一组柯萨奇A组6型病毒突变株及其应用的制作方法
本发明属于医药生物领域，具体而言，涉及一组柯萨奇a组6型病毒突变株及其应用。
背景技术：
手足口病(hand-foot-and-mouthdisease，hfmd)是全球性传染病，以婴幼儿发病为主。该病的主要症状表现为手、足、口腔、臀部的皮疹、斑丘疹、疱疹，发热或不发热[1]。绝大多数hfmd患儿感染5至7天后可自愈，小部分患儿病情发展快，在发病1至5天后发展为重症病例，出现严重的中枢神经系统并发症，包括急性无菌性脑膜炎、脑炎、急性弛缓性麻痹等与肺水肿、心肌炎等呼吸及循环功能障碍，甚至可导致死亡[2-3]。。
引起hfmd的病毒属于小rna病毒科(picornaviridae)肠道病毒属(enteroviruse)，包括柯萨奇病毒(coxsackievirus)a组的2、4、5、6、7、9、10、16型，b组的1、2、3、4、5、13型，肠道病毒71型(humanenterovirus71,ev-71)，埃可病毒(echo-viruses)等[11-14]。2008年5月2日，中国卫生部将hfmd列入传染病防治法规定的丙类传染病进行管理。根据文献报道，ev-71、cv-a6、cv-a10、cv-a16和cv-a4是引起目前hfmd的主要病原体[15]。
早期研究表明cv-a16和ev-71是hfmd流行的主要病原体[16-21]。但是自2008年cv-a6引起芬兰hfmd暴发后，数年内在欧洲、美洲和亚洲等多个国家和地区均有cv-a6相关hfmd的流行[22-25]。特别是，cv-a6已成为中国大陆主要的循环血清型[26-29]。本实验室2016-2017年在湖北省襄阳市部分地区收集了3703份hfmd患儿临床样本，通过核酸分型确诊cv-a6阳性样本共1938份，占比60.26％，已经成为导致该地区发生hfmd的最主要血清型。目前，市面上现存的ev-71疫苗对cv-a6引起的hfmd无法形成有效的交叉保护。为了更全面的预防hfmd，需要研发包含cv-a6在内的多种肠道病毒抗原的多价hfmd疫苗。
在疫苗的研发过程中，需要建立合适的感染模型和疾病模型对候选疫苗的效力进行评价，最常用的感染模型仍然是小鼠模型。国际学术期刊已发表的文章更多采用的是小日龄乳鼠的被动免疫保护模型，包括使用抗血清的预防性和治疗性被动保护模型和母传抗体被动免疫保护模型。其中，ev-71、cv-a16、cv-a6、cv-a10全病毒灭活疫苗及相应的病毒样颗粒的保护效力已有相关报道[30-31]。被动免疫保护模型虽证实了中和抗体的保护作用，但不能模拟自然状态下疫苗免疫和病毒感染的过程，不能反映动物接种疫苗后的体液和细胞免疫应答及攻毒后的保护效力。所以更好的用于评价疫苗效力的动物模型是主动免疫保护模型，即使用候选疫苗直接对小鼠进行免疫并对小鼠进行攻毒。而大日龄小鼠对包括cv-a6在内的柯萨奇a组病毒不敏感，使攻毒前没有初次免疫和加强免疫的窗口期，是主动免疫保护模型建立的技术障碍。
参考文献
1.who.aguidetoclinicalmanagementandpublichealthresponseforhand,footandmouthdisease[m].who,2011.
2.pragerp,nolanm,andrewsip,etal.neurogenicpulmonaryedemainenterovirus71encephalitisisnotuniformlyfatalbutcausesseveremorbidityinsurvivors[j].pediatrcritcaremed,2003,4(3):377–381.
3.hsuch,lucy,shaopl,etal.epidemiologicandclinicalfeaturesofnon-polioenteroviralinfectionsinnortherntaiwanin2008[j].jmicrobiolimmunolinfect,2011,44(4):265–273.
4.ishimaruy,nakanos,yamaokak,etal.outbreaksofhand,foot,andmouthdiseasebyenterovirus71：highincidenceofcomplicationdisordersofcentralnervoussystem[j].archdischild,1980,55(8):583–588.
5.yipcc,lausk,woopc,etal.humanenterovirus71epidemics:what'snext？[j].emerghealththreatsj,2013,6:19780.
6.nagyg,takatsys,kukane,etal.virologicaldiagnosisofenterovirustype71infections:experiencesgainedduringanepidemicofacutecnsdiseasesinhungaryin1978[j].archvirol,1982,71(3):217–227.
7.chanlg,parasharud,lyems,etal.deathsofchildrenduringanoutbreakofhand,foot,andmouthdiseaseinsarawak,malaysia:clinicalandpathologicalcharacteristicsofthedis-ease.fortheoutbreakstudygroup[j].clininfectdis,2000,31(3):678–683.
8.anglw,kohbk,chankp,etal.epidemiologyandcontrolofhand,footandmouthdiseaseinsingapore,2001-2007[j].annacadmedsingapore,2009,38(2):106–112.
9.chankp,gohkt,chongcy,etal.epidemichand,footandmouthdiseasecausedbyhumanenterovirus71,singapore[j].emerginfectdis,2003,9(1):78–85.
10.tupv,thaont,pererad,etal.epidemiologicandvirologicinvestigationofhand,foot,andmouthdisease,southernvietnam,2005[j].emerginfectdis,2007,13(11):1733–1741.
11.oberstems,maherk,kilpatrickdr,etal.molecularevolutionofthehuman:correlationofserotypewithvp1sequenceandapplicationtopicornavirusclassification[j].jvirol,1999,73(3):1941–1948.
12.wangyr,sunll,xiaowl,etal.epidemiologyandclinicalcharacteristicsofhandfoot,andmouthdiseaseinashenzhensentinelhospitalfrom2009to2011[j].bmcinfectdis,2013,13:539–542.
13.yipcc,lausk,zhoub,etal.emergenceofenterovirus71"double-recombinant"strainsbelongingtoanovelgenotypedoriginatingfromsouthernchina:firstevidenceforcombinationofintratypicandintertypicrecombinationeventsinev71[j].archvirol,2010,155(9):1413–1424.
14.zhanghm,licr,liuyj,etal.toinvestigatepathogenofhand,footandmouthdiseaseinshenzhenin2008[j].zhonghuashiyanhelinchuangbingduxuezazhi,2009,23(5):334–336.
15.chenkt,changhl,wangst,etal.epidemiologicfeaturesofhand-foot-mouthdiseaseandherpanginacausedbyenterovirus71intaiwan,1998-2005[j].pediatrics,2007,120(2):e244-52.
16.pourianfarhr,kirkk,grollol.initialevidenceondifferencesamongenterovirus71,coxsackievirusa16andcoxsackievirusb4inbindingtocellsurfaceheparansulphate[j].virusdisease,2014,25(3):277–284.
17.liw,yil,suj,etal.seroprevalenceofhumanenterovirus71andcoxsackievirusa16inguangdong,china,inpre-andpost-2010hfmdepidemicperiod[j].plosone,2013,8(12):e80515.
18.yanxf,gaos,xiajf,etal.epidemiccharacteristicsofhand,foot,andmouthdiseaseinshanghaifrom2009to2010:enterovirus71subgenotypec4astheprimarycausativeagentandahighincidenceofmixedinfectionswithcoxsackievirusa16[j].scandjinfectdis,2012,44(4):297–305.
19.mae,fungc,yipsh,etal.estimationofthebasicreproductionnumberofenterovirus71andcoxsackievirusa16inhand,foot,andmouthdiseaseoutbreaks[j].pediatrinfectdisj,2011,30(8):675–679.
20.chenl,moux,zhangq,etal.detectionofhumanenterovirus71andcoxsackievirusa16inchildrenwithhand,footandmouthdiseaseinchina[j].molmedrep,2012,5(4):1001–1004.
