CN113621058B - 黄热病毒抗体及其应用 - Google Patents

黄热病毒抗体及其应用 Download PDF

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CN113621058B
CN113621058B CN202110955433.2A CN202110955433A CN113621058B CN 113621058 B CN113621058 B CN 113621058B CN 202110955433 A CN202110955433 A CN 202110955433A CN 113621058 B CN113621058 B CN 113621058B
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高福
严景华
李燕
仵丽丽
王奇慧
马素芳
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Abstract

本发明公开了黄热病毒抗体，包括重链可变区和轻链可变区，其中所述重链可变区及轻链可变区的氨基酸序列如序列表中所示。还公开了编码所述重链可变区和轻链可变区的核苷酸酸序列以及本发明抗体在制备用于检测、治疗和/或预防黄热病毒药物或试剂上的应用、及包含本发明抗体的药物组合物或试剂。该抗体对于黄热病毒具有高亲和力和特异性。

Description

黄热病毒抗体及其应用
技术领域
本发明涉及生物医药技术领域，具体涉及黄热病毒抗体及其应用。
背景技术
黄热病毒(YFV)是以伊蚊为媒介在虫媒传染病毒，与寨卡病毒(ZIKV)、西尼罗河病毒(WNV)和登革热病毒(DENV)等同属黄病毒科黄病毒属。黄热病毒的传播媒介是蚊子，因此，在蚊子流行地区和发展中国家中黄热病的暴发率很高。当前主要流行于非洲和南美洲的热带、亚热带地区。黄热病毒可引起严重危害人类健康的黄热病，表现为黄疸，出血、甚至多***的器官衰竭。因黄热病症状与同科病毒引起的疾病症状类似，且同一地区还可能同时流行黄热病毒和其它黄病毒，因此在临床上凭借临床症状难以鉴别。此外，由于黄病毒科的病毒在抗原性上具有相似的特征，抗体可能会具有交叉反应性。而且，登革病毒还会引起ADE(抗体依赖的增强作用)，这些情况使得临床诊断更为复杂。以上问题也对开发黄热病的血清学诊断方法提出了巨大挑战。
诊断黄热病毒感染的实验室方法主要包括病毒分离、核酸检测、抗原检测和血清学检测等。目前最可靠的诊断方法主要是提取病毒RNA后进行qRT-PCR，该方法费时、成本高昂且需要在特定的实验室由专业人员进行操作，使用的仪器也较复杂，无法广泛便捷的应用。因此，迫切需要开发一种操作简单、可在感染早期即可检测产品和方法，且课在偏远地区使用。
黄热病毒的非结构蛋白1(nonstructural protein 1，NS1)是比较保守的糖蛋白，有膜型和分泌型两种形式，在感染机体中表达量高且时间长，是检测黄热病毒感染的良好靶点。
发明内容
为解决上述技术问题，本发明的目的在于提供一种黄热病毒抗体，能与黄热病毒NS1 蛋白结合，具有较高的亲和力和特异性，以及较好的稳定性。
一种黄热病毒抗体，所述抗体包含如下(1)-(5)中任一氨基酸序列：
(1)如氨基酸序列SEQ ID NO：1所示的氨基酸重链可变区，如氨基酸序列SEQ IDNO：2所示的氨基酸轻链可变区；
(2)如氨基酸序列SEQ ID NO：3所示的氨基酸重链可变区，如氨基酸序列SEQ IDNO：4所示的氨基酸轻链可变区；
(3)如氨基酸序列SEQ ID NO：5所示的氨基酸重链可变区，如氨基酸序列SEQ IDNO：6所示的氨基酸轻链可变区；
(4)如氨基酸序列SEQ ID NO：7所示的氨基酸重链可变区，如氨基酸序列SEQ IDNO：8所示的氨基酸轻链可变区；
(5)如氨基酸序列SEQ ID NO：9所示的氨基酸重链可变区，如氨基酸序列SEQ IDNO：10所示的氨基酸轻链可变区。
以上所述的抗体，其中，所述氨基酸序列SEQ ID NO：11所示的抗体重链恒定区，如氨基酸序列SEQ ID NO：12所示的抗体轻链恒定区。
以上所述的抗体，其中，所述抗体重链恒定区的编码核苷酸序列如SEQ ID NO：13所示，所述抗体轻链恒定区的编码核苷酸序列如SEQ ID NO：14所示。
以上所述的抗体，其中，所述抗体还包括信号肽序列，所述信号肽的氨基酸序列如SEQ ID NO：15所示，所述信号肽序列的编码核苷酸序列如SEQ ID NO：16所示。
以上所述的抗体，其中，所述氨基酸轻链均为K型轻链。
本发明的一种核酸分子，其中，所述核酸分子包含编码以上所述抗体的核苷酸序列，所述核苷酸序列如下：
(1)如核苷酸序列SEQ ID NO：17所示的氨基酸重链可变区，如核苷酸序列SEQ IDNO：18所示的氨基酸轻链可变区；
(2)如核苷酸序列SEQ ID NO：19所示的氨基酸重链可变区，如核苷酸序列SEQ IDNO：20所示的氨基酸轻链可变区；
(3)如核苷酸序列SEQ ID NO：21所示的氨基酸重链可变区，如核苷酸序列SEQ IDNO：22所示的氨基酸轻链可变区；
(4)如核苷酸序列SEQ ID NO：23所示的氨基酸重链可变区，如核苷酸序列SEQ IDNO：24所示的氨基酸轻链可变区；
(5)如核苷酸序列SEQ ID NO：25所示的氨基酸重链可变区，如核苷酸序列SEQ IDNO：26所示的氨基酸轻链可变区。
本发明还包含以上所述核酸分子的表达载体。
本发明还包含以上所述表达载体的宿主细胞。
本发明的一种药物组合物，其中，包含以上任一所述的抗体。
本发明的一种检测试剂，其中，包含以上权利要求中任一所述的抗体。
本发明的一种检测试剂盒，其中，包含以上权利要求中任一所述的抗体。
以上任一所述抗体在制备检测、治疗和/或预防黄热病毒药物、试剂或试剂盒上的应用。
有益效果
本发明提供的黄热病毒抗体可以特异性识别黄热病毒，与黄病毒科的其他病毒无交叉反应，本发明的抗体与黄热病毒具有高亲和力，对于黄热病毒的检测以及检测产品的制备都具有较高的应用价值。
附图说明
图1为黄热病毒YFV-NS1蛋白的亲和层析及SDS-PAGE鉴定结果图；
图2为寨卡病毒ZIKV-NS1蛋白的亲和层析及SDS-PAGE鉴定结果图；
图3为血清型登革热病毒DV1-NS1/DV2-NS1/DV3-NS1/DV4-NS1蛋白的亲和层析及SDS-PAGE鉴定结果图；
图4为西尼罗病毒WN-NS1蛋白的亲和层析及SDS-PAGE鉴定结果图；
图5为抗体蛋白通过Western Blotting和SDS-PAGE鉴定的结果图；
图6为测定黄热病毒抗体的结合活性的结果图；
图7为测定黄热病毒抗体亲和力的结果图。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
实施例1人源抗体的制备
一、黄热病毒非结构蛋白l(YFV-NS1)的表达与纯化
YFV-NS1的胞外区基因片段通过Nde I和Xho I克隆至pFastBac I载体中，转化至大肠杆菌DH10，获得重组质粒Bacmid-NS1。然后，转染sf9细胞，获得含有NS1基因的杆状病毒。杆状病毒感染Hi5细胞后，可分泌表达NS1蛋白至培养上清。通过亲和层析和分子筛层析的方法进行纯化，通过SDS-PAGE鉴定蛋白纯度。结果显示，NS1蛋白存在单体和二聚体形式，单体大小为43kDa，如SEQ ID NO：27所示，结果如图1。将YFV-NS1 浓缩至1mg/mL，冷冻保存于-80℃备用。
二、其他黄病毒科NS1蛋白的表达与纯化
其他黄病毒属病毒：西尼罗病毒WNV-NS1(WNV-NS1单体蛋白，大小为92kDa，如SEQID NO：28所示，如图2所示)、寨卡病毒ZIKV-NS1(ZIKV-NS1单体蛋白，90kDa，如SEQ ID NO：29所示，如图3所示)、四种血清型登革热病毒DENV-NS1(DV1-NS1/ DV2-NS1/DV3-NS1/DV4-NS1单体蛋白，大小分别为92kDa/86kDa/43kDa/43kDa，如SEQ ID NO：30-33所示，如图4所示)蛋白表达纯化过程参考上述YFV-NS1蛋白表达纯化方法。
三、人源单克隆抗体的制备
1单细胞分选和克隆构建
取得康复者知情同意的情况下，采集两名感染YFV 6个月的康复者的外周血样本，分离外周血单核细胞(PBMCs)，加入各类血细胞特异性标记抗体和特异性YFV-NS1抗原一起孵育。经FACSAria II(BD Biosciences)分选，收集YFV-NS1阳性的记忆型B 细胞至96孔板中，每孔1个细胞。
