表达非洲猪瘟病毒p72、B602L蛋白的重组腺病毒载体、重组腺病毒及构建方法
阅读说明:本技术 表达非洲猪瘟病毒p72、B602L蛋白的重组腺病毒载体、重组腺病毒及构建方法 (Recombinant adenovirus vector for expressing African swine fever virus p72 and B602L proteins, recombinant adenovirus and construction method ) 是由 周远成 邝声耀 阴文奇 于 2020-06-15 设计创作,主要内容包括:本发明公开了一种表达非洲猪瘟病毒p72、B602L蛋白的重组腺病毒载体、重组腺病毒及构建方法,该重组腺病毒感染细胞后可以同步表达非洲猪瘟p72、B602L蛋白,B602L通过自裂解2A信号肽与p72串联表达。本发明构建了能同时表达p72、B602L蛋白的重组腺病毒载体,使用该载体与腺病毒骨架系统共转染细胞后获得可表达非洲猪瘟病毒p72、B602L蛋白的重组腺病毒,使用该重组腺病毒免疫试验动物后可以刺激试验动物产生特异性的抗体,为非洲猪瘟疫苗的开发提供了新的试验数据。(The invention discloses a recombinant adenovirus vector for expressing African swine fever virus p72 and B602L proteins, a recombinant adenovirus and a construction method thereof, wherein the recombinant adenovirus can synchronously express African swine fever p72 and B602L proteins after infecting cells, and B602L is expressed in series with p72 through self-cleavage 2A signal peptide. The recombinant adenovirus vector capable of expressing the p72 and B602L proteins is constructed, the recombinant adenovirus capable of expressing the African swine fever virus p72 and B602L proteins is obtained after the vector and an adenovirus skeleton system are used for cotransfecting cells, and after the recombinant adenovirus is used for immunizing a test animal, the test animal can be stimulated to generate specific antibodies, so that new test data are provided for the development of an African swine fever vaccine.)
技术领域
本发明涉及基因工程技术领域,具体涉及一种表达非洲猪瘟病毒p72、B602L蛋白的重组腺病毒载体、重组腺病毒及构建方法。
背景技术
非洲猪瘟病毒(African swine fever virus,ASFV)是一种核质大DNA病毒,也是唯一虫媒DNA病毒,可感染家猪和野猪,具有较强的传染性和致病性。猪感染非洲猪瘟的临床症状与猪瘟和猪丹毒等疾病类似,其症状的严重程度与ASFV的毒力、感染计量和感染途径的不同而不同,根据临床症状的严重程度可以分为急性感染、亚急性感染和隐形感染等。急性感染表现为食欲减退、高热、白细胞减少、皮肤以及内脏器官的出血为主要特征,死亡几率较高,有的可达100%。亚急性感染表现为暂时性的血小板和包细胞减少,可观察到出血灶,同时可导致呼吸改变、流产和死亡,死亡率较急性感染低。到目前位置尚无针对非洲猪瘟的特效疫苗和治疗药物,因此国内外均采用强制捕杀的方式处理发病猪群。
非洲猪瘟于2018传入我国,截至目前在全国范围内报道的疫情超过150起。非洲猪瘟的爆发给我国生猪养殖业造成了巨大的经济损失。非洲猪瘟在我国出现的一年多时间内,我国能繁种猪存栏量由疫情出现前的约4000万头降低至最低峰的约1900万头,据不完全按统计,因非洲猪瘟导致的直接和间接经济损失可能超过1万亿元。非洲猪瘟爆发后,国内多个单位开展了非洲猪瘟疫苗研究,包括亚单位疫苗、多肽疫苗和基因缺失疫苗等,但截至目前尚无有效的疫苗问世。
发明内容
针对现有技术中的上述不足,本发明提供了一种表达非洲猪瘟病毒p72、B602L蛋白的重组腺病毒载体、重组腺病毒及构建方法。
为实现上述目的,本发明解决其技术问题所采用的技术方案是:
一种编码非洲猪瘟病毒p72、B602L蛋白的核苷酸序列,该核苷酸序列如SEQ IDNO:1所示。
一种表达非洲猪瘟病毒p72、B602L蛋白的重组腺病毒载体,包含SEQ ID NO:1所示的核苷酸序列。
