CN113801208B - 一种新型冠状病毒的t细胞免疫检测方法 - Google Patents

一种新型冠状病毒的t细胞免疫检测方法 Download PDF

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CN113801208B
CN113801208B CN202111150064.6A CN202111150064A CN113801208B CN 113801208 B CN113801208 B CN 113801208B CN 202111150064 A CN202111150064 A CN 202111150064A CN 113801208 B CN113801208 B CN 113801208B
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刘军
张�杰
林浩
武桂珍
高福
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National Institute for Viral Disease Control and Prevention Chinese Center for Disease Control and Prevention
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Abstract

本发明公开了一种新型冠状病毒的T细胞免疫检测方法，属于免疫检测领域。本发明设计了271条肽段，其中S1、S2、M和N肽库分别包含92、93、29和57个肽。本发明可以依照具体情况进行肽库的选择：为提高检测灵敏度，可选择总肽库、S1或M肽库；如需提高检测特异度，可以将S1和M肽库结果相结合。同时，本发明通过多肽抗原的刺激，并在体外培养细胞9天，使病毒特异性T细胞增殖，进一步提高检测的灵敏度。经过总肽库刺激的新冠病人康复6个月后的T细胞反应阳性率为：93.42％；康复12个月后的T细胞反应阳性率为：91.78％；利用S1肽库和M肽库配伍的结果为：灵敏度71.43％，特异度96.43％。

Description

一种新型冠状病毒的T细胞免疫检测方法
技术领域
本发明涉及一种新型冠状病毒的T细胞免疫检测方法，属于免疫检测领域。
背景技术
新型冠状病毒的出现和传播对全球人民的健康及生活产生重大影响。关于恢复期新冠病毒(SARS-CoV-2)患者及疫苗接种个体的适应性免疫反应持续时间的研究，可能有助于了解在SARS-CoV-2感染的自然后及接种疫苗后免疫保护如何发展和持续，并为疫苗研发提供有用的信息。全面的机体免疫功能评价应当从体液免疫和细胞免疫两个方面来进行，体液免疫评估主要包括ELISA试验检测新冠特异性抗体含量、抗体中和试验检测中和抗体滴度等；细胞免疫评估则主要通过酶联免疫斑点检测技术检测T细胞分泌γ-干扰素(IFN-γ)的细胞频率、通过流式技术检测胞内细胞因子量等。
酶联免疫斑点检测(Enzyme-linked Immunospot Assay，ELISpot)结合了细胞培养技术与酶联免疫吸附技术，能够检测到单个细胞分泌的细胞因子情况。其原理是：用PVDF膜为底的96孔板包被上特异性的单克隆抗体以捕获细胞分泌的细胞因子，在培养板的孔内加入待检测的抗原刺激物和细胞进行培养。在刺激物的刺激下，T细胞就会在对应的时间段分泌对应的细胞因子，此时细胞因子就被包被在膜上的抗体所捕获。洗去细胞后，被捕获的细胞因子可以与生物素标记的第二抗体结合，然后用酶标亲和素与生物素结合，进行化学酶联显色，就可以在膜的局部形成一个个圆形的斑点，每一个斑点就对应了当初一个分泌细胞因子的细胞。
当前，国内外在新冠病毒T细胞免疫功能检测领域上所采用的ELISpot检测方法一般为合成新型冠状病毒全序列短肽(15-mer)作为肽库，并用该抗原刺激物刺激外周血单个核细胞(Peripheral blood mononuclear cell,PBMC)隔夜培养或立即进行ELISpot检测，该方法只能检测康复初期免疫记忆较强的康复者，对于康复后较长时间的患者，由于免疫记忆随着康复时间的延长逐渐减弱减弱，这种方法进行的免疫评价灵敏度降低，故一般不用于实际检测，多用于T细胞免疫功能的评估。
发明内容
为了解决现有ELISpot检测方法对新冠患者、新冠康复者或新冠疫苗接种者的细胞免疫水平的监测上存在准确性差，灵敏度低的问题，本发明提供了一种多肽组合物，利用该多肽组合物结合ELISpot可以是使ELISpot的检测灵敏度达到71.43％，特异度达到96.43％；本发明还提供了一种检测试剂盒，用于评估抗新冠免疫活性，从而辅助判断治疗效果，为临床治疗的选择提供一定的参考。
本发明的第一个目的是提供一种多肽组合物，所述多肽组合物由S1多肽库、S2多肽库、M多肽库、N多肽库中的任意一个以上的多肽库组成。
在一种实施方式中，所述S1多肽库由氨基酸序列如SEQ ID NO.87～SEQ IDNO.178所示的多肽组成，S2多肽库由氨基酸序列如SEQ ID NO.179～SEQ ID NO.271所示的多肽组成，M多肽库由氨基酸序列如SEQ ID NO.1～SEQ ID NO.29所示的多肽组成，N多肽库由氨基酸序列如SEQ ID NO.30～SEQ ID NO.86所示的多肽组成。
在一种实施方式中，所述多肽组合物由能力与MHC-I或MHC-II类分子结合，其中，当多肽组合物与MHC结合时能被CD4+或CD8+T细胞识别。
本发明的第二个目的是提供所述多肽组合物在制备用于评估新冠患者、新冠康复者或新冠疫苗接种者细胞免疫水平的产品中的应用。
本发明的第三个目的是提供一种评估新冠患者、新冠康复者或新冠疫苗接种者细胞免疫水平的试剂盒，含有上述多肽组合物。
在一种实施方式中，所述试剂盒中还含有IFN-γ单克隆抗体。
在一种实施方式中，所述多肽组合物以肽库储液的形式存在于产品中。
在一种实施方式中，所述试剂盒中还设置有ELISpot板，ELISpot上预包被捕获T细胞分泌的细胞因子的单克隆抗体。
在一种实施方式中，所述ELISpot板上预包被有捕获IFN-γ的单克隆抗体。
本发明的第四个目的是提供一种评估新冠患者、新冠康复者或新冠疫苗接种者细胞免疫水平的检测方法，使用所述多肽组合物刺激新冠患者、新冠康复者或新冠疫苗接种者的PBMC细胞，然后使用酶联免疫斑点技术检测多肽刺激后是否出现斑点，即IFN-γ的释放。
在一种实施方式中，所述使用方法具体步骤如下：
(1)分离样品中的外周血单个核细胞；
(2)培养扩增步骤(1)的外周血单个核细胞；
(3)取IFN-γ捕获抗体进行包被，孵育，加入封闭液封闭；
(4)去除封闭液，加入多肽组合物和步骤(2)的外周血单个核细胞，孵育，洗涤，去除液体；
(5)加入IFN-γ单克隆抗体，孵育，去除上清，洗涤；
(6)加入亲和素-HRP结合物，孵育，去除上清，洗涤；
(7)检测并读取斑点。
在一种实施方式中，所述方法中设置有阴性对照；所述阳性对照的刺激溶液为佛波酯(PMA)。
在一种实施方式中，所述封闭液为含10％胎牛血清的RPMI1640培养基。
在一种实施方式中，所述细胞因子为IFN-γ。
在一种实施方式中，所述样品包括脐带血、骨髓、外周血。
通过所述显色反应后的斑点形成细胞数量判断所述T细胞肽库筛选结果；若显色反应后的斑点形成细胞数量为阴性对照中的斑点形成细胞数量的两倍或以上，则结果为阳性。
在一种实施方式中，所述培养扩增所述的外周血单个核细胞包括以下步骤：
(1)取外周血单个核细胞，加入培养基和肽库溶液，孵育；
(2)加入含有白介素7的培养基，孵育；
(3)加入含有白介素2的培养基，孵育；
(4)离心，收集沉淀的细胞，洗涤，静息，即得扩增的外周血单个核细胞；。
在一种实施方式中，所述培养基为含10％胎牛血清的RPMI1640培养基；所述IL-7的浓度含量为15-25ng/mL；所述IL-2的浓度含量为175～225U/mL。
有益效果：
本发明设计了271条由10个氨基酸重叠而成的15-18聚体SARS-CoV-2肽段，这些肽段跨越了整个S、M和N蛋白，根据SARS-CoV-2S蛋白S1区和S2区的自然分裂位点，将S蛋白的多肽分为S1区和S2区，分别包含92和93个肽，M和N蛋白库包含29和57个肽，肽段作为抗原刺激物用于复苏后的PBMC的刺激培养；期间加入白介素2(IL-2)和白介素7(IL-7)等生物因子，培养9天后进行ELISpot检测，为了量化抗原特异性反应，从实验孔中减去阴性对照孔中的斑点数量，结果以每106个PBMCs表达斑点形成细胞(SFC)。评价标准如下:若阴性对照SFC<20个/106个，则阳性反应定义为SFC>40个/106个；否则，阳性反应被定义为结果至少是阴性对照组的两倍。
经过总肽库刺激的新冠病人康复6个月后的T细胞反应阳性率为：93.42％；康复12个月后的T细胞反应阳性率为：91.78％。而利用S1肽库和M肽库配伍评价的方法则可以得到更高的灵敏度和特异度(金标准为病毒核酸检测)：灵敏度71.43％，特异度96.43％。
本发明通过多肽抗原的刺激，并在体外培养细胞9天，使病毒特异性T细胞增殖，有效提高检测的灵敏度。
本发明可以依照具体情况选择判定标准进行肽库的选择，并对不同肽库刺激样本得到的ELISpot结果进行选择或组合：如需要提高检测灵敏度，尽量减少漏判，可选择总肽库或S1或M肽库任一阳性即判定为最终阳性；如需要减少误判(排除非阳性病人)，需要提高特异度，可以将S1肽库和M肽库结果相结合(双阳判定为最终阳性)。
具体实施方式
以下结合具体实施例来进一步说明本发明，但实施例并不对本发明做任何形式的限定。除非特别说明，本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明，以下实施例所用试剂和材料均为市售商品或者可以通过已知方法制备。
实施例1肽库的制备
设计了271条由10个氨基酸重叠而成的15-18聚体SARS-CoV-2肽段，这些肽段跨越了整个S、M和N蛋白，根据SARS-CoV-2S蛋白S1区和S2区的自然分裂位点，将S蛋白的多肽分为S1区和S2区，分别包含92和93个肽，M和N蛋白库包含29和57个肽。将肽段混合成为肽库，并作为抗原刺激物用于复苏后的PBMC的刺激培养。
将271条肽段混合制备成四个肽库，其中，S1肽库包含多肽92条，S2、M、N肽库分别包含多肽93、29、57条。具体制备方法是，用DMSO溶解合成的多肽，目标浓度为20μg/μl，充分混合均匀，每条多肽取1μl混合成肽库。
实施例2肽库在ELISpot检测中的应用
(1)外周血PBMC分离
(a)主要试剂耗材
10ml抗凝管与促凝管(BD)、10ml移液管、淋巴细胞分离管(Cat#DKW-LST-24015SK)、1640培养基、DMSO、15ml离心管、50ml离心管、细胞冻存盒、细胞冻存管(Thermo)、国产细胞冻存管(江苏海门，黄盖/蓝盖)、液氮冻存盒、电动移液器、异丙醇、小牛血清(FBS)、巴斯德消毒吸管，具有升降加速度配有15mL离心管吊桶的水平离心机。
(b)样本处理
采用达科为人外周血淋巴细胞分离管Cat#DKW-LST-24015SK。
(c)步骤
1)预处理：取出淋巴细胞分离管，用离心机800g，20℃，离心1min。将存储有抗凝血的试管400g离心5min(或经过长时间放置后直接吸取)，吸取上层血浆2ml，分2管(蓝盖国产细胞冻存管)冻存。
2)样本离心：向含有抗凝血的试管中补加RPMI 1640培养基至10ml，颠倒混匀后，缓缓倒入淋巴细胞分离管中，800g，离心25min，20℃。
3)吸取PBMC：离心后管底是红细胞，中间层是分离液，最上层是血浆，血浆和分离液中间是致密的白膜，含单个核细胞(包括淋巴细胞和单核粒细胞)。在细胞操作台里，用巴斯德吸管吸取中间的白色细胞层，转移至新的15ml离心管内，补加RPMI 1640培养液至12ml。400g离心10min。
4)PBMC清洗：离心完毕，在细胞操作台里，倒掉上清，晃动离心管，利用残余的约200微升的液体重悬细胞。补加RPMI 1640培养基10ml，400g，10min清洗细胞。
5)细胞冻存：最后尽量吸弃上清，按照上述操作重悬细胞，先加入1ml的FBS，然后再补加1ml的冻存液(含有20％DMSO的FBS)，分装两管，放入冻存盒内，置-80度冻存过夜。促凝血3000rpm离心10min，血清分装3管(黄盖国产冻存管)，-80℃冻存。
(2)肽库刺激和PBMC培养
(a)主要试剂耗材
15ml离心管、1.5ml ep管、1ml移液枪、10ul移液枪、细胞计数板、细胞计数器、24孔细胞培养板、RPMI 1640培养基、白介素-2(IL-2)、白介素-7(IL-7)、小牛血清(FBS)、青链霉素、台盼蓝染液、水浴箱、配有15mL离心管吊桶的水平离心机、细胞培养箱。
(b)步骤
1、细胞复苏：复苏细胞混匀在10ml完全培养基(10％FBS，RPMI 1640培养基)里，混匀1500r离心10min，弃上清，加入x ml的完全培养基重悬。
2、细胞计数：ep管内加10μl台盼蓝，10μl混匀后的细胞，读两个九宫格的细胞数y。
细胞浓度为z＝(y个÷2格*2倍*104)/ml
细胞数为w＝z*x＝(y个÷2格*2倍*104)/ml*x ml
根据具体细胞浓度要求计算所需毫升数并定容(24孔板：2ml/孔，细胞3×106/孔)。
