针对甲型肝炎病毒的单域抗体及其衍生蛋白的制作方法

本发明涉及生物技术或免疫学技术领域，涉及针对甲型肝炎病毒的单域抗体及其衍生蛋白。

背景技术：

抗体(antibody,ab)即免疫球蛋白(immunoglobulin,ig)是血液和组织液中的一类糖蛋白，由b细胞接收抗原刺激后增殖分化生成的浆细胞产生，主要存在与血清等体液中，能与相应抗原特异性地结合，是介导体液免疫的重要效应分子。抗体除了因其作为重要效应分子介导特异性体液免疫应答外，抗体在疾病的防治特别是感染性疾病的防治方面扮演者重要角色，behring创立血清疗法因此获得医学与生理学诺贝尔奖。此后，以多克隆、单克隆及基因工程抗体技术人工制备的抗体逐步应用于临床。仅2011年抗药药物就以480亿美元的销售额领跑全球药品市场，占整个生物制药市场的34.4％。至2017年，美国fda和欧盟ema批准的治疗性抗体药物有76个，主要用于肿瘤、自身免疫病等疾病的治疗。

抗体作为可以特异性识别某一个蛋白(抗原)的分子，除了在疾病预防和治疗方面的作用强大之外，其作为检测工具在检验、科学研究方面的用途也不言而喻。酶联免疫吸附实验(elisa)正是利用抗体/抗原结合以实现检测目的一个重要方法。目前常用的elisa检测方法有直接法、间接法、竞争法等，其基本原理均为抗原抗体结合反应。以此方法为基础elisa试剂盒在检验检疫等场合的用途也十分广泛。部分检验检疫方法对于抗体的亲和力和稳定性要求非常苛刻，这就要求所使用的抗体在与其特异性结合的抗原亲和力要非常高，稳定性要非常好，并且这些抗体的使用和储存环境要更为广泛才更有利于检测方法的推广。传统单抗在抗原亲和力方面表现良好，并且虽然现在可以通过添加保护剂或改变缓冲液的方法提升其稳定性，但是其储存条件要求依然相对苛刻，一般应在低温运输或保存。而单域抗体在亲和力和稳定性方面就表现的更为优秀，单域抗体在未进行亲和力成熟之前，其与抗原的亲和力可达到pm级别，并且其室温稳定性良好，可以降低运输和储存成本。此外，单域抗体可以在原核表达系统中可以大量可溶性表达，降低生产成本，并且提高抗体批件稳定性。

甲型病毒性肝炎简称甲肝，是一种由甲型肝炎病毒(hav)感染引起的以肝脏炎症病变为主的传染病。1974年feinstone等通过免疫电镜技术在甲肝患者粪便中发现甲肝病毒(hav)。甲肝病毒是直径为27～32nm的小rna病毒，其结构含有vp1～vp4四种多肽，基因组为单股正链rna，约7500个核苷酸。hav的传播途径为粪－口途径，主要在发展中国家流行。我国是甲肝的高流行国家。提高人群的免疫水平是预防甲肝的主要措施，随着甲肝灭活疫苗应用覆盖面不断扩大，甲肝的暴发流行得到有效的控制。因其临床表现与其他肝炎相似，因此甲肝的实验室诊断结果就显得尤为重要。在甲肝急性期患者血清中可以查出抗hav-igm及igg，患者患病后2周抗hav-igm阳性率为100％，持续2～3周，然后迅速下降。因此，检测抗hav-igm可作为急性期的诊断指标。抗hav-igg是甲肝的主要中和抗体，一般可从病人血清中查出，此种抗体存在于患者体内很长时间甚至终身。甲肝的实验室诊断方法主要有rt-pcr法、rt-pcr-elisa法、间接elisa法、spa协同凝集试验等。rt-pcr法灵敏度高，特异性强。但需专门的仪器设备，操作过程复杂，且对rna的提取过程要求较严格；rt-pcr-elisa法先通过rt-pcr扩增甲肝病毒rna，再用elisa法检测扩增产物。在保证了特异性的同时，也提高了检测的灵敏度。但是试验必须建立在rt-pcr的基础上，检测成本高；spa协同凝集试验利用金黄色葡萄球菌细胞壁中的a蛋白(spa)能特异性的与hav-igg的fc段结合，当待检样本中含有hav-ag时，即出现反向间接凝集反应。该试验灵敏度和特异性较低，结果判断人为影响因素大，仅可作为设备受限时的初步检测方法；一般的elisa法利用固相载体有吸附免疫活性蛋白的特性，在已包被抗人μ链的酶标板中，加入hav抗体待检血清，若待检血清为甲肝感染急性期，则血清中的igm抗体被其抗体人μ链抗体捕获，再加入酶标抗体，底物显色后，判断待检血清者为甲肝感染患者。但该方法只能检测已感染甲肝病毒的患者血清的igm抗体，不能直接定量或半定量患者体内甲肝病毒；所以直接用甲肝病毒抗体检测待检样品中甲肝病毒含量可以更直接、准确的反映患者血清内或者其他样品中的甲肝病毒含量，但目前市面上直接检测甲肝病毒的定量或半定量elisa试剂盒产品很少，并且已有的直接检测试剂盒的捕获抗体选择的是兔源多克隆抗体，由于兔多抗的制备批间差异很大，导致抗体的亲和力差异很大，这就造成了试剂盒检测的生产批间稳定性很差，重复性不好。所以选择生产工艺稳定并且亲和力更高的甲肝病毒抗体是准确检测样本甲肝病毒含量的重点。

技术实现要素：

为了克服上述缺陷，本发明的目的是提供特异性针对甲型肝炎病毒的单域抗体并建立基于抗甲型肝炎病毒单域抗体及其衍生蛋白，首先，在获得高特异性的单域抗体后通过原核表达或者酵母表达便可以获得，产量和纯化效率高并且批间质量稳定，其次，单域抗体的亲和力比传统抗体有一定优势，可以提升抗原捕获能力和检测效果，提高检测的灵敏度，此外，单域抗体属于单克隆抗体，这就能保证检测试剂的单一性和可控性，更好的控制检测试剂盒的检测标准。

单域抗体作为单克隆抗体的一类，其比传统单抗的亲和力普遍更高，稳定性更好，并且它也具备传统单抗的优点。因此，本发明就是利用上述优势，获得针对甲型肝炎病毒的抗体，这些获得的抗体既能够在普通的elisa实验中作为检测工具对各类样品中的甲型肝炎病毒进行定性分析，也可以被开发成elisa检测试剂盒，对人群中的甲型肝炎病毒进行定量分析，用于甲肝的临床诊断。

为解决上述技术问题，本发明采用如下技术方案：针对甲型肝炎病毒的单域抗体,其特征在于，所述的单域抗体的序列包括互补决定区cdr；所述互补决定区cdr包括cdr1、cdr2和cdr3的氨基酸序列；所述的cdr1的氨基酸序列如seqidno:83-seqidno:117中的任意一条所示；所述的cdr2的氨基酸序列如seqidno:118-seqidno:143中的任意一条所示；所述的cdr3的氨基酸序列如seqidno:144-seqidno:174中的任意一条所示。

上述所有序列，可以替换为与该序列具有“至少80％同源性”的序列或仅一个或少数几个氨基酸替换的序列；优选为“至少85％同源性”，更优选为“至少90％同源性”，更优选为“至少95％同源性”，最优选为“至少98％同源性”。

针对甲型肝炎病毒的单域抗体,所述的单域抗体的序列包括互补决定区cdr；所述互补决定区cdr包括cdr1、cdr2和cdr3的氨基酸序列；所述的单域抗体的互补决定区cdr的序列为下述(1)-(39)中的一种：

(1)seqidno:83所示的cdr1，seqidno:118所示的cdr2，seqidno:144所示的cdr3(对应于抗体1a2、1b4)；

(2)seqidno:84所示的cdr1，seqidno:119所示的cdr2，seqidno:145所示的cdr3(对应于抗体1a4)；

(3)seqidno:84所示的cdr1，seqidno:119所示的cdr2，seqidno:156所示的cdr3(对应于抗体4a1)；

(4)seqidno:84所示的cdr1，seqidno:119所示的cdr2，seqidno:169所示的cdr3(对应于抗体4a9)；

(5)seqidno:85所示的cdr1，seqidno:120所示的cdr2，seqidno:146所示的cdr3(对应于抗体1a6)；

(6)seqidno:86所示的cdr1，seqidno:121所示的cdr2，seqidno:147所示的cdr3(对应于抗体1a7)；

(7)seqidno:87所示的cdr1，seqidno:122所示的cdr2，seqidno:148所示的cdr3(对应于抗体1b1)；

(8)seqidno:88所示的cdr1，seqidno:123所示的cdr2，seqidno:149所示的cdr3(对应于抗体1b10)；

(9)seqidno:89所示的cdr1，seqidno:124所示的cdr2，seqidno:150所示的cdr3(对应于抗体1c4)；

(10)seqidno:90所示的cdr1，seqidno:124所示的cdr2，seqidno:150所示的cdr3(对应于抗体1c9)；

(11)seqidno:91所示的cdr1，seqidno:125所示的cdr2，seqidno:151所示的cdr3(对应于抗体1d6)；

(12)seqidno:92所示的cdr1，seqidno:126所示的cdr2，seqidno:152所示的cdr3(对应于抗体1d7)；

(13)seqidno:92所示的cdr1，seqidno:126所示的cdr2，seqidno:153所示的cdr3(对应于抗体1e5)；

(14)seqidno:92所示的cdr1，seqidno:126所示的cdr2，seqidno:173所示的cdr3(对应于抗体4f11)；

(15)seqidno:93所示的cdr1，seqidno:126所示的cdr2，seqidno:152所示的cdr3(对应于抗体1f4；

(16)seqidno:94所示的cdr1，seqidno:127所示的cdr2，seqidno:154所示的cdr3(对应于抗体1g4)；

(17)seqidno:95所示的cdr1，seqidno:128所示的cdr2，seqidno:155所示的cdr3(对应于抗体1g11)；

(18)seqidno:96所示的cdr1，seqidno:129所示的cdr2，seqidno:156所示的cdr3(对应于抗体1h1)；

(19)seqidno:97所示的cdr1，seqidno:126所示的cdr2，seqidno:152所示的cdr3(对应于抗体1h2)；

(20)seqidno:98所示的cdr1，seqidno:130所示的cdr2，seqidno:157所示的cdr3(对应于抗体1h3)；

(21)seqidno:99所示的cdr1，seqidno:126所示的cdr2，seqidno:158所示的cdr3(对应于抗体1h9)；

(22)seqidno:100所示的cdr1，seqidno:131所示的cdr2，seqidno:159所示的cdr3(对应于抗体2a10)；

(23)seqidno:101所示的cdr1，seqidno:132所示的cdr2，seqidno:160所示的cdr3(对应于抗体2b2、4e5)；

(24)seqidno:102所示的cdr1，seqidno:126所示的cdr2，seqidno:152所示的cdr3(对应于抗体2b8；

(25)seqidno:103所示的cdr1，seqidno:133所示的cdr2，seqidno:161所示的cdr3(对应于抗体2b12)；

(26)seqidno:104所示的cdr1，seqidno:134所示的cdr2，seqidno:162所示的cdr3(对应于抗体2c3)；

(27)seqidno:105所示的cdr1，seqidno:135所示的cdr2，seqidno:163所示的cdr3(对应于抗体2e2)；

(28)seqidno:106所示的cdr1，seqidno:136所示的cdr2，seqidno:164所示的cdr3(对应于抗体2g1)；

(29)seqidno:107所示的cdr1，seqidno:137所示的cdr2，seqidno:165所示的cdr3(对应于抗体3b10)；

(30)seqidno:108所示的cdr1，seqidno:138所示的cdr2，seqidno:166所示的cdr3(对应于抗体3d7)；

(31)seqidno:109所示的cdr1，seqidno:139所示的cdr2，seqidno:167所示的cdr3(对应于抗体3d11)；

(32)seqidno:110所示的cdr1，seqidno:140所示的cdr2，seqidno:168所示的cdr3(对应于抗体4a3)；

(33)seqidno:111所示的cdr1，seqidno:118所示的cdr2，seqidno:144所示的cdr3(对应于抗体4a11)；

(34)seqidno:112所示的cdr1，seqidno:126所示的cdr2，seqidno:152所示的cdr3(对应于抗体4d1)；

(35)seqidno:113所示的cdr1，seqidno:141所示的cdr2，seqidno:170所示的cdr3(对应于抗体4d10)；

(36)seqidno:114所示的cdr1，seqidno:142所示的cdr2，seqidno:171所示的cdr3(对应于抗体4e7)；

(37)seqidno:115所示的cdr1，seqidno:126所示的cdr2，seqidno:172所示的cdr3(对应于抗体4f5)；

(38)seqidno:116所示的cdr1，seqidno:143所示的cdr2，seqidno:174所示的cdr3(对应于抗体4g5)；

(39)seqidno:117所示的cdr1，seqidno:118所示的cdr2，seqidno:144所示的cdr3(对应于抗体4h4)。

优选的，针对甲型肝炎病毒的单域抗体，所述单域抗体为seqidno:1-41中的任意一条所示的抗体链。

单域抗体链的编码序列分别如seqidno：42-82所示。

所述的抗体序列还包括框架区fr；所述框架区fr包括fr1、fr2、fr3和fr4的氨基酸序列；框架区fr的氨基酸序列分别为：seqidno:175-195任意一条所示的fr1；seqidno:196-222任意一条所示的fr2；seqidno:223-252任意一条所示的fr3；seqidno:253-254任意一条所示的fr4。上述所有fr1、fr2、fr3和fr4的序列可以替换为其保守序列变体。