21.panh,zhuyf,qix,etal.[analysisontheepidemiologicalandgeneticcharacteristicsofenterovirustype71andcoxsackiea16virusinfectioninjiangsu,china][j].zhonghualiuxingbingxuezazhi,2009,30(4):339–343.
22.osterbackr,vuorinent,linnam,etal.coxsackievirusa6andhand,foot,andmouthdisease,finland[j].emerginfectdis,2009,15(9):1485–1488.
23.weish,huangyp,liumc,etal.anoutbreakofcoxsackievirusa6hand,foot,andmouthdiseaseassociatedwithonychomadesisintaiwan,2010[j].bmcinfectdis,2011,11:346.
24.montesm,artiedaj,pieirold,etal.hand,foot,andmouthdiseaseoutbreakandcox-sackievirusa6,northernspain,2011[j].emerginfectdis,2013,19(4).
25.fujimotot,iizukas,enomotom,etal.hand,foot,andmouthdiseasecausedbycox-sackievirusa6,japan,2011[j].emerginfectdis,2012,18(2):337–339.
26.lijl,yuanj,yangf,etal.epidemiccharacteristicsofhand,foot,andmouthdiseaseinsouthernchina,2013.coxsackievirusa6hasemergedasthepredominantcausativeagent[j].jinfect,2014,69:299–303.
27.dib,zhangy,xieh,etal.circulationofcoxsackievirusa6inhand-foot-mouthdiseaseinguangzhou,2010-2012[j].virolj,2014,11:157–163.
28.gaof,maoqy,chenp,etal.seroepidemiologyofcoxsackievirusa6,coxsackievirusa16,andenterovirus71infectionsininfantsandchildren:aprospectivecohortstudyinjiangsu,china[j].jinfect,2016,73:509–512.
29.lij,suny,duy,etal.characterizationofcoxsackievirusa6-andenterovirus71-as-sociatedhandfootandmouthdiseaseinbeijing,china,from2013to2015[j].frontmi,2016,7:391.
30.kuz,liuq,yex,etal.avirus-likeparticlebasedbivalentvaccineconfersdualprotec-tionagainstenterovirus71andcoxsackievirusa16infectionsinmice[j].vaccine,2014,32(34):4296–303.
31.limh,inhj,leeja,etal.theimmunogenicityandprotectioneffectofaninactivatedcoxsackievirusa6,a10anda16vaccineagainsthand,foot,andmouthdisease[j].vaccine,2018,36(24):3445–452.
技术实现要素：
基于此，本发明的核心是获得能感染14日龄小鼠的攻毒株，使得新生乳鼠获得间隔一周的2次免疫，加强免疫后第5日中和抗体出现后进行攻毒，观察攻毒后14日龄小鼠的发病及致死率，与未免疫攻毒的对照组比较，以免疫攻毒组的小鼠存活率或发病保护率，计算候选疫苗保护效力。
为获得能够致死14日龄小鼠的攻毒株，本研究使用基因组已测序、遗传背景清晰一致、可在rd细胞传代的克隆纯化株cv-a6-r10和cv-a6-r5，依次感染5日龄、10日龄昆明鼠，确定cv-a6-r10为可使10日龄小鼠致病、致死的强毒力株，而cv-a6-r5为弱毒力株。通过腹腔和颅内两种不同途径感染12日龄昆明鼠，确定高剂量cv-a6-r10经腹腔感染途径能够成功感染并致死12日龄昆明鼠。将高剂量cv-a6-r10采用腹腔注射途径感染14日龄昆明鼠时，同样能够成功感染并致死14日龄昆明鼠。对cv-a6-r10和cv-a6-r5分别进行全基因组序列测定，确定与小鼠体内毒力相关的核苷酸、氨基酸突变位点。
本发明首先涉及一种柯萨奇a组6型病毒强毒力突变株，其特征在于，所述的强毒力突变株中单独或任意组合的含有如下位点：
vp1蛋白：124m、242v。
优选的，所述的强毒力突变株还包含如下任一或任意组合的位点：
vp2蛋白：57d、vp3蛋白：135p。
优选的，所述的强毒力突变株为cv-a6-r10，其氨基酸序列如seqidno.1所示。
本发明还涉及编码所述的强毒力突变株的核苷酸序列，优选的，所述的强毒力突变株的核苷酸序列如seqidno.3所示。
本发明还涉及一种柯萨奇a组6型病毒弱毒力突变株，其特征在于，所述的弱毒力突变株中单独或任意组合的含有如下位点：
vp1蛋白：124v、242i。
优选的，所述的弱毒力突变株还包含如下任一或任意组合的位点：
vp2蛋白：57d、vp3蛋白：135p。
优选的，所述的弱毒力突变株为cv-a6-r5，其氨基酸序列如seqidno.2所示。
本发明还涉及编码所述的弱毒力突变株的核苷酸序列，优选的，所述的弱毒力突变株的核苷酸序列如seqidno.4所示。
本发明还涉及所述的柯萨奇a组6型强/弱毒力突变株在制备动物模型中的应用。
优选的，所述的动物模型为：评价抗柯萨奇病毒的疫苗保护效力的小鼠模型、评价抗柯萨奇病毒的药物的治疗效果的小鼠模型。
本发明还涉及所述的柯萨奇a组6型强/弱毒力突变株在制备疫苗中的应用。
本发明的有益效果在于：
1.发明人采用未经小鼠体内传代适应的cv-a6克隆株，获得感染14日龄乳鼠的强毒力株cv-a6-r10。因此，相较小鼠适应攻毒株而言，未经小鼠体内传代适应的攻毒株与疫苗株的核苷酸和氨基酸序列更为接近，同源性更强。
2.该强毒力株具有较强的生长能力，能有效地感染rd细胞并有明显的细胞病变效应。
3.发明人用强、弱毒株(分别为cv-a6-r10，cv-a6-r5)，采用相同的感染剂量对小鼠采用颅内途径感染病毒，比较了两种途径攻毒的毒力，结果证明强、弱毒力株致病率、致死率存在差异。cv-a6-r10感染对应的大日龄昆明鼠出现精神萎靡、竖毛、体重下降症状及后肢麻痹、瘫痪、死亡等典型神经症状和较高的死亡率。
4.发明人通过对cv-a6强、弱毒力株的基因组序列分析、比对，发现若干影响毒力的位点，为研究该类病毒的致病机理奠定基础。同时阐明可能相关的重要毒力、神经毒力位点，具有潜在药物靶点的应用价值。
附图说明
图1、cv-a6-r10和cv-a6-r5颅内感染5日龄及10日龄昆明鼠生存曲线及临床评分：图1a和1b，强毒株cv-a6-r10和弱毒株cv-a6-r5感染5日龄昆明鼠观察期间生存曲线和临床评分，感染剂量7.6x104ccid50/20μl/dose，10只/组。图1c和1d，强毒株cv-a6-r10和弱毒株cv-a6-r5感染10日龄昆明鼠观察期间生存曲线和临床评分，感染剂量1.1x105ccid50/30μl/dose，10只/组。
图2、cv-a6-r10和cv-a6-r5颅内感染10日龄昆明鼠第7天的临床症状：图2a，强毒株cv-a6-r10感染10日龄昆明鼠；图2b，弱毒株cv-a6-r5感染10日龄昆明鼠；图2c，未感染对照组(mem注射组)。感染剂量为1.1x105ccid50/30μl/dose，10只/组。
图3、cv-a6-r10腹腔和颅内感染12日龄昆明鼠生存曲线及临床评分：图3a和3b，强毒株cv-a6-r10腹腔感染12日龄昆明鼠观察期间生存曲线和临床评分，感染剂量分别为1.6x107ccid50/300μl/dose和1.7x108ccid50/300μl/dose，6只/组。图3c和3d，强毒株cv-a6-r10颅内感染12日龄昆明鼠观察期间生存曲线和临床评分，感染剂量分别为1.6x106ccid50/30μl/dose和1.7x107ccid50/30μl/dose，6只/组。
图4、cv-a6-r10腹腔感染14日龄昆明鼠生存曲线(a)及临床评分(b)。感染剂量为1.7x108ccid50/300μl/dose，6只/组。。
具体实施方式
实施例1：cv-a6rd细胞适应株的分离和纯化
在rd细胞分离cv-a6-hev69/xy/chn/2017病毒株，培养8代后通过3次连续末端稀释法纯化得到19株克隆株。设计引物进行病毒全长基因组扩增，产物送至生工测序。分析各个克隆株的全基因组序列，比对核苷酸及氨基酸序列，依据氨基酸差异，将其分为7个基因型。
有限稀释纯化病毒：
(1)细胞培养板的准备：将rd细胞接种于96孔细胞培养板中，置37℃、5％co2细胞培养箱中备用。
(2)病毒样品的稀释与接种：将cv-a6-hev69/xy/chn/2017病毒样品进行十倍梯度稀释并接种于96孔板中，培养7d。
(3)病毒的收获：
1)镜检各孔cpe情况，从最后一个cpe阳性的稀释度对应的复孔中挑取克隆株。吸取孔中200μl病毒收获液移至1.5ml离心管中。
2)取100μl收获液进行十倍稀释，重复以上步骤进行三次连续的有限稀释，最终获得纯化的病毒克隆株。
(4)病毒克隆株的扩大培养：将经过三次连续纯化得到的病毒克隆株，按moi＝0.01～0.1接种于rd细胞中进行扩大培养。
实施例2：扩大培养cv-a6病毒克隆株
1、将生长状态良好、100％汇合的rd细胞(t25瓶)，弃细胞培养液，pbs清洗三次，补加mem维持液，接种200μl克隆株病毒液，置于37℃，5％二氧化碳培养箱孵育2h，孵育结束补加相应体积维持液。
2、待细胞病变达到80-90％时,反复冻融三次后离心收获病毒液，分装冻存，即为p2代。
3、重复以上步骤，获得cv-a6病毒克隆株19株，选择7株克隆株通过在14日龄乳鼠上的感染试验判定其中两株毒力存在差异的克隆株即：cv-a6-r10，cv-a6-r5。
实施例3：cv-a6-r10及cv-a6-r5全基因组序列测定
1、病毒rna提取
使用takara公司的minibestviralrna/dnaextractionkitver.5.0试剂盒(货号：9766)抽提纯化病毒rna。
2、逆转录合成cdna
逆转录合成cdna反应体系如表6及表7所示
表6
表7
(1)按照表6所示在pcr管中加入相应试剂，混合均匀后置于pcr仪中，65℃反应5min，然后迅速放在冰上骤冷。
(2)往上述反应液中加入表7所示的试剂，混合均匀后置于pcr仪，30℃，10min(使用random6mers时)；42℃，30～60min；95℃，5min；4℃，5min。
3、pcr扩增
(1)扩增引物：cv-a6全序列测定和扩展引物见表8。
表8：cv-a6全基因组序列扩增和测序引物
(2)采用toyobo公司生产的kod-plus-neo(codeno.kod-401)进行pcr扩增，98℃10min→98℃30sec，57℃3min30sec，68℃(延伸速度依据扩增片段大小，30sec/kb)，循环数30cycles。
4、琼脂糖凝胶电泳
待反应结束后，进行1％琼脂糖凝胶电泳，鉴定pcr扩增条带。待pcr产物鉴定正确，将剩余扩增产物送至生工生物工程技术服务有限公司进行测序，将测序各片段拼接得到全基因组序列。
