势表位融合蛋白优选通过荧光免疫层析平台进行筛选评价确定等。
本发明还提供了上述技术方案所述优势表位融合蛋白在制备新型冠状病毒诊断用试剂中的应用。
本发明还提供了一种新型冠状病毒诊断试剂，所述诊断试剂包括上述技术方案所述新型冠状病毒优势表位融合蛋白。
本发明还提供了一种双抗夹心体外诊断试剂，所述双抗夹心体外诊断试剂包括上述技术方案所述新型冠状病毒优势表位融合蛋白。
下面结合具体实施例对本发明所述的新型冠状病毒优势表位融合蛋白、诊断试剂及应用做进一步详细的介绍，本发明的技术方案包括但不限于以下实施例。
实施例1
新型冠状病毒2019-nCoV多优势表位融合重组蛋白的制备:
通过计算预测筛选新型冠状病毒2019-nCoV N蛋白、S蛋白、E蛋白的优势抗原表位，并通过柔性多肽（GGGS）连接到一起，组成2019-nCoV病毒多优势表位融合蛋白。委托北京德奥平生物技术有限公司生产制备，纯度大于90%。
本发明实施例中构建了8种重组蛋白，其中4种采用本发明技术方案所述融合蛋白方案，另外4种采用传统根据检索序列进行蛋白构建，并与市面重组蛋白进行对比基本序列一致。
融合序列1
N蛋白348-406，S蛋白452-611，E蛋白30-75，下划线为linker GGGS
DQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAADLDDFSKQGGGSDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHAGGGSTLAILTALRLCAYCCNIVNVSLVKPSFYVYSRVKNLNSSRVPDLLV（SEQ ID NO.11）
Theoretical pI/Mw: 8.32 / 29663.80
融合序列2
N蛋白21-43，348-406，S蛋白452-611，E蛋白30-75，下划线为linker GGGS
SDSTGSNQNGERSGARSKQRRPQGGGSDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAADLDDFSKQGGGSDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHAGGGSTLAILTALRLCAYCCNIVNVSLVKPSFYVYSRVKNLNSSRVPDLLV（SEQ ID NO.8）
Theoretical pI/Mw: 8.77 / 32407.61
融合序列3
N蛋白21-43，65-86，348-406，S蛋白452-611，E蛋白30-75，下划线为linker GGGS
SDSTGSNQNGERSGARSKQRRPQGGGSKFPRGQGVPINTNSSPDDQIGYGGGSDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAADLDDFSKQGGGSDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHAGGGSTLAILTALRLCAYCCNIVNVSLVKPSFYVYSRVKNLNSSRVPDLLV（SEQ ID NO.9）
Theoretical pI/Mw: 8.76 / 35038.43
融合序列4
N蛋白21-43，65-86，248-271，348-406，S蛋白452-611，E蛋白30-75，下划线为linker GGGS
SDSTGSNQNGERSGARSKQRRPQGGGSKFPRGQGVPINTNSSPDDQIGYGGGSKKSAAEASKKPRQKRTATKAYNVTGGGSDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAADLDDFSKQGGGSDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHAGGGSTLAILTALRLCAYCCNIVNVSLVKPSFYVYSRVKNLNSSRVPDLLV（SEQ ID NO.10）
Theoretical pI/Mw: 9.27 / 37941.72
对比其他几种蛋白序列及原料，以下为其他人常规设计的蛋白原料序列
N蛋白
MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAADLDDFSKQLQQSMSSADSTQA（SEQ ID NO.12）
Theoretical pI/Mw: 10.07 / 45625.70
S1-RBD
RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFA（SEQ ID NO.13）
Theoretical pI/Mw: 8.91 / 25169.48
S1
VNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRAR（SEQ IDNO.14）
Theoretical pI/Mw: 8.27 / 75012.62
S2
SVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT（SEQ IDNO.15）
Theoretical pI/Mw: 5.25 / 64505.74
实施例2
8种重组蛋白原料的性能验证
获得以上4种融合蛋白及4种常规表达重组蛋白，分别进行稳定性，抗体滴度检测。
稳定性测试：将获得的8种重组蛋白加入保护液PBS+0.2%牛血清白蛋白，放置于室温（大约20℃）和37℃，活性检测活力变化情况，确定重组蛋白稳定性。通过基因重组融合改造，尤其对N蛋白的等电点进行优化，从下表数据可看出，融合蛋白稳定性相比于单独的重组蛋白在室温和37℃环境下放置，活性下降比例降低，稳定性大幅度提升。
表1 8种重组蛋白生物活性对比表
Figure DEST_PATH_IMAGE002
抗体滴度检测：分别采用8种重组蛋白免疫兔子，目前阶段仅免疫一次，观察免疫14天抗体滴度变化，比较8种重组蛋白免疫原性。通过融合病毒蛋白的优势抗原表位，可提高融合蛋白的免疫原性，从而监测免疫14天看题滴度变化，发现融合蛋白免疫14天后，滴度相较重组蛋白整体要高，尤其融合蛋白1显示了较好的免疫原性。
表2 重组蛋白免疫原性对比表
Figure DEST_PATH_IMAGE004
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 北京丹大生物技术有限公司
<120> 新型冠状病毒优势表位融合蛋白、诊断试剂及应用
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<170> SIPOSequenceListing 1.0
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<211> 23
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<213> 人工序列(Artificial Sequence)
<400> 1
Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly Glu Arg Ser Gly Ala Arg
1 5 10 15
Ser Lys Gln Arg Arg Pro Gln
20
<210> 2
<211> 22
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro
1 5 10 15
Asp Asp Gln Ile Gly Tyr
20
<210> 3
<211> 24
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Lys Lys Ser Ala Ala Glu Ala Ser Lys Lys Pro Arg Gln Lys Arg Thr
1 5 10 15
Ala Thr Lys Ala Tyr Asn Val Thr
20
<210> 4
<211> 59
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Asp Gln Val Ile Leu Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe
1 5 10 15
Pro Pro Thr Glu Pro Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr
20 25 30
Gln Ala Leu Pro Gln Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu
35 40 45
Pro Ala Ala Asp Leu Asp Asp Phe Ser Lys Gln
50 55
<210> 5
<211> 160
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly
1 5 10 15
Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln
20 25 30
Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser
35 40 45
Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser
50 55 60
Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn Gly Leu
65 70 75 80
Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu Pro Phe
85 90 95
Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val Arg Asp
100 105 110
Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe Gly Gly
115 120 125
Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val Ala Val
130 135 140
Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile His Ala
145 150 155 160
<210> 6
<211> 46
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Thr Leu Ala Ile Leu Thr Ala Leu Arg Leu Cys Ala Tyr Cys Cys Asn
1 5 10 15
Ile Val Asn Val Ser Leu Val Lys Pro Ser Phe Tyr Val Tyr Ser Arg
20 25 30
Val Lys Asn Leu Asn Ser Ser Arg Val Pro Asp Leu Leu Val
35 40 45
<210> 7
<211> 4
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Gly Gly Gly Ser
1
<210> 8
<211> 300
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly Glu Arg Ser Gly Ala Arg