上述表达非洲猪瘟病毒p72、B602L蛋白的重组腺病毒载体的构建方法,包括以下步骤:人工合成SEQ ID NO:1所示的核苷酸序列,并将其插入到pDC316-mCMV-EGFP载体中,制得。
一种表达非洲猪瘟病毒p72、B602L蛋白的重组腺病毒,包含SEQ ID NO:1所示的核苷酸序列或上述重组腺病毒载体。
上述表达非洲猪瘟病毒p72、B602L蛋白的重组腺病毒的构建方法,包括以下步骤:
(1)人工合成SEQ ID NO:1所示的核苷酸序列,并将其插入到pDC316-mCMV-EGFP载体中;
(2)将步骤(1)构建的载体与pBHGloxdelE13cre载体共转染293A细胞后进行培养,制得。
上述重组腺病毒可以用于制备非洲猪瘟疫苗。
本发明提供的表达非洲猪瘟病毒p72、B602L蛋白的重组腺病毒载体、重组腺病毒及构建方法,具有以下有益效果:
本发明构建了能同时表达非洲猪瘟病毒p72、B602L蛋白的重组腺病毒载体,使用该载体与腺病毒骨架系统共转染细胞后获得可表达非洲猪瘟病毒p72、B602L蛋白的重组腺病毒,使用该腺病毒免疫试验动物后可以刺激试验动物产生特异性的抗体,为非洲猪瘟疫苗的开发提供了新的试验数据。
附图说明
图1为构建pDC316-p72-B602L载体的结构图。
图2为制备的重组腺病毒形成的荧光嗜斑结果图。
具体实施方式
实施例1重组腺病毒载体的构建
表达非洲猪瘟病毒p72蛋白的氨基酸序列见SEQ ID NO:2,表达非洲猪瘟病毒B602L蛋白的氨基酸序列见SEQ ID NO:3,在p72蛋白末端添加了组氨酸标签HHHHHH,并且在添加了组氨酸标签HHHHHH的p72蛋白和B602L蛋白之间添加T2A自切割信号肽,T2A的氨基酸序列见SEQ ID NO:4。
根据上述氨基酸序列,人工设计能够表达p72和B602L蛋白的核苷酸序列,该核苷酸序列见SEQ ID NO:1,设计好的核苷酸序列由南京金斯瑞生物科技有限公司合成,将合成的序列插入到pDC316-mCMV-EGFP载体的NheⅠ和HindⅢ酶切位点之间,构建好的载体命名为pDC316-p72-B602L,其结构图见图1。
实施例2重组腺病毒的制备
按照常规方法传代293A细胞至12孔板,传代后第二天使用转染试剂(HD,购于Promega公司)转染pDC316-p72-B602L质粒和pBHGloxdelE13cre质粒,转染试剂使用6ul,pDC316-p72-B602L质粒和pBHGloxdelE13cre质粒各1ug。转染后将细胞置37℃,5%二氧化碳培养箱中培养。每日观察细胞状态。转染后12小时可观察到绿色荧光蛋白的表达,转然后7天观察到绿色荧光嗜斑(结果图见图2)。出现荧光斑后继续培养48小时,将细胞置-80℃反复冻融三次,12000rpm离心,-80℃保存,计为ADV p72/B602L P1代病毒。
按照常规方法传代293A细胞至96孔板中,取-80℃保存的ADV p72/B602L P1代病毒使用无血清DMEM细胞培养液进行10倍梯度稀释,取101-108稀释度的接种于加有293A细胞悬液的培养孔中,每个孔添加100ul病毒稀释液,每个稀释梯度24孔。加入病毒稀释液后置37℃,5%二氧化碳培养箱中培养每日观察。待出现绿色荧光斑后取最低稀释度单荧光斑孔病毒液接种293A进行扩大培养,培养至细胞病变至80%时收获病毒,-80℃反复冻融1次,12000rpm离心5min,取上清分装后-80℃保存,记为ADV p72/B602L P3代病毒。取ADV p72/B602L P3代病毒按照常规方法接种293A细胞继续培养扩繁至ADV p72/B602L P5代病毒,所有收获病毒均置于-80℃保存。
实施例3 ADV p72/B602L小量制备和纯化
按常规方法传代293A细胞至T175细胞瓶,待细胞生长成致密单层之后除去细胞培养液,接种0.5ml ADV p72/B602L P3代病毒,37℃细胞培养箱吸附30分钟,然后往接种后的细胞瓶中加入30ml 2%小牛血清(购于GIBCO公司)的DMEM(购于GIBCO公司)培养液。37℃,5%二氧化碳培养箱中培养。待细胞90%以上细胞出现细胞病变后收获病毒液,12000rpm离心10分钟,取上清加入到100KD超滤离心管中(购于MERCK公司),5000rpm离心至原来体积的1/10后加入0.1M磷酸盐缓冲液至原体积,5000rpm离心至原体积的1/100,最后按照5:1的比例添加甘油后分装并置于-80℃保存。
实施例4 ADV p72/B602L不同代次以及纯化病毒的病毒含量测定
按照常规方法传代293A细胞至96孔板,置37℃,5%二氧化碳培养箱中培养。取ADVp72/B602L P4、P5代病毒和纯化病毒使用2%血清DMEM进行10倍梯度稀释至1010,稀释后接种传代的293A细胞,每个稀释度接种8孔。接种后置37℃,5%二氧化碳培养箱中培养6天。将培养后的96孔板置于倒置荧光显微镜下观察各稀释度孔是否出现细胞病变和绿色荧光斑,记录各稀释度细胞绿色荧光斑和细胞病变孔数量,根据Reed-Muench方法计算ADV p72/B602L P4、ADV p72/B602L P5代和ADV p72/B602L纯化病毒的TCID50分别为109.5TCID50/ml、1010.2TCID50/ml、1011.5TCID50/ml。