本发明的又一个目的是提供一种核苷酸分子，该核苷酸分子编码所述的针对甲型肝炎病毒的单域抗体，其核苷酸序列如seqidno：42-82中的任意一条所示。

本发明还提供一种表达载体，其包含所述核苷酸分子。

本发明还涉及一种宿主细胞，其可以表达出所述的针对甲型肝炎病毒的单域抗体，或其包含所述的表达载体。

本发明还提供针对甲型肝炎病毒的单域抗体的fc融合抗体。

本发明还公开特异性针对甲型肝炎病毒的单域抗体在hav-elisa检测试剂盒的制备中的应用，由前述针对甲型肝炎病毒的单域抗体在检测人群中甲型肝炎病毒含量的半定量和定量检测方法。

相对于现有技术，本发明的有益效果是：单域抗体作为甲型肝炎病毒检测抗体，其与传统单克隆抗体相比，其亲和力更高，与现有检测试剂盒中多克隆抗体相比，其成分更加单一，批间稳定性更好，质量控制更容易实施。从而使实际的甲型肝炎病毒含量检测结果可信度更高。

附图说明

图1是对所构建的单域抗体噬菌体展示文库进行目的片段插入率检测的pcr产物电泳图，从左至右，泳道1和泳道17是dna分子marker，泳道2-16、泳道18-33是从构建的针对甲型肝炎病毒单域抗体文库中随机挑取的不同克隆的pcr产物；

图2是对所构建的单域抗体文库进行生物淘选的具体结果，其中p/n＝生物淘选中阳性孔洗脱下的噬菌体感染tg1细菌后生长的单克隆细菌数/阳性孔洗脱下的噬菌体感染tg1细菌后生长的单克隆细菌数，该参数在富集发生后会逐渐增大；i/e＝生物淘选中每轮加入阳性孔的噬菌体总量/生物淘选中每轮从阳性孔洗脱出的噬菌体总量，该参数在富集发生后会逐渐趋近于1；

图3是部分表达纯化后的针对甲型肝炎病毒的单域抗体，蛋白粗液经镍柱树脂亲和层析纯化后的sds-page电泳图，从左至右，泳道1是蛋白marker，泳道2-14是经镍离子耦联的琼脂糖树脂一步亲和层析纯化后的单域抗体，其中vhh1-13与seqidno.1-13一一对应；

图4是纯化后的纳米抗体与甲肝病毒结合的elisa检测结果；

图5是纯化后的纳米抗体对甲肝病毒衣壳蛋白的配对elisa检测结果；

图6是融合表达载体的示意图。

具体实施方式

下面结合实施例对本发明做进一步的详细说明，以令本领域技术人员参照说明书文字能够据以实施。

针对甲型肝炎病毒的纳米抗体

单域抗体(sdab，也被研发者ablynx称为纳米抗体或vhh)是本领域技术人员公知的。单域抗体为其互补决定区是单域多肽的一部分的抗体。因此，单域抗体包含单个互补决定区(单个cdr1、单个cdr2和单个cdr3)。单域抗体的实例为仅有重链的抗体(该抗体天然不包含轻链)、衍生自常规抗体的单域抗体和工程化抗体。

单域抗体可以衍生自任何物种，包括小鼠、人、骆驼、美洲驼、山羊、兔和牛。例如，天然存在的vhh分子可以衍生自骆驼科物种(例如骆驼、单峰骆驼、美洲驼和原驼)提供的抗体。像完整的抗体一样，单域抗体能够选择性地结合特定抗原。单域抗体可以仅含有免疫球蛋白链的可变结构域，该结构域具有cdr1、cdr2和cdr3以及框架区。纳米抗体的分子量仅为约12-15kda，比由两条重链和两条轻链组成的普通抗体的分子量(150-160kda)小得多。

值得一提的是，本发明中，与本发明公开的cdr1-3的序列同源性高的序列，也可以得到针对甲肝病毒的纳米抗体。在一些实施例中，与(1)-(39)中的序列具有“至少80％同源性”的序列，或者“至少85％同源性”、“至少90％同源性”、“至少95％同源性”、“至少98％同源性”的序列都可以实现发明目的(即衍生蛋白)。

在一些实施例中，与(1)-(39)中的序列相比仅仅替换一个或少数几个氨基酸的序列，例如，包含1、2、3、4、5、6、7、8、9或10个保守氨基酸取代，也可以实现发明目的。实际上，在确定两个氨基酸序列之间的序列同一性程度或在确定单域抗体中的cdr1、cdr2和cdr3组合时，技术人员可以考虑所谓的“保守”氨基酸取代，在取代情况下，所述取代将优选为保守氨基酸取代，所述保守氨基酸，其通常可以被描述为氨基酸残基被具有相似化学结构的另一个氨基酸残基替代的氨基酸取代，并且该取代对多肽的功能、活性或其它生物学性质几乎没有或基本上没有影响。所述保守氨基酸取代在本领域是通用的，例如保守氨基酸取代是以下(a)-(d)组内的一个或少数几个氨基酸被同一组内另一个或少数几个氨基酸所取代：(a)极性带负电残基及其不带电酰胺：asp、asn、glu、gln；(b)极性带正电残基：his、arg、lys；(c)芳香族残基：phe、trp、tyr；(d)脂肪族非极性或弱极性残基：ala、ser、thr、gly、pro、met、leu、ile、val、cys。特别优选的保守氨基酸取代如下：asp被glu取代；asn被gln或his取代；glu被asp取代；gln被asn取代；his被asn或gln取代；arg被lys取代；lys被arg、gln取代；phe被met、leu、tyr取代；trp被tyr取代；tyr被phe、trp取代；ala被gly或ser取代；ser被thr取代；thr被ser取代；gly被ala或pro取代；met被leu、tyr或ile取代；leu被ile或val取代；ile被leu或val取代；val被ile或leu取代；cys被ser取代。另外，本领域技术人员知晓，单域抗体的创造性体现在cdr1-3区，而框架区序列fr1-4并非是不可改变的，fr1-4的序列可以采取本发明公开的序列的保守序列变体。

本发明采用甲型肝炎病毒灭活疫苗来免疫阿拉善双峰驼，经过7次免疫之后提取该双峰驼外周血淋巴细胞并建立甲型肝炎病毒的单域抗体文库。在抗体筛选过程中，将甲型肝炎病毒衣壳蛋白偶联生物素(vp2-vp3-vp1-biotin)，将中性亲和素蛋白偶联在酶标板上，利用生物素和中性亲和素结合的特性，使甲型肝炎病毒衣壳蛋白间接展示在酶标板表面，使其抗原表位得以暴露，然后利用噬菌体展示技术筛选甲型肝炎病毒灭活疫苗免疫后的单域抗体基因库(骆驼重链抗体噬菌体展示基因库)，获得了能在大肠杆菌中高效表达的单域抗体株。

将筛选获得的单域抗体在大肠杆菌中表达后通过镍离子偶联的琼脂糖进行纯化，将纯化后的纳米抗体进行分析。

下面结合具体实施例，进一步阐述本发明。

实施例1：针对甲型肝炎病毒的单域抗体文库的构建：

(1)使用1ml甲型肝炎病毒灭活疫苗免疫一只内蒙古阿拉善双峰驼，每周一次，共连续免疫7次，免疫过程中刺激b细胞表达特异性的纳米抗体；(2)免疫结束后，提取骆驼外周血淋巴细胞100ml并提取总rna；(3)合成cdna并利用套式pcr扩增vhh；(4)利用限制性内切酶psti及noti酶切20μgpmecs噬菌体展示载体及10μgvhh并连接两种片段；(5)将连接产物转化至电转感受态细胞tg1中，构建针对甲肝病毒衣壳蛋白噬菌体展示文库并测定库容，文库库容的大小约为2×10⁹；同时，通过菌落pcr检测所建文库中目的片段的正确插入率，图1显示菌落pcr结果，随机的选取30个克隆做菌落pcr，结果显示插入率达到93.3％。

实施例2：甲型肝炎病毒衣壳蛋白的表达：

(1)从ncbi的蛋白数据库中查到甲型肝炎病毒衣壳蛋白的序列信息(accesionnumber:p08617.1)；(2)经过uniprot分析，选取vp2-vp3-vp1作为目的表达片段；(3)在vp2-vp3-vp1的5’端添加kozak增强序列(5’-ggatcgaaccctt-3’)及iggkappa的信号肽(metdtlllwvlllwvpgstgd)，3’端添加6*his标签融合表达；(4)使用实施例8中的所述方案进行真核表达；(5)参照实施例9所述方案使用镍柱亲和层析进行纯化；(6)参照实施例9方法进行sds-page分析，确定蛋白纯度在95％以上，且浓度在0.5mg/ml以上后，低温保存备用。

实施例3：针对甲型肝炎病毒单域抗体的筛选：

(1)取200μl重组tg1细胞至2×ty培养基中培养，期间加入40μl辅助噬菌体vcsm13侵染tg1细胞，并培养过夜以扩增噬菌体，次日利用peg/nacl沉淀噬菌体，离心收集扩增噬菌体；(2)将稀释在100mmph8.3的nahco3中的中性亲和素蛋白500ug偶联在酶标板上，4℃放置过夜，同时设立阴性对照孔；(3)第二天加入100μl生物素标记的甲型肝炎病毒衣壳蛋白(vp2-vp3-vp1-biotin)，室温孵育2小时，阴性对照孔加入100μlpbs；(4)2小时后，加入100μl的3％的脱脂乳，室温封闭2h；(5)封闭结束后，加入100μl扩增后噬菌体文库(大约2×10¹¹个噬菌体颗粒)，室温作用1h；(6)作用1小时后，用pbs+0.05％tween-20洗5遍，以洗掉未结合的噬菌体；(7)用终浓度为25mg/ml的胰蛋白酶将与甲型肝炎病毒衣壳蛋白特异性结合的噬菌体解离下，并感染处于对数生长期的大肠杆菌tg1细胞，37℃培养1h，产生并收集噬菌体用于下一轮的筛选，相同筛选过程重复3轮，逐步得到富集，富集效果如图2所示，p/n值从第一轮的2.2到第三轮约为30，i/e质从第一轮的13333到第三轮约为333，证明文库针对甲型肝炎病毒衣壳蛋白具有非常明显的富集。

实施例4：用噬菌体的酶联免疫方法(elisa)筛选特异性阳性克隆:

(1)根据上述单域抗体筛选方法对甲型肝炎病毒纳米抗体进行3轮筛选，筛选结束后，针对甲型肝炎病毒衣壳蛋白的噬菌体富集因子达到10以上(约为30)，从筛选获得的阳性克隆中挑选400个单菌落分别接种于含100μg/ml氨苄青霉素的tb培养基的96深孔板中，并设置空白对照，37℃培养至对数期后，加入终浓度为1mm的iptg，28℃培养过夜；(2)利用渗透涨破法获得粗提抗体；将中性亲和素蛋白稀释至100mmph8.3的nahco3中并将100μg中性亲和素蛋白在酶标板中4℃包被过夜，次日向酶标板内加入100ugvp2-vp3-vp1-biotin蛋白；(3)将上述步骤中获得的抗体粗提液取100ul转移至加入抗原的elisa板上，室温孵育1h；(4)用pbst洗去未结合的抗体，加入100ul经1:2000稀释后的mouseanti-hatagantibody(鼠抗ha抗体，thermofisher)，在室温孵育1h；(5)用pbst洗去未结合的抗体，加入100ul经1:20000稀释后的anti-rabbithrpconjugate(山羊抗兔辣根过氧化物酶标记抗体，购自于thermofisher)，在室温孵育1h；(6)用pbst洗去未结合的抗体，加入辣根过氧化物酶显色液，37℃下反应15min后，加入中止液，于酶标仪上450nm波长处，读取吸收值；(7)当样品孔od值大于对照孔5倍以上时，判定为阳性克隆孔；(8)将阳性克隆孔的菌转摇在含有100μg/ul氨苄青霉素的lb培养基中以便提取质粒并进行测序。

根据序列比对软件vectornti分析各个克隆株的基因序列，把cdr1，cdr2，cdr3序列相同的株视为同一克隆株，而序列不同的株视为不同克隆株，最终获得特异性针对甲型肝炎病毒的单域抗体。其抗体的氨基酸序列为fr1-cdr1-fr2-cdr2-fr3-cdr3-fr4结构，构成整个vhh。获得的单域抗体重组质粒可以在原核系统中进行表达，最终获得单域抗体蛋白。

其中，其中seqidno.1-41依次为抗体编号1a2、1a4、1a6、1a7、1b1、1b4、1b10、1c4、1c9、1d6、1d7、1e5、1f4、1g4、1g11、1h1、1h2、1h3、1h9、2a10、2b2、2b8、2b12、2c3、2e2、2g1、3b10、3d7、3d11、4a1、4a3、4a9、4a11、4d1、4d10、4e5、4e7、4f5、4f11、4g5、4h4。