3、PBMC刺激：将24孔板放入培养箱静置沉淀3-5h后，用实施例1中的肽库刺激45min(肽库：条数×0.2μl+100μl培养基/孔)，随后加入900μl含有IL-7(20ug/ml)的完全培养基。
4、细胞培养：培养9天，培养期间每隔两天进行半量换液。更换的培养基为含有200U/ml的IL-2的完全培养基。
(3)ELISpot检测
(a)主要试剂耗材
PVDF膜为底的96孔板、1.5mlep管、1ml移液枪、10ul移液枪、细胞计数板、细胞计数器、分液槽、包被抗体(purified anti-IFN-γ)、1XPBS、RPMI 1640培养基、小牛血清(FBS)、DMSO、佛波酯(PMA)、去离子水、吐温、检测抗体(Biotinylated anti-IFN-γ)、亲和素-HRP结合物(streptavidin-HRP)、AEC底物溶液(AEC substrate)、AEC染料(AEC chromogen)、细胞培养箱、冰箱、ELISpot读板仪ELISpot Reader System(CTL-Immunospot S5 Versa)等。
(b)步骤
1.包被抗体：准备PVDF膜为底的96孔板(100μl/孔)，包被抗体(purified anti-IFN-γ)与1×PBS混合，比例1:200。
2.孵育：盖盖平放，4℃过夜。
3.封闭：轻倒出溶液，1640培养基洗2次(180μl/孔)后，加入每孔180μl完全培养基(RPMI 1640培养基+10％小牛血清+1-2％青链霉素)封闭。
4.孵育：室温孵育2h。
5.多肽和细胞：
5.1多肽：单条肽刺激：肽的终浓度10μg/ml，每孔2μg；肽库刺激：单肽终浓度2μg/ml，每孔0.4μg。
5.2细胞：轻吸出1ml上清，使用剩余液体吹起细胞混匀并加入10ml完全培养基，1500r离心10min，弃上清并重悬，加入完全培养基计数。
6.加入多肽并接种：轻倒出封闭液，实验组设2个复孔，并设无刺激物的阴性对照组(mock)及PHA阳性对照组(PMA)(a、实验组：肽库+细胞；b、阴性对照组(mock)：培养基+细胞；c、阳性对照组：细胞+PMA)。
依次加入实施例1中的肽库(0.02*条数μl+100μl完全培养基/孔)、mock(100μl完全培养基/孔)、细胞(1×105/孔)、PMA(10μl PMA+90μl完全培养基/孔，粉末PMA每瓶用500μl RPMI1640培养基或PBS溶解)。
7.孵育：37℃，5％CO2，平放孵育18h。
8.洗板：去除细胞，常温去离子水洗3次，每次5分钟，PBST洗3次，每次2分钟，每次清洗后用力甩掉清洗液后在吸水纸上用力扣干。
9.孵抗体：检测抗体(Biotinylated anti-IFN-γ)1:250稀释到含10％FBS的PBS中充分混匀。每孔100μl，室温孵育2h。去除原液体，常温去离子水洗3次，每次5分钟，PBST清洗3次，每次间隔2分钟，吸水纸上用力扣干。亲和素-HRP结合物(streptavidin-HRP)1:100稀释到含10％血清的PBS中充分混匀。每孔100μl，室温孵育1h。去除原液体，PBST清洗3次，PBS清洗2次，每次间隔2min，吸水纸上用力扣干。
10.显色：体系：100μl/孔AEC底物溶液(AEC substrate)+1滴/ml AEC染料(AECchromogen)避光混匀，每孔加100μl。
11.洗板：避光孵育7.5min分钟后看到清晰的斑点，用水冲洗膜的双面以中止反应，室温下晾干(开盖并解开底膜反扣过夜)、使用ELISpot Reader System(CTL-Immunospot S5 Versa)计数斑点。
根据结果显示，经过总肽库刺激的新冠病人康复6个月后的T细胞反应阳性率为：93.42％；康复12个月后的T细胞反应阳性率为：91.78％。而利用S1肽库和M肽库配伍评价的方法则可以得到更高的灵敏度和特异度(金标准为病毒核酸检测)：灵敏度71.43％，特异度96.43％。
在实际应用中，可以依照具体情况选择判定标准，如需要提高检测灵敏度，尽量减少漏判，那么可选择总肽库或S1或M肽库任一阳性即判定为最终阳性，如果实际情况是希望减少误判，需要提高特异度，那么将S1肽库和M肽库结果相结合(双阳判定为最终阳性)的方法无疑是最好的选择。
表1不同肽库在ELISpot检测中的应用结果
虽然本发明已以较佳实施例公开如上，但其并非用以限定本发明，任何熟悉此技术的人，在不脱离本发明的精神和范围内，都可做各种的改动与修饰，因此本发明的保护范围应该以权利要求书所界定的为准。
SEQUENCE LISTING
<110> 中国疾病预防控制中心病毒病预防控制所
<120> 一种新型冠状病毒的T细胞免疫检测方法
<130> BAA211317A
<160> 271
<170> PatentIn version 3.3
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Met Ala Asp Ser Asn Gly Thr Ile Thr Val Glu Glu Leu Lys Lys Leu
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Leu
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Ile Thr Val Glu Glu Leu Lys Lys Leu Leu Glu Gln Trp Asn Leu Val
1 5 10 15
Ile
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Trp Ile Cys Leu Leu Gln Phe Ala Tyr Ala Asn Arg Asn Arg Phe Leu
1 5 10 15
Tyr Ile
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Tyr Ala Asn Arg Asn Arg Phe Leu Tyr Ile Ile Lys Leu Ile Phe Leu
1 5 10 15
Trp Leu
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Tyr Ile Ile Lys Leu Ile Phe Leu Trp Leu Leu Trp Pro Val Thr Leu
1 5 10 15
Ala
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<212> PRT
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Leu Trp Leu Leu Trp Pro Val Thr Leu Ala Cys Phe Val Leu Ala Ala
1 5 10 15
Val Tyr
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Leu Ala Cys Phe Val Leu Ala Ala Val Tyr Arg Ile Asn Trp Ile
1 5 10 15
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<212> PRT
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<400> 8
Leu Ala Ala Val Tyr Arg Ile Asn Trp Ile Thr Gly Gly Ile Ala Ile
1 5 10 15
Ala Met
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<400> 9
Trp Ile Thr Gly Gly Ile Ala Ile Ala Met Ala Cys Leu Val Gly Leu
1 5 10 15
Met Trp
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Ala Met Ala Cys Leu Val Gly Leu Met Trp Leu Ser Tyr Phe Ile Ala
1 5 10 15
Ser Phe
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<212> PRT
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<400> 11
Met Trp Leu Ser Tyr Phe Ile Ala Ser Phe Arg Leu Phe Ala Arg Thr
1 5 10 15
Arg
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Ala Ser Phe Arg Leu Phe Ala Arg Thr Arg Ser Met Trp Ser Phe
1 5 10 15
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Phe Ala Arg Thr Arg Ser Met Trp Ser Phe Asn Pro Glu Thr Asn Ile
1 5 10 15
Leu Leu
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Ser Phe Asn Pro Glu Thr Asn Ile Leu Leu Asn Val Pro Leu His Gly
1 5 10 15
Thr Ile
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Leu Leu Asn Val Pro Leu His Gly Thr Ile Leu Thr Arg Pro Leu Leu
1 5 10 15
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<212> PRT
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<400> 16
His Gly Thr Ile Leu Thr Arg Pro Leu Leu Glu Ser Glu Leu Val Ile
1 5 10 15
Gly Ala
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<212> PRT
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<400> 17
Leu Leu Glu Ser Glu Leu Val Ile Gly Ala Val Ile Leu Arg Gly His
1 5 10 15
Leu Arg
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<400> 18
Gly Ala Val Ile Leu Arg Gly His Leu Arg Ile Ala Gly His His Leu
1 5 10 15
Gly Arg
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<400> 19
Leu Arg Ile Ala Gly His His Leu Gly Arg Cys Asp Ile Lys Asp Leu
1 5 10 15
Pro Lys
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<400> 20
Gly Arg Cys Asp Ile Lys Asp Leu Pro Lys Glu Ile Thr Val Ala Thr
1 5 10 15
Ser Arg
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<400> 21
Pro Lys Glu Ile Thr Val Ala Thr Ser Arg Thr Leu Ser Tyr Tyr Lys
1 5 10 15
Leu
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<211> 17
<212> PRT
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<400> 22
Thr Ser Arg Thr Leu Ser Tyr Tyr Lys Leu Gly Ala Ser Gln Arg Val
1 5 10 15
Ala
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<212> PRT
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<400> 23
Tyr Lys Leu Gly Ala Ser Gln Arg Val Ala Gly Asp Ser Gly Phe Ala
1 5 10 15
Ala Tyr
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<400> 24