测序结果显示：
cv-a6-r10氨基酸序列如seqidno.1所示，其中，1至69为vp4、70至325为vp2、326至565为vp3、566至870为vp1、871至1020为2a、1021至1119为2b、1120至1448为2c、1449至1534为3a、1535至1556为3b、1557至1739为3c、1740至2201为3d；
cv-a6-r10核苷酸序列如seqidno.3所示，其中，1至748为5’utr、749至7354为连续的编码区、7355至7435为3’utr。
cv-a6-r5氨基酸序列如seqidno.2所示，其中，1至69为vp4、70至325为vp2、326至565为vp3、566至870为vp1、871至1020为2a、1021至1119为2b、1120至1448为2c、1449至1534为3a、1535至1556为3b、1557至1739为3c、1740至2201为3d；
cv-a6-r5核苷酸序列如seqidno.4所示，其中，1至748为5’utr、749至7354为连续的编码区、7355至7435为3’utr。
实施例4：颅内注射cv-a6-r10能有效感染5日龄和10日龄昆明鼠
图1a和1b显示,用相同的病毒剂量即7.6x104ccid50/20μl/dose，将cv-a6-r10和cv-a6-r5采用颅内接种感染5日龄昆明鼠，小鼠发病率分别为100％、60％，死亡率分别为100％、40％；
图1c和图1d显示，当用相同的病毒剂量即1.1x105ccid50/30μl/dose，将cv-a6-r10和cv-a6-r5采用颅内接种感染10日龄昆明鼠，小鼠发病率分别为100％、60％，死亡率分别为70％、0％。
10日龄昆明鼠在接种cv-a6-r10后第七天出现死亡，而在接种cv-a6-r5后14天观察期结束时并未有死亡。
小鼠在被cv-a6-r10感染后表现出体重下降，共济失调，后肢瘫痪等临床症状(图2a)。cv-a6-r10实验组临床症状明显严重于cv-a6-r5实验组小鼠(图2b)。
以上试验的未感染对照组小鼠(图2c)均为正常，无相应的临床症状。
动物实验表明，在同一感染剂量下，cv-a6-r10能导致10日龄乳鼠出现更严重的临床症状和较高的死亡率，为强毒力适应株。而cv-a6-r5接种10日龄乳鼠，乳鼠部分出现致病症状，无死亡，为弱毒力适应株。
实施例5：颅内和腹腔注射cv-a6-r10能有效感染12日龄昆明鼠
图3a和3b显示，分别用不同的cv-a6-r10病毒剂量1.6x107ccid50/300μl/dose和1.7x108ccid50/300μl/dose采用腹腔途径感染12日龄昆明鼠，昆明鼠发病率分别为100％、100％，死亡率分别为100％、100％；
图3c和3d显示，分别用不同的cv-a6-r10病毒剂量1.6x106ccid50/30μl/dose和1.7x107ccid50/30μl/dose采用颅内途径感染12日龄昆明鼠，昆明鼠发病率分别为100％、100％，死亡率分别为0％、100％。
腹腔感染高、低剂量以及颅内感染高剂量的cv-a6-r10组12日龄乳鼠均出现精神萎靡、体重下降、共济失调、后肢瘫痪、无力、直至死亡等神经症状。
颅内感染低剂量的cv-a6-r10组12日龄乳鼠均出现精神萎靡、体重下降、共济失调和后肢瘫痪症状，但是至观察期结束，症状均出现减轻并逐渐趋向健康。
实验结果证明了高剂量的cv-a6-r10经过腹腔注射途径能够100％致死12日龄乳鼠。
实施例6：腹腔注射高剂量cv-a6-r10能有效感染14日龄昆明鼠
图4显示，将cv-a6-r10以高感染剂量即1.7x108ccid50/300μl/dose采用腹腔注射方式感染14日龄昆明鼠，昆明鼠发病率和死亡率分别为100％和100％。
感染cv-a6-r10组14日龄乳鼠均出现精神萎靡、体重下降、共济失调、后肢瘫痪，在感染后第4天全部死亡。
注射mem维持液的对照组小鼠在观察期14天内均无发病症状和死亡。
实验结果表明高剂量cv-a6-r10可以有效使14日龄昆明鼠腹腔感染发病并导致全部死亡。
实施例7：cv-a6-r10和cv-a6-r5全序列分析
为了研究cv-a6-r10毒力的分子机制，将cv-a6-r10和cv-a6-r5基因组序列进行比对。
cv-a6-r10株中：
5’utr基因第627位核苷酸为腺嘌呤(a)；
vp2基因1126位核苷酸为胸腺嘧啶(t)；
vp3基因第2128位核苷酸为腺嘌呤(a)；
vp1基因第2813位核苷酸为腺嘌呤(a)，编码的第124位氨基酸为蛋氨酸(m)；第3167位核苷酸为鸟嘌呤(g)，编码的第242位氨基酸为缬氨酸(v)。
cv-a6-r5株中：
5’utr基因第627位核苷酸为鸟嘌呤(g)；
vp2基因1126位核苷酸为胞嘧啶(c)；
vp3基因第2128位核苷酸为鸟嘌呤(g)；
vp1基因第2813位核苷酸为鸟嘌呤(g),编码的第124位氨基酸为缬氨酸(v)；第3167位核苷酸为腺嘌呤(a)，编码的第242位氨基酸为异亮氨酸(i)。
以上强、弱毒力毒株各位点的核苷酸、氨基酸差异与感染小鼠的毒力相关。
表1：影响毒株感染小鼠的相关毒力核苷酸(nt)及氨基酸(aa)位点
最后需要说明的是，以上实施例仅用于帮助本领域技术人员理解本发明的实质，不用于限定本发明的保护范围。
sequencelisting
<110>武汉生物制品研究所有限责任公司
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180185190
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195200205
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225230235240
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argthrasnasncysalathrileilemetprotyrvalasnalaleu
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645650655
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660665670
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675680685
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thrthrproglymetleuleuglntyrmettyrvalproproglyala
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proserilephealalysleuseraspproproproglnvalserval
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prophemetserproalathralatyrglntrpphetyraspglytyr
755760765
prothrpheglygluhislysglnalathrasnleuglntyrglygln
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785790795800
serthrthrglylysasnvalhisvalargvaltyrmetargilelys
805810815
hisvalargalatrpvalproargproleuargserglnalatyrmet
820825830
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835840845
aspargalaserilethrthrthrasptyrgluglyglyvalproala
850855860
asnproglnargthrserglyargleuglyglnglnserglyalaile
865870875880
tyrvalglyasnpheargvalvalasnarghisleualathrargasn
885890895
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900905910
serserthrthralaglnglycysaspthrilealaargcysaspcys
915920925
glnthrglyvaltyrtyrcysasnserlysarglyshistyrproval
930935940
serpheserlysproserleuvalphevalglualaserglutyrtyr
945950955960
proalaargtyrglnserhisleumetleualalysglyhisserglu
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proglyaspcysglyglyileleuargcysglnhisglyvalilegly
980985990
ilevalserthrglyglyasnglyleuvalglyphealaaspvalarg
99510001005
aspleuleutrpleuaspgluglualametgluglnglyvalser
101010151020
asptyrilelysglyleuglyaspalapheglythrglyphethr
102510301035
aspalavalalaarggluvalglualaleulysasntyrleuile
104010451050
glysergluglyalavalglulysileleulysasnleuilelys
105510601065
leuileseralaleuvalilevalileargserasptyraspmet
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valthrleuthralathrleualaleuileglycyshisglyser
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113011351140
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114511501155
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117511801185
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119011951200
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120512101215
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122012251230
lysserlyshisargilegluprovalcysleuileilearggly
123512401245
serproglythrglylysserleualathrglyileilealaarg
125012551260
alailealaasplystyrhisserservaltyrserleupropro
126512701275
aspproasphispheaspglytyrlysglnglnvalvalthrval
128012851290
metaspaspleucysglnasnproaspglylysaspmetserleu
129513001305
phecysglnmetvalserthrvalasppheileproprometala
