一种黑素瘤纳米抗体库的构建方法与流程

本发明涉及生物工程
技术领域：
，具体涉及一种黑素瘤纳米抗体库的构建方法。
背景技术：
：传统单克隆抗体分子量大，在临床诊疗过程中无法穿透动物机体内的血脑屏障、血睾屏障等保护性组织，大大限制了传统抗体在特定诊断和治疗领域的应用。羊驼外周血液中存在一种天然缺失轻链但仍具有活性的重链抗体。体外表达该抗体的抗原结合区(vhh)发现仍然具有生物学功能，可以与特定抗原识别。与传统抗体相比，vhh抗体具有以下几个方面的优势：1、分子量小，可穿透血脑等生物屏障；2、易进行筛选分析，可在原核或真核系统高效表达；3、特异性强且亲和力高；4、免疫原性弱，临床使用时副作用小；5、稳定性强，易运输和保存。同时因为纳米抗体的分子量小，具有更多工程学改造的空间，为改善抗体的功能特征(如稳定性、亲和性、特异性和体积大小等)和药代动力学属性等更多应用方向提供了无限潜能。纳米抗体库为筛选特异性纳米抗体提供材料。技术实现要素：本发明的目的在于提供一种黑素瘤纳米抗体库的构建方法，采用本发明的方法构建得到的黑素瘤纳米抗体库是一个特性很好的免疫文库，适用于特异性地筛选与黑素瘤生长、发育、迁移、增殖等相关的纳米抗体，大大节省了免疫成本和羊驼资源。本发明提供了一种黑素瘤纳米抗体库的构建方法，包括以下步骤：1)黑色素瘤细胞b16培养到80％丰度，破碎细胞后，作为抗原共合物；2)将所述步骤1)得到的抗原共和物与佐剂按1:1进行配制，将得到的配制物乳化后以200ug/次分点皮下免疫羊驼四次，每次间隔两周，第一次免疫时，所述佐剂为完全弗氏佐剂，第二至第四免疫时，所述佐剂为不完全弗氏佐剂；3)第四次免疫后1周采集50ml羊驼抗凝血液，分离得到淋巴细胞，提取淋巴细胞总rna，将所述rna反转录为cdna；4)以所述步骤3)得到的cdna为模版，用call001-f和call002-r引物进行第一轮pcr扩增，分离纯化700bp的扩增产物；所述call001-f引物具有seqidno.1所示的核苷酸序列；所述call002-r引物具有seqidno.2所示的核苷酸序列；5)以步骤4)得到的700bp的扩增产物为模版，用vhh2-f和vhh2-r引物pcr扩增vhh片段，分离纯化vhh片段；所述vhh2-f引物具有seqidno.3所示的核苷酸序列；所述vhh2-r引物具有seqidno.4所示的核苷酸序列；6)将所述步骤5)得到的vhh片段和pcantab5e分别用sfii酶进行两次酶切，连接酶连接，构建得到pcantab5e-vhh重组表达载体；7)将pcantab5e-vhh重组表达载体电转化到tg1感受态细胞中，得到黑素瘤纳米抗体库。优选的，所述步骤1)培养使用的培养基为含有体积分数为8～12％fbs的dmem高糖培养基。优选的，所述步骤3)反转录包括：将5μg总rna、1μlrandom6primer、1μloligo(dt)18primer、1μldntpmix和7μlddh2o混合后在65℃下处理5min，将得到的处理物在冰上静置5min，将所述静置物与4μl5xprimescriptiibuffer、0.5μlrnaseinhibiter、1μlprimerscripiireversetranscriptase和4.5μlddh2o混合后，依次40℃处理40min和70℃处理15min，得到cdna。优选的，所述步骤4)第一pcr扩增的体系每50μl包括：2μlcdna、浓度为10umol/μl的call001-f1μl、浓度为10umol/ul的call001-r1μl、25μltaqgreenpcrmix和21μlddh2o。优选的，所述步骤4)第一轮pcr扩增的程序包括：95℃5min；95℃30s，53℃30s，72℃40s，30个循环；72℃5min。优选的，所述步骤5)pcr扩增vhh片段的体系每50μl包括：浓度为800ug/ul的700bp扩增产物6μl、浓度为10μmol/ul的vhh2-f2μl、浓度为10μmol/ul的vhh2-r2μl、taqgreenpcrmix25μl和ddh2o15μl。优选的，所述步骤5)pcr扩增vhh片段的程序包括：95℃5min；95℃30s，56℃30s，72℃30s，30个循环；72℃5min。优选的，所述步骤6)连接的体系每200μl包括：vhh片段1μg、pcantab5e载体3μg、t4dnaligase10μl、10×buffer20μl，ddh2o补充至200μl。优选的，所述步骤7)电转化包括：所述电转化的电压为1.5～2.0kv，所述电转化的电阻为180～220ω，所述电转化的电容为20～30μf，所述电转化的时间为4～6ms。本发明提供了一种黑素瘤纳米抗体库的构建方法，将黑色素瘤细胞b16培养到80％丰度，反复冻融结合超声波破碎仪破碎细胞(超声破碎参数为在30w,10min)，以200ug/次总蛋白分点皮下免疫羊驼四次，每次间隔两周，第一次免疫时，所述佐剂为完全弗氏佐剂，第二至第四次免疫时，所述佐剂为不完全弗氏佐剂；最后一次免疫后1周采集50ml羊驼血液，分离淋巴细胞提取总rna，反转录为cdna；以cdna为模版，用第一对引物进行pcr扩增，得到700bp扩增产物；以700bp扩增产物为模版，用第二对引物进行pcr扩增，将得到的vhh片段和pcantab5e分别经sfii两次酶切后连接；将得到的连接产物电转化到tg1感受态细胞，得到黑素瘤纳米库。采用本发明的构建方法构建得到的黑素瘤纳米抗体库，是一个特性很好的免疫文库，适用于特异性地筛选与黑素瘤生长、发育、迁移、增殖等相关的纳米抗体。附图说明图1为第一轮pcr-vhh凝胶电泳图；图2为第二轮pcr-vhh电泳图；图3为平板检测b16-vhh纳米文库库容结果；图4为平板检测b16-vhh纳米文库丰度结果；图5为pcr检测b16-vhh纳米文库插入率结果。具体实施方式本发明提供了一种小鼠黑素瘤蛋白纳米抗体库的构建方法，包括以下步骤：1)黑色素瘤细胞b16培养到80％丰度，破碎细胞，得到抗原共合物。2)将步骤1)得到的抗原共和物与佐剂按体积比为1:1进行配制，将得到的配制物乳化后按200ug/次分点皮下免疫羊驼四次，每次间隔两周，第一次免疫时，所述佐剂为完全弗氏佐剂，第二至第四次免疫时，所述佐剂为不完全弗氏佐剂。3)第4次免疫后1周采集50ml羊驼抗凝血液，分离得到淋巴细胞，提取淋巴细胞总rna，将所述rna反转录为cdna；4)以所述步骤3)得到的cdna为模版，用call001-f和call002-r引物进行第一轮pcr扩增，分离纯化700bp的扩增产物；所述call001-f引物具有seqidno.1所示的核苷酸序列；所述call002-r引物具有seqidno.2所示的核苷酸序列；5)以所述步骤4)得到的700bp的扩增产物为模版，用vhh2-f和vhh2-r引物pcr扩增vhh片段，分离纯化vhh片段；所述vhh2-f引物具有seqidno.3所示的核苷酸序列；所述vhh2-r引物具有seqidno.4所示的核苷酸序列；6)将所述步骤5)得到的vhh片段和pcantab5e分别用sfii酶进行两次酶切，连接酶连接，构建得到pcantab5e-vhh重组表达载体；7)将所述步骤6)得到的pcantab5e-vhh重组表达载体电转化tg1感受态细胞，得到黑素瘤纳米抗体库。在本发明中，所述培养使用的培养基优选为含有体积分数为8～12％fbs的dmem高糖培养基，更优选为含有体积分数为10％fbs的dmem高糖培养基。本发明对所述破碎细胞的方法没有特殊限定，采用常规方法即可，如超声破碎，所述超声破碎的参数优选包括：所述超声破碎的功率优选为30w，所述超声破碎的时间优选为10min。本发明将得到的抗原共和物与佐剂按体积比为1:1进行配制，将得到的配制物乳化后按200ug/次分点皮下免疫羊驼四次，每次间隔两周，第一次免疫时，所述佐剂为完全弗氏佐剂，第二至第四次免疫时，所述佐剂为不完全弗氏佐剂。本发明在第4次免疫后1周采集50ml羊驼抗凝血液，分离得到淋巴细胞，提取淋巴细胞总rna，将所述rna反转录为cdna。本发明对所述提取淋巴细胞总rna的方法没有特殊限定，采用常规rna提取的方法即可。在本发明中，所述反转录优选包括：将5μg总rna、1μlrandom6primer、1μloligo(dt)18primer、1μldntpmix和7μlddh2o混合后在65℃下处理5min，将得到的处理物在冰上静置5min，将所述静置物与4μl5xprimescriptiibuffer、0.5μlrnaseinhibiter、1μlprimerscripiirtase和4.5μlddh2o混合后，依次40℃处理40min和70℃处理15min，得到cdna。本发明以cdna为模版，用call001-f和call002-r引物对进行第一pcr扩增，分离纯化700bp的扩增产物；所述call001-f引物具有seqidno.1所示的核苷酸序列；所述call002-r引物具有seqidno.2所示的核苷酸序列。在本发明中，所述call001-f引物具有seqidno.1所示的核苷酸序列，具体序列如下：gtcctggctgctcttctacaagg；所述call002-r引物具有seqidno.2所示的核苷酸序列，具体序列如下：ggtacgtgctgttgaactgttcc。在本发明中，所述call001-f引物对应羊驼抗体的leader区，所述call002-r引物对应于羊驼抗体的第二恒定区(ch2)，可分别对常规抗体(900bp)及重链抗体(700bp)的leader区及ch2区域进行扩增。本发明将扩增得到的700bp扩增产物进行pcr扩增vhh片段。