我们通过RT-PCR和巢式PCR的方法从单个B细胞中扩增抗体可变区基因序列(VH和VL)。首先，将获得的B细胞通过SuperScript III reverse transcriptase试剂盒进行逆转录。获得cDNA后，采用HotStar plus酶(QIAGEN)进行巢式PCR来扩增抗体可变区序列。第一轮PCR反应条件如下：95℃ 5min；95℃ 30s，55℃(VH和VK)或50℃ (Vλ)60s，72℃ 90s，35个循环；72℃ 7min。将获得的产物作为模板进行第二轮PCR，条件如下：95℃ 5min；95℃30s，58℃(重链)/60℃(K链)/64℃(λ链)30s，72℃ 90s， 35个循环；72℃ 7min。
采用琼脂糖凝胶电泳，分离PCR产物，用凝胶提取试剂盒(CWBIO)提取分子量约为400bp的PCR条带，并将纯化后的产物进行测序。利用IMGT(internationalImMunoGeneTics)信息***和IgBlast对获得的靶序列进行分析。可变区序列与相对应的应的恒定区通过搭桥PCR连接，克隆至表达载体pCAGGS中，其中重链与λ链采用EcoRI 和XhoI酶切位点，κ链采用SacI与XhoI酶切位点。
2蛋白表达和纯化
将重链和轻链构建质粒转染HEK-293T细胞。转染6小时后用更换为新鲜的无血清的培养基进行培养。第3天和第7天，收集细胞培养上清。将收集的上清与含有20mM磷酸钠(pH 7.0)的缓冲液等体积混合，6500rpm离心30min去掉碎片，经过0.22um的滤膜过滤后，上清液流过HiTrap Protein A亲和层析柱，然后使用0.1M甘氨酸(pH 3.0)洗脱结合的抗体。收集洗脱液浓缩后进行分子筛层析。目的峰通过SDS-PAGE，分离出11种抗体，重链约为45kDa，轻链约为25kDa，说明目标蛋白得到正常表达。
实施例2抗体的功能鉴定
一、ELISA检测抗体特异性结合YFV-NS11)使用包被液稀释抗原2μg/mL(YFV-NS1、YFV-NS1 C terminal、WNV-NS1、ZIKV-NS1、DENV1-NS1、DENV2-NS1、DENV3-NS1、 DENV4-NS1)，加速至酶标板中，100μL/孔，4℃过夜。
2)第二天，加入PBST200μL/孔，洗板机(BIO-TEK，405LS)洗板5次后，加入 5％的脱脂奶粉封闭液，100μL/孔，37℃孵箱孵育l小时。
3)PBST漂洗5次，加入抗体YB5、YB17、YB19、YB33或YD30，400ng/孔，37℃孵箱孵育1小时。
4)PBST漂洗5次，将HPR标记的羊抗人IgG，100μL/孔，37℃孵箱孵育45min。
5)PBST漂洗5次，加入TMB显色液，100μL/孔，室温避光，反应2分钟，之后加入2MH2SO4终止反应，100μL/孔。
6)检测450nm波长处的OD值，保存记录原始数据。
结果如图6所示，抗体YB5、YB17、YB19、YB33和YD30均为YFV的特异性单抗；与其他黄病毒科NS1蛋白均无结合。
二、表面等离子共振技术(SPR)检测抗体与YFV-NS1的结合能力
此项实验采用BIAcoreTM 8K机器完成。具体步骤如下：
首先，将抗人的二抗以氨基偶联的方式固定在CM5芯片的通道(flow cell，Fc)。固定量控制在10,000响应值(response units，RU)左右。以抗体捕获的方式固定YB5、YB17、YB19、 YB33或YD30，捕获量在60RU左右。缓冲液(10mM HEPES，150mM NaCl，pH7.4) 倍比稀释YFV-NS1蛋白，依次流过各通道，流速为30μL/min。利用BIAevaluation software(Biacore，Inc.)软件计算结合动力学常数。
SPR的结果显示，抗体均可以与YFV-NS1蛋白结合(图7)。得到的各相互作用的结合常数(ka)、解离常数(kd)和平衡解离常数(kD)，如表1所示。
表1
Figure BDA0003220033340000031
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
序列表
<110> 中国科学院微生物研究所
<120> 黄热病毒抗体及其应用
<130> P210072
<160> 33
<170> SIPOSequenceListing 1.0
<210> 1
<211> 124
<212> PRT
<213> 人工序列(Synthetic sequences)
<400> 1
Glu Val Gln Leu Val Gln Ser Gly Gly Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Tyr
20 25 30
Gly Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Ser Val Tyr Lys Lys Val Thr Thr Lys Tyr Ala Gln Lys
50 55 60
Phe Gln Gly Arg Val Thr Met Thr Ala Asp Thr Ser Thr Ser Thr Ala
65 70 75 80
Tyr Met Glu Leu Arg Ser Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr
85 90 95
Cys Ala Arg Ala Glu Phe Val Pro Leu Leu Ala Pro Gly Asp Phe Asp
100 105 110
Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 2
<211> 112
<212> PRT
<213> 人工序列(Synthetic sequences)
<400> 2
Glu Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Ser Val Ala Pro Gly
1 5 10 15
Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu His Ser
20 25 30
Asp Gly Lys Ala Tyr Leu Tyr Trp Phe Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Gln Leu Leu Ile Tyr Glu Ala Ser Ser Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Asp Asp Val Gly Val Tyr Tyr Cys Met Gln Cys
85 90 95
Ile Asp Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys
100 105 110
<210> 3
<211> 118
<212> PRT
<213> 人工序列(Synthetic sequences)
<400> 3
Glu Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Val Ser Gly Tyr Thr Leu Thr Glu Phe
20 25 30
Ser Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Met
35 40 45
Gly Ser Phe Asp Pro Glu Asp Gly Glu Thr Ile Tyr Val Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Arg Gly Tyr Asp Gly Leu Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 4
<211> 107
<212> PRT
<213> 人工序列(Synthetic sequences)
<400> 4
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Asn Asn
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Arg Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Pro Gly Ser Gly Thr Tyr Phe Thr Leu Thr Ile Thr Gly Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Thr Tyr Ser Thr Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 5
<211> 125
<212> PRT
<213> 人工序列(Synthetic sequences)
<400> 5
Glu Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ser