131013151320
serleugluglulysglyvalserphethrserlysphevalile
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valthraspsertyrlysthraspleuglyargleuaspalagly
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140014051410
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141514201425
leuileargglutyrasnasnargseralaileglyasnthrile
143014351440
glualaleupheglnglyproprolyspheargproileargile
144514501455
serleugluglualaproalaproaspvalileseraspleuleu
146014651470
alaservalaspserglugluvalargglntyrcysargaspgln
147514801485
glytrpileileprogluthrprothrasnvalgluarghisleu
149014951500
serargalavalleuilemetglnserilealathrvalvalala
150515101515
valvalserleuvaltyrvaliletyrlysleuphealaglyphe
152015251530
glnglyalatyrserglyalaprolysglnvalleulyslyspro
153515401545
ileleuargthralathrvalglnglyproserleuasppheala
155015551560
leuserleuleuargargasnileargglnvalglnthraspgln
156515701575
glyhisphethrmetleuglyvalargaspargleualavalleu
158015851590
proarghisserglnproglylysthriletrpvalgluhislys
159516001605
leuvalasnileleuaspalavalgluleuvalaspgluglngly
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valasnleugluleuthrleuilethrleuaspthrasnglulys
162516301635
pheargaspilethrlyspheileprogluasnileseralaala
164016451650
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phevalprovalglyaspvalvalglntyrglypheleuasnleu
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serglylysprothrhisargthrmetmettyrasnpheprothr
168516901695
lysalaglyglncysglyglyvalvalthrservalglylysval
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ileglyilehisileglyglyasnglyargglnglyphecysala
171517201725
glyleulysargsertyrphealasergluglnglygluilegln
173017351740
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176017651770
gluglyasnlysgluproalavalleuhisserlysaspproarg
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leugluvalaspphegluglnalaleupheserlystyrvalgly
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asnthrilehisgluproaspglutyrilelysglualaalaleu
180518101815
histyralaasnglnleulysglnleuasnileaspthrsergln
182018251830
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ileaspleuhisthrseralaglytyrprotyrseralaleugly
185018551860
ilelyslysargaspileleuaspprothrthrargaspvalser
186518701875
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serthrtyrvallysaspgluleuargserileasplysilelys
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210521102115
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212021252130
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213521402145
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505560
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245250255
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260265270
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275280285
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290295300
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305310315320
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385390395400
asnvalaspglnleucysalaserpheglnvalaspproglyargasn
405410415
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465470475480
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485490495
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500505510
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545550555560
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565570575
valseralaleualaaspthrthrileserargvalthralaalaasn
580585590
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ilegluthrargcysvalmetasnargasnglyvalasnglualaser
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valgluhisphetyrserargalaglyleuvalglyvalvalgluval
645650655
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660665670
valmetglyphevalglnglnargarglysleugluleuserthrtyr
675680685
valargpheaspalagluphethrphevalserasnleuasnasnser
690695700
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705710715720
prolysproaspserarglyssertyrglntrpglnthralathrasn
725730735
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740745750
prophemetserproalathralatyrglntrpphetyraspglytyr
755760765
prothrpheglygluhislysglnalathrasnleuglntyrglygln
770775780
cysproasnasnmetmetglyhisphealaileargthrvalserglu
785790795800
serthrthrglylysasnilehisvalargvaltyrmetargilelys
805810815
hisvalargalatrpvalproargproleuargserglnalatyrmet
820825830
vallysasntyrprothrtyrserglnthrilethrasnthralaala
835840845
aspargalaserilethrthrthrasptyrgluglyglyvalproala
850855860
asnproglnargthrserglyargleuglyglnglnserglyalaile
865870875880
tyrvalglyasnpheargvalvalasnarghisleualathrargasn
885890895
asptrpalaasnleuvaltrpgluserserserargaspleuleuval
900905910
serserthrthralaglnglycysaspthrilealaargcysaspcys
915920925
glnthrglyvaltyrtyrcysasnserlysarglyshistyrproval
930935940
serpheserlysproserleuvalphevalglualaserglutyrtyr
945950955960
proalaargtyrglnserhisleumetleualalysglyhisserglu
965970975
proglyaspcysglyglyileleuargcysglnhisglyvalilegly
980985990
ilevalserthrglyglyasnglyleuvalglyphealaaspvalarg
99510001005
aspleuleutrpleuaspgluglualametgluglnglyvalser
101010151020
asptyrilelysglyleuglyaspalapheglythrglyphethr
102510301035
aspalavalalaarggluvalglualaleulysasntyrleuile
104010451050
glysergluglyalavalglulysileleulysasnleuilelys
105510601065