实施例5 ADV p72/B602L免疫仔猪后的体液免疫效果测定
1、仔猪免疫试验
购买4周龄断奶仔猪15头,将该15头仔猪随机分为3组,取纯化后的ADV p72/B602L纯化病毒使用无血清DMEM进行稀释,然后分别注射108TCID50、109TCID50的ADV p72/B602L纯化病毒和DMEM,注射方式为颈部肌肉注射,注射量为2ml/头,分别标记为1、2、3组,然后对免疫前、免疫后2周、免疫后4周采集血清进行血清抗体的测定。
2、血清抗体测定
选择p72蛋白多肽FPENSHNIQTAGKQDC,由南京金斯瑞生物科技有限公司合成,并在碳末端C上标记BSA蛋白。取合成并偶联BSA的多肽(纯度99%),使用无菌注射用水溶解至1mg/ml,然后使用0.05M pH9.6的碳酸盐缓冲液稀释至4ug/ml。取酶标板,每孔加入100ul稀释后的多肽溶液,2-8℃包被12-16小时;去除含多肽的包被液,每孔加入0.1ml封闭液(0.15M PBS,0.05%吐温20,3%BSA,pH 7.4),37℃封闭3h;去除封闭液,使用PBST(0.15MPBS,0.05%吐温20,pH 7.4)洗涤5次;将采集的血清进行100倍稀释,取稀释后的血清每孔加入100ul,设立稀释液对照孔后室温孵育60min;去除血清,使用PBST洗涤5次;每孔加入100ul 1:5000稀释的HRP标记的山羊抗猪IgG,室温孵育30min,使用PBST洗涤5次;加入100ul TMB显色液,室温孵育15min,加入50ul 2M硫酸终止反应,测定OD450光吸收值,以测定孔与阴性对照孔A450nm>2.1判为阳性,测定结果见表1所示。
表1免疫ADV p72/B602L后抗体测定结果
由表1可知,免疫组抗体均为阳性,并且ADV p72/B602L 109TCID50组高于108TCID50组,由此说明ADV p72/B602L可以诱导仔猪产生特异性的体液免疫应答。
序列表
<110> 畜科生物工程有限公司
<120> 表达非洲猪瘟病毒p72、B602L蛋白的重组腺病毒载体、重组腺病毒及构建方法
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3600
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atggcctccg gcggcgcctt ctgcctgatc gccaatgacg gcaaggctga caagattatc 60
ctggcccagg acctgctgaa cagcaggatc agcaatatca agaacgtcaa taagagctac 120
gggaagccgg accccgagcc aaccctgagc cagatcgagg agacccacct ggtgcacttc 180
aacgcccatt tcaagcctta cgtgcccgtg gggtttgagt acaacaaggt gcgcccccac 240
accggaaccc ccaccctggg caataagctc accttcggga tcccacagta cggagacttc 300
ttccacgaca tggtggggca tcacatcctg ggagcctgcc acagcagctg gcaggacgcc 360
cccatccagg ggacatccca gatgggagcc cacggccagc tgcagacctt tccaagaaat 420
gggtacgact gggacaatca gaccccgctg gagggcgccg tgtataccct ggtggaccca 480
ttcggccgcc ccatcgtgcc tgggaccaag aacgcctacc ggaacctcgt ctactactgt 540
gagtaccccg gcgagaggct gtacgagaat gtcaggttcg atgtgaacgg gaatagcctg 600
gacgagtact ccagcgacgt gaccaccctg gtcaggaagt tttgtatccc tggcgacaaa 660
atgacaggct acaagcatct ggtgggacag gaggtgtccg tggagggaac cagcgggcct 720
ctgctgtgta atatccacga cctgcacaag ccacaccagt ctaagccgat cctgaccgat 780
gagaatgata cccagcgcac ctgctctcac acaaacccca agtttctgtc tcagcacttc 840
cctgagaata gccacaacat ccagaccgcc gggaagcagg acatcacccc catcaccgac 900
gccacctacc tggacatccg gaggaacgtg cactactcgt gcaacggccc tcagacccca 960
aagtactacc agcccccact ggctctgtgg atcaagctgc ggttctggtt caacgagaat 1020
