CN1216902C - 非典型性肺炎病毒特异蛋白质和临床检测的方法及试剂盒 - Google Patents
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Abstract
本发明公开了一种非典型性肺炎病毒特异蛋白质和临床检测的方法及试剂盒。它提供了非典型性肺炎病毒特异蛋白质，或其具有结合人免疫球蛋白之免疫活性的片段或衍生物；并利用该通式蛋白质免疫检测人非典型性肺炎病毒的方法及试剂盒。本发明解决了临床对非典型性肺炎病毒感染者的早期快速诊断问题，具有较高的准确性，为临床检测本病提供了快速、准确的诊断方法和试剂盒。
Description
技术领域
本发明涉及人类医学卫生领域，具体地说是一种非典型性肺炎病毒特异蛋白质和临床检测的方法及试剂盒。
背景技术
非典型性肺炎病毒，即SARS病毒是一种引起严重急性呼吸道疾病综合症(Severe Acute Respiratory Syndrome，SARS)，又称非典型性肺炎的病毒，属冠状病毒(coronavirus)类病毒，基因组共含29，737个核苷酸。造成感染严重时可致人于死，病死率高达4.9％。主要通过近距离空气飞沫和密切接触传播。该病毒目前还没有有效的免疫化学检测方法，只能根据临床症状(高热、干咳、肺部X光检测有阴影等)来诊断，受到很大的局限，只能在受感染者发病后才能作出诊断，且不易与其它肺炎和一般流感区别。本发明根据该病毒的基因组序列分析结果，选取该病毒特有蛋白质和多肽片断，建立一种检测非典型性肺炎病毒抗体的免疫学方法和含有它们的试剂盒。
发明内容
本发明的目的是提供一种非典型性肺炎病毒特异蛋白质和临床检测的方法及试剂盒。
本发明采用的技术方案如下：
1、一种非典型性肺炎病毒特异蛋白质，具有Seq.ID No.1--Seq.ID No.118的氨基酸序列之一。
所述的蛋白质相对应的抗是单克隆抗体，或是多克隆抗体。
2、一种非典型性肺炎病毒特异蛋白质临床检测方法，其具体步骤如下：
1)制备上述的蛋白质；
通过本领域技术人员周知的PCR扩增技术和重组DNA技术，流程见附图1，具体如下：
A、设计相应的PCR引物，同时在所用PCR引物中添加编码相应数目氨基酸残基对应的碱基，分别从临床非典型性肺炎病毒标准株中扩增编码上述蛋白质的DNA片段；
B、将所得PCR产物导入合适的表达载体，构建重组DNA；
C、用所得重组DNA转化合适的宿主细胞，在适于目标蛋白质表达的条件下培养如此转化的宿主细胞；
D、回收并纯化目标蛋白质；
2)用纯化的如以上第1点所述的蛋白质，或以上第1点所述的抗体包被一种固相支持物；
3)用比色法、分光光度法或荧光法测定的酶标记如权利要求1所述的蛋白质或以上所述的抗体，或以上所述的抗体的第二抗体，或使用胶体金，或放射性同位素作为标记；
4)将待检测个体的生物学样本及由步骤3)标记的蛋白质加入经包被的固相支持物上，在适合形成抗原抗体免疫结合复合物的条件下孵育一段时间；所述的“适合形成抗原抗体免疫复合物的条件”是指在适当的温度及时间条件下孵育抗原与含抗体的样品的混合物，例如在约0-50摄氏度，标记约30分钟至约48小时。
5)清洗去除任何未结合的非典型性肺炎病毒抗体；所用清洗液一般为pH约5-8的磷酸盐缓冲溶液。
6)定量检测孵育混合物中抗原抗体免疫复合物的存在。可采用多种本领域周知的方法定量检测抗原抗体复合物的形成，如以过氧化物溶液为A液及联苯胺类物质为B液的底物与带有含标记的辣根过氧化物酶的抗原抗体免疫复合物作用，显色后在波长为450nm(参比波长630nm)酶标仪读取吸光值；将所得读数与阴性对照的读数加以比较，判断所测样本的结果。
所用的标记酶包括氧化还原酶类，该氧化还原酶为催化酶、过氧化物酶、辣根过氧化物酶、葡萄糖氧化酶或β-半乳糖苷酶；碱性磷酸酶；乙酰胆碱酯酶。
所用的第二抗体是IgG和/或IgM和/或IgA。
所述的固相支持物包括本领域周知的有机和无机多聚物，或用胶体金的相关技术包被到试纸上；
所述的有机多聚物为葡聚糖、天然或经修饰的纤维素、聚乙烯、聚苯乙烯。
3、一种非典型性肺炎病毒特异蛋白质临床检测的试剂盒，包括：
1)包被有纯化的蛋白质的固相支持物；相应的经标记的的蛋白质和使用该试剂盒的说明(双抗原夹心法)；
或2)包被有纯化的抗体的固相支持物；相应的经标记的抗体和使用该试剂盒的说明(双抗体夹心法)；
或3)包被有纯化的蛋白质的固相支持物；相应的第二抗体和使用该试剂盒的说明(Elisa法)。
所说的快速检测非典型性肺炎病毒的试纸，是由喷涂过的蛋白或抗体，及喷涂有胶体金的试纸基质粘合而成；
在上述检测试剂盒中，经标记的非典型性肺炎病毒蛋白质是液体的形式包装在容器中，或便于重构的冻干物的形式置于的包装中。在后一种情况中，检测试剂盒中还优选含有用于重构经标记的非典型性肺炎病毒蛋白质的缓冲液；
在上述检测试剂盒中，还含有提供判断检测结果的相应的范围。
本发明与背景技术相比具有的有益效果是：它解决了临床对非典型性肺炎病毒感染者的早期快速诊断问题，具有较高的准确性，为临床检测本病提供了快速、准确的诊断方法和试剂盒。
附图说明
图1是将PCR产物与载体连接构建重组DNA示意图；
图2是高压液相色谱(HPLC)图。
具体实施方式
实施例1、
一、制备蛋白质：
A、设计相应的PCR引物，同时在所用PCR引物中添加编码相应数目氨基酸残基对应的碱基，其中之一的引物如下：AlL：CGGGATCCGAACTTTAAAATCTGTGTAGC AlR：CCGCTCGAGATTTCTGCAACCAGCTCAAC
分别从临床非典型性肺炎病毒标准株中提取RNA和扩增编码上述蛋白质的DNA片段；提取RNA的方法可采用普通分子生物学实验手册，如“分子克隆”介绍的方法，也可以采用商业试剂盒，如TrizolR，按说明书进行，然后加入反向引物和逆转录酶，合成cDNA；采用商品化试剂盒，如InvitrogenTM公司的SuperScript试剂盒，按说明书步骤合成cDNA；再利用PCR技术扩增编码上述蛋白质的DNA片段。PCR反应体系的组成为一般的标准组成，包括：PCR反应缓冲液(Tris.Cl 125mM PH8.1；(NH4)2SO4 125mM；MgCl2 12.5mM；BSA 1mg/ml；Formamide 25％)；反应条件指：模板变性温度为92℃-98℃，优选的：95℃，时间为10秒-10分钟，优选的：30秒；模板退火温度为45℃--65℃，优选的：52℃，时间为10秒-10分钟，优选的：30秒；模板延伸温度为65℃--85℃，优选的：70℃，时间为10秒-10分钟，优选的：40秒；循环20--40次，优选的：30次。
B、将所得PCR产物导入合适的表达载体，如附图1所示的pET-28质粒，构建重组DNA；
1μg的质粒DNA于37℃下经NdeI和XhoI酶完全酶切，酚/氯仿抽提，-20℃下无水乙醇沉淀过夜。离心后沉淀溶于13μl H2O。经过NdeI和XhoI酶充分酶切的PCR产物与上述预酶切的质粒连接。
C、用所得重组DNA转化合适的宿主细胞，在适于目标蛋白质表达的条件下培养如此转化的宿主细胞；
转化至大肠杆菌ER2566，涂布含有200μg/ml氨苄青霉素的LB选择平板。37℃过夜培养。构建过程参见图1。所述连接及转化的方法和条件参见，J.萨姆布雷奇等著的《分子克隆实验指南》(Molecular Cloning：A LaboratoryManual)P672-849(科学出版社，1998)
从转化平板上挑取抗性菌落，提取质粒并电泳检查，筛选重组子。通过限制性内切酶NdeI和XhoI酶切分析，筛选含有正确插入的目的基因的重组子。对所得含有插入片段的重组质粒测序，确证在PCR过程中目的基因片段未发生变异或出现差错。
D、回收并纯化目标蛋白质；
将选定的重组子菌株接种于含200μg/ml氨苄青霉素的LB培养基中，置于恒温摇床培养，37℃培养至菌液OD值约0.6后加入1mM IPTG诱导，30℃继续培养2小时。然后将发酵培养物离心收集菌体沉淀。用超声波将所得细菌菌体破碎后再次离心，收集上清液。利用金属镍-Sepharose(Pharmacia公司)的亲和层析柱进行蛋白的分离纯化(洗脱条件0.02M pH7.4磷酸盐缓冲溶液(PBS)，洗脱速度1ml/分钟)。经充分洗涤去除非特异性结合的蛋白质后，分离并收集与金属镍-Sepharose结合的表达蛋白，进行SDS-PAGE电泳和高压液相色谱分析。
附图2为经纯化的蛋白质的高压液相色谱图。
二、间接法检测非典型性肺炎病毒抗体
1)每次试验设空白对照、阴性对照和阳性对照各1孔。空白孔不加液体；阴、阳性对照无需稀释直接取50μl加入阴、阳性对照孔中；其余检测孔加入样品稀释液，每孔100μl，再加入待测样品10μl。充分混匀，贴上封口胶，置37℃温育30分钟。
2)将50倍浓缩洗板液用双蒸水50倍稀释后用于洗涤板孔。
3)弃去各孔中样品，每孔加满洗液，静置数秒后弃去，重复5次，拍干。
4)每孔加入酶标抗人IgG工作液100μl，贴上封口胶。置37℃温育20分钟。
5)弃去各孔中液体，用洗板液反复洗涤5次，拍干。
6)每孔加入底物液A 50μl，再加入底物液B 50μl，轻拍混匀，置37℃避光温育10分钟。
7)显色完毕后，每孔加入终止液50μl，轻拍混匀，立即以空白孔调零，置酶标仪450nm波长下测定OD值(参考波长为630nm)。
实施例2、双抗原夹心法检测非典型性肺炎病毒抗体
在96孔微孔反应板上加入预先用0.02M pH9.6的碳酸盐缓冲液(CB)稀释抗原至最适浓度的包被液，4℃静置24小时，加入含0.1％TW-20的0.02M pH7.4 PBS洗涤液(PBS-T)加满各孔，静置数秒后弃去，重复清洗两次。每孔加入200μl入1％牛血清白蛋白(BSA)和1％脱脂奶粉的0.02M pH7.4磷酸盐缓冲液(PBS)，4℃下封闭2小时。弃去封闭液，室温下晾干，用铝薄袋封板，4℃保藏待用。在微孔反应板孔中加入预先用酶标抗原稀释液(20％山羊血清，1％酪蛋白，0.02MPBS，pH7.4)以优化好的浓度稀释的SARS冠状病毒抗原相应的酶标抗原，每孔50μl，再加入15个来自不同待检测的受试者血清，37℃温育60分钟。然后用含0.1％TW-20的0.02M PBS-T洗涤液加满各孔，静置数秒后弃去，重复清洗5次。每孔中加入50μl由0.06％过氧化脲(Sigma)0.01M pH4.5的醋酸盐缓冲液组成的酶作用底物A液以及50μl由0.06％TMB(Sigma)0.01M pH4.5的柠檬酸盐缓冲液组成的酶作用底物B液，37℃温育15分钟。每孔中加入50μl的2MH2SO4终止反应。在酶标仪(Dynatech MR 4100)上读取450nm处的吸光值(以630为参比波长)。其中阳性对照和阴性对照均为经过临床确诊及其他可能的方法复核的人血清，根据实预先确定的阈值判定样本结果，高于阈值的样本为非典型性肺炎病毒阳性。
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<210>18
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<212>PRT
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Ser Phe Ser Ala Ser Thr Ser Ala Phe Ile Asp Thr Ile Lys Ser Leu
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Val Ile Arg Ser Ile Phe Ala Arg Thr Leu Asp Ala Ala Asn His Ser
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Ile Pro Asp Leu Gln Arg Ala Ala Val Thr Ile Leu Asp Gly Ile Ser
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<212>PRT
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Thr Asn Asp Asn Thr Ser Arg Tyr Trp Glu Pro Glu Phe Tyr Glu Ala
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Asp Val Thr Asp Val Thr Gln Leu Tyr Leu Gly Gly Met Ser Tyr Tyr
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Cys Lys Ser His Lys Pro Pro Ile Ser Phe Pro Leu Cys Ala Asn Gly
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20 25 30
Glu Glu Thr Phe Lys Leu Ser Tyr Gly Ile Ala Thr Val Arg Glu Val
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50 55 60
Pro Pro Leu Asn Arg Asn Tyr Val Phe Thr Gly Tyr Arg Val Thr Lys
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Gly Asp Tyr Phe Val Leu Thr Ser His Thr Val Met Pro Leu Ser Ala
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Pro Thr Leu Val Pro Gln Glu His Tyr Val Arg Ile Thr Gly Leu Tyr
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Pro Thr Leu Asn
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<212>PRT
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35 40 45
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65 70 75 80
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100 105 110
Val Phe Cys Thr Val Asn Ala Leu Pro Glu Thr Thr Ala Asp Ile Val
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Asn Ala Arg
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<212>PRT
