一种CD123结合蛋白、含其的CAR及其应用的制作方法

本申请涉及生物医药领域，具体涉及一种cd123结合蛋白、含其的car及其应用。
背景技术：
：一、急性髓系白血病治疗现状：急性髓系白血病(acutemyeloidleukemia，aml)是一种血液系统的恶性肿瘤，即髓系造血干细胞(hscs)/祖细胞恶性疾病，以骨髓与外周血中原始和幼稚髓性细胞异常增生为主要特征(hopekj,jinl,dickje.humanacutemyeloidleukemiastemcells.archmedres,2003,34(6):507-514.)。在我国aml是最常见的恶性血液病，每年的发病率为1.8/10万人，65岁以上老年人的发病率已增加到17.2/10万人。传统治疗药物疗效比较低，且副作用较强，患者五年生存率很低(10％左右)(tamamyang,kadiat,ravandif,etal.frontlinetreatmentofacutemyeloidleukemiainadults.critrevoncolhematol,2017,110:20-34)。另外对于大于65岁的老年患者一般很难耐受传统高剂量的化疗和干细胞移植治疗方案，一般预后也较差，大部分在确诊后1年内死亡(dekouchkovskyi,abdul-haym.acutemyeloidleukemia:acomprehensivereviewand2016update.bloodcancerj,2016,6(7):e441.)。因此急切需要探索新型的治疗药物或治疗技术。cart(chimericantigenreceptortcellimmunotherapy)治疗为复发或难治性白血病治疗提供了另外一条途径。如近年报道的mustang’smb-102cd123cart，2019年被美国fda授予孤儿药物认定，用于治疗aml，这一成果的产生也为国内外研究基于aml抗原靶点的cart的其他可行方案奠定了基础。二、母细胞性浆细胞样树突细胞肿瘤(bpdcn)治疗现状：母细胞性浆细胞样树突细胞肿瘤(bpdcn)，即cd4+cd56+造血系统肿瘤，为急性白血病的亚型。发病人群约占急性白血病患者的1％，阳性标志物主要为cd56,cd36,cd38,cd45ra,cd68,cd123,以及hladr等标记物。目前用于bpdcn的治疗方法是基于高剂量的化学疗法，但一般在2年内会复发，主要表现在骨髓、皮肤或者中枢神经系统等部位。另一种治疗方法基于造血细胞的同种异体移植，但由于患病人群大多年老体弱，很难耐受治疗，实现长期存活很难。近几年基于特异抗原cd123的发现，有望改变这种状况。2018年12月美国食品药品监督管理局批准stemlinetherapeutics公司的sl-401(tagraxofusp，一种与白喉毒素连接的cd123注射剂)用于治疗bpdcn。临床反应中最严重的副反应为毛细管渗漏综合症(economidesmp,mccued,laneaa,pemmarajun,etal.tagraxofusp,thefirstcd123-targetedtherapyandfirsttargetedtreatmentforblasticplasmacytoiddendriticcellneoplasm.expertrevclinpharmacol,2019,12(10):941-946.)。虽然基于cd123的特异性疗法的药物1期临床试验(如nct00401739、nct00397579)结果取得很好的结果，但还需要评估这些靶向药物的作用，最大程度的减少癌细胞残留。鉴于cd123靶向药物的早期临床实验结果，基于cd123的cart也为这一疾病的彻底治疗提供了更多的希望。三、cd123靶点及靶向药物cd123即为人白介素3受体α链(interleukin-3receptorsubunitalpha，il-3rα)。aml的发生与白血病干细胞(leukemiastemcells,lscs)或肿瘤干细胞密切相关(mardirosa,dossantosc,mcdonaldt,etal.tcellsexpressingcd123-specificchimericantigenreceptorsexhibitspecificcytolyticeffectorfunctionsandantitumoreffectsagainsthumanacutemyeloidleukemia.blood,2013,122(18):3138-48.)。其中表面抗原cd123在不同的血液癌中均过表达，如aml、b细胞系急性淋巴白血病(b-all)、bpdcn、毛细胞白血病(testau,pelosie,frankela.cd123isamembranebiomarkerandatherapeutictargetinhematologicmalignancies.biomarkres,2014,2(1):4.)。另外在其他细胞类型如单核细胞、树突状细胞、内皮细胞、正常的hsc亚群细胞等正常细胞仅有低水平的表达(suny,wangs,zhaol,etal.ifn-γandtnf-αaggravateendothelialdamagecausedbycd123-targetedcartcell.oncotargetsther,2019,12:4907-4925.)。因此cd123是靶向治疗血液癌的理想靶点。目前基于cd123的靶向药物的研究也比较多，主要有sl-401(tagraxofusp)、csl362(taclotuzumab)、抗体药组合物、抗cd123与抗cd3的双特异性抗体、cd123嵌合抗原受体细胞等药物(testau,pelosie,castellig.cd123asatherapeutictargetinthetreatmentofhematologicalmalignancies.cancers,2019,11(9):1358.)，这些药物虽然有一定的应答率，但是aml患者的长久存活很难。因此急迫需要寻找更优选的基于该靶点的其他治疗方法。四、cd123-cart细胞免疫治疗研究情况cart技术的突破已经在临床上显示了令人满意的结果，尤其是在b细胞癌症中。基于cd123特异性靶点的car细胞免疫治疗目前也取得一定的进展，如mustang’smb-102(cd123cart)，曾先后于2018年，2019年分别被美国fda授予孤儿药物认定，用于治疗bpdcn和aml。已有研究报道虽然基于该靶点的car细胞具有特异性抗aml能力，但saargill等人发现所构建细胞对正常造血细胞具有一定毒性(gills,tasiansk,ruellam,etal.preclinicaltargetingofhumanacutemyeloidleukemiaandmyeloablationusingchimericantigenreceptor-modifiedtcells.blood,2014,123(15):2343-2354.)；suny等人发现基于克隆号32716产生的cd123-cart对内皮细胞有损伤的报告(suny,wangs,zhaol,etal.ifn-γandtnf-αaggravateendothelialdamagecausedbycd123-targetedcartcell.oncotargetsther,2019,12:4907-4925.)；tettamanti等人报道一种cd123-carcik对正常的骨髓前体细胞和骨髓干细胞有一定程度的抑制效应(testau,pelosie,castellig.cd123asatherapeutictargetinthetreatmentofhematologicalmalignancies.cancers,2019,11(9):1358.)。为了减少cd123-cart对正常造血细胞的毒性，更多研究者意识到基于靶点表位高亲和力的单链抗体可能并不是最好的选择，而需要进一步寻找亲和力较温和的单链抗体用于cart的设计中。如arcangelis等优化了cd123-car的表达的一种方法，通过降低car的结合亲和力使效应细胞功能较弱，从而减少对正常表达细胞的损害(arcangelis,rotirotimc,bardellim,etal.balanceofanti-cd123chimericantigenreceptorbindingaffinityanddensityforthetargetingofacutemyeloidleukemia.molther,2017,25(8):1933-1945.)。cartellierim等通过设计一种通用cart解决cd123的脱靶效应(cartellierim,feldmanna,koristkas,etal.switchingcartcellsonandoff:anovelmodularplatformforretargetingoftcellstoamlblasts.bloodcancerj,2016,6(8):e458.)。虽然cd123-cart在体外显示了良好的杀瘤效果，但是临床安全性是非常关键的，尽管cd123具有作为白血病免疫治疗靶点的巨大潜力，但目前还没有cd123相关cart治疗正式应用于临床中。大部分还处在临床前及临床i期研究中，如nct02159495、nct02623582、nct03766126、nct03114670nct03222674、nct03631576、nct04010877、nct03796390等。以cd123为靶标的靶向药物的临床试验，目前功效还很有限并且仍有安全性问题的发生，因此靶向cd123-cart的探索还有很大的发展空间。cd123作为lscs的特异性抗原，基于已有研究，研发新的cd123-cart对应用于血液肿瘤免疫治疗具有重要意义。因此，本领域技术人员致力于通过从源头开发，寻找到亲和力适当、特异性较好的cd123的单克隆抗体，并应用与cd123-cart中，为临床研究提供更多的参考。五、现有技术现有技术1：suny等人构建产生了一款cd123-cart(suny,wangs,zhaol,etal.ifn-γandtnf-αaggravateendothelialdamagecausedbycd123-targetedcartcell.oncotargetsther,2019,12:4907-4925.)。该cd123-cart的单链抗体序列来自克隆号32716(杂交瘤技术产生的单克隆抗体)。具有cd8a铰链区，cd8跨膜区，4-1bb(即cd137)共刺激结构域、cd3ζ胞内区。首先cd3/cd28磁珠与t细胞按照1：1比例分选供体阳性t细胞，t细胞激活48小时后使用逆转录载体进行基因转导，使用100u/ml白介2(il-2)使t细胞扩增15天，获得一定数量的cd123-cart细胞，实验表明对aml有杀伤能力，但其体外实验说明对人脐静脉内皮细胞和人真皮微血管内皮细胞有损伤。现有技术2：saargill等人构建了一款anti-cd123-cart，其中单链抗体可变区来自克隆号32716和26292。使用pelns慢病毒质粒载体，构建了pelnsanti-cd123-4-1bb-cd3ζ(car123)质粒dna，并于293t中包装含有pelns-anti-cd123-4-1bb-cd3ζ质粒的慢病毒。该款cart细胞的制备流程基本为：首先使用抗cd4/cd8磁珠分选阳性t细胞，然后使用cd3/cd28磁珠激活，激活第二天，按照病毒与t细胞的5：1的感染复数感染t细胞。并使用100u/ml的il-2使t细胞扩增，培养15天，获得一定数量的cd123-cart细胞。该款cart细胞具备体内外特异性抗aml能力，但是对正常造血细胞具有一定毒性。技术实现要素：为解决现有技术中靶向cd123的cart方案对正常细胞的细胞毒性的技术问题，本发明提供了一种cd123结合蛋白、含其的car及其应用。本发明通过自主构建cd123抗体库筛选并优化得到十八种阳性单克隆，并测序鉴定得到相应的单克隆轻链可变区和重链可变区序列，为优化cd123-cart提供了更多scfv序列的筛选范围；本发明所得序列应用于cart时，体外细胞杀伤实验验证了该cart具有积极的抗癌能力，其中具有显著性差异的一个单克隆的亲和力相对阳性对照较弱或者杀伤的作用机制与其他报告的cd123-cart的作用机制有差异，并且对对正常低水平表达细胞的杀伤作用较弱，具有更高的安全性。为解决上述问题，本发明提供的技术方案之一是：一种cd123结合蛋白或其变体，其包括重链可变区和/或轻链可变区；所述重链可变区包括氨基酸序列分别如seqidno:141、seqidno:150、seqidno:157所示的hcdr1、hcdr2和hcdr3；或，如seqidno:137、seqidno:146、seqidno:156所示的hcdr1、hcdr2和hcdr3；或，如seqidno:145、seqidno:153、seqidno:161所示的hcdr1、hcdr2和hcdr3；所述轻链可变区包括氨基酸序列分别如seqidno:121、seqidno:129、seqidno:135所示的lcdr1、lcdr2和lcdr3；或，如seqidno:125、seqidno:130、seqidno:133所示的lcdr1、lcdr2和lcdr3；所述变体在所述cd123结合蛋白的氨基酸序列具有一至多个优选一至三个氨基酸的增加、缺失或取代并保持所述cd123结合蛋白的功能。较佳地，如上所述的cd123结合蛋白或其变体，所述重链可变区包括氨基酸序列分别如seqidno:141、seqidno:150、seqidno:157所示的hcdr1、hcdr2和hcdr，所述轻链可变区包括氨基酸序列分别如seqidno:125、seqidno:130、seqidno:133所示的lcdr1、lcdr2和lcdr；或，所述重链可变区包括氨基酸序列分别如seqidno:137、seqidno:146、seqidno:156所示的hcdr1、hcdr2和hcdr，所述轻链可变区包括氨基酸序列分别如seqidno:121、seqidno:129、seqidno:135所示的lcdr1、lcdr2和lcdr；或，所述重链可变区包括氨基酸序列分别如seqidno:145、seqidno:153、seqidno:161所示的hcdr1、hcdr2和hcdr，所述轻链可变区包括氨基酸序列分别如seqidno:125、seqidno:130、seqidno:133所示的lcdr1、lcdr2和lcdr；优选地，所述变体为在如seqidno:141所示hcdr1的氨基酸序列上发生t1p、n2s、y3f和/或v5i取代，在如seqidno:150所示的hcdr2的氨基酸序列上发生t7s、n9h、a13s和/或t8r取代，在如seqidno:157所示的hcdr3的氨基酸序列上发生f3y和/或d9a取代，在如seqidno:125所示的lcdr1的氨基酸序列上发生s3n、k4e、s7t、t8s和/或m14i/l取代，和/或，在如seqidno:133所示的lcdr3的氨基酸序列上发生e5d取代；或，所述变体为在如seqidno:137所示hcdr1的氨基酸序列上发生v5a取代，在如seqidno:146所示的hcdr2的氨基酸序列上发生y4f、s5g、s7v/t和/或t15k取代，在如seqidno:156所示的hcdr3的氨基酸序列上发生t2l取代，在如seqidno:121所示的lcdr1的氨基酸序列上发生t1s和/或h12y取代，在如seqidno:129所示的lcdr2的氨基酸序列上发生n4k取代，和/或，在如seqidno:135所示的lcdr3的氨基酸序列上发生h1q、f3y/n、h4s、r5g、s6y和/或r8p/y/f取代。较佳地，如上所述的cd123结合蛋白或其变体，在所述变体中：所述重链可变区包括氨基酸序列分别如seqidno:137、seqidno:146、seqidno:156所示的hcdr1、hcdr2和hcdr3，所述轻链可变区包括氨基酸序列分别如seqidno:121、seqidno:129、seqidno:132所示的lcdr1、lcdr2和lcdr3；或，所述重链可变区包括氨基酸序列分别如seqidno:138、seqidno:147、seqidno:157所示的hcdr1、hcdr2和hcdr3，所述轻链可变区包括氨基酸序列分别如seqidno:124、seqidno:130、seqidno:133所示的lcdr1、lcdr2和lcdr3；或，所述重链可变区包括氨基酸序列分别如seqidno:139、seqidno:148、seqidno:158所示的hcdr1、hcdr2和hcdr3，所述轻链可变区包括氨基酸序列分别如seqidno:122、seqidno:129、seqidno:134所示的lcdr1、lcdr2和lcdr3；或，所述重链可变区包括氨基酸序列分别如seqidno:140、seqidno:149、seqidno:157所示的hcdr1、hcdr2和hcdr3，所述轻链可变区包括氨基酸序列分别如seqidno:125、seqidno:130、seqidno:133所示的lcdr1、lcdr2和lcdr3；或，所述重链可变区包括氨基酸序列分别如seqidno:141、seqidno:150、seqidno:157所示的hcdr1、hcdr2和hcdr3，所述轻链可变区包括氨基酸序列分别如seqidno:125、seqidno:130、seqidno:133所示的lcdr1、lcdr2和lcdr3；或，所述重链可变区包括氨基酸序列分别如seqidno:137、seqidno:146、seqidno:156所示的hcdr1、hcdr2和hcdr3，所述轻链可变区包括氨基酸序列分别如seqidno:121、seqidno:129、seqidno:135所示的lcdr1、lcdr2和lcdr3；或，所述重链可变区包括氨基酸序列分别如seqidno:142、seqidno:147、seqidno:159所示的hcdr1、hcdr2和hcdr3，所述轻链可变区包括氨基酸序列分别如seqidno:125、seqidno:130、seqidno:133所示的lcdr1、lcdr2和lcdr3；或，所述重链可变区包括氨基酸序列分别如seqidno:139、seqidno:148、seqidno:158所示的hcdr1、hcdr2和hcdr3，所述轻链可变区包括氨基酸序列分别如seqidno:123、seqidno:131、seqidno:162所示的lcdr1、lcdr2和lcdr3；或，所述重链可变区包括氨基酸序列分别如seqidno:138、seqidno:147、seqidno:157所示的hcdr1、hcdr2和hcdr3，所述轻链可变区包括氨基酸序列分别如seqidno:126、seqidno:130、seqidno:136所示的lcdr1、lcdr2和lcdr3；或，所述重链可变区包括氨基酸序列分别如seqidno:141、seqidno:151、seqidno:157所示的hcdr1、hcdr2和hcdr3，所述轻链可变区包括氨基酸序列分别如seqidno:127、seqidno:130、seqidno:133所示的lcdr1、lcdr2和lcdr3；或，所述重链可变区包括氨基酸序列分别如seqidno:141、seqidno:151、seqidno:157所示的hcdr1、hcdr2和hcdr3，所述轻链可变区包括氨基酸序列分别如seqidno:128、seqidno:130、seqidno:133所示的lcdr1、lcdr2和lcdr3；或，所述重链可变区包括氨基酸序列分别如seqidno:143、seqidno:147、seqidno:157所示的hcdr1、hcdr2和hcdr3，所述轻链可变区包括氨基酸序列分别如seqidno:125、seqidno:130、seqidno:133所示的lcdr1、lcdr2和lcdr3；或，所述重链可变区包括氨基酸序列分别如seqidno:137、seqidno:152、seqidno:156所示的hcdr1、hcdr2和hcdr3，所述轻链可变区包括氨基酸序列分别如seqidno:123、seqidno:129、seqidno:135所示的lcdr1、lcdr2和lcdr3；或，所述重链可变区包括氨基酸序列分别如seqidno:144、seqidno:147、seqidno:159所示的hcdr1、hcdr2和hcdr3，所述轻链可变区包括氨基酸序列分别如seqidno:125、seqidno:130、seqidno:133所示的lcdr1、lcdr2和lcdr3；或，所述重链可变区包括氨基酸序列分别如seqidno:141、seqidno:147、seqidno:160所示的hcdr1、hcdr2和hcdr3，所述轻链可变区包括氨基酸序列分别如seqidno:125、seqidno:130、seqidno:133所示的lcdr1、lcdr2和lcdr3；或，所述重链可变区包括氨基酸序列分别如seqidno:145、seqidno:153、seqidno:161所示的hcdr1、hcdr2和hcdr3，所述轻链可变区包括氨基酸序列分别如seqidno:125、seqidno:130、seqidno:133所示的lcdr1、lcdr2和lcdr3；或，所述重链可变区包括氨基酸序列分别如seqidno:138、seqidno:154、seqidno:157所示的hcdr1、hcdr2和hcdr3，所述轻链可变区包括氨基酸序列分别如seqidno:125、seqidno:130、seqidno:133所示的lcdr1、lcdr2和lcdr3；或，所述重链可变区包括氨基酸序列分别如seqidno:137、seqidno:155、seqidno:156所示的hcdr1、hcdr2和hcdr3，所述轻链可变区包括氨基酸序列分别如seqidno:121、seqidno:129、seqidno:135所示的lcdr1、lcdr2和lcdr3。较佳地，如上所述的cd123结合蛋白或其变体，所述重链可变区的氨基酸序列如seqidno:4所示，所述轻链可变区的氨基酸序列如seqidno:2所示；或，所述重链可变区的氨基酸序列如seqidno:8所示，所述轻链可变区的氨基酸序列如seqidno:6所示；或，所述重链可变区的氨基酸序列如seqidno:12所示，所述轻链可变区的氨基酸序列如seqidno:10所示；或，所述重链可变区的氨基酸序列如seqidno:16所示，所述轻链可变区的氨基酸序列如seqidno:14所示；或，所述重链可变区的氨基酸序列如seqidno:20所示，所述轻链可变区的氨基酸序列如seqidno:18所示；或，所述重链可变区的氨基酸序列如seqidno:24所示，所述轻链可变区的氨基酸序列如seqidno:22所示；或，所述重链可变区的氨基酸序列如seqidno:28所示，所述轻链可变区的氨基酸序列如seqidno:26所示；或，所述重链可变区的氨基酸序列如seqidno:32所示，所述轻链可变区的氨基酸序列如seqidno:30所示；或，所述重链可变区的氨基酸序列如seqidno:36所示，所述轻链可变区的氨基酸序列如seqidno:34所示；或，所述重链可变区的氨基酸序列如seqidno:40所示，所述轻链可变区的氨基酸序列如seqidno:38所示；或，所述重链可变区的氨基酸序列如seqidno:44所示，所述轻链可变区的氨基酸序列如seqidno:42所示；或，所述重链可变区的氨基酸序列如seqidno:48所示，所述轻链可变区的氨基酸序列如seqidno:46所示；或，所述重链可变区的氨基酸序列如seqidno:52所示，所述轻链可变区的氨基酸序列如seqidno:50所示；或，所述重链可变区的氨基酸序列如seqidno:56所示，所述轻链可变区的氨基酸序列如seqidno:54所示；或，所述重链可变区的氨基酸序列如seqidno:60所示，所述轻链可变区的氨基酸序列如seqidno:58所示；或，所述重链可变区的氨基酸序列如seqidno:64所示，所述轻链可变区的氨基酸序列如seqidno:62所示；或，所述重链可变区的氨基酸序列如seqidno:68所示，所述轻链可变区的氨基酸序列如seqidno:66所示；或，所述重链可变区的氨基酸序列如seqidno:72所示，所述轻链可变区的氨基酸序列如seqidno:70所示；所述变体的轻链可变区和/或重链可变区分别与所述cd123结合蛋白的轻链可变区和/或重链可变区的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％、99％的同一性。优选地，编码所述重链可变区的核苷酸序列如seqidno:3所示，编码所述轻链可变区的核苷酸序列如seqidno:1所示；或，编码所述重链可变区的核苷酸序列如seqidno:7所示，编码所述轻链可变区的核苷酸序列如seqidno:5所示；或，编码所述重链可变区的核苷酸序列如seqidno:11所示，编码所述轻链可变区的核苷酸序列如seqidno:9所示；或，编码所述重链可变区的核苷酸序列如seqidno:15所示，编码所述轻链可变区的核苷酸序列如seqidno:13所示；或，编码所述重链可变区的核苷酸序列如seqidno:19所示，编码所述轻链可变区的核苷酸序列如seqidno:17所示；或，编码所述重链可变区的核苷酸序列如seqidno:23所示，编码所述轻链可变区的核苷酸序列如seqidno:21所示；或，编码所述重链可变区的核苷酸序列如seqidno:27所示，编码所述轻链可变区的核苷酸序列如seqidno:25所示；或，编码所述重链可变区的核苷酸序列如seqidno:31所示，编码所述轻链可变区的核苷酸序列如seqidno:29所示；或，编码所述重链可变区的核苷酸序列如seqidno:35所示，编码所述轻链可变区的核苷酸序列如seqidno:33所示；或，编码所述重链可变区的核苷酸序列如seqidno:39所示，编码所述轻链可变区的核苷酸序列如seqidno:37所示；或，编码所述重链可变区的核苷酸序列如seqidno:43所示，编码所述轻链可变区的核苷酸序列如seqidno:41所示；或，编码所述重链可变区的核苷酸序列如seqidno:47所示，编码所述轻链可变区的核苷酸序列如seqidno:45所示；或，编码所述重链可变区的核苷酸序列如seqidno:51所示，编码所述轻链可变区的核苷酸序列如seqidno:49所示；或，编码所述重链可变区的核苷酸序列如seqidno:55所示，编码所述轻链可变区的核苷酸序列如seqidno:53所示；或，编码所述重链可变区的核苷酸序列如seqidno:59所示，编码所述轻链可变区的核苷酸序列如seqidno:57所示；或，编码所述重链可变区的核苷酸序列如seqidno:63所示，编码所述轻链可变区的核苷酸序列如seqidno:61所示；或，编码所述重链可变区的核苷酸序列如seqidno:67所示，编码所述轻链可变区的核苷酸序列如seqidno:65所示；或，编码所述重链可变区的核苷酸序列如seqidno:71所示，编码所述轻链可变区的核苷酸序列如seqidno:69所示。较佳地，如上所述的cd123结合蛋白或其变体，所述cd123结合蛋白或其变体的结构包括免疫球蛋白、fab、fab’、f(ab’)2、fv例如单链fv片段(scfv)。较佳地，如上所述的cd123结合蛋白或其变体，所述cd123结合蛋白或其变体为免疫球蛋白，且其包括鼠或人的抗体恒定区或其突变；所述鼠的抗体恒定区包括鼠igg1、igg2a、igg2b3或igg3的重链恒定区和κ或λ型轻链恒定区，所述人的抗体恒定区包括人igg1、igg2、igg3或igg4的重链恒定区和κ或λ型轻链恒定区。优选地，所述突变与所述鼠或人抗体恒定区的氨基酸序列具有至少80％、85％、90％、95％、96％、97％、98％、99％的同一性。为解决上述问题，本发明提供的技术方案之二是：一种抗体药物偶联体(adc)，其结构如下式i所示：ab-[(l2)n-l1-d]y式i其中，d为具有细胞毒性的小分子药物，l1和l2为分别连接所述药物和所述抗体的接头；n为0或1；y表示偶联于ab的d的平均数，且0<y≤10，优选2≤y≤7；更优选3≤y≤6；所述ab为如本发明所述的cd123结合蛋白或其变体。优选地，所述小分子药物为mmae、mmaf、dm1、dm3和dm4中的一种或多种；所述接头为spp、siab、smcc、mp、vc、ala-phe、pab和mc-vc-pab中的一种或多种。为解决上述问题，本发明提供的技术方案之三是：一种嵌合抗原受体(car)，其特征在于，所述car包括：前导链、特异性识别cd123的胞外结合结构域、铰链区、跨膜区、共刺激分子和胞内信号区；所述胞外结合结构域包括如本发明所述的cd123结合蛋白或其变体，优选为scfv结构。较佳地，如上所述的car中，所述前导链的氨基酸序列如：seqidno:74所示，优选编码该前导链的核苷酸序列如seqidno:73所示。较佳地，如上所述的car中，所述scfv中重链可变区与轻链可变区的连接方式为重链可变区-linker-轻链可变区或者轻链可变区-linker-重链可变区的顺序。优选地，所述linker的氨基酸序列如seqidno:76所示，优选编码所述linker的核苷酸序列如seqidno:75所示。较佳地，如上所述的car中，所述铰链区选自以下蛋白：igg1、igg4、igd或cd8。优选地，所述cd8铰链区氨基酸序列如seqidno:78所示。更优选地，编码所述铰链区的核苷酸序列如seqidno:77所示。较佳地，如上所述的car中，所述跨膜区选自以下蛋白：cd3、cd4、cd5、cd8、cd9、cd16、cd22、cd27、cd28、cd33、cd37、cd45、cd64、cd80、cd86、cd134、cd137、cd152、cd154和pd1。优选地，所述cd8的跨膜区的氨基酸序列如seqidno:80所示。更优选地，编码所述跨膜区的核苷酸序列如seqidno:79所示。较佳地，如上所述的car中，所述共刺激分子选自以下蛋白：4-1bb、cd2、cd7、cd27、cd28、cd30、cd40、cd54、cd83、cd134、cd137、cd150、cd152、cd223、cd270、cd273、cd274、cd278、card11、nkd2c、dap10、lat、slp76和zap70。优选地，所述4-1bb共刺激区域氨基酸序列如seqidno:82所示。更优选地，编码所述共刺激分子的核苷酸序列如seqidno:81所示。较佳地，如上所述的car中，所述胞内信号区选自：cd3ζ、fcεriγ、cd28、4-1bb和cd134或其组合。优选地，所述cd3ζ的氨基酸序列如seqidno:84所示；更优选地，编码所述cd3ζ的核苷酸序列如seqidno:83所示。较佳地，如上所述的car中，所述car的氨基酸序列如seqidno:86、88、90、92、94、96、98、100、102、104、106、108、110、112、114、116、118或120所示。优选地，编码所述car的核苷酸序列如seqidno:85、87、89、91、93、95、97、99、101、103、105、107、109、111、113、115、117或119所示。为解决上述问题，本发明提供的技术方案之四是：一种分离的核酸，所述分离的核酸编码如本发明所述的cd123结合蛋白或其变体或如本发明所述的car。优选地，编码所述cd123结合蛋白或其变体的核苷酸序列中，编码所述重链可变区的核苷酸序列如seqidno:3所示，编码所述轻链可变区的核苷酸序列如seqidno:1所示；或，编码所述重链可变区的核苷酸序列如seqidno:7所示，编码所述轻链可变区的核苷酸序列如seqidno:5所示；或，编码所述重链可变区的核苷酸序列如seqidno:11所示，编码所述轻链可变区的核苷酸序列如seqidno:9所示；或，编码所述重链可变区的核苷酸序列如seqidno:15所示，编码所述轻链可变区的核苷酸序列如seqidno:13所示；或，编码所述重链可变区的核苷酸序列如seqidno:19所示，编码所述轻链可变区的核苷酸序列如seqidno:17所示；或，编码所述重链可变区的核苷酸序列如seqidno:23所示，编码所述轻链可变区的核苷酸序列如seqidno:21所示；或，编码所述重链可变区的核苷酸序列如seqidno:27所示，编码所述轻链可变区的核苷酸序列如seqidno:25所示；或，编码所述重链可变区的核苷酸序列如seqidno:31所示，编码所述轻链可变区的核苷酸序列如seqidno:29所示；或，编码所述重链可变区的核苷酸序列如seqidno:35所示，编码所述轻链可变区的核苷酸序列如seqidno:33所示；或，编码所述重链可变区的核苷酸序列如seqidno:39所示，编码所述轻链可变区的核苷酸序列如seqidno:37所示；或，编码所述重链可变区的核苷酸序列如seqidno:43所示，编码所述轻链可变区的核苷酸序列如seqidno:41所示；或，编码所述重链可变区的核苷酸序列如seqidno:47所示，编码所述轻链可变区的核苷酸序列如seqidno:45所示；或，编码所述重链可变区的核苷酸序列如seqidno:51所示，编码所述轻链可变区的核苷酸序列如seqidno:49所示；或，编码所述重链可变区的核苷酸序列如seqidno:55所示，编码所述轻链可变区的核苷酸序列如seqidno:53所示；或，编码所述重链可变区的核苷酸序列如seqidno:59所示，编码所述轻链可变区的核苷酸序列如seqidno:57所示；或，编码所述重链可变区的核苷酸序列如seqidno:63所示，编码所述轻链可变区的核苷酸序列如seqidno:61所示；或，编码所述重链可变区的核苷酸序列如seqidno:67所示，编码所述轻链可变区的核苷酸序列如seqidno:65所示；或，编码所述重链可变区的核苷酸序列如seqidno:71所示，编码所述轻链可变区的核苷酸序列如seqidno:69所示；编码所述car的核苷酸序列如seqidno:85、87、89、91、93、95、97、99、101、103、105、107、109、111、113、115、117或119所示。