在本发明中，所述第一轮pcr扩增的体系优选每50μl包括：2μlcdna、浓度为10umol/μl的call001-f1μl、浓度为10umol/ul的call001-r1μl、25μltaqgreenpcrmix和21μlddh2o。在本发明中，所述第一pcr扩增的程序优选包括：95℃5min；95℃30s，53℃30s，72℃40s，30个循环；72℃5min。本发明以所述700bp扩增产物为模版，用vhh2-f和vhh2-r引物进行pcr扩增vhh片段，分离纯化vhh片段；所述vhh2-f引物具有seqidno.3所示的核苷酸序列；所述vhh2-r引物具有seqidno.4所示的核苷酸序列。在本发明中，所述vhh2-f引物具有seqidno.3所示的核苷酸序列，具体序列如下所示：tttctattactaggcccagccggccagktgcagctcgtggagtcnggngg；所述vhh2-r引物具有seqidno.4所示的核苷酸序列，具体序列如下所示：aaaccgttggccataatggcctggagctggggtcttcgctgtggt。根据第一轮pcr产物的测序结果，在羊驼单域抗体(vhh结构域)的两端恒定区fr1、fr4区域分别设计第二轮扩增引物vhh2-f和vhh2-r，以扩增vhh序列。本发明将得到的vhh片段和pcantab5e分别用sfii酶进行两次酶切，连接酶连接，构建得到pcantab5e-vhh重组表达载体。本发明对所述vhh片段和pcantab5e的酶切是根据pcantab5e的酶切位点，采用常规酶切的方法即可。在本发明中，经sfii两次酶切后能够消除空载率。在本发明中，所述连接使用的酶优选为t4连接酶。在本发明中，所述连接的体系优选每200μl包括：vhh片段1μg、pcantab5e载体3μg、t4dnaligase10μl、10×buffer20μl，ddh2o补充至200μl。本发明对所述连接的条件没有特殊限定，采用常规连接条件即可。本发明将所述pcantab5e-vhh重组表达载体电转化tg1感受态细胞，得到黑素瘤纳米库。在本发明中，所述电转化优选包括：所述电转化的电压优选为1.5～2.0kv，更优选为1.8kv；所述电转化的电阻优选为180～220ω，更优选为200ω；所述电转化的电容优选为20～30μf，更优选为25μf；所述电转化的时间优选为4～6ms，更优选为5ms。本发明电转化后还包括：将得到的电转化物在37℃下孵育1h，将得到的孵育物在5000g下离心5min，将得到的沉淀加入soc液体培养基后，得到黑素瘤纳米库。下面结合具体实施例对本发明所述的一种黑素瘤纳米抗体库的构建方法做进一步详细的介绍，本发明的技术方案包括但不限于以下实施例。实施例1细胞培养及抗原制备：b16培养到80％时进行细胞破碎：-70℃和37℃反复冻融，冻融时间为15min/次，超生破碎30min，离心取上清。用bca检测蛋白浓度。待作抗原。免疫程序及鉴定：挑选体况良好的成年雄性羊驼，免疫之前采集5ml实验羊驼的促凝血液，分离未免血清，保存备用。将制备的小鼠黑色素瘤细胞蛋白根据浓度分装成4管，每管200μg。采取肩胛部皮下多点注射的方式进行免疫，免疫间隔为2周，共免疫4次，每次免疫的蛋白量为200μg，其中初次免疫使用完全弗氏佐剂(sigama)，第二到第四次免疫使用不完全弗氏佐剂(sigama)，第三次免疫后采集5ml羊驼促凝血，分离血清elisa检测免疫效果，elisa按说明书进行，在此过程中，使用的二抗来源于llama。淋巴细胞总rna的提取和vhh模板cdna的合成按rna提取试剂盒说明书提取rna后，并按长链cdna反转录试剂盒说明书合成vhh第一条链，反应体系如表1：表1反应体系总rna5ugrandom6/oligo(dt)18primer1/1μldntpmix1μlrnasewaterupto10μl65℃5min冰上5min5xprimescriptiibuffer4ulrnaseinhibiter0.5ulprimerscripiirtase1ulrnasewaterupto20ul反应体系瞬时涡旋，pcr仪按40℃、40min；70℃、15min；12℃保存的反应体系进行反转录。第一轮pcr扩增vhh片段：纳米抗体免疫文库构建采用巢式pcr法，第一轮pcr扩增的引物call001-f和call002-r，其中call001-f对应羊驼抗体的leader区，call002-r对应于羊驼抗体的第二恒定区(ch2)，可分别对常规抗体(900bp)及重链抗体(700bp)的leader区及ch2区域进行扩增，具体方法如下：第一轮pcr引物见表2：表2引物序列call001-f：gtcctggctgctcttctacaaggcall002-r：ggtacgtgctgttgaactgttcc以合成的第一链cdna为模板进行pcr扩增，反转录产物分成30个反应，每个pcr反应体系为50ul，反应所用试剂为康为世纪的2xtap酶，反应体系见表3：表3反应体系cdna2μlcall001-f(10umol/ul)1μlcall002-r10umol/ul)1μltaqgreenpcrmix25μlwater21μl总体积50μlpcr扩增反应程序见表4：表4扩增程序琼脂糖凝胶电泳分析第一轮pcr反应产物主要包含大小为900bp和700bp的两种扩增产物，紫外灯下切胶回收700bp的核酸，胶回收试剂盒(康为世纪)回收纯化700bp的核酸片段，1ul胶回收产物与pmd19-tsimple载体相连，反应体系见表5：表5反应体系pmd19-tsimple1μl胶回收产物1μlsolutioni5μlwaterupto10ul总体积10μl瞬时涡旋反应体系，4℃反应过夜，热激连接产物与dh5α感受态细胞混匀，冰上孵育25min，42℃精确热激90s，冰上孵育4min，加入400ul的lb培养基37℃、200rpm孵育40min，50ul转化后的菌液均匀的涂布到含有氨苄青霉素(amp)的lb固体培养基表面，培养板倒置，37℃过夜培养。次日挑取20个单克隆菌落接种到5ml含有amp抗性的lb液体培养基过夜培养后测定其碱基序列。vectornti软件分析测序结果，设计建库用的第二轮pcr引物vhh2-f和vhh2-r。第二轮pcr扩增vhh：以第一轮pcr产生的700bp的回收片段为模板，以测序设计的引物vhh2-f和vhh2-r进行vhh片段扩增。50μl的反应体系，共做24个pcr反应，具体反应体系见表6：表6反应体系pcr扩增反应条件见表7：表7扩增程序取2ul二轮pcr反应产物进行琼脂糖凝胶电泳检测，扩增产物为单一条带。二轮vhh-pcr产物可直接使用核酸纯化试剂盒说明书进行纯化。vhh片段及载体酶切纯化：摇菌质粒小提含有pcantab5e质粒的大肠杆菌。抽取10ul的质粒进行质粒测序，检测所用质粒质量，有无突变碱基等。vhh片段及质粒pcantab5e进行二轮酶切，反应所用酶为sfii(thermo)，酶切体系见表8：表8酶切体系vhh片段及pcantab5e载体进行2轮酶切以消除空载率，第一轮vhh和载体都做20个反应的酶切，反应条件：50℃水浴酶切1h。pcr产物纯化试剂盒纯化vhh体系；载体酶切体系先进行琼脂糖纯化，然后采用胶回收试剂盒纯化。一轮酶切后的纯化产物按照上面的酶切体系再度50℃水浴、酶切1h，两种产物再度纯化。酶切产物连接及纯化：两轮酶切纯化后的vhh片段和载体pcantab5e进行连接，连接采用t4连接酶(thermo)，反应体系见表9：表9连接体系vhh片段1ugpcantab5e载体3ugt4dnaligase10μl10×buffer20μlwaterupto200μl总体积200μl反应体系瞬时涡旋，4℃反应过夜。次日按说明书进行连接产物的纯化。黑素瘤纳米抗体库(b16-vhh)的构建及特性鉴定：将纯化后的pcantab5e-vhh连接产物(总体积100ul)加入刚制备的新鲜tg1感受态细胞，吹打混匀，冰浴10min；在电压1.8kv，电阻200ω，电容25μf，时间5ms的条件下，在电转仪中进行转化后，将电转杯中点击后的tg1细胞转移至50ml无菌离心管中，37℃振荡孵育1h；孵育过的菌液5000g离心5min，弃净上清，重新加入8ml新鲜的soc培养基重悬沉淀；取100μl菌液梯度稀释后涂布到16个2×ytag的固体培养板(90mm)测定库容，每个梯度两个平板，共做8个梯度平板，剩余菌液涂布到30个2×ytag固体培养板(150mm平板)，25℃静置培养箱培养过夜；次日待培养板菌落长好后，39个150mm的平板中每个平板加入5ml的2×yt液体培养基清洗菌落收集清洗液；5000g、4℃、离心12min，加入含15％甘油的2×yt液体培养基90ml重悬沉淀，分管密封后即为制备的初级文库菌，命名为b16-vhh，即为黑素瘤纳米抗体库，取100μl初级文库菌用于测定文库丰度，其余分装冻存于-70℃。文库库容的测定：100ul电转后的菌液梯度稀释，稀释度从10-1～10-8；每个稀释度各取100ul涂布2个2×ytag固体培养板，25℃培养过夜；次日对梯度板的菌落进行统计，计算库容。文库丰度的测定：取100ul初级文库菌液进行梯度稀释，稀释度从10-4～10-10；每个稀释度各取100ul菌液涂布2个2×ytag固体培养板，25℃培养过夜；次日对梯度板的菌落进行统计，计算文库丰度。文库vhh片段插入率及插入多样性的测定：待文库库容大小测定后，从测定库容的固体培养板中随机挑取50个单克隆菌落接种于1ml2×ytag培养基，37℃振荡培养过夜；次日对50个克隆进行菌液pcr鉴定，并将剩余的菌液送测序公司进行测序；分析pcr鉴定结果及测序结果，计算vhh片段的插入率及插入的多样性。