41种单域抗体序列的cdr1-3的序列如表1-2所示，41种单域抗体的fr1-3序列如表3-4所示。其中41种单域抗体中，除了抗体1b1的fr4序列为seqidno:254(grgtqvtvss)以外，其它40种抗体的fr4序列均为seqidno:253(gqgtqvtvss)。

表141种单域抗体的cdr1-2序列

表241种单域抗体的cdr3序列

表341种单域抗体的fr1、fr2序列

表441种单域抗体的fr3序列

实施例5：甲型肝炎病毒的特异性单域抗体在宿主菌大肠杆菌中的纯化及表达

(1)将上述测序分析所获得不同克隆株的质粒(pmecs-vhh)电转化到大肠杆菌hb2151中，并将其涂布在lb+amp+glucose即含有氨苄青霉素和葡萄糖的培养平板上，37℃培养过夜；(2)挑选单个菌落接种在5ml含有岸边青霉素的lb培养液中，37℃摇床培养过夜；(3)接种1ml的过夜培养菌种至330mltb培养液中，37℃摇床培养，培养到od600nm值达到0.6-0.9时，加入1miptg，28℃摇床培养过夜；(4)离心，收集大肠杆菌，利用渗透胀破法，获得抗体粗提液；(5)通过镍柱亲和层析法纯化出抗体，纯化后的单域抗体，如图3所示。

实施例6：甲型肝炎病毒的特异性单域抗体的fc融合抗体真核表达载体的构建

将实施例4中获得的目标序列亚克隆至真核表达载体中：(1)将实施例4中筛选出来的抗体经sanger测序得到其核苷酸序列；(2)通过序列合成的方式将密码子优化后的上述核苷酸序列(seqidno.42-82)合成至本公司设计改造的载体rjk-v4-hfc中(即seqidno.42-82分别插入载体rjk-v4-hfc的多克隆位点mcs，rjk-v4-hfc载体的构建过程如实施例11所述)；(3)将构建好的重组真核表达载体转化至dh5α大肠杆菌中，培养进行质粒大提，去除内毒素；(4)将大提后的质粒再进行序列测序鉴定；(5)将确定无误后的重组载体准备后续真核细胞转染表达。

实施例7：甲型肝炎病毒的特异性单域抗体的fc融合抗体在悬浮expicho-s细胞中表达

(1)转染前3天以2.5×10⁵/ml细胞传代和扩大培养expicho-stm细胞，计算出的所需的细胞体积转移至装有新鲜的已预热的120ml(终体积)的expichotm表达培养基的500ml摇瓶中；使细胞浓度达到约4×10⁶-6×10⁶活细胞/ml；(2)在转染前一天，将expicho-stm细胞稀释浓度至3.5×10⁶活细胞/ml，使细胞过夜培养；(3)转染当天，测定细胞密度和活细胞百分比。转染之前细胞密度应达到约7×10⁶-10×10⁶活细胞/ml；(4)用预热至37℃新鲜的expichotm表达培养基将细胞稀释至6×10⁶个活细胞/ml。计算出的所需的细胞体积转移至装有新鲜的已预热的100ml(终体积)的expichotm表达培养基的500ml摇瓶中；(5)使轻轻颠倒混匀expifectaminetmcho试剂，用3.7mloptiprotm培养基稀释expifectaminetmcho试剂，回荡或混匀；(6)用冷藏的4mloptiprotm培养基稀释质粒dna，回荡混匀；(7)将expifectaminecho/质粒dna复合物室温孵育1-5分钟，然后轻轻加入制备的细胞悬液中，加入过程中轻轻回荡摇瓶，所述的质粒dna为实施例6制得的甲型肝炎病毒的特异性单域抗体的fc融合抗体真核表达载体；(8)将细胞在37℃、8％co2、加湿的空气中震荡培养；(9)转染后第1天(18-22小时后)添加600ulexpifectaminetmchoenhancer和24mlexpichofeed。(10)在转染后约8天(细胞活率低于70％)收集上清。

实施例8：甲型肝炎病毒的特异性单域抗体的fc融合抗体在悬浮293f细胞中的表达

重组单域抗体表达实验流程(以500ml摇瓶为例)：

1、转染前3天以2.5×10⁵/ml细胞传代和扩大培养293f细胞，计算出的所需的细胞体积转移至装有新鲜的已预热的120ml(终体积)的opm-293cd05medium培养基的500ml摇瓶中。使细胞浓度达到约2×10⁶-3×10⁶活细胞/ml。

2、转染当天，测定细胞密度和活细胞百分比。转染之前细胞密度应达到约2×10⁶-3×10⁶活细胞/ml。

3、用预热的opm-293cd05medium将细胞稀释至1×10⁶个活细胞/ml。计算出所需的细胞体积转移至装有新鲜的已预热的100ml(终体积)的培养基的500ml摇瓶中。

4、用4mlopti-mem培养基稀释pei(1mg/ml)试剂，回荡或吹打混匀；用4mlopt-mem培养基稀释质粒dna，回荡混匀，并用0.22um的滤头过滤。室温孵育5min；所述的质粒dna为实施例6制得的甲型肝炎病毒的特异性单域抗体的fc融合抗体真核表达载体。

5、将稀释的pei试剂加入稀释的dna中，颠倒混匀。将pei/质粒dna复合物室温孵育15-20分钟，然后轻轻加入制备的细胞悬液中，加入过程中轻轻回荡摇瓶。

6、将细胞在37℃、5％co2、120rpm震荡培养。

7、转染后第24h、72h添加5mlopm-chopff05补料。

8、在转染后约7天(细胞活率低于70％)收集上清。

实施例9：人源fc重组单域抗体的纯化

1、将实施例7或8中获得蛋白表达上清用0.45μm的一次性滤头过滤除掉不可溶杂质；

2、将上述滤液使用蛋白纯化仪进行亲和层析纯化，利用人源fc与proteina结合的能力，使用偶联proteina的琼脂糖填料进行纯化；

3、将滤液通过1ml/分钟的流速流穿proteina预装柱，该步骤中滤液中的目标蛋白会与填料结合；

4、通过低盐和高盐缓冲液将柱上结合的杂质蛋白洗涤；

5、用低ph缓冲液将柱上结合的目标蛋白进行系统；

6、将洗脱液迅速加入ph9.0的tris-hcl溶液，进行中和；

7、将上述中和后的蛋白溶液透析后，进行sds-page分析，确定蛋白纯度在95％以上，且浓度在0.5mg/ml以上后，低温保存备用。

实施例10：特异性针对甲型肝炎病毒衣壳蛋白的重组单域抗体与甲型肝炎病毒亲和力的elisa检测以及检测标准曲线的绘制

(1)将实施例9中纯化获得重组抗体(实施例7制得的特异性单域抗体的fc融合抗体经实施例9纯化而得)对甲型肝炎病毒的亲和力进行初步检测；

(2)首先使用定量的标准品测定重组单域抗体与甲肝病毒的亲和力步骤如下(即下面的步骤(3)-(9)；

(3)将100ng/100μl甲型肝炎病毒衣壳蛋白样品包被到elisa板材上；

(4)使用脱脂奶粉对上述包被的板材进行封闭；

(5)加入实施例9中获得的针对甲型肝炎病毒的重组单域抗体；

(6)加入特异性针对人源fc的检测抗体(hrp标记)

(7)加入显色底物tmb；

(8)加入终止液终止反应；

(9)测量od450值(结果如图4所示，可见针对甲型肝炎病毒的单域抗体均与甲型肝炎病毒具有较强的亲和力)；

(10)然后挑选elisa结合力最好的重组抗体进行甲型肝炎病毒含量标准曲线的绘制。

(11)将0-100ng/100μl的甲型肝炎病毒样品包被到elisa板材上；

(12)使用脱脂奶粉对步骤(11)的包被板材进行封闭；

(13)加入步骤(10)中获得的效果最佳的针对甲型肝炎病毒的重组单域抗体；

(14)加入特异性针对人源fc的检测抗体(hrp标记)

(15)加入显色底物tmb；

(16)加入终止液终止反应；

(17)测量od450值，根据结果绘制甲型肝炎病毒含量和od450关系曲线图，选择曲线图线性范围区段进行线性回归计算，作为标准曲线进行其他样品中甲型肝炎病毒含量的测定。

实施例10：甲型肝炎病毒的特异性单域抗体在hav-elisa试剂盒中的应用(双抗夹心法)

(1)将实施例9和实施例5中获得单域抗体通过双抗夹心法检测目标样品中甲型肝炎病毒的含量；

(2)首先使用标准品测定并绘制甲型肝炎病毒含量与od450的标准曲线，步骤如下；

(3)将实施例9中鉴定、纯化得到单域抗体作为捕获抗体包被到elisa板材上；

(4)使用脱脂奶粉对上述包被的板材进行封闭；

(5)加入标准样品孵育；

(6)加入实施例5中获得的单域抗体作为检测抗体(带有ha标签)，孵育；

(7)加入抗ha的抗体孵育(hrp标记)；

(8)加入显色底物tmb；

(9)加入终止液终止反应；

(10)测量od450值，并作出比值分析，结果如图5所示；

(11)然后基于上述步骤获得的标准曲线对待检样品进行检测。

实施例11：纳米抗体真核表达载体rjk-v4-hfc的构建

所提及纳米抗体通用的目标载体rjk-v4-hfc，为本公司在invitrogen商业化载体pcdna3.4(载体资料链接

https://assets.thermofisher.com/tfs-assets/lsg/manuals/pcdna3_4_topo_ta_cloning_kit_man.pdf)的基础上融合了人源igg的重链编码序列(ncbiaccessionno.:ab776838.1)中的fc区段后改造而来的，即该载体包含了igg重链的铰链区(hinge)ch2和ch3区。具体改造方案如下：

(1)选取pcdna3.4上的限制性酶切位点xbai和agei；

(2)在fc片段编码序列的5’端和3’端通过重叠pcr的方式分别引入多克隆位点(mcs，multiplecloningsite)和6×his标签，如图6所示；

(3)使用分别带有xbai和agei酶切位点的一对引物通过pcr的方式将上述片段扩增；

(4)使用限制性内切酶xbai和agei分别酶切pcdna3.4和(3)中的重组dna片段；

(5)将酶切后的载体和插入片段在t4连接酶的作用下连接，然后将连接产物转化至大肠杆菌，扩增，测序核实，获得重组质粒。

尽管本发明的实施方案已公开如上，但其并不仅仅限于说明书和实施方式中所列运用，它完全可以被适用于各种适合本发明的领域，对于熟悉本领域的人员而言，可容易地实现另外的修改，因此在不背离权利要求及等同范围所限定的一般概念下，本发明并不限于特定的细节。

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<211>127

<212>prt

<213>人工序列(artificialsequence)

<400>26

glnvalglnleuvalgluserglyglyglyservalglnalaglygly

151015

serleuargleusercysalaalaserglytyrthrtyrserserser

202530

cysmetglytrppheargglnalaproglylysgluarggluglyval

354045

alathrileaspserpheglyasnthrlystyralaaspservallys

505560

glyargphethrileserlysaspasnalalysasnthrleutyrleu

65707580

glnmetasnserleulysprogluaspthralamettyrtyrcysthr

859095

alaaspmetaspglyargtrpglyglytyrcysvalargseralaglu

100105110

aspphealatyrtrpglyglnglythrglnvalthrvalserser

115120125

<210>27

<211>121

<212>prt

<213>人工序列(artificialsequence)

<400>27

glnvalglnleuvalgluserglyglyglyservalglnthrglygly

151015

serleuargleusercysalaalaserglyhisglysercysthrtyr

202530

aspmetsertrptyrargglnalaproglylysserargglupheval

354045

servalilemetseraspglythrthralatyrthrserservallys

505560

aspargphethrileserglnaspasnalalysserthrvaltyrleu

65707580

glnmetasnserleulysprogluaspthralaargtyrtyrcysglu

859095

alavalleuargglyargcysthrargserglyphethrtyrtrpgly

100105110

glnglythrglnvalthrvalserser

115120

<210>28

<211>126

<212>prt

<213>人工序列(artificialsequence)

<400>28

glnvalglnleuvalgluserglyglyglyservalglnalaglygly

151015

serleuargleusercysthralaserglyserthrtyrserargarg

202530

tyrileglytrppheargglnalaproglylysgluarggluglyval

354045

alathrileserthrglyglyglypheargsertyralaaspserval

505560

lysglyargphethrileserglnaspasnalalysasnthrvaltyr

65707580

leuglnmetasnserleulysprogluaspthralamettyrtyrcys

859095

alaalaglyserasnvalaspargargtrpserleuleuserargasp

100105110

tyrasntyrtrpglyglnglythrglnvalthrvalserser

115120125

<210>29

<211>120

<212>prt

<213>人工序列(artificialsequence)

<400>29

glnvalglnleuvalgluserglyglyglyservalglnproglygly

151015

serleuargleusercysvalvalserglytyrthrileaspargleu

202530

cysthrglytrppheargglnalaproglylysgluargglupheval

354045

serserthrserthraspglyilethrargtyralaaspservallys

505560

glyargphethrvalserserasplysalalysaspthrvaltyrleu

65707580

glnmetasnserleulysprogluaspthralamettyrphecyshis

859095

pheglyglyargtrpleuthrproargalacysasptyrtrpglygln

100105110

glythrglnvalthrvalserser

115120

<210>30

<211>121

<212>prt

<213>人工序列(artificialsequence)