1 5 10 15
Ser Lys Gln Arg Arg Pro Gln Gly Gly Gly Ser Asp Gln Val Ile Leu
20 25 30
Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu Pro
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Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr Gln Ala Leu Pro Gln
50 55 60
Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala Ala Asp Leu
65 70 75 80
Asp Asp Phe Ser Lys Gln Gly Gly Gly Ser Asp Ile Ser Thr Glu Ile
85 90 95
Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys
100 105 110
Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly
115 120 125
Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala
130 135 140
Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn
145 150 155 160
Lys Cys Val Asn Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu
165 170 175
Thr Glu Ser Asn Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp
180 185 190
Ile Ala Asp Thr Thr Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile
195 200 205
Leu Asp Ile Thr Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro
210 215 220
Gly Thr Asn Thr Ser Asn Gln Val Ala Val Leu Tyr Gln Asp Val Asn
225 230 235 240
Cys Thr Glu Val Pro Val Ala Ile His Ala Gly Gly Gly Ser Thr Leu
245 250 255
Ala Ile Leu Thr Ala Leu Arg Leu Cys Ala Tyr Cys Cys Asn Ile Val
260 265 270
Asn Val Ser Leu Val Lys Pro Ser Phe Tyr Val Tyr Ser Arg Val Lys
275 280 285
Asn Leu Asn Ser Ser Arg Val Pro Asp Leu Leu Val
290 295 300
<210> 9
<211> 326
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly Glu Arg Ser Gly Ala Arg
1 5 10 15
Ser Lys Gln Arg Arg Pro Gln Gly Gly Gly Ser Lys Phe Pro Arg Gly
20 25 30
Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro Asp Asp Gln Ile Gly
35 40 45
Tyr Gly Gly Gly Ser Asp Gln Val Ile Leu Leu Asn Lys His Ile Asp
50 55 60
Ala Tyr Lys Thr Phe Pro Pro Thr Glu Pro Lys Lys Asp Lys Lys Lys
65 70 75 80
Lys Ala Asp Glu Thr Gln Ala Leu Pro Gln Arg Gln Lys Lys Gln Gln
85 90 95
Thr Val Thr Leu Leu Pro Ala Ala Asp Leu Asp Asp Phe Ser Lys Gln
100 105 110
Gly Gly Gly Ser Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr
115 120 125
Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser
130 135 140
Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val
145 150 155 160
Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly
165 170 175
Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn
180 185 190
Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys
195 200 205
Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp
210 215 220
Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys
225 230 235 240
Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn
245 250 255
Gln Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val
260 265 270
Ala Ile His Ala Gly Gly Gly Ser Thr Leu Ala Ile Leu Thr Ala Leu
275 280 285
Arg Leu Cys Ala Tyr Cys Cys Asn Ile Val Asn Val Ser Leu Val Lys
290 295 300
Pro Ser Phe Tyr Val Tyr Ser Arg Val Lys Asn Leu Asn Ser Ser Arg
305 310 315 320
Val Pro Asp Leu Leu Val
325
<210> 10
<211> 354
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly Glu Arg Ser Gly Ala Arg
1 5 10 15
Ser Lys Gln Arg Arg Pro Gln Gly Gly Gly Ser Lys Phe Pro Arg Gly
20 25 30
Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro Asp Asp Gln Ile Gly
35 40 45
Tyr Gly Gly Gly Ser Lys Lys Ser Ala Ala Glu Ala Ser Lys Lys Pro
50 55 60
Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr Gly Gly Gly
65 70 75 80
Ser Asp Gln Val Ile Leu Leu Asn Lys His Ile Asp Ala Tyr Lys Thr
85 90 95
Phe Pro Pro Thr Glu Pro Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu
100 105 110
Thr Gln Ala Leu Pro Gln Arg Gln Lys Lys Gln Gln Thr Val Thr Leu
115 120 125
Leu Pro Ala Ala Asp Leu Asp Asp Phe Ser Lys Gln Gly Gly Gly Ser
130 135 140
Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly
145 150 155 160
Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln
165 170 175
Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser
180 185 190
Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser
195 200 205
Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn Gly Leu
210 215 220
Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu Pro Phe
225 230 235 240
Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val Arg Asp
245 250 255
Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe Gly Gly
260 265 270
Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val Ala Val
275 280 285
Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile His Ala
290 295 300
Gly Gly Gly Ser Thr Leu Ala Ile Leu Thr Ala Leu Arg Leu Cys Ala
305 310 315 320
Tyr Cys Cys Asn Ile Val Asn Val Ser Leu Val Lys Pro Ser Phe Tyr
325 330 335
Val Tyr Ser Arg Val Lys Asn Leu Asn Ser Ser Arg Val Pro Asp Leu
340 345 350
Leu Val
<210> 11
<211> 273
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Asp Gln Val Ile Leu Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe
1 5 10 15
Pro Pro Thr Glu Pro Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr
20 25 30
Gln Ala Leu Pro Gln Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu
35 40 45