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Glu Ile Ser Met Ala Thr Asn Tyr Asp Leu Ser Val Val Asn Ala Arg
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Ala Pro Arg Thr Leu Leu Thr Lys Gly Thr Leu Glu Pro Glu Tyr Phe
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50 55 60
Gly Thr Cys Arg Arg Cys Pro Ala Glu Ile Val Asp Thr Val Ser Ala
65 70 75 80
Leu Val Tyr Asp Asn Lys Leu Lys Ala His Lys Asp Lys Ser Ala Gln
85 90 95
Cys Phe Lys Met Phe Tyr Lys Gly Val Ile Thr His Asp Val Ser Ser
100 105 110
Ala Ile Asn Arg Pro Gln Ile Gly Val Val Arg Glu Phe Leu Thr Arg
115 120 125
Asn Pro Ala Trp Arg Lys Ala Val Phe Ile Ser Pro Tyr Asn Ser Gln
130 135 140
Asn Ala Val Ala Ser
145 149
<210>23
<211>150
<212>PRT
<213>SARS coronavirus
<400>23
Ala Val Phe Ile Ser Pro Tyr Asn Ser Gln Asn Ala Val Ala Ser Lys
1 5 10 15
Ile Leu Gly Leu Pro Thr Gln Thr Val Asp Ser Ser Gln Gly Ser Glu
20 25 30
Tyr Asp Tyr Val Ile Phe Thr Gln Thr Thr Glu Thr Ala His Ser Cys
35 40 45
Asn Val Asn Arg Phe Asn Val Ala Ile Thr Arg Ala Lys Ile Gly Ile
50 55 60
Leu Cys Ile Met Ser Asp Arg Asp Leu Tyr Asp Lys Leu Gln Phe Thr
65 70 75 80
Ser Leu Glu Ile Pro Arg Arg Asn Val Ala Thr Leu Gln Ala Glu Asn
85 90 95
Val Thr Gly Leu Phe Lys Asp Cys Ser Lys Ile Ile Thr Gly Leu His
100 105 110
Pro Thr Gln Ala Pro Thr His Leu Ser Val Asp Ile Lys Phe Lys Thr
115 120 125
Glu Gly Leu Cys Val Asp Ile Pro Gly Ile Pro Lys Asp Met Thr Tyr
130 135 140
Arg Arg Leu Ile Ser Met
145 150
<210>24
<211>158
<212>PRT
<213>SARS coronavirus
<400>24
Pro Gly Ile Pro Lys Asp Met Thr Tyr Arg Arg Leu Ile Ser Met Met
1 5 10 15
Gly Phe Lys Met Asn Tyr Gln Val Asn Gly Tyr Pro Asn Met Phe Ile
20 25 30
Thr Arg Glu Glu Ala Ile Arg His Val Arg Ala Trp Ile Gly Phe Asp
35 40 45
Val Glu Gly Cys His Ala Thr Arg Asp Ala Val Gly Thr Asn Leu Pro
50 55 60
Leu Gln Leu Gly Phe Ser Thr Gly Val Asn Leu Val Ala Val Pro Thr
65 70 75 80
Gly Tyr Val Asp Thr Glu Asn Asn Thr Glu Phe Thr Arg Val Asn Ala
85 90 95
Lys Pro Pro Pro Gly Asp Gln Phe Lys His Leu Ile Pro Leu Met Tyr
100 105 110
Lys Gly Leu Pro Trp Asn Val Val Arg Ile Lys Ile Val Gln Met Leu
115 120 125
Ser Asp Thr Leu Lys Gly Leu Ser Asp Arg Val Val Phe Val Leu Trp
130 135 140
Ala His Gly Phe Glu Leu Thr Ser Met Lys Tyr Phe Val Lys
145 150 155 158
<210>25
<211>146
<212>PRT
<213>SARS coronavirus
<400>25
Leu Ser Asp Arg Val Val Phe Val Leu Trp Ala His Gly Phe Glu Leu
1 5 10 15
Thr Ser Met Lys Tyr Phe Val Lys Ile Gly Pro Glu Arg Thr Cys Cys
20 25 30
Leu Cys Asp Lys Arg Ala Thr Cys Phe Ser Thr Ser Ser Asp Thr Tyr
35 40 45
Ala Cys Trp Asn His Ser Val Gly Phe Asp Tyr Val Tyr Asn Pro Phe
50 55 60
Met Ile Asp Val Gln Gln Trp Gly Phe Thr Gly Asn Leu Gln Ser Asn
65 70 75 80
His Asp Gln His Cys Gln Val His Gly Asn Ala His Val Ala Ser Cys
85 90 95
Asp Ala Ile Met Thr Arg Cys Leu Ala Val His Glu Cys Phe Val Lys
100 105 110
Arg Val Asp Trp Ser Val Glu Tyr Pro Ile Ile Gly Asp Glu Leu Arg
115 120 125
Val Asn Ser Ala Cys Arg Lys Val Gln His Met Val Val Lys Ser Ala
130 135 140
Leu Leu
145 146
<210>26
<211>146
<212>PRT
<213>SARS coronavirus
<400>26
Arg Lys Val Gln His Met Val Val Lys Ser Ala Leu Leu Ala Asp Lys
1 5 10 15
Phe Pro Val Leu His Asp Ile Gly Asn Pro Lys Ala Ile Lys Cys Val
20 25 30
Pro Gln Ala Glu Val Glu Trp Lys Phe Tyr Asp Ala Gln Pro Cys Ser
35 40 45
Asp Lys Ala Tyr Lys Ile Glu Glu Leu Phe Tyr Ser Tyr Ala Thr His
50 55 60
His Asp Lys Phe Thr Asp Gly Val Cys Leu Phe Trp Asn Cys Asn Val
65 70 75 80
Asp Arg Tyr Pro Ala Asn Ala Ile Val Cys Arg Phe Asp Thr Arg Val
85 90 95
Leu Ser Asn Leu Asn Leu Pro Gly Cys Asp Gly Gly Ser Leu Tyr Val
100 105 110
Asn Lys His Ala Phe His Thr Pro Ala Phe Asp Lys Ser Ala Phe Thr
115 120 125
Asn Leu Lys Gln Leu Pro Phe Phe Tyr Tyr Ser Asp Ser Pro Cys Glu
130 135 140
Ser His
145 146
<210>27
<211>151
<212>PRT
<213>SARS coronavirus
<400>27
Pro Phe Phe Tyr Tyr Ser Asp Ser Pro Cys Glu Ser His Gly Lys Gln
1 5 10 15
Val Val Ser Asp Ile Asp Tyr Val Pro Leu Lys Ser Ala Thr Cys Ile
20 25 30
Thr Arg Cys Asn Leu Gly Gly Ala Val Cys Arg His His Ala Asn Glu
35 40 45
Tyr Arg Gln Tyr Leu Asp Ala Tyr Asn Met Met Ile Ser Ala Gly Phe
50 55 60
Ser Leu Trp Ile Tyr Lys Gln Phe Asp Thr Tyr Asn Leu Trp Asn Thr
65 70 75 80
Phe Thr Arg Leu Gln Ser Leu Glu Asn Val Ala Tyr Asn Val Val Asn
85 90 95
Lys Gly His Phe Asp Gly His Ala Gly Glu Ala Pro Val Ser Ile Ile
100 105 110
Asn Asn Ala Val Tyr Thr Lys Val Asp Gly Ile Asp Val Glu Ile Phe
115 120 125
Glu Asn Lys Thr Thr Leu Pro Val Asn Val Ala Phe Glu Leu Trp Ala
130 135 140
Lys Arg Asn Ile Lys Pro Val
145 150 151
<210>28
<211>148
<212>PRT
<213>SARS coronavirus
<400>28
Pro Val Asn Val Ala Phe Glu Leu Trp Ala Lys Arg Asn Ile Lys Pro
1 5 10 15
Val Pro GIu Ile Lys Ile Leu Asn Asn Leu Gly Val Asp Ile Ala Ala
20 25 30
Asn Thr Val Ile Trp Asp Tyr Lys Arg Glu Ala Pro Ala His Val Ser
35 40 45
Thr Ile Gly Val Cys Thr Met Thr Asp Ile Ala Lys Lys Pro Thr Glu
50 55 60
Ser Ala Cys Ser Ser Leu Thr Val Leu Phe Asp Gly Arg Val Glu Gly
65 70 75 80
Gln Val Asp Leu Phe Arg Asn Ala Arg Asn Gly Val Leu Ile Thr Glu
85 90 95
Gly Ser Val Lys Gly Leu Thr Pro Ser Lys Gly Pro Ala Gln Ala Ser
100 105 110
Val Asn Gly Val Thr Leu Ile Gly Glu Ser Val Lys Thr Gln Phe Asn
115 120 125
Tyr Phe Lys Lys Val Asp Gly Ile Ile Gln Gln Leu Pro Glu Thr Tyr
130 135 140
Phe Thr Gln Ser
145 148
<210>29
<211>144
<212>PRT
<213>SARS coronavirus
<400>29
Ser Gly Cys Leu Ile Phe Trp Ala Leu Leu Leu Lys Asn Ser Gly Leu
1 5 10 15
Ser Leu Asn Gly Leu Arg Arg Asn Leu Val Gln Glu Leu Asn Phe Ser
20 25 30
Arg Met Leu Gly Arg Phe Ser Asn Phe Ser Leu Gln Val Phe Leu Thr
35 40 45
Ser Ser Arg Val Lys Tyr Arg Leu Leu Gln Ile Thr Ser Arg Ile Val
50 55 60
Asn Ala Ser Leu Met Leu Leu Thr Arg His Ser Lys Cys Ala Leu Ile
65 70 75 80
Lys Ser Leu Ser Leu Ala Gln Ser Cys Asp His Ser Thr Val Lys Ser
85 90 95
Ser Ser Leu Lys Ala Arg Asp Phe Thr Val Ser Val Tyr Val Ala Arg
100 105 110
Ser Ser Cys Asn Tyr Ser Cys Leu Leu Arg His Gln Lys Lys Pro Phe
115 120 125
Leu Lys Val Ile His Met Thr Gln Tyr Leu Pro Leu Arg Arg Leu Phe
144
<210>30
<211>150
<212>PRT
<213>SARS coronavirus
<400>30
Asp Gly Ile Ile Gln Gln Leu Pro Glu Thr Tyr Phe Thr Gln Ser Arg
1 5 10 15
Asp Leu Glu Asp Phe Lys Pro Arg Ser Gln Met Glu Thr Asp Phe Leu
20 25 30
Glu Leu Ala Met Asp Glu Phe Ile Gln Arg Tyr Lys Leu Glu Gly Tyr
35 40 45
Ala Phe Glu His Ile Val Tyr Gly Asp Phe Ser His Gly Gln Leu Gly
50 55 60
Gly Leu His Leu Met Ile Gly Leu Ala Lys Arg Ser Gln Asp Ser Pro
65 70 75 80
Leu Lys Leu Glu Asp Phe Ile Pro Met Asp Ser Thr Val Lys Asn Tyr