<400>30

glnvalglnleuvalgluserglyglyglyservalglnalaglygly

151015

serleuargleusercysalaalaserglyalaalatyrserasptyr

202530

cysmetglytrppheargglnalaproglylysgluarggluargval

354045

alathrpheleuargaspglyseralaasptyralaalaservalthr

505560

glyargphethrileserlysaspasnvallysasnthrleutyrleu

65707580

glnmetasnserleulysprogluaspthralamettyrtyrcysala

859095

alaasptyrtrpglytyrglntyrasncysargpheglutyrtrpgly

100105110

glnglythrglnvalthrvalserser

115120

<210>31

<211>132

<212>prt

<213>人工序列(artificialsequence)

<400>31

glnvalglnleuvalgluserglyglyglyservalglnalaglygly

151015

serleuargleusercysalavalserglytyrthrvalserargser

202530

cysmetalatrppheargglnvalproglylysgluarggluglyval

354045

alaserileserseraspglyglyilethrthrtyrvalaspserval

505560

lysglyargphethrileserglnaspasnalalysasnthrleutyr

65707580

leuglnmetasnservallysprogluaspthralavaltyrtyrcys

859095

alathrglyvalleuglyphepheserasnleucysargserthrval

100105110

valproprothralaargpheargtyrtrpglyglnglythrglnval

115120125

thrvalserser

130

<210>32

<211>121

<212>prt

<213>人工序列(artificialsequence)

<400>32

glnvalglnleuvalgluserglyglyglyservalglnalaglygly

151015

serleuargleusercysalaalaserglyalaalatyrserasptyr

202530

cysmetglytrppheargglnalaproglylysgluarggluargval

354045

alathrpheleuargaspglyseralaasptyralaalaservalthr

505560

glyargphethrileserlysaspasnvallysasnthrleutyrleu

65707580

glnmetasnserleulysprogluaspthralamettyrtyrcysala

859095

valasptyrtrpglytyrglytyrasncysargpheglutyrtrpgly

100105110

glnglythrglnvalthrvalserser

115120

<210>33

<211>127

<212>prt

<213>人工序列(artificialsequence)

<400>33

glnvalglnleuvalgluserglyglyglyservalglnalaglygly

151015

serleuargleuthrcysalavalserglytyrsertyrserserasn

202530

hismetglytrppheargglnalaproglylysgluarggluglyval

354045

alavalilephethrglyglyglyargthrtrptyralaasnserval

505560

lysglyargphethrileserhisaspasnalalysasnthrvaltyr

65707580

leuglnmetasnserleulysleugluaspthralamettyrtyrcys

859095

alaalaasptyrvalprovalglyargargtyralavalleuasppro

100105110

asptyrthrtyrtrpglyglnglythrglnvalthrvalserser

115120125

<210>34

<211>125

<212>prt

<213>人工序列(artificialsequence)

<400>34

glnvalglnleuvalgluserglyglyglyservalglnalaglygly

151015

serleuargleusercysalaalaserglytyrthrtrpserserthr

202530

cysmetglytrppheargglnalaproglylysgluargglnglyval

354045

alaalaileserproglyglyserphelystyrtyralaaspserval

505560

lysglyargphethrileserargaspasnalalysasnthrvaltyr

65707580

leuglnmetasnserleuglnprogluaspthralamettyrtyrcys

859095

alaalaalaprophealacysaspglyasptrptyrserglyargtrp

100105110

asnasntrpglyglnglythrglnvalthrvalserser

115120125

<210>35

<211>126

<212>prt

<213>人工序列(artificialsequence)

<400>35

glnvalglnleuvalgluserglyglyglyleuvalglnproglygly

151015

serprolysleusercysalaalaserglyphethrpheserasntyr

202530

alametsertrpvalargglnalaproglylysvalleuglutrpval

354045

serpheileasnserglyglyglyglythralatyralaaspproval

505560

lysglyargphethrileserargaspasnalalysasnthrleutyr

65707580

leuglnleuasnserleulysthrgluaspthralamettyrtyrcys

859095

alalysglyleuglntyraspthrtyrcysserserglntyrcystyr

100105110

alaasnproglyglyglnglythrglnvalthrvalserser

115120125

<210>36

<211>126

<212>prt

<213>人工序列(artificialsequence)

<400>36

glnvalglnleuvalgluserglyglyglythrvalglnalaglygly

151015

serleuargleusercysalaalaserglytyrthrtyrserserala

202530

cysmetglytrppheargglnalaproglylysgluarggluglyval

354045

alathrileglyseraspglyserthrsertyrthraspservallys

505560

glyargphethrileserlysaspasnvalhisasnasnleutyrleu

65707580

glnmetasnserleulysprogluaspthralamettyrtyrcysala

859095

alathrilethrthrvalthrmetcysargargargaspglyargleu

100105110

pheglyhistrpglyglnglythrglnvalthrvalserser

115120125

<210>37

<211>126

<212>prt

<213>人工序列(artificialsequence)

<400>37

glnvalglnleuvalgluserglyglyglyservalglnalaglygly

151015

serleuargleusercysalavalserglypheprotyrserthrtyr

202530

tyrmetglytrppheargglnalaproglylysgluarggluglyval

354045

alaalavaltyrproglyvalglyleulysargtyralaaspserval

505560

lysglyargphethrileserglnaspaspalalysasnthrleutyr

65707580

leuglnmetasnserleulysprogluaspthralaleutyrtyrcys

859095

alavalaspglnalavalserleuglytrpgluleuserproglyarg

100105110

tyrargtyrtrpglyglnglythrglnvalthrvalserser

115120125

<210>38

<211>125

<212>prt

<213>人工序列(artificialsequence)

<400>38

glnvalglnleuvalgluserglyglyglyservalglnthrglygly

151015

serleulysleusercysvalhisserglytyrsertyrserserval

202530

cysmetalatrppheargglnalaproglylysgluarggluglyval

354045

alaalaileserproglyglyserphelystyrtyrvalaspserval

505560

lysglyargphethrileserargaspasnserlysasnthrglytyr

65707580

leuglnmetasnserleulysprogluaspthralamettyrtyrcys

859095

alathrserargserglycysargaspaspphepheglntyrglutyr

100105110

asptyrtrpglyglnglythrglnvalthrvalserser

115120125

<210>39

<211>125

<212>prt

<213>人工序列(artificialsequence)

<400>39

glnvalglnleuvalgluserglyglyglyseralaglnalaglygly

151015

serleuargleusercysalaalaserglyasnsertyrserargasn

202530

cysmetglytrppheargglnalaproglylysgluargglnglyval

354045

alaalaileserproglyglyserphelystyrtyralaaspserval

505560

lysglyargphethrileserargaspasnalalysasnthrvaltyr

65707580

leuglnmetasnserleuglnprogluaspthralamettyrtyrcys

859095

alaalaalaproleualacysaspglyasptrptyrserglyargtrp

100105110

asnasntrpglyglnglythrglnvalthrvalserser

115120125

<210>40

<211>128

<212>prt

<213>人工序列(artificialsequence)

<400>40

glnvalglnleuvalgluserglyglyglyservalproalaglygly

151015

serleuargleusercysalaalaserglytyrthrtyrserglyser

202530

cysmetglytrppheargglnalaproglylysgluargglugluval

354045

alathriletyrthraspasnglyphelystyrtyrthrgluserval

505560

lysglyargphethrileserglnasplysalalysasnthrvaltyr

65707580

leuglnmetasnserleulysprogluaspthralavaltyrtyrcys

859095

alaalaaspglyglythrglyleutrptyrthrcysalaargglygln

100105110

ileserhisasntyrtrpglyglnglythrglnvalthrvalserser

115120125

<210>41

<211>127

<212>prt

<213>人工序列(artificialsequence)

<400>41

glnvalglnleuvalgluserglyglyglyservalglnalaglygly

151015

serleuargleusercysthrvalserglytyrsertyrserserasp

202530

tyrmetglytrppheargglnalaproglylysgluarggluglyval

354045

alavalilephethrglyglyglyargthrtrptyralaasnserval

505560

lysglyargphethrileserglnaspasnalalysasnthrvaltyr

65707580

leuglnmetasnserleulysleugluaspthralamettyrtyrcys

859095

alaalaasptyrvalprovalglyargargtyralavalleuasppro

100105110

asptyrthrtyrtrpglyglnglythrglnvalthrvalserser

115120125

<210>42

<211>381

<212>dna

<213>人工序列(artificialsequence)

<400>42

caggtgcagctggtggagagcggcggcggcagcgtgcaggccggcggcagcctgaggctg60

agctgcgccgtgagcggctacagcggcaggagcgactacatgggctggttcaggcaggcc120

cccggcaaggagagggagggcgtggccgtgatcttcaccggcggcggcaggacctggtac180

gccaacagcgtgaagggcaggttcaccatcagccagggcaacgccgagaacaccgtgtac240

ctgcagatgaacagcctgaagctggaggacaccgccatgtactactgcgccgccgactac300

gtgcccgtgggcaggaggtacgccgtgctggaccccgactacacctactggggccagggc360

acccaggtgaccgtgagcagc381

<210>43

<211>363

<212>dna

<213>人工序列(artificialsequence)

<400>43

caggtgcagctggtggagagcggcggcggcctggtgcagcccggcggcagcctgaggctg60

agctgcgccgccagcggcgccgcctacagcgactactgcatgggctggttcaggcaggcc120

cccggcaaggagagggagagggtggccaccttcctgagggacggcagcgccgactacgcc180

gccagcgtgaccggcaggttcaccatcagcaaggacaacgtgaagaacaccctgtacctg240

cagatgaacagcctgaagcccgaggacaccgccatgtactactgcgccgccgactactgg300

ggctacggctacaactgcaggttcgagtactggggccagggcacccaggtgaccgtgagc360

agc363

<210>44

<211>381

<212>dna

<213>人工序列(artificialsequence)

<400>44

caggtgcagctggtggagagcggcggcggcagcgtgcaggccggcggcagcctgaggctg60

agctgcgccgccagcggctacgactacagcacctactgcatgggctggttcaggcaggcc120

cccggcaaggagagggagggcgtggccagcatcagcaccgtgggcaccaccagctacgcc180

gacagcgtgaagggcaggttcaccatcagcagggacaacgccaagaacaccgtgtacctg240

cagatgaacagcctgaagaccgaggacaccaacatgtactactgcgccagcctggacagg300

aggatgtacggcggcatctggtactgcgacctgaggaggtacaactactggggccagggc360

acccaggtgaccgtgagcagc381

<210>45

<211>345

<212>dna

<213>人工序列(artificialsequence)

<400>45

caggtgcagctggtggagagcggcggcggcagcgtgcagcccggcggcagcctgaggctg60

agctgcgccgccagcggcttcaccttcagcagctaccccatgaactgggtgaggcaggcc120

cccggcaagggcctggagtgggtgagcgccatcaacagcggcggcggcagcacctactac180

gccgacagcgtgaagggcaggttcaccatcagcagggacaacgccaagaacaccctgtac240

ctgcagctgaacggcctgaagaccgaggacaccgccatgtactactgcgccctggacagc300

aggggcacccagaggggccagggcacccaggtgaccgtgagcagc345

<210>46

<211>363

<212>dna

<213>人工序列(artificialsequence)

<400>46

caggtgcagctggtggagagcggcggcggcctggtgcagcccggcggcagcctgaggctg60

agctgcgccgccagcggcttcaccttcagcaggtacgacatgagctgggtgaggcagccc120

cccggcaagggcctggagtgggtgagcgccatctacagcggcagcggcaacacctactac180

gccgacagcgtgaagggcaggttcaccatcagcagggacaacgccaagaacaccctgtac240

ctgcagatgaacagcctgaagaccgaggacaccgccgtgtactactgcgccgccgactgg300

accggcaggaccctgcacgagtacaactacctgggcaggggcacccaggtgaccgtgagc360

agc363

<210>47

<211>381

<212>dna

<213>人工序列(artificialsequence)

<400>47

caggtgcagctggtggagagcggcggcggcagcgtgcaggccggcggcagcctgaggctg60

agctgcgccgtgagcggctacagcggcaggagcgactacatgggctggttcaggcaggcc120

cccggcaaggagagggagggcgtggccgtgatcttcaccggcggcggcaggacctggtac180

gccaacagcgtgaagggcaggttcaccatcagccaggacaacgccaagaacaccgtgtac240

ctgcagatgaacagcctgaagctggaggacaccgccatgtactactgcgccgccgactac300

gtgcccgtgggcaggaggtacgccgtgctggaccccgactacacctactggggccagggc360

acccaggtgaccgtgagcagc381

<210>48

<211>369

<212>dna

<213>人工序列(artificialsequence)

<400>48

caggtgcagctggtggagagcggcggcggcagcgtgcaggccggcggcagcctgaggctg60

agctgcgccgtgagcggctacatcagccacccctactgcatggcctggttcaggcaggcc120

cccggcaaggagagggagggcgtggccggcatcagcatcggcggcggcggcaccttcacc180

gccgacagcgtgaagggcaggttcaccatcagccaggacaacgccaagaacaccgtgcac240

ctgaggatgaacagcctgaagcccgaggacaccgccatgtactactgcgccgccaggggc300

ggctactactgcaccgccagcgccagctacaactactggggccagggcacccaggtgacc360

gtgagcagc369

<210>49

<211>378

<212>dna

<213>人工序列(artificialsequence)