为解决上述问题，本发明提供的技术方案之五是：一种表达载体，所示表达载体包括如本发明所述的分离的核酸。优选地，所述表达载体的出发载体为慢病毒质粒表达载体，例如plvx-ef1、lenticrisprv2或pelns，由此获得plvx-ef1-car、lenticrisprv2-car或pelns-car。为解决上述问题，本发明提供的技术方案之六是：一种转化体，其包括如本发明所述的表达载体。优选地，所述转化体为基因修饰的慢病毒或免疫效应细胞。更优选地，所述慢病毒为含有plvx-ef1-car、lenticrisprv2-car或pelns-car的慢病毒，所述免疫效应细胞为t淋巴细胞、nk细胞或nkt细胞。为解决上述问题，本发明提供的技术方案之七是：一种药物组合物，所述药物组合物包含如本发明所述的cd123结合蛋白或其变体、所述的抗体药物偶联体、所述的转化体以及药学上可接受的载体。优选地，所述药物组合物还包括免疫检查点抗体。为解决上述问题，本发明提供的技术方案之八是：一种如本发明所述的cd123结合蛋白或其变体、所述的抗体药物偶联体、所述的car、所述的分离的核酸、所述的表达载体、所述的转化体或所述的药物组合物在制备抑制和/或治疗cd123阳性肿瘤的药物中的应用；所述肿瘤优选为急性髓细胞白血病、慢性髓细胞白血病、骨髓增生异常综合征、急性淋巴细胞白血病、慢性淋巴细胞白血病、母细胞性浆细胞样树突细胞肿瘤、多毛细胞白血病、霍奇金淋巴瘤等。为解决上述问题，本发明提供的技术方案之九是：一种套装药盒，其包含药盒a和药盒b，所述药盒a包括如本发明所述的cd123结合蛋白或其变体、所述的抗体药物偶联体、所述的car、所述的分离的核酸、所述的表达载体、所述的转化体或所述的药物组合物中的一种，所述药盒b包括其他靶向cd123或非cd123靶向的药物。所述药盒a和药盒b可同时施用，也可以先后施用；所述先后施用中，所述药盒a先于所述药盒b施用，或者所述药盒b先于所述药盒a施用。在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。本发明所用试剂和原料均市售可得。本发明的积极进步效果在于：本发明的cd123单克隆抗体序列的单链抗体识别抗原更加温和，产生的cart具有抗癌能力。本发明优选的克隆产生的cd123-cart特异性杀伤相对稍弱，可以减轻或者避免对正常细胞(尤其是低表达cd123的正常细胞)的损伤。对于临床治疗的安全性方面(如改善毛细血管渗漏综合征、白血球减少等症状)更有参考价值。基于本发明产生的cart为免疫细胞治疗提供更多的参考价值，可以用作多种癌症的治疗，如急性髓细胞白血病、慢性髓细胞白血病、骨髓增生异常综合征、急性淋巴细胞白血病、慢性淋巴细胞白血病、母细胞性浆细胞样树突细胞肿瘤、多毛细胞白血病、霍奇金淋巴瘤。本发明的抗体序列为基于本发明cd123抗体的开发、研究及应用提供更多的参考价值。附图说明图1为新型cd123-cart制备及功能验证技术路线图；图2为三轮免疫的三只小鼠血清elisa检测结果；图3为四轮免疫的三只小鼠血清elisa检测结果；图4为小鼠脾脏rna抽提鉴定结果；图5为抗体轻重链基因扩增鉴定；图6为lc-rbs与hc-rbs扩增鉴定；图7为轻链重链重组鉴定；图8为轻链重链重组后的全长序列酶切鉴定；图9为fab抗体库构建载体简图；图10为酶切位点所在部分序列；其中a为apali酶切位点所在部分序列，b为noti酶切位点所在部分序列；图11为抗体基因种源分析图；图12为elisa验证噬菌体抗体库质量图；图13为cd123-k562细胞系cd123表达情况；图14为18种新型cd123-car-t转染阳性表达情况；图15为18种新型cd123-car-t细胞靶向k562的细胞毒性情况；图16为18种新型cd123-car-t细胞靶向k562-cd123的细胞毒性情况。具体实施方式下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。术语解释：1.aml：acutemyeloidleukemia，简写aml。急性髓系白血病，是一种血液系统的恶性肿瘤，即髓系造血干(hscs)/祖细胞恶性疾病,以骨髓与外周血中原始和幼稚髓性细胞异常增生为主要特征。2.cd123：即人白介素3受体α链(interleukin-3receptorsubunitalpha，il-3rα)，是il-3受体的α链的跨膜片段，全长378个氨基酸。cd123除了在aml中高表达以外，在其它多种血液肿瘤如血浆树突细胞肿瘤、多毛细胞白血病、霍奇金淋巴瘤中也有表达。3.vh：variableregionofheavychain，简称vh。4.vl：variableregionoflightchain，简称vl。5.抗体:指包含通过二硫键互联的至少两条重链和两条轻链或其抗原结合部分的蛋白质。每条重链由重链可变区(本文缩写为vh)和重链恒定区组成。每条轻链由轻链可变区(本文缩写为vl)和轻链恒定区组成。vh和vl区可进一步细分为称为互补性决定区(cdr)的高变区，它们散布在称为构架区(fr)的更保守区域中。6.免疫球蛋白：是由两条相同的轻链和两条相同的重链通过链间二硫键连接而成的四肽链结构。轻链分为κ型和λ型，重链分为μ、δ、γ、α和ε链等五种类型。7.单克隆抗体：单分子组成的抗体分子制备物。单克隆抗体显示对特定表位的单结合特异性和亲和性。8.噬菌体展示技术：将外源基因插入噬菌体的基因组中(一般外源基因插入到噬菌体基因viii或基因iii的5’端)，使产物与其衣壳蛋白融合表达，外源肽的结构与功能通过活病毒的形式表现出来。可以应用于随机肽文库的构建、表位文库构建及人源抗体库的构建等，通过文库提供的大量结构与功能的信息，实现了高通量筛选，在研究分子间相互作用，新药设计，寻找新配体，寻找并制备人源单抗等方面提供了全新的策略。用于噬菌体展示技术的噬菌体主要包括m13、fd、f1等。9.elisa:enzymelinkedimmunosorbentassay，简写elisa。酶联免疫吸附测定，指将可溶性的抗原或抗体结合到聚苯乙烯等固相载体上，利用抗原抗体特异性结合进行免疫反应的定性和定量检测方法。12.fab片段：fragmentofantigenbinding，简写fab，抗原结合片段。由vl、vh、cl和ch结构域组成的一价片段。13.scfv：singlechainantibodyfragment，scfv。单链抗体是由抗体重链可变区(vh,variableregionoftheheavychain)和轻链可变区(vl,variableregionoflight)通过一个短肽(linker连接子)连接而成的单链抗体。14.linker：连接肽。目前最常用的linker是由4个甘氨酸和1个丝氨酸重复3次排列组合成的十五肽序列。在scfv中，linker使重链可变区和轻链可变区自由折叠，从而维持重链可变区和轻链可变区间的构象。15.car：chimericantigenreceptor，简写car。嵌合抗原受体是工程化受体，其将单克隆抗体的单链可变片段(scfv)与包含用于t细胞激活的一种或多种信号转导结构域的细胞内部分联合。如嵌合抗原受体一般包括胞外结合区，跨膜区和胞内信号区。通常胞外区包含能够识别肿瘤相关抗原的scfv，跨膜区采用cd8，cd28等分子的跨膜区，胞内信号区采用免疫受体酪氨酸活化基序(itam)cd3ζ或fcεriγ及共刺激信号分子cd28、cd27、cd137、cd134等的胞内信号区。16.hscs：hematopoieticstemcells，hscs，髓系造血干细胞。17.lscs：leukemiastemcells，简称lscs，白血病干细胞。18.bpdcn：blasticplasmacytoiddendriticcellneoplasm，bpdcn，母细胞性浆细胞样树突细胞肿瘤，一种非常罕见的造血系统恶性肿瘤，曾被命名为母细胞性nk细胞淋巴瘤或cd4+cd56造血细胞肿瘤等。19.egfrt：epidermalgrowthfactorreceptor，截短的表皮生长因子受体。20.t2a：2a肽，核糖体跳跃序列。一种可“自我剪切的短小肽链”，最初在手足口病毒(foot-and-mouthdiseasevirus，fmda)中发现，平均长度为18-22个氨基酸，2a肽可在蛋白翻译时通过核糖体跳跃从自身最后2个氨基酸c末端断裂。21.4-1bb：即cd137，属于肿瘤坏死因子受体超家族的重要成员，主要表达于活化的cd4+和cd8+t细胞。22.nk：naturalkillercell，简称nk，自然杀伤细胞，来源于骨髓淋巴样干细胞，其分化、发育依赖于骨髓及胸腺微环境，主要分布于骨髓、外周血、肝、脾、肺和淋巴结。nk细胞不同于t、b细胞，是一类无需预先致敏就能非特异性杀伤肿瘤细胞和病毒感染细胞的淋巴细胞。23.cd20：一种b细胞分化抗原，仅位于前b细胞和成熟b细胞，它在95％以上的b细胞性淋巴瘤中表达，而在造血干细胞、血浆细胞和其他正常组织中不表达。24.itam：immunoreceptortyrosine-basedactivationmotif，简称itam。免疫受体酪氨酸激活基序，基本组成是：yxxl/v，活化受体与相应配体结合后，在细胞膜相连的一类蛋白酪氨酸激酶ptk的作用下，其酪氨酸被磷酸化，进一步招募胞内游离的其他蛋白激酶或衔接蛋白，向细胞内传导活化信号。25.tm：transmembraneprotein，简称tm，跨膜蛋白。26.g4s1:fourglycinesfollowedbyoneserine，简称g4s1。27.5b9：核蛋白la/ss-b中一段连续的10个氨基酸序列，简称5b9。28.293t：由293细胞派生，表达sv40大t抗原的人肾上皮细胞系，被广泛应用于瞬时转染以过表达各种目标蛋白，或是用以包装病毒。29.il-2：interleukin-2，简称il-2，白介素。30.ifn-γ：interferon-γ，简称ifn-γ，γ-干扰素。31.噬菌体：phage，是侵袭细菌的病毒。主要有双链噬菌体和单链丝状噬菌体两大类。双链噬菌体为λ类噬菌体，单链丝状噬菌体有m13、f1、fd噬菌体。其中m13一类特异的雄性大肠埃希菌噬菌体，m13子代噬菌体通过细胞壁挤出，并不杀死细菌，但细菌生长速度缓慢。32.噬菌粒：带有丝状噬菌体大间隔区，集质粒和丝状噬菌体有利特征一身的载体，具有复制起点，抗生素选择性标记以及丝状噬菌体的间隔区。33.vcsm13:辅助噬菌体的一种，一类自身dna复制效率极低的丝状噬菌体的突变型，其基因组dna具有m13噬菌体所有的功能基因，但是间隔区是缺陷的。34.hat培养基：其中h为hypoxanthine次黄嘌呤，a为aminopterin氨基蝶呤，t为thymidine胸腺嘧啶核苷。根据次黄嘌呤核苷酸和嘧啶核苷酸生物合成途径设计的选择培养基。35.flt3：fms-liketyrosinekinase-3，fms样的酪氨酸激酶3，简称flt3。36.pbsm：含脱脂奶粉的pbs，一种封闭液。37.tbst:tbs和tween的缩写。tbs为tris-hci缓冲盐溶液。tween为非离子型去污剂。38.tmb：tetramethylbenzidine，四甲基联苯胺。39.hrp：horseradishperoxidase，hrp，辣根过氧化物酶。40.pfu：plaqueformingunit，空斑形成单位。41.pcr：polymerasechainreaction，多聚酶链式反应。42.rna：ribonucleicacid，核糖核酸。43.cdna:complementarydna，互补dna。44.overlappcr:重叠pcr，由于采用具有互补末端的引物，使pcr产物形成了重叠链，从而在随后的扩增反应中通过重叠链的延伸，将不同来源的扩增片段重叠拼接起来的一项技术。45.rbs：本专利所用rbs特指抗体轻链重链overlap连接时重叠的一段核苷酸序列，命名为rbs。46.2yt培养基：其中主要成分蛋白胨和酵母浸出汁的一种培养基，其中y指蛋白胨，t指酵母浸出汁。47.dt388il3：il-3分子和白喉类毒素(diphtheriatoxin)的融合蛋白。48.csl360：澳大利亚csl一家公司开发的一种来源于单克隆抗体7g3的igg1嵌合形式。49.nt:nucleotide，核苷酸。50.aa：aminoacid，氨基酸。51.panning：即盘选，噬菌体随机肽库中挑选抗原表位的过程。52.car：carbenicillin，羧苄霉素。53.tet:tetracycline，四环素。54.kan：kanamycin，卡那霉素。图1为本发明新型cd123-cart制备及功能验证技术路线图，其基本过程包括：①免疫小鼠：通过cd123抗原五次免疫小鼠，使免疫小鼠血清效价大于12万，用于抗体库构建。②抗体库构建：提取相应高效价的小鼠脾脏rna，反转录rna为cdna，使用通用简并引物将免疫后小鼠抗体的轻重链基因扩增出来，再通过轻重链重叠连接进行重组，最后将重组后的抗体fab基因序列插入到fab载体中，从而获得小鼠fab免疫抗体库，库容量>5×10^7pfu；③阳性克隆筛选：噬菌体展示小鼠抗体库，经elisa方法3轮筛选，获得富集鼠阳性单克隆；④测序鉴定：选择阳性克隆送测序公司测序，获得fab阳性克隆重链可变区及轻链可变区序列；⑤car合成：使用阳性克隆fab抗体重链可变区和轻链可变区序列，根据二代car技术总体设计结构，人工合成car序列，包括前导链、anti-cd123-scfv单链抗体链、铰链区、跨膜区、一个共刺激结构域、cd3ζ信号链；⑥慢病毒制备：利用合成的car序列，构建包含anti-cd123-scfv嵌合抗原受体目的基因片段的慢病毒质粒用于病毒制备；⑦cart制备：制备好的病毒液感染分选激活后的t细胞，培养一段时间获得cart细胞；⑧功能验证：体外细胞杀伤实验验证cart的抗肿瘤效果。实施例1：鼠源单克隆抗体的产生及免疫检测1.1免疫小鼠及血清亲和力elisa检测为了产生针对cd123的抗体，本实施例使用cd123蛋白质(编号sns001-002，购自北京百普赛斯生物科技有限公司acro)免疫小鼠。采用皮下注射免疫三只小鼠，进行三轮免疫后，通过尾静脉取血获得血清样品。对第三次免疫小鼠血清进行亲和力elisa检测。具体步骤如下：1)抗原包被：采用亲和力elisa板，在96孔半孔酶标板中，加入用pbs稀释至2μg/ml的sns001-002，每孔30μl，4度包被过夜。2)洗板：pbst洗板3次。3)封闭：5％pbsm，室温，2小时。4)洗板：tbst洗板3次。5)样品准备及加样：样品稀释：在稀释板中，1：500，1：1500，1：4500，1：13500，1：40500，1：121500，1:364500，1:1093500等稀释度稀释，未免疫小鼠血清和5％的奶粉做阴性对照。加样：30μl/孔加入到各孔中，室温孵育1小时。6)洗板：tbst洗板6次。7)加二抗：1:4000稀释二抗(anti-mouse-fab-hrp)，室温，1小时。8)洗板：tbst洗板6次。9)加tmb，室温，5分钟。10)终止反应：加2mhcl，终止反应。11)数据采集：采用酶标仪(thermo，multiskanfc)，读取od450吸光值。三轮免疫后elisa检测实验结果见图2显示，经分析有三只小鼠血清效价均大于10万。对上述三只小鼠继续免疫，第四轮免疫后继续进行小鼠血清亲和力elisa检测。实验结果见图3显示，经分析有三只小鼠血清效价均大于12万，血清免疫合格。然后对筛选的三只小鼠进行一次加强免疫，之后取小鼠脾脏，为构建抗体库做准备。1.2fab抗体文库构建根据1.1中筛选三只小鼠的血清效价情况，选择编号为1#小鼠构建免疫库，构建库容量为>5×107pfu(plaqueformingunit)的抗体库。本实验方法为：在获得了免疫小鼠脾脏的基础上，首先通过通用简并引物将免疫后小鼠抗体轻重链基因扩增出来，再通过体外overlap连接进行重组，最后将重组后的抗体fab基因序列插入到fab载体中，最终获得了>5×107pfu小鼠免疫抗体库；并分别从抗体基因正确插入率和终止密码子等角度对所构建抗体库进行质控与分析。总体流程为：提取小鼠脾脏rna，反转录rna为cdna，以cdna为模板随机扩增抗体轻/重链基因，鉴定合格后以该扩增产物为模板进一步pcr分别扩增重链rbs序列和轻链rbs序列，最后采用重叠pcr法获得重链rbs序列和轻链rbs序列的连接产物，经酶切连接及电转转化，获得fab抗体库。具体实验及结果如下：1.2.1提取小鼠脾脏细胞rna分离小鼠脾脏，按照e.z.n.a.hptotalrnakit(r6734-01)的提供的方法对小鼠脾脏组织进行rna抽提，抽提之后，通过琼脂糖凝胶电泳(tanoneps300)鉴定，见图4，rna主条带28s和18s较清晰完整，od260/280在1.9-2.1之间，所抽提rna质量良好，最终得到rna浓度为190ng/ul，总体积100ul。可以进行反转录实验。1.2.2rna逆转录具体步骤按照thermo反转录试剂盒(roche,11939823001)进行rna反转录，得到cdna。1.2.3抗体轻重链基因扩增及鉴定以cdna为模板，根据常规引物设计方案进行核苷酸序列扩增，扩增之后，轻链和重链各随机抽取(轻链4条重链8条)所扩增的产物进行琼脂糖凝胶电泳鉴定，结果(见图5)显示轻重链基因扩增良好，且大小约为750bp左右，符合预期大小。之后进行割胶回收，用于后续酶切反应。胶回收获得如下结果sns001-002-lc-gene:112ng/μl，100μlsns001-002-hc-gene:104ng/μl，100μl1.2.4lc-rbs(重叠区)/hc-rbs(重叠区)扩增分别以所获得的抗体轻重链基因为模板进行扩增(见表1、表2和表3)，获得产物lc-rbs和hc-rbs如图6，产物鉴定后将pcr产物进行回收。表1.lc-rbs扩增反应体系组成单反应需要量混合体系需要量(5×)2×pcrmix25μl125μlsns001-002-lc-gene0.1μl0.5μlol-bflc-super-f1.2μl6μlol-middle-super-r1.2μl6μlddh2o添加至50μl添加至250μl表2.hc-rbs扩增反应体系组成单反应需要量混合体系需要量(5×)2×pcrmix25μl125μlsns001-002-hc-gene0.1μl0.5μlol-middle-super-f1.2μl6μlol-afhc-super-r1.2μl6μlddh2o添加至50μl添加至250μl表3.lc-rbs/hc-rbs扩增反应程序扩增的产物进行琼脂糖凝胶电泳鉴定，如图6，结果显示轻重链基因扩增良好，且大小约为800bp左右，符合预期大小。之后进行割胶回收。胶回收结果如下sns001-002-lc-rbs:63ng/μl，100μlsns001-002-hc-rbs:51ng/μl，100μl1.2.5轻重链overlap重组以所获得的lc-rbs和hc-rbs样品为模板(表4)，进行体外连接(表5)。表4.overlap反应体系组成单反应需要量2×pcrmix25μlsns001-002-lc-rbs8μlsns001-002-hc-rbs10μlddh2o添加至50μl表5.overlappcr反应程序得到结果如图7，显示特异连接片段在1900bp左右。后续进行该位置胶回收。1.2.6酶切参照内切酶试剂盒提供的方法将上述产物进行hindiii和noti双酶切，切下的片段约75bp，鉴定后进行胶回收接近1800bp处特异条带(如图8)。1.2.7连接参照连接酶试剂盒提供(takara，ai91067a)的方法，结合目的库容大小，进行足量的连接反应。获得含有该片段的质粒载体。1.2.8转化使用电转感受态细胞dh5α-e(货号11319019)进行电转转化。具体为首先将连接产物脱盐纯化后，然后按照按1μg连接产物/电转进行转化，电转转化结束后，取10μl进行稀释法测定和计算库容；同时涂布单克隆平板以测序；剩余菌液涂平板，37℃过夜培养，第二天刮取平板菌苔，加甘油保存于-80℃。1.2.9质量鉴定1)fab库库容测定：采用稀释点板法测定本实施例所构建免疫库的fab文库均为5.4×107pfu。进行单克隆测序验证与结果分析：共测序单克隆序列10个(测序商：金斯瑞；测序方法：二代测序技术)，测序成功9个，一个空载体或载体自连。序列分析表明正确插入率大于80％。2)fab抗体库抗体基因完整性检测抗体库构建载体信息，图9为fab抗体库构建载体简图，抗体库构建完成之后，apali和noti之间为抗体fab全长序列。3)抗体基因完整性通过分析(dnastar分析软件)测序结果获得抗体基因的完整性：根据成功测序的序列，分别从正向测序选择apali序列(图10，a)，并从反向测序结果选取noti序列(图10，b)进行展示，如图10，分析结果表明抗体基因完整性大于80％。(注：本轮共建2批次库，实验测序分为1、2各测序5条)4)插入抗体基因种源分析将测序获得的抗体序列在ncbi中进行blast，检查所插入抗体序列是否为小鼠抗体。并分析其轻重链cdr3，查看unique数量。如图11结果，表明所插入抗体序列均为鼠源性抗体。从cdr3可以体现出较高的丰富度，本次建库质量合格。1.3阳性抗体克隆筛选以生物素化的sns001-002作为抗原，使用1.2构建的免疫库继续筛选，通过elisa方法筛选阳性抗体并对阳性抗体进行亲和力排序。具体实验步骤如下：1.3.1抗体库海选①将抗体库接种到2yt培养基(实验室自制，每1升培养基包含蛋白胨16g，酵母提取物10g，氯化钠5g)中(抗性car+/tet+含有终浓度2％的葡萄糖)。②220rpm，37℃摇床中培养直到od600值达到0.5-0.6。③加入vscm13辅助噬菌体，37℃静止30分钟后，220rpm37℃摇床中培养1小时。④置换成2yt(抗性为car+/kan+)，225rpm30℃培养过夜。⑤13,500×g离心5分钟，将上清与peg6000/nacl溶液混合，冰上孵育1-2小时或过夜，目的是将噬菌体颗粒从溶液中沉淀下来，从而达到富集噬菌体的作用。⑥13,500×g，4℃，离心10分钟，去上清；适当体积的pbs重悬，获得噬菌体悬液。⑦将噬菌体悬液与生物素-抗原包被得免疫磁珠孵育，并按照kingfisher磁珠筛选系统方法进行结合和清洗。⑧用胰蛋白酶洗脱噬菌体。⑨洗脱后噬菌体溶液与对数期大肠杆菌ss320细胞充分混合后37℃静置培养。涂布在2yt(car+/tet+)平板上，同时进行噬菌体滴度检测，37℃培养箱过夜培养。⑩计算噬菌体output(输出噬菌体滴度)，input(输入噬菌体的滴度)等，刮板并进行2-3轮筛选.1.3.2单克隆elisa初筛1.3.2.1准备2yt培养基(抗性：car+/tet+)，在96孔深孔板中加500μl，挑选第三轮的单克隆接入到96孔深孔板中培养，220rpm，37℃过夜培养。1.3.2.2离心取上清进行elisa，方法如下：①抗原用pbs稀释到2μg/ml后，每孔加入30ul，4℃过夜。pbst洗板3次。②1％bsa(sigma-aldrich,v900933)封闭1小时，pbst洗板3次。③加入离心后得上清30μl，室温2小时，pbst洗板3次。④加入1:5000稀释的二抗，即辣根过氧化物酶标记的鼠抗fab(anti-mouse-fab-hrp)(revmabbioscience,41-1043-00)30μl，室温1小时，pbst洗板3次。⑤加入30μltmb显色，之后加入30μl2mhcl终止反应。酶标仪od450读取数据。1.3.3亲和力排序采用elisa方法进行亲和力排序①抗原用pbs稀释到2μg/ml每孔加入30μl，4℃过夜。pbst洗板3次。②1％bsa封闭1小时后pbst洗板3次。③将elisa初筛中的疑似阳性克隆接种于50ml培养基中，诱导表达后，离心收集菌体，将菌体裂解后离心收集的上清稀释8个浓度梯度加入包被的elisa板中，室温2小时，pbst洗板3次。④加入1:4000稀释的二抗anti-mouse-fab-hrp30μl，室温1小时。pbst洗板9次。⑤加入30μltmb，加入30μl2mhcl终止。酶标仪od450读取数据进行后续分析。1.3.4实验结果1.3.4.1三轮海选的噬菌体滴度测定，如表6表6三轮海选的噬菌体滴度测定结果输入输出输出/输入ck输出/ck第1次盘洗2.25e+133.9e+090.0001733332.85e+091.3684211第2次盘洗2.50e+134.20+090.000173.30e+091.2727第3次盘洗1.75e+131.02e+100.000581.65e+096.181.3.4.2三轮盘洗后筛选的克隆富集测序样品分析，如表7表7三轮盘洗后筛选克隆富集测序样品数量统计分析1.3.4.3噬菌体elisa验证库质量综合实验结果比对，如图12，通过噬菌体elisa盘洗筛选，到第三轮时已经体现出了较好的富集，2、3轮测序结果显示出现了克隆上的富集。从盘洗滴度上看在第三轮有富集。因此挑选盘洗三轮的克隆进行elisa初筛。1.3.4.4单克隆elisa初筛结果共挑选352个单克隆，显示阳性克隆的数量为74个。选数值较高的克隆送测序公司测序。1.3.4.5测序结果将测序回来的序列与克隆编号一一对应，用软件分析正确序列，用ncbi汇总序列，找出同一序列的代表序列进行汇总，共得到18条单一序列。其中轻重链可变区序列信息如下表8所示，cdr信息如表9和表10所示：表8单链抗体轻重链可变区序列信息表表9抗体轻链cdr编号表10抗体重链cdr编号1.3.4.6亲和力排序结果将上述18个阳性克隆进行50ml诱导表达和裂解浓缩，得到的上清进行梯度稀释，进行定量elisa，提前包被目标抗原及定量fab一抗(jackson，115-007-003)，进行亲和力elisa测定。将elisa数据用graphpad+prism作图(包含ec50值)。根据检测的结果，对18条阳性克隆相对亲和力的结果进行排序，按照亲和力从强到弱，对应编码1-18顺序如下表11：表11阳性克隆相对亲和力排序结果根据亲和定量结果，可知初筛共得到18条阳性克隆都具有较好的亲和力，说明阳性克隆表达的抗体能特异性识别目的抗原且量效关系明显。实施例2：含有嵌合抗原受体的慢病毒质粒的构建及病毒包装2.1嵌合抗原受体核酸序列合成本实施例设计嵌合抗原受体序列的排列顺序为：从5端到3端依次为：前导链-scfv-cd8铰链区-cd8跨膜区-4-1bb-cd3ζ。利用实施例1中筛选的18种阳性克隆的轻链可变区和重链可变区序列(表11中vl、vh序列号)并同时提交以下序列，分别为编号为32716克隆(来自美国希望之城)的轻重链可变区序列(来自专利cn201480024929.6)、前导链区(seqidno:73)、linker区(seqidno:75)、cd8铰链区(seqidno:77)，cd8跨膜区(seqidno:79)、4-1bb共刺激区域(seqidno:81)和cd3ζ序列(seqidno:83)，委托南京金斯瑞公司合成。其中前导链、linker、铰链区、跨膜区、4-1bb共刺激区、cd3ζ区序列编码如下表显示，具体序列编号见附表12。表12：car其他相关序列信息汇总表名称nt序列aa序列前导链seqidno:73seqidno:74linkerseqidno:75seqidno:76cd8铰链区seqidno:77seqidno:78cd8跨膜区seqidno:79seqidno:804-1bb共刺激区域seqidno:81seqidno:82cd3ζ区aaseqidno:83seqidno:84合成后的完整核苷酸序列依此命名为010-car-nt、021-car-nt、027-car-nt、062-car-nt、064-car-nt、070-car-nt、110-car-nt、127-car-nt、137-car-nt、208-car-nt、209-car-nt、227-car-nt、236-car-nt、245-car-nt、255-car-nt、257-car-nt、273-car-nt、290-car-nt和0033-car-nt。0033-car-nt在本发明中作为对照，其抗体可变区序列来自专利cn201480024929.6，除了scfv之外的区域采用表12所示的序列，其轻链可变区氨基酸序列如seqidno:169所示，其重链可变区氨基酸序列如seqidno:170所示。本发明的car的氨基酸序列如seqidno:86、88、90、92、94、96、98、100、102、104、106、108、110、112、114、116、118、120所示。编码car的核苷酸序列如seqidno:85、87、89、91、93、95、97、99、101、103、105、107、109、111、113、115、117、119所示。然后将上述合成的car序列插入到慢病毒载体中plvx-ef1中，按照常规酶切连接、转化、测序等步骤方法，构建含有嵌合抗原受体的慢病毒质粒载体plvx-ef1-car共19种(分别为plvx-ef1-010-car、plvx-ef1-021-car、plvx-ef1-027-car、plvx-ef1-062-car、plvx-ef1-064-car、plvx-ef1-070-car、plvx-ef1-110-car、plvx-ef1-127-car、plvx-ef1-137-car、plvx-ef1-208-car、plvx-ef1-209-car、plvx-ef1-227-car、plvx-ef1-236-car、plvx-ef1-245-car、plvx-ef1-255-car、plvx-ef1-257-car、plvx-ef1-273-car、plvx-ef1-290-car和plvx-ef1-0033-car)，其中plvx-ef1-0033-car在本实验中作为阳性对照使用。2.2包含嵌合抗原受体基因片段的慢病毒质粒载体的提取使用质粒小提中量试剂盒tianprepminiplasmidkit(cat.#dp103-03)。2.3慢病毒的包装及病毒收获将第6～10代的293t细胞(atcc：crl-11268)以5×106的密度接种培养至于10cm培养皿中，37℃，5％co2培养过夜以为转染准备。培养基为含10％胎牛血清(购自英潍捷基)的dmem(购自英潍捷基)，次日，在转染前约2小时更换培养液为无血清dmem。转染的步骤如下：1)分别制备19种car(其中的单链抗体序列，包括前述实施例提供的18种单链抗体序列及专利cn201480024929.6中32716单克隆抗体的单链抗体序列)病毒包装需要的质粒混合液，每种病毒包装一小盘病毒(培养皿规格100mm*20mm)。每份需要的质粒按照如下比例制备混合液：6μg的目的基因质粒plvx-ef1-car，分别与6μg包装质粒pspax2和3μg包膜质粒pmdg.2，以及60ulpei脂质体(购自polysiences)，溶入1mldmem中，混匀。重复以上操作分别制备含其他目的质粒的质粒混合液。(质粒的来源：金斯瑞)2)静置15分钟后，按照不同病毒的标记名称，取相应标记的质粒混合液对应逐滴分别加入到19个培养皿中，然后轻轻摇匀，再于37℃，5％co2培养箱培养4～6h后，更换为含10％胎牛血清的dmem。在转染48小时或72小时后收集病毒上清液，使用0.45μm滤器(购自millipore公司)过滤收集病毒，然后采用beckmanoptimal-100xp超速离心机28000rpm，4℃离心2小时，弃离心上清，离心所得沉淀用1/10～1/50原液体积的x-vivotm15培养基(购买自lonza)进行重悬，经0.22μm除菌过滤后，以1000μl/管分装冻存于-80℃，以待病毒滴定或感染t淋巴细胞。2.4测定慢病毒滴度(1)第一天铺板：以4×105/ml接种293t细胞于96孔培养板，培养液为含10％胎牛血清的dmem。接种体积100μl/孔，37℃，5％co2培养箱培养。(2)第二天感染：①制备待感染病毒液：取2.3得到的19种浓缩病毒各取10μl用无血清培养基分别稀释100倍、1000倍感染293t。即10μl原病毒与90μl无血清培养基混合得到稀释10倍的病毒稀释液，然后取稀释10倍的病毒稀释液10μl与90μl无血清培养基混合得到稀释100倍的病毒稀释液，依此类推得到稀释1000倍的病毒稀释液。②选择96孔板中需要感染的孔，将其中的完全培养基弃掉。根据标记添加上一步制备的不同稀释倍数的病毒稀释液100μl。每个梯度两个复孔，37℃，5％co2培养箱培养。(3)第三天：将无血清培养基更换为完全培养基(10％胎牛血清的dmem)。(4)第四天检测：感染24小时后，流式细胞仪检测fab，以阳性率为5～20％的细胞数为宜，计算滴度(pfu/ml)＝细胞数×阳性率×稀释倍数。经过计算，所测的病毒滴度平均约为4×107pfu/ml。实施例3：k562-cd123的稳定表达细胞系的构建和验证首先ncbi(https://www.ncbi.nlm.nih.gov/)查找cd123蛋白序列(xp_016884980.1)，委托南京金斯瑞合成含有该蛋白基因的慢病毒质粒的载体，转染于感受态细胞stb13，按照实施例2.2进行质粒的提取，提取的目的质粒与包装质粒按照一定比例转染至293t细胞，分别于转染48小时及72小时后收集cd123病毒液并过滤，并参考实施例2.4进行病毒滴定。按照moi＝5:1，使用cd123病毒液感染k562细胞(来自北大医学部)。感染2天后进行嘌呤霉素筛选(筛选浓度为5μg/ml)，筛选一周后，以未染色k562细胞为对照细胞，使用cd123抗体对k562细胞和构建的cd123-k562染色，流式检测cd123表达情况。