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Asp Thr Phe Ser Ser Tyr
20 25 30
Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Arg Ile Ile Pro Val Leu Val Thr Pro Asn Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Lys Ile Pro Val Ala Gly Arg Pro Leu Tyr Tyr Asp Ala Phe
100 105 110
Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser
115 120 125
<210> 6
<211> 107
<212> PRT
<213> 人工序列(Synthetic sequences)
<400> 6
Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Val Pro Lys Leu Leu Ile
35 40 45
Ser Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Val Ala Thr Tyr Tyr Cys Gln Lys Tyr Asn Ser Ala Pro Asn
85 90 95
Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys
100 105
<210> 7
<211> 119
<212> PRT
<213> 人工序列(Synthetic sequences)
<400> 7
Gln Val Gln Leu Gln Glu Ser Gly Ala Gly Leu Leu Lys Pro Ser Glu
1 5 10 15
Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr
20 25 30
Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Asn His Thr Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys
50 55 60
Ser Arg Leu Thr Ile Ser Val Asp Arg Ser Lys Asn Gln Phe Ser Leu
65 70 75 80
Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Phe Tyr Cys Ala
85 90 95
Arg Trp Gly Tyr Thr Ser Gly Trp Tyr Ala Asp Ser Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 8
<211> 107
<212> PRT
<213> 人工序列(Synthetic sequences)
<400> 8
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Arg Trp
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln Asn Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Ala Gly Phe Pro Arg
85 90 95
Ala Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 9
<211> 122
<212> PRT
<213> 人工序列(Synthetic sequences)
<400> 9
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Thr
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Tyr
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Ile Ile Ser Tyr Asp Gly Arg Asn Thr His Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Pro Lys Asn Lys Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ser Glu Asp Thr Ala Phe Tyr Tyr Cys
85 90 95
Ala Arg Asp Thr Ala Ala Ala Gly Asp Trp Gly Val Phe Asp Ser Trp
100 105 110
Gly Gln Gly Thr Leu Val Ile Val Ser Ser
115 120
<210> 10
<211> 108
<212> PRT
<213> 人工序列(Synthetic sequences)
<400> 10
Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Thr Tyr Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Phe Asp Ser Ser Asn Leu Glu Pro Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ala Asn Val Pro Pro
85 90 95
Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 11
<211> 330
<212> PRT
<213> 人工序列(Synthetic sequences)
<400> 11
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
225 230 235 240
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330
<210> 12
<211> 108
<212> PRT
<213> 人工序列(Synthetic sequences)
<400> 12
Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu
1 5 10 15
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
20 25 30
Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln
35 40 45
Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
50 55 60
Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu
65 70 75 80
Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser
85 90 95
Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys Ser
100 105
<210> 13
<211> 993
<212> DNA
<213> 人工序列(Synthetic sequences)
<400> 13
gccagcacca aaggcccgag cgtgtttccg ctggcgccga gcagcaaaag caccagcggc 60
ggcaccgcgg cgctgggctg cctggtgaaa gattattttc cggaaccggt gaccgtgagc 120
tggaacagcg gcgcgctgac cagcggcgtg catacctttc cggcggtgct gcagagcagc 180
ggcctgtata gcctgagcag cgtggtgacc gtgccgagca gcagcctggg cacccagacc 240
tatatttgca acgtgaacca taaaccgagc aacaccaaag tggataaacg cgtggagccc 300
aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360
ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480
tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540
agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660
aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 720
ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 780
gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840
ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 900
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960
cagaagagcc tctccctgtc tccgggtaaa tga 993
<210> 14
<211> 327
<212> DNA
<213> 人工序列(Synthetic sequences)
<400> 14
cgaactgtgg ctgcaccatc tgtgttcatc ttccctccca gcgacgagca gctgaagagc 60
ggcaccgcca gcgtggtctg tctcctgaac aacttctatc ccagggaggc caaggtccag 120
tggaaagtgg acaacgccct gcaaagcggc aatagccagg agtccgtcac agagcaggac 180
agcaaggaca gcacctacag cctgtccagc accctgaccc tcagcaaggc cgactacgag 240
aagcacaagg tgtacgcttg cgaggtgacc catcagggcc tgtccagccc cgtgaccaag 300
tccttcaaca ggggcgaatg cagctaa 327
<210> 15
<211> 21
<212> PRT
<213> 人工序列(Synthetic sequences)
<400> 15
Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro
1 5 10 15
Gly Ser Thr Gly Asp
20
<210> 16
<211> 63
<212> DNA
<213> 人工序列(Synthetic sequences)
<400> 16
atggagacgg atacgctgct cctgtgggtt ttgctgctgt gggttccagg ttccactggt 60
gac 63
<210> 17
<211> 372
<212> DNA
<213> 人工序列(Synthetic sequences)
<400> 17
gaggtgcagc tggtgcagtc tggaggtgag gtgaagaagc ctggggcctc agtgaaggtc 60
tcctgcaagg cttctggtta cacctttagc agttatggaa tcagctgggt gcgacaggcc 120
cctggacaag ggcttgagtg gatgggatgg atcagcgttt ataaaaaagt taccacaaaa 180
tatgcacaga agttccaggg cagagtcacc atgaccgcag acacatccac gagcacagcg 240
tacatggaac tgaggagcct gagatctgac gacacggccg tgtattattg tgcgagagca 300
gaatttgtcc cacttttagc ccccggggac tttgactact ggggccaggg aaccctggtc 360
accgtctcct ca 372
<210> 18
<211> 336
<212> DNA
<213> 人工序列(Synthetic sequences)
<400> 18
gaaattgtgt tgacgcagtc tccactctct ctgtccgtcg cccctggaca gccggcctcc 60
atctcctgca agtctagtca gagcctcctg catagtgatg gaaaggccta tttgtattgg 120
ttcttgcaga agccaggcca gtctccacaa ctcctaatct atgaagcttc gagccggttc 180
tctggagtgc cagataggtt cactggcagc gggtcaggga cagatttcac actgaaaatc 240
agccgggtgg aggctgacga tgttggagtt tattattgca tgcaatgtat agaccttcct 300
ctcactttcg gcggagggac caaggtggag atcaaa 336
<210> 19
<211> 354
<212> DNA
<213> 人工序列(Synthetic sequences)
<400> 19
gaggtgcagc tggtggagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 60
tcctgcaagg tttccggata caccctcact gaattttcca tacactgggt gcgacaggct 120
cctggaaaag ggcttgagtg gatgggaagt tttgatcctg aagatggtga aacaatctac 180
gtacagaagt tccagggcag agttaccatg accgcggaca catctacaga cacagcctac 240
atggagctga gcagcctgag atctgaggac acggccgtgt attattgtgc aagaagacgt 300
ggctacgatg ggctggacta ctggggccag ggaaccctgg tcaccgtctc ctca 354
<210> 20
<211> 321
<212> DNA
<213> 人工序列(Synthetic sequences)
<400> 20
gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgttggaga cagagtcacc 60
atcacttgcc gggcaagtca gagcattagc aacaacttaa attggtatca gcagaaacca 120
gggagagccc ctaagttgct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180
aggttcagtg gccctggatc tgggacatat ttcactctca ccatcaccgg tctgcaacct 240
gaagattttg caacttacta ctgtcaacag acttacagta ccccgtacac ttttggccag 300
gggaccaagc tggagatcaa a 321
<210> 21
<211> 375
<212> DNA
<213> 人工序列(Synthetic sequences)
<400> 21
gaggttcagc tggtggagtc tggggctgag gtgaagaagc ctgggtcctc ggtgaaggtc 60
tcctgcaagg cttctggaga caccttcagc agctatgcta tcagctgggt gcggcaggcc 120
cctggacaag ggcttgagtg gatgggaagg atcatccctg tccttgttac accaaactac 180
gcacagaagt tccagggcag agtcacgata accgcggaca aatccacgag cacagcctac 240
atggagctga gcagcctgac atctgaggac acggccgtgt attattgtgc gactaagata 300
ccagtggctg gcagaccact ttactatgat gcttttgata tctggggcca agggacaatg 360
gtcaccgtct cctca 375
<210> 22
<211> 321
<212> DNA
<213> 人工序列(Synthetic sequences)
<400> 22
gacatccagt tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60