<400>49

caggtgcagctggtggagagcggcggcggcagcgtgcagcccggcggcagcctgaggctg60

agctgcgccgccagcggctacacctacagcaggtacagcatgggctggttcaggcaggcc120

cccggcaaggagagggagggcgtggccagcctggacagcgacggcgccaccatctacacc180

gacagcgtgaagggcaggttcaccatcgccaaggacaacgccaagaacaccctgtacctg240

cagatgaacagcctgaagcccgaggacaccggcatgtactactgcgccgcccagaggatc300

aggtgggtggccttcgtgagcctgaggagcgccgacttcgactactggggccagggcacc360

caggtgaccgtgagcagc378

<210>50

<211>378

<212>dna

<213>人工序列(artificialsequence)

<400>50

caggtgcagctggtggagagcggcggcggcagcgtgcaggccggcggcagcctgaggctg60

agctgcgccgccagcggctacgcctacagcaggtacagcatgggctggttcaggcaggcc120

cccggcaaggagagggagggcgtggccagcctggacagcgacggcgccaccatctacacc180

gacagcgtgaagggcaggttcaccatcgccaaggacaacgccaagaacaccctgtacctg240

cagatgaacagcctgaagcccgaggacaccggcatgtactactgcgccgcccagaggatc300

aggtgggtggccttcgtgagcctgaggagcgccgacttcgactactggggccagggcacc360

caggtgaccgtgagcagc378

<210>51

<211>387

<212>dna

<213>人工序列(artificialsequence)

<400>51

caggtgcagctggtggagagcggcggcggctgggtgcaggccggcggcagcctgaggctg60

agctgcgccgccagcggcgtgaccagcaagagggcctgcatgggctggttcaggcaggcc120

cccggcaaggagagggagtgggtggccaccatcagcaggcccggcggcgccacctacagc180

gccgacagcgtgaagggcaggttcaccatcagccaggacaacgccgagaacatggccttc240

ctgatcctgaacagcctgaaggccgaggacgccgccatctactactgcgccttcagggtg300

atcgaccccttcgacacctgcagcagcaggaggagggacggcgacttcccctactggggc360

cagggcacccaggtgaccgtgagcagc387

<210>52

<211>375

<212>dna

<213>人工序列(artificialsequence)

<400>52

caggtgcagctggtggagagcggcggcggcagcgcccaggccggcggcagcctgaggctg60

agctgcgccgccagcggcaacagctacagcaggaactgcatgggctggttcaggcaggcc120

cccggcaaggagaggcagggcgtggccgccatcagccccggcggcagcttcaagtactac180

gccgacagcgtgaagggcaggttcaccatcagcagggacaacgccaagaacaccgtgtac240

ctgcagatgaacagcctgcagcccgaggacaccgccatgtactactgcgccgccgccccc300

ttcgcctgcgacggcgactggtacagcggcaggtggaacaactggggccagggcacccag360

gtgaccgtgagcagc375

<210>53

<211>375

<212>dna

<213>人工序列(artificialsequence)

<400>53

caggtgcagctggtggagagcggcggcggcagcgcccaggccggcggcagcctgaggctg60

agctgcgccgccagcggcaacagctacagcaggaactgcatgggctggttcaggcaggcc120

cccggcaaggagaggcagggcgtggccgccatcagccccggcggcagcttcaagtactac180

gccgacagcgtgaagggcaggttcaccatcagcagggacaacgccaagaacaccgtgtac240

ctgcagatgaacagcctgcagcccgaggacaccgccatgtactactgcgccgccgccccc300

ttcgcctgcgacggcgactggcacagcggcaggtggaacaactggggccagggcacccag360

gtgaccgtgagcagc375

<210>54

<211>375

<212>dna

<213>人工序列(artificialsequence)

<400>54

caggtgcagctggtggagagcggcggcggcagcgtgcaggccggcggcagcctgaggctg60

agctgcgccgccagcggctacacccacagcagctactgcatgggctggttcaggcaggcc120

cccggcaaggagaggcagggcgtggccgccatcagccccggcggcagcttcaagtactac180

gccgacagcgtgaagggcaggttcaccatcagcagggacaacgccaagaacaccgtgtac240

ctgcagatgaacagcctgcagcccgaggacaccgccatgtactactgcgccgccgccccc300

ttcgcctgcgacggcgactggtacagcggcaggtggaacaactggggccagggcacccag360

gtgaccgtgagcagc375

<210>55

<211>390

<212>dna

<213>人工序列(artificialsequence)

<400>55

caggtgcagctggtggagagcggcggcggcagcgtgcaggccggcggcagcctgaggctg60

agctgcgccgccagcggctacacctacagcagcaactgcatggcctggttcaggcaggcc120

cccggcaaggagagggagggcgtggccgtgatctacaccggcggcggcagcacctactac180

gccgacagcgtgaagggcaggttcaccatcagccaggacaacgccaagaacaccgtgtac240

ctgcagatgaacagcctgaagcccgaggacaccgccatgtactactgcgccgccgacctg300

agggccgccccctacggcggcatcggctacttccccgccgccaccgacttcggctactgg360

ggccagggcacccaggtgaccgtgagcagc390

<210>56

<211>393

<212>dna

<213>人工序列(artificialsequence)

<400>56

caggtgcagctggtggagagcggcggcggcagcgtgcaggccggcggcagcctgaggctg60

gcctgcgccgccagcggctacgcctacagcaggtactacatgggctggttcaggcaggtg120

cccggcaaggagagggagggcgtggccaccatcagcaccggcggcggcaccacctactac180

gccgacagcgtgaagggcaggttcaccgtgagcagggacaacgccaagaacaccctgtac240

ctgcagatgaacagcctgacccccgaggacaccgccatctactactgcgccgccagggtg300

ggcagccccttcggcggcgcccccgacctgagcagcctgaggcccgagaggtacagctac360

tggggccagggcacccaggtgaccgtgagcagc393

<210>57

<211>354

<212>dna

<213>人工序列(artificialsequence)

<400>57

caggtgcagctggtggagagcggcggcggcagcggccaacccggcggcagcctgaggctg60

agctgcgtggtgagcggctacaccatcagcagcctgtgcatgggctggttcaggaggacc120

gagagggaggccgtggccaggatcgacagggacggcggcaccaggtacgccgaccccgtg180

aagggcaggttcaccatcagcaaggacggcgccaagaacaccctgtacctgcagatgaac240

agcctgaagcccgaggacaccgccatgtactactgcgccgccgactactggggctaccag300

tacaactgcaggttcgagtactggggccagggcacccaggtgaccgtgagcagc354

<210>58

<211>375

<212>dna

<213>人工序列(artificialsequence)

<400>58

caggtgcagctggtggagagcggcggcggcagcgtgcaggccggcggcagcctgaggctg60

agctgcgccgccagcggcgacaccaacaggcagaggtgcatgggctggttcaggcaggcc120

cccggcgagggcagggagggcgtggccgccatcagccccggcggcagcttcaagtactac180

gccgacagcgtgaagggcaggttcaccatcagcagggacaacgccaagaacaccgtgtac240

ctgcagatgaacagcctgcagcccgaggacaccgccatgtactactgcgccgccgccccc300

ttcgcctgcgacggcgactggtacagcggcaggtggaacaactggggccagggcacccag360

gtgaccgtgagcagc375

<210>59

<211>381

<212>dna

<213>人工序列(artificialsequence)

<400>59

caggtgcagctggtggagagcggcggcggcagcgtgcagaccggcggcagcctgaggctg60

agctgcgccgccagcggctacaccgtgaacaccaagtgcatgggctggttcaggcaggcc120

cccggcaaggagagggagggcgtggccaccatctacaccggcaccagcaccacctactac180

gccgacagcgtgaagggcaggttcaccatcagcagggacaacgccaggaacaccatctac240

ctgcagatggacagcgtgaagcccgacgacaccgccatgtactactgcgccgccaccacc300

tacatcggcagctggtgcggcctgaggagccccctggactacaacaactggggccagggc360

acccaggtgaccgtgagcagc381

<210>60

<211>375

<212>dna

<213>人工序列(artificialsequence)

<400>60

caggtgcagctggtggagagcggcggcggcagcgcccaggccggcggcagcctgaggctg60

agctgcgccgccagcggctacacctacagcaccaactgcatgggctggctgaggcaggcc120

cccggcaaggagaggcagggcgtggccgccatcagccccggcggcagcttcaagtactac180

gccgacagcgtgaagggcaggttcaccatcagcagggacaacgccaagaacaccgtgtac240

ctgcagatgaacagcctgaagcccgaggacaccgccatgtactactgcgccgccgccccc300

ttcgcctgcgacggcgactggcacagcaggaggtggaacaactggggccagggcacccag360

gtgaccgtgagcagc375

<210>61

<211>378

<212>dna

<213>人工序列(artificialsequence)

<400>61

caggtgcagctggtggagagcggcggcggcagcgtgcaggccggcggcagcctgaggctg60

agctgcgtggccagcggctacaccggcgacaccagcgccatggcctggttcaggcaggcc120

cccggcaaggagagggagggcgtggccagcatctacagggtgagcagcgccaccttctac180

gccgacagcgtgaagggcaggttcaccatcagccaggacaaggccaacgacagcctgtac240

ctgcagatgaacaacctgaagcccgaggacaccgccgtgtactactgcgccgccaggagc300

accgccgtgaccctgaggcagctgacccagcaggcctacaactactggggccagggcacc360

caggtgaccgtgagcagc378

<210>62

<211>378

<212>dna

<213>人工序列(artificialsequence)

<400>62

caggtgcagctggtggagagcggcggcggcagcgtgcaggccggcggcagcctgaggctg60

agctgcgccgcccccggctacacctacagcagcgcctgcatgggctggttcaggcaggcc120

cccggcaaggagagggagggcgtggccaccatcggcagcgacggcagcaccagctacacc180

gacagcgtgaagggcaggttcaccatcagcaaggacaacgtgcacaacaacctgtacctg240

cagatgaacagcctgaagcccgaggacaccgccatgtactactgcgccgccaccatcacc300

accgtgaccatgtgcaggaggagggacggcaggctgttcggccactggggccagggcacc360

caggtgaccgtgagcagc378

<210>63

<211>375

<212>dna

<213>人工序列(artificialsequence)

<400>63

caggtgcagctggtggagagcggcggcggcagcgtgcaggccggcggcagcctgaggctg60

agctgcagcgtgagcggctacacctacagctacaactgcgtgggctggttcaggcaggcc120

cccggcaaggagaggcagggcgtggccgccatcagccccggcggcagcttcaagtactac180

gccgacagcgtgaagggcaggttcaccatcagcagggacaacgccaagaacaccgtgtac240

ctgcagatgaacagcctgcagcccgaggacaccgccatgtactactgcgccgccgccccc300

ttcgcctgcgacggcgactggtacagcggcaggtggaacaactggggccagggcacccag360

gtgaccgtgagcagc375

<210>64

<211>357

<212>dna

<213>人工序列(artificialsequence)

<400>64

caggtgcagctggtggagagcggcggcggcctggtgcagcccggcggcagcctgaggctg60

agctgcgccgccgccggcttcaggttcagcagggtggacatgagctgggtgaggcaggcc120

cccggcaagggcctggagtgggtggccaggatcaacgacgacggcagctggagggacgcc180

gccgacagcgtgaagggcaggttcaccatcagcagggacaacgccaagaacaccctgtac240

ctgcagatgaacagcctgaagaccgaggacaccgccgtgtactactgcgccaccggcttc300

cccagcggcggcaactggatccagaggggccagggcacccaggtgaccgtgagcagc357

<210>65

<211>384

<212>dna

<213>人工序列(artificialsequence)

<400>65

caggtgcagctggtggagagcggcggcggcagcgtgcaggccggcggcagcctgaggctg60

agctgcgccgtgagcggcgacaccgccaacagcaactgcatgggctggttcaggcaggcc120

cccggcgaggagagggagtgggtggccagcatcgtgagcgccagcggcttcatctactac180

gccgacgccgtgaggggcaggttcaccgtgagccaggacaagagcaagaacaccgtgtac240

ctgcagatgaacagcctgaagcccgaggacaccgccatgtactactgcgccgccaagccc300

aggcccaggttcctgaccgtgcccgccggctgcggctacgagtacaactactggggccag360

ggcacccaggtgaccgtgagcagc384

<210>66

<211>375

<212>dna

<213>人工序列(artificialsequence)

<400>66

caggtgcagctggtggagagcggcggcggcagcgtgcaggccggcggcagcctgaggctg60

agctgcgccgccagcggcagcacctacaccatcgcctggttcaggcaggcccccggcaag120

gagagggagggcatcgccgccatctacaccctggccggcggcacctactacgccgacagc180

gtgaagggcaggttcaccatcagcagggacaacgccaagaacaccctgagcctgcagatg240

aacagcctgaagcccgaggacaccgccatgtactactgcgccgccgccatcggcgccggc300

tacggcaggaactggctgctgagcgagaggtggtacaactactggggccagggcacccag360

gtgaccgtgagcagc375

<210>67

<211>381

<212>dna

<213>人工序列(artificialsequence)