结果如图13所示：cd123-k562+fab样品的cd123阳性表达率为85％左右，k562本身cd123阳性表达率接近0，k562+fab样品为本底染色背景噪音1％左右。因此所构建cd123-k562细胞系cd123阳性表达率为84％左右，表明细胞系构建成功，同时也表明k562细胞不表达cd123，后续实验可以以k562作为阴性对照。所构建k562-cd123可以用于下一步实验。实施例4：t细胞的分离与激活培养取健康供者的新鲜外周血，通过ficoll密度梯度离心法分离新鲜的外周血单核细胞；再利用偶联了cd3-cd28抗体磁珠(购买自美国thermolfisherscientific公司，产品名称为humant-activatorcd3/cd28)来富集cd3+t细胞。具体如下：①将分离的外周血单核细胞经检测后稀释至浓度为(10-30)×106个单个细胞/ml；②pbs洗涤后，300g，5min离心，弃掉上清；③按照cd3-cd28抗体磁珠与单核细胞3:1的比例计算需要的磁珠量。用1mlx-vivo-15培养基重悬磁珠，然后将磁珠悬液重悬以上细胞沉淀，再将磁珠和细胞的混合悬液转移到1.5mlep管中，室温下低速转动孵育1小时左右；④磁珠与细胞孵育结束后，转移至磁微粒收集器(magneticparticlesconcentrator，简称mpc，购买自美国invitrogen公司)，将上清弃掉，使用x-vivo-15培养基富集cd3+t细胞。⑤将富集的cd3+t细胞重悬按照接种的细胞数均匀接种到6孔板中培养，使细胞浓度为1×106个/ml。⑥最后在37℃、5％co2培养箱中培养1-2天，根据细胞生长情况进行下一步病毒感染实验。实施例5:重组慢病毒感染t细胞用上述实施例2获得的重组慢病毒感染实施例4获得激活的t细胞，获得cart细胞。具体如下：①一般在t细胞激活的前三天进行感染，本实验在t细胞激活第二天进行。按照病毒与t细胞为5：1比例进行感染。②感染条件：2000g/min离心2小时；③感染结束后，将细胞用新鲜培养基重悬，使细胞浓度1×106个/ml，然后于37℃培养箱中培养。④感染后的细胞每隔一天以5×105/ml的密度进行传代，同时补加终浓度100u/ml的重组人il-2(peprotech,200-02-1000)。⑤感染的t细胞在培养第5天时通过流式细胞检测嵌合抗原受体表达情况。实验结果如下：以未感染的t淋巴细胞作为阴性对照细胞，以0033-car-t为阳性对照，表达嵌合抗原受体t细胞的阳性率如图14所示，cd123-k562+fab样品的cd123阳性表达率为85％左右，k562本身cd123阳性表达率接近0，k562+fab样品为本底染色背景噪音1％左右。因此cd123-k562细胞系cd123阳性表达率为84％左右。通过慢病毒感染的方法能够获得一定阳性率的cart细胞，本实施例中表达率在90％以上有7个阳性克隆，分别为062-car-t、064-car-t、208-car-t、227-car-t、245-car-t和257-car-t，273-car-t；表达率在80％-90％有4个阳性克隆，010-car-t、137-car-t、209-car-t和255-car-t；表达率在70％-80％有4个阳性克隆，027-car-t、070-car-t、110-car-t和236-car-t；表达率在60％-70％有2个阳性克隆：021-car-t及290-car-t；表达率在60％以下有1个阳性克隆：127-car-t。其中阴性对照几乎没有表达，阳性对照(033-car-t)表达率为85％，以上数据表明本次筛选的阳性克隆在应用于car表达方面，于阳性对照比较，差别不明显，表明基于阳性克隆的cart构建成功，可以进行下一步的功能验证分析。继续培养第10天约有20～40倍的扩增，表明表达嵌合抗原受体的t细胞在体外能够进行一定数量的扩增，为后续体外毒性试验提供了保证。实施例6表达嵌合抗原受体细胞的体外毒性效果实验用k562-123(使用实施例3中构建的细胞系)作为靶细胞，以t为阴性对照，以构建的0033-cart为阳性对照细胞，以含有筛选的18种阳性克隆单链抗体-cart为效应细胞。效靶比为5：1。靶细胞数量为40000/孔。各组均设3个复孔，取3个复孔的平均值。检测时间为第16-20h。其中各实验组和各对照组如下：对照组1：靶细胞最大释放ldh对照组2：靶细胞自发释放ldh对照组3：效应细胞自发释放ldh对照组4：t自发释放ldh实验组1：靶细胞+表达嵌合抗原受体的t实验组2：靶细胞+未感染t细胞采用cytotox96非放射性细胞毒性检测试剂盒(promega公司)进行。定量地测量乳酸脱氢酶(ldh)。ldh是一种稳定的胞质酶，在细胞裂解时会释放出来，释放出的ldh在培养基上清中，可通过30分钟偶联的酶反应来检测，在酶反应中ldh可使一种四唑盐(int)转化为红色的甲臜(formazan)。生成的红色产物的量与裂解的细胞数成正比。具体步骤参照cytotox96非放射性细胞毒性检测试剂盒说明书。car-t细胞毒性计算公式为：(实验组1-对照组2-对照组3)/(对照组1-对照组2)*100％t细胞毒性计算公式为：(实验组2-对照组2-对照组4)/(对照组1-对照组2)*100％检测结果汇总整理如图15-16所示。从结果可以看出，基于筛选的18种克隆的新型cd123-cart对k562细胞没有杀伤作用，杀伤结果为负值，这与k562细胞本身不表达cd123的情况比较相符。但这18种融合表达cd123单链抗体car-t细胞对表达cd123抗原的靶细胞k562-123则都表现出杀伤作用，其中与阴性对照比较具有显著性差异的为236-car-t；但与阳性对照相比，236-car-t的杀伤肿瘤作用稍弱或较温和。基于目前cd123-cart的进展，虽然cd123-cart在体内外实验表明均具有显著的抗肿瘤效应，但也普遍存在对正常低水平表达细胞的杀伤作用。如loffs等人曾体外实验模拟了cd123-cart的对高水平表达和低表达水平细胞的杀伤能力。实验表明传统cd123-cart对这两类细胞都是持续杀伤，高水平细胞在120小时内就完全被杀伤，而低水平表达细胞在120小时后逐渐缓慢被完全杀伤。另外体内实验也说明对正常低水平表达细胞有损伤，如给免疫缺陷小鼠模型注射cd123-cart细胞，发现小鼠体内白血球是持续减少的，说明传统cd123-cart对骨髓抑制过程比较持久，也就是对造血干细胞有细胞毒性(loffs,dietrichj,meyerje,etal.rapidlyswitchableuniversalcar-tcellsfortreatmentofcd123-positiveleukemia.moltheroncolytics,2020,17:408-420.)。另有文献报告基于编号为32716克隆(来自美国希望之城)的scfv序列(来自专利cn201480024929.6)的cart细胞对低水平内皮细胞也有损伤，研究发现主要是由于cart分泌的干扰素γ和肿瘤坏死因子-α导致内皮细胞cd123表达上调引起(suny,wangs,zhaol,etal.ifn-γandtnf-αaggravateendothelialdamagecausedbycd123-targetedcartcell.oncotargetsther,2019,12:4907-4925.)。从cart作用机制上来说，本专利优选的克隆特异性杀伤比阳性对照克隆0033-car-t稍弱，推测可以减轻或者避免对正常细胞(尤其是低表达cd123的正常细胞)的损伤，从安全性方面综合考虑，上述抗体克隆安全性更高。sequencelisting<110>西安桑尼赛尔生物医药有限公司<120>一种cd123结合蛋白、含其的car及其应用<130>bqp20014237c<160>170<170>patentinversion3.5<210>1<211>324<212>dna<213>artificialsequence<220><223>p31-010-vl-nt<400>1gacattgttctcaaccagtctccagcaatcatgtctgcatctctaggggaacgggtcacc60atgacctgcactgccagctcaagtgtaagttccagttacttgcactggtaccagcagaag120ccaggatcctcccccaaactctggatttatagcacatccaacctggcttctggagtcccg180gctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcaacatggag240gctgaagatgctgccacttattactgccaccagtttcatcgttccccacccacgttcggt300gctgggaccaagctggagctgaaa324<210>2<211>108<212>prt<213>artificialsequence<220><223>p31-010-vl-aa<400>2aspilevalleuasnglnserproalailemetseralaserleugly151015gluargvalthrmetthrcysthralaserserservalserserser202530tyrleuhistrptyrglnglnlysproglyserserprolysleutrp354045iletyrserthrserasnleualaserglyvalproalaargpheser505560glyserglyserglythrsertyrserleuthrileserasnmetglu65707580alagluaspalaalathrtyrtyrcyshisglnphehisargserpro859095prothrpheglyalaglythrlysleugluleulys100105<210>3<211>339<212>dna<213>artificialsequence<220><223>p31-010-vh-nt<400>3gaggtgcagcttgttgagtctggacctagcctcgtgaaaccttctcagtctctgtccctc60acctgcactgtcactggctactcaatcaccagtgattatgtctggaactggatccggcaa120tttccaggaaacaaactggagtggatgggctacataagctacagtggtagcactagttac180aatccatctctcacaagtcgaatctctatcactcgagacacatccaagaaccagttcttc240ctgcagttgaattctgtgactactgaggacacagccacatattactgtacaagaggaacg300gactactggggtcaaggaacctcagtcaccgtctcctca339<210>4<211>113<212>prt<213>artificialsequence<220><223>p31-010-vh-aa<400>4gluvalglnleuvalgluserglyproserleuvallysprosergln151015serleuserleuthrcysthrvalthrglytyrserilethrserasp202530tyrvaltrpasntrpileargglnpheproglyasnlysleuglutrp354045metglytyrilesertyrserglyserthrsertyrasnproserleu505560thrserargileserilethrargaspthrserlysasnglnphephe65707580leuglnleuasnservalthrthrgluaspthralathrtyrtyrcys859095thrargglythrasptyrtrpglyglnglythrservalthrvalser100105110ser<210>5<211>333<212>dna<213>artificialsequence<220><223>p31-021-vl-nt<400>5gacattgtgttgacacagtctcctgcttccttagctgtatctctggggcagagggccacc60atctcatgcagggccagcaaaagtgtcagttcatctggctatagttatatacactggtac120caacagaaaccaggacagccacccaaactcctcatctatcttgcatccaacctagaatct180ggggtccctgccaggttcagtggcagtgggtctgggacagacttcaccctcaacatccat240cctgtggaggaggaggatgctgcaacctattactgtcagcacagtagggagcttccgtac300acgttcggaggggggaccaagctggaaataaaa333<210>6<211>111<212>prt<213>artificialsequence<220><223>p31-021-vl-aa<400>6aspilevalleuthrglnserproalaserleualavalserleugly151015glnargalathrilesercysargalaserlysservalserserser202530glytyrsertyrilehistrptyrglnglnlysproglyglnpropro354045lysleuleuiletyrleualaserasnleugluserglyvalproala505560argpheserglyserglyserglythraspphethrleuasnilehis65707580provalgluglugluaspalaalathrtyrtyrcysglnhisserarg859095gluleuprotyrthrpheglyglyglythrlysleugluilelys100105110<210>7<211>354<212>dna<213>artificialsequence<220><223>p31-021-vh-nt<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ervalserthrsergly202530tyrsertyrmethistrptyrglnglnlysproglyglnproprolys354045leuleuiletyrleualaserasnleugluserglyvalproalaarg505560pheserglyserglyserglythraspphethrleuasnilehispro65707580valgluglugluaspalaalathrtyrtyrcysglnhisserargglu859095leuprotyrthrpheglyglyglythrlysleugluilelys100105110<210>19<211>354<212>dna<213>artificialsequence<220><223>p31-064-vh-nt<400>19caggtgcagctgaaggagtcaggacctggcctggtggcgccctcacagagcctgtccatt60atttgcactgtctctgggttttccttaaccaactatggtgtacactgggttcgccagcct120ccaggaaagggtctggagtggctgggagtaatatgggctgatggaaccacaaattataat180tcggctctcatgtccagactgagcatcagcaaagacaactccaagagccaagttttctta240aaaatgaacagtctgcaaactgatgacacagccatgtactactgtgccagagaagggttc300gacaccacctggtttgattattggggccaagggactctggtcactgtctctgca354<210>20<211>118<212>prt<213>artificialsequence<220><223>p31-064-vh-aa<400>20glnvalglnleulysgluserglyproglyleuvalalaprosergln151015serleuserileilecysthrvalserglypheserleuthrasntyr202530glyvalhistrpvalargglnproproglylysglyleuglutrpleu354045glyvaliletrpalaaspglythrthrasntyrasnseralaleumet505560serargleuserileserlysaspasnserlysserglnvalpheleu65707580lysmetasnserleuglnthraspaspthralamettyrtyrcysala859095arggluglypheaspthrthrtrppheasptyrtrpglyglnglythr100105110leuvalthrvalserala115<210>21<211>324<212>dna<213>artificialsequence<220><223>p31-070-vl-nt<400>21gacattgttctctcccagtctccagcaatcatgtctgcatctctaggggaacgggtctcc60atgacctgcactgccagctcaagtgtaagttccagttacttgcactggtaccagcagaag120ccaggatcctcccccaaactctggatttatagcacatccaacctggcttctggagtccca180actcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagcatggag240gctgaagatgctgccacttattactgccaccagtttcatcgttccccacggacgttcggt300ggaggcaccaagctggaaatcaaa324<210>22<211>108<212>prt<213>artificialsequence<220><223>p31-070-vl-aa<400>22aspilevalleuserglnserproalailemetseralaserleugly151015gluargvalsermetthrcysthralaserserservalserserser202530tyrleuhistrptyrglnglnlysproglyserserprolysleutrp354045iletyrserthrserasnleualaserglyvalprothrargpheser505560glyserglyserglythrsertyrserleuthrilesersermetglu65707580alagluaspalaalathrtyrtyrcyshisglnphehisargserpro859095argthrpheglyglyglythrlysleugluilelys100105<210>23<211>339<212>dna<213>artificialsequence<220><223>p31-070-vh-nt<400>23gaggtgcagcttcaggagtccggaccaagcctcgtgaaaccttctcagactctgtccctc60acctgttctgtcactggcgactccatcaccagtgattatgtctggaactggatccggcaa120tttccaggaaacaaactggagtggatgggctacataagctacagtggtagcactagttac180aatccatctctcacaagtcgaatctctatcactcgagacacatccaagaaccagttcttc240ctgcagttgaattctgtgactactgaggacacagccacatattactgtacaagaggaacg300gactactggggtcaaggaacctcagtcaccgtctcctca339<210>24<211>113<212>prt<213>artificialsequence<220><223>p31-070-vh-aa<400>24gluvalglnleuglngluserglyproserleuvallysprosergln151015thrleuserleuthrcysservalthrglyaspserilethrserasp202530tyrvaltrpasntrpileargglnpheproglyasnlysleuglutrp354045metglytyrilesertyrserglyserthrsertyrasnproserleu505560thrserargileserilethrargaspthrserlysasnglnphephe65707580leuglnleuasnservalthrthrgluaspthralathrtyrtyrcys859095thrargglythrasptyrtrpglyglnglythrservalthrvalser100105110ser<210>25<211>330<212>dna<213>artificialsequence<220><223>p31-110-vl-nt<400>25gacattgtgatgacacaacctgcttccttagctgtatctctggggcagagggccaccatc60tcatgcagggccagcaaaagtgtcagtacatctggctatagttatatgcactggtaccaa120cagaaaccaggacagccacccaaactcctcatctatcttgcatccaacctagaatctggg180gtccctgccaggttcagtggcagtgggtctgggacagacttcaccctcaacatccatcct240gtggaggaggaggatgctgcaacctattactgtcagcacagtagggagcttccgtacacg300ttcggaggggggaccaagctggaaataaaa330<210>26<211>110<212>prt<213>artificialsequence<220><223>p31-110-vl-aa<400>26aspilevalmetthrglnproalaserleualavalserleuglygln151015argalathrilesercysargalaserlysservalserthrsergly202530tyrsertyrmethistrptyrglnglnlysproglyglnproprolys354045leuleuiletyrleualaserasnleugluserglyvalproalaarg505560pheserglyserglyserglythraspphethrleuasnilehispro65707580valgluglugluaspalaalathrtyrtyrcysglnhisserargglu859095leuprotyrthrpheglyglyglythrlysleugluilelys100105110<210>27<211>354<212>dna<213>artificialsequence<220><223>p31-110-vh-nt<400>27gaggtgcagcttcagcagtcaggacctggcctggtggcgccctcacagagcctgtccatc60acttgcactgtctctggcttttcattaaccagctatggtatacactgggttcgccagcct120ccaggaaagggtctggagtggctgggagtaatatgggctgatggaagcacaaattataat180tcggctctcatgtccagactgagcatccgcaaagacaactccaagagccaaattttctta240aaaatgaacagtctgcaaaatgatgacatagccatgtactactgtgccagagaagggtac300gacaccacctggtttgattactggggccaagggactctggtcactgtctctgca354<210>28<211>118<212>prt<213>artificialsequence<220><223>p31-110-vh-aa<400>28gluvalglnleuglnglnserglyproglyleuvalalaprosergln151015serleuserilethrcysthrvalserglypheserleuthrsertyr202530glyilehistrpvalargglnproproglylysglyleuglutrpleu354045glyvaliletrpalaaspglyserthrasntyrasnseralaleumet505560serargleuserilearglysaspasnserlysserglnilepheleu65707580lysmetasnserleuglnasnaspaspilealamettyrtyrcysala859095arggluglytyraspthrthrtrppheasptyrtrpglyglnglythr100105110leuvalthrvalserala115<210>29<211>324<212>dna<213>artificialsequence<220><223>p31-127-vl-nt<400>29gacattgttctcacccagtctccagcaatcatgtctgcatctccaggggagaaggtcacc60atgacctgcagtgccagctcaagtgtaagttccagttacttgcactggtaccagcagaag120tcaggtgcctcccccaaactctggatttatagcacatccaaattggcttctggagtccct180gctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagtgtggaa240gctgaagatgctgccacttatttctgccagcagaacagtggttacccattcacgttcggc300tcggggacaaagttggaaataaaa324<210>30<211>108<212>prt<213>artificialsequence<220><223>p31-127-vl-aa<400>30aspilevalleuthrglnserproalailemetseralaserprogly151015glulysvalthrmetthrcysseralaserserservalserserser202530tyrleuhistrptyrglnglnlysserglyalaserprolysleutrp354045iletyrserthrserlysleualaserglyvalproalaargpheser505560glyserglyserglythrsertyrserleuthrileserservalglu65707580alagluaspalaalathrtyrphecysglnglnasnserglytyrpro859095phethrpheglyserglythrlysleugluilelys100105<210>31<211>339<212>dna<213>artificialsequence<220><223>p31-127-vh-nt<400>31gaagtgatgcttcaggagtcgggacctggcctggtgaaaccttctcagtctctgtccctc60acctgcactgtcactggcttctcaatcaccagtgattatgcctggaactggatccggcag120tttccaggaaacaagctggagtggatgggctacataagttttggtggtactacttcctac180aacccatctctcaaaagtcgaatctctatcactcgagacacatccaagaaccagttcttc240ctgcagttgaattctgtgacttctgaagacacagccacatattactgtacaagagggttg300gactactggggccaaggcaccattctcacagtctcctca339<210>32<211>113<212>prt<213>artificialsequence<220><223>p31-127-vh-aa<400>32gluvalmetleuglngluserglyproglyleuvallysprosergln151015serleuserleuthrcysthrvalthrglypheserilethrserasp202530tyralatrpasntrpileargglnpheproglyasnlysleuglutrp354045metglytyrileserpheglyglythrthrsertyrasnproserleu505560lysserargileserilethrargaspthrserlysasnglnphephe65707580leuglnleuasnservalthrsergluaspthralathrtyrtyrcys859095thrargglyleuasptyrtrpglyglnglythrileleuthrvalser100105110ser<210>33<211>330<212>dna<213>artificialsequence<220><223>p31-137-vl-nt<400>33gacattgtgatgacacaacctgcttccttagctgtatctctggggcagagggccaccatc60tcatgcagggccagcaaaagtgtcagtacatctggctatagttatttacactggtaccaa120cagaaaccaggacagccacccaaactcctcatctatcttgcatccaacctagaatctggg180gtccctgccaggttcagtggcagtgggtctgggacagacttcaccctcaacatccatcct240gtggaggaggaggatgctgcaacctattactgtcagcacagtagggaccttccgtacacg300ttcggaggggggaccaagctggaaataaaa330<210>34<211>110<212>prt<213>artificialsequence<220><223>p31-137-vl-aa<400>34aspilevalmetthrglnproalaserleualavalserleuglygln151015argalathrilesercysargalaserlysservalserthrsergly202530tyrsertyrleuhistrptyrglnglnlysproglyglnproprolys354045leuleuiletyrleualaserasnleugluserglyvalproalaarg505560pheserglyserglyserglythraspphethrleuasnilehispro65707580valgluglugluaspalaalathrtyrtyrcysglnhisserargasp859095leuprotyrthrpheglyglyglythrlysleugluilelys100105110<210>35<211>354<212>dna<213>artificialsequence<220><223>p31-137-vh-nt<400>35caggtgcagctgaaggagtcaggacctggcctggtggcgccctcacagagcctgtccatc60acttgcactgtctctggcttttcattacccagctatggtgtacactgggttcgccagcct120ccaggaaagggtctggagtggctgggagtaatctgggctgatggaagcacaaattataat180tcggctctcatgtccagactgagcatcagcaaagacaactccaagaaccaagttttcttg240aaaatgaacagtctgcaaaatgatgatacagccatgtattactgtgccagagaagggttc300gacaccacctggtttgattactggggccaagggactctggtcactgtctctgca354<210>36<211>118<212>prt<213>artificialsequence<220><223>p31-137-vh-aa<400>36glnvalglnleulysgluserglyproglyleuvalalaprosergln151015serleuserilethrcysthrvalserglypheserleuprosertyr202530glyvalhistrpvalargglnproproglylysglyleuglutrpleu354045glyvaliletrpalaaspglyserthrasntyrasnseralaleumet505560serargleuserileserlysaspasnserlysasnglnvalpheleu65707580lysmetasnserleuglnasnaspaspthralamettyrtyrcysala859095arggluglypheaspthrthrtrppheasptyrtrpglyglnglythr100105110leuvalthrvalserala115<210>37<211>330<212>dna<213>artificialsequence<220><223>p31-