leuileseralaleuvalilevalileargserasptyraspmet
107010751080
valthrleuthralathrleualaleuileglycyshisglyser
108510901095
protrpalatrpilelysalalysthralaserileleuglyile
110011051110
proilealaglnlysglnseralasertrpleulyslyspheasn
111511201125
aspmetalaasnalaalalysglypheglutrpileserasnlys
113011351140
ileserlyspheileasptrpleulysglulysileileproala
114511501155
alaargglulysvalglupheleuasnasnleulysglnleupro
116011651170
leuleugluasnglnileserasnleugluglnseralaalaser
117511801185
glngluaspleuglualametpheglyasnvalsertyrleuala
119011951200
hisphecysarglystyrglnproleutyralathrglualalys
120512101215
argvaltyralaleuglulysargmetasnasntyrmetglnphe
122012251230
lysserlyshisargilegluprovalcysleuileilearggly
123512401245
serproglythrglylysserleualathrglyileilealaarg
125012551260
alailealaasplystyrhisserservaltyrserleupropro
126512701275
aspproasphispheaspglytyrlysglnglnvalvalthrval
128012851290
metaspaspleucysglnasnproaspglylysaspmetserleu
129513001305
phecysglnmetvalserthrvalasppheileproprometala
131013151320
serleugluglulysglyvalserphethrserlysphevalile
132513301335
alaserthrasnalaserasnileilevalprothrvalserasp
134013451350
seraspalaileargargargphetyrmetaspcysaspileglu
135513601365
valthraspsertyrlysthraspleuglyargleuaspalagly
137013751380
argalaalalysleucyssergluasnasnthralaasnphelys
138513901395
argcysserproleuvalcysglylysalaileglnleuargasp
140014051410
arglysserlysvalargtyrservalaspthrvalvalserglu
141514201425
leuileargglutyrasnasnargseralaileglyasnthrile
143014351440
glualaleupheglnglyproprolyspheargproileargile
144514501455
serleugluglualaproalaproaspvalileseraspleuleu
146014651470
alaservalaspserglugluvalargglntyrcysargaspgln
147514801485
glytrpileileprogluthrprothrasnvalgluarghisleu
149014951500
serargalavalleuilemetglnserilealathrvalvalala
150515101515
valvalserleuvaltyrvaliletyrlysleuphealaglyphe
152015251530
glnglyalatyrserglyalaprolysglnvalleulyslyspro
153515401545
ileleuargthralathrvalglnglyproserleuasppheala
155015551560
leuserleuleuargargasnileargglnvalglnthraspgln
156515701575
glyhisphethrmetleuglyvalargaspargleualavalleu
158015851590
proarghisserglnproglylysthriletrpvalgluhislys
159516001605
leuvalasnileleuaspalavalgluleuvalaspgluglngly
161016151620
valasnleugluleuthrleuilethrleuaspthrasnglulys
162516301635
pheargaspilethrlyspheileprogluasnileseralaala
164016451650
seraspalathrleuvalileasnthrgluhismetprosermet
165516601665
phevalprovalglyaspvalvalglntyrglypheleuasnleu
167016751680
serglylysprothrhisargthrmetmettyrasnpheprothr
168516901695
lysalaglyglncysglyglyvalvalthrservalglylysval
170017051710
ileglyilehisileglyglyasnglyargglnglyphecysala
171517201725
glyleulysargsertyrphealasergluglnglygluilegln
173017351740
trpvallysproasnlysgluthrglyargleuasnileasngly
174517501755
prothrargthrlysleugluproservalphehisaspilephe
176017651770
gluglyasnlysgluproalavalleuhisserlysaspproarg
177517801785
leugluvalaspphegluglnalaleupheserlystyrvalgly
179017951800
asnthrilehisgluproaspglutyrilelysglualaalaleu
180518101815
histyralaasnglnleulysglnleuasnileaspthrsergln
182018251830
metsermetgluglualacystyrglythraspasnleugluala
183518401845
ileaspleuhisthrseralaglytyrprotyrseralaleugly
185018551860
ilelyslysargaspileleuaspprothrthrargaspvalser
186518701875
lysmetlysphetyrmetasplystyrglyleuaspleuprotyr
188018851890
serthrtyrvallysaspgluleuargserileasplysilelys
189519001905
lysglylysserargleuileglualaserserleuasnaspser
191019151920
valtyrleuargmetalapheglyhisleutyrgluthrphehis
192519301935
alaasnproglythrvalthrglyseralavalglycysasnpro
194019451950
aspvalphetrpserlysleuproileleuleuproglyserleu
195519601965
phealapheasptyrserglytyraspalaserleuserproval
197019751980
trppheargalaleugluleuvalleuarggluileglytyrgly
198519901995
aspglualaileserleuilegluglyileasnhisthrhishis
200020052010
valtyrargasnlysthrtyrcysvalleuglyglymetproser
201520202025
glycysserglythrserilepheasnsermetileasnasnile
203020352040
ileileargserleuleuilelysthrphelysglyvalaspleu
204520502055
aspgluleuasnmetvalalatyrglyaspaspvalleualaser
206020652070
tyrpropheproileaspcyssergluleualaargthrglylys
207520802085
glutyrglyleuthrmetthrproalaasplysserprocysphe
209020952100
asngluvalasntrpgluasnalathrpheleulysargglyphe
210521102115
leuproaspgluglnphepropheleuilehisprothrmetpro
212021252130
metlysgluilehisgluserileargtrpthrlysaspalaarg
213521402145
asnthrglnasphisvalargserleucysleuleualatrphis
215021552160
asnglylysglnglutyrglulysphevalseralaileargser
216521702175
valproileglylysalaleualaileproasntyrgluasnleu
218021852190
argargasntrpleugluleuphe
21952200
<210>3
<211>7435
<212>dna
<213>人工序列
<400>3
ttaaaacagcttgtgggttgcacccacccacagggcccactgggcgctagcacactgatt60
ctacggaatctttgtgcgcctgttttataaccccttcccccaaaactgtaacttagaaga120
atattacactactgatcaatagcaggcatggcgcgccagtcatgtctagatcaagcactt180
ctgtctccccggactgagtatcaatagactgctagcgcggttgaaggagaaaacgtccgt240
tacccggctaactacttcgagaaacttagtagcaccattgaagctgcggagtgtttcgtt300
cagcactcccccagtgtagatcaggtcgatgagtcactgcactccccacgggcgaccgtg360
gcagtgactgcgttggcggcctgcctatggggcaacccataggacgctctaaagtggaca420
tggtgcgaagagtctattgagctagttagtagtcctccggcccctgaatgcggctaatcc480
caactgcggagcacatgccctcaatccagagggtggtgtgtcgtaacgggcaactctgca540
gcggaaccgactactttgggtgtccgtgtttccttttattcttatattggctgcttatgg600