gtgaatctgg ccattccctc tgtgagcatc cccttcgggg agcgcttcat caccataaag 1080
ctggcctccc agaaggacct ggtgaatgag ttccccggcc tgttcgtccg gcagagcaga 1140
ttcatcgccg gccgcccctc aagacgaaac attcgcttca aaccctggtt catccctggt 1200
gtgatcaatg agatcagcct gaccaacaac gagctgtata tcaacaacct gttcgtgact 1260
cctgagatcc ataatctctt cgtgaagcgg gtgcgatttt ccctcatccg ggtgcacaag 1320
acccaggtga cccacaccaa taacaaccac cacgacgaga agctgatgag cgccctgaag 1380
tggcctatcg aatacatgtt catcggcctg aagcccacct ggaacatctc cgaccagaac 1440
ccccatcagc accgggactg gcataagttc ggccacgtgg tgaacgccat catgcagcca 1500
acccaccacg ccgagattag cttccaggac agagataccg ccctgcctga cgcctgctcc 1560
agcatttccg acatcagtcc tgtgacctac cctatcaccc tgcccatcat caagaatatt 1620
agcgtgaccg cccacggaat taacctgatt gacaaattcc ccagcaaatt ctgctccagc 1680
tacatcccct tccactacgg aggcaacgcc atcaagaccc ctgacgatcc cggcgccatg 1740
atgattactt tcgccctgaa gcccagggag gagtaccagc cctctggcca catcaacgtg 1800
agccgggcta gagagttcta catcagctgg gacaccgact acgtgggatc catcacaacc 1860
gccgacctgg tggtgagcgc ttccgccatc aacttcctgc tgctgcaaaa tgggtctgcc 1920
gtgctgagat actccaccca ccaccaccat caccacgaag gcaggggctc actgctgacc 1980
tgcggggacg tggaggagaa cccaggcccc gccgagttta acatcgacga gctgctcaag 2040
aacgtgctgg aggacccatc aactgagatc tccgaggaga ccctgaagca gctctaccag 2100
cgcaccaacc catacaagca gtttaagaac gatagccggg tcgccttctg ttctttcact 2160
aatctgaggg agcagtatat ccggagactg attatgacca gctttatcgg ctacgtgttc 2220
aaggccctgc aggagtggat gccttcttac agcaagccca cccacaccac caaaaccctg 2280
ctgagcgagc tgatcaccct ggtggacacc ctgaaacagg agaccaatga cgtcccaagc 2340
gagagcgtgg tcaacacaat cctgagcatc gccgacagtt gtaagacaca gacccagaag 2400
tccaaggagg ctaagaccac catcgacagc ttcctgcgcg agcacttcgt gttcgacccc 2460
aacctccacg ctcagagtgc ctacacctgc gccgacacca acgtcgacac ctgcgcaagc 2520
atgtgcgctg acacaaatgt ggacacctgc gcctccatgt gcgcagacac caatgtcgac 2580
acctgtgcca gcacctgcac ctccactgaa tacacagacc tggctgaccc cgagcgcatc 2640
cctctccaca tcatgcagaa gaccctgaac gtgcccaacg agctgcaggc cgacatcgac 2700
gccatcaccc agacccctca gggatatcgg gccgccgccc acatcctgca gaacattgag 2760
ctccaccagt ccattaagca catgctggag aaccccaggg ctttcaaacc catcctgttt 2820
aacactaaga tcacgagata cctgagccag cacatccccc cccaggatac tttctataaa 2880