85 90 95
Phe Ile Thr Asp Ala Gln Thr Gly Ser Ser Lys Cys Val Cys Ser Val
100 105 110
Ile Asp Leu Leu Leu Asp Asp Phe Val Glu Ile Ile Lys Ser Gln Asp
115 120 125
Leu Ser Val Ile Ser Lys Val Val Lys Val Thr Ile Asp Tyr Ala Glu
130 135 140
Ile Ser Phe Met Leu Trp
145 150
<210>31
<211>146
<212>PRT
<213>SARS coronavirus
<400>31
Val Val Lys Val Thr Ile Asp Tyr Ala Glu Ile Ser Phe Met Leu Trp
1 5 10 15
Cys Lys Asp Gly His Val Glu Thr Phe Tyr Pro Lys Leu Gln Ala Ser
20 25 30
Arg Ala Trp Gln Pro Gly Val Ala Met Pro Asn Leu Tyr Lys Met Gln
35 40 45
Arg Met Leu Leu Glu Lys Cys Asp Leu Gln Asn Tyr Gly Glu Asn Ala
50 55 60
Val Ile Pro Lys Gly Ile Met Met Asn Val Ala Lys Tyr Thr Gln Leu
65 70 75 80
Cys Gln Tyr Leu Asn Thr Leu Thr Leu Ala Val Pro Tyr Asn Met Arg
85 90 95
Val Ile His Phe Gly Ala Gly Ser Asp Lys Gly Val Ala Pro Gly Thr
100 105 110
Ala Val Leu Arg Gln Trp Leu Pro Thr Gly Thr Leu Leu Val Asp Ser
115 120 125
Asp Leu Asn Asp Phe Val Ser Asp Ala Tyr Ser Thr Leu Ile Gly Asp
130 135 140
Cys Ala
145 146
<210>32
<211>151
<212>PRT
<213>SARS coronavirus
<400>32
Asp Leu Asn Asp Phe Val Ser Asp Ala Tyr Ser Thr Leu Ile Gly Asp
1 5 10 15
Cys Ala Thr Val His Thr Ala Asn Lys Trp Asp Leu Ile Ile Ser Asp
20 25 30
Met Tyr Asp Pro Arg Thr Lys His Val Thr Lys Glu Asn Asp Ser Lys
35 40 45
Glu Gly Phe Phe Thr Tyr Leu Cys Gly Phe Ile Lys Gln Lys Leu Ala
50 55 60
Leu Gly Gly Ser Ile Ala Val Lys Ile Thr Glu His Ser Trp Asn Ala
65 70 75 80
Asp Leu Tyr Lys Leu Met Gly His Phe Ser Trp Trp Thr Ala Phe Val
85 90 95
Thr Asn Val Asn Ala Ser Ser Ser Glu Ala Phe Leu Ile Gly Ala Asn
100 105 110
Tyr Leu Gly Lys Pro Lys Glu Gln Ile Asp Gly Tyr Thr Met His Ala
115 120 125
Asn Tyr Ile Phe Trp Arg Asn Thr Asn Pro Ile Gln Leu Ser Ser Tyr
130 135 140
Ser Leu Phe Asp Met Ser Lys
145 150 151
<210>33
<211>149
<212>PRT
<213>SARS coronavirus
<400>33
Ile Gln Leu Ser Ser Tyr Ser Leu Phe Asp Met Ser Lys Phe Pro Leu
1 5 10 15
Lys Leu Arg Gly Thr Ala Val Met Ser Leu Lys Glu Asn Gln Ile Asn
20 25 30
Asp Met Ile Tyr Ser Leu Leu Glu Lys Gly Arg Leu Ile Ile Arg Glu
35 40 45
Asn Asn Arg Val Val Val Ser Ser Asp Ile Leu Val Asn Asn Thr Asn
50 55 60
Met Phe Ile Phe Leu Leu Phe Leu Thr Leu Thr Ser Gly Ser Asp Leu
65 70 75 80
Asp Arg Cys Thr Thr Phe Asp Asp Val Gln Ala Pro Asn Tyr Thr Gln
85 90 95
His Thr Ser Ser Met Arg Gly Val Tyr Tyr Pro Asp Glu Ile Phe Arg
100 105 110
Ser Asp Thr Leu Tyr Leu Thr Gln Asp Leu Phe Leu Pro Phe Tyr Ser
115 120 125
Asn Val Thr Gly Phe His Thr Ile Asn His Thr Phe Gly Asn Pro Val
130 135 140
Ile Pro Phe Lys Asp
145 149
<210>34
<211>152
<212>PRT
<213>SARS coronavirus
<400>34
Asn Val Thr Gly Phe His Thr Ile Asn His Thr Phe Gly Asn Pro Val
1 5 10 15
Ile Pro Phe Lys Asp Gly Ile Tyr Phe Ala Ala Thr Glu Lys Ser Asn
20 25 30
Val Val Arg Gly Trp Val Phe Gly Ser Thr Met Asn Asn Lys Ser Gln
35 40 45
Ser Val Ile Ile Ile Asn Asn Ser Thr Asn Val Val Ile Arg Ala Cys
50 55 60
Asn Phe Glu Leu Cys Asp Asn Pro Phe Phe Ala Val Ser Lys Pro Met
65 70 75 80
Gly Thr Gln Thr His Thr Met Ile Phe Asp Asn Ala Phe Asn Cys Thr
85 90 95
Phe Glu Tyr Ile Ser Asp Ala Phe Ser Leu Asp Val Ser Glu Lys Ser
100 105 110
Gly Asn Phe Lys His Leu Arg Glu Phe Val Phe Lys Asn Lys Asp Gly
115 120 125
Phe Leu Tyr Val Tyr Lys Gly Tyr Gln Pro Ile Asp Val Val Arg Asp
130 135 140
Leu Pro Ser Gly Phe Asn Thr Leu
145 150 152
<210>35
<211>152
<212>PRT
<213>SARS coronavirus
<400>35
Tyr Gln Pro Ile Asp Val Val Arg Asp Leu Pro Ser Gly Phe Asn Thr
1 5 10 15
Leu Lys Pro Ile Phe Lys Leu Pro Leu Gly Ile Asn Ile Thr Asn Phe
20 25 30
Arg Ala Ile Leu Thr Ala Phe Ser Pro Ala Gln Asp Ile Trp Gly Thr
35 40 45
Ser Ala Ala Ala Tyr Phe Val Gly Tyr Leu Lys Pro Thr Thr Phe Met
50 55 60
Leu Lys Tyr Asp Glu Asn Gly Thr Ile Thr Asp Ala Val Asp Cys Ser
65 70 75 80
Gln Asn Pro Leu Ala Glu Leu Lys Cys Ser Val Lys Ser Phe Glu Ile
85 90 95
Asp Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val Val Pro Ser Gly
100 105 110
Asp Val Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Gly Glu
115 120 125
Val Phe Asn Ala Thr Lys Phe Pro Ser Val Tyr Ala Trp Glu Arg Lys
130 135 140
Lys Ile Ser Asn Cys Val Ala Asp
145 150 152
<210>36
<211>153
<212>PRT
<213>SARS coronavirus
<400>36
Lys Phe Pro Ser Val Tyr Ala Trp Glu Arg Lys Lys Ile Ser Asn Cys
1 5 10 15
Val Ala Asp Tyr Ser Val Leu Tyr Asn Ser Thr Phe Phe Ser Thr Phe
20 25 30
Lys Cys Tyr Gly Val Ser Ala Thr Lys Leu Asn Asp Leu Cys Phe Ser
35 40 45
Asn Val Tyr Ala Asp Ser Phe Val Val Lys Gly Asp Asp Val Arg Gln
50 55 60
Ile Ala Pro Gly Gln Thr Gly Val Ile Ala Asp Tyr Asn Tyr Lys Leu
65 70 75 80
Pro Asp Asp Phe Met Gly Cys Val Leu Ala Trp Asn Thr Arg Asn Ile
85 90 95
Asp Ala Thr Ser Thr Gly Asn Tyr Asn Tyr Lys Tyr Arg Tyr Leu Arg
100 105 110
His Gly Lys Leu Arg Pro Phe Glu Arg Asp Ile Ser Asn Val Pro Phe
115 120 125
Ser Pro Asp Gly Lys Pro Cys Thr Pro Pro Ala Leu Asn Cys Tyr Trp
130 135 140
Pro Leu Asn Asp Tyr Gly Phe Tyr Thr
145 150 153
<210>37
<211>146
<212>PRT
<213>SARS coronavirus
<400>37
Pro Ala Leu Asn Cys Tyr Trp Pro Leu Asn Asp Tyr Gly Phe Tyr Thr
1 5 10 15
Thr Thr Gly Ile Gly Tyr Gln Pro Tyr Arg Val Val Val Leu Ser Phe
20 25 30
Glu Leu Leu Asn Ala Pro Ala Thr Val Cys Gly Pro Lys Leu Ser Thr
35 40 45
Asp Leu Ile Lys Asn Gln Cys Val Asn Phe Asn Phe Asn Gly Leu Thr
50 55 60
Gly Thr Gly Val Leu Thr Pro Ser Ser Lys Arg Phe Gln Pro Phe Gln
65 70 75 80
Gln Phe Gly Arg Asp Val Ser Asp Phe Thr Asp Ser Val Arg Asp Pro
85 90 95
Lys Thr Ser Glu Ile Leu Asp Ile Ser Pro Cys Ala Phe Gly Gly Val
100 105 110
Ser Val Ile Thr Pro Gly Thr Asn Ala Ser Ser Glu Val Ala Val Leu
115 120 125
Tyr Gln Asp Val Asn Cys Thr Asp Val Ser Thr Ala Ile His Ala Asp
130 135 140
Gln Leu
145 146
<210>38
<211>147
<212>PRT
<213>SARS coronavirus
<400>38
Cys Thr Asp Val Ser Thr Ala Ile His Ala Asp Gln Leu Thr Pro Ala
1 5 10 15
Trp Arg Ile Tyr Ser Thr Gly Asn Asn Val Phe Gln Thr Gln Ala Gly
20 25 30
Cys Leu Ile Gly Ala Glu His Val Asp Thr Ser Tyr Glu Cys Asp Ile
35 40 45
Pro Ile Gly Ala Gly Ile Cys Ala Ser Tyr His Thr Val Ser Leu Leu
50 55 60
Arg Ser Thr Ser Gln Lys Ser Ile Val Ala Tyr Thr Met Ser Leu Gly
65 70 75 80
Ala Asp Ser Ser Ile Ala Tyr Ser Asn Asn Thr Ile Ala Ile Pro Thr
85 90 95
Asn Phe Ser Ile Ser Ile Thr Thr Glu Val Met Pro Val Ser Met Ala
100 105 110
Lys Thr Ser Val Asp Cys Asn Met Tyr Ile Cys Gly Asp Ser Thr Glu
115 120 125
Cys Ala Asn Leu Leu Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn
130 135 140
Arg Ala Leu
145 147
<210>39
<211>147
<212>PRT
<213>SARS coronavirus
<400>39
Leu Gln Tyr Gly Ser Phe Cys Thr Gln Leu Asn Arg Ala Leu Ser Gly