Val Ala Gly Asp Ser Gly Phe Ala Ala Tyr Ser Arg Tyr Arg Ile Gly
1 5 10 15
Asn Tyr
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Ala Tyr Ser Arg Tyr Arg Ile Gly Asn Tyr Lys Leu Asn Thr Asp His
1 5 10 15
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Ile Gly Asn Tyr Lys Leu Asn Thr Asp His Ser Ser Ser Ser Asp Asn
1 5 10 15
Ile Ala
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Asp His Ser Ser Ser Ser Asp Asn Ile Ala Leu Leu Val Gln
1 5 10
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<400> 28
Met Ser Asp Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro Arg Ile Thr
1 5 10 15
Phe
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Asn Gln Arg Asn Ala Pro Arg Ile Thr Phe Gly Gly Pro Ser Asp Ser
1 5 10 15
Thr Gly
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Thr Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly Glu
1 5 10 15
Arg
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Ser Thr Gly Ser Asn Gln Asn Gly Glu Arg Ser Gly Ala Arg Ser Lys
1 5 10 15
Gln Arg
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Glu Arg Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu
1 5 10 15
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<400> 33
Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn Thr Ala Ser
1 5 10 15
Trp Phe
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Gly Leu Pro Asn Asn Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His
1 5 10 15
Gly Lys
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Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Asp Leu Lys Phe Pro
1 5 10 15
Arg
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His Gly Lys Glu Asp Leu Lys Phe Pro Arg Gly Gln Gly Val Pro Ile
1 5 10 15
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Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro
1 5 10 15
Asp Asp
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Pro Ile Asn Thr Asn Ser Ser Pro Asp Asp Gln Ile Gly Tyr Tyr Arg
1 5 10 15
Arg Ala
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Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg
1 5 10 15
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Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly Gly Asp Gly Lys Met
1 5 10 15
Lys
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Arg Ile Arg Gly Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp
1 5 10 15
Tyr Phe
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Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr Leu Gly Thr Gly
1 5 10 15
Pro Glu
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Tyr Phe Tyr Tyr Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly
1 5 10 15
Ala
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Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn Lys Asp Gly Ile Ile
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Trp Val
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Gly Ala Asn Lys Asp Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu
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Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp His
1 5 10 15
Ile
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Gly Ala Leu Asn Thr Pro Lys Asp His Ile Gly Thr Arg Asn Pro Ala
1 5 10 15
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Lys Asp His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val
1 5 10 15
Leu
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Asn Pro Ala Asn Asn Ala Ala Ile Val Leu Gln Leu Pro Gln Gly Thr
1 5 10 15
Thr Leu
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Val Leu Gln Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala
1 5 10 15
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Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly Ser Arg Gly Gly
1 5 10 15
Ser Gln
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Tyr Ala Glu Gly Ser Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser
1 5 10 15
Ser Arg
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Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg Asn Ser Ser Arg
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Ser Ser Ser Arg Ser Arg Asn Ser Ser Arg Asn Ser Thr Pro Gly Ser
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Ser Arg
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Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Thr Ser Pro Ala Arg
1 5 10 15
Met Ala
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Ser Arg Gly Thr Ser Pro Ala Arg Met Ala Gly Asn Gly Gly Asp Ala
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Ala Leu
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Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu Leu Asp
1 5 10 15
Arg Leu
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Ala Leu Ala Leu Leu Leu Leu Asp Arg Leu Asn Gln Leu Glu Ser Lys
1 5 10 15
Met
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Asp Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys Gly Gln Gln
1 5 10 15
Gln Gln
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Lys Met Ser Gly Lys Gly Gln Gln Gln Gln Gly Gln Thr Val Thr Lys
1 5 10 15
Lys
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Gly Gln Gln Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu
1 5 10 15
Ala
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<400> 62
Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys Lys Pro Arg Gln
1 5 10 15
Lys Arg
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<400> 63
Glu Ala Ser Lys Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr
1 5 10 15
Asn Val
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Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr Gln Ala Phe Gly Arg
1 5 10 15
Arg