tggaactact acattgagga taactatgag gagctgaggg ccgccaccga atccatctac 2940
ccagagaagc ccgacctgga gtttgccttt atcatctacg acgtcgtgga ctcgagcaac 3000
cagcagaagg tggacgagtt ttactacaaa tataaggacc agatctttag cgaggtgtcc 3060
tccatccagc tggggaactg gaccctcctg ggctccttca aggccaaccg cgaaaggtat 3120
aactacttca accagaacaa cgaaatcatc aagagaatcc tggaccgcca cgaggaggac 3180
ctgaaaatcg gcaaggagat cctgcgcaac accatctacc ataagaaggc caaaaacatc 3240
caggagaccg ggcccgacgc ccccggcctg tccatctaca acagcacctt ccacactgat 3300
agcggcatta agggcctgct gagctttaag gagctgaaga atctggagaa ggccagcggc 3360
aacatcaaga aggcccgaga atacgacttc atcgacgact gtgaggagaa gatcaagcag 3420
ctgctgagca aggagaatct gacccccgac gaggagagcg agctgatcaa gaccaagaag 3480
cagctggaca acgccctgga gatgctgaac gtgcctgacg ataccatcag agtggatatg 3540
tgggtgaata acaataacaa gctggagaag gagatcctgt acaccaaggc cgagctgtaa 3600
<210> 2
<211> 652
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Ala Ser Gly Gly Ala Phe Cys Leu Ile Ala Asn Asp Gly Lys Ala
1 5 10 15
Asp Lys Ile Ile Leu Ala Gln Asp Leu Leu Asn Ser Arg Ile Ser Asn
20 25 30
Ile Lys Asn Val Asn Lys Ser Tyr Gly Lys Pro Asp Pro Glu Pro Thr
35 40 45
Leu Ser Gln Ile Glu Glu Thr His Leu Val His Phe Asn Ala His Phe
50 55 60
Lys Pro Tyr Val Pro Val Gly Phe Glu Tyr Asn Lys Val Arg Pro His
65 70 75 80
Thr Gly Thr Pro Thr Leu Gly Asn Lys Leu Thr Phe Gly Ile Pro Gln
85 90 95
Tyr Gly Asp Phe Phe His Asp Met Val Gly His His Ile Leu Gly Ala
100 105 110
Cys His Ser Ser Trp Gln Asp Ala Pro Ile Gln Gly Thr Ser Gln Met
115 120 125
Gly Ala His Gly Gln Leu Gln Thr Phe Pro Arg Asn Gly Tyr Asp Trp
130 135 140
Asp Asn Gln Thr Pro Leu Glu Gly Ala Val Tyr Thr Leu Val Asp Pro
145 150 155 160
Phe Gly Arg Pro Ile Val Pro Gly Thr Lys Asn Ala Tyr Arg Asn Leu
165 170 175
Val Tyr Tyr Cys Glu Tyr Pro Gly Glu Arg Leu Tyr Glu Asn Val Arg
180 185 190
Phe Asp Val Asn Gly Asn Ser Leu Asp Glu Tyr Ser Ser Asp Val Thr
195 200 205
Thr Leu Val Arg Lys Phe Cys Ile Pro Gly Asp Lys Met Thr Gly Tyr
210 215 220
Lys His Leu Val Gly Gln Glu Val Ser Val Glu Gly Thr Ser Gly Pro
225 230 235 240
Leu Leu Cys Asn Ile His Asp Leu His Lys Pro His Gln Ser Lys Pro
245 250 255
Ile Leu Thr Asp Glu Asn Asp Thr Gln Arg Thr Cys Ser His Thr Asn
260 265 270
Pro Lys Phe Leu Ser Gln His Phe Pro Glu Asn Ser His Asn Ile Gln