1 5 10 15
Ile Ala Ala Glu Gln Asp Arg Asn Thr Arg Glu Val Phe Ala Gln Val
20 25 30
Lys Gln Met Tyr Lys Thr Pro Thr Leu Lys Tyr Phe Gly Gly Phe Asn
35 40 45
Phe Ser Gln Ile Leu Pro Asp Pro Leu Lys Pro Thr Lys Arg Ser Phe
50 55 60
Ile Glu Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly Phe
65 70 75 80
Met Lys Gln Tyr Gly Glu Cys Leu Gly Asp Ile Asn Ala Arg Asp Leu
85 90 95
Ile Cys Ala Gln Lys Phe Asn Gly Leu Thr Val Leu Pro Pro Leu Leu
100 105 110
Thr Asp Asp Met Ile Ala Ala Tyr Thr Ala Ala Leu Val Ser Gly Thr
115 120 125
Ala Thr Ala Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro
130 135 140
Phe Ala Met
145 147
<210>40
<211>146
<212>PRT
<213>SARS coronavirus
<400>40
Asp Ser His Asp Thr Val Leu Thr Ser Glu Glu Val Val Leu Lys Asn
1 5 10 15
Gly Glu Leu Glu Ala Leu Glu Thr Pro Val Asp Ser Phe Thr Asn Gly
20 25 30
Ala Ile Val Gly Thr Pro Val Cys Val Asn Gly Leu Met Leu Leu Glu
35 40 45
Ile Lys Asp Lys Glu Gln Tyr Cys Ala Leu Ser Pro Gly Leu Leu Ala
50 55 60
Thr Asn Asn Val Phe Arg Leu Lys Gly Gly Ala Pro Ile Lys Gly Val
65 70 75 80
Thr Phe Gly Glu Asp Thr Val Trp Glu Val Gln Gly Tyr Lys Asn Val
85 90 95
Arg Ile Thr Phe Glu Leu Asp Glu Arg Val Asp Lys Val Leu Asn Glu
100 105 110
Lys Cys Ser Val Tyr Thr Val Glu Ser Gly Thr Glu Val Thr Glu Phe
115 120 125
Ala Cys Val Val Ala Glu Ala Val Val Lys Thr Leu Gln Pro Val Ser
130 135 140
Asp Leu
145 146
<210>41
<211>149
<212>PRT
<213>SARS coronavirus
<400>41
Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe Ala Met Gln
1 5 10 15
Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn Val Leu Tyr
20 25 30
Glu Asn Gln Lys Gln Ile Ala Asn Gln Phe Asn Lys Ala Ile Ser Gln
35 40 45
Ile Gln Glu Ser Leu Thr Thr Thr Ser Thr Ala Leu Gly Lys Leu Gln
50 55 60
Asp Val Val Asn Gln Asn Ala Gln Ala Leu Asn Thr Leu Val Lys Gln
65 70 75 80
Leu Ser Ser Asn Phe Gly Ala Ile Ser Ser Val Leu Asn Asp Ile Leu
85 90 95
Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln Ile Asp Arg Leu Ile
100 105 110
Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln Gln Leu Ile
115 120 125
Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala Thr Lys Met
130 135 140
Ser Glu Cys Val Leu
145 149
<210>42
<211>150
<212>PRT
<213>SARS coronavirus
<400>42
Arg Ala Ser Ala Asn Leu Ala Ala Thr Lys Met Ser Glu Cys Val Leu
1 5 10 15
Gly Gln Ser Lys Arg Val Asp Phe Cys Gly Lys Gly Tyr His Leu Met
20 25 30
Ser Phe Pro Gln Ala Ala Pro His Gly Val Val Phe Leu His Val Thr
35 40 45
Tyr Val Pro Ser Gln Glu Arg Asn Phe Thr Thr Ala Pro Ala Ile Cys
50 55 60
His Glu Gly Lys Ala Tyr Phe Pro Arg Glu Gly Val Phe Val Phe Asn
65 70 75 80
Gly Thr Ser Trp Phe Ile Thr Gln Arg Asn Phe Phe Ser Pro Gln Ile
85 90 95
Ile Thr Thr Asp Asn Thr Phe Val Ser Gly Asn Cys Asp Val Val Ile
100 105 110
Gly Ile Ile Asn Asn Thr Val Tyr Asp Pro Leu Gln Pro Glu Leu Asp
115 120 125
Ser Phe Lys Glu Glu Leu Asp Lys Tyr Phe Lys Asn His Thr Ser Pro
130 135 140
Asp Val Asp Leu Gly Asp
145 150
<210>43
<211>120
<212>PRT
<213>SARS coronavirus
<400>43
Lys Tyr Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile
1 5 10 15
Ser Gly Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg
20 25 30
Leu Asn Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln
35 40 45
Glu Leu Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Val Trp
50 55 60
Leu Gly Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Leu
65 70 75 80
Leu Cys Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Ala Cys Ser
85 90 95
Cys Gly Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro Val Leu
100 105 110
Lys Gly Val Lys Leu His Tyr Thr
115 120
<210>44
<211>151
<212>PRT
<213>SARS coronavirus
<400>44
Thr Leu Gly Ser Ile Thr Ala Gln Pro Val Lys Ile Asp Asn Ala Ser
1 5 10 15
Pro Ala Ser Thr Val His Ala Thr Ala Thr Ile Pro Leu Gln Ala Ser
20 25 30
Leu Pro Phe Gly Trp Leu Val Ile Gly Val Ala Phe Leu Ala Val Phe
35 40 45
Gln Ser Ala Thr Lys Ile Ile Ala Leu Asn Lys Arg Trp Gln Leu Ala
50 55 60
Leu Tyr Lys Gly Phe Gln Phe Ile Cys Asn Leu Leu Leu Leu Phe Val
65 70 75 80
Thr Ile Tyr Ser His Leu Leu Leu Val Ala Ala Gly Met Glu Ala Gln
85 90 95
Phe Leu Tyr Leu Tyr Ala Leu Ile Tyr Phe Leu Gln Cys Ile Asn Ala
100 105 110
Cys Arg Ile Ile Met Arg Cys Trp Leu Cys Trp Lys Cys Lys Ser Lys
115 120 125
Asn Pro Leu Leu Tyr Asp Ala Asn Tyr Phe Val Cys Trp His Thr His
130 135 140
Asn Tyr Asp Tyr Cys Ile Pro
145 150 151
<210>45
<211>133
<212>PRT
<213>SARS coronavirus
<400>45
Asp Ala Asn Tyr Phe Val Cys Trp His Thr His Asn Tyr Asp Tyr Cys
1 5 10 15
Ile Pro Tyr Asn Ser Val Thr Asp Thr Ile Val Val Thr Glu Gly Asp
20 25 30
Gly Ile Ser Thr Pro Lys Leu Lys Glu Asp Tyr Gln Ile Gly Gly Tyr
35 40 45
Ser Glu Asp Arg His Ser Gly Val Lys Asp Tyr Val Val Val His Gly
50 55 60
Tyr Phe Thr Glu Val Tyr Tyr Gln Leu Glu Ser Thr Gln Ile Thr Thr
65 70 75 80
Asp Thr Gly Ile Glu Asn Ala Thr Phe Phe Ile Phe Asn Lys Leu Val
85 90 95
Lys Asp Pro Pro Asn Val Gln Ile His Thr Ile Asp Gly Ser Ser Gly
100 105 110
Val Ala Asn Pro Ala Met Asp Pro Ile Tyr Asp Glu Pro Thr Thr Thr
115 120 125
Thr Ser Val Pro Leu
130 133
<210>46
<211>83
<212>PRT
<213>SARS coronavirus
<400>46
Gln Glu Ser Glu Tyr Glu Leu Met Tyr Ser Phe Val Ser Glu Glu Thr
1 5 10 15
Gly Thr Leu Ile Val Asn Ser Val Leu Leu Phe Leu Ala Phe Val Val
20 25 30
Phe Leu Leu Val Thr Leu Ala Ile Leu Thr Ala Leu Arg Leu Cys Ala
35 40 45
Tyr Cys Cys Asn Ile Val Asn Val Ser Leu Val Lys Pro Thr Val Tyr
50 55 60
Val Tyr Ser Arg Val Lys Asn Leu Asn Ser Ser Glu Gly Val Pro Asp
65 70 75 80
Leu Leu Val
83
<210>47
<211>150
<212>PRT
<213>SARS coronavirus
<400>47
Leu Leu Gln Phe Ala Tyr Ser Asn Arg Asn Arg Phe Leu Tyr Ile Ile
1 5 10 15
Lys Leu Val Phe Leu Trp Leu Leu Trp Pro Val Thr Leu Ala Cys Phe
20 25 30
Val Leu Ala Ala Val Tyr Arg Ile Asn Trp Val Thr Gly Gly Ile Ala
35 40 45
Ile Ala Met Ala Cys Ile Val Gly Leu Met Trp Leu Ser Tyr Phe Val
50 55 60
Ala Ser Phe Arg Leu Phe Ala Arg Thr Arg Ser Met Trp Ser Phe Asn
65 70 75 80
Pro Glu Thr Asn Ile Leu Leu Asn Val Pro Leu Arg Gly Thr Ile Val
85 90 95
Thr Arg Pro Leu Met Glu Ser Glu Leu Val Ile Gly Ala Val Ile Ile
100 105 110
Arg Gly His Leu Arg Met Ala Gly His Ser Leu Gly Arg Cys Asp Ile
115 120 125
Lys Asp Leu Pro Lys Glu Ile Thr Val Ala Thr Ser Arg Thr Leu Ser
130 135 140
Tyr Tyr Lys Leu Gly Ala
145 150
<210>48
<211>55
<212>PRT
<213>SARS coronavirus
<400>48
Ile Thr Val Ala Thr Ser Arg Thr Leu Ser Tyr Tyr Lys Leu Gly Ala
1 5 10 15
Ser Gln Arg Val Gly Thr Asp Ser Gly Phe Ala Ala Tyr Asn Arg Tyr
20 25 30
Arg Ile Gly Asn Tyr Lys Leu Asn Thr Asp His Ala Gly Ser Asn Asp
35 40 45