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Tyr Asn Val Thr Gln Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln
1 5 10 15
Gly Asn
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<400> 66
Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp Gln Glu Leu
1 5 10 15
Ile Arg
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<212> PRT
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<400> 67
Gly Asn Phe Gly Asp Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys
1 5 10 15
His Trp
<210> 68
<211> 17
<212> PRT
<213> 人工序列
<400> 68
Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln Ile Ala Gln Phe
1 5 10 15
Ala
<210> 69
<211> 17
<212> PRT
<213> 人工序列
<400> 69
Lys His Trp Pro Gln Ile Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe
1 5 10 15
Phe
<210> 70
<211> 18
<212> PRT
<213> 人工序列
<400> 70
Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile
1 5 10 15
Gly Met
<210> 71
<211> 18
<212> PRT
<213> 人工序列
<400> 71
Ala Phe Phe Gly Met Ser Arg Ile Gly Met Glu Val Thr Pro Ser Gly
1 5 10 15
Thr Trp
<210> 72
<211> 18
<212> PRT
<213> 人工序列
<400> 72
Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr Gly Ala
1 5 10 15
Ile Lys
<210> 73
<211> 18
<212> PRT
<213> 人工序列
<400> 73
Thr Trp Leu Thr Tyr Thr Gly Ala Ile Lys Leu Asp Asp Lys Asp Pro
1 5 10 15
Asn Phe
<210> 74
<211> 17
<212> PRT
<213> 人工序列
<400> 74
Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln Val Ile Leu
1 5 10 15
Leu
<210> 75
<211> 18
<212> PRT
<213> 人工序列
<400> 75
Pro Asn Phe Lys Asp Gln Val Ile Leu Leu Asn Lys His Ile Asp Ala
1 5 10 15
Tyr Lys
<210> 76
<211> 18
<212> PRT
<213> 人工序列
<400> 76
Leu Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu
1 5 10 15
Pro Lys
<210> 77
<211> 17
<212> PRT
<213> 人工序列
<400> 77
Tyr Lys Thr Phe Pro Pro Thr Glu Pro Lys Lys Asp Lys Lys Lys Lys
1 5 10 15
Ala
<210> 78
<211> 16
<212> PRT
<213> 人工序列
<400> 78
Glu Pro Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr Gln Ala Leu
1 5 10 15
<210> 79
<211> 16
<212> PRT
<213> 人工序列
<400> 79
Lys Lys Lys Ala Asp Glu Thr Gln Ala Leu Pro Gln Arg Gln Lys Lys
1 5 10 15
<210> 80
<211> 17
<212> PRT
<213> 人工序列
<400> 80
Thr Gln Ala Leu Pro Gln Arg Gln Lys Lys Gln Gln Thr Val Thr Leu
1 5 10 15
Leu
<210> 81
<211> 16
<212> PRT
<213> 人工序列
<400> 81
Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala Ala Asp Leu
1 5 10 15
<210> 82
<211> 17
<212> PRT
<213> 人工序列
<400> 82
Thr Val Thr Leu Leu Pro Ala Ala Asp Leu Asp Asp Phe Ser Lys Gln
1 5 10 15
Leu
<210> 83
<211> 17
<212> PRT
<213> 人工序列
<400> 83
Ala Asp Leu Asp Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser
1 5 10 15
Ala
<210> 84
<211> 15
<212> PRT
<213> 人工序列
<400> 84
Lys Gln Leu Gln Gln Ser Met Ser Ser Ala Asp Ser Thr Gln Ala
1 5 10 15
<210> 85
<211> 17
<212> PRT
<213> 人工序列
<400> 85
Leu Pro Leu Val Ser Ser Gln Cys Val Asn Leu Thr Thr Arg Thr Gln
1 5 10 15
Leu
<210> 86
<211> 15
<212> PRT
<213> 人工序列
<400> 86
Gln Cys Val Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr
1 5 10 15
<210> 87
<211> 17
<212> PRT
<213> 人工序列
<400> 87
Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe Thr
1 5 10 15
Arg
<210> 88
<211> 18
<212> PRT
<213> 人工序列
<400> 88
Leu Pro Pro Ala Tyr Thr Asn Ser Phe Thr Arg Gly Val Tyr Tyr Pro
1 5 10 15
Asp Lys
<210> 89
<211> 18
<212> PRT
<213> 人工序列
<400> 89
Ser Phe Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser
1 5 10 15
Val Leu
<210> 90
<211> 18
<212> PRT
<213> 人工序列
<400> 90
Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu His Ser Thr Gln Asp
1 5 10 15
Leu Phe
<210> 91
<211> 18
<212> PRT
<213> 人工序列
<400> 91
Ser Ser Val Leu His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser
1 5 10 15
Asn Val
<210> 92
<211> 18
<212> PRT
<213> 人工序列
<400> 92
Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp Phe His Ala
1 5 10 15
Ile His
<210> 93
<211> 18
<212> PRT
<213> 人工序列
<400> 93
Phe Ser Asn Val Thr Trp Phe His Ala Ile His Val Ser Gly Thr Asn
1 5 10 15
Gly Thr
<210> 94
<211> 18
<212> PRT
<213> 人工序列
<400> 94
His Ala Ile His Val Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp Asn
1 5 10 15
Pro Val
<210> 95
<211> 18
<212> PRT
<213> 人工序列
<400> 95
Thr Asn Gly Thr Lys Arg Phe Asp Asn Pro Val Leu Pro Phe Asn Asp
1 5 10 15
Gly Val
<210> 96
<211> 18
<212> PRT
<213> 人工序列
<400> 96
Asp Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr
1 5 10 15
Glu Lys
<210> 97
<211> 18
<212> PRT
<213> 人工序列
<400> 97
Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu Lys Ser Asn Ile Ile Arg
1 5 10 15
Gly Trp
<210> 98
<211> 17
<212> PRT
<213> 人工序列
<400> 98
Ser Thr Glu Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr
1 5 10 15
Leu
<210> 99
<211> 18
<212> PRT
<213> 人工序列
<400> 99
Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser Lys Thr Gln
1 5 10 15
Ser Leu
<210> 100
<211> 17
<212> PRT
<213> 人工序列
<400> 100
Gly Thr Thr Leu Asp Ser Lys Thr Gln Ser Leu Leu Ile Val Asn Asn
1 5 10 15
Ala
<210> 101
<211> 17
<212> PRT
<213> 人工序列
<400> 101
Asn Val Val Ile Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe
1 5 10 15
Leu
<210> 102
<211> 17
<212> PRT
<213> 人工序列
<400> 102
Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr Tyr His
1 5 10 15
Lys
<210> 103
<211> 17
<212> PRT
<213> 人工序列
<400> 103
Asn Asp Pro Phe Leu Gly Val Tyr Tyr His Lys Asn Asn Lys Ser Trp
1 5 10 15
Met
<210> 104
<211> 18
<212> PRT
<213> 人工序列
<400> 104
Val Tyr Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg
1 5 10 15
Val Tyr
<210> 105
<211> 18
<212> PRT
<213> 人工序列
<400> 105
Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr Ser Ser Ala Asn Asn
1 5 10 15
Cys Thr
<210> 106
<211> 15
<212> PRT
<213> 人工序列
<400> 106
Phe Arg Val Tyr Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val
1 5 10 15
<210> 107
<211> 17
<212> PRT
<213> 人工序列
<400> 107
Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu
1 5 10 15
Met
<210> 108
<211> 16
<212> PRT
<213> 人工序列