tgacaattgagagattgttaccatatagctattggattggccatccagtgacaaacagag660
ctttgatatacttgtttgtgggttttgttccacttaccagtcgtacagttcatactctaa720
agtacattctgattctgaacaatagaaaatgggcgcccaagtctcaacagaaaaatctgg780
gtcgcacgagacaaagaatgtagcgaccgaagggtctactatcaatttcaccaacatcaa840
ttactataaggattcttatgcagcgtcagctagtagacaggactttgcacaagaccccgc900
aaagttcacacgccccgtcttggataccatcagggaggttgcagccccgctgcaatcccc960
ttctgttgaggcgtgcggttatagtgaccgagttgcacagttgactgtgggcaactcaac1020
cattactacccaagaggcagccaacattgtattgagttacggagagtggccagaatattg1080
tccctccacggatgctacagctgtggacaaacctactcgccctgatgtgtcagtgaatag1140
gttctacacactgtcaactaagagttggaagacagaatctactggctggtactggaaatt1200
ccctgatgtgctaaatgacacaggagtatttggtcaaaacgcccaattccactacttgta1260
ccgctcaggtttctgcatgcacgttcagtgtaatgcaagcaagttccatcagggggccct1320
tttagtggctgcaatccccgaatttgtggttgccgcaagcagccctgccgcgaagcctaa1380
tggacaagggttgtacccagatttcgcccacactaacccaggtaaaaatggccaagagtt1440
tcgagatccttatgtcttggatgctggtgtccccctaagtcaagcactggtttaccccca1500
tcaatggatcaatctacgaactaataactgcgcgaccattattatgccatatgtcaatgc1560
gcttccatttgattcagcgctcaaccactcaaattttggattggttgtgatccctattag1620
ccccttaaaatattgtaatggggctaccacagaagtgccaatcacactaactattgcccc1680
acttaactcggagtttagcggtctccgacaagcaataaaacaagggttccccacagagct1740
caagcctggtaccaatcaatttctcacaactgatgacgggacgtccccaccaatactgcc1800
cggttttgaaccaactccattgattcacattcctggtgagttcacctccttgttagattt1860
gtgtcaaatagaaaccatactagaagtcaataacaccactggcaccactggagtcagtag1920
attactaatccccgttcgagcacagaacaatgtggaccagttgtgcgcatcattccaagt1980
agaccctgggcgcaatggcccgtggcaatccacaatggtcggtcagatctgcaggtatta2040
cactcaatggtcaggttcccttaaggtaacctttatgttcacaggttcttttatggctac2100
agggaaaatgctgatagcctatacaccacctggtagtgctcagcccgctacaagggaagc2160
agcaatgcttgggactcatatagtgtgggattttggtttgcaatcatcagttaccctggt2220
cataccttggattagtaatacccattttagagcagttaagactggaggggtatacgacta2280
ctacgcaaccgggatcgtcaccatttggtaccaaaccaactttgtagtgccaccagacac2340
ccccactgaggctaatattatagctcttggagcagcacagaaaaactttaccctaaagtt2400
gtgtaaggacactgacgagatccagcaaacagcagagtaccaaaatgatcccattacaaa2460
tgcagtggaaagcgctgtgagcgcgcttgctgacaccacaatatcccgggtgaccgcagc2520
caacactgcagctagcacccactccctgggaacagggcgtgtaccagcattgcaagccgc2580
agaaacgggagcaagctctaatgccagtgatgagaaccttattgagacccgctgtgtgat2640
gaatcgaaacggggttaatgaggcgagtgtggaacacttttactctcgtgcagggctggt2700
aggagttgtggaggtgaaggactcgggcactagcctggatgggtacacagtttggcccat2760
agatgtgatgggcttcgtgcaacagcggcgcaagctagagttatcaacatacatgcgctt2820
tgatgccgagttcacttttgtgtccaacctcaataacagcacgacacccgggatgctgct2880
gcagtacatgtatgtgccaccaggggcccctaagccagatagcaggaaatcataccaatg2940
gcagactgctactaacccgtcgatattcgcaaaattgagtgatccacccccccaggtatc3000
tgtcccgttcatgtcgccagcaacggcttatcagtggttttatgatggttaccctacatt3060
tggtgaacacaaacaagccaccaatttgcaatatgggcaatgtcctaataacatgatggg3120
ccattttgctatccgaacagtcagtgaatctaccaccgggaaaaacgtccacgttcgggt3180
gtacatgagaattaagcacgtgagagcttgggtacctagaccccttcgatcccaagcata3240
tatggtcaagaactacccgacatacagccaaacaataactaacactgcagctgaccgtgc3300
aagcataaccaccacggattatgaaggcggggtaccagcaaacccacagaggacatctgg3360
taggttaggtcaacaatccggggctatctatgtaggcaacttcagagtggtaaaccgaca3420
tctcgccactcgtaatgattgggcaaatctagtatgggaaagtagctcacgagatctttt3480
ggtgtcctccaccactgctcagggatgtgataccattgcccgatgtgattgtcaaacagg3540
agtgtattactgcaactctaaaaggaaacactacccggttagtttttctaagcccagcct3600
cgtcttcgtggaagctagtgagtattaccctgccaggtatcagtcacaccttatgcttgc3660
aaagggacattctgaacccggggactgtggcggcattcttaggtgccaacatggcgtgat3720
tggtatcgtgtccactggtggtaatggacttgttggatttgcagatgtcagagacctttt3780
gtggctggatgaagaagctatggaacagggtgtgtcagattacatcaaagggctcggtga3840
cgcatttggaactggctttactgatgcagtagctagggaggtggaggctcttaagaacta3900
ccttataggatctgaaggggctgttgaaaagatcttaaagaatttaattaagctgatctc3960
agcattagtcatagtgatcagaagtgattatgacatggtaaccctcacagcaaccttggc4020
actcatagggtgtcatggcagcccctgggcgtggatcaaggctaagacagcatccatcct4080
aggcatccctatcgcccagaagcagagtgcgtcatggctcaagaagtttaatgacatggc4140
caatgctgccaagggatttgagtggatttccaataaaatcagcaaatttattgattggct4200
taaggagaaaattataccagcagctagagagaaggttgaatttttgaacaacctaaaaca4260
actgccattgttggagaaccaaatctcaaacctggagcagtccgccgcttcgcaagaaga4320
ccttgaggcaatgtttgggaacgtatcgtacctcgctcacttctgccgtaaataccaacc4380
actttatgctacagaagccaaaagagtttatgctttggaaaagaggatgaataattacat4440
gcagttcaagagcaaacaccgtattgaacctgtatgtcttatcatcagaggctccccagg4500
cactggaaagtccttggcaaccggtataattgcccgagcaatagctgacaaataccactc4560
tagtgtgtactcactcccgccagatccagaccactttgatggatacaaacagcaagtggt4620
cacagttatggacgatctatgccaaaatcctgatggcaaggatatgtcactcttttgtca4680
gatggtatccaccgtagatttcatcccaccaatggcttctttggaagagaaaggggtttc4740
attcacatctaaatttgttattgcatccactaatgccagcaacatcatagtgccaacagt4800
gtctgattctgatgctattcgccgcaggttctacatggactgcgacatcgaggtaacgga4860
ctcgtataaaacagatttgggtaggttagatgctggaagagctgccaaattatgctctga4920
aaataacacagcaaacttcaaacgctgtagcccactagtgtgtgggaaggccatccaatt4980
aagagataggaagtccaaagttagatacagtgtggatacggtggtttcagagctcataag5040
ggaatacaataacaggtctgccattggaaacacaattgaagcgttgttccaggggccacc5100
caagtttagacctattagaattagtcttgaggaggcgccagcaccagatgttattagtga5160
tctacttgccagtgtggatagtgaagaggtgcgccaatactgtagagaccaaggttggat5220
cataccagaaacccctaccaacgttgagcgacatttaagtagggccgtgctaatcatgca5280
atccattgccacggtcgttgcagtggtctcactggtgtatgttatctacaagctttttgc5340
tggattccagggtgcgtattctggcgctcctaagcaagtgctcaagaaacccatcctccg5400