Asn Ile Ala Leu Leu Val Gln
50 55
<210>49
<211>117
<212>PRT
<213>SARS coronavirus
<400>49
Phe Leu Thr Leu Ile Val Phe Thr Ser Cys Glu Leu Tyr His Tyr Gln
1 5 10 15
Glu Cys Val Arg Gly Thr Thr Val Leu Leu Lys Glu Pro Cys Pro Ser
20 25 30
Gly Thr Tyr Glu Gly Asn Ser Pro Phe His Pro Leu Ala Asp Asn Lys
35 40 45
Phe Ala Leu Thr Cys Thr Ser Thr His Phe Ala Phe Ala Cys Ala Asp
50 55 60
Gly Thr Arg His Thr Tyr Gln Leu Arg Ala Arg Ser Val Ser Pro Lys
65 70 75 80
Leu Phe Ile Arg Gln Glu Glu Val Gln Gln Glu Leu Tyr Ser Pro Leu
85 90 95
Phe Leu Ile Val Ala Ala Leu Val Phe Leu Ile Leu Cys Phe Thr Ile
100 105 110
Lys Arg Lys Thr Glu
115 117
<210>50
<211>55
<212>PRT
<213>SARS coronavirus
<400>50
Ala Ile Pro Cys Phe Asn Asn Ala Tyr Tyr Ile Leu Val Phe Thr Arg
1 5 10 15
Asn Pro Gly Ser Arg Arg Thr Leu Tyr Gln Ser Leu Asn Glu His Glu
20 25 30
Thr Ser His Cys Phe Asp Leu Tyr Phe Ser Met Gln Leu His Met His
35 40 45
Cys Ser Thr Ala Leu Cys Ile
50 55
<210>51
<211>147
<212>PRT
<213>SARS coronavirus
<400>51
Ala Val Val Lys Thr Leu Gln Pro Val Ser Asp Leu Leu Thr Asn Met
1 5 10 15
Gly Ile Asp Leu Asp Glu Trp Ser Val Ala Thr Phe Tyr Leu Phe Asp
20 25 30
Asp Ala Gly Glu Glu Asn Phe Ser Ser Arg Met Tyr Cys Ser Phe Tyr
35 40 45
Pro Pro Asp Glu Glu Glu Glu Asp Asp Ala Glu Cys Glu Glu Glu Glu
50 55 60
Ile Asp Glu Thr Cys Glu His Glu Tyr Gly Thr Glu Asp Asp Tyr Gln
65 70 75 80
Gly Leu Pro Leu Glu Phe Gly Ala Ser Ala Glu Thr Val Arg Val Glu
85 90 95
Glu Glu Glu Glu Glu Asp Trp Leu Asp Asp Thr Thr Glu Gln Ser Glu
100 105 110
Ile Glu Pro Glu Pro Glu Pro Thr Pro Glu Glu Pro Val Asn Gln Phe
115 120 125
Thr Gly Tyr Leu Lys Leu Thr Asp Asn Val Ala Ile Lys Cys Val Asp
130 135 140
Ile Val Lys
145 147
<210>52
<211>146
<212>PRT
<213>SARS coronavirus
<400>52
Asn Glu Gln Ile Lys Met Ser Asp Asn Gly Pro Gln Ser Asn Gln Arg
1 5 10 15
Ser Ala Pro Arg Ile Thr Phe Gly Gly Pro Thr Asp Ser Thr Asp Asn
20 25 30
Asn Gln Asn Gly Gly Arg Asn Gly Ala Arg Pro Lys Gln Arg Arg Pro
35 40 45
Gln Gly Leu Pro Asn Asn Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln
50 55 60
His Gly Lys Glu Glu Leu Arg Phe Pro Arg Gly Gln Gly Val Pro Ile
65 70 75 80
Asn Thr Asn Ser Gly Pro Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala
85 90 95
Thr Arg Arg Val Arg Gly Gly Asp Gly Lys Met Lys Glu Leu Ser Pro
100 105 110
Arg Trp Tyr Phe Tyr Tyr Leu Gly Thr Gly Pro Glu Ala Ser Leu Pro
115 120 125
Tyr Gly Ala Asn Lys Glu Gly Ile Val Trp Val Ala Thr Glu Gly Ala
130 135 140
Leu Asn
145 146
<210>53
<211>147
<212>PRT
<213>SARS coronavirus
<400>53
Asn Lys Glu Gly Ile Val Trp Val Ala Thr Glu Gly Ala Leu Asn Thr
1 5 10 15
Pro Lys Asp His Ile Gly Thr Arg Asn Pro Asn Asn Asn Ala Ala Thr
20 25 30
Val Leu Gln Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala
35 40 45
Glu Gly Ser Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg
50 55 60
Ser Arg Gly Asn Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Asn
65 70 75 80
Ser Pro Ala Arg Met Ala Ser Gly Gly Gly Glu Thr Ala Leu Ala Leu
85 90 95
Leu Leu Leu Asp Arg Leu Asn Gln Leu Glu Ser Lys Val Ser Gly Lys
100 105 110
Gly Gln Gln Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu
115 120 125
Ala Ser Lys Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Gln Tyr Asn
130 135 140
Val Thr Gln
145 147
<210>54
<211>146
<212>PRT
<213>SARS coronavirus
<400>54
Arg Gln Lys Arg Thr Ala Thr Lys Gln Tyr Asn Val Thr Gln Ala Phe
1 5 10 15
Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp Gln Asp
20 25 30
Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln Ile Ala Gln
35 40 45
Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile Gly Met
50 55 60
Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr His Gly Ala Ile Lys
65 70 75 80
Leu Asp Asp Lys Asp Pro Gln Phe Lys Asp Asn Val Ile Leu Leu Asn
85 90 95
Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu Pro Lys Lys
100 105 110
Asp Lys Lys Lys Lys Thr Asp Glu Ala Gln Pro Leu Pro Gln Arg Gln
115 120 125
Lys Lys Gln Pro Thr Val Thr Leu Leu Pro Ala Ala Asp Met Asp Asp
130 135 140
Phe Ser
145 146
<210>55
<211>51
<212>PRT
<213>SARS coronavirus
<400>55
Asp Lys Lys Lys Lys Thr Asp Glu Ala Gln Pro Leu Pro Gln Arg Gln
1 5 10 15
Lys Lys Gln Pro Thr Val Thr Leu Leu Pro Ala Ala Asp Met Asp Asp
20 25 30
Phe Ser Arg Gln Leu Gln Asn Ser Met Ser Gly Ala Ser Ala Asp Ser
35 40 45
Thr Gln Ala
50 51
<210>56
<211>51
<212>PRT
<213>SARS coronavirus
<400>56
Asp Lys Lys Lys Lys Thr Asp Glu Ala Gln Pro Leu Pro Gln Arg Gln
1 5 10 15
Lys Lys Gln Pro Thr Val Thr Leu Leu Pro Ala Ala Asp Met Asp Asp
20 25 30
Phe Ser Arg Gln Leu Gln Asn Ser Met Ser Gly Ala Ser Ala Asp Ser
35 40 45
Thr Gln Ala
50 51
<210>57
<211>150
<212>PRT
<213>SARS coronavirus
<400>57
Tyr Leu Lys Leu Thr Asp Asn Val Ala Ile Lys Cys Val Asp Ile Val
1 5 10 15
Lys Glu Ala Gln Ser Ala Asn Pro Met Val Ile Val Asn Ala Ala Asn
20 25 30
Ile His Leu Lys His Gly Gly Gly Val Ala Gly Ala Leu Asn Lys Ala
35 40 45
Thr Asn Gly Ala Met Gln Lys Glu Ser Asp Asp Tyr Ile Lys Leu Asn
50 55 60
Gly Pro Leu Thr Val Gly Gly Ser Cys Leu Leu Ser Gly His Asn Leu
65 70 75 80
Ala Lys Lys Cys Leu His Val Val Gly Pro Asn Leu Asn Ala Gly Glu
85 90 95
Asp Ile Gln Leu Leu Lys Ala Ala Tyr Glu Asn Phe Asn Ser Gln Asp
100 105 110
Ile Leu Leu Ala Pro Leu Leu Ser Ala Gly Ile Phe Gly Ala Lys Pro
115 120 125
Leu Gln Ser Leu Gln Val Cys Val Gln Thr Val Arg Thr Gln Val Tyr
130 135 140
Ile Ala Val Asn Asp Lys
145 150
<210>58
<211>155
<212>PRT
<213>SARS coronavirus
<400>58
Leu Gln Ser Leu Gln Val Cys Val Gln Thr Val Arg Thr Gln Val Tyr
1 5 10 15
Ile Ala Val Asn Asp Lys Ala Leu Tyr Glu Gln Val Val Met Asp Tyr
20 25 30
Leu Asp Asn Leu Lys Pro Arg Val Glu Ala Pro Lys Gln Glu Glu Pro
35 40 45
Pro Asn Thr Glu Asp Ser Lys Thr Glu Glu Lys Ser Val Val Gln Lys
50 55 60
Pro Val Asp Val Lys Pro Lys Ile Lys Ala Cys Ile Asp Glu Val Thr
65 70 75 80
Thr Thr Leu Glu Glu Thr Lys Phe Leu Thr Asn Lys Leu Leu Leu Phe
85 90 95
Ala Asp Ile Asn Gly Lys Leu Tyr His Asp Ser Gln Asn Met Leu Arg
100 105 110
Gly Glu Asp Met Ser Phe Leu Glu Lys Asp Ala Pro Tyr Met Val Gly
115 120 125
Asp Val Ile Thr Ser Gly Asp Ile Thr Cys Val Val Ile Pro Ser Lys
130 135 140
Lys Ala Gly Gly Thr Thr Glu Met Leu Ser Arg
145 150 155
<210>59
<211>149
<212>PRT
<213>SARS coronavirus
<400>59
Leu Ser Thr Asn His Gly His Lys Val Val Glu Leu Val Ala Glu Met
1 5 10 15
Asp Gly Ile Gln Tyr Gly Arg Ser Gly Ile Thr Leu Gly Val Leu Val
20 25 30
Pro His Val Gly Glu Thr Pro Ile Ala Tyr Arg Asn Val Leu Leu Arg
35 40 45
Lys Asn Gly Asn Lys Gly Ala Gly Gly His Ser Tyr Gly Ile Asp Leu
50 55 60