<400> 108
Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu Met Asp Leu Glu Gly Lys
1 5 10 15
<210> 109
<211> 18
<212> PRT
<213> 人工序列
<400> 109
Ser Gln Pro Phe Leu Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys
1 5 10 15
Asn Leu
<210> 110
<211> 17
<212> PRT
<213> 人工序列
<400> 110
Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe Val Phe
1 5 10 15
Lys
<210> 111
<211> 18
<212> PRT
<213> 人工序列
<400> 111
Asn Phe Lys Asn Leu Arg Glu Phe Val Phe Lys Asn Ile Asp Gly Tyr
1 5 10 15
Phe Lys
<210> 112
<211> 16
<212> PRT
<213> 人工序列
<400> 112
Phe Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His
1 5 10 15
<210> 113
<211> 18
<212> PRT
<213> 人工序列
<400> 113
Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr Pro Ile Asn Leu
1 5 10 15
Val Arg
<210> 114
<211> 17
<212> PRT
<213> 人工序列
<400> 114
Tyr Ser Lys His Thr Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly
1 5 10 15
Phe
<210> 115
<211> 18
<212> PRT
<213> 人工序列
<400> 115
Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu Pro
1 5 10 15
Leu Val
<210> 116
<211> 17
<212> PRT
<213> 人工序列
<400> 116
Pro Gln Gly Phe Ser Ala Leu Glu Pro Leu Val Asp Leu Pro Ile Gly
1 5 10 15
Ile
<210> 117
<211> 16
<212> PRT
<213> 人工序列
<400> 117
Leu Glu Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe
1 5 10 15
<210> 118
<211> 18
<212> PRT
<213> 人工序列
<400> 118
Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr Leu Leu Ala
1 5 10 15
Leu His
<210> 119
<211> 15
<212> PRT
<213> 人工序列
<400> 119
Ile Thr Arg Phe Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu
1 5 10 15
<210> 120
<211> 18
<212> PRT
<213> 人工序列
<400> 120
Gln Thr Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser
1 5 10 15
Ser Ser
<210> 121
<211> 18
<212> PRT
<213> 人工序列
<400> 121
Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser Gly Trp Thr Ala Gly
1 5 10 15
Ala Ala
<210> 122
<211> 18
<212> PRT
<213> 人工序列
<400> 122
Asp Ser Ser Ser Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly
1 5 10 15
Tyr Leu
<210> 123
<211> 18
<212> PRT
<213> 人工序列
<400> 123
Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro Arg Thr Phe
1 5 10 15
Leu Leu
<210> 124
<211> 18
<212> PRT
<213> 人工序列
<400> 124
Val Gly Tyr Leu Gln Pro Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn
1 5 10 15
Gly Thr
<210> 125
<211> 16
<212> PRT
<213> 人工序列
<400> 125
Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala Val
1 5 10 15
<210> 126
<211> 18
<212> PRT
<213> 人工序列
<400> 126
Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala Val Asp Cys Ala Leu Asp
1 5 10 15
Pro Leu
<210> 127
<211> 18
<212> PRT
<213> 人工序列
<400> 127
Thr Asp Ala Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys
1 5 10 15
Thr Leu
<210> 128
<211> 18
<212> PRT
<213> 人工序列
<400> 128
Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys Ser Phe Thr Val
1 5 10 15
Glu Lys
<210> 129
<211> 18
<212> PRT
<213> 人工序列
<400> 129
Lys Cys Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr
1 5 10 15
Ser Asn
<210> 130
<211> 18
<212> PRT
<213> 人工序列
<400> 130
Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val Gln Pro
1 5 10 15
Thr Glu
<210> 131
<211> 16
<212> PRT
<213> 人工序列
<400> 131
Gln Thr Ser Asn Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe
1 5 10 15
<210> 132
<211> 17
<212> PRT
<213> 人工序列
<400> 132
Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn
1 5 10 15
Leu
<210> 133
<211> 18
<212> PRT
<213> 人工序列
<400> 133
Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu
1 5 10 15
Val Phe
<210> 134
<211> 17
<212> PRT
<213> 人工序列
<400> 134
Ile Thr Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe
1 5 10 15
Ala
<210> 135
<211> 18
<212> PRT
<213> 人工序列
<400> 135
Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn
1 5 10 15
Cys Val
<210> 136
<211> 18
<212> PRT
<213> 人工序列
<400> 136
Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val
1 5 10 15
Leu Tyr
<210> 137
<211> 16
<212> PRT
<213> 人工序列
<400> 137
Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Ala Ser Phe
1 5 10 15
<210> 138
<211> 18
<212> PRT
<213> 人工序列
<400> 138
Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr
1 5 10 15
Lys Leu
<210> 139
<211> 16
<212> PRT
<213> 人工序列
<400> 139
Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe
1 5 10 15
<210> 140
<211> 16
<212> PRT
<213> 人工序列
<400> 140
Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala
1 5 10 15
<210> 141
<211> 18
<212> PRT
<213> 人工序列
<400> 141
Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg
1 5 10 15
Gln Ile
<210> 142
<211> 18
<212> PRT
<213> 人工序列
<400> 142
Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr
1 5 10 15
Gly Lys
<210> 143
<211> 18
<212> PRT
<213> 人工序列
<400> 143
Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Lys Ile Ala Asp Tyr Asn
1 5 10 15
Tyr Lys
<210> 144
<211> 16
<212> PRT
<213> 人工序列
<400> 144
Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe
1 5 10 15
<210> 145
<211> 18
<212> PRT
<213> 人工序列
<400> 145
Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile
1 5 10 15
Ala Trp
<210> 146
<211> 16
<212> PRT
<213> 人工序列
<400> 146
Pro Asp Asp Phe Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu
1 5 10 15
<210> 147
<211> 15
<212> PRT
<213> 人工序列
<400> 147
Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val
1 5 10 15
<210> 148
<211> 18
<212> PRT
<213> 人工序列
<400> 148
Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn Tyr Asn
1 5 10 15
Tyr Leu
<210> 149
<211> 17
<212> PRT
<213> 人工序列
<400> 149
Asp Ser Lys Val Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg
1 5 10 15
Lys
<210> 150
<211> 17
<212> PRT
<213> 人工序列
<400> 150
Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro
1 5 10 15
Phe
<210> 151
<211> 15
<212> PRT
<213> 人工序列
<400> 151
Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile
1 5 10 15
<210> 152
<211> 18
<212> PRT
<213> 人工序列
<400> 152
Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr
1 5 10 15
Gln Ala
<210> 153
<211> 18
<212> PRT
<213> 人工序列
<400> 153
Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys
1 5 10 15
Asn Gly
<210> 154
<211> 18
<212> PRT
<213> 人工序列
<400> 154
Ile Tyr Gln Ala Gly Ser Thr Pro Cys Asn Gly Val Glu Gly Phe Asn
1 5 10 15
Cys Tyr
<210> 155
<211> 17
<212> PRT
<213> 人工序列
<400> 155
Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser
1 5 10 15
Tyr
<210> 156
<211> 18
<212> PRT
<213> 人工序列
<400> 156
Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly Phe Gln Pro Thr
1 5 10 15
Asn Gly
<210> 157
<211> 18
<212> PRT
<213> 人工序列
<400> 157
Leu Gln Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro
1 5 10 15
Tyr Arg
<210> 158
<211> 17
<212> PRT
<213> 人工序列
<400> 158
Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser
1 5 10 15
Phe
<210> 159
<211> 18
<212> PRT
<213> 人工序列
<400> 159
Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe Glu Leu Leu His Ala
1 5 10 15
Pro Ala
<210> 160
<211> 18
<212> PRT
<213> 人工序列
<400> 160
Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro
1 5 10 15
Lys Lys
<210> 161
<211> 17
<212> PRT
<213> 人工序列
<400> 161
His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr Asn Leu Val
1 5 10 15
Lys
<210> 162
<211> 17
<212> PRT
<213> 人工序列
<400> 162
Cys Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn
1 5 10 15
Phe
<210> 163
<211> 16
<212> PRT
<213> 人工序列
<400> 163
Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn Gly Leu
1 5 10 15
<210> 164
<211> 17
<212> PRT
<213> 人工序列
<400> 164
Asn Lys Cys Val Asn Phe Asn Phe Asn Gly Leu Thr Gly Thr Gly Val
1 5 10 15
Leu
<210> 165
<211> 18
<212> PRT
<213> 人工序列
<400> 165
Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys
1 5 10 15
Lys Phe
<210> 166
<211> 17
<212> PRT
<213> 人工序列
<400> 166
Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu Pro Phe Gln Gln
1 5 10 15
Phe
<210> 167
<211> 16
<212> PRT
<213> 人工序列
<400> 167
Ser Asn Lys Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala
1 5 10 15
<210> 168
<211> 18
<212> PRT
<213> 人工序列
<400> 168
Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala
1 5 10 15
Val Arg
<210> 169
<211> 18
<212> PRT
<213> 人工序列
<400> 169
Arg Asp Ile Ala Asp Thr Thr Asp Ala Val Arg Asp Pro Gln Thr Leu
1 5 10 15
Glu Ile
<210> 170
<211> 18
<212> PRT
<213> 人工序列
<400> 170
Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro
1 5 10 15
Cys Ser
<210> 171
<211> 18
<212> PRT
<213> 人工序列
<400> 171
Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe Gly Gly Val Ser
1 5 10 15
Val Ile
<210> 172
<211> 18
<212> PRT
<213> 人工序列
<400> 172
Thr Pro Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn
1 5 10 15
Thr Ser
<210> 173
<211> 18
<212> PRT
<213> 人工序列
<400> 173
Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val Ala Val
1 5 10 15
Leu Tyr
<210> 174
<211> 18
<212> PRT
<213> 人工序列
<400> 174
Asn Cys Thr Glu Val Pro Val Ala Ile His Ala Asp Gln Leu Thr Pro
1 5 10 15
Thr Trp
<210> 175
<211> 18
<212> PRT
<213> 人工序列
<400> 175
Ala Ile His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr
1 5 10 15
Gly Ser
<210> 176
<211> 18
<212> PRT
<213> 人工序列
<400> 176
Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser Asn Val Phe Gln Thr
1 5 10 15
Arg Ala
<210> 177
<211> 17
<212> PRT
<213> 人工序列
<400> 177
Ser Thr Gly Ser Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly
1 5 10 15
Ala
<210> 178
<211> 18
<212> PRT
<213> 人工序列
<400> 178
Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val Asn Asn
1 5 10 15
Ser Tyr
<210> 179
<211> 17
<212> PRT
<213> 人工序列
<400> 179
Leu Ile Gly Ala Glu His Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro
1 5 10 15
Ile
<210> 180
<211> 17
<212> PRT
<213> 人工序列
<400> 180
Val Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys
1 5 10 15
Ala
<210> 181
<211> 17
<212> PRT
<213> 人工序列
<400> 181
Gly Ile Cys Ala Ser Tyr Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala
1 5 10 15
Arg
<210> 182
<211> 18
<212> PRT
<213> 人工序列
<400> 182
Gln Thr Gln Thr Asn Ser Pro Arg Arg Ala Arg Ser Val Ala Ser Gln
1 5 10 15
Ser Ile
<210> 183
<211> 18
<212> PRT
<213> 人工序列
<400> 183
Arg Arg Ala Arg Ser Val Ala Ser Gln Ser Ile Ile Ala Tyr Thr Met
1 5 10 15
Ser Leu
<210> 184
<211> 18
<212> PRT
<213> 人工序列
<400> 184
Ser Gln Ser Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser
1 5 10 15
Val Ala
<210> 185
<211> 18
<212> PRT
<213> 人工序列
<400> 185
Thr Met Ser Leu Gly Ala Glu Asn Ser Val Ala Tyr Ser Asn Asn Ser
1 5 10 15
Ile Ala
<210> 186
<211> 18
<212> PRT
<213> 人工序列
<400> 186
Asn Ser Val Ala Tyr Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe
1 5 10 15
Thr Ile
<210> 187
<211> 18
<212> PRT
<213> 人工序列
<400> 187
Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile Ser Val Thr Thr Glu
1 5 10 15
Ile Leu
<210> 188
<211> 17
<212> PRT
<213> 人工序列
<400> 188
Asn Phe Thr Ile Ser Val Thr Thr Glu Ile Leu Pro Val Ser Met Thr
1 5 10 15
Lys
<210> 189
<211> 18
<212> PRT
<213> 人工序列
<400> 189
Thr Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val Asp Cys
1 5 10 15
Thr Met
<210> 190
<211> 18
<212> PRT
<213> 人工序列
<400> 190
Ser Met Thr Lys Thr Ser Val Asp Cys Thr Met Tyr Ile Cys Gly Asp
1 5 10 15
Ser Thr
<210> 191
<211> 18
<212> PRT
<213> 人工序列
<400> 191
Asp Cys Thr Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu
1 5 10 15
Leu Leu
<210> 192
<211> 16
<212> PRT
<213> 人工序列
<400> 192
Gly Asp Ser Thr Glu Cys Ser Asn Leu Leu Leu Gln Tyr Gly Ser Phe
1 5 10 15
<210> 193
<211> 18
<212> PRT
<213> 人工序列
<400> 193
Cys Ser Asn Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn
1 5 10 15
Arg Ala
<210> 194
<211> 17
<212> PRT
<213> 人工序列
<400> 194
Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Thr Gly Ile Ala
1 5 10 15
Val
<210> 195
<211> 15
<212> PRT
<213> 人工序列
<400> 195
Gln Leu Asn Arg Ala Leu Thr Gly Ile Ala Val Glu Gln Asp Lys