atcacttgcc gggcgagtca gggcattagg aattatttag cctggtatca gcagaaacca 120
gggaaagttc ctaaactcct gatctctgct gcatccactt tgcaatcagg ggtcccatct 180
cggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240
gaagatgttg caacttatta ctgtcaaaag tataacagtg ccccgaacac tttcggccct 300
gggaccaaag tggatatcaa a 321
<210> 23
<211> 357
<212> DNA
<213> 人工序列(Synthetic sequences)
<400> 23
caggtgcagc tgcaggagtc gggcgcagga ctgttgaagc cttcggagac cctgtccctc 60
acctgcgctg tctatggtgg gtccttcagt ggttactact ggagctggat ccgccagccc 120
ccagggaagg ggctggagtg gattggggaa atcaatcata ctggaagcac caactacaac 180
ccgtccctca agagtcgact caccatatca gtagacaggt ccaagaacca attctccctg 240
aagctgagct ctgtgaccgc cgcggacacg gctgtgtttt actgtgcgag atgggggtat 300
accagtggct ggtacgctga ctcctggggc cagggaaccc tggtcaccgt ctcctca 357
<210> 24
<211> 321
<212> DNA
<213> 人工序列(Synthetic sequences)
<400> 24
gacatccaga tgacccagtc tccatcttcc gtgtctgcat ctgtaggaga cagagtcacc 60
atcacttgtc gggcgagtca gggtattagc aggtggttag cctggtatca gcagaaacca 120
gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaaatgg ggtcccatca 180
aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240
gaagattttg caacttacta ttgtcaacag ggtgccggtt tccctcgggc gttcggccaa 300
gggaccaagg tggaaatcaa a 321
<210> 25
<211> 366
<212> DNA
<213> 人工序列(Synthetic sequences)
<400> 25
gaggttcagc tggtggagtc tgggggaggc gtggtccagc ctgggacgtc cctgagactc 60
tcctgtgcag cctctggatt caccttccgt agctatggca tgcactgggt ccgccaggct 120
cccggcaagg ggctggagtg ggtggcaatt atatcatatg atggaagaaa tacacactac 180
gcagattcag tgaagggccg attcaccatc tccagagaca atcccaagaa caagttgtat 240
ctgcaaatga acagcctgag aagtgaggac acggcttttt attactgtgc gagagatact 300
gcagcagctg gcgattgggg ggtctttgac tcctggggcc agggaaccct ggtcatcgtc 360
tcctca 366
<210> 26
<211> 324
<212> DNA
<213> 人工序列(Synthetic sequences)
<400> 26
gacatccagt tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60
atcacttgcc aggcgagtca ggacattacc tactatttga attggtatca gcagaaacca 120
gggaaagccc ctaagctcct gatctttgat tcatccaatt tggaaccagg ggtcccatca 180
aggttcagtg gaagtggatc tggaacagat tttactttca ccatcagcag cctgcagcct 240
gaagatattg caacatatta ctgtcaacag tatgctaatg tccctccgta cacttttggc 300
caggggacca aactggagat caaa 324
<210> 27
<211> 351
<212> PRT
<213> 人工序列(Synthetic sequences)
<400> 27
Gln Gly Cys Ala Val Asn Phe Gly Lys Arg Glu Leu Lys Cys Gly Asp
1 5 10 15
Gly Ile Phe Val Phe Arg Asp Ser Asp Asp Trp Leu Thr Lys Tyr Ser
20 25 30
Tyr Tyr Pro Glu Asp Pro Val Lys Leu Ala Ser Ile Ile Lys Ala Ser
35 40 45
His Glu Glu Gly Lys Cys Gly Leu Asn Ser Val Asp Ser Leu Glu His
50 55 60
Glu Met Trp Arg Ser Arg Ala Asp Glu Ile Asn Ala Ile Phe Glu Glu
65 70 75 80
Asn Glu Val Asp Ile Ser Val Val Val Gln Asp Pro Lys Asn Ile Tyr
85 90 95
Gln Arg Gly Thr His Pro Phe Ser Arg Ile Arg Asp Gly Leu Gln Tyr
100 105 110
Gly Trp Lys Thr Trp Gly Lys Asn Leu Val Phe Ser Pro Gly Arg Lys
115 120 125
Asn Gly Ser Phe Ile Ile Asp Gly Lys Ser Arg Lys Glu Cys Pro Phe
130 135 140
Ser Asn Arg Val Trp Asn Ser Phe Gln Ile Glu Glu Phe Gly Met Gly
145 150 155 160
Val Phe Thr Thr Arg Val Phe Met Asp Ala Thr Phe Asp Tyr Ser Val
165 170 175
Asp Cys Asp Gly Ala Ile Leu Gly Ala Ala Val Asn Gly Lys Lys Ser
180 185 190
Ala His Gly Ser Pro Thr Phe Trp Met Gly Ser His Glu Val Asn Gly
195 200 205
Thr Trp Met Ile His Thr Leu Glu Thr Leu Asp Tyr Lys Glu Cys Glu
210 215 220
Trp Pro Leu Thr His Thr Ile Gly Thr Ser Val Glu Glu Ser Asp Met
225 230 235 240
Phe Met Pro Arg Ser Ile Gly Gly Pro Val Ser Ser His Asn Arg Ile
245 250 255
Pro Gly Tyr Lys Val Gln Thr Asn Gly Pro Trp Met Gln Val Pro Leu
260 265 270
Glu Val Lys Arg Glu Val Cys Pro Gly Thr Ser Val Val Val Asp Ser
275 280 285
Asn Cys Asp Gly Arg Gly Lys Ser Thr Arg Ser Thr Thr Asp Ser Gly
290 295 300
Lys Ile Ile Pro Glu Trp Cys Cys Arg Ser Cys Thr Met Pro Pro Val
305 310 315 320