Lys Ser Tyr Asp Leu Gly Asp Glu Leu Gly Thr Asp Pro Ile Glu Asp
65 70 75 80
Tyr Glu Gln Asn Trp Asn Thr Lys His Gly Ser Gly Ala Leu Arg Glu
85 90 95
Leu Thr Arg Glu Leu Asn Gly Gly Ala Val Thr Arg Tyr Val Asp Asn
100 105 110
Asn Phe Cys Gly Pro Asp Gly Tyr Pro Leu Asp Cys Ile Lys Asp Phe
115 120 125
Leu Ala Arg Ala Gly Lys Ser Met Cys Thr Leu Ser Glu Gln Leu Asp
130 135 140
Tyr Ile Glu Ser Lys
145 149
<210>60
<211>148
<212>PRT
<213>SARS coronavirus
<400>60
Val Ile Pro Ser Lys Lys Ala Gly Gly Thr Thr Glu Met Leu Ser Arg
1 5 10 15
Ala Leu Lys Lys Val Pro Val Asp Glu Tyr Ile Thr Thr Tyr Pro Gly
20 25 30
Gln Gly Cys Ala Gly Tyr Thr Leu Glu Glu Ala Lys Thr Ala Leu Lys
35 40 45
Lys Cys Lys Ser Ala Phe Tyr Val Leu Pro Ser Glu Ala Pro Asn Ala
50 55 60
Lys Glu Glu Ile Leu Gly Thr Val Ser Trp Asn Leu Arg Glu Met Leu
65 70 75 80
Ala His Ala Glu Glu Thr Arg Lys Leu Met Pro Ile Cys Met Asp Val
85 90 95
Arg Ala Ile Met Ala Thr Ile Gln Arg Lys Tyr Lys Gly Ile Lys Ile
100 105 110
Gln Glu Gly Ile Val Asp Tyr Gly Val Arg Phe Phe Phe Tyr Thr Ser
115 120 125
Lys Glu Pro Val Ala Ser Ile Ile Thr Lys Leu Asn Ser Leu Asn Glu
130 135 140
Pro Leu Val Thr
145 148
<210>61
<211>147
<212>PRT
<213>SARS coronavirus
<400>61
Thr Lys Leu Asn Ser Leu Asn Glu Pro Leu Val Thr Met Pro Ile Gly
1 5 10 15
Tyr Val Thr His Gly Phe Asn Leu Glu Glu Ala Ala Arg Cys Met Arg
20 25 30
Ser Leu Lys Ala Pro Ala Val Val Ser Val Ser Ser Pro Asp Ala Val
35 40 45
Thr Thr Tyr Asn Gly Tyr Leu Thr Ser Ser Ser Lys Thr Ser Glu Glu
50 55 60
His Phe Val Glu Thr Val Ser Leu Ala Gly Ser Tyr Arg Asp Trp Ser
65 70 75 80
Tyr Ser Gly Gln Arg Thr Glu Leu Gly Val Glu Phe Leu Lys Arg Gly
85 90 95
Asp Lys Ile Val Tyr His Thr Leu Glu Ser Pro Val Glu Phe His Leu
100 105 110
Asp Gly Glu Val Leu Ser Leu Asp Lys Leu Lys Ser Leu Leu Ser Leu
115 120 125
Arg Glu Val Lys Thr Ile Lys Val Phe Thr Thr Val Asp Asn Thr Asn
130 135 140
Leu His Thr
145 147
<210>62
<211>147
<212>PRT
<213>SARS coronavirus
<400>62
Thr Lys Leu Asn Ser Leu Asn Glu Pro Leu Val Thr Met Pro Ile Gly
1 5 10 15
Tyr Val Thr His Gly Phe Asn Leu Glu Glu Ala Ala Arg Cys Mer Arg
20 25 30
Ser Leu Lys Ala Pro Ala Val Val Ser Val Ser Ser Pro Asp Ala Val
35 40 45
Thr Thr Tyr Asn Gly Tyr Leu Thr Ser Ser Ser Lys Thr Ser Glu Glu
50 55 60
His Phe Val Glu Thr Val Ser Leu Ala Gly Ser Tyr Arg Asp Trp Ser
65 70 75 80
Tyr Ser Gly Gln Arg Thr Glu Leu Gly Val Glu Phe Leu Lys Arg Gly
85 90 95
Asp Lys Ile Val Tyr His Thr Leu Glu Ser Pro Val Glu Phe His Leu
100 105 110
Asp Gly Glu Val Leu Ser Leu Asp Lys Leu Lys Ser Leu Leu Ser Leu
115 120 125
Arg Glu Val Lys Thr Ile Lys Val Phe Thr Thr Val Asp Asn Thr Asn
130 135 140
Leu His Thr
145 147
<210>63
<211>149
<212>PRT
<213>SARS coronavirus
<400>63
Tyr Arg Ala Arg Ala Gly Asp Ala Ala Asn Phe Cys Ala Leu Ile Leu
1 5 10 15
Ala Tyr Ser Asn Lys Thr Val Gly Glu Leu Gly Asp Val Arg Glu Thr
20 25 30
Met Thr His Leu Leu Gln His Ala Asn Leu Glu Ser Ala Lys Arg Val
35 40 45
Leu Asn Val Val Cys Lys His Cys Gly Gln Lys Thr Thr Thr Leu Thr
50 55 60
Gly Val Glu Ala Val Met Tyr Met Gly Thr Leu Ser Tyr Asp Asn Leu
65 70 75 80
Lys Thr Gly Val Ser Ile Pro Cys Val Cys Gly Arg Asp Ala Thr Gln
85 90 95
Tyr Leu Val Gln Gln Glu Ser Ser Phe Val Met Met Ser Ala Pro Pro
100 105 110
Ala Glu Tyr Lys Leu Gln Gln Gly Thr Phe Leu Cys Ala Asn Glu Tyr
115 120 125
Thr Gly Asn Tyr Gln Cys Gly His Tyr Thr His Ile Thr Ala Lys Glu
130 135 140
Thr Leu Tyr Arg Ile
145 149
<210>64
<211>148
<212>PRT
<213>SARS coronavirus
<400>64
Cys Gly His Tyr Thr His Ile Thr Ala Lys Glu Thr Leu Tyr Arg Ile
1 5 10 15
Asp Gly Ala His Leu Thr Lys Met Ser Glu Tyr Lys Gly Pro Val Thr
20 25 30
Asp Val Phe Tyr Lys Glu Thr Ser Tyr Thr Thr Thr Ile Lys Pro Val
35 40 45
Ser Tyr Lys Leu Asp Gly Val Thr Tyr Thr Glu Ile Glu Pro Lys Leu
50 55 60
Asp Gly Tyr Tyr Lys Lys Asp Asn Ala Tyr Tyr Thr Glu Gln Pro Ile
65 70 75 80
Asp Leu Val Pro Thr Gln Pro Leu Pro Asn Ala Ser Phe Asp Asn Phe
85 90 95
Lys Leu Thr Cys Ser Asn Thr Lys Phe Ala Asp Asp Leu Asn Gln Met
100 105 110
Thr Gly Phe Thr Lys Pro Ala Ser Arg Glu Leu Ser Val Thr Phe Phe
115 120 125
Pro Asp Leu Asn Gly Asp Val Val Ala Ile Asp Tyr Arg His Tyr Ser
130 135 140
Ala Ser Phe Lys
145 148
<210>65
<211>151
<212>PRT
<213>SARS coronavirus
<400>65
Val Val Ala Ile Asp Tyr Arg His Tyr Ser Ala Ser Phe Lys Lys Gly
1 5 10 15
Ala Lys Leu Leu His Lys Pro Ile Val Trp His Ile Asn Gln Ala Thr
20 25 30
Thr Lys Thr Thr Phe Lys Pro Asn Thr Trp Cys Leu Arg Cys Leu Trp
35 40 45
Ser Thr Lys Pro Val Asp Thr Ser Asn Ser Phe Glu Val Leu Ala Val
50 55 60
Glu Asp Thr Gln Gly Met Asp Asn Leu Ala Cys Glu Ser Gln Gln Pro
65 70 75 80
Thr Ser Glu Glu Val Val Glu Asn Pro Thr Ile Gln Lys Glu Val Ile
85 90 95
Glu Cys Asp Val Lys Thr Thr Glu Val Val Gly Asn Val Ile Leu Lys
100 105 110
Pro Ser Asp Glu Gly Val Lys Val Thr Gln Glu Leu Gly His Glu Asp
115 120 125
Leu Met Ala Ala Tyr Val Glu Asn Thr Ser Ile Thr Ile Lys Lys Pro
130 135 140
Asn Glu Leu Ser Leu Ala Leu
145 150 151
<210>66
<211>146
<212>PRT
<213>SARS coronavirus
<400>66
Asn Thr Ser Ile Thr Ile Lys Lys Pro Asn Glu Leu Ser Leu Ala Leu
1 5 10 15
Gly Leu Lys Thr Ile Ala Thr His Gly Ile Ala Ala Ile Asn Ser Val
20 25 30
Pro Trp Ser Lys Ile Leu Ala Tyr Val Lys Pro Phe Leu Gly Gln Ala
35 40 45
Ala Ile Thr Thr Ser Asn Cys Ala Lys Arg Leu Ala Gln Arg Val Phe
50 55 60
Asn Asn Tyr Met Pro Tyr Val Phe Thr Leu Leu Phe Gln Leu Cys Thr
65 70 75 80
Phe Thr Lys Ser Thr Asn Ser Arg Ile Arg Ala Ser Leu Pro Thr Thr
85 90 95
Ile Ala Lys Asn Ser Val Lys Ser Val Ala Lys Leu Cys Leu Asp Ala
100 105 110
Gly Ile Asn Tyr Val Lys Ser Pro Lys Phe Ser Lys Leu Phe Thr Ile
115 120 125
Ala Met Trp Leu Leu Leu Leu Ser Ile Cys Leu Gly Ser Leu Ile Cys
130 135 140
Val Thr
145 146
<210>67
<211>151
<212>PRT
<213>SARS coronavirus
<400>67
Leu Leu Leu Leu Ser Ile Cys Leu Gly Ser Leu Ile Cys Val Thr Ala
1 5 10 15
Ala Phe Gly Val Leu Leu Ser Asn Phe Gly Ala Pro Ser Tyr Cys Asn
20 25 30
Gly Val Arg Glu Leu Tyr Leu Asn Ser Ser Asn Val Thr Thr Met Asp
35 40 45
Phe Cys Glu Gly Ser Phe Pro Cys Ser Ile Cys Leu Ser Gly Leu Asp
50 55 60
Ser Leu Asp Ser Tyr Pro Ala Leu Glu Thr Ile Gln Val Thr Ile Ser
65 70 75 80
Ser Tyr Lys Leu Asp Leu Thr Ile Leu Gly Leu Ala Ala Glu Trp Val
85 90 95
Leu Ala Tyr Met Leu Phe Thr Lys Phe Phe Tyr Leu Leu Gly Leu Ser
100 105 110
Ala Ile Met Gln Val Phe Phe Gly Tyr Phe Ala Ser His Phe Ile Ser
115 120 125
Asn Ser Trp Leu Met Trp Phe Ile Ile Ser Ile Val Gln Met Ala Pro
130 135 140
Val Ser Ala Met Val Arg Met
145 150 151
<210>68
<211>150
<212>PRT
<213>SARS coronavirus