实验结果：1.elisa法检测免疫效果空白孔和阴性孔中都添加10-1稀释的血清，根据elisa数据结果发现：未免血清中不含b16细胞蛋白的抗体，而免疫后的全血清中的阳性反应十分强烈，在稀释10-2后仍然具有阳性反应，而免疫后分离的纳米抗体在稀释到10-2后同样具有阳性反应，但是阳性率低于全血清，结果说明免疫b16蛋白后，羊驼体内不光产生了针对b16蛋白的纳米抗体，同时也产生了抗b16蛋白的传统抗体。elisa结果显示免疫效果可用于后续文库构建。实验结果如下表10。表10elisa法检测免疫效果未免血清0.0380.0660.3130.1930.097免疫血清0.0520.0812.5211.9361.012纯化血清0.0400.0791.9821.2130.8762.淋巴细胞分离及vhh片段扩增细胞计数仪检测分离得到的羊驼免疫后淋巴细胞为3.6×107个，将淋巴细胞分成10管，每管添加1ml的trizol，按步骤提取细胞总rna，两步法反转录合成长片段cdna，call001和calloo2引物扩增vhh的leader区到ch2区域，凝胶电泳检测条带，由下图1发现，第一轮pcr引物可以扩增传统抗体(900bp)和重链抗体(700bp)的vhh片段。第一轮pcr产物连接pmd19t载体后，挑选20个单克隆菌斑进行测序分析，vectornti软件(vectornti11.5.1)对测序结果进行分析比对，设计第二轮pcr引物vhh2-f和vhh2-r二次体外扩增vhh区域。第二轮pcr扩增体系同上一轮pcr扩增体系，琼脂糖凝胶电泳检测第二轮pcr产物电泳图如下图2所示：第二轮pcr产物大小为400bp左右，条带单一，大小与预期相符。3.黑素瘤纳米抗体库(b16-vhh)构建梯度稀释法检测b16-vhh纳米抗体文库库容：10-6稀释平板上有72个单克隆菌落，800x72x10-6＝为5.76×1010个(图3)，文库的丰度为365x10x107＝3.65×1010个/ml(图4)。50个菌液pcr样品中仅有2个样品片段长度与vhh片段不符(图5)，50个测序样品有16，26号克隆测序结果不合格，并非vhh片段，软件分析48个样品序列的相似性发现48个插入片段均不相同，测序合格的样品序列ncbi分析发现其都是vhh序列，因此可计算文库vhh片段的插入率为98％。软件转化48个样品序列对应的氨基酸序列，黑素瘤纳米库中的氨基酸序列如下所示：seqidno.5：esggglvqpggsltlscvasgftlnghaigwfrqapgkeregvaclsnsggstlyaesvkgrfaiskdndkdmmylrmnslrpddtatyyclgssdidhwgkgilvtvsa；seqidno.6：esgggdmqpgealrlscaasgsifanyimgwyrqapgkerellitiktirshtnyshtvkdrftasrdnakntvslrindlkpedtavyycntdpplpnywgqgtqvtvss；seqidno.7：esggglvqpggslrlscdetssrtlghyigwfrqapgkeregvaclsnsggstlyadfvkgrftisrynagntlplqmnnlevddtavyychaderesdtvikshwgqgtqvtvsp；seqidno.8：esggglvqpggslrlscaasgftfddygmswvrqapgkerevvsettprtrcicahsvegrftisrdnakntvwlqmnllepedtavyycrshcgdpkfgswgqgtqvavss；seqidno.9：esggglvqpggslrlscavsgsidsffymawhrqapgkglewvasihsggnrtyyqdfvkgrftisrdnakntiylqmtnlkpedtavyyclaritsdidhwgkgilvtvsa；seqidno.10：esggglvqpggslrlscdetssrtldyykigwfrqapgkeregvscsnsggstlyadsvkgrftvsrdiaenkvylqmknlkpedtgiyycraritsscsgpfgsyglgaqvtvts；seqidno.11：esggglvqpggslrlscvasgsifanyimnwvrlap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rydttnyadsvkgrftistgnaentlylqmnslkpedtaiyycaatiyearglrfwglgtqvtvss；seqidno.51：esggglvqpggsltlsciasgttfsitksawyrqapgnqrervlvaaitsrgtttyyassvegrftisrdnaknavylqmnslnaedtavyaanfahdgscpfherkywgkgtlvivss；seqidno.52：esggglvqagdsltlscaasafnlddyamswvrqapgkgrewtcsiyedsstycdsvkgrftistdsttdtvylqmnslgpedtaiyycicnaragagrtvgpdqynywgqgtqvtvss。以上均说明黑素瘤纳米抗体库(b16-vhh)是一个特性很好的免疫文库，适合用于特异性纳米抗体的筛选。以上所述仅是本发明的优选实施方式，应当指出，对于本
技术领域：
的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。序列表<110>山西农业大学<120>一种黑素瘤纳米抗体库的构建方法<160>52<170>siposequencelisting1.0<210>1<211>23<212>dna<213>人工序列(artificialsequence)<400>1gtcctggctgctcttctacaagg23<210>2<211>23<212>dna<213>人工序列(artificialsequence)<400>2ggtacgtgctgttgaactgttcc23<210>3<211>50<212>dna<213>人工序列(artificialsequence)<400>3tttctattactaggcccagccggccagktgcagctcgtggagtcnggngg50<210>4<211>45<212>dna<213>人工序列(artificialsequence)<400>4aaaccgttggccataatggcctggagctggggtcttcgctgtggt45<210>5<211>110<212>prt<213>人工序列(artificialsequence)<400>5gluserglyglyglyleuvalglnproglyglyserleuthrleuser151015cysvalalaserglyphethrleuasnglyhisalaileglytrpphe202530argglnalaproglylysgluarggluglyvalalacysleuserasn354045serglyglyserthrleutyralagluservallysglyargpheala505560ileserlysaspasnasplysaspmetmettyrleuargmetasnser65707580leuargproaspaspthralathrtyrtyrcysleuglyserserasp859095ileasphistrpglylysglyileleuvalthrvalserala100105110<210>6<211>111<212>prt<213>人工序列(artificialsequence)<400>6gluserglyglyglyaspmetglnproglyglualaleuargleuser151015cysalaalaserglyserilephealaasntyrilemetglytrptyr202530argglnalaproglylysgluarggluleuleuilethrilelysthr354045ileargserhisthrasntyrserhisthrvallysaspargphethr505560alaserargaspasnalalysasnthrvalserleuargileasnasp65707580leulysprogluaspthralavaltyrtyrcysasnthrasppropro859095leuproasntyrtrpglyglnglythrglnvalthrvalserser100105110<210>7<211>116<212>prt<213>人工序列(artificialsequence)<400>7gluserglyglyglyleuvalglnproglyglyserleuargleuser151015cysaspgluthrserserargthrleuglyhistyrileglytrpphe202530argglnalaproglylysgluarggluglyvalalacysleuserasn354045serglyglyserthrleutyralaaspphevallysglyargphethr505560ileserargtyrasnalaglyasnthrleuproleuglnmetasnasn65707580leugluvalaspaspthralavaltyrtyrcyshisalaaspgluarg859095gluseraspthrvalilelysserhistrpglyglnglythrglnval