cacggcaacagtgcaaggacctagccttgattttgccctatccctactgagaaggaacat5460
caggcaggttcagacagatcaagggcacttcactatgctgggtgtcagggatcgcttagc5520
agttctcccgcgccactcgcagcccggaaaaacaatctgggtggaacacaaactcgtgaa5580
catcctggatgctgtcgagttggtggatgagcaaggggttaacctagagctcactctaat5640
cactcttgataccaatgagaaattcagagatatcaccaagttcattccagaaaacatcag5700
cgctgctagtgacgccaccctagtgattaatacagaacacatgccctcaatgtttgtacc5760
tgtgggagatgtcgtacaatacggtttcctgaatctcagtggaaagcccacccatcgcac5820
catgatgtacaacttccctactaaggcaggacagtgtggaggggtggtgacatcagttgg5880
gaaagttattggaattcacataggaggcaatggtaggcaaggtttctgtgcgggacttaa5940
gaggagctactttgccagtgagcaaggggagatccaatgggtaaagcctaacaaagaaac6000
tgggagactcaacatcaacgggccaactcgcactaagctcgaacctagtgtgttccatga6060
tatctttgagggcaacaaggaaccagcggtcttacacagcaaagaccctcgtctcgaggt6120
ggattttgagcaggcattgttctccaagtatgtaggaaacactatacatgagcctgatga6180
atatatcaaggaggcagccttacattatgcaaatcagttgaagcagctaaatatagacac6240
ttctcaaatgagcatggaagaggcttgctacggcacagacaaccttgaagctattgacct6300
tcacactagtgcaggctacccctacagcgccttggggatcaagaagagggatatcttaga6360
ccccaccaccagggatgtgagtaagatgaagttctacatggacaagtatggtcttgatct6420
cccttactctacttatgttaaggatgagctacgctcaatagataagatcaaaaaggggaa6480
atcccgcttaattgaagctagcagtttgaacgactcagtttacctcagaatggccttcgg6540
acatctctatgaaactttccatgcaaaccctgggactgtgactggttcggctgtgggatg6600
taacccggacgtgttctggagcaagttgccaatcctgctccctggttccctctttgcttt6660
tgactactcgggctatgatgctagtctcagcccagtttggttcagagcattggagctagt6720
tcttagagagataggctacggtgacgaggcaatctcgctcattgaagggatcaatcatac6780
acaccatgtatatcgcaacaaaacttattgcgtacttggtgggatgccatcaggctgttc6840
aggaacatccatttttaactcaatgattaacaacatcatcattagatcattgcttatcaa6900
aacatttaagggtgttgacctggatgaactcaacatggttgcttatggggacgatgtact6960
tgctagttacccttttcctattgactgctcagaactagcaagaacaggcaaggagtatgg7020
tttaaccatgacccccgcagataagtctccttgcttcaatgaagttaattgggaaaatgc7080
aacctttcttaagaggggtttcttgcctgatgaacaatttccatttttgattcaccccac7140
catgccaatgaaggagattcacgaatccattcggtggaccaaggatgcacgcaatactca7200
agatcacgtgcgatccttgtgtctattggcgtggcacaacggcaaacaagaatatgaaaa7260
atttgtaagtgcaattaggtctgtcccaataggaaaggcactggctattccaaattatga7320
aaacctgagacgcaattggctcgaattattttagaggtcgaatacacctcaaccccacca7380
ggaatctggtcgtgaatatgactggtgggggtaaatttgttataaccagaatagc7435
<210>4
<211>7435
<212>dna
<213>人工序列
<400>4
ttaaaacagcttgtgggttgcacccacccacagggcccactgggcgctagcacactgatt60
ctacggaatctttgtgcgcctgttttataaccccttcccccaaaactgtaacttagaaga120
atattacactactgatcaatagcaggcatggcgcgccagtcatgtctagatcaagcactt180
ctgtctccccggactgagtatcaatagactgctagcgcggttgaaggagaaaacgtccgt240
tacccggctaactacttcgagaaacttagtagcaccattgaagctgcggagtgtttcgtt300
cagcactcccccagtgtagatcaggtcgatgagtcactgcactccccacgggcgaccgtg360
gcagtgactgcgttggcggcctgcctatggggcaacccataggacgctctaaagtggaca420
tggtgcgaagagtctattgagctagttagtagtcctccggcccctgaatgcggctaatcc480
caactgcggagcacatgccctcaatccagagggtggtgtgtcgtaacgggcaactctgca540
gcggaaccgactactttgggtgtccgtgtttccttttattcttatattggctgcttatgg600
tgacaattgagagattgttaccatatggctattggattggccatccagtgacaaacagag660
ctttgatatacttgtttgtgggttttgttccacttaccagtcgtacagttcatactctaa720
agtacattctgattctgaacaatagaaaatgggcgcccaagtctcaacagaaaaatctgg780
gtcgcacgagacaaagaatgtagcgaccgaagggtctactatcaatttcaccaacatcaa840
ttactataaggattcttatgcagcgtcagctagtagacaggactttgcacaagaccccgc900
aaagttcacacgccccgtcttggataccatcagggaggttgcagccccgctgcaatcccc960
ttctgttgaggcgtgcggttatagtgaccgagttgcacagttgactgtgggcaactcaac1020
cattactacccaagaggcagccaacattgtattgagttacggagagtggccagaatattg1080
tccctccacggatgctacagctgtggacaaacctactcgccctgacgtgtcagtgaatag1140
gttctacacactgtcaactaagagttggaagacagaatctactggctggtactggaaatt1200
ccctgatgtgctaaatgacacaggagtatttggtcaaaacgcccaattccactacttgta1260
ccgctcaggtttctgcatgcacgttcagtgtaatgcaagcaagttccatcagggggccct1320
tttagtggctgcaatccccgaatttgtggttgccgcaagcagccctgccgcgaagcctaa1380
tggacaagggttgtacccagatttcgcccacactaacccaggtaaaaatggccaagagtt1440
tcgagatccttatgtcttggatgctggtgtccccctaagtcaagcactggtttaccccca1500
tcaatggatcaatctacgaactaataactgcgcgaccattattatgccatatgtcaatgc1560
gcttccatttgattcagcgctcaaccactcaaattttggattggttgtgatccctattag1620
ccccttaaaatattgtaatggggctaccacagaagtgccaatcacactaactattgcccc1680
acttaactcggagtttagcggtctccgacaagcaataaaacaagggttccccacagagct1740
caagcctggtaccaatcaatttctcacaactgatgacgggacgtccccaccaatactgcc1800
cggttttgaaccaactccattgattcacattcctggtgagttcacctccttgttagattt1860
gtgtcaaatagaaaccatactagaagtcaataacaccactggcaccactggagtcagtag1920
attactaatccccgttcgagcacagaacaatgtggaccagttgtgcgcatcattccaagt1980
agaccctgggcgcaatggcccgtggcaatccacaatggtcggtcagatctgcaggtatta2040
cactcaatggtcaggttcccttaaggtaacctttatgttcacaggttcttttatggctac2100
agggaaaatgctgatagcctatacaccgcctggtagtgctcagcccgctacaagggaagc2160
agcaatgcttgggactcatatagtgtgggattttggtttgcaatcatcagttaccctggt2220
cataccttggattagtaatacccattttagagcagttaagactggaggggtatacgacta2280
ctacgcaaccgggatcgtcaccatttggtaccaaaccaactttgtagtgccaccagacac2340
ccccactgaggctaatattatagctcttggagcagcacagaaaaactttaccctaaagtt2400
gtgtaaggacactgacgagatccagcaaacagcagagtaccaaaatgatcccattacaaa2460
tgcagtggaaagcgctgtgagcgcgcttgctgacaccacaatatcccgggtgaccgcagc2520
caacactgcagctagcacccactccctgggaacagggcgtgtaccagcattgcaagccgc2580
agaaacgggagcaagctctaatgccagtgatgagaaccttattgagacccgctgtgtgat2640
gaatcgaaacggggttaatgaggcgagtgtggaacacttttactctcgtgcagggctggt2700