208-vl-nt<400>37gacattgtgatgacacaacctgcttccttagctgtatctctggggcagagggccaccatc60tcatgcagggccagcgaaagtgtcagtacatctggctatagttatatgcactggtaccaa120cagaaaccaggacagccacccaaactcctcatctatcttgcatccaacctagaatctggg180gtccctgccaggttcagtggcagtgggtctgggacagacttcaccctcaacatccatcct240gtggaggaggaggatgctgcaacctattactgtcagcacagtagggaacttccgtacacg300ttcggaggggggaccaagctggaaataaaa330<210>38<211>110<212>prt<213>artificialsequence<220><223>p31-208-vl-aa<400>38aspilevalmetthrglnproalaserleualavalserleuglygln151015argalathrilesercysargalasergluservalserthrsergly202530tyrsertyrmethistrptyrglnglnlysproglyglnproprolys354045leuleuiletyrleualaserasnleugluserglyvalproalaarg505560pheserglyserglyserglythraspphethrleuasnilehispro65707580valgluglugluaspalaalathrtyrtyrcysglnhisserargglu859095leuprotyrthrpheglyglyglythrlysleugluilelys100105110<210>39<211>354<212>dna<213>artificialsequence<220><223>p31-208-vh-nt<400>39caggtgcagctgaaggagtcaggacctggcctggtggcgccctcacagagcctgtccatc60acttgcactgtctctgacttttcattaaccaactatggtgtacactgggttcgccagcct120ccaggaaagggtctggagtggctgggggtgatatgggctgatggaagcacacattataat180tcggctctcatgtccagactgagcatcagcaaagacaactccaagagccaagttttctta240aaaatgagcagtctgcaaaatgatgacacagccatatactactgtgccagagaagggttc300gacaccacctggtttgattactggggccaagggactctggtcactgtctctgca354<210>40<211>118<212>prt<213>artificialsequence<220><223>p31-208-vh-aa<400>40glnvalglnleulysgluserglyproglyleuvalalaprosergln151015serleuserilethrcysthrvalserasppheserleuthrasntyr202530glyvalhistrpvalargglnproproglylysglyleuglutrpleu354045glyvaliletrpalaaspglyserthrhistyrasnseralaleumet505560serargleuserileserlysaspasnserlysserglnvalpheleu65707580lysmetserserleuglnasnaspaspthralailetyrtyrcysala859095arggluglypheaspthrthrtrppheasptyrtrpglyglnglythr100105110leuvalthrvalserala115<210>41<211>333<212>dna<213>artificialsequence<220><223>p31-209-vl-nt<400>41gacattgtgctcactcagtctcctgcttccttagctgtatctctggggcagagggccacc60atctcatgcagggccaacaaaagtgtcactacatctggctatagttatatgcactggtac120caacagaaaccaggacagccacccaaactcctcatctatcttgcatccaacctagaatct180ggggtccctgccaggttcagtggcagtgggtctgggacagacttcaccctcaacatccat240cctgtggaggaggaggatgctgcaacctattactgtcagcacagtagggagcttccgtac300acgttcggaggggggaccaagctggaaataaaa333<210>42<211>111<212>prt<213>artificialsequence<220><223>p31-209-vl-aa<400>42aspilevalleuthrglnserproalaserleualavalserleugly151015glnargalathrilesercysargalaasnlysservalthrthrser202530glytyrsertyrmethistrptyrglnglnlysproglyglnpropro354045lysleuleuiletyrleualaserasnleugluserglyvalproala505560argpheserglyserglyserglythraspphethrleuasnilehis65707580provalgluglugluaspalaalathrtyrtyrcysglnhisserarg859095gluleuprotyrthrpheglyglyglythrlysleugluilelys100105110<210>43<211>354<212>dna<213>artificialsequence<220><223>p31-209-vh-nt<400>43caggtgcagctgaagcagtcaggacctggcctggtggcgccctcacagagcctgtccatc60acttgcactgtctctgacttttcattaaccaactatggtgtacactgggttcgccagcct120ccaggaaagggtctggagtggctgggggtgatatgggctgatggaagcacacattataat180tcggctctcatgtccagactgagcatcagcaaagacaactccaagagccaagttttctta240aaaatgagcagtctgcaaaatgatgacacagccatatactactgtggcagagaagggttc300gacaccacctggtttgattactggggccaagggactctggtcactgtctctgca354<210>44<211>118<212>prt<213>artificialsequence<220><223>p31-209-vh-aa<400>44glnvalglnleulysglnserglyproglyleuvalalaprosergln151015serleuserilethrcysthrvalserasppheserleuthrasntyr202530glyvalhistrpvalargglnproproglylysglyleuglutrpleu354045glyvaliletrpalaaspglyserthrhistyrasnseralaleumet505560serargleuserileserlysaspasnserlysserglnvalpheleu65707580lysmetserserleuglnasnaspaspthralailetyrtyrcysgly859095arggluglypheaspthrthrtrppheasptyrtrpglyglnglythr100105110leuvalthrvalserala115<210>45<211>330<212>dna<213>artificialsequence<220><223>p31-227-vl-nt<400>45gacattgtgatgacacaacctgcttccttagctgtatctctggggcagagggccaccatc60tcatgcagggccagcaaaagtgtcagtacatctggctatagttatatgcactggtaccaa120cagaaaccaggacagccacccaaactcctcatctatcttgcatccaacctagaatctggg180gtccctgccaggttcagtggcagtgggtctgggacagacttcaccctcaacatccatcct240gtggaggaggaggatgctgcaacctattactgtcagcacagtagggagcttccgtacacg300ttcggaggggggaccaagctggaaataaaa330<210>46<211>110<212>prt<213>artificialsequence<220><223>p31-227-vl-aa<400>46aspilevalmetthrglnproalaserleualavalserleuglygln151015argalathrilesercysargalaserlysservalserthrsergly202530tyrsertyrmethistrptyrglnglnlysproglyglnproprolys354045leuleuiletyrleualaserasnleugluserglyvalproalaarg505560pheserglyserglyserglythraspphethrleuasnilehispro65707580valgluglugluaspalaalathrtyrtyrcysglnhisserargglu859095leuprotyrthrpheglyglyglythrlysleugluilelys100105110<210>47<211>354<212>dna<213>artificialsequence<220><223>p31-227-vh-nt<400>47caggtgcagctgaaggagtcaggacctggcctggtggcgccctcacagagcctgtccatc60acttgcactgtctctggcttttcattacccaactatggtgtacactgggttcgccagcct120ccaggaaagggtctggagtggctgggagtaatatgggctgatggaagcacaaattataat180tcggctctcatgtccagactgagcatcagcaaagacaactccaagaaccaagttttctta240aaaatgaacagtctgcaaaatgatgacacagccatgtactactgtgccagagaaggtttc300gacaccacctggtttgattactggggccaagggactctggtcactgtctctgca354<210>48<211>118<212>prt<213>artificialsequence<220><223>p31-227-vh-aa<400>48glnvalglnleulysgluserglyproglyleuvalalaprosergln151015serleuserilethrcysthrvalserglypheserleuproasntyr202530glyvalhistrpvalargglnproproglylysglyleuglutrpleu354045glyvaliletrpalaaspglyserthrasntyrasnseralaleumet505560serargleuserileserlysaspasnserlysasnglnvalpheleu65707580lysmetasnserleuglnasnaspaspthralamettyrtyrcysala859095arggluglypheaspthrthrtrppheasptyrtrpglyglnglythr100105110leuvalthrvalserala115<210>49<211>324<212>dna<213>artificialsequence<220><223>p31-236-vl-nt<400>49gacattgttctcaaccagtctccagcaatcatgtctgcatctccaggggagaaggtcacc60atgacctgcagtgccagctcaagtgtaagttccagttacttgcactggtaccagcagaag120ccaggatcctcccccaaactctggatttatagcacatccaacctggcttctggagtccca180gctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagcatggag240gctgaagatgctgccacttattactgccaccagtttcatcgttccccacggacgttcggt300ggaggcaccaagctggaaatcaaa324<210>50<211>108<212>prt<213>artificialsequence<220><223>p31-236-vl-aa<400>50aspilevalleuasnglnserproalailemetseralaserprogly151015glulysvalthrmetthrcysseralaserserservalserserser202530tyrleuhistrptyrglnglnlysproglyserserprolysleutrp354045iletyrserthrserasnleualaserglyvalproalaargpheser505560glyserglyserglythrsertyrserleuthrilesersermetglu65707580alagluaspalaalathrtyrtyrcyshisglnphehisargserpro859095argthrpheglyglyglythrlysleugluilelys100105<210>51<211>339<212>dna<213>artificialsequence<220><223>p31-236-vh-nt<400>51gaggtgcagcttcaggagtcgggacctggcctggtgaaaccttctcagtctctgtccctc60acctgcactgtcactggctactcaatcaccagtgattatgtctggaactggatccggcag120tttccaggaaacaaactggagtggatgggctacataagctacagtggtgtcactagttac180aacccatctctcaaaagtcgaatctctatcactcgagacacatccaagaaccagttcttc240ctgcagttgaattctgtgactactgaggacacagccacatattactgtgcaagaggaacg30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<211>348<212>dna<213>artificialsequence<220><223>p31-257-vh-nt<400>63caggtccaactgcagcaacctggggctgaactggtgaggcctggggcttcagtgaagctg60tcctgcaaggcttctggctacacgttcaccaactactggatgcactgggtgaagcagagg120cctggacaaggtcttgagtggattggcatgattgatccttccgatagtgaaactaggtta180aatcagaagttcaaggacaaggccacattgactgtcgacaaatcctccagcacagcctac240atgcaactccgcagcccgacatctgaggactctgcggtctattactgtgcaagaagggac300tatagccttgactactggggccaaggcaccactctcacagtctcctca348<210>64<211>116<212>prt<213>artificialsequence<220><223>p311-257-vh-aa<400>64glnvalglnleuglnglnproglyalagluleuvalargproglyala151015servallysleusercyslysalaserglytyrthrphethrasntyr202530trpmethistrpvallysglnargproglyglnglyleuglutrpile354045glymetileaspproseraspsergluthrargleuasnglnlysphe505560lysasplysalathrleuthrvalasplysserserserthralatyr65707580metglnleuargserprothrsergluaspseralavaltyrtyrcys859095alaargargasptyrserleuasptyrtrpglyglnglythrthrleu100105110thrvalserser115<210>65<211>333<212>dna<213>artificialsequence<220><223>p31-273-vl-nt<400>65gacattgtgctcactcagtctcctgcttccttagctgtatctctggggcagagggccacc60atctcatgcagggccagcaaaagtgtcagtacatctggctatagttatatgcactggtac120caacagaaaccaggacagccacccaaactcctcatctatcttgcatccaacctagaatct180ggggtccctgccaggttcagtggcagtgggtctgggacagacttcaccctcaacatccat240cctgtggaggaggaggatgctgcaacctattactgtcagcacagtagggagcttccgtac300acgttcggaggggggaccaagctggaaataaaa333<210>66<211>111<212>prt<213>artificialsequence<220><223>p31-273-vl-aa<400>66aspilevalleuthrglnserproalaserleualavalserleugly151015glnargalathrilesercysargalaserlysservalserthrser202530glytyrsertyrmethistrptyrglnglnlysproglyglnpropro354045lysleuleuiletyrleualaserasnleugluserglyvalproala505560argpheserglyserglyserglythraspphethrleuasnilehis65707580provalgluglugluaspalaalathrtyrtyrcysglnhisserarg859095gluleuprotyrthrpheglyglyglythrlysleugluilelys100105110<210>67<211>354<212>dna<213>artificialsequence<220><223>p31-273-vh-nt<400>67caggtgcagctgaaggagtcaggacctggcctggtggcgccctcacagagcctgtccatc60acttgcactgtctctggcttttcattacccagctatggtgtacactgggttcgccagcct120ccaggaaagggtctggagtggctgggagtaatatgggctgatggaagcagaaattataat180tcggctctcatgtccagactgagcatcagcaaagacaactccaagaaccaagttttctta240aaaatgaacagtctgcaaaatgatgatacagccatgtattactgtgtcagagaagggttc300gacaccacctggtttgattactggggccaagggactctggtcactgtctctgca354<210>68<211>118<212>prt<213>artificialsequence<220><223>p31-273-vh-aa<400>68glnvalglnleulysgluserglyproglyleuvalalaprosergln151015serleuserilethrcysthrvalserglypheserleuprosertyr202530glyvalhistrpvalargglnproproglylysglyleuglutrpleu354045glyvaliletrpalaaspglyserargasntyrasnseralaleumet505560serargleuserileserlysaspasnserlysasnglnvalpheleu65707580lysmetasnserleuglnasnaspaspthralamettyrtyrcysval859095arggluglypheaspthrthrtrppheasptyrtrpglyglnglythr100105110leuvalthrvalserala115<210>69<211>324<212>dna<213>artificialsequence<220><223>p31-290-vl-nt<400>69gacattgttctctcccagtctccagcaatcatgtctgcatctctaggggaacgggtcacc60atgacctgcactgccagctcaagtgtaagttccagttacttgcactggtaccagcagaag120ccaggatcctcccccaaactctggatttatagcacatccaacctggcttctggagtccca180cctcgcttcagtggcagtgggtctgggacctcttactctctcacaatcagcagcatggag240gctgaagatgctgccacttattactgccaccagtttcatcgttccccacggacgttcggt300ggaggcaccaagctggaaatcaaa324<210>70<211>108<212>prt<213>artificialsequence<220><223>p31-290-vl-aa<400>70aspilevalleuserglnserproalailemetseralaserleugly151015gluargvalthrmetthrcysthralaserserservalserserser202530tyrleuhistrptyrglnglnlysproglyserserprolysleutrp354045iletyrserthrserasnleualaserglyvalproproargpheser505560glyserglyserglythrsertyrserleuthrilesersermetglu65707580alagluaspalaalathrtyrtyrcyshisglnphehisargserpro859095argthrpheglyglyglythrlysleugluilelys100105<210>71<211>339<212>dna<213>artificialsequence<220><223>p31-290-vh-nt<400>71gaggtgcagcttcaggagtcgggacctggcctggtgaaaccttctcagtctctgtccctc60acctgcactgtcactggctactcaatcaccagtgattatgtctggaactggatccggcag120tttccaggaaacaaactggagtggatgggctacataagctacagtggtagcactagttac180aacccatctctcaaaagtcgaatctctatcactcgagacacatccaagaaccagttcttc240ctgcagttgaattctgtgactactgaggacacagccacatattactgtgcaagaggaacg300gactattggggtcaaggaacctcagtcaccgtctcctca339<210>72<211>113<212>prt<213>artificialsequence<220><223>p31-290-vh-aa<400>72gluvalglnleuglngluserglyproglyleuvallysprosergln151015serleuserleuthrcysthrvalthrglytyrserilethrserasp202530tyrvaltrpasntrpileargglnpheproglyasnlysleuglutrp354045metglytyrilesertyrserglyserthrsertyrasnproserleu505560lysserargileserilethrargaspthrserlysasnglnphephe65707580leuglnleuasnservalthrthrgluaspthralathrtyrtyrcys859095alaargglythrasptyrtrpglyglnglythrservalthrvalser100105110ser<210>73<211>63<212>dna<213>artificialsequence<220><223>前导链nt<400>73atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccagg60ccg63<210>74<211>21<212>prt<213>artificialsequence<220><223>前导链aa<400>74metalaleuprovalthralaleuleuleuproleualaleuleuleu151015hisalaalaargpro20<210>75<211>45<212>dna<213>artificialsequence<220><223>linkernt<400>75ggtggcggtggctcgggcggtggtgggtcgggtggcggcggatct45<210>76<211>15<212>prt<213>artificialsequence<220><223>linkeraa<400>76glyglyglyglyserglyglyglyglyserglyglyglyglyser151015<210>77<211>135<212>dna<213>artificialsequence<220><223>cd8铰链区nt<400>77accacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctg60tccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctg120gacttcgcctgtgat135<210>78<211>45<212>prt<213>artificialsequence<220><223>cd8铰链区aa<400>78thrthrthrproalaproargproprothrproalaprothrileala151015serglnproleuserleuargproglualacysargproalaalagly202530glyalavalhisthrargglyleuaspphealacysasp354045<210>79<211>72<212>dna<213>artificialsequence<220><223>cd8跨膜区nt<400>79atctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatc60accctttactgc72<210>80<211>24<212>prt<213>artificialsequence<220><223>cd8跨膜区aa<400>80iletyriletrpalaproleualaglythrcysglyvalleuleuleu151015serleuvalilethrleutyrcys20<210>81<211>126<212>dna<213>artificialsequence<220><223>4-1bb共刺激区域nt<400>81aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaa60actactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgt120gaactg126<210>82<211>42<212>prt<213>artificialsequence<220><223>4-1bb共刺激区域aa<400>82lysargglyarglyslysleuleutyrilephelysglnprophemet151015argprovalglnthrthrglnglugluaspglycyssercysargphe202530proglugluglugluglyglycysgluleu3540<210>83<211>336<212>dna<213>artificialsequence<220><223>cd3ζ区nt<400>83agagtgaagttcagcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctc60tataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggc120cgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaat180gaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgc240cggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacc300tacgacgcccttcacatgcaggccctgccccctcgc336<210>84<211>112<212>prt<213>artificialsequence<220><223>cd3ζ区aa<400>84argvallyspheserargseralaaspalaproalatyrglnglngly151015glnasnglnleutyrasngluleuasnleuglyargarggluglutyr202530aspvalleuasplysargargglyargaspproglumetglyglylys354045proargarglysasnproglngluglyleutyrasngluleuglnlys505560asplysmetalaglualatyrsergluileglymetlysglygluarg65707580argargglylysglyhisaspglyleutyrglnglyleuserthrala859095thrlysaspthrtyraspalaleuhismetglnalaleuproproarg100105110<210>85<211>1440<212>dna<213>artificialsequence<220><223>010-car-nt<400>85atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccagg60ccggaggtgcagcttgttgagtctggacctagcctcgtgaaaccttctcagtctctgtcc120ctcacctgcactgtcactggctactcaatcaccagtgattatgtctggaactggatccgg180caatttccaggaaacaaactggagtggatgggctacataagctacagtggtagcactagt240tacaatccatctctcacaagtcgaatctctatcactcgagacacatccaagaaccagttc300ttcctgcagttgaattctgtgactactgaggacacagccacatattactgtacaagagga360acggactactggggtcaaggaacctcagtcaccgtctcctcaggtggcggtggctcgggc420ggtggtgggtcgggtggcggcggatctgacattgttctcaaccagtctccagcaatcatg480tctgcatctctaggggaacgggtcaccatgacctgcactgccagctcaagtgtaagttcc540agttacttgcactggtaccagcagaagccaggatcctcccccaaactctggatttatagc600acatccaacctggcttctggagtcccggctcgcttcagtggcagtgggtctgggacctct660tactctctcacaatcagcaacatggaggctgaagatgctgccacttattactgccaccag720tttcatcgttccccacccacgttcggtgctgggaccaagctggagctgaaaaccacgacg780ccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgc840ccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcc900tgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactg960gttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaacca1020tttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaa1080gaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcg1140taccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtac1200gatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaag1260aaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagt1320gagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggt1380ctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc1440<210>86<211>480<212>prt<213>artificialsequence<220><223>010-car-aa<400>86metalaleuprovalthralaleuleuleuproleualaleuleuleu151015hisalaalaargprogluvalglnleuvalgluserglyproserleu202530vallysproserglnserleuserleuthrcysthrvalthrglytyr354045serilethrserasptyrvaltrpasntrpileargglnpheprogly505560asnlysleuglutrpmetglytyrilesertyrserglyserthrser65707580tyrasnproserleuthrserargileserilethrargaspthrser859095lysasnglnphepheleuglnleuasnservalthrthrgluaspthr100105110alathrtyrtyrcysthrargglythrasptyrtrpglyglnglythr115120125servalthrvalserserglyglyglyglyserglyglyglyglyser130135140glyglyglyglyseraspilevalleuasnglnserproalailemet145150155160seralaserleuglygluargvalthrmetthrcysthralaserser165170175servalsersersertyrleuhistrptyrglnglnlysproglyser180185190serprolysleutrpiletyrserthrserasnleualaserglyval195200205proalaargpheserglyserglyserglythrsertyrserleuthr210215220ileserasnmetglualagluaspalaalathrtyrtyrcyshisgln225230235240phehisargserproprothrpheglyalaglythrlysleugluleu245250255lysthrthrthrproalaproargproprothrproalaprothrile260265270alaserglnproleuserleuargproglualacysargproalaala275280285glyglyalavalhisthrargglyleuaspphealacysaspiletyr290295300iletrpalaproleualaglythrcysglyvalleuleuleuserleu305310315320valilethrleutyrcyslysargglyarglyslysleuleutyrile325330335phelysglnprophemetargprovalglnthrthrglnglugluasp340345350glycyssercysargpheproglugluglugluglyglycysgluleu355360365argvallysphes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gatggctgtagctgccgatttccagaa1080gaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcg1140taccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtac1200gatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaag1260aaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagt1320gagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggt1380ctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc1440<210>90<211>480<212>prt<213>artificialsequence<220><223>p31-027-aa<400>90metalaleuprovalthralaleuleuleuproleualaleuleuleu151015hisalaalaargproglnvalglnleulysgluserglyproglyleu202530vallysproserglnserleuserleuthrcysthrvalthrglyphe354045serilethrserasptyralatrpasntrpileargglnpheprogly505560asnlysleuglutrpmetglytyrileserpheglyglythrthrser65707580tyrasnproserleulysserargileserilethrargaspthrser859095lysasnglnphepheleuglnleuasnservalthrsergluaspthr100105110alathrtyrtyrcysthrargglyleuasptyrtrpglyglnglythr115120125ileleuthrvalserserglyglyglyglyserglyglyglyglyser130135140glyglyglyglyseraspilevalleuthrglnserproalailemet145150155160seralaserleuglygluargvalthrmetthrcysthralaserser165170175servalsersersertyrleutyrtrptyrglnglnlysproglyser180185190serprolysleutrpiletyrserthrserasnleualaserglyval195200205proalaargpheserglyserglyserglythrsertyrserleuthr210215220ilesersermetglualagluaspalaalathrtyrtyrcyshisgln225230235240tyrhisargserprotyrthrpheglyglyglythrlysleugluile245250255lysthrthrthrproalaproargproprothrproalaprothrile260265270alaserglnproleuserleuargproglualacysargproalaala275280285glyglyalavalhisthrargglyleuaspphealacysaspiletyr290295300iletrpalaproleualaglythrcysglyvalleuleuleuserleu305310315320valilethrleutyrcyslysargglyarglyslysleuleutyrile325330335phelysglnprophemetargprovalglnthrthrglnglugluasp340345350glycyssercysargpheproglugluglugluglyglycysgluleu355360365argvallyspheserargseralaaspalaproalatyrglnglngly370375380glnasnglnleutyrasngluleuasnleuglyargarggluglutyr385390395400aspvalleuasplysargargglyargaspproglumetglyglylys405410415proargarglysasnproglngluglyleutyrasngluleuglnlys420425430asplysmetalaglualatyrsergluileglymetlysglygluarg435440445argargglylysglyhisaspglyleutyrglnglyleuserthrala450455460thrlysaspthrtyraspalaleuhismetglnalaleuproproarg465470475480<210>91<211>1464<212>dna<213>artificialsequence<220><223>p31-062-nt<400>91atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccagg60ccggaggtgcagcttcaggagtcgggacctggcctggtggcgccctcacagagcctgtcc120atcacttgcactgtctctgacttttcattaaccaactttggtgtacactgggttcgccag180cctccaggaaagggtctggagtggctgggggtgatatgggctgatggaagcacacattat240aattcgtctctcatgtccagactgagcatcagcaaagacaactccaagagccaagttttc300ttaaaaatgagcagtctgcaaaatgatgacacagccatatactattgtgccagagagggg360ttcgacaccacctggtttgattactggggccaagggactctggtcactgtctctgcaggt420ggcggtggctcgggcggtggtgggtcgggtggcggcggatctgacattgtgatgacacag480tctcctgcttccttagctgtatctctggggcagagggccaccatctcatgcagggccagc540aaaagtgtcagtacatctggctatagttatatgcactggtaccaacagaaaccaggacag600ccacccaaactcctcatctatcttgcatccaacctagaatctggggtccctgccaggttc660agtggcagtgggtctgggacagacttcaccctcaacatccatcctgtggaggaggaggat720gctgcaacctattactgtcagcacagtagggagcttccgtacacgttcggaggggggacc780aagctggaaataaaaaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatc840gcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtg900cacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggact960tgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaa1020ctcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagat1080ggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttc1140agcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctc1200aatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgag1260atggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaa1320gataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaag1380gggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgccctt1440cacatgcaggccctgccccctcgc1464<210>92<211>488<212>prt<213>artificialsequence<220><223>p31-062-aa<400>92metalaleuprovalthralaleuleuleuproleualaleuleuleu151015hisalaalaargprogluvalglnleuglngluserglyproglyleu202530valalaproserglnserleuserilethrcysthrvalseraspphe354045serleuthrasnpheglyvalhistrpvalargglnproproglylys505560glyleuglutrpleuglyvaliletrpalaaspglyserthrhistyr65707580asnserserleumetserargleuserileserlysaspasnserlys859095serglnvalpheleulysmetserserleuglnasnaspaspthrala100105110iletyrtyrcysalaarggluglypheaspthrthrtrppheasptyr115120125trpglyglnglythrleuvalthrvalseralaglyglyglyglyser130135140glyglyglyglyserglyglyglyglyseraspilevalmetthrgln145150155160serproalaserleualavalserleuglyglnargalathrileser165170175cysargalaserlysservalserthrserglytyrsertyrmethis180185190trptyrglnglnlysproglyglnproprolysleuleuiletyrleu195200205alaserasnleugluserglyvalproalaargpheserglysergly210215220serglythraspphethrleuasnilehisprovalgluglugluasp225230235240alaalathrtyrtyrcysglnhisserarggluleuprotyrthrphe245250255glyglyglythrlysleugluilelysthrthrthrproalaproarg260265270proprothrproalaprothrilealaserglnproleuserleuarg275280285proglualacysargproalaalaglyglyalavalhisthrarggly290295300leuaspphealacysaspiletyriletrpalaproleualaglythr305310315320cysglyvalleuleuleuserleuvalilethrleutyrcyslysarg325330335glyarglyslysleuleutyrilephelysglnprophemetargpro340345350valglnthrthrglnglugluaspglycyssercysargpheproglu355360365gluglugluglyglycysgluleuargvallyspheserargserala370375380aspalaproalatyrglnglnglyglnasnglnleutyrasngluleu385390395400asnleuglyargarggluglutyraspvalleuasplysargarggly405410415argaspproglumetglyglylysproargarglysasnproglnglu420425430glyleutyrasngluleuglnlysasplysmetalaglualatyrser435440445gluileglymetlysglygluargargargglylysglyhisaspgly450455460leutyrglnglyleuserthralathrlysaspthrtyraspalaleu465470475480hismetglnalaleuproproarg485<210>93<211>1461<212>dna<213>artificialsequence<220><223>064-car-nt<400>93atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccagg60ccgcaggtgcagctgaaggagtcaggacctggcctggtggcgccctcacagagcctgtcc120attatttgcactgtctctgggttttccttaaccaactatggtgtacactgggttcgccag180cctccaggaaagggtctggagtggctgggagtaatatgggctgatggaaccacaaattat240aattcggctctcatgtccagactgagcatcagcaaagacaactccaagagccaagttttc300ttaaaaatgaacagtctgcaaactgatgacacagccatgtactactgtgccagagaaggg360ttcgacaccacctggtttgattattggggccaagggactctggtcactgtctctgcaggt420ggcggtggctcgggcggtggtgggtcgggtggcggcggatctgacattgtgatgacacaa480cctgcttccttagctgtatctctggggcagagggccaccatctcatgcagggccagcaaa540agtgtcagtacatctggctatagttatatgcactggtaccaacagaaaccaggacagcca600cccaaactcctcatctatcttgcatccaacctagaatctggggtccctgccaggttcagt660ggcagtgggtctgggacagacttcaccctcaacatccatcctgtggaggaggaggatgct720gcaacctattactgtcagcacagtagggagcttccgtacacgttcggaggggggaccaag780ctggaaataaaaaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcg840tcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcac900acgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgt960ggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactc1020ctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggc1080tgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagc1140aggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaat1200ctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatg1260gggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagat1320aagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaagggg1380cacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcac1440atgcaggccctgccccctcgc1461<210>94<211>487<212>prt<213>artificialsequence<220><223>064-car-aa<400>94metalaleuprovalthralaleuleuleuproleualaleuleuleu151015hisalaalaargproglnvalglnleulysgluserglyproglyleu202530valalaproserglnserleuserileilecysthrvalserglyphe354045serleuthrasntyrglyvalhistrpvalargglnproproglylys505560glyleuglutrpleuglyvaliletrpalaaspglythrthrasntyr65707580asnseralaleumetserargleuserileserlysaspasnserlys859095serglnvalpheleulysmetasnserleuglnthraspaspthrala100105110mettyrtyrcysalaarggluglypheaspthrthrtrppheasptyr115120125trpglyglnglythrleuvalthrvalseralaglyglyglyglyser130135140glyglyglyglyserglyglyglyglyseraspilevalmetthrgln145150155160proalaserleualavalserleuglyglnargalathrilesercys165170175argalaserlysservalserthrserglytyrsertyrmethistrp180185190tyrglnglnlysproglyglnproprolysleuleuiletyrleuala195200205serasnleugluserglyvalproalaargpheserglyserglyser210215220glythraspphethrleuasnilehisprovalgluglugluaspala225230235240alathrtyrtyrcysglnhisserarggluleuprotyrthrphegly245250255glyglythrlysleugluilelysthrthrthrproalaproargpro260265270prothrproalaprothrilealaserglnproleuserleuargpro275280285glualacysargproalaalaglyglyalavalhisthrargglyleu290295300aspphealacysaspiletyriletrpalaproleualaglythrcys305310315320glyvalleuleuleuserleuvalilethrleutyrcyslysarggly325330335arglyslysleuleutyrilephelysglnprophemetargproval340345350glnthrthrglnglugluaspglycyssercysargpheprogluglu355360365glugluglyglycysgluleuargvallyspheserargseralaasp370375380alaproalatyrglnglnglyglnasnglnleutyrasngluleuasn385390395400leuglyargarggluglutyraspvalleuasplysargargglyarg405410415aspproglumetglyglylysproargarglysasnproglnglugly420425430leutyrasngluleuglnlysasplysmetalaglualatyrserglu435440445ileglymetlysglygluargargargglylysglyhisaspglyleu450455460tyrglnglyleuserthralathrlysaspthrtyraspalaleuhis465470475480metglnalaleuproproarg485<210>95<211>1440<212>dna<213>artificialsequence<220><223>070-car-nt<400>95atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccagg60ccggaggtgcagcttcaggagtccggaccaagcctcgtgaaaccttctcagactctgtcc120ctcacctgttctgtcactggcgactccatcaccagtgattatgtctggaactggatccgg180caatttccaggaaacaaactggagtggatgggctacataagctacagtggtagcactagt240tacaatccatctctcacaagtcgaatctctatcactcgagacacatccaagaaccagttc300ttcctgcagttgaattctgtgactactgaggacacagccacatattactgtacaagagga360acggactactggggtcaaggaacctcagtcaccgtctcctcaggtggcggtggctcgggc420ggtggtgggtcgggtggcggcggatctgacattgttctctcccagtctccagcaatcatg480tctgcatctctaggggaacgggtctccatgacctgcactgccagctcaagtgtaagttcc540agttacttgcactggtaccagcagaagccaggatcctcccccaaactctggatttatagc600acatccaacctggcttctggagtcccaactcgcttcagtggcagtgggtctgggacctct660tactctctcacaatcagcagcatggaggctgaagatgctgccacttattactgccaccag720tttcatcgttccccacggacgttcggtggaggcaccaagctggaaatcaaaaccacgacg780ccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgc840ccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcc900tgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactg960gttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaacca1020tttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaa1080gaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcg1140taccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtac1200gatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaag1260aaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagt1320gagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggt1380ctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc1440<210>96<211>480<212>prt<213>artificialsequence<220><223>070-car-aa<400>96metalaleuprovalthralaleuleuleuproleualaleuleuleu151015hisalaalaargprogluvalglnleuglngluserglyproserleu202530vallysproserglnthrleuserleuthrcysservalthrglyasp354045serilethrserasptyrvaltrpasntrpileargglnpheprogly505560asnlysleuglutrpmetglytyrilesertyrserglyserthrser65707580tyrasnproserleuthrserargileserilethrargaspthrser859095lysasnglnphepheleuglnleuasnservalthrthrgluaspthr100105110alathrtyrtyrcysthrargglythrasptyrtrpglyglnglythr115120125servalthrvalserserglyglyglyglyserglyglyglyglyser130135140glyglyglyglyseraspilevalleuserglnserproalailemet145150155160seralaserleuglygluargvalsermetthrcysthralaserser165170175servalsersersertyrleuhistrptyrglnglnlysproglyser180185190serprolysleutrpiletyrserthrserasnleualaserglyval195200205prothrargpheserglyserglyserglythrsertyrserleuthr210215220ilesersermetglualagluaspalaalathrtyrtyrcyshisgln225230235240phehisargserproargthrpheglyglyglythrlysleugluile245250255lysthrthrthrproalaproargproprothrproalaprothrile260265270alaserglnproleuserleuargproglualacysargproalaala275280285glyglyalavalhisthrargglyleuaspphealacysaspiletyr290295300iletrpalaproleualaglythrcysglyvalleuleuleuserleu305310315320valilethrleutyrcyslysargglyarglyslysleuleutyrile325330335phelysglnprophemetargprovalglnthrthrglnglugluasp340345350glycyssercysargpheproglugluglugluglyglycysgluleu355360365argvallyspheserargseralaaspalaproalatyrglnglngly370375380glnasnglnleutyrasngluleuasnleuglyargarggluglutyr385390395400aspvalleuasplysargargglyargaspproglumetglyglylys405410415proargarglysasnproglngluglyleutyrasngluleuglnlys420425430asplysmetalaglualatyrsergluileglymetlysglygluarg435440445argargglylysglyhisaspglyleutyrglnglyleuserthrala450455