<400>67

caggtgcagctggtggagagcggcggcggcagcgtgcaggccggcggcagcctgaggctg60

agctgcgccgccagcggctacacctacagcagcagctgcatgggctggttcaggcaggcc120

cccggcaaggagagggagggcgtggccaccatcgacagcttcggcaacaccaagtacgcc180

gacagcgtgaagggcaggttcaccatcagcaaggacaacgccaagaacaccctgtacctg240

cagatgaacagcctgaagcccgaggacaccgccatgtactactgcaccgccgacatggac300

ggcaggtggggcggctactgcgtgaggagcgccgaggacttcgcctactggggccagggc360

acccaggtgaccgtgagcagc381

<210>68

<211>363

<212>dna

<213>人工序列(artificialsequence)

<400>68

caggtgcagctggtggagagcggcggcggcagcgtgcagaccggcggcagcctgaggctg60

agctgcgccgccagcggccacggcagctgcacctacgacatgagctggtacaggcaggcc120

cccggcaagagcagggagttcgtgagcgtgatcatgagcgacggcaccaccgcctacacc180

agcagcgtgaaggacaggttcaccatcagccaggacaacgccaagagcaccgtgtacctg240

cagatgaacagcctgaagcccgaggacaccgccaggtactactgcgaggccgtgctgagg300

ggcaggtgcaccaggagcggcttcacctactggggccagggcacccaggtgaccgtgagc360

agc363

<210>69

<211>378

<212>dna

<213>人工序列(artificialsequence)

<400>69

caggtgcagctggtggagagcggcggcggcagcgtgcaggccggcggcagcctgaggctg60

agctgcaccgccagcggcagcacctacagcaggaggtacatcggctggttcaggcaggcc120

cccggcaaggagagggagggcgtggccaccatcagcaccggcggcggcttcaggagctac180

gccgacagcgtgaagggcaggttcaccatcagccaggacaacgccaagaacaccgtgtac240

ctgcagatgaacagcctgaagcccgaggacaccgccatgtactactgcgccgccggcagc300

aacgtggacaggaggtggagcctgctgagcagggactacaactactggggccagggcacc360

caggtgaccgtgagcagc378

<210>70

<211>360

<212>dna

<213>人工序列(artificialsequence)

<400>70

caggtgcagctggtggagagcggcggcggcagcgtgcagcccggcggcagcctgaggctg60

agctgcgtggtgagcggctacaccatcgacaggctgtgcaccggctggttcaggcaggcc120

cccggcaaggagagggagttcgtgagcagcaccagcaccgacggcatcaccaggtacgcc180

gacagcgtgaagggcaggttcaccgtgagcagcgacaaggccaaggacaccgtgtacctg240

cagatgaacagcctgaagcccgaggacaccgccatgtacttctgccacttcggcggcagg300

tggctgacccccagggcctgcgactactggggccagggcacccaggtgaccgtgagcagc360

<210>71

<211>363

<212>dna

<213>人工序列(artificialsequence)

<400>71

caggtgcagctggtggagagcggcggcggcagcgtgcaggccggcggcagcctgaggctg60

agctgcgccgccagcggcgccgcctacagcgactactgcatgggctggttcaggcaggcc120

cccggcaaggagagggagagggtggccaccttcctgagggacggcagcgccgactacgcc180

gccagcgtgaccggcaggttcaccatcagcaaggacaacgtgaagaacaccctgtacctg240

cagatgaacagcctgaagcccgaggacaccgccatgtactactgcgccgccgactactgg300

ggctaccagtacaactgcaggttcgagtactggggccagggcacccaggtgaccgtgagc360

agc363

<210>72

<211>396

<212>dna

<213>人工序列(artificialsequence)

<400>72

caggtgcagctggtggagagcggcggcggcagcgtgcaggccggcggcagcctgaggctg60

agctgcgccgtgagcggctacaccgtgagcaggagctgcatggcctggttcaggcaggtg120

cccggcaaggagagggagggcgtggccagcatcagcagcgacggcggcatcaccacctac180

gtggacagcgtgaagggcaggttcaccatcagccaggacaacgccaagaacaccctgtac240

ctgcagatgaacagcgtgaagcccgaggacaccgccgtgtactactgcgccaccggcgtg300

ctgggcttcttcagcaacctgtgcaggagcaccgtggtgccccccaccgccaggttcagg360

tactggggccagggcacccaggtgaccgtgagcagc396

<210>73

<211>363

<212>dna

<213>人工序列(artificialsequence)

<400>73

caggtgcagctggtggagagcggcggcggcagcgtgcaggccggcggcagcctgaggctg60

agctgcgccgccagcggcgccgcctacagcgactactgcatgggctggttcaggcaggcc120

cccggcaaggagagggagagggtggccaccttcctgagggacggcagcgccgactacgcc180

gccagcgtgaccggcaggttcaccatcagcaaggacaacgtgaagaacaccctgtacctg240

cagatgaacagcctgaagcccgaggacaccgccatgtactactgcgccgtggactactgg300

ggctacggctacaactgcaggttcgagtactggggccagggcacccaggtgaccgtgagc360

agc363

<210>74

<211>381

<212>dna

<213>人工序列(artificialsequence)

<400>74

caggtgcagctggtggagagcggcggcggcagcgtgcaggccggcggcagcctgaggctg60

acctgcgccgtgagcggctacagctacagcagcaaccacatgggctggttcaggcaggcc120

cccggcaaggagagggagggcgtggccgtgatcttcaccggcggcggcaggacctggtac180

gccaacagcgtgaagggcaggttcaccatcagccacgacaacgccaagaacaccgtgtac240

ctgcagatgaacagcctgaagctggaggacaccgccatgtactactgcgccgccgactac300

gtgcccgtgggcaggaggtacgccgtgctggaccccgactacacctactggggccagggc360

acccaggtgaccgtgagcagc381

<210>75

<211>375

<212>dna

<213>人工序列(artificialsequence)

<400>75

caggtgcagctggtggagagcggcggcggcagcgtgcaggccggcggcagcctgaggctg60

agctgcgccgccagcggctacacctggagcagcacctgcatgggctggttcaggcaggcc120

cccggcaaggagaggcagggcgtggccgccatcagccccggcggcagcttcaagtactac180

gccgacagcgtgaagggcaggttcaccatcagcagggacaacgccaagaacaccgtgtac240

ctgcagatgaacagcctgcagcccgaggacaccgccatgtactactgcgccgccgccccc300

ttcgcctgcgacggcgactggtacagcggcaggtggaacaactggggccagggcacccag360

gtgaccgtgagcagc375

<210>76

<211>378

<212>dna

<213>人工序列(artificialsequence)

<400>76

caggtgcagctggtggagagcggcggcggcctggtgcagcccggcggcagccccaagctg60

agctgcgccgccagcggcttcaccttcagcaactacgccatgagctgggtgaggcaggcc120

cccggcaaggtgctggagtgggtgagcttcatcaacagcggcggcggcggcaccgcctac180

gccgaccccgtgaagggcaggttcaccatcagcagggacaacgccaagaacaccctgtac240

ctgcagctgaacagcctgaagaccgaggacaccgccatgtactactgcgccaagggcctg300

cagtacgacacctactgcagcagccagtactgctacgccaaccccggcggccagggcacc360

caggtgaccgtgagcagc378

<210>77

<211>378

<212>dna

<213>人工序列(artificialsequence)

<400>77

caggtgcagctggtggagagcggcggcggcaccgtgcaggccggcggcagcctgaggctg60

agctgcgccgccagcggctacacctacagcagcgcctgcatgggctggttcaggcaggcc120

cccggcaaggagagggagggcgtggccaccatcggcagcgacggcagcaccagctacacc180

gacagcgtgaagggcaggttcaccatcagcaaggacaacgtgcacaacaacctgtacctg240

cagatgaacagcctgaagcccgaggacaccgccatgtactactgcgccgccaccatcacc300

accgtgaccatgtgcaggaggagggacggcaggctgttcggccactggggccagggcacc360

caggtgaccgtgagcagc378

<210>78

<211>378

<212>dna

<213>人工序列(artificialsequence)

<400>78

caggtgcagctggtggagagcggcggcggcagcgtgcaggccggcggcagcctgaggctg60

agctgcgccgtgagcggcttcccctacagcacctactacatgggctggttcaggcaggcc120

cccggcaaggagagggagggcgtggccgccgtgtaccccggcgtgggcctgaagaggtac180

gccgacagcgtgaagggcaggttcaccatcagccaggacgacgccaagaacaccctgtac240

ctgcagatgaacagcctgaagcccgaggacaccgccctgtactactgcgccgtggaccag300

gccgtgagcctgggctgggagctgagccccggcaggtacaggtactggggccagggcacc360

caggtgaccgtgagcagc378

<210>79

<211>375

<212>dna

<213>人工序列(artificialsequence)

<400>79

caggtgcagctggtggagagcggcggcggcagcgtgcagaccggcggcagcctgaagctg60

agctgcgtgcacagcggctacagctacagcagcgtgtgcatggcctggttcaggcaggcc120

cccggcaaggagagggagggcgtggccgccatcagccccggcggcagcttcaagtactac180

gtggacagcgtgaagggcaggttcaccatcagcagggacaacagcaagaacaccggctac240

ctgcagatgaacagcctgaagcccgaggacaccgccatgtactactgcgccaccagcagg300

agcggctgcagggacgacttcttccagtacgagtacgactactggggccagggcacccag360

gtgaccgtgagcagc375

<210>80

<211>375

<212>dna

<213>人工序列(artificialsequence)

<400>80

caggtgcagctggtggagagcggcggcggcagcgcccaggccggcggcagcctgaggctg60

agctgcgccgccagcggcaacagctacagcaggaactgcatgggctggttcaggcaggcc120

cccggcaaggagaggcagggcgtggccgccatcagccccggcggcagcttcaagtactac180

gccgacagcgtgaagggcaggttcaccatcagcagggacaacgccaagaacaccgtgtac240

ctgcagatgaacagcctgcagcccgaggacaccgccatgtactactgcgccgccgccccc300

ctggcctgcgacggcgactggtacagcggcaggtggaacaactggggccagggcacccag360

gtgaccgtgagcagc375

<210>81

<211>384

<212>dna

<213>人工序列(artificialsequence)

<400>81

caggtgcagctggtggagagcggcggcggcagcgtgcccgccggcggcagcctgaggctg60

agctgcgccgccagcggctacacctacagcggcagctgcatgggctggttcaggcaggcc120

cccggcaaggagagggaggaggtggccaccatctacaccgacaacggcttcaagtactac180

accgagagcgtgaagggcaggttcaccatcagccaggacaaggccaagaacaccgtgtac240

ctgcagatgaacagcctgaagcccgaggacaccgccgtgtactactgcgccgccgacggc300

ggcaccggcctgtggtacacctgcgccaggggccagatcagccacaactactggggccag360

ggcacccaggtgaccgtgagcagc384

<210>82

<211>381

<212>dna

<213>人工序列(artificialsequence)

<400>82

caggtgcagctggtggagagcggcggcggcagcgtgcaggccggcggcagcctgaggctg60

agctgcaccgtgagcggctacagctacagcagcgactacatgggctggttcaggcaggcc120

cccggcaaggagagggagggcgtggccgtgatcttcaccggcggcggcaggacctggtac180

gccaacagcgtgaagggcaggttcaccatcagccaggacaacgccaagaacaccgtgtac240

ctgcagatgaacagcctgaagctggaggacaccgccatgtactactgcgccgccgactac300

gtgcccgtgggcaggaggtacgccgtgctggaccccgactacacctactggggccagggc360

acccaggtgaccgtgagcagc381

<210>83

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>83

glytyrserglyargserasptyr

15

<210>84

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>84

glyalaalatyrserasptyrcys

15

<210>85

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>85

glytyrasptyrserthrtyrcys

15

<210>86

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>86

glyphethrphesersertyrpro

15

<210>87

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>87

glyphethrpheserargtyrasp

15

<210>88

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>88

glytyrileserhisprotyrcys

15

<210>89

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>89

glytyrthrtyrserargtyrser

15

<210>90

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>90

glytyralatyrserargtyrser

15

<210>91

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>91

glyvalthrserlysargalacys

15

<210>92

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>92

glyasnsertyrserargasncys

15

<210>93

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>93

glytyrthrhissersertyrcys

15

<210>94

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>94

glytyrthrtyrserserasncys

15

<210>95

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>95

glytyralatyrserargtyrtyr

15

<210>96

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>96

glytyrthrileserserleucys

15

<210>97

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>97

glyaspthrasnargglnargcys

15

<210>98

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>98

glytyrthrvalasnthrlyscys

15

<210>99

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>99

glytyrthrtyrserthrasncys

15

<210>100

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>100

glytyrthrglyaspthrserala

15

<210>101

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>101

glytyrthrtyrserseralacys

15

<210>102

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>102

glytyrthrtyrsertyrasncys

15

<210>103

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>103

glypheargpheserargvalasp

15

<210>104

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>104

glyaspthralaasnserasncys

15

<210>105

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>105

glyserthrtyrthrilealatrp

15

<210>106

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>106

glytyrthrtyrsersersercys

15

<210>107

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>107

glyhisglysercysthrtyrasp

15

<210>108

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>108

glyserthrtyrserargargtyr

15

<210>109

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>109

glytyrthrileaspargleucys

15

<210>110

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>110

glytyrthrvalserargsercys

15

<210>111

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>111

glytyrsertyrserserasnhis

15

<210>112

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>112

glytyrthrtrpserserthrcys

15

<210>113

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>113

glyphethrpheserasntyrala

15

<210>114

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>114

glypheprotyrserthrtyrtyr

15

<210>115

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>115

glytyrsertyrserservalcys

15

<210>116

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>116

glytyrthrtyrserglysercys

15

<210>117

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>117

glytyrsertyrserserasptyr

15

<210>118

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>118

ilephethrglyglyglyargthr

15

<210>119

<211>7

<212>prt

<213>人工序列(artificialsequence)