<400>68
Leu Ser His Phe Val Asn Leu Asp Asn Leu Arg Ala Asn Asn Thr Lys
1 5 10 15
Gly Ser Leu Pro Ile Asn Val Ile Val Phe Asp Gly Lys Ser Lys Cys
20 25 30
Asp Glu Ser Ala Ser Lys Ser Ala Ser Val Tyr Tyr Ser Gln Leu Met
35 40 45
Cys Gln Pro Ile Leu Leu Leu Asp Gln Ala Leu Val Ser Asp Val Gly
50 55 60
Asp Ser Thr Glu Val Ser Val Lys Met Phe Asp Ala Tyr Val Asp Thr
65 70 75 80
Phe Ser Ala Thr Phe Ser Val Pro Met Glu Lys Leu Lys Ala Leu Val
85 90 95
Ala Thr Ala His Ser Glu Leu Ala Lys Gly Val Ala Leu Asp Gly Val
100 105 110
Leu Ser Thr Phe Val Ser Ala Ala Arg Gln Gly Val Val Asp Thr Asp
115 120 125
Val Asp Thr Lys Asp Val Ile Glu Cys Leu Lys Leu Ser His His Ser
130 135 140
Asp Leu Glu Val Thr Gly
145 150
<210>69
<211>146
<212>PRT
<213>SARS coronavirus
<400>69
Cys Leu Lys Leu Ser His His Ser Asp Leu Glu Val Thr Gly Asp Ser
1 5 10 15
Cys Asn Asn Phe Met Leu Thr Tyr Asn Lys Val Glu Asn Met Thr Pro
20 25 30
Arg Asp Leu Gly Ala Cys Ile Asp Cys Asn Ala Arg His Ile Asn Ala
35 40 45
Gln Val Ala Lys Ser His Asn Val Ser Leu Ile Trp Asn Val Lys Asp
50 55 60
Tyr Met Ser Leu Ser Glu Gln Leu Arg Lys Gln Ile Arg Ser Ala Ala
65 70 75 80
Lys Lys Asn Asn Ile Pro Phe Arg Leu Thr Cys Ala Thr Thr Arg Gln
85 90 95
Val Val Asn Val Ile Thr Thr Lys Ile Ser Leu Lys Gly Gly Lys Ile
100 105 110
Val Ser Thr Cys Phe Lys Leu Met Leu Lys Ala Thr Leu Leu Cys Val
115 120 125
Leu Ala Ala Leu Val Cys Tyr Ile Val Met Pro Val His Thr Leu Ser
130 135 140
Ile His
145 146
<210>70
<211>150
<212>PRT
<213>SARS coronavirus
<400>70
Ser Met Cys Thr Leu Ser Glu Gln Leu Asp Tyr Ile Glu Ser Lys Arg
1 5 10 15
Gly Val Tyr Cys Cys Arg Asp His Glu His Glu Ile Ala Trp Phe Thr
20 25 30
Glu Arg Ser Asp Lys Ser Tyr Glu His Gln Thr Pro Phe Glu Ile Lys
35 40 45
Ser Ala Lys Lys Phe Asp Thr Phe Lys Gly Glu Cys Pro Lys Phe Val
50 55 60
Phe Pro Leu Asn Ser Lys Val Lys Val Ile Gln Pro Arg Val Glu Lys
65 70 75 80
Lys Lys Thr Glu Gly Phe Met Gly Arg Ile Arg Ser Val Tyr Pro Val
85 90 95
Ala Ser Pro Gln Glu Cys Asn Asn Met His Leu Ser Thr Leu Met Lys
100 105 110
Cys Asn His Cys Asp Glu Val Ser Trp Gln Thr Cys Asp Phe Leu Lys
115 120 125
Ala Thr Cys Glu His Cys Gly Thr Glu Asn Leu Val Ile Glu Gly Pro
130 135 140
Thr Thr Cys Gly Tyr Leu
145 150
<210>71
<211>150
<212>PRT
<213>SARS coronavirus
<400>71
Leu Leu Leu Ser Ile Cys Leu Gly Ser Leu Ile Cys Val Thr Ala Ala
1 5 10 15
Phe Gly Val Leu Leu Ser Asn Phe Gly Ala Pro Ser Tyr Cys Asn Gly
20 25 30
Val Arg Glu Leu Tyr Leu Asn Ser Ser Asn Val Thr Thr Met Asp Phe
35 40 45
Cys Glu Gly Ser Phe Pro Cys Ser Ile Cys Leu Ser Gly Leu Asp Ser
50 55 60
Leu Asp Ser Tyr Pro Ala Leu Glu Thr Ile Gln Val Thr Ile Ser Ser
65 70 75 80
Tyr Lys Leu Asp Leu Thr Ile Leu Gly Leu Ala Ala Glu Trp Val Leu
85 90 95
Ala Tyr Met Leu Phe Thr Lys Phe Phe Tyr Leu Leu Gly Leu Ser Ala
100 105 110
Ile Met Gln Val Phe Phe Gly Tyr Phe Ala Ser His Phe Ile Ser Asn
115 120 125
Ser Trp Leu Met Trp Phe Ile Ile Ser Ile Val Gln Met Ala Pro Val
130 135 140
Ser Ala Met Val Arg Met
145 150
<210>72
<211>149
<212>PRT
<213>SARS coronavirus
<400>72
Ser Thr Cys Phe Lys Leu Met Leu Lys Ala Thr Leu Leu Cys Val Leu
1 5 10 15
Ala Ala Leu Val Cys Tyr Ile Val Met Pro Val His Thr Leu Ser Ile
20 25 30
His Asp Gly Tyr Thr Asn Glu Ile Ile Gly Tyr Lys Ala Ile Gln Asp
35 40 45
Gly Val Thr Arg Asp Ile Ile Ser Thr Asp Asp Cys Phe Ala Asn Lys
50 55 60
His Ala Gly Phe Asp Ala Trp Phe Ser Gln Arg Gly Gly Ser Tyr Lys
65 70 75 80
Asn Asp Lys Ser Cys Pro Val Val Ala Ala Ile Ile Thr Arg Glu Ile
85 90 95
Gly Phe Ile Val Pro Gly Leu Pro Gly Thr Val Leu Arg Ala Ile Asn
100 105 110
Gly Asp Phe Leu His Phe Leu Pro Arg Val Phe Ser Ala Val Gly Asn
115 120 125
Ile Cys Tyr Thr Pro Ser Lys Leu Ile Glu Tyr Ser Asp Phe Ala Thr
130 135 140
Ser Ala Cys Val Leu
145 149
<210>73
<211>151
<212>PRT
<213>SARS coronavirus
<400>73
Thr Pro Ser Lys Leu Ile Glu Tyr Ser Asp Phe Ala Thr Ser Ala Cys
1 5 10 15
Val Leu Ala Ala Glu Cys Thr Ile Phe Lys Asp Ala Met Gly Lys Pro
20 25 30
Val Pro Tyr Cys Tyr Asp Thr Asn Leu Leu Glu Gly Ser Ile Ser Tyr
35 40 45
Ser Glu Leu Arg Pro Asp Thr Arg Tyr Val Leu Met Asp Gly Ser Ile
50 55 60
Ile Gln Phe Pro Asn Thr Tyr Leu Glu Gly Ser Val Arg Val Val Thr
65 70 75 80
Thr Phe Asp Ala Glu Tyr Cys Arg His Gly Thr Cys Glu Arg Ser Glu
85 90 95
Val Gly Ile Cys Leu Ser Thr Ser Gly Arg Trp Val Leu Asn Asn Glu
100 105 110
His Tyr Arg Ala Leu Ser Gly Val Phe Cys Gly Val Asp Ala Met Asn
115 120 125
Leu Ile Ala Asn Ile Phe Thr Pro Leu Val Gln Pro Val Gly Ala Leu
130 135 140
Asp Val Ser Ala Ser Val Val
145 150 151
<210>74
<211>154
<212>PRT
<213>SARS coronavirus
<400>74
Pro Leu Val Gln Pro Val Gly Ala Leu Asp Val Ser Ala Ser Val Val
1 5 10 15
Ala Gly Gly Ile Ile Ala Ile Leu Val Thr Cys Ala Ala Tyr Tyr Phe
20 25 30
Met Lys Phe Arg Arg Val Phe Gly Glu Tyr Asn His Val Val Ala Ala
35 40 45
Asn Ala Leu Leu Phe Leu Met Ser Phe Thr Ile Leu Cys Leu Val Pro
50 55 60
Ala Tyr Ser Phe Leu Pro Gly Val Tyr Ser Val Phe Tyr Leu Tyr Leu
65 70 75 80
Thr Phe Tyr Phe Thr Asn Asp Val Ser Phe Leu Ala His Leu Gln Trp
85 90 95
Phe Ala Met Phe Ser Pro Ile Val Pro Phe Trp Ile Thr Ala Ile Tyr
100 105 110
Val Phe Cys Ile Ser Leu Lys His Cys His Trp Phe Phe Asn Asn Tyr
115 120 125
Leu Arg Lys Arg Val Met Phe Asn Gly Val Thr Phe Ser Thr Phe Glu
130 135 140
Glu Ala Ala Leu Cys Thr Phe Leu Leu Asn
145 150 154
<210>75
<211>147
<212>PRT
<213>SARS coronavirus
<400>75
Phe Asn Gly Val Thr Phe Ser Thr Phe Glu Glu Ala Ala Leu Cys Thr
1 5 10 15
Phe Leu Leu Asn Lys Glu Met Tyr Leu Lys Leu Arg Ser Glu Thr Leu
20 25 30
Leu Pro Leu Thr Gln Tyr Asn Arg Tyr Leu Ala Leu Tyr Asn Lys Tyr
35 40 45
Lys Tyr Phe Ser Gly Ala Leu Asp Thr Thr Ser Tyr Arg Glu Ala Ala
50 55 60
Cys Cys His Leu Ala Lys Ala Leu Asn Asp Phe Ser Asn Ser Gly Ala
65 70 75 80
Asp Val Leu Tyr Gln Pro Pro Gln Thr Ser Ile Thr Ser Ala Val Leu
85 90 95
Gln Ser Gly Phe Arg Lys Met Ala Phe Pro Ser Gly Lys Val Glu Gly
100 105 110
Cys Met Val Gln Val Thr Cys Gly Thr Thr Thr Leu Asn Gly Leu Trp
115 120 125
Leu Asp Asp Thr Val Tyr Cys Pro Arg His Val Ile Cys Thr Ala Glu
130 135 140
Asp Met Leu
145 147
<210>76
<211>22
<212>PRT
<213>SARS coronavirus
<400>76
Lys Lys Met Ala Asp Asn Gly Thr Ile Thr Val Glu Glu Leu Lys Gln
1 5 10 15
Leu Leu Glu Gln Trp Asn
20 22
<210>77
<211>24
<212>PRT
<213>SARS coronavirus
<400>77
Lys Lys Leu Asn Val Pro Leu Arg Gly Thr Ile Val Thr Arg Pro Leu
1 5 10 15
Met Glu Ser Glu Leu Val Ile Gly
20 24
<210>78
<211>20
<212>PRT
<213>SARS coronavirus
<400>78
Lys Lys Arg Met Ala Gly His Ser Leu Gly Arg Cys Asp Ile Lys Asp
1 5 10 15