275 280 285
Thr Ala Gly Lys Gln Asp Ile Thr Pro Ile Thr Asp Ala Thr Tyr Leu
290 295 300
Asp Ile Arg Arg Asn Val His Tyr Ser Cys Asn Gly Pro Gln Thr Pro
305 310 315 320
Lys Tyr Tyr Gln Pro Pro Leu Ala Leu Trp Ile Lys Leu Arg Phe Trp
325 330 335
Phe Asn Glu Asn Val Asn Leu Ala Ile Pro Ser Val Ser Ile Pro Phe
340 345 350
Gly Glu Arg Phe Ile Thr Ile Lys Leu Ala Ser Gln Lys Asp Leu Val
355 360 365
Asn Glu Phe Pro Gly Leu Phe Val Arg Gln Ser Arg Phe Ile Ala Gly
370 375 380
Arg Pro Ser Arg Arg Asn Ile Arg Phe Lys Pro Trp Phe Ile Pro Gly
385 390 395 400
Val Ile Asn Glu Ile Ser Leu Thr Asn Asn Glu Leu Tyr Ile Asn Asn
405 410 415
Leu Phe Val Thr Pro Glu Ile His Asn Leu Phe Val Lys Arg Val Arg
420 425 430
Phe Ser Leu Ile Arg Val His Lys Thr Gln Val Thr His Thr Asn Asn
435 440 445
Asn His His Asp Glu Lys Leu Met Ser Ala Leu Lys Trp Pro Ile Glu
450 455 460
Tyr Met Phe Ile Gly Leu Lys Pro Thr Trp Asn Ile Ser Asp Gln Asn
465 470 475 480
Pro His Gln His Arg Asp Trp His Lys Phe Gly His Val Val Asn Ala
485 490 495
Ile Met Gln Pro Thr His His Ala Glu Ile Ser Phe Gln Asp Arg Asp
500 505 510
Thr Ala Leu Pro Asp Ala Cys Ser Ser Ile Ser Asp Ile Ser Pro Val
515 520 525
Thr Tyr Pro Ile Thr Leu Pro Ile Ile Lys Asn Ile Ser Val Thr Ala
530 535 540
His Gly Ile Asn Leu Ile Asp Lys Phe Pro Ser Lys Phe Cys Ser Ser
545 550 555 560
Tyr Ile Pro Phe His Tyr Gly Gly Asn Ala Ile Lys Thr Pro Asp Asp
565 570 575
Pro Gly Ala Met Met Ile Thr Phe Ala Leu Lys Pro Arg Glu Glu Tyr
580 585 590
Gln Pro Ser Gly His Ile Asn Val Ser Arg Ala Arg Glu Phe Tyr Ile
595 600 605
Ser Trp Asp Thr Asp Tyr Val Gly Ser Ile Thr Thr Ala Asp Leu Val
610 615 620
Val Ser Ala Ser Ala Ile Asn Phe Leu Leu Leu Gln Asn Gly Ser Ala
625 630 635 640
Val Leu Arg Tyr Ser Thr His His His His His His
645 650
<210> 3
<211> 529
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Ala Glu Phe Asn Ile Asp Glu Leu Leu Lys Asn Val Leu Glu Asp Pro
1 5 10 15
Ser Thr Glu Ile Ser Glu Glu Thr Leu Lys Gln Leu Tyr Gln Arg Thr
20 25 30
Asn Pro Tyr Lys Gln Phe Lys Asn Asp Ser Arg Val Ala Phe Cys Ser
35 40 45
Phe Thr Asn Leu Arg Glu Gln Tyr Ile Arg Arg Leu Ile Met Thr Ser
50 55 60
Phe Ile Gly Tyr Val Phe Lys Ala Leu Gln Glu Trp Met Pro Ser Tyr
65 70 75 80