Pro Ala Ala Asp Leu Asp Asp Phe Ser Lys Gln Gly Gly Gly Ser Asp
50 55 60
Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val
65 70 75 80
Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro
85 90 95
Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe
100 105 110
Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr
115 120 125
Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn Gly Leu Thr
130 135 140
Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu Pro Phe Gln
145 150 155 160
Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val Arg Asp Pro
165 170 175
Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe Gly Gly Val
180 185 190
Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val Ala Val Leu
195 200 205
Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala Ile His Ala Gly
210 215 220
Gly Gly Ser Thr Leu Ala Ile Leu Thr Ala Leu Arg Leu Cys Ala Tyr
225 230 235 240
Cys Cys Asn Ile Val Asn Val Ser Leu Val Lys Pro Ser Phe Tyr Val
245 250 255
Tyr Ser Arg Val Lys Asn Leu Asn Ser Ser Arg Val Pro Asp Leu Leu
260 265 270
Val
<210> 12
<211> 419
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Met Ser Asp Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro Arg Ile Thr
1 5 10 15
Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly Glu Arg
20 25 30
Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn
35 40 45
Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Asp Leu
50 55 60
Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro
65 70 75 80
Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly
85 90 95
Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr
100 105 110
Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn Lys Asp
115 120 125
Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp
130 135 140
His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val Leu Gln
145 150 155 160
Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly Ser
165 170 175
Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg Asn
180 185 190
Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Thr Ser Pro Ala
195 200 205
Arg Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu Leu
210 215 220
Asp Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys Gly Gln Gln
225 230 235 240
Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys
245 250 255
Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr Gln
260 265 270
Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp
275 280 285
Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln Ile
290 295 300
Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile
305 310 315 320
Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr Gly Ala
325 330 335
Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln Val Ile Leu
340 345 350
Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu Pro
355 360 365
Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr Gln Ala Leu Pro Gln
370 375 380
Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala Ala Asp Leu
385 390 395 400
Asp Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser Ala Asp Ser
405 410 415
Thr Gln Ala
<210> 13
<211> 224
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
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 Ala
210 215 220
<210> 14
<211> 670
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 14
Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser
1 5 10 15
Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val
20 25 30
Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr
35 40 45
Trp Phe His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe
50 55 60
Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr
65 70 75 80
Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp
85 90 95
Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val
100 105 110
Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val
115 120 125
Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val
130 135 140
Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe
145 150 155 160
Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu
165 170 175
Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His
180 185 190
Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu
195 200 205
Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln
210 215 220
Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser
225 230 235 240
Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln
245 250 255
Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp
260 265 270
Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu
275 280 285
Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg
290 295 300
Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu
305 310 315 320
Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr
325 330 335
Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val
340 345 350
Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser
355 360 365
Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser
370 375 380
Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr
385 390 395 400
Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly
405 410 415
Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly
420 425 430
Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro
435 440 445
Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro
450 455 460
Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr
465 470 475 480
Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val
485 490 495
Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro
500 505 510
Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe
515 520 525
Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe
530 535 540
Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala
545 550 555 560
Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser
565 570 575
Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln
580 585 590
Val Ala Val Leu Tyr Gln Asp Val Asn Cys Thr Glu Val Pro Val Ala
595 600 605
Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly
610 615 620
Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His
625 630 635 640
Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys
645 650 655
Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg
660 665 670
<210> 15
<211> 588
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 15
Ser Val Ala Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala
1 5 10 15
Glu Asn Ser Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn
20 25 30
Phe Thr Ile Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys
35 40 45
Thr Ser Val Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys
50 55 60
Ser Asn Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg
65 70 75 80
Ala Leu Thr Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val
85 90 95
Phe Ala Gln Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe
100 105 110
Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser
115 120 125
Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala
130 135 140
Asp Ala Gly Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala
145 150 155 160
Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu
165 170 175
Pro Pro Leu Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu
180 185 190
Leu Ala Gly Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala
195 200 205
Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile
210 215 220
Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn
225 230 235 240
Gln Phe Asn Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr
245 250 255
Ala Ser Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln
260 265 270
Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile
275 280 285
Ser Ser Val Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala
290 295 300
Glu Val Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln
305 310 315 320
Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser
325 330 335
Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu Gly Gln Ser
340 345 350
Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro
355 360 365
Gln Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro
370 375 380
Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp Gly
385 390 395 400
Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly Thr His
405 410 415
Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile Ile Thr Thr
420 425 430
Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly Ile Val
435 440 445
Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys
450 455 460
Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp
465 470 475 480
Leu Gly Asp Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys
485 490 495
Glu Ile Asp Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu
500 505 510
Ile Asp Leu Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro
515 520 525
Trp Tyr Ile Trp Leu Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met
530 535 540
Val Thr Ile Met Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys
545 550 555 560
Gly Cys Cys Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser
565 570 575
Glu Pro Val Leu Lys Gly Val Lys Leu His Tyr Thr
580 585

Claims (2)

1.新型冠状病毒优势表位融合蛋白，其特征在于，所述优势表位融合蛋白的氨基酸序列如SEQ ID NO.8所示；或所述优势表位融合蛋白的氨基酸序列如SEQ ID NO.9所示；或所述优势表位融合蛋白的氨基酸序列如SEQ ID NO.10所示。
2.权利要求1所述优势表位融合蛋白在制备新型冠状病毒诊断用试剂中的应用。
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CN113321715B (zh) * 2020-02-28 2023-02-17 北京万泰生物药业股份有限公司 新型冠状病毒抗原及其检测用途
CN113087777B (zh) * 2020-04-17 2023-05-05 成都威斯克生物医药有限公司 抗SARS-CoV-2感染的蛋白以及用该蛋白制备的疫苗
CN111848753B (zh) * 2020-07-20 2022-03-15 中国科学院过程工程研究所 新型冠状病毒抗原表位及其应用
CN112409496B (zh) * 2020-11-26 2021-07-13 焦顺昌 一种跨膜表达新型冠状病毒抗原s2的融合蛋白、重组载体、重组树突状细胞及其应用
CN112229994B (zh) * 2020-12-10 2021-03-16 丹娜(天津)生物科技股份有限公司 一种基于磁微粒化学发光的新型冠状病毒抗体检测试剂盒
CN114751964B (zh) * 2020-12-29 2024-03-08 苏州方舟生物科技有限公司 β属冠状病毒通用疫苗蛋白片段及其筛选方法和应用
CN112415189B (zh) * 2021-01-22 2021-03-30 北京百普赛斯生物科技股份有限公司 偶联新型冠状病毒s2蛋白的磁珠及其制备方法与应用
EP4291212A1 (en) 2021-02-15 2023-12-20 LivingMed Biotech S.R.L. Genetically clostridium modifiedstrains expressing recombinant antigens and uses thereof
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