100105110thrvalserpro115<210>8<211>112<212>prt<213>人工序列(artificialsequence)<400>8gluserglyglyglyleuvalglnproglyglyserleuargleuser151015cysalaalaserglyphethrpheaspasptyrglymetsertrpval202530argglnalaproglylysgluarggluvalvalsergluthrthrpro354045argthrargcysilecysalahisservalgluglyargphethrile505560serargaspasnalalysasnthrvaltrpleuglnmetasnleuleu65707580gluprogluaspthralavaltyrtyrcysargserhiscysglyasp859095prolyspheglysertrpglyglnglythrglnvalalavalserser100105110<210>9<211>112<212>prt<213>人工序列(artificialsequence)<400>9gluserglyglyglyleuvalglnproglyglyserleuargleuser151015cysalavalserglyserileaspserphephetyrmetalatrphis202530argglnalaproglylysglyleuglutrpvalalaserilehisser354045glyglyasnargthrtyrtyrglnaspphevallysglyargphethr505560ileserargaspasnalalysasnthriletyrleuglnmetthrasn65707580leulysprogluaspthralavaltyrtyrcysleualaargilethr859095seraspileasphistrpglylysglyileleuvalthrvalserala100105110<210>10<211>116<212>prt<213>人工序列(artificialsequence)<400>10gluserglyglyglyleuvalglnproglyglyserleuargleuser151015cysaspgluthrserserargthrleuasptyrtyrlysileglytrp202530pheargglnalaproglylysgluarggluglyvalsercysserasn354045serglyglyserthrleutyralaaspservallysglyargphethr505560valserargaspilealagluasnlysvaltyrleuglnmetlysasn65707580leulysprogluaspthrglyiletyrtyrcysargalaargilethr859095sersercysserglypropheglysertyrglyleuglyalaglnval100105110thrvalthrser115<210>11<211>121<212>prt<213>人工序列(artificialsequence)<400>11gluserglyglyglyleuvalglnproglyglyserleuargleuser151015cysvalalaserglyserilephealaasntyrilemetasntrpval202530argleualaproglylysgluargglugluvalsercysileserpro354045serglyaspserthrasntyrglyaspservallysglyargphethr505560ileserargaspasnalalysserthrvalpheleuglnmetaspasn65707580leuserprogluaspthralailetyrtyrcysglyalaargilethr859095valpheargargcysserphevalserasnglutyrasptyrtrpgly100105110glnglythrglnvalthrvalserser115120<210>12<211>116<212>prt<213>人工序列(artificialsequence)<400>12gluserglyglyglyvalvalglnproglyglyserleuargleuser151015cysalaalaserglyserilephealaasntyrilemetsertrpval202530argglnvalproglylysargarggluglyvalsercysthrgluser354045asnasnglyserthrthrtyrargaspservallysglyargphethr505560ileserargaspasnvallysasnglnleutyrleuglnmetasnser65707580leulysprogluaspthralavaltyrtyrcysalalysglyaspile859095valilealasertyrileleuaspthrtrpglyglnglythrleuval100105110thrvalserser115<210>13<211>115<212>prt<213>人工序列(artificialsequence)<400>13gluserglyglyglyleuvalglnproglyglyserleuargleuser151015cysalaalaserglyserilephealaasntyrilemetasntrpval202530argglnalaproglylysglygluvalsercysileglyprosergly354045aspserthrargtyralaaspservalthrglyargphethrileser505560argaspasnalalysasnthrleutyrleuglnmetasnserleulys65707580progluaspthralaleutyrphecysalaargaspargglyglutyr859095serargaspaspasptyrasptyrtrpglyglnglythrglnvalthr100105110valserser115<210>14<211>121<212>prt<213>人工序列(artificialsequence)<400>14gluserglyglyaspservalglnproglyglyserleuargleuser151015cysalaalaserglyserilephealaasntyrilemetsertrpval202530argglnalaproglylysglyleuglutrpilealaalailehisser354045glyglyasnargthrsertyralathrservallysglyargphethr505560ileserargaspasnalalysasnthrleutyrleuglnmetasnser65707580leulysprogluaspthralailetyrtyrcysglyalaargilethr859095valpheargargcysserphevalserasnglutyrasptyrtrpgly100105110glnglythrglnvalthrvalserser115120<210>15<211>112<212>prt<213>人工序列(artificialsequence)<400>15gluserglyglyglygluvalglnproglyglyserleuargleuser151015cysvalalaserglyserthrpheserasntyrlysmetsertrptyr202530argglnthrproglyglugluarggluleuvalalaglyilelysser354045leuglyaspthrglntyrseraspservallysglyargphealaile505560serargaspaspserargasnvalmettyrleuglnmetasnserleu65707580lysphegluaspthralavaltyrtyrcysasnalagluthrpheglu859095argasntyrgluphetrpglyglnglyileargvalthrvalserleu100105110<210>16<211>111<212>prt<213>人工序列(artificialsequence)<400>16gluserglyglyglyleuvalglnproglyglyserleuargleuser151015cysalaalaserglyserilephealaargtyrilemetsertrpval202530argglnalaproglylysglyleuglugluvalsercysileglypro354045serglyaspserthralatyrseraspservallysglyargphethr505560ileserargaspasnalalysasnthrvaltyrleuglnmetaspser65707580leulysleuaspaspthrglyvaltyrtyrcysargalaargthrarg859095gluasnvaltyrtrpglyglnglythrglnvalthrvalserser100105110<210