Ser Phe His Gly Ser Asp Gly Cys Trp Tyr Pro Met Glu Ile Arg Pro
325 330 335
Met Lys Thr Ser Asp Ser His Leu Val Arg Ser Trp Val Thr Ala
340 345 350
<210> 28
<211> 351
<212> PRT
<213> 人工序列(Synthetic sequences)
<400> 28
Thr Gly Cys Ala Ile Asp Ile Ser Arg Gln Glu Leu Arg Cys Gly Ser
1 5 10 15
Gly Val Phe Ile His Asn Asp Val Glu Ala Trp Met Asp Arg Tyr Lys
20 25 30
Tyr Tyr Pro Glu Thr Pro Gln Gly Leu Ala Lys Ile Ile Gln Lys Ala
35 40 45
His Lys Glu Gly Val Cys Gly Leu Arg Ser Val Ser Arg Leu Glu His
50 55 60
Gln Met Trp Glu Ala Val Lys Asp Glu Leu Asn Thr Leu Leu Lys Glu
65 70 75 80
Asn Gly Val Asp Leu Ser Val Val Val Glu Lys Gln Glu Gly Met Tyr
85 90 95
Lys Ser Ala Pro Lys Arg Leu Thr Ala Thr Thr Glu Lys Leu Glu Ile
100 105 110
Gly Trp Lys Ala Trp Gly Lys Ser Ile Leu Phe Ala Pro Glu Leu Ala
115 120 125
Asn Asn Thr Phe Val Val Asp Gly Pro Glu Thr Lys Glu Cys Pro Thr
130 135 140
Gln Asn Arg Ala Trp Asn Ser Leu Glu Val Glu Asp Phe Gly Phe Gly
145 150 155 160
Leu Thr Ser Thr Arg Met Phe Leu Lys Val Arg Glu Ser Asn Thr Thr
165 170 175
Glu Cys Asp Ser Lys Ile Ile Gly Thr Ala Val Lys Asn Asn Leu Ala
180 185 190
Ile His Ser Asp Leu Ser Tyr Trp Ile Glu Ser Arg Leu Asn Asp Thr
195 200 205
Trp Lys Leu Glu Arg Ala Val Leu Gly Glu Val Lys Ser Cys Thr Trp
210 215 220
Pro Glu Thr His Thr Leu Trp Gly Asp Gly Ile Leu Glu Ser Asp Leu
225 230 235 240
Ile Ile Pro Val Thr Leu Ala Gly Pro Arg Ser Asn His Asn Arg Arg
245 250 255
Pro Gly Tyr Lys Thr Gln Asn Gln Gly Pro Trp Asp Glu Gly Arg Val
260 265 270
Glu Ile Asp Phe Asp Tyr Cys Pro Gly Thr Thr Val Thr Leu Ser Glu
275 280 285
Ser Cys Gly His Arg Gly Pro Ala Thr Arg Thr Thr Thr Glu Ser Gly
290 295 300
Lys Leu Ile Thr Asp Trp Cys Cys Arg Ser Cys Thr Leu Pro Pro Leu
305 310 315 320
Arg Tyr Gln Thr Asp Ser Gly Cys Trp Tyr Gly Met Glu Ile Arg Pro
325 330 335
Gln Arg His Asp Glu Lys Thr Leu Val Gln Ser Gln Val Asn Ala
340 345 350
<210> 29
<211> 351
<212> PRT
<213> 人工序列(Synthetic sequences)
<400> 29
Val Gly Cys Ser Val Asp Phe Ser Lys Lys Glu Thr Arg Cys Gly Thr
1 5 10 15
Gly Val Phe Val Tyr Asn Asp Val Glu Ala Trp Arg Asp Arg Tyr Lys
20 25 30
Tyr His Pro Asp Ser Pro Arg Arg Leu Ala Ala Ala Val Lys Gln Ala
35 40 45
Trp Glu Asp Gly Ile Cys Gly Ile Ser Ser Val Ser Arg Met Glu Asn
50 55 60
Ile Met Trp Arg Ser Val Glu Gly Glu Leu Asn Ala Ile Leu Glu Glu
65 70 75 80
Asn Gly Val Gln Leu Thr Val Val Val Gly Ser Val Lys Asn Pro Met
85 90 95
Trp Arg Gly Pro Gln Arg Leu Pro Val Pro Val Asn Glu Leu Pro His
100 105 110
Gly Trp Lys Ala Trp Gly Lys Ser Tyr Phe Val Arg Ala Ala Lys Thr
115 120 125
Asn Asn Ser Phe Val Val Asp Gly Asp Thr Leu Lys Glu Cys Pro Leu
130 135 140
Lys His Arg Ala Trp Asn Ser Phe Leu Val Glu Asp His Gly Phe Gly
145 150 155 160
Val Phe His Thr Ser Val Trp Leu Lys Val Arg Glu Asp Tyr Ser Leu
165 170 175
Glu Cys Asp Pro Ala Val Ile Gly Thr Ala Val Lys Gly Lys Glu Ala
180 185 190
Val His Ser Asp Leu Gly Tyr Trp Ile Glu Ser Glu Lys Asn Asp Thr
195 200 205
Trp Arg Leu Lys Arg Ala His Leu Ile Glu Met Lys Thr Cys Glu Trp
210 215 220
Pro Lys Ser His Thr Leu Trp Thr Asp Gly Ile Glu Glu Ser Asp Leu
225 230 235 240
Ile Ile Pro Lys Ser Leu Ala Gly Pro Leu Ser His His Asn Thr Arg
245 250 255
Glu Gly Tyr Arg Thr Gln Met Lys Gly Pro Trp His Ser Glu Glu Leu
260 265 270
Glu Ile Arg Phe Glu Glu Cys Pro Gly Thr Lys Val His Val Glu Glu
275 280 285
Thr Cys Gly Thr Arg Gly Pro Ser Leu Arg Ser Thr Thr Ala Ser Gly
290 295 300
Arg Val Ile Glu Glu Trp Cys Cys Arg Glu Cys Thr Met Pro Pro Leu
305 310 315 320
Ser Phe Arg Ala Lys Asp Gly Cys Trp Tyr Gly Met Glu Ile Arg Pro
325 330 335
Arg Lys Glu Pro Glu Ser Asn Leu Val Arg Ser Met Val Thr Ala
340 345 350
<210> 30
<211> 351
<212> PRT
<213> 人工序列(Synthetic sequences)
<400> 30
Ser Gly Cys Val Ile Asn Trp Lys Gly Arg Glu Leu Lys Cys Gly Ser
1 5 10 15