460thrlysaspthrtyraspalaleuhismetglnalaleuproproarg465470475480<210>97<211>1461<212>dna<213>artificialsequence<220><223>110-car-nt<400>97atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccagg60ccggaggtgcagcttcagcagtcaggacctggcctggtggcgccctcacagagcctgtcc120atcacttgcactgtctctggcttttcattaaccagctatggtatacactgggttcgccag180cctccaggaaagggtctggagtggctgggagtaatatgggctgatggaagcacaaattat240aattcggctctcatgtccagactgagcatccgcaaagacaactccaagagccaaattttc300ttaaaaatgaacagtctgcaaaatgatgacatagccatgtactactgtgccagagaaggg360tacgacaccacctggtttgattactggggccaagggactctggtcactgtctctgcaggt420ggcggtggctcgggcggtggtgggtcgggtggcggcggatctgacattgtgatgacacaa480cctgcttccttagctgtatctctggggcagagggccaccatctcatgcagggccagcaaa540agtgtcagtacatctggctatagttatatgcactggtaccaacagaaaccaggacagcca600cccaaactcctcatctatcttgcatccaacctagaatctggggtccctgccaggttcagt660ggcagtgggtctgggacagacttcaccctcaacatccatcctgtggaggaggaggatgct720gcaacctattactgtcagcacagtagggagcttccgtacacgttcggaggggggaccaag780ctggaaataaaaaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcg840tcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcac900acgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgt960ggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactc1020ctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggc1080tgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagc1140aggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaat1200ctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatg1260gggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagat1320aagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaagggg1380cacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcac1440atgcaggccctgccccctcgc1461<210>98<211>487<212>prt<213>artificialsequence<220><223>110-car-aa<400>98metalaleuprovalthralaleuleuleuproleualaleuleuleu151015hisalaalaargprogluvalglnleuglnglnserglyproglyleu202530valalaproserglnserleuserilethrcysthrvalserglyphe354045serleuthrsertyrglyilehistrpvalargglnproproglylys505560glyleuglutrpleuglyvaliletrpalaaspglyserthrasntyr65707580asnseralaleumetserargleuserilearglysaspasnserlys859095serglnilepheleulysmetasnserleuglnasnaspaspileala100105110mettyrtyrcysalaarggluglytyraspthrthrtrppheasptyr115120125trpglyglnglythrleuvalthrvalseralaglyglyglyglyser130135140glyglyglyglyserglyglyglyglyseraspilevalmetthrgln145150155160proalaserleualavalserleuglyglnargalathrilesercys165170175argalaserlysservalserthrserglytyrsertyrmethistrp180185190tyrglnglnlysproglyglnproprolysleuleuiletyrleuala195200205serasnleugluserglyvalproalaargpheserglyserglyser210215220glythraspphethrleuasnilehisprovalgluglugluaspala225230235240alathrtyrtyrcysglnhisserarggluleuprotyrthrphegly245250255glyglythrlysleugluilelysthrthrthrproalaproargpro260265270prothrproalaprothrilealaserglnproleuserleuargpro275280285glualacysargproalaalaglyglyalavalhisthrargglyleu290295300aspphealacysaspiletyriletrpalaproleualaglythrcys305310315320glyvalleuleuleuserleuvalilethrleutyrcyslysarggly325330335arglyslysleuleutyrilephelysglnprophemetargproval340345350glnthrthrglnglugluaspglycyssercysargpheprogluglu355360365glugluglyglycysgluleuargvallyspheserargseralaasp370375380alaproalatyrglnglnglyglnasnglnleutyrasngluleuasn385390395400leuglyargarggluglutyraspvalleuasplysargargglyarg405410415aspproglumetglyglylysproargarglysasnproglnglugly420425430leutyrasngluleuglnlysasplysmetalaglualatyrserglu435440445ileglymetlysglygluargargargglylysglyhisaspglyleu450455460tyrglnglyleuserthralathrlysaspthrtyraspalaleuhis465470475480metglnalaleuproproarg485<210>99<211>1440<212>dna<213>artificialsequence<220><223>127-car-nt<400>99atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccagg60ccggaagtgatgcttcaggagtcgggacctggcctggtgaaaccttctcagtctctgtcc120ctcacctgcactgtcactggcttctcaatcaccagtgattatgcctggaactggatccgg180cagtttccaggaaacaagctggagtggatgggctacataagttttggtggtactacttcc240tacaacccatctctcaaaagtcgaatctctatcactcgagacacatccaagaaccagttc300ttcctgcagttgaattctgtgacttctgaagacacagccacatattactgtacaagaggg360ttggactactggggccaaggcaccattctcacagtctcctcaggtggcggtggctcgggc420ggtggtgggtcgggtggcggcggatctgacattgttctcacccagtctccagcaatcatg480tctgcatctccaggggagaaggtcaccatgacctgcagtgccagctcaagtgtaagttcc540agttacttgcactggtaccagcagaagtcaggtgcctcccccaaactctggatttatagc600acatccaaattggcttctggagtccctgctcgcttcagtggcagtgggtctgggacctct660tactctctcacaatcagcagtgtggaagctgaagatgctgccacttatttctgccagcag720aacagtggttacccattcacgttcggctcggggacaaagttggaaataaaaaccacgacg780ccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgc840ccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcc900tgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactg960gttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaacca1020tttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaa1080gaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcg1140taccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtac1200gatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaag1260aaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagt1320gagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggt1380ctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc1440<210>100<211>480<212>prt<213>artificialsequence<220><223>127-car-aa<400>100metalaleuprovalthralaleuleuleuproleualaleuleuleu151015hisalaalaargprogluvalmetleuglngluserglyproglyleu202530vallysproserglnserleuserleuthrcysthrvalthrglyphe354045serilethrserasptyralatrpasntrpileargglnpheprogly505560asnlysleuglutrpmetglytyrileserpheglyglythrthrser65707580tyrasnproserleulysserargileserilethrargaspthrser859095lysasnglnphepheleuglnleuasnservalthrsergluaspthr100105110alathrtyrtyrcysthrargglyleuasptyrtrpglyglnglythr115120125ileleuthrvalserserglyglyglyglyserglyglyglyglyser130135140glyglyglyglyseraspilevalleuthrglnserproalailemet145150155160seralaserproglyglulysvalthrmetthrcysseralaserser165170175servalsersersertyrleuhistrptyrglnglnlysserglyala180185190serprolysleutrpiletyrserthrserlysleualaserglyval195200205proalaargpheserglyserglyserglythrsertyrserleuthr210215220ileserservalglualagluaspalaalathrtyrphecysglngln225230235240asnserglytyrprophethrpheglyserglythrlysleugluile245250255lysthrthrthrproalaproargproprothrproalaprothrile260265270alaserglnproleuserleuargproglualacysargproalaala275280285glyglyalavalhisthrargglyleuaspphealacysaspiletyr290295300iletrpalaproleualaglythrcysglyvalleuleuleuserleu305310315320valilethrleutyrcyslysargglyarglyslysleuleutyrile325330335phelysglnprophemetargprovalglnthrthrglnglugluasp340345350glycyssercysargpheproglugluglugluglyglycysgluleu355360365argvallyspheserargseralaaspalaproalatyrglnglngly370375380glnasnglnleutyrasngluleuasnleuglyargarggluglutyr385390395400aspvalleuasplysargargglyargaspproglumetglyglylys405410415proargarglysasnproglngluglyleutyrasngluleuglnlys420425430asplysmetalaglualatyrsergluileglymetlysglygluarg435440445argargglylysglyhisaspglyleutyrglnglyleuserthrala450455460thrlysaspthrtyraspalaleuhismetglnalaleuproproarg465470475480<210>101<211>1461<212>dna<213>artificialsequence<220><223>137-car-nt<400>101atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccagg60ccgcaggtgcagctgaaggagtcaggacctggcctggtggcgccctcacagagcctgtcc120atcacttgcactgtctctggcttttcattacccagctatggtgtacactgggttcgccag180cctccaggaaagggtctggagtggctgggagtaatctgggctgatggaagcacaaattat240aattcggctctcatgtccagactgagcatcagcaaagacaactccaagaaccaagttttc300ttgaaaatgaacagtctgcaaaatgatgatacagccatgtattactgtgccagagaaggg360ttcgacaccacctggtttgattactggggccaagggactctggtcactgtctctgcaggt420ggcggtggctcgggcggtggtgggtcgggtggcggcggatctgacattgtgatgacacaa480cctgcttccttagctgtatctctggggcagagggccaccatctcatgcagggccagcaaa540agtgtcagtacatctggctatagttatttacactggtaccaacagaaaccaggacagcca600cccaaactcctcatctatcttgcatccaacctagaatctggggtccctgccaggttcagt660ggcagtgggtctgggacagacttcaccctcaacatccatcctgtggaggaggaggatgct720gcaacctattactgtcagcacagtagggaccttccgtacacgttcggaggggggaccaag780ctggaaataaaaaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcg840tcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcac900acgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgt960ggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactc1020ctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggc1080tgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagc1140aggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaat1200ctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatg1260gggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagat1320aagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaagggg1380cacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcac1440atgcaggccctgccccctcgc1461<210>102<211>487<212>prt<213>artificialsequence<220><223>137-car-aa<400>102metalaleuprovalthralaleuleuleuproleualaleuleuleu151015hisalaalaargproglnvalglnleulysgluserglyproglyleu202530valalaproserglnserleuserilethrcysthrvalserglyphe354045serleuprosertyrglyvalhistrpvalargglnproproglylys505560glyleuglutrpleuglyvaliletrpalaaspglyserthrasntyr65707580asnseralaleumetserargleuserileserlysaspasnserlys859095asnglnvalpheleulysmetasnserleuglnasnaspaspthrala100105110mettyrtyrcysalaarggluglypheaspthrthrtrppheasptyr115120125trpglyglnglythrleuvalthrvalseralaglyglyglyglyser130135140glyglyglyglyserglyglyglyglyseraspilevalmetthrgln145150155160proalaserleualavalserleuglyglnargalathrilesercys165170175argalaserlysservalserthrserglytyrsertyrleuhistrp180185190tyrglnglnlysproglyglnproprolysleuleuiletyrleuala195200205serasnleugluserglyvalproalaargpheserglyserglyser210215220glythraspphethrleuasnilehisprovalgluglugluaspala225230235240alathrtyrtyrcysglnhisserargaspleuprotyrthrphegly245250255glyglythrlysleugluilelysthrthrthrproalaproargpro260265270prothrproalaprothrilealaserglnproleuserleuargpro275280285glualacysargproalaalaglyglyalavalhisthrargglyleu290295300aspphealacysaspiletyriletrpalaproleualaglythrcys305310315320glyvalleuleuleuserleuvalilethrleutyrcyslysarggly325330335arglyslysleuleutyrilephelysglnprophemetargproval340345350glnthrthrglnglugluaspglycyssercysargpheprogluglu355360365glugluglyglycysgluleuargvallyspheserargseralaasp370375380alaproalatyrglnglnglyglnasnglnleutyrasngluleuasn385390395400leuglyargarggluglutyraspvalleuasplysargargglyarg405410415aspproglumetglyglylysproargarglysasnproglnglugly420425430leutyrasngluleuglnlysasplysmetalaglualatyrserglu435440445ileglymetlysglygluargargargglylysglyhisaspglyleu450455460tyrglnglyleuserthralathrlysaspthrtyraspalaleuhis465470475480metglnalaleuproproarg485<210>103<211>1461<212>dna<213>artificialsequence<220><223>208-car-nt<400>103atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccagg60ccgcaggtgcagctgaaggagtcaggacctggcctggtggcgccctcacagagcctgtcc120atcacttgcactgtctctgacttttcattaaccaactatggtgtacactgggttcgccag180cctccaggaaagggtctggagtggctgggggtgatatgggctgatggaagcacacattat240aattcggctctcatgtccagactgagcatcagcaaagacaactccaagagccaagttttc300ttaaaaatgagcagtctgcaaaatgatgacacagccatatactactgtgccagagaaggg360ttcgacaccacctggtttgattactggggccaagggactctggtcactgtctctgcaggt420ggcggtggctcgggcggtggtgggtcgggtggcggcggatctgacattgtgatgacacaa480cctgcttccttagctgtatctctggggcagagggccaccatctcatgcagggccagcgaa540agtgtcagtacatctggctatagttatatgcactggtaccaacagaaaccaggacagcca600cccaaactcctcatctatcttgcatccaacctagaatctggggtccctgccaggttcagt660ggcagtgggtctgggacagacttcaccctcaacatccatcctgtggaggaggaggatgct720gcaacctattactgtcagcacagtagggaacttccgtacacgttcggaggggggaccaag780ctggaaataaaaaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcg840tcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcac900acgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgt960ggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactc1020ctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggc1080tgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagc1140aggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaat1200ctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatg1260gggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagat1320aagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaagggg1380cacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcac1440atgcaggccctgccccctcgc1461<210>104<211>487<212>prt<213>artificialsequence<220><223>208-car-aa<400>104metalaleuprovalthralaleuleuleuproleualaleuleuleu151015hisalaalaargproglnvalglnleulysgluserglyproglyleu202530valalaproserglnserleuserilethrcysthrvalseraspphe354045serleuthrasntyrglyvalhistrpvalargglnproproglylys505560glyleuglutrpleuglyvaliletrpalaaspglyserthrhistyr65707580asnseralaleumetserargleuserileserlysaspasnserlys859095serglnvalpheleulysmetserserleuglnasnaspaspthrala100105110iletyrtyrcysalaarggluglypheaspthrthrtrppheasptyr115120125trpglyglnglythrleuvalthrvalseralaglyglyglyglyser130135140glyglyglyglyserglyglyglyglyseraspilevalmetthrgln145150155160proalaserleualavalserleuglyglnargalathrilesercys165170175argalasergluservalserthrserglytyrsertyrmethistrp180185190tyrglnglnlysproglyglnproprolysleuleuiletyrleuala195200205serasnleugluserglyvalproalaargpheserglyserglyser210215220glythraspphethrleuasnilehisprovalgluglugluaspala225230235240alathrtyrtyrcysglnhisserarggluleuprotyrthrphegly245250255glyglythrlysleugluilelysthrthrthrproalaproargpro260265270prothrproalaprothrilealaserglnproleuserleuargpro275280285glualacysargproalaalaglyglyalavalhisthrargglyleu290295300aspphealacysaspiletyriletrpalaproleualaglythrcys305310315320glyvalleuleuleuserleuvalilethrleutyrcyslysarggly325330335arglyslysleuleutyrilephelysglnprophemetargproval340345350glnthrthrglnglugluaspglycyssercysargpheprogluglu355360365glugluglyglycysgluleuargvallyspheserargseralaasp370375380alaproalatyrglnglnglyglnasnglnleutyrasngluleuasn385390395400leuglyargarggluglutyraspvalleuasplysargargglyarg405410415aspproglumetglyglylysproargarglysasnproglnglugly420425430leutyrasngluleuglnlysasplysmetalaglualatyrserglu435440445ileglymetlysglygluargargargglylysglyhisaspglyleu450455460tyrglnglyleuserthralathrlysaspthrtyraspalaleuhis465470475480metglnalaleuproproarg485<210>105<211>1464<212>dna<213>artificialsequence<220><223>209-car-nt<400>105atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccagg60ccgcaggtgcagctgaagcagtcaggacctggcctggtggcgccctcacagagcctgtcc120atcacttgcactgtctctgacttttcattaaccaactatggtgtacactgggttcgccag180cctccaggaaagggtctggagtggctgggggtgatatgggctgatggaagcacacattat240aattcggctctcatgtccagactgagcatcagcaaagacaactccaagagccaagttttc300ttaaaaatgagcagtctgcaaaatgatgacacagccatatactactgtggcagagaaggg360ttcgacaccacctggtttgattactggggccaagggactctggtcactgtctctgcaggt420ggcggtggctcgggcggtggtgggtcgggtggcggcggatctgacattgtgctcactcag480tctcctgcttccttagctgtatctctggggcagagggccaccatctcatgcagggccaac540aaaagtgtcactacatctggctatagttatatgcactggtaccaacagaaaccaggacag600ccacccaaactcctcatctatcttgcatccaacctagaatctggggtccctgccaggttc660agtggcagtgggtctgggacagacttcaccctcaacatccatcctgtggaggaggaggat720gctgcaacctattactgtcagcacagtagggagcttccgtacacgttcggaggggggacc780aagctggaaataaaaaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatc840gcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtg900cacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggact960tgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaa1020ctcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagat1080ggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttc1140agcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctc1200aatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgag1260atggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaa1320gataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaag1380gggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgccctt1440cacatgcaggccctgccccctcgc1464<210>106<211>488<212>prt<213>artificialsequence<220><223>209-car-aa<400>106metalaleuprovalthralaleuleuleuproleualaleuleuleu151015hisalaalaargproglnvalglnleulysglnserglyproglyleu202530valalaproserglnserleuserilethrcysthrvalseraspphe354045serleuthrasntyrglyvalhistrpvalargglnproproglylys505560glyleuglutrpleuglyvaliletrpalaaspglyserthrhistyr65707580asnseralaleumetserargleuserileserlysaspasnserlys859095serglnvalpheleulysmetserserleuglnasnaspaspthrala100105110iletyrtyrcysglyarggluglypheaspthrthrtrppheasptyr115120125trpglyglnglythrleuvalthrvalseralaglyglyglyglyser130135140glyglyglyglyserglyglyglyglyseraspilevalleuthrgln145150155160serproalaserleualavalserleuglyglnargalathrileser165170175cysargalaasnlysservalthrthrserglytyrsertyrmethis180185190trptyrglnglnlysproglyglnproprolysleuleuiletyrleu195200205alaserasnleugluserglyvalproalaargpheserglysergly210215220serglythraspphethrleuasnilehisprovalgluglugluasp225230235240alaalathrtyrtyrcysglnhisserarggluleuprotyrthrphe245250255glyglyglythrlysleugluilelysthrthrthrproalaproarg260265270proprothrproalaprothrilealaserglnproleuserleuarg275280285proglualacysargproalaalaglyglyalavalhisthrarggly290295300leuaspphealacysaspiletyriletrpalaproleualaglythr305310315320cysglyvalleuleuleuserleuvalilethrleutyrcyslysarg325330335glyarglyslysleuleutyrilephelysglnprophemetargpro340345350valglnthrthrglnglugluaspglycyssercysargpheproglu355360365gluglugluglyglycysgluleuargvallyspheserargserala370375380aspalaproalatyrglnglnglyglnasnglnleutyrasngluleu385390395400asnleuglyargarggluglutyraspvalleuasplysargarggly405410415argaspproglumetglyglylysproargarglysasnproglnglu420425430glyleutyrasngluleuglnlysasplysmetalaglualatyrser435440445gluileglymetlysglygluargargargglylysglyhisaspgly450455460leutyrglnglyleuserthralathrlysaspthrtyraspalaleu465470475480hismetglnalaleuproproarg485<210>107<211>1461<212>dna<213>artificialsequence<220><223>227-car-nt<400>107atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccagg60ccgcaggtgcagctgaaggagtcaggacctggcctggtggcgccctcacagagcctgtcc120atcacttgcactgtctctggcttttcattacccaactatggtgtacactgggttcgccag180cctccaggaaagggtctggagtggctgggagtaatatgggctgatggaagcacaaattat240aattcggctctcatgtccagactgagcatcagcaaagacaactccaagaaccaagttttc300ttaaaaatgaacagtctgcaaaatgatgacacagccatgtactactgtgccagagaaggt360ttcgacaccacctggtttgattactggggccaagggactctggtcactgtctctgcaggt420ggcggtggctcgggcggtggtgggtcgggtggcggcggatctgacattgtgatgacacaa480cctgcttccttagctgtatctctggggcagagggccaccatctcatgcagggccagcaaa540agtgtcagtacatctggctatagttatatgcactggtaccaacagaaaccaggacagcca600cccaaactcctcatctatcttgcatccaacctagaatctggggtccctgccaggttcagt660ggcagtgggtctgggacagacttcaccctcaacatccatcctgtggaggaggaggatgct720gcaacctattactgtcagcacagtagggagcttccgtacacgttcggaggggggaccaag780ctggaaataaaaaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcg840tcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcac900acgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgt960ggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactc1020ctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggc1080tgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagc1140aggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaat1200ctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatg1260gggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagat1320aagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaagggg1380cacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcac1440atgcaggccctgccccctcgc1461<210>108<211>487<212>prt<213>artificialsequence<220><223>227-car-aa<400>108metalaleuprovalthralaleuleuleuproleualaleuleuleu151015hisalaalaargproglnvalglnleulysgluserglyproglyleu202530valalaproserglnserleuserilethrcysthrvalserglyphe354045serleuproasntyrglyvalhistrpvalargglnproproglylys505560glyleuglutrpleuglyvaliletrpalaaspglyserthrasntyr65707580asnseralaleumetserargleuserileserlysaspasnserlys859095asnglnvalpheleulysmetasnserleuglnasnaspaspthrala100105110mettyrtyrcysalaarggluglypheaspthrthrtrppheasptyr115120125trpglyglnglythrleuvalthrvalseralaglyglyglyglyser130135140glyglyglyglyserglyglyglyglyseraspilevalmetthrgln145150155160proalaserleualavalserleuglyglnargalathrilesercys165170175argalaserlysservalserthrserglytyrsertyrmethistrp180185190tyrglnglnlysproglyglnproprolysleuleuiletyrleuala195200205serasnleugluserglyvalproalaargpheserglyserglyser210215220glythraspphethrleuasnilehisprovalgluglugluaspala225230235240alathrtyrtyrcysglnhisserarggluleuprotyrthrphegly245250255glyglythrlysleugluilelysthrthrthrproalaproargpro260265270prothrproalaprothrilealaserglnproleuserleuargpro275280285glualacysargproalaalaglyglyalavalhisthrargglyleu290295300aspphealacysaspiletyriletrpalaproleualaglythrcys305310315320glyvalleuleuleuserleuvalilethrleutyrcyslysarggly325330335arglyslysleuleutyrilephelysglnprophemetargproval340345350glnthrthrglnglugluaspglycyssercysargpheprogluglu355360365glugluglyglycysgluleuargvallyspheserargseralaasp370375380alaproalatyrglnglnglyglnasnglnleutyrasngluleuasn385390395400leuglyargarggluglutyraspvalleuasplysargargglyarg405410415aspproglumetglyglylysproargarglysasnproglnglugly420425430leutyrasngluleuglnlysasplysmetalaglualatyrserglu435440445ileglymetlysglygluargargargglylysglyhisaspglyleu450455460tyrglnglyleuserthralathrlysaspthrtyraspalaleuhis465470475480metglnalaleuproproarg485<210>109<211>1440<212>dna<213>artificialsequence<220><223>236-car-nt<400>109atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccagg60ccggaggtgcagcttcaggagtcgggacctggcctggtgaaaccttctcagtctctgtcc120ctcacctgcactgtcactggctactcaatcaccagtgattatgtctggaactggatccgg180cagtttccaggaaacaaactggagtggatgggctacataagctacagtggtgtcactagt240tacaacccatctctcaaaagtcgaatctctatcactcgagacacatccaagaaccagttc300ttcctgcagttgaattctgtgactactgaggacacagccacatattactgtgcaagagga360acggactactggggtcaaggaacctcagtcaccgtctcctcaggtggcggtggctcgggc420ggtggtgggtcgggtggcggcggatctgacattgttctcaaccagtctccagcaatcatg480tctgcatctccaggggagaaggtcaccatgacctgcagtgccagctcaagtgtaagttcc540agttacttgcactggtaccagcagaagccaggatcctcccccaaactctggatttatagc600acatccaacctggcttctggagtcccagctcgcttcagtggcagtgggtctgggacctct660tactctctcacaatcagcagcatggaggctgaagatgctgccacttattactgccaccag720tttcatcgttccccacggacgttcggtggaggcaccaagctggaaatcaaaaccacgacg780ccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgc840ccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcc900tgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactg960gttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaacca1020tttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaa1080gaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcg1140taccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtac1200gatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaag1260aaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagt1320gagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggt1380ctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc1440<210>110<211>480<212>prt<213>artificialsequence<220><223>236-car-aa<400>110metalaleuprovalthralaleuleuleuproleualaleuleuleu151015hisalaalaargprogluvalglnleuglngluserglyproglyleu202530vallysproserglnserleuserleuthrcysthrvalthrglytyr354045serilethrserasptyrvaltrpasntrpileargglnpheprogly505560asnlysleuglutrpmetglytyrilesertyrserglyvalthrser65707580tyrasnproserleulysserargileserilethrargaspthrser859095lysasnglnphepheleuglnleuasnservalthrthrgluaspthr100105110alathrtyrtyrcysalaargglythrasptyrtrpglyglnglythr115120125servalthrvalserserglyglyglyglyserglyglyglyglyser130135140glyglyglyglyseraspilevalleuasnglnserproalailemet145150155160seralaserproglyglulysvalthrmetthrcysseralaserser165170175servalsersersertyrleuhistrptyrglnglnlysproglyser180185190serprolysleutrpiletyrserthrserasnleualaserglyval195200205proalaargpheserglyserglyserglythrsertyrserleuthr210215220ilesersermetglualagluaspalaalathrtyrtyrcyshisgln225230235240phehisargserproargthrpheglyglyglythrlysleugluile245250255lysthrthrthrproalaproargproprothrproalaprothrile260265270alaserglnproleuserleuargproglualacysargproalaala275280285glyglyalavalhisthrargglyleuaspphealacysaspiletyr290295300iletrpalaproleualaglythrcysglyvalleuleuleuserleu305310315320valilethrleutyrcyslysargglyarglyslysleuleutyrile325330335phelysglnprophemetargprovalglnthrthrglnglugluasp340345350glycyssercysargpheproglugluglugluglyglycysgluleu355360365argvallyspheserargseralaaspalaproalatyrglnglngly370375380glnasnglnleutyrasngluleuasnleuglyargarggluglutyr385390395400aspvalleuasplysargargglyargaspproglumetglyglylys405410415proargarglysasnproglngluglyleutyrasngluleuglnlys420425430asplysmetalaglualatyrsergluileglymetlysglygluarg435440445argargglylysglyhisaspglyleutyrglnglyleuserthrala450455460thrlysaspthrtyraspalaleuhismetglnalaleuproproarg465470475480<210>111<211>1461<212>dna<213>artificialsequence<220><223>245-car-nt<400>111atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccagg60ccgcaggtgcagctgaaggagtcaggacctggcctggtggcgccctcacagagcctgtcc120atcacttgcactgtctctgggttttcattaaccagctatggtgtacactgggttcgccag180cctccaggaaagggtctggagtggctgggagtaatatgggctgatggaagcacaaattat240aattcggctctcatgtccagactgagcatcagcaaagacaactccaagagccaagttttc300ttaaaaatgaacagtctgcgaactgatgacacagccatgtactactgtgccagagaaggt360tacgacaccacctggtttgattattggggccaagggactctggtcactgtctctgcaggt420ggcggtggctcgggcggtggtgggtcgggtggcggcggatctgacattgtgatgacacaa480cctgcttccttagctgtatctctggggcagagggccaccatctcatgcagggccagcaaa540agtgtcagtacatctggctatagttatatgcactggtaccaacagaaaccaggacagcca600cccaaactcctcatctatcttgcatccaacctagaatctggggtccctgccaggttcagt660ggcagtgggtctgggacagacttcaccctcaacatccatcctgtggaggaggaggatgct720gcaacctattactgtcagcacagtagggagcttccgtacacgttcggaggggggaccaag780ctggaaataaaaaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcg840tcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcac900acgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgt960ggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactc1020ctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggc1080tgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagc1140aggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaat1200ctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatg1260gggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagat1320aagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaagggg1380cacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcac1440atgcaggccctgccccctcgc1461<210>112<211>487<212>prt<213>artificialsequence<220><223>245-car-aa<400>112metalaleuprovalthralaleuleuleuproleualaleuleuleu151015hisalaalaargproglnvalglnleulysgluserglyproglyleu202530valalaproserglnserleuserilethrcysthrvalserglyphe354045serleuthrsertyrglyvalhistrpvalargglnproproglylys505560glyleuglutrpleuglyvaliletrpalaaspglyserthrasntyr65707580asnseralaleumetserargleuserileserlysaspasnserlys859095serglnvalpheleulysmetasnserleuargthraspaspthrala100105110mettyrtyrcysalaarggluglytyraspthrthrtrppheasptyr115120125trpglyglnglythrleuvalthrvalseralaglyglyglyglyser130135140glyglyglyglyserglyglyglyglyseraspilevalmetthrgln145150155160proalaserleualavalserleuglyglnargalathrilesercys165170175argalaserlysservalserthrserglytyrsertyrmethistrp180185190tyrglnglnlysproglyglnproprolysleuleuiletyrleuala195200205serasnleugluserglyvalproalaargpheserglyserglyser210215220glythraspphethrleuasnilehisprovalgluglugluaspala225230235240alathrtyrtyrcysglnhisserarggluleuprotyrthrphegly245250255glyglythrlysleugluilelysthrthrthrproalaproargpro260265270prothrproalaprothrilealaserglnproleuserleuargpro275280285glualacysargproalaalaglyglyalavalhisthrargglyleu290295300aspphealacysaspiletyriletrpalaproleualaglythrcys305310315320glyvalleuleuleuserleuvalilethrleutyrcyslysarggly325330335arglyslysleuleutyrilephelysglnprophemetargproval340345350glnthrthrglnglugluaspglycyssercysargpheprogluglu355360365glugluglyglycysgluleuargvallyspheserargseralaasp370375380alaproalatyrglnglnglyglnasnglnleutyrasngluleuasn385390395400leuglyargarggluglutyraspvalleuasplysargargglyarg405410415aspproglumetglyglylysproargarglysasnproglnglugly420425430leutyrasngluleuglnlysasplysmetalaglualatyrserglu435440445ileglymetlysglygluargargargglylysglyhisaspglyleu450455460tyrglnglyleuserthralathrlysaspthrtyraspalaleuhis465470475480metglnalaleuproproarg485<210>113<211>1461<212>dna<213>artificialsequence<220><223>255-car-nt<400>113atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccagg60ccgcaggtgcagctgaaggagtcaggacctggcctggtggcgccctcacagagcctgtcc120atcacttgcactgtctctgggttttcattaaccaactatggtgtacactgggttcgccag180cctccaggaaagggtctggagtggctgggagtaatatgggctgatggaagcacaaattac240aattcggctctcatgtccagactgaccatcaccaaagacacctccaagagccaagttttc300ttaaaaatgaacagtctggaaactgatgacacagccatataccactgtgccagagaaggg360ttcgacaccacctggtttgcttactggggccaagggactctggtcactgtctctgcaggt420ggcggtggctcgggcggtggtgggtcgggtggcggcggatctgacattgtgatgacacaa480cctgcttccttagctgtatctctggggcagagggccaccatctcatgcagggccagcaaa540agtgtcagtacatctggctatagttatatgcactggtaccaacagaaaccaggacagcca600cccaaactcctcatctatcttgcatccaacctagaatctggggtccctgccaggttcagt660ggcagtgggtctgggacagacttcaccctcaacatccatcctgtggaggaggaggatgct720gcaacctattactgtcagcacagtagggagcttccgtacacgttcggaggggggaccaag780ctggaaataaaaaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcg840tcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcac900acgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgt960ggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactc1020ctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggc1080tgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagc1140aggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaat1200ctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatg1260gggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagat1320aagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaagggg1380cacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcac1440atgcaggccctgccccctcgc1461<210>114<211>487<212>prt<213>artificialsequence<220><223>255-car-aa<400>114