>17<211>117<212>prt<213>人工序列(artificialsequence)<400>17gluserglyglyglyservalgluproglyserileleuargleuser151015cysvalalaserglyserthrpheserasntyrlysmetsertrptyr202530argglnthrproglyglugluarggluleuvalthralaserglyala354045tyrlysasntyrseraspservalargglyargphethrileserarg505560valasnalaasnasnthrmettyrleuglnmetserasnleuglupro65707580gluaspthrglyvaltyrtyrcysalaalaarghisilesertyrcys859095procysglythrasptyrargaspgluleutrpglyglnglythrgln100105110valthrvalserpro115<210>18<211>122<212>prt<213>人工序列(artificialsequence)<400>18gluserglyglyglyleuvalglnproglyglyserleuargleuser151015cysthralaserglyphethrphethrasptyrglymetglytrpphe202530argglnalaproglylysgluargglugluvalsercysileargpro354045serglyaspserthrasntyrleuaspservallysglyargphethr505560ileserargaspasnthrlysserthrvalhisleuhismetasnser65707580leulysprogluaspthralailetyrphecysalaalaargilethr859095alavalargglymetcysleumetasnaspargtrpphegluhistrp100105110glyglnglythrglnvalthrvalserser115120<210>19<211>114<212>prt<213>人工序列(artificialsequence)<400>19gluserglyglyglyservalglnproglyglyserleuargleuser151015cysglnalaserglyphethrpheserlystyrargmetargtrptyr202530argglnalaproglylysglyarggluleuvalalaglugluvalser354045glyglyilethrasntyralaaspservallysglyargphethrile505560pheargaspasnthrlysalaleuvalaspleuglnmetasnserleu65707580lysprogluaspthrglyvaltyrphecysalaleuaspargvaltyr859095proglncysvalphesertyraspvalglnglythrglnvalthrval100105110serser<210>20<211>117<212>prt<213>人工序列(artificialsequence)<400>20gluserglyglyglyleuvalglnproglyglyserleuargleuser151015cysvalalaserglypheasppheserthrtyralametsertrptyr202530argglnalaproglylystrpleuglutrpvalseralaalailethr354045alaasnalaaspthrserasntyrglyaspservallysglyargphe505560thrileserargaspasnalalysserthrvalpheleuglnmetasp65707580asnleuserprogluaspthralailetyrtyrcysargargserala859095glysersercysserglypropheglysertyrglyleuglyalagln100105110valthrvalthrser115<210>21<211>113<212>prt<213>人工序列(artificialsequence)<400>21gluserglyglyglyleuvalglnalaglyglyserleuargleuser151015cysthrglyphethrpheseralatyralametalatrptyrarggln202530alaproglylysgluarggluleuvalalavalilethralaaspgly354045serthrtrptyralagluservallysglyargphethrileserarg505560aspasnalalysargthrleuhisleuglnmetasnasnleulyspro65707580gluaspthralamettyrtyrcysalaileglnasnargaspasptyr859095lysproileserasptyrleuglyargglythrglnvalthrvalser100105110pro<210>22<211>112<212>prt<213>人工序列(artificialsequence)<400>22argalatyrglucysserhisglyglyserleuargleusercyslys151015alaserglypheilepheserasntyralametsertrptyrarggln202530alaproglyglngluarggluglyvalsercysileasnglyarggly354045glytyrthrasntyralaaspservallysglyargphethrileser505560thrglyasnalagluasnthrleutyrleuglnmetasnserleulys65707580progluaspthralailetyrtyrcysalaalathriletyrgluala859095argglyleuargphetrpglyglnglythrglnvalthrvalserser100105110<210>23<211>116<212>prt<213>人工序列(artificialsequence)<400>23gluserglyglyglyleuvalasnthrglyglyserleuargleuser151015cysvalalaserglyilealaalaserthrserthrilealatrpphe202530argglnalaproglylysgluarggluleuvalalathrilesergly354045serglyasnthrtyrtyrthraspservallysglyargpheserile505560serargaspasnvalglnasnleuleutyrleuglnmetasnserleu65707580lysproaspaspthralamettyrtyrcysalaglycysserglytyr859095valcysleuproaspglytyrasntyrtrpglyglnglythrglnval100105110thrvalserser115<210>24<211>113<212>prt<213>人工序列(artificialsequence)<400>24gluserglyglyargleuvalglnproglylysserleulysleuser151015cysalaalaserglyphethrpheaspasptyrglymetglytrpphe202530argglnthrproglylysgluleuglugluvalsercysileglypro354045serglyaspserthralatyralaasnservallysglyargphethr505560ileserargaspasnalathrasnthrvaltyrleuglnmetasnser65707580leuasnsergluaspthrglyiletyrtyrcysargserhiscysgly859095aspprolyspheglysertrpglyglnglythrglnvalalavalser100105110ser<210>25<211>119<212>prt<213>人工序列(artificialsequence)<400>25gluserglyglyglyleuvalglnproglyglyserleuargleuser151015cysalaalaserglyserilephealaasntyrilemetglytrptyr202530argglnalaproglygluglnarggluleuleuilethrilelysthr354045ileargserhisthralatyralaaspservallysglyargphethr505560valserargaspasnvalargasnthrvalasnleuglnmetasnser65707580leugluprogluaspthralavaltyrtyrcystyralaasnileile859095leumetglygluleuaspserglyleuhisasptyrtrpglyglngly100105110thrglnvalthrvalserser115<210>26<211