aggagttgtggaggtgaaggactcgggcactagcctggatgggtacacagtttggcccat2760
agatgtgatgggcttcgtgcaacagcggcgcaagctagagttatcaacatacgtgcgctt2820
tgatgccgagttcacttttgtgtccaacctcaataacagcacgacacccgggatgctgct2880
gcagtacatgtatgtgccaccaggggcccctaagccagatagcaggaaatcataccaatg2940
gcagactgctactaacccgtcgatattcgcaaaattgagtgatccacccccccaggtatc3000
tgtcccgttcatgtcgccagcaacggcttatcagtggttttatgatggttaccctacatt3060
tggtgaacacaaacaagccaccaatttgcaatatgggcaatgtcctaataacatgatggg3120
ccattttgctatccgaacagtcagtgaatctaccaccgggaaaaacatccacgttcgggt3180
gtacatgagaattaagcacgtgagagcttgggtacctagaccccttcgatcccaagcata3240
tatggtcaagaactacccgacatacagccaaacaataactaacactgcagctgaccgtgc3300
aagcataaccaccacggattatgaaggcggggtaccagcaaacccacagaggacatctgg3360
taggttaggtcaacaatccggggctatctatgtaggcaacttcagagtggtaaaccgaca3420
tctcgccactcgtaatgattgggcaaatctagtatgggaaagtagctcacgagatctttt3480
ggtgtcctccaccactgctcagggatgtgataccattgcccgatgtgattgtcaaacagg3540
agtgtattactgcaactctaaaaggaaacactacccggttagtttttctaagcccagcct3600
cgtcttcgtggaagctagtgagtattaccctgccaggtatcagtcacaccttatgcttgc3660
aaagggacattctgaacccggggactgtggcggcattcttaggtgccaacatggcgtgat3720
tggtatcgtgtccactggtggtaatggacttgttggatttgcagatgtcagagacctttt3780
gtggctggatgaagaagctatggaacagggtgtgtcagattacatcaaagggctcggtga3840
cgcatttggaactggctttactgatgcagtagctagggaggtggaggctcttaagaacta3900
ccttataggatctgaaggggctgttgaaaagatcttaaagaatttaattaagctgatctc3960
agcattagtcatagtgatcagaagtgattatgacatggtaaccctcacagcaaccttggc4020
actcatagggtgtcatggcagcccctgggcgtggatcaaggctaagacagcatccatcct4080
aggcatccctatcgcccagaagcagagtgcgtcatggctcaagaagtttaatgacatggc4140
caatgctgccaagggatttgagtggatttccaataaaatcagcaaatttattgattggct4200
taaggagaaaattataccagcagctagagagaaggttgaatttttgaacaacctaaaaca4260
actgccattgttggagaaccaaatctcaaacctggagcagtccgccgcttcgcaagaaga4320
ccttgaggcaatgtttgggaacgtatcgtacctcgctcacttctgccgtaaataccaacc4380
actttatgctacagaagccaaaagagtttatgctttggaaaagaggatgaataattacat4440
gcagttcaagagcaaacaccgtattgaacctgtatgtcttatcatcagaggctccccagg4500
cactggaaagtccttggcaaccggtataattgcccgagcaatagctgacaaataccactc4560
tagtgtgtactcactcccgccagatccagaccactttgatggatacaaacagcaagtggt4620
cacagttatggacgatctatgccaaaatcctgatggcaaggatatgtcactcttttgtca4680
gatggtatccaccgtagatttcatcccaccaatggcttctttggaagagaaaggggtttc4740
attcacatctaaatttgttattgcatccactaatgccagcaacatcatagtgccaacagt4800
gtctgattctgatgctattcgccgcaggttctacatggactgcgacatcgaggtaacgga4860
ctcgtataaaacagatttgggtaggttagatgctggaagagctgccaaattatgctctga4920
aaataacacagcaaacttcaaacgctgtagcccactagtgtgtgggaaggccatccaatt4980
aagagataggaagtccaaagttagatacagtgtggatacggtggtttcagagctcataag5040
ggaatacaataacaggtctgccattggaaacacaattgaagcgttgttccaggggccacc5100
caagtttagacctattagaattagtcttgaggaggcgccagcaccagatgttattagtga5160
tctacttgccagtgtggatagtgaagaggtgcgccaatactgtagagaccaaggttggat5220
cataccagaaacccctaccaacgttgagcgacatttaagtagggccgtgctaatcatgca5280
atccattgccacggtcgttgcagtggtctcactggtgtatgttatctacaagctttttgc5340
tggattccagggtgcgtattctggcgctcctaagcaagtgctcaagaaacccatcctccg5400
cacggcaacagtgcaaggacctagccttgattttgccctatccctactgagaaggaacat5460
caggcaggttcagacagatcaagggcacttcactatgctgggtgtcagggatcgcttagc5520
agttctcccgcgccactcgcagcccggaaaaacaatctgggtggaacacaaactcgtgaa5580
catcctggatgctgtcgagttggtggatgagcaaggggttaacctagagctcactctaat5640
cactcttgataccaatgagaaattcagagatatcaccaagttcattccagaaaacatcag5700
cgctgctagtgacgccaccctagtgattaatacagaacacatgccctcaatgtttgtacc5760
tgtgggagatgtcgtacaatacggtttcctgaatctcagtggaaagcccacccatcgcac5820
catgatgtacaacttccctactaaggcaggacagtgtggaggggtggtgacatcagttgg5880
gaaagttattggaattcacataggaggcaatggtaggcaaggtttctgtgcgggacttaa5940
gaggagctactttgccagtgagcaaggggagatccaatgggtaaagcctaacaaagaaac6000
tgggagactcaacatcaacgggccaactcgcactaagctcgaacctagtgtgttccatga6060
tatctttgagggcaacaaggaaccagcggtcttacacagcaaagaccctcgtctcgaggt6120
ggattttgagcaggcattgttctccaagtatgtaggaaacactatacatgagcctgatga6180
atatatcaaggaggcagccttacattatgcaaatcagttgaagcagctaaatatagacac6240
ttctcaaatgagcatggaagaggcttgctacggcacagacaaccttgaagctattgacct6300
tcacactagtgcaggctacccctacagcgccttggggatcaagaagagggatatcttaga6360
ccccaccaccagggatgtgagtaagatgaagttctacatggacaagtatggtcttgatct6420
cccttactctacttatgttaaggatgagctacgctcaatagataagatcaaaaaggggaa6480
atcccgcttaattgaagctagcagtttgaacgactcagtttacctcagaatggccttcgg6540
acatctctatgaaactttccatgcaaaccctgggactgtgactggttcggctgtgggatg6600
taacccggacgtgttctggagcaagttgccaatcctgctccctggttccctctttgcttt6660
tgactactcgggctatgatgctagtctcagcccagtttggttcagagcattggagctagt6720
tcttagagagataggctacggtgacgaggcaatctcgctcattgaagggatcaatcatac6780
acaccatgtatatcgcaacaaaacttattgcgtacttggtgggatgccatcaggctgttc6840
aggaacatccatttttaactcaatgattaacaacatcatcattagatcattgcttatcaa6900
aacatttaagggtgttgacctggatgaactcaacatggttgcttatggggacgatgtact6960
tgctagttacccttttcctattgactgctcagaactagcaagaacaggcaaggagtatgg7020
tttaaccatgacccccgcagataagtctccttgcttcaatgaagttaattgggaaaatgc7080
aacctttcttaagaggggtttcttgcctgatgaacaatttccatttttgattcaccccac7140
catgccaatgaaggagattcacgaatccattcggtggaccaaggatgcacgcaatactca7200
agatcacgtgcgatccttgtgtctattggcgtggcacaacggcaaacaagaatatgaaaa7260
atttgtaagtgcaattaggtctgtcccaataggaaaggcactggctattccaaattatga7320
aaacctgagacgcaattggctcgaattattttagaggtcgaatacacctcaaccccacca7380
ggaatctggtcgtgaatatgactggtgggggtaaatttgttataaccagaatagc7435
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