<400>119

pheleuargaspglyserala

15

<210>120

<211>7

<212>prt

<213>人工序列(artificialsequence)

<400>120

ileserthrvalglythrthr

15

<210>121

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>121

ileasnserglyglyglyserthr

15

<210>122

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>122

iletyrserglyserglyasnthr

15

<210>123

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>123

ileserileglyglyglyglythr

15

<210>124

<211>7

<212>prt

<213>人工序列(artificialsequence)

<400>124

leuaspseraspglyalathr

15

<210>125

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>125

ileserargproglyglyalathr

15

<210>126

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>126

ileserproglyglyserphelys

15

<210>127

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>127

iletyrthrglyglyglyserthr

15

<210>128

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>128

ileserthrglyglyglythrthr

15

<210>129

<211>7

<212>prt

<213>人工序列(artificialsequence)

<400>129

ileaspargaspglyglythr

15

<210>130

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>130

iletyrthrglythrserthrthr

15

<210>131

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>131

iletyrargvalserseralathr

15

<210>132

<211>7

<212>prt

<213>人工序列(artificialsequence)

<400>132

ileglyseraspglyserthr

15

<210>133

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>133

ileasnaspaspglysertrparg

15

<210>134

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>134

ilevalseralaserglypheile

15

<210>135

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>135

iletyrthrleualaglyglythr

15

<210>136

<211>7

<212>prt

<213>人工序列(artificialsequence)

<400>136

ileaspserpheglyasnthr

15

<210>137

<211>7

<212>prt

<213>人工序列(artificialsequence)

<400>137

ilemetseraspglythrthr

15

<210>138

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>138

ileserthrglyglyglyphearg

15

<210>139

<211>7

<212>prt

<213>人工序列(artificialsequence)

<400>139

thrserthraspglyilethr

15

<210>140

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>140

ileserseraspglyglyilethr

15

<210>141

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>141

ileasnserglyglyglyglythr

15

<210>142

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>142

valtyrproglyvalglyleulys

15

<210>143

<211>8

<212>prt

<213>人工序列(artificialsequence)

<400>143

iletyrthraspasnglyphelys

15

<210>144

<211>21

<212>prt

<213>人工序列(artificialsequence)

<400>144

alaalaasptyrvalprovalglyargargtyralavalleuasppro

151015

asptyrthrtyrtrp

20

<210>145

<211>16

<212>prt

<213>人工序列(artificialsequence)

<400>145

alaalaasptyrtrpglytyrglytyrasncysargpheglutyrtrp

151015

<210>146

<211>22

<212>prt

<213>人工序列(artificialsequence)

<400>146

alaserleuaspargargmettyrglyglyiletrptyrcysaspleu

151015

argargtyrasntyrtrp

20

<210>147

<211>9

<212>prt

<213>人工序列(artificialsequence)

<400>147

alaleuaspserargglythrglnarg

15

<210>148

<211>15

<212>prt

<213>人工序列(artificialsequence)

<400>148

alaalaasptrpthrglyargthrleuhisglutyrasntyrleu

151015

<210>149

<211>17

<212>prt

<213>人工序列(artificialsequence)

<400>149

alaalaargglyglytyrtyrcysthralaseralasertyrasntyr

151015

trp

<210>150

<211>21

<212>prt

<213>人工序列(artificialsequence)

<400>150

alaalaglnargileargtrpvalalaphevalserleuargserala

151015

asppheasptyrtrp

20

<210>151

<211>23

<212>prt

<213>人工序列(artificialsequence)

<400>151

alapheargvalileasppropheaspthrcysserserargargarg

151015

aspglyasppheprotyrtrp

20

<210>152

<211>19

<212>prt

<213>人工序列(artificialsequence)

<400>152

alaalaalaprophealacysaspglyasptrptyrserglyargtrp

151015

asnasntrp

<210>153

<211>19

<212>prt

<213>人工序列(artificialsequence)

<400>153

alaalaalaprophealacysaspglyasptrphisserglyargtrp

151015

asnasntrp

<210>154

<211>24

<212>prt

<213>人工序列(artificialsequence)

<400>154

alaalaaspleuargalaalaprotyrglyglyileglytyrphepro

151015

alaalathrasppheglytyrtrp

20

<210>155

<211>25

<212>prt

<213>人工序列(artificialsequence)

<400>155

alaalaargvalglyserpropheglyglyalaproaspleuserser

151015

leuargprogluargtyrsertyrtrp

2025

<210>156

<211>16

<212>prt

<213>人工序列(artificialsequence)

<400>156

alaalaasptyrtrpglytyrglntyrasncysargpheglutyrtrp

151015

<210>157

<211>21

<212>prt

<213>人工序列(artificialsequence)

<400>157

alaalathrthrtyrileglysertrpcysglyleuargserproleu

151015

asptyrasnasntrp

20

<210>158

<211>19

<212>prt

<213>人工序列(artificialsequence)

<400>158

alaalaalaprophealacysaspglyasptrphisserargargtrp

151015

asnasntrp

<210>159

<211>20

<212>prt

<213>人工序列(artificialsequence)

<400>159

alaalaargserthralavalthrleuargglnleuthrglnglnala

151015

tyrasntyrtrp

20

<210>160

<211>21

<212>prt

<213>人工序列(artificialsequence)

<400>160

alaalathrilethrthrvalthrmetcysargargargaspglyarg

151015

leupheglyhistrp

20

<210>161

<211>13

<212>prt

<213>人工序列(artificialsequence)

<400>161

alathrglypheproserglyglyasntrpileglnarg

1510

<210>162

<211>22

<212>prt

<213>人工序列(artificialsequence)

<400>162

alaalalysproargproargpheleuthrvalproalaglycysgly

151015

tyrglutyrasntyrtrp

20

<210>163

<211>22

<212>prt

<213>人工序列(artificialsequence)

<400>163

alaalaalaileglyalaglytyrglyargasntrpleuleuserglu

151015

argtrptyrasntyrtrp

20

<210>164

<211>22

<212>prt

<213>人工序列(artificialsequence)

<400>164

thralaaspmetaspglyargtrpglyglytyrcysvalargserala

151015

gluaspphealatyrtrp

20

<210>165

<211>16

<212>prt

<213>人工序列(artificialsequence)

<400>165

glualavalleuargglyargcysthrargserglyphethrtyrtrp

151015

<210>166

<211>20

<212>prt

<213>人工序列(artificialsequence)

<400>166

alaalaglyserasnvalaspargargtrpserleuleuserargasp

151015

tyrasntyrtrp

20

<210>167

<211>15

<212>prt

<213>人工序列(artificialsequence)

<400>167

hispheglyglyargtrpleuthrproargalacysasptyrtrp

151015

<210>168

<211>26

<212>prt

<213>人工序列(artificialsequence)

<400>168

alathrglyvalleuglyphepheserasnleucysargserthrval

151015

valproprothralaargpheargtyrtrp

2025

<210>169

<211>16

<212>prt

<213>人工序列(artificialsequence)

<400>169

alavalasptyrtrpglytyrglytyrasncysargpheglutyrtrp

151015

<210>170

<211>20

<212>prt

<213>人工序列(artificialsequence)

<400>170

alalysglyleuglntyraspthrtyrcysserserglntyrcystyr

151015

alaasnprogly

20

<210>171

<211>20

<212>prt

<213>人工序列(artificialsequence)

<400>171

alavalaspglnalavalserleuglytrpgluleuserproglyarg

151015

tyrargtyrtrp

20

<210>172

<211>19

<212>prt

<213>人工序列(artificialsequence)

<400>172

alathrserargserglycysargaspaspphepheglntyrglutyr

151015

asptyrtrp

<210>173

<211>19

<212>prt

<213>人工序列(artificialsequence)

<400>173

alaalaalaproleualacysaspglyasptrptyrserglyargtrp

151015

asnasntrp

<210>174

<211>22

<212>prt

<213>人工序列(artificialsequence)

<400>174

alaalaaspglyglythrglyleutrptyrthrcysalaargglygln

151015

ileserhisasntyrtrp

20

<210>175

<211>25

<212>prt

<213>人工序列(artificialsequence)

<400>175

glnvalglnleuvalgluserglyglyglyservalglnalaglygly

151015

serleuargleusercysalavalser

2025

<210>176

<211>25

<212>prt

<213>人工序列(artificialsequence)

<400>176

glnvalglnleuvalgluserglyglyglyleuvalglnproglygly

151015

serleuargleusercysalaalaser

2025

<210>177

<211>25

<212>prt

<213>人工序列(artificialsequence)

<400>177

glnvalglnleuvalgluserglyglyglyservalglnalaglygly

151015

serleuargleusercysalaalaser

2025

<210>178

<211>25

<212>prt

<213>人工序列(artificialsequence)

<400>178

glnvalglnleuvalgluserglyglyglyservalglnproglygly

151015

serleuargleusercysalaalaser

2025

<210>179

<211>25

<212>prt

<213>人工序列(artificialsequence)

<400>179

glnvalglnleuvalgluserglyglyglytrpvalglnalaglygly

151015

serleuargleusercysalaalaser

2025

<210>180

<211>25

<212>prt

<213>人工序列(artificialsequence)

<400>180

glnvalglnleuvalgluserglyglyglyseralaglnalaglygly

151015

serleuargleusercysalaalaser

2025

<210>181

<211>25

<212>prt

<213>人工序列(artificialsequence)

<400>181

glnvalglnleuvalgluserglyglyglyservalglnalaglygly

151015

serleuargleualacysalaalaser

2025

<210>182

<211>25

<212>prt

<213>人工序列(artificialsequence)

<400>182

glnvalglnleuvalgluserglyglyglyserglyglnproglygly

151015

serleuargleusercysvalvalser

2025

<210>183

<211>25

<212>prt

<213>人工序列(artificialsequence)

<400>183

glnvalglnleuvalgluserglyglyglyservalglnthrglygly

151015

serleuargleusercysalaalaser

2025

<210>184

<211>25

<212>prt

<213>人工序列(artificialsequence)

<400>184

glnvalglnleuvalgluserglyglyglyservalglnalaglygly

151015

serleuargleusercysvalalaser

2025

<210>185

<211>25

<212>prt

<213>人工序列(artificialsequence)

<400>185

glnvalglnleuvalgluserglyglyglyservalglnalaglygly

151015

serleuargleusercysalaalapro

2025

<210>186

<211>25

<212>prt

<213>人工序列(artificialsequence)

<400>186

glnvalglnleuvalgluserglyglyglyservalglnalaglygly

151015

serleuargleusercysservalser

2025

<210>187

<211>25

<212>prt

<213>人工序列(artificialsequence)

<400>187

glnvalglnleuvalgluserglyglyglyleuvalglnproglygly

151015

serleuargleusercysalaalaala

2025

<210>188

<211>25

<212>prt

<213>人工序列(artificialsequence)

<400>188

glnvalglnleuvalgluserglyglyglyservalglnalaglygly

151015

serleuargleusercysthralaser

2025

<210>189

<211>25

<212>prt

<213>人工序列(artificialsequence)

<400>189

glnvalglnleuvalgluserglyglyglyservalglnproglygly

151015

serleuargleusercysvalvalser

2025

<210>190

<211>25

<212>prt

<213>人工序列(artificialsequence)

<400>190

glnvalglnleuvalgluserglyglyglyservalglnalaglygly

151015

serleuargleuthrcysalavalser

2025

<210>191

<211>25

<212>prt

<213>人工序列(artificialsequence)

<400>191

glnvalglnleuvalgluserglyglyglyleuvalglnproglygly

151015

serprolysleusercysalaalaser

2025

<210>192

<211>25

<212>prt

<213>人工序列(artificialsequence)

<400>192

glnvalglnleuvalgluserglyglyglythrvalglnalaglygly

151015

serleuargleusercysalaalaser

2025

<210>193

<211>25

<212>prt

<213>人工序列(artificialsequence)

<400>193

glnvalglnleuvalgluserglyglyglyservalglnthrglygly

151015

serleulysleusercysvalhisser

2025

<210>194

<211>25

<212>prt

<213>人工序列(artificialsequence)

<400>194

glnvalglnleuvalgluserglyglyglyservalproalaglygly

151015

serleuargleusercysalaalaser

2025

<210>195

<211>25

<212>prt

<213>人工序列(artificialsequence)

<400>195

glnvalglnleuvalgluserglyglyglyservalglnalaglygly

151015

serleuargleusercysthrvalser

2025

<210>196

<211>17

<212>prt

<213>人工序列(artificialsequence)