Ser Lys Pro Thr His Thr Thr Lys Thr Leu Leu Ser Glu Leu Ile Thr
85 90 95
Leu Val Asp Thr Leu Lys Gln Glu Thr Asn Asp Val Pro Ser Glu Ser
100 105 110
Val Val Asn Thr Ile Leu Ser Ile Ala Asp Ser Cys Lys Thr Gln Thr
115 120 125
Gln Lys Ser Lys Glu Ala Lys Thr Thr Ile Asp Ser Phe Leu Arg Glu
130 135 140
His Phe Val Phe Asp Pro Asn Leu His Ala Gln Ser Ala Tyr Thr Cys
145 150 155 160
Ala Asp Thr Asn Val Asp Thr Cys Ala Ser Met Cys Ala Asp Thr Asn
165 170 175
Val Asp Thr Cys Ala Ser Met Cys Ala Asp Thr Asn Val Asp Thr Cys
180 185 190
Ala Ser Thr Cys Thr Ser Thr Glu Tyr Thr Asp Leu Ala Asp Pro Glu
195 200 205
Arg Ile Pro Leu His Ile Met Gln Lys Thr Leu Asn Val Pro Asn Glu
210 215 220
Leu Gln Ala Asp Ile Asp Ala Ile Thr Gln Thr Pro Gln Gly Tyr Arg
225 230 235 240
Ala Ala Ala His Ile Leu Gln Asn Ile Glu Leu His Gln Ser Ile Lys
245 250 255
His Met Leu Glu Asn Pro Arg Ala Phe Lys Pro Ile Leu Phe Asn Thr
260 265 270
Lys Ile Thr Arg Tyr Leu Ser Gln His Ile Pro Pro Gln Asp Thr Phe
275 280 285
Tyr Lys Trp Asn Tyr Tyr Ile Glu Asp Asn Tyr Glu Glu Leu Arg Ala
290 295 300
Ala Thr Glu Ser Ile Tyr Pro Glu Lys Pro Asp Leu Glu Phe Ala Phe
305 310 315 320
Ile Ile Tyr Asp Val Val Asp Ser Ser Asn Gln Gln Lys Val Asp Glu
325 330 335
Phe Tyr Tyr Lys Tyr Lys Asp Gln Ile Phe Ser Glu Val Ser Ser Ile
340 345 350
Gln Leu Gly Asn Trp Thr Leu Leu Gly Ser Phe Lys Ala Asn Arg Glu
355 360 365
Arg Tyr Asn Tyr Phe Asn Gln Asn Asn Glu Ile Ile Lys Arg Ile Leu
370 375 380
Asp Arg His Glu Glu Asp Leu Lys Ile Gly Lys Glu Ile Leu Arg Asn
385 390 395 400
Thr Ile Tyr His Lys Lys Ala Lys Asn Ile Gln Glu Thr Gly Pro Asp
405 410 415
Ala Pro Gly Leu Ser Ile Tyr Asn Ser Thr Phe His Thr Asp Ser Gly
420 425 430
Ile Lys Gly Leu Leu Ser Phe Lys Glu Leu Lys Asn Leu Glu Lys Ala
435 440 445
Ser Gly Asn Ile Lys Lys Ala Arg Glu Tyr Asp Phe Ile Asp Asp Cys
450 455 460
Glu Glu Lys Ile Lys Gln Leu Leu Ser Lys Glu Asn Leu Thr Pro Asp
465 470 475 480
Glu Glu Ser Glu Leu Ile Lys Thr Lys Lys Gln Leu Asp Asn Ala Leu
485 490 495
Glu Met Leu Asn Val Pro Asp Asp Thr Ile Arg Val Asp Met Trp Val
500 505 510
Asn Asn Asn Asn Lys Leu Glu Lys Glu Ile Leu Tyr Thr Lys Ala Glu
515 520 525
Leu
<210> 4
<211> 18
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro
1 5 10 15
Gly Pro