Gly Ile Phe Val Thr Asn Glu Val His Thr Trp Thr Glu Gln Tyr Lys
20 25 30
Phe Gln Ala Asp Ser Pro Lys Arg Leu Ser Ala Ala Ile Gly Lys Ala
35 40 45
Trp Glu Glu Gly Val Cys Gly Ile Arg Ser Ala Thr Arg Leu Glu Asn
50 55 60
Ile Met Trp Lys Gln Ile Ser Asn Glu Leu Asn His Ile Leu Leu Glu
65 70 75 80
Asn Asp Met Lys Leu Thr Val Val Val Gly Asp Val Thr Gly Ile Leu
85 90 95
Ala Gln Gly Lys Lys Met Ile Arg Pro Gln Pro Met Glu His Lys Tyr
100 105 110
Ser Trp Lys Ser Trp Gly Lys Ala Lys Ile Thr Gly Ala Asp Val Gln
115 120 125
Asn Thr Thr Phe Ile Ile Asp Gly Pro Asn Thr Pro Glu Cys Pro Asp
130 135 140
Asn Gln Arg Ala Trp Asn Ile Trp Glu Val Glu Asp Tyr Gly Phe Gly
145 150 155 160
Val Phe Thr Thr Asn Ile Trp Leu Lys Leu Arg Asp Ser Tyr Thr Gln
165 170 175
Val Cys Asp His Arg Leu Met Ser Ala Ala Ile Lys Asp Ser Lys Ala
180 185 190
Val His Ala Asp Met Gly Tyr Trp Ile Glu Ser Glu Lys Asn Glu Thr
195 200 205
Trp Lys Leu Glu Arg Ala Ser Phe Ile Glu Val Lys Thr Cys Ile Trp
210 215 220
Pro Arg Ser His Thr Leu Trp Ser Asn Gly Val Leu Glu Ser Glu Met
225 230 235 240
Ile Ile Pro Lys Ile Tyr Gly Gly Pro Val Ser Gln His Asn Tyr Arg
245 250 255
Pro Gly Tyr Phe Thr Gln Thr Ala Gly Pro Trp His Leu Gly Lys Leu
260 265 270
Glu Leu Asp Phe Asp Leu Cys Glu Gly Thr Thr Val Val Val Asp Glu
275 280 285
Asn Cys Gly Asn Arg Gly Pro Ser Leu Arg Thr Thr Thr Val Thr Gly
290 295 300
Lys Thr Ile His Glu Trp Cys Cys Arg Ser Cys Thr Leu Pro Pro Leu
305 310 315 320
Arg Phe Lys Gly Glu Asp Gly Cys Trp Tyr Gly Met Glu Ile Arg Pro
325 330 335
Val Lys Glu Lys Glu Glu Asn Leu Val Lys Ser Met Val Ser Ala
340 345 350
<210> 31
<211> 351
<212> PRT
<213> 人工序列(Synthetic sequences)
<400> 31
Ser Gly Cys Val Val Ser Trp Lys Asn Lys Glu Leu Lys Cys Gly Ser
1 5 10 15
Gly Ile Phe Ile Thr Asp Asn Val His Thr Trp Thr Glu Gln Tyr Lys
20 25 30
Phe Gln Pro Glu Ser Pro Ser Lys Leu Ala Ser Ala Ile Gln Lys Ala
35 40 45
His Glu Glu Gly Ile Cys Gly Ile Arg Ser Val Thr Arg Leu Glu Asn
50 55 60
Leu Met Trp Lys Gln Ile Thr Pro Glu Leu Asn His Ile Leu Ser Glu
65 70 75 80
Asn Glu Val Lys Leu Thr Ile Met Thr Gly Asp Ile Lys Gly Ile Met
85 90 95
Gln Ala Gly Lys Arg Ser Leu Arg Pro Gln Pro Thr Glu Leu Lys Tyr
100 105 110
Ser Trp Lys Thr Trp Gly Lys Ala Lys Met Leu Ser Thr Glu Ser His
115 120 125
Asn Gln Thr Phe Leu Ile Asp Gly Pro Glu Thr Ala Glu Cys Pro Asn
130 135 140
Thr Asn Arg Ala Trp Asn Ser Leu Glu Val Glu Asp Tyr Gly Phe Gly
145 150 155 160
Val Phe Thr Thr Asn Ile Trp Leu Lys Leu Lys Glu Lys Gln Asp Val
165 170 175
Phe Cys Asp Ser Lys Leu Met Ser Ala Ala Ile Lys Asp Asn Arg Ala
180 185 190
Val His Ala Asp Met Gly Tyr Trp Ile Glu Ser Ala Leu Asn Asp Thr
195 200 205
Trp Lys Ile Glu Lys Ala Ser Phe Ile Glu Val Lys Asn Cys His Trp
210 215 220
Pro Lys Ser His Thr Leu Trp Ser Asn Gly Val Leu Glu Ser Glu Met
225 230 235 240
Ile Ile Pro Lys Asn Leu Ala Gly Pro Val Ser Gln His Asn Tyr Arg
245 250 255
Pro Gly Tyr His Thr Gln Ile Thr Gly Pro Trp His Leu Gly Lys Leu
260 265 270
Glu Met Asp Phe Asp Phe Cys Asp Gly Thr Thr Val Val Val Thr Glu
275 280 285
Asp Cys Gly Asn Arg Gly Pro Ser Leu Arg Thr Thr Thr Ala Ser Gly
290 295 300
Lys Leu Ile Thr Glu Trp Cys Cys Arg Ser Cys Thr Leu Pro Pro Leu
305 310 315 320
Arg Tyr Arg Gly Glu Asp Gly Cys Trp Tyr Gly Met Glu Ile Arg Pro
325 330 335
Leu Lys Glu Lys Glu Glu Asn Leu Val Asn Ser Leu Val Thr Ala
340 345 350
<210> 32
<211> 351
<212> PRT
<213> 人工序列(Synthetic sequences)
<400> 32
Met Gly Cys Val Ile Asn Trp Lys Gly Lys Glu Leu Lys Cys Gly Asn
1 5 10 15
Gly Ile Phe Val Thr Asn Glu Val His Thr Trp Thr Glu Gln Tyr Lys
20 25 30
Phe Gln Ala Asp Ser Pro Lys Arg Leu Ala Thr Ala Ile Ala Gly Ala
35 40 45
Trp Glu Asn Gly Val Cys Gly Ile Arg Ser Thr Thr Arg Met Glu Asn
50 55 60
Leu Leu Trp Lys Gln Ile Ala Asn Glu Leu Asn Tyr Ile Leu Trp Glu
65 70 75 80