<400>196

metglytrppheargglnalaproglylysgluarggluglyvalala

151015

val

<210>197

<211>17

<212>prt

<213>人工序列(artificialsequence)

<400>197

metglytrppheargglnalaproglylysgluarggluargvalala

151015

thr

<210>198

<211>17

<212>prt

<213>人工序列(artificialsequence)

<400>198

metglytrppheargglnalaproglylysgluarggluglyvalala

151015

ser

<210>199

<211>17

<212>prt

<213>人工序列(artificialsequence)

<400>199

metasntrpvalargglnalaproglylysglyleuglutrpvalser

151015

ala

<210>200

<211>17

<212>prt

<213>人工序列(artificialsequence)

<400>200

metsertrpvalargglnproproglylysglyleuglutrpvalser

151015

ala

<210>201

<211>17

<212>prt

<213>人工序列(artificialsequence)

<400>201

metalatrppheargglnalaproglylysgluarggluglyvalala

151015

gly

<210>202

<211>17

<212>prt

<213>人工序列(artificialsequence)

<400>202

metglytrppheargglnalaproglylysgluargglutrpvalala

151015

thr

<210>203

<211>17

<212>prt

<213>人工序列(artificialsequence)

<400>203

metglytrppheargglnalaproglylysgluargglnglyvalala

151015

ala

<210>204

<211>17

<212>prt

<213>人工序列(artificialsequence)

<400>204

metalatrppheargglnalaproglylysgluarggluglyvalala

151015

val

<210>205

<211>17

<212>prt

<213>人工序列(artificialsequence)

<400>205

metglytrppheargglnvalproglylysgluarggluglyvalala

151015

thr

<210>206

<211>14

<212>prt

<213>人工序列(artificialsequence)

<400>206

metglytrppheargargthrgluargglualavalalaarg

1510

<210>207

<211>17

<212>prt

<213>人工序列(artificialsequence)

<400>207

metglytrppheargglnalaproglygluglyarggluglyvalala

151015

ala

<210>208

<211>17

<212>prt

<213>人工序列(artificialsequence)

<400>208

metglytrppheargglnalaproglylysgluarggluglyvalala

151015

thr

<210>209

<211>17

<212>prt

<213>人工序列(artificialsequence)

<400>209

metglytrpleuargglnalaproglylysgluargglnglyvalala

151015

ala

<210>210

<211>17

<212>prt

<213>人工序列(artificialsequence)

<400>210

metalatrppheargglnalaproglylysgluarggluglyvalala

151015

ser

<210>211

<211>17

<212>prt

<213>人工序列(artificialsequence)

<400>211

valglytrppheargglnalaproglylysgluargglnglyvalala

151015

ala

<210>212

<211>17

<212>prt

<213>人工序列(artificialsequence)

<400>212

metsertrpvalargglnalaproglylysglyleuglutrpvalala

151015

arg

<210>213

<211>17

<212>prt

<213>人工序列(artificialsequence)

<400>213

metglytrppheargglnalaproglyglugluargglutrpvalala

151015

ser

<210>214

<211>17

<212>prt

<213>人工序列(artificialsequence)

<400>214

ilealatrppheargglnalaproglylysgluarggluglyileala

151015

ala

<210>215

<211>17

<212>prt

<213>人工序列(artificialsequence)

<400>215

metsertrptyrargglnalaproglylysserarggluphevalser

151015

val

<210>216

<211>17

<212>prt

<213>人工序列(artificialsequence)

<400>216

ileglytrppheargglnalaproglylysgluarggluglyvalala

151015

thr

<210>217

<211>17

<212>prt

<213>人工序列(artificialsequence)

<400>217

thrglytrppheargglnalaproglylysgluarggluphevalser

151015

ser

<210>218

<211>17

<212>prt

<213>人工序列(artificialsequence)

<400>218

metalatrppheargglnvalproglylysgluarggluglyvalala

151015

ser

<210>219

<211>17

<212>prt

<213>人工序列(artificialsequence)

<400>219

metsertrpvalargglnalaproglylysvalleuglutrpvalser

151015

phe

<210>220

<211>17

<212>prt

<213>人工序列(artificialsequence)

<400>220

metglytrppheargglnalaproglylysgluarggluglyvalala

151015

ala

<210>221

<211>17

<212>prt

<213>人工序列(artificialsequence)

<400>221

metalatrppheargglnalaproglylysgluarggluglyvalala

151015

ala

<210>222

<211>17

<212>prt

<213>人工序列(artificialsequence)

<400>222

metglytrppheargglnalaproglylysgluargglugluvalala

151015

thr

<210>223

<211>38

<212>prt

<213>人工序列(artificialsequence)

<400>223

trptyralaasnservallysglyargphethrileserglnglyasn

151015

alagluasnthrvaltyrleuglnmetasnserleulysleugluasp

202530

thralamettyrtyrcys

35

<210>224

<211>38

<212>prt

<213>人工序列(artificialsequence)

<400>224

asptyralaalaservalthrglyargphethrileserlysaspasn

151015

vallysasnthrleutyrleuglnmetasnserleulysprogluasp

202530

thralamettyrtyrcys

35

<210>225

<211>38

<212>prt

<213>人工序列(artificialsequence)

<400>225

sertyralaaspservallysglyargphethrileserargaspasn

151015

alalysasnthrvaltyrleuglnmetasnserleulysthrgluasp

202530

thrasnmettyrtyrcys

35

<210>226

<211>38

<212>prt

<213>人工序列(artificialsequence)

<400>226

tyrtyralaaspservallysglyargphethrileserargaspasn

151015

alalysasnthrleutyrleuglnleuasnglyleulysthrgluasp

202530

thralamettyrtyrcys

35

<210>227

<211>38

<212>prt

<213>人工序列(artificialsequence)

<400>227

tyrtyralaaspservallysglyargphethrileserargaspasn

151015

alalysasnthrleutyrleuglnmetasnserleulysthrgluasp

202530

thralavaltyrtyrcys

35

<210>228

<211>38

<212>prt

<213>人工序列(artificialsequence)

<400>228

trptyralaasnservallysglyargphethrileserglnaspasn

151015

alalysasnthrvaltyrleuglnmetasnserleulysleugluasp

202530

thralamettyrtyrcys

35

<210>229

<211>38

<212>prt

<213>人工序列(artificialsequence)

<400>229

phethralaaspservallysglyargphethrileserglnaspasn

151015

alalysasnthrvalhisleuargmetasnserleulysprogluasp

202530

thralamettyrtyrcys

35

<210>230

<211>38

<212>prt

<213>人工序列(artificialsequence)

<400>230

iletyrthraspservallysglyargphethrilealalysaspasn

151015

alalysasnthrleutyrleuglnmetasnserleulysprogluasp

202530

thrglymettyrtyrcys

35

<210>231

<211>38

<212>prt

<213>人工序列(artificialsequence)

<400>231

tyrseralaaspservallysglyargphethrileserglnaspasn

151015

alagluasnmetalapheleuileleuasnserleulysalagluasp

202530

alaalailetyrtyrcys

35

<210>232

<211>38

<212>prt

<213>人工序列(artificialsequence)

<400>232

tyrtyralaaspservallysglyargphethrileserargaspasn

151015

alalysasnthrvaltyrleuglnmetasnserleuglnprogluasp

202530

thralamettyrtyrcys

35

<210>233

<211>38

<212>prt

<213>人工序列(artificialsequence)

<400>233

tyrtyralaaspservallysglyargphethrileserglnaspasn

151015

alalysasnthrvaltyrleuglnmetasnserleulysprogluasp

202530

thralamettyrtyrcys

35

<210>234

<211>38

<212>prt

<213>人工序列(artificialsequence)

<400>234

tyrtyralaaspservallysglyargphethrvalserargaspasn

151015

alalysasnthrleutyrleuglnmetasnserleuthrprogluasp

202530

thralailetyrtyrcys

35

<210>235

<211>38

<212>prt

<213>人工序列(artificialsequence)

<400>235

argtyralaaspprovallysglyargphethrileserlysaspgly

151015

alalysasnthrleutyrleuglnmetasnserleulysprogluasp

202530

thralamettyrtyrcys

35

<210>236

<211>38

<212>prt

<213>人工序列(artificialsequence)

<400>236

tyrtyralaaspservallysglyargphethrileserargaspasn

151015

alaargasnthriletyrleuglnmetaspservallysproaspasp

202530

thralamettyrtyrcys

35

<210>237

<211>38

<212>prt

<213>人工序列(artificialsequence)

<400>237

tyrtyralaaspservallysglyargphethrileserargaspasn

151015

alalysasnthrvaltyrleuglnmetasnserleulysprogluasp

202530

thralamettyrtyrcys

35

<210>238

<211>38

<212>prt

<213>人工序列(artificialsequence)

<400>238

phetyralaaspservallysglyargphethrileserglnasplys

151015

alaasnaspserleutyrleuglnmetasnasnleulysprogluasp

202530

thralavaltyrtyrcys

35

<210>239

<211>38

<212>prt

<213>人工序列(artificialsequence)

<400>239

sertyrthraspservallysglyargphethrileserlysaspasn

151015

valhisasnasnleutyrleuglnmetasnserleulysprogluasp

202530

thralamettyrtyrcys

35

<210>240

<211>38

<212>prt

<213>人工序列(artificialsequence)

<400>240

aspalaalaaspservallysglyargphethrileserargaspasn

151015

alalysasnthrleutyrleuglnmetasnserleulysthrgluasp

202530

thralavaltyrtyrcys

35

<210>241

<211>38

<212>prt

<213>人工序列(artificialsequence)

<400>241

tyrtyralaaspalavalargglyargphethrvalserglnasplys

151015

serlysasnthrvaltyrleuglnmetasnserleulysprogluasp

202530

thralamettyrtyrcys

35

<210>242

<211>38

<212>prt

<213>人工序列(artificialsequence)

<400>242

tyrtyralaaspservallysglyargphethrileserargaspasn

151015

alalysasnthrleuserleuglnmetasnserleulysprogluasp

202530

thralamettyrtyrcys

35

<210>243

<211>38

<212>prt

<213>人工序列(artificialsequence)

<400>243

lystyralaaspservallysglyargphethrileserlysaspasn

151015

alalysasnthrleutyrleuglnmetasnserleulysprogluasp

202530

thralamettyrtyrcys

35

<210>244

<211>38

<212>prt

<213>人工序列(artificialsequence)

<400>244

alatyrthrserservallysaspargphethrileserglnaspasn

151015

alalysserthrvaltyrleuglnmetasnserleulysprogluasp

202530

thralaargtyrtyrcys

35

<210>245

<211>38

<212>prt

<213>人工序列(artificialsequence)

<400>245

sertyralaaspservallysglyargphethrileserglnaspasn

151015

alalysasnthrvaltyrleuglnmetasnserleulysprogluasp

202530

thralamettyrtyrcys

35

<210>246

<211>38

<212>prt

<213>人工序列(artificialsequence)

<400>246

argtyralaaspservallysglyargphethrvalserserasplys

151015

alalysaspthrvaltyrleuglnmetasnserleulysprogluasp

202530

thralamettyrphecys

35

<210>247

<211>38

<212>prt

<213>人工序列(artificialsequence)

<400>247

thrtyrvalaspservallysglyargphethrileserglnaspasn

151015

alalysasnthrleutyrleuglnmetasnservallysprogluasp

202530

thralavaltyrtyrcys

35

<210>248

<211>38

<212>prt

<213>人工序列(artificialsequence)

<400>248

trptyralaasnservallysglyargphethrileserhisaspasn

151015

alalysasnthrvaltyrleuglnmetasnserleulysleugluasp

202530

thralamettyrtyrcys

35

<210>249

<211>38

<212>prt

<213>人工序列(artificialsequence)

<400>249

alatyralaaspprovallysglyargphethrileserargaspasn

151015

alalysasnthrleutyrleuglnleuasnserleulysthrgluasp

202530

thralamettyrtyrcys

35

<210>250

<211>38

<212>prt

<213>人工序列(artificialsequence)

<400>250

argtyralaaspservallysglyargphethrileserglnaspasp

151015

alalysasnthrleutyrleuglnmetasnserleulysprogluasp

202530

thralaleutyrtyrcys

35

<210>251

<211>38

<212>prt

<213>人工序列(artificialsequence)

<400>251

tyrtyrvalaspservallysglyargphethrileserargaspasn

151015

serlysasnthrglytyrleuglnmetasnserleulysprogluasp

202530

thralamettyrtyrcys

35

<210>252

<211>38

<212>prt

<213>人工序列(artificialsequence)

<400>252

tyrtyrthrgluservallysglyargphethrileserglnasplys

151015

alalysasnthrvaltyrleuglnmetasnserleulysprogluasp

202530

thralavaltyrtyrcys

35

<210>253

<211>10

<212>prt

<213>人工序列(artificialsequence)

<400>253

glyglnglythrglnvalthrvalserser

1510

<210>254

<211>10

<212>prt

<213>人工序列(artificialsequence)

<400>254

glyargglythrglnvalthrvalserser

1510

<210>255

<211>13

<212>dna

<213>人工序列(artificialsequence)

<400>255

ggatcgaaccctt13

<210>256

<211>21

<212>prt

<213>人工序列(artificialsequence)

<400>256

metgluthraspthrleuleuleutrpvalleuleuleutrpvalpro

151015

glyserthrglyasp

20