1 5 10 15
<210> 196
<211> 18
<212> PRT
<213> 人工序列
<400> 196
Ala Leu Thr Gly Ile Ala Val Glu Gln Asp Lys Asn Thr Gln Glu Val
1 5 10 15
Phe Ala
<210> 197
<211> 18
<212> PRT
<213> 人工序列
<400> 197
Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln Val Lys Gln Ile
1 5 10 15
Tyr Lys
<210> 198
<211> 18
<212> PRT
<213> 人工序列
<400> 198
Glu Val Phe Ala Gln Val Lys Gln Ile Tyr Lys Thr Pro Pro Ile Lys
1 5 10 15
Asp Phe
<210> 199
<211> 16
<212> PRT
<213> 人工序列
<400> 199
Gln Ile Tyr Lys Thr Pro Pro Ile Lys Asp Phe Gly Gly Phe Asn Phe
1 5 10 15
<210> 200
<211> 15
<212> PRT
<213> 人工序列
<400> 200
Pro Pro Ile Lys Asp Phe Gly Gly Phe Asn Phe Ser Gln Ile Leu
1 5 10 15
<210> 201
<211> 16
<212> PRT
<213> 人工序列
<400> 201
Asp Phe Gly Gly Phe Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys
1 5 10 15
<210> 202
<211> 18
<212> PRT
<213> 人工序列
<400> 202
Asn Phe Ser Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser
1 5 10 15
Phe Ile
<210> 203
<211> 18
<212> PRT
<213> 人工序列
<400> 203
Asp Pro Ser Lys Pro Ser Lys Arg Ser Phe Ile Glu Asp Leu Leu Phe
1 5 10 15
Asn Lys
<210> 204
<211> 17
<212> PRT
<213> 人工序列
<400> 204
Arg Ser Phe Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp
1 5 10 15
Ala
<210> 205
<211> 17
<212> PRT
<213> 人工序列
<400> 205
Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly Phe Ile Lys Gln
1 5 10 15
Tyr
<210> 206
<211> 18
<212> PRT
<213> 人工序列
<400> 206
Thr Leu Ala Asp Ala Gly Phe Ile Lys Gln Tyr Gly Asp Cys Leu Gly
1 5 10 15
Asp Ile
<210> 207
<211> 17
<212> PRT
<213> 人工序列
<400> 207
Ile Lys Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp Leu
1 5 10 15
Ile
<210> 208
<211> 16
<212> PRT
<213> 人工序列
<400> 208
Cys Leu Gly Asp Ile Ala Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe
1 5 10 15
<210> 209
<211> 17
<212> PRT
<213> 人工序列
<400> 209
Ala Ala Arg Asp Leu Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val
1 5 10 15
Leu
<210> 210
<211> 15
<212> PRT
<213> 人工序列
<400> 210
Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu
1 5 10 15
<210> 211
<211> 17
<212> PRT
<213> 人工序列
<400> 211
Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu Thr Asp Glu Met Ile
1 5 10 15
Ala
<210> 212
<211> 18
<212> PRT
<213> 人工序列
<400> 212
Leu Pro Pro Leu Leu Thr Asp Glu Met Ile Ala Gln Tyr Thr Ser Ala
1 5 10 15
Leu Leu
<210> 213
<211> 15
<212> PRT
<213> 人工序列
<400> 213
Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly Thr Ile
1 5 10 15
<210> 214
<211> 17
<212> PRT
<213> 人工序列
<400> 214
Gln Tyr Thr Ser Ala Leu Leu Ala Gly Thr Ile Thr Ser Gly Trp Thr
1 5 10 15
Phe
<210> 215
<211> 17
<212> PRT
<213> 人工序列
<400> 215
Leu Ala Gly Thr Ile Thr Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala
1 5 10 15
Leu
<210> 216
<211> 17
<212> PRT
<213> 人工序列
<400> 216
Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe Ala
1 5 10 15
Met
<210> 217
<211> 17
<212> PRT
<213> 人工序列
<400> 217
Ala Gly Ala Ala Leu Gln Ile Pro Phe Ala Met Gln Met Ala Tyr Arg
1 5 10 15
Phe
<210> 218
<211> 16
<212> PRT
<213> 人工序列
<400> 218
Ile Pro Phe Ala Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val
1 5 10 15
<210> 219
<211> 17
<212> PRT
<213> 人工序列
<400> 219
Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn Val Leu
1 5 10 15
Tyr
<210> 220
<211> 18
<212> PRT
<213> 人工序列
<400> 220
Asn Gly Ile Gly Val Thr Gln Asn Val Leu Tyr Glu Asn Gln Lys Leu
1 5 10 15
Ile Ala
<210> 221
<211> 18
<212> PRT
<213> 人工序列
<400> 221
Asn Val Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn Ser
1 5 10 15
Ala Ile
<210> 222
<211> 18
<212> PRT
<213> 人工序列
<400> 222
Lys Leu Ile Ala Asn Gln Phe Asn Ser Ala Ile Gly Lys Ile Gln Asp
1 5 10 15
Ser Leu
<210> 223
<211> 18
<212> PRT
<213> 人工序列
<400> 223
Asn Ser Ala Ile Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser
1 5 10 15
Ala Leu
<210> 224
<211> 18
<212> PRT
<213> 人工序列
<400> 224
Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala Leu Gly Lys Leu Gln Asp
1 5 10 15
Val Val
<210> 225
<211> 18
<212> PRT
<213> 人工序列
<400> 225
Ala Ser Ala Leu Gly Lys Leu Gln Asp Val Val Asn Gln Asn Ala Gln
1 5 10 15
Ala Leu
<210> 226
<211> 18
<212> PRT
<213> 人工序列
<400> 226
Gln Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu Val Lys
1 5 10 15
Gln Leu
<210> 227
<211> 18
<212> PRT
<213> 人工序列
<400> 227
Ala Gln Ala Leu Asn Thr Leu Val Lys Gln Leu Ser Ser Asn Phe Gly
1 5 10 15
Ala Ile
<210> 228
<211> 18
<212> PRT
<213> 人工序列
<400> 228
Val Lys Gln Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val Leu Asn
1 5 10 15
Asp Ile
<210> 229
<211> 18
<212> PRT
<213> 人工序列
<400> 229
Phe Gly Ala Ile Ser Ser Val Leu Asn Asp Ile Leu Ser Arg Leu Asp
1 5 10 15
Lys Val
<210> 230
<211> 17
<212> PRT
<213> 人工序列
<400> 230
Leu Asn Asp Ile Leu Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln
1 5 10 15
Ile
<210> 231
<211> 18
<212> PRT
<213> 人工序列
<400> 231
Arg Leu Asp Lys Val Glu Ala Glu Val Gln Ile Asp Arg Leu Ile Thr
1 5 10 15
Gly Arg
<210> 232
<211> 18
<212> PRT
<213> 人工序列
<400> 232
Glu Val Gln Ile Asp Arg Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln
1 5 10 15
Thr Tyr
<210> 233
<211> 18
<212> PRT
<213> 人工序列
<400> 233
Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln Gln Leu
1 5 10 15
Ile Arg
<210> 234
<211> 17
<212> PRT
<213> 人工序列
<400> 234
Leu Gln Thr Tyr Val Thr Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg
1 5 10 15
Ala
<210> 235
<211> 17
<212> PRT
<213> 人工序列
<400> 235
Gln Gln Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala
1 5 10 15
Ala
<210> 236
<211> 18
<212> PRT
<213> 人工序列
<400> 236
Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala Thr Lys Met Ser Glu
1 5 10 15
Tyr Val
<210> 237
<211> 18
<212> PRT
<213> 人工序列
<400> 237
Asn Leu Ala Ala Thr Lys Met Ser Glu Tyr Val Leu Gly Gln Ser Lys
1 5 10 15
Arg Val
<210> 238
<211> 18
<212> PRT
<213> 人工序列
<400> 238
Ser Glu Tyr Val Leu Gly Gln Ser Lys Arg Val Asp Phe Cys Gly Lys