Asn Asn Ile Lys Leu Thr Val Val Val Gly Asp Ile Thr Gly Val Leu
85 90 95
Glu Gln Gly Lys Arg Thr Leu Thr Pro Gln Pro Met Glu Leu Lys Tyr
100 105 110
Ser Trp Lys Thr Trp Gly Lys Ala Lys Ile Val Thr Ala Glu Thr Gln
115 120 125
Asn Ser Ser Phe Ile Ile Asp Gly Pro Asn Thr Pro Glu Cys Pro Ser
130 135 140
Ala Ser Arg Ala Trp Asn Val Trp Glu Val Glu Asp Tyr Gly Phe Gly
145 150 155 160
Val Phe Thr Thr Asn Ile Trp Leu Lys Leu Arg Glu Met Tyr Thr Gln
165 170 175
Leu Cys Asp His Arg Leu Met Ser Ala Ala Val Lys Asp Glu Arg Ala
180 185 190
Val His Ala Asp Met Gly Tyr Trp Ile Glu Ser Gln Lys Asn Gly Ser
195 200 205
Trp Lys Leu Glu Lys Ala Ser Leu Ile Glu Val Lys Thr Cys Thr Trp
210 215 220
Pro Lys Ser His Thr Leu Trp Ser Asn Gly Val Leu Glu Ser Asp Met
225 230 235 240
Ile Ile Pro Lys Ser Leu Ala Gly Pro Ile Ser Gln His Asn Tyr Arg
245 250 255
Pro Gly Tyr His Thr Gln Thr Ala Gly Pro Trp His Leu Gly Lys Leu
260 265 270
Glu Leu Asp Phe Asn Tyr Cys Glu Gly Thr Thr Val Val Ile Thr Glu
275 280 285
Asn Cys Gly Thr Arg Gly Pro Ser Leu Arg Thr Thr Thr Val Ser Gly
290 295 300
Lys Leu Ile His Glu Trp Cys Cys Arg Ser Cys Thr Leu Pro Pro Leu
305 310 315 320
Arg Tyr Met Gly Glu Asp Gly Cys Trp Tyr Gly Met Glu Ile Arg Pro
325 330 335
Ile Asn Glu Lys Glu Glu Asn Met Val Lys Ser Leu Val Ser Ala
340 345 350
<210> 33
<211> 351
<212> PRT
<213> 人工序列(Synthetic sequences)
<400> 33
Met Gly Cys Val Val Ser Trp Asn Gly Lys Glu Leu Lys Cys Gly Ser
1 5 10 15
Gly Ile Phe Val Ile Asp Asn Val His Thr Arg Thr Glu Gln Tyr Lys
20 25 30
Phe Gln Pro Glu Ser Pro Ala Arg Leu Ala Ser Ala Ile Leu Asn Ala
35 40 45
His Lys Asp Gly Val Cys Gly Val Arg Ser Thr Thr Arg Leu Glu Asn
50 55 60
Val Met Trp Lys Gln Ile Thr Asn Glu Leu Asn Tyr Val Leu Trp Glu
65 70 75 80
Gly Gly His Asp Leu Thr Val Val Ala Gly Asp Val Lys Gly Val Leu
85 90 95
Thr Glu Gly Lys Arg Ala Leu Thr Pro Pro Val Asn Asp Leu Lys Tyr
100 105 110
Ser Trp Lys Thr Trp Gly Lys Ala Lys Ile Phe Thr Leu Glu Ala Arg
115 120 125
Asn Ser Thr Phe Leu Ile Asp Gly Pro Asp Thr Ser Glu Cys Pro Asn
130 135 140
Glu Arg Arg Ala Trp Asn Phe Leu Glu Val Glu Asp Tyr Gly Phe Gly
145 150 155 160
Met Phe Thr Thr Asn Ile Trp Met Lys Phe Arg Glu Gly Ser Ser Glu
165 170 175
Val Cys Asp His Arg Leu Met Ser Ala Ala Ile Lys Asp Gln Lys Ala
180 185 190
Val His Ala Asp Met Gly Tyr Trp Ile Glu Ser Ser Lys Asn Gln Thr
195 200 205
Trp Gln Ile Glu Lys Ala Ser Leu Ile Glu Val Lys Thr Cys Leu Trp
210 215 220
Pro Lys Thr His Thr Leu Trp Ser Asn Gly Val Leu Glu Ser Gln Met
225 230 235 240
Leu Ile Pro Arg Ser Tyr Ala Gly Pro Phe Ser Gln His Asn Tyr Arg
245 250 255
Gln Gly Tyr Ala Thr Gln Thr Met Gly Pro Trp His Leu Gly Lys Leu
260 265 270
Glu Ile Asn Phe Gly Glu Cys Pro Gly Thr Thr Val Ala Ile Gln Glu
275 280 285
Asp Cys Gly His Arg Gly Pro Ser Leu Arg Thr Thr Thr Ala Ser Gly
290 295 300
Lys Leu Val Thr Gln Trp Cys Cys Arg Ser Cys Ala Met Pro Pro Leu
305 310 315 320
Arg Phe Leu Gly Glu Asp Gly Cys Trp Tyr Gly Met Glu Ile Arg Pro
325 330 335
Leu Ser Glu Lys Glu Glu Asn Met Val Lys Ser Gln Val Thr Ala
340 345 350

Claims (9)

1.一种黄热病毒抗体， 其特征在于， 所述抗体包含：
如氨基酸序列 SEQ ID NO：1 所示的氨基酸重链可变区， 如氨基酸序列 SEQ IDNO：2
所示的氨基酸轻链可变区。
2.如权利要求 1 所述的抗体，其特征在于，包含如SEQ ID NO ：11 所示的抗体重链恒定区，如SEQ ID NO：12 所示的抗体轻链恒定区。
3.如权利要求 1 所述的抗体，其特征在于，所述氨基酸轻链均为 K 型轻链。
4.一种核酸分子，其中，所述核酸分子包含编码以上所述抗体的核苷酸序列。
5.包含权利要求 4 中所述核酸分子的表达载体。
6.包含权利要求 5 中所述表达载体的宿主细胞。
7.一种检测试剂，其特征在于，包含权利要求 1 至 3 中任一所述的抗体。
8.一种检测试剂盒，其特征在于，包含权利要求 1 至 3 中任一所述的抗体。
9.权利要求 1 至 3 中任一所述抗体在制备检测试剂或试剂盒上的应用。
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FR2794865B1 (fr) * 1999-06-09 2003-04-18 Pasteur Institut Methode de detection precoce des flavivirus et ses applications
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CN102827260B (zh) * 2012-08-31 2014-05-14 任瑞文 一种检测黄热病毒抗体的重组抗原蛋白、试剂盒及其应用
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