>110<212>prt<213>人工序列(artificialsequence)<400>26gluserglyglyglyleuvalglnproglyglyserleuasnleuser151015cysvalalaserglyphethrpheargasptyrglymetglytrpphe202530argglnalaproglylysgluarggluleugluthrthrproargthr354045argcysilecysseraspservallysglyargphethrileserarg505560aspasnalalysasnthrvaltyrleuglnmetaspserleulysleu65707580aspaspthrglyvaltyrtyrcysargalaargthrargaspaspglu859095asnvaltyrtrpglyglnglythrglnvalthrvalserser100105110<210>27<211>109<212>prt<213>人工序列(artificialsequence)<400>27gluserglyglyglyleuvalglnalaglyglyserleuargleuser151015cysalaalaserglyserilephealaargtyrilemetasntrpphe202530argglnalaproglylysglutyrgluphevalileglyprosergly354045aspserthrargtyralaaspservallysglyargphethrileser505560argaspasnalalysasnthrvaltyrproglnmetasnserleuglu65707580progluaspthralavaltyrtyrcysasnalaalaalathrproglu859095asptyrtrpglylysglythrleuvalthrvalserser100105<210>28<211>112<212>prt<213>人工序列(artificialsequence)<400>28gluserglyglyglyleuvalglnalaglyalaserleuargleuser151015cysalaalaserglyargserthrlysvaltyrsermetglytrptyr202530argglnproproglylysglyargglutrpvalserilethrsergly354045glyglytyrthrasptyralaaspservallysglyargphethrile505560serargaspasnalalysasnthrmettyrleuglnmetasnserleu65707580lysprogluaspthralavaltyrtyrcysasnalaglusertyrgly859095seraspserilevaltrpglyglnglythrglnvalthrvalserser100105110<210>29<211>118<212>prt<213>人工序列(artificialsequence)<400>29gluserglyglyglyleuvalglnproglyglyserleuargleuser151015cysthrthrserargpheserthrasptyrhisalametglytrptyr202530argglnalaproglylysglnarggluphevalalaasnserilethr354045serglyglyservalasntyrseraspservallysglyargphethr505560ileserargaspasnalalysasnthrleutyrleuglnmetasnasn65707580leulysprogluaspthralaleutyrtyrcysalaargalaleuthr859095argaspglntrpsertyrglyasnmetasptyrtrpglylysglyala100105110leuilethrvalserser115<210>30<211>110<212>prt<213>人工序列(artificialsequence)<400>30gluserglyglyglyleuvalglnproglyglyserleuthrleuser151015cystyrargglnalaproglylysglnarggluleuilealaserile202530argglnalaproglylysglyarggluglyvalglycysilesertyr354045argglylysilethrvaltyralaaspservallysglyargphethr505560valpheargaspaspalalysthrthrvalpheleuglnmetasnval65707580leulysprogluaspseralailetyrphecyscysprophehisglu859095trplystyrtrpglyglnglythrglnvalthrvalserser100105110<210>31<211>115<212>prt<213>人工序列(artificialsequence)<400>31gluserglyglyglyleuvalglnproglyglyserleuserleuser151015cysalavalserglyargvalseralaileseralametglytrptyr202530argglnalaproglulysglnarggluleualacysgluglythrarg354045aspthrargthrlysalathrprovallysglyargphethrileser505560argaspasnalalysasnthrvaltyrleuglnmetasnserleulys65707580progluaspthralavaltyrtyrcysaspleutrpglyaspcysser859095thrproaspprolystyrasptyrtrpglyglnglythrglnvalthr100105110valserser115<210>32<211>116<212>prt<213>人工序列(artificialsequence)<400>32gluserglyglyglyleuvalglnproglyglyserleuthrleuser151015cysalaasnmetglntyrthrleuasptyrtyralavalalatrpphe202530argglnserproglylysglyargglualavalalacysileserser354045thraspgluthrthralaalaaspprovallysglyargphethrile505560serargaspasnasplysserthrmettyrleuglnserasnserleu65707580lyssergluaspthralavaltyrtyrcysasnglnglytyrtyrala859095serserargtyrtyralametalatyrleuglyglnglythrglnval100105110thrvalserser115<210>33<211>119<212>prt<213>人工序列(artificialsequence)<400>33gluserglyglyglyleuvalargproglyglyserleuargleuser151015cysvalalathrglyserglupheargleuphetyrmetalatrptyr202530argglnalaproglylysgluarggluglnvalalathrthrthrleu354045argcysaspleuthrargtyrthrtyralaaspservallysglyarg505560phealaileserargaspasnalalysasnthrvaltyrleuglnmet65707580asnserleulysprogluaspthralaleutyrtyrcysglyaspcys859095serthrproaspcysvalpheserglyglysertyrtrpglylysgly100105110thrleuvalthrvalserser115<210>34<211>117<212>prt<213>人工序列(artificialsequence)<400>34gluserglyglyglyleuvalglnproglyserserleuthrleuser151015cysalavalserglyargalapheserasntyrlysmetsertrptyr202530argglnalaproglygluthrproglualavalserglyilelysser354045thrglyglyserthrtyrtyralagluphevalglnglyargpheser505560glyserargaspalaalalysasnthrvaltyrleuglnmetasnasn65707580leulysprogluaspthralavaltyrtyrcysalaleuaspargval859095tyrproglncysvalphesertyraspvalargglyglnglythrgln100105110valthrvalserser115<210>35<211>117<212>prt<213>人工序列(artificialsequence)<400>35gluserglyglyglyleuvalglnproglyglyserleuargleuser151015cysthrthrserargpheserleuasptyrhisaspileglytrptyr202530argglnalaproglylysglyleuglutrpvalserserileasnarg354045thrglyglyserthrtyrtyrseraspservallysglyargphethr505560metserargaspasnalalysasnthrtrphisleuglnmetasnser65707580leulysprogluaspserglyvaltyrtyrcysargvalaspserlys859095valproglncysvalpheserglyglysertrpglyproglythrgln100105110valthrvalserthr115<210>36<211>118<212>prt<213>人工序列(artificialsequence)<400>36gluserglyglyglyleuvalglnproglyglyserleuargleuser151015cysalaalaserglyargserthrlysargtyrsermetglytrptyr202530argglnproproglylysleuvalalathrileglythraspglygly354045alathrasntyralaaspservallysglyargphethrileserarg505560aspasnalalysasnthralatyrleuglnmetasnlysvalglupro65707580glyaspthralavaltyrilecysalaalaaspleutrpglyserala859095glyvaltrpthrprogluglyglutyrasptyrtrpglyglnglythr100105110glnvalthrvalserser115<210>37<211>116<212>prt<213>人工序列(artificialsequence)<400>37gluserglyglyaspleuvalhisalaglygluserleuargleuser151015cysglualaserglyserargpheargglutyrserleualatrpphe202530argglnthrproglyaspgluargglumetilealagluvalthrala354045serglyalatyrlysasntyralaaspvalvallysglyargphethr505560ileserargaspasnalalyslysthrleualaleuglumetasnasn65707580leulysprogluaspthralathrtyrtyrcysasnalatrpaspgln859095lysglyleuargargglutyrasptyrtrpglyglnglythrglnval100105110thrvalserser115<210>38<211>118<212>prt<213>人工序列(artificialsequence)<400>38gluserglyglyglyleuvalglnproglyglyserleuargleuser151015cysthralaserglyglythrpheasptyrtyrhisileglytrpphe202530argglnalaproglylysgluargglugluilesercysileserile354045thrserglyargserthrtyrtyralaaspservallysglyargphe505560thrileserargaspasnalalysasnthralatyrleuglnmetasn65707580asnleulysprogluaspthrglyvaltyrtyrcysglyaspcysser859095thrproglncysvalpheserglyglysertyrtrpglyglnglythr100105110glnvalthrvalserser115<210>39<211>117<212>prt<213>人工序列(artificialsequence)<400>39gluserglyglyglyprovalglnproglyglyserleuargleuser151015cysalaalaserglyproglypheaspaspserglymetglytrptyr202530argglnalaproglylysgluarggluglyleusercysaspargasp354045thrgluprothrlysthrtyrtyrileaspalavallysglyargphe505560thrileserargaspasnalalysasnthralatyrleuglnmetasn65707580aspleulysprogluaspthralavaltyrtyrcysasnalatrpasp859095lyslysargleuargargglutyrasptyrtrpglyglnglythrgln100105110valthrvalserser115<210>40<211>117<212>prt<213>人工序列(artificialsequence)<400>40gluserglyglyglyleuvalglnproglyglyserleuargleuser151015cysthrthrserlyspheserleuasptyrhisaspileglytrpphe202530argglnalaproglylysgluglyargglutrpvalserilethrarg354045thrglyglyserthrtyrtyrvalaspservallysglyargphethr505560ileserargasplysasnthrvaltyrleuglnmetasnserleulys65707580progluaspthrglyiletyrphecysalaalagluhisthrglyleu859095gluthrcysaspleuthrargtyrthrtyrtrpglyglnglythrgln100105110valthrvalserser115<210>41<211>115<212>prt<213>人工序列(artificialsequence)<400>41gluserglyglyglytrpvalglnproglygluserleuargleuser151015cysalaalaserglyphethrphetyrargphealametasntrpphe202530argglnalaproglylysglualaalagluhisthrglyleugluthr354045argaspserileleutyralaaspthrvallysaspargphelysile505560serargaspasnglymetasnthrvaltyrleuglnmetasnasnleu65707580lysprogluaspthrglyiletyrtyrcysasnalatrpaspglngly859095leuglyglyargglutyrasptyrtrpglyglnglythrglnvalile100105110valserser115<210>42<211>119<212>prt<213>人工序列(artificialsequence)<400>42gluserglyglyglyservalgluproglyserileleuargleuser151015cysvalalaserglypheglyleuaspalaleualaileglytrpphe202530argglnalaproglyasnglnglnarggluleuvalalaservalser354045argtyraspthrthrasntyralaasnservallysglyargphethr505560ileserargaspasnalalysserthrvaltyrleuglnmetasnlys65707580leulysprogluaspthralavaltyrtyrcysalagluaspserthr859095aspglygluthrcysgluproglyasppheargsertrpglyglngly100105110thrglnvalthrvalserser115<210>43<211>118<212>prt<213>人工序列(artificialsequence)<400>43gluserglyglyglyleuvalglnproglyglyserleuargleuser151015cysvalalaserglypheglyleuaspalaleualaileglytrpphe202530argtrpalaproglylysgluargargleuvalsercysileargmet354045aspaspserthrasntyralaaspvalvallysglyargphethrile505560serargaspasnalalyslysthrleualaleuglumetasnasnleu65707580lysprogluaspthralathrtyrtyrcysalaleuhisilesertyr859095cysprocysglythrasptyrargaspgluleutrpglyglnglythr100105110glnvalthrvalserpro115<210>44<211>115<212>prt<213>人工序列(artificialsequence)<400>44gluserglyglyglyleuvalglnalaglyglyserleuargleuser151015cysglntyrserglyleuthrgluasphistyralailealatrpphe202530argglnthrproglyglugluarggluleuvalglualaserleuala354045tyrlysasntyralaaspphevalargglyargphethrlysserlys505560aspalaalalysasnthrvaltyrleuglnmetasnasnleulyspro65707580gluaspthralavaltyrtyrcysalaleuasptyrargserargcys859095gluleutrpalaargtyrasptyrargglyglnglythrglnvalthr100105110valserser115<210>45<211>120<212>prt<213>人工序列(artificialsequence)<400>45gluserglyglyglyservalglnproglyglyserleuargleuser151015cysalaalaserglypheserleuasptyrtyrasnileglytrpphe202530argglnalaproglylysgluarggluglyvalsercysileserarg354045argvalseraspglyseralatyrtyralaasnservallysglyarg505560phethrileserargaspasnalalysserthrvaltyrleuglnmet65707580asnlysleulysprogluaspthralavaltyrtyrcysalaleugly859095glythraspprocysglythrasptyrargaspgluleuproglyarg100105110glythrglnvalthrvalserser115120<210>46<211>120<212>prt<213>人工序列(artificialsequence)<400>46gluserglyglyglyglnvalglnproglyglyserleuargleuser151015cysalaalaserglyproglypheaspaspserglyleuglytrpphe202530argglnargproglylysalaargglualailesercysileserser354045argserthrtyrilehistyralaaspservallysglyargphethr505560ilepheargaspasnthrlysalaleuvalaspleuglnmetasnser65707580leulysprogluaspthrglyvaltyrphecysalaalaarghisile859095sertyrcysprocysglythrasptyrargaspgluleutrpglygln100105110glythrglnvalthrvalserpro115120<210>47<211>116<212>prt<213>人工序列(artificialsequence)<400>47gluserglyglyglyvalvalglnproglyglyserleulysleupro151015cysalaalaserglyproglypheaspaspserglymetglytrptyr202530argglnalaproglylysgluarggluglyleusercysaspargasp354045thrgluprothrlysthrtyrtyrileaspalaaspservallysgly505560argphethrileserarglysseraspleuasnthrmettyrleugln65707580metasnserleulysprogluaspthralavaltyrtyrcysalaleu859095asptyrargserasnserargglytyrtrpglyglnglythrglnval100105110thrvalserser115<210>48<211>121<212>prt<213>人工序列(artificialsequence)<400>48gluserglyglyglyleuvalglnproglyglyserleuargleuser151015cysalaalaserglyargserthrlysargtyrsermetglytrptyr202530argglnproproglylysglyargglutrpargserilethrsergly354045glyglytyrthrasptyrglyaspphevallysglyargphethrile505560serargaspasnalalysasnthrleutyrleuglumetasnaspleu65707580lysprogluaspthralaleutyrphecysalaalaalaalaproarg859095argleupheglnasncysgluleuglythralatyrasptyrtrpgly100105110glnglythrglnvalthrvalserser115120<210>49<211>108<212>prt<213>人工序列(artificialsequence)<400>49gluserglyglyglyvalvalglnproglyglyserleulysleupro151015cysthralaserglyserasnleuaspphehisaspvalglytrpphe202530argglnvalproglylysgluleuglutrpvalserseriletyrser354045alaserserasnthrphetyrglyaspphevallysglyargphethr505560ileserargaspasnalalysasnthrvaltyrleuglnmetsergly65707580leuleuthrgluaspthrglyvaltyrcysprophehisglutrplys859095tyrtrpglyglnglythrglnvalthrvalserser100105<210>50<211>114<212>prt<213>人工序列(artificialsequence)<400>50gluserglyglyglyservalgluproglyserileleuargleuser151015cysvalalaserglypheglyleuaspalaleualaileglytrpphe202530argglnalaproglyasnglnglnarggluleuvalalaservalser354045argtyraspthrthrasntyralaaspservallysglyargphethr505560ileserthrglyasnalagluasnthrleutyrleuglnmetasnser65707580leulysprogluaspthralailetyrtyrcysalaalathriletyr859095glualaargglyleuargphetrpglyleuglythrglnvalthrval100105110serser<210>51<211>119<212>prt<213>人工序列(artificialsequence)<400>51gluserglyglyglyleuvalglnproglyglyserleuthrleuser151015cysilealaserglythrthrpheserilethrlysseralatrptyr202530argglnalaproglyasnglnarggluargvalleuvalalaalaile354045thrserargglythrthrthrtyrtyralaserservalgluglyarg505560phethrileserargaspasnalalysasnalavaltyrleuglnmet65707580asnserleuasnalagluaspthralavaltyralaalaasnpheala859095hisaspglysercysprophehisgluarglystyrtrpglylysgly100105110thrleuvalilevalserser115<210>52<211>119<212>prt<213>人工序列(artificialsequence)<400>52gluserglyglyglyleuvalglnalaglyaspserleuthrleuser151015cysalaalaseralapheasnleuaspasptyralametsertrpval202530argglnalaproglylysglyargglutrpthrcysseriletyrglu354045aspserserthrtyrcysaspservallysglyargphethrileser505560thraspserthrthraspthrvaltyrleuglnmetasnserleugly65707580progluaspthralailetyrtyrcysilecysasnalaargalagly859095alaglyargthrvalglyproaspglntyrasntyrtrpglyglngly100105110thrglnvalthrvalserser115当前第1页12