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V_K)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)信息系统和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BIAcore^TM 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

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。

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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