Leu Pro Lys Glu
20
<210>79
<211>18
<212>PRT
<213>SARS coronavirus
<400>79
Lys Lys Asn Thr Asp His Ala Gly Ser Asn Asp Asn Ile Ala Leu Leu
1 5 10 15
Val Gln
18
<210>80
<211>27
<212>PRT
<213>SARS coronavirus
<400>80
Lys Lys Lys Asp Gly Ile Tyr Phe Ala Ala Thr Glu Lys Ser Asn Val
1 5 10 15
Val Arg Gly Trp Val Phe Gly Ser Thr Met Asn
20 27
<210>81
<211>24
<212>PRT
<213>SARS coronavirus
<400>81
Lys Lys Trp Val Phe Gly Ser Thr Met Asn Asn Lys Ser Gln Ser Val
1 5 10 15
Ile Ile Ile Asn Asn Ser Thr Asn
20 24
<210>82
<211>24
<212>PRT
<213>SARS coronavirus
<400>82
Lys Lys Asp Cys Ser Gln Asn Pro Leu Ala Glu Leu Lys Cys Ser Val
1 5 10 15
Lys Ser Phe Glu Ile Asp Lys Gly
20 24
<210>83
<211>24
<212>PRT
<213>SARS coronavirus
<400>83
Lys Lys Gln Thr Ser Asn Phe Arg Val Val Pro Ser Gly Asp Val Val
1 5 10 15
Arg Phe Pro Asn Ile Thr Asn Leu
20 24
<210>84
<211>23
<212>PRT
<213>SARS coronavirus
<400>84
Lys Lys Pro Phe Gly Glu Val Phe Asn Ala Thr Lys Phe Pro Ser Val
1 5 10 15
Tyr Ala Trp Glu Arg Lys Lys
20 23
<210>85
<211>24
<212>PRT
<213>SARS coronavirus
<400>85
Lys Lys Gln Asp Val Asn Cys Thr Asp Val Ser Thr Ala Ile His Ala
1 5 10 15
Asp Gln Leu Thr Pro Ala Trp Arg
20 24
<210>86
<211>24
<212>PRT
<213>SARS coronavirus
<400>86
Lys Lys Thr Gly Asn Asn Val Phe Gln Thr Gln Ala Gly Cys Leu Ile
1 5 10 15
Gly Ala Glu His Val Asp Thr Ser
20 24
<210>87
<211>24
<212>PRT
<213>SARS coronavirus
<400>87
Lys Lys Lys Gly Ala Cys Ser Cys Gly Ser Cys Cys Lys Phe Asp Glu
1 5 10 15
Asp Asp Ser Glu Pro Val Leu Lys
20 24
<210>88
<211>25
<212>PRT
<213>SARS coronavirus
<400>88
Lys Lys Met Ser Asp Asn Gly Pro Gln Ser Asn Gln Arg Ser Ala Pro
1 5 10 15
Arg Ile Thr Phe Gly Gly Pro Thr Asp
20 25
<210>89
<211>24
<212>PRT
<213>SARS coronavirus
<400>89
Lys Lys Pro Thr Asp Ser Thr Asp Asn Asn Gln Asn Gly Gly Arg Asn
1 5 10 15
Gly Ala Arg Pro Lys Gln Arg Arg
20 24
<210>90
<211>24
<212>PRT
<213>SARS coronavirus
<400>90
Lys Lys Ser Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg
1 5 10 15
Ser Arg Gly Asn Ser Arg Asn Ser
20 24
<210>91
<211>24
<212>PRT
<213>SARS coronavirus
<400>91
Lys Lys Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Asn Ser Pro Ala
1 5 10 15
Arg Met Ala Ser Gly Gly Gly Glu
20 24
<210>92
<211>19
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Lys Lys Lys Val Ser Gly Lys Gly Gln Gln Gln Gln Gly Gln Thr Val
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Thr Lys Lys
19
<210>93
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1 5 10 15
Gln Arg Gln Lys Lys Gln
20 22
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<211>24
<212>PRT
<213>SARS coronavirus
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Lys Lys Met Asp Asp Phe Ser Arg Gln Leu Gln Asn Ser Met Ser Gly
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Ala Ser Ala Asp Ser Thr Gln Ala
20 24
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<212>PRT
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Met Trp Ser Phe Asn Pro Glu Thr Asn
20 25
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<212>PRT
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1 5 10 15
Pro Asp Val Asp
20
<210>98
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<212>PRT
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1 5 10 15
Tyr Leu Gly Thr Gly Pro Glu Ala
20 24
<210>99
<211>24
<212>PRT
<213>SARS coronavirus
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1 5 10 15
Thr Gln Ala Phe Gly Arg Arg Gly
20 24
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<211>27
<212>PRT
<213>SARS coronavirus
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Lys Lys Glu Gln Trp Asn Leu Val Ile Gly Phe Leu Phe Leu Ala Trp
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Ile Met Leu Leu Gln Phe Ala Tyr Ser Asn Arg
20 25 27
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<211>24
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1 5 10 15
lle Leu Leu Asn Val Pro Leu Arg
20 24
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20 23
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20 25
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20 24
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20 25
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Claims (8)
1、一种非典型性肺炎病毒特异蛋白质，其特征在于：具有序列Seq.ID No.1--Seq.ID No.118的氨基酸序列之一。
2、根据权利要求1所述的蛋白质相对应的抗体，其特征在于：所述的蛋白质相对应的抗体是单克隆抗体，或是多克隆抗体。
3、一种非典型性肺炎病毒特异蛋白质临床检测方法，其特征在于其具体步骤如下：
1)制备如权利要求1所述的蛋白质；
通过本领域技术人员周知的PCR扩增技术和重组DNA技术，具体如下：
A、设计相应的PCR引物，同时在所用PCR引物中添加编码相应数目氨基酸残基对应的碱基，分别从临床非典型性肺炎病毒标准株中扩增编码上述蛋白质的DNA片段；
B、将所得PCR产物导入合适的表达载体，构建重组DNA；
C、用所得重组DNA转化合适的宿主细胞，在适于目标蛋白质表达的条件下培养如此转化的宿主细胞；
D、回收并纯化目标蛋白质；
2)用纯化的如权利要求1所述的蛋白质，或如权利要求2所述的抗体包被一种固相支持物；
3)用比色法、分光光度法或荧光法测定的酶标记如权利要求1所述的蛋白质或权利要求2所述的抗体，或权利要求2所述的抗体的第二抗体，或使用胶体金，或放射性同位素作为标记；
4)将待检测个体的生物学样本及由步骤3)标记的蛋白质加入经包被的固相支持物上，在适合形成抗原抗体免疫结合复合物的条件下孵育一段时间；
5)清洗去除任何未结合的非典型性肺炎病毒抗体；
6)定量检测孵育混合物中抗原抗体免疫复合物的存在。
4、根据权利要求3所述的一种非典型性肺炎病毒特异蛋白质临床检测方法，其特征在于：所用的标记酶包括氧化还原酶类，该氧化还原酶为催化酶、过氧化物酶、辣根过氧化物酶、葡萄糖氧化酶或β-半乳糖苷酶；碱性磷酸酶；乙酰胆碱酯酶。
5、根据权利要求3所述的非典型性肺炎病毒特异蛋白质临床检测方法，其特征在于：所用的第二抗体是IgG和/或IgM和/或IgA。
6、根据权利要求3所述的非典型性肺炎病毒特异蛋白质临床检测方法，其特征在于：所述的固相支持物包括本领域周知的有机和无机多聚物，或用胶体金的相关技术包被到试纸上。
7、根据权利要求6所述的一种非典型性肺炎病毒特异蛋白质临床检测方法，其特征在于：所述的有机多聚物为葡聚糖、天然或经修饰的纤维素、聚乙烯、聚苯乙烯。
8、一种非典型性肺炎病毒特异蛋白质临床检测的试剂盒，其特征在于包括：
1)包被有纯化的如权利要求1所述的蛋白质的固相支持物；相应的经标记的如权利要求1所述的蛋白质和使用该试剂盒的说明；
或2)包被有纯化的如权利要求2所述的抗体的固相支持物；相应的经标记的如权利要求2所述的抗体和使用该试剂盒的说明；
或3)包被有纯化的如权利要求1所述的蛋白质的固相支持物；如权利要求3中所述的经标记的第二抗体和使用该试剂盒的说明。
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| CN 03116657 CN1216902C (zh) | 2003-04-26 | 2003-04-26 | 非典型性肺炎病毒特异蛋白质和临床检测的方法及试剂盒 |
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Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2005025407A2 (en) | 2003-09-12 | 2005-03-24 | Becton, Dickinson And Company | Assay for sars coronavirus by amplification and detection of the replicase sequence |
| EP1756146A2 (en) * | 2004-06-17 | 2007-02-28 | Becton, Dickinson and Company | Immunogenic domains of sars coronavirus |
| WO2010061919A1 (ja) * | 2008-11-28 | 2010-06-03 | 日油株式会社 | Ｓａｒｓコロナウイルスの細胞傷害性ｔ細胞エピトープペプチド及びその用途 |
| CN107290527A (zh) * | 2017-07-31 | 2017-10-24 | 南京医科大学第附属医院 | 用于检测乙酰胆碱酯酶的胶体金层析快速诊断试纸条及其制备方法 |
| CN113214374B (zh) * | 2020-02-06 | 2022-08-26 | 深圳华大基因股份有限公司 | 一种包虫病新抗原Cystatin蛋白 |
| CN112592385B (zh) * | 2020-12-02 | 2023-02-03 | 杭州百凌生物科技有限公司 | 一种人工多肽组合物、其抗体及在病理检测中的应用 |
| CN113238048B (zh) * | 2021-05-11 | 2024-03-15 | 抗码(苏州)生物科技有限公司 | 一种诊断标志物及其在区分新冠病毒感染和新冠病毒灭活疫苗接种中的应用 |
| CN114605506B (zh) * | 2022-04-08 | 2024-05-07 | 湖南大学 | 冠状病毒m蛋白胞外域多肽及其应用 |
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