1 5 10 15
Gly Tyr
<210> 239
<211> 16
<212> PRT
<213> 人工序列
<400> 239
Ser Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe
1 5 10 15
<210> 240
<211> 17
<212> PRT
<213> 人工序列
<400> 240
Phe Cys Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ser Ala Pro
1 5 10 15
His
<210> 241
<211> 18
<212> PRT
<213> 人工序列
<400> 241
His Leu Met Ser Phe Pro Gln Ser Ala Pro His Gly Val Val Phe Leu
1 5 10 15
His Val
<210> 242
<211> 16
<212> PRT
<213> 人工序列
<400> 242
Ser Ala Pro His Gly Val Val Phe Leu His Val Thr Tyr Val Pro Ala
1 5 10 15
<210> 243
<211> 16
<212> PRT
<213> 人工序列
<400> 243
Val Val Phe Leu His Val Thr Tyr Val Pro Ala Gln Glu Lys Asn Phe
1 5 10 15
<210> 244
<211> 17
<212> PRT
<213> 人工序列
<400> 244
Val Thr Tyr Val Pro Ala Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala
1 5 10 15
Ile
<210> 245
<211> 18
<212> PRT
<213> 人工序列
<400> 245
Gln Glu Lys Asn Phe Thr Thr Ala Pro Ala Ile Cys His Asp Gly Lys
1 5 10 15
Ala His
<210> 246
<211> 18
<212> PRT
<213> 人工序列
<400> 246
Ala Pro Ala Ile Cys His Asp Gly Lys Ala His Phe Pro Arg Glu Gly
1 5 10 15
Val Phe
<210> 247
<211> 18
<212> PRT
<213> 人工序列
<400> 247
Gly Lys Ala His Phe Pro Arg Glu Gly Val Phe Val Ser Asn Gly Thr
1 5 10 15
His Trp
<210> 248
<211> 18
<212> PRT
<213> 人工序列
<400> 248
Glu Gly Val Phe Val Ser Asn Gly Thr His Trp Phe Val Thr Gln Arg
1 5 10 15
Asn Phe
<210> 249
<211> 17
<212> PRT
<213> 人工序列
<400> 249
Gly Thr His Trp Phe Val Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile
1 5 10 15
Ile
<210> 250
<211> 18
<212> PRT
<213> 人工序列
<400> 250
Thr Gln Arg Asn Phe Tyr Glu Pro Gln Ile Ile Thr Thr Asp Asn Thr
1 5 10 15
Phe Val
<210> 251
<211> 18
<212> PRT
<213> 人工序列
<400> 251
Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp
1 5 10 15
Val Val
<210> 252
<211> 15
<212> PRT
<213> 人工序列
<400> 252
Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val Ile Gly Ile Val
1 5 10 15
<210> 253
<211> 18
<212> PRT
<213> 人工序列
<400> 253
Val Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro
1 5 10 15
Glu Leu
<210> 254
<211> 18
<212> PRT
<213> 人工序列
<400> 254
Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys
1 5 10 15
Asn His
<210> 255
<211> 18
<212> PRT
<213> 人工序列
<400> 255
Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr Ser Pro Asp Val
1 5 10 15
Asp Leu
<210> 256
<211> 17
<212> PRT
<213> 人工序列
<400> 256
Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly
1 5 10 15
Ile
<210> 257
<211> 18
<212> PRT
<213> 人工序列
<400> 257
Pro Asp Val Asp Leu Gly Asp Ile Ser Gly Ile Asn Ala Ser Val Val
1 5 10 15
Asn Ile
<210> 258
<211> 18
<212> PRT
<213> 人工序列
<400> 258
Ile Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp
1 5 10 15
Arg Leu
<210> 259
<211> 18
<212> PRT
<213> 人工序列
<400> 259
Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn Glu Val Ala Lys
1 5 10 15
Asn Leu
<210> 260
<211> 18
<212> PRT
<213> 人工序列
<400> 260
Ile Asp Arg Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile
1 5 10 15
Asp Leu
<210> 261
<211> 17
<212> PRT
<213> 人工序列
<400> 261
Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu Gly Lys
1 5 10 15
Tyr
<210> 262
<211> 17
<212> PRT
<213> 人工序列
<400> 262
Ser Leu Ile Asp Leu Gln Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys
1 5 10 15
Trp
<210> 263
<211> 17
<212> PRT
<213> 人工序列
<400> 263
Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp
1 5 10 15
Leu
<210> 264
<211> 18
<212> PRT
<213> 人工序列
<400> 264
Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu Gly Phe Ile Ala Gly
1 5 10 15
Leu Ile
<210> 265
<211> 18
<212> PRT
<213> 人工序列
<400> 265
Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys Ser
1 5 10 15
Cys Gly
<210> 266
<211> 16
<212> PRT
<213> 人工序列
<400> 266
Cys Ser Cys Leu Lys Gly Cys Cys Ser Cys Gly Ser Cys Cys Lys Phe
1 5 10 15
<210> 267
<211> 17
<212> PRT
<213> 人工序列
<400> 267
Gly Cys Cys Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser
1 5 10 15
Glu
<210> 268
<211> 18
<212> PRT
<213> 人工序列
<400> 268
Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro Val Leu Lys Gly
1 5 10 15
Val Lys
<210> 269
<211> 15
<212> PRT
<213> 人工序列
<400> 269
Asp Asp Ser Glu Pro Val Leu Lys Gly Val Lys Leu His Tyr Thr
1 5 10 15
<210> 270
<211> 18
<212> PRT
<213> 人工序列
<400> 270
Lys Leu Leu Glu Gln Trp Asn Leu Val Ile Gly Phe Leu Phe Leu Thr
1 5 10 15
Trp Ile
<210> 271
<211> 18
<212> PRT
<213> 人工序列
<400> 271
Val Ile Gly Phe Leu Phe Leu Thr Trp Ile Cys Leu Leu Gln Phe Ala
1 5 10 15
Tyr Ala

Claims (10)

1.一种多肽组合物，其特征在于，所述多肽组合物由S1多肽库、S2多肽库、M多肽库、N多肽库中的任意一个以上的多肽库组成；
所述S1多肽库由氨基酸序列如SEQ ID NO.87～SEQ ID NO.178所示的多肽组成，S2多肽库由氨基酸序列如SEQ ID NO.179～SEQ ID NO.271所示的多肽组成，M多肽库由氨基酸序列如SEQ ID NO.1～SEQ ID NO.29所示的多肽组成，N多肽库由氨基酸序列如SEQ IDNO.30～SEQ ID NO.86所示的多肽组成。
2.根据权利要求1所述的多肽组合物，其特征在于，所述多肽组合物能与MHC-I或MHC-II类分子结合，其中，当多肽组合物与MHC结合时能被CD4+或CD8+T细胞识别。
3.权利要求1所述的多肽组合物在制备用于评估新冠患者、新冠康复者或新冠疫苗接种者细胞免疫水平的产品中的应用。
4.一种评估新冠患者、新冠康复者或新冠疫苗接种者细胞免疫水平的试剂盒，其特征在于，含有权利要求1或2所述多肽组合物。
5.根据权利要求4所述的试剂盒，其特征在于，所述试剂盒中还设置有酶联免疫斑点技术检测板，检测板上预包被捕获T细胞分泌的细胞因子的单克隆抗体。
6.一种评估新冠患者细胞、新冠康复者或新冠疫苗接种者免疫水平的非疾病诊断目的的检测方法，其特征在于，使用权利要求1或2所述多肽组合物刺激新冠患者的PBMC细胞，细胞在体外扩增之后，然后使用ELISpot检测多肽刺激后是否出现斑点，即IFN-γ的释放。
7.根据权利要求6所述的检测方法，其特征在于，所述方法的步骤如下：
(1)分离样品中的外周血单个核细胞；
(2)培养扩增步骤(1)的外周血单个核细胞；
(3)取IFN-γ捕获抗体进行包被，孵育，加入封闭液封闭；
(4)去除封闭液，加入多肽组合物和步骤(2)的外周血单个核细胞，孵育，洗涤，去除液体；
(5)加入IFN-γ单克隆抗体，孵育，去除上清，洗涤；
(6)加入亲和素-HRP结合物，孵育，去除上清，洗涤；
(7)检测并读取斑点。
8.根据权利要求7所述的检测方法，其特征在于，所述方法中设置有阴性对照；阳性对照的刺激溶液为佛波酯。
9.根据权利要求7所述的检测方法，其特征在于，所述方法步骤(2)中培养扩增所述的外周血单个核细胞，包括以下步骤：
(1)取外周血单个核细胞，加入培养基和肽库溶液，孵育；
(2)加入含有白介素7的培养基，孵育；
(3)加入含有白介素2的培养基，孵育；
(4)离心，收集沉淀的细胞，洗涤，静息，即得扩增的外周血单个核细胞。
10.根据权利要求9所述的检测方法，其特征在于，所述培养基为含10％胎牛血清的RPMI1640培养基；所述IL-7的浓度含量为15-25ng/mL；所述IL-2的浓度含量为175～225U/mL。
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新型冠状病毒肺炎（COVID-19）和严重急性呼吸综合征（SARS）药物治疗中的共性和差异探讨;梁楠等;《中国药事》;第530-535页 *

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