metalaleuprovalthralaleuleuleuproleualaleuleuleu151015hisalaalaargproglnvalglnleulysgluserglyproglyleu202530valalaproserglnserleuserilethrcysthrvalserglyphe354045serleuthrasntyrglyvalhistrpvalargglnproproglylys505560glyleuglutrpleuglyvaliletrpalaaspglyserthrasntyr65707580asnseralaleumetserargleuthrilethrlysaspthrserlys859095serglnvalpheleulysmetasnserleugluthraspaspthrala100105110iletyrhiscysalaarggluglypheaspthrthrtrpphealatyr115120125trpglyglnglythrleuvalthrvalseralaglyglyglyglyser130135140glyglyglyglyserglyglyglyglyseraspilevalmetthrgln145150155160proalaserleualavalserleuglyglnargalathrilesercys165170175argalaserlysservalserthrserglytyrsertyrmethistrp180185190tyrglnglnlysproglyglnproprolysleuleuiletyrleuala195200205serasnleugluserglyvalproalaargpheserglyserglyser210215220glythraspphethrleuasnilehisprovalgluglugluaspala225230235240alathrtyrtyrcysglnhisserarggluleuprotyrthrphegly245250255glyglythrlysleugluilelysthrthrthrproalaproargpro260265270prothrproalaprothrilealaserglnproleuserleuargpro275280285glualacysargproalaalaglyglyalavalhisthrargglyleu290295300aspphealacysaspiletyriletrpalaproleualaglythrcys305310315320glyvalleuleuleuserleuvalilethrleutyrcyslysarggly325330335arglyslysleuleutyrilephelysglnprophemetargproval340345350glnthrthrglnglugluaspglycyssercysargpheprogluglu355360365glugluglyglycysgluleuargvallyspheserargseralaasp370375380alaproalatyrglnglnglyglnasnglnleutyrasngluleuasn385390395400leuglyargarggluglutyraspvalleuasplysargargglyarg405410415aspproglumetglyglylysproargarglysasnproglnglugly420425430leutyrasngluleuglnlysasplysmetalaglualatyrserglu435440445ileglymetlysglygluargargargglylysglyhisaspglyleu450455460tyrglnglyleuserthralathrlysaspthrtyraspalaleuhis465470475480metglnalaleuproproarg485<210>115<211>1458<212>dna<213>artificialsequence<220><223>257-car-nt<400>115atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccagg60ccgcaggtccaactgcagcaacctggggctgaactggtgaggcctggggcttcagtgaag120ctgtcctgcaaggcttctggctacacgttcaccaactactggatgcactgggtgaagcag180aggcctggacaaggtcttgagtggattggcatgattgatccttccgatagtgaaactagg240ttaaatcagaagttcaaggacaaggccacattgactgtcgacaaatcctccagcacagcc300tacatgcaactccgcagcccgacatctgaggactctgcggtctattactgtgcaagaagg360gactatagccttgactactggggccaaggcaccactctcacagtctcctcaggtggcggt420ggctcgggcggtggtgggtcgggtggcggcggatctgacattgtaatgacacagtctcct480gcttccttagctgtatctctggggcagagggccaccatctcatgcagggccagcaaaagt540gtcagtacatctggctatagttatatgcactggtaccaacagaaaccaggacagccaccc600aaactcctcatctatcttgcatccaacctagaatctggggtccctgccaggttcagtggc660agtgggtctgggacagacttcaccctcaacatccatcctgtggaggaggaggatgctgca720acctattactgtcagcacagtagggagcttccgtacacgttcggaggggggaccaagctg780gaaataaaaaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcg840cagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacg900agggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggg960gtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctg1020tatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgt1080agctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcagg1140agcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctcaatcta1200ggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatgggg1260ggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataag1320atggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcac1380gatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatg1440caggccctgccccctcgc1458<210>116<211>486<212>prt<213>artificialsequence<220><223>257-car-aa<400>116metalaleuprovalthralaleuleuleuproleualaleuleuleu151015hisalaalaargproglnvalglnleuglnglnproglyalagluleu202530valargproglyalaservallysleusercyslysalaserglytyr354045thrphethrasntyrtrpmethistrpvallysglnargproglygln505560glyleuglutrpileglymetileaspproseraspsergluthrarg65707580leuasnglnlysphelysasplysalathrleuthrvalasplysser859095serserthralatyrmetglnleuargserprothrsergluaspser100105110alavaltyrtyrcysalaargargasptyrserleuasptyrtrpgly115120125glnglythrthrleuthrvalserserglyglyglyglyserglygly130135140glyglyserglyglyglyglyseraspilevalmetthrglnserpro145150155160alaserleualavalserleuglyglnargalathrilesercysarg165170175alaserlysservalserthrserglytyrsertyrmethistrptyr180185190glnglnlysproglyglnproprolysleuleuiletyrleualaser195200205asnleugluserglyvalproalaargpheserglyserglysergly210215220thraspphethrleuasnilehisprovalgluglugluaspalaala225230235240thrtyrtyrcysglnhisserarggluleuprotyrthrpheglygly245250255glythrlysleugluilelysthrthrthrproalaproargpropro260265270thrproalaprothrilealaserglnproleuserleuargproglu275280285alacysargproalaalaglyglyalavalhisthrargglyleuasp290295300phealacysaspiletyriletrpalaproleualaglythrcysgly305310315320valleuleuleuserleuvalilethrleutyrcyslysargglyarg325330335lyslysleuleutyrilephelysglnprophemetargprovalgln340345350thrthrglnglugluaspglycyssercysargpheproglugluglu355360365gluglyglycysgluleuargvallyspheserargseralaaspala370375380proalatyrglnglnglyglnasnglnleutyrasngluleuasnleu385390395400glyargarggluglutyraspvalleuasplysargargglyargasp405410415proglumetglyglylysproargarglysasnproglngluglyleu420425430tyrasngluleuglnlysasplysmetalaglualatyrsergluile435440445glymetlysglygluargargargglylysglyhisaspglyleutyr450455460glnglyleuserthralathrlysaspthrtyraspalaleuhismet465470475480glnalaleuproproarg485<210>117<211>1464<212>dna<213>artificialsequence<220><223>273-car-nt<400>117atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccagg60ccgcaggtgcagctgaaggagtcaggacctggcctggtggcgccctcacagagcctgtcc120atcacttgcactgtctctggcttttcattacccagctatggtgtacactgggttcgccag180cctccaggaaagggtctggagtggctgggagtaatatgggctgatggaagcagaaattat240aattcggctctcatgtccagactgagcatcagcaaagacaactccaagaaccaagttttc300ttaaaaatgaacagtctgcaaaatgatgatacagccatgtattactgtgtcagagaaggg360ttcgacaccacctggtttgattactggggccaagggactctggtcactgtctctgcaggt420ggcggtggctcgggcggtggtgggtcgggtggcggcggatctgacattgtgctcactcag480tctcctgcttccttagctgtatctctggggcagagggccaccatctcatgcagggccagc540aaaagtgtcagtacatctggctatagttatatgcactggtaccaacagaaaccaggacag600ccacccaaactcctcatctatcttgcatccaacctagaatctggggtccctgccaggttc660agtggcagtgggtctgggacagacttcaccctcaacatccatcctgtggaggaggaggat720gctgcaacctattactgtcagcacagtagggagcttccgtacacgttcggaggggggacc780aagctggaaataaaaaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatc840gcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtg900cacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggact960tgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaa1020ctcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagat1080ggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttc1140agcaggagcgcagacgcccccgcgtaccagcagggccagaaccagctctataacgagctc1200aatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgag1260atggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaa1320gataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaag1380gggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgccctt1440cacatgcaggccctgccccctcgc1464<210>118<211>488<212>prt<213>artificialsequence<220><223>273-car-aa<400>118metalaleuprovalthralaleuleuleuproleualaleuleuleu151015hisalaalaargproglnvalglnleulysgluserglyproglyleu202530valalaproserglnserleuserilethrcysthrvalserglyphe354045serleuprosertyrglyvalhistrpvalargglnproproglylys505560glyleuglutrpleuglyvaliletrpalaaspglyserargasntyr65707580asnseralaleumetserargleuserileserlysaspasnserlys859095asnglnvalpheleulysmetasnserleuglnasnaspaspthrala100105110mettyrtyrcysvalarggluglypheaspthrthrtrppheasptyr115120125trpglyglnglythrleuvalthrvalseralaglyglyglyglyser130135140glyglyglyglyserglyglyglyglyseraspilevalleuthrgln145150155160serproalaserleualavalserleuglyglnargalathrileser165170175cysargalaserlysservalserthrserglytyrsertyrmethis180185190trptyrglnglnlysproglyglnproprolysleuleuiletyrleu195200205alaserasnleugluserglyvalproalaargpheserglysergly210215220serglythraspphethrleuasnilehisprovalgluglugluasp225230235240alaalathrtyrtyrcysglnhisserarggluleuprotyrthrphe245250255glyglyglythrlysleugluilelysthrthrthrproalaproarg260265270proprothrproalaprothrilealaserglnproleuserleuarg275280285proglualacysargproalaalaglyglyalavalhisthrarggly290295300leuaspphealacysaspiletyriletrpalaproleualaglythr305310315320cysglyvalleuleuleuserleuvalilethrleutyrcyslysarg325330335glyarglyslysleuleutyrilephelysglnprophemetargpro340345350valglnthrthrglnglugluaspglycyssercysargpheproglu355360365gluglugluglyglycysgluleuargvallyspheserargserala370375380aspalaproalatyrglnglnglyglnasnglnleutyrasngluleu385390395400asnleuglyargarggluglutyraspvalleuasplysargarggly405410415argaspproglumetglyglylysproargarglysasnproglnglu420425430glyleutyrasngluleuglnlysasplysmetalaglualatyrser435440445gluileglymetlysglygluargargargglylysglyhisaspgly450455460leutyrglnglyleuserthralathrlysaspthrtyraspalaleu465470475480hismetglnalaleuproproarg485<210>119<211>1440<212>dna<213>artificialsequence<220><223>290-car-nt<400>119atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccagg60ccggaggtgcagcttcaggagtcgggacctggcctggtgaaaccttctcagtctctgtcc120ctcacctgcactgtcactggctactcaatcaccagtgattatgtctggaactggatccgg180cagtttccaggaaacaaactggagtggatgggctacataagctacagtggtagcactagt240tacaacccatctctcaaaagtcgaatctctatcactcgagacacatccaagaaccagttc300ttcctgcagttgaattctgtgactactgaggacacagccacatattactgtgcaagagga360acggactattggggtcaaggaacctcagtcaccgtctcctcaggtggcggtggctcgggc420ggtggtgggtcgggtggcggcggatctgacattgttctctcccagtctccagcaatcatg480tctgcatctctaggggaacgggtcaccatgacctgcactgccagctcaagtgtaagttcc540agttacttgcactggtaccagcagaagccaggatcctcccccaaactctggatttatagc600acatccaacctggcttctggagtcccacctcgcttcagtggcagtgggtctgggacctct660tactctctcacaatcagcagcatggaggctgaagatgctgccacttattactgccaccag720tttcatcgttccccacggacgttcggtggaggcaccaagctggaaatcaaaaccacgacg780ccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgc840ccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcc900tgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactg960gttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaacca1020tttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaa1080gaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcg1140taccagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtac1200gatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaag1260aaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagt1320gagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggt1380ctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc1440<210>120<211>480<212>prt<213>artificialsequence<220><223>290-car-aa<400>120metalaleuprovalthralaleuleuleuproleualaleuleuleu151015hisalaalaargprogluvalglnleuglngluserglyproglyleu202530vallysproserglnserleuserleuthrcysthrvalthrglytyr354045serilethrserasptyrvaltrpasntrpileargglnpheprogly505560asnlysleuglutrpmetglytyrilesertyrserglyserthrser65707580tyrasnproserleulysserargileserilethrargaspthrser859095lysasnglnphepheleuglnleuasnservalthrthrgluaspthr100105110alathrtyrtyrcysalaargglythrasptyrtrpglyglnglythr115120125servalthrvalserserglyglyglyglyserglyglyglyglyser130135140glyglyglyglyseraspilevalleuserglnserproalailemet145150155160seralaserleuglygluargvalthrmetthrcysthralaserser165170175servalsersersertyrleuhistrptyrglnglnlysproglyser180185190serprolysleutrpiletyrserthrserasnleualaserglyval195200205proproargpheserglyserglyserglythrsertyrserleuthr210215220ilesersermetglualagluaspalaalathrtyrtyrcyshisgln225230235240phehisargserproargthrpheglyglyglythrlysleugluile245250255lysthrthrthrproalaproargproprothrproalaprothrile260265270alaserglnproleuserleuargproglualacysargproalaala275280285glyglyalavalhisthrargglyleuaspphealacysaspiletyr290295300iletrpalaproleualaglythrcysglyvalleuleuleuserleu305310315320valilethrleutyrcyslysargglyarglyslysleuleutyrile325330335phelysglnprophemetargprovalglnthrthrglnglugluasp340345350glycyssercysargpheproglugluglugluglyglycysgluleu355360365argvallyspheserargseralaaspalaproalatyrglnglngly370375380glnasnglnleutyrasngluleuasnleuglyargarggluglutyr385390395400aspvalleuasplysargargglyargaspproglumetglyglylys405410415proargarglysasnproglngluglyleutyrasngluleuglnlys420425430asplysmetalaglualatyrsergluileglymetlysglygluarg435440445argargglylysglyhisaspglyleutyrglnglyleuserthrala450455460thrlysaspthrtyraspalaleuhismetglnalaleuproproarg465470475480<210>121<211>12<212>prt<213>artificialsequence<220><223>vlcdr1-1<400>121thralaserserservalsersersertyrleuhis1510<210>122<211>12<212>prt<213>artificialsequence<220><223>vlcdr1-2<400>122thralaserserservalsersersertyrleutyr1510<210>123<211>12<212>prt<213>artificialsequence<220><223>vlcdr1-3<400>123seralaserserservalsersersertyrleuhis1510<210>124<211>15<212>prt<213>artificialsequence<220><223>vlcdr1-4<400>124argalaserlysservalserserserglytyrsertyrilehis151015<210>125<211>15<212>prt<213>artificialsequence<220><223>vlcdr1-5<400>125argalaserlysservalserthrserglytyrsertyrmethis151015<210>126<211>15<212>prt<213>artificialsequence<220><223>vlcdr1-6<400>126argalaserlysservalserthrserglytyrsertyrleuhis151015<210>127<211>15<212>prt<213>artificialsequence<220><223>vlcdr1-7<400>127argalasergluservalserthrserglytyrsertyrmethis151015<210>128<211>15<212>prt<213>artificialsequence<220><223>vlcdr1-8<400>128argalaasnlysservalthrthrserglytyrsertyrmethis151015<210>129<211>7<212>prt<213>artificialsequence<220><223>vlcdr2-1<400>129serthrserasnleualaser15<210>130<211>7<212>prt<213>artificialsequence<220><223>vlcdr2-2<400>130leualaserasnleugluser15<210>131<211>7<212>prt<213>artificialsequence<220><223>vlcdr2-3<400>131serthrserlysleualaser15<210>132<211>9<212>prt<213>artificialsequence<220><223>vlcdr3-1<400>132hisglnphehisargserproprothr15<210>133<211>9<212>prt<213>artificialsequence<220><223>vlcdr3-2<400>133glnhisserarggluleuprotyrthr15<210>134<211>9<212>prt<213>artificialsequence<220><223>vlcdr3-3<400>134hisglntyrhisargserprotyrthr15<210>135<211>9<212>prt<213>artificialsequence<220><223>vlcdr3-4<400>135hisglnphehisargserproargthr15<210>136<211>9<212>prt<213>artificialsequence<220><223>vlcdr3-5<400>136glnhisserargaspleuprotyrthr15<210>137<211>7<212>prt<213>artificialsequence<220><223>vhcdr1-1<400>137thrserasptyrvaltrpasn15<210>138<211>6<212>prt<213>artificialsequence<220><223>vhcdr1-2<400>138prosertyrglyvalhis15<210>139<211>7<212>prt<213>artificialsequence<220><223>vhcdr1-3<400>139thrserasptyralatrpasn15<210>140<211>6<212>prt<213>artificialsequence<220><223>vhcdr1-4<400>140thrasnpheglyvalhis15<210>141<211>6<212>prt<213>artificialsequence<220><223>vhcdr1-5<400>141thrasntyrglyvalhis15<210>142<211>6<212>prt<213>artificialsequence<220><223>vhcdr1-6<400>142thrsertyrglyilehis15<210>143<211>6<212>prt<213>artificialsequence<220><223>vhcdr1-7<400>143proasntyrglyvalhis15<210>144<211>6<212>prt<213>artificialsequence<220><223>vhcdr1-8<400>144thrsertyrglyvalhis15<210>145<211>6<212>prt<213>artificialsequence<220><223>vhcdr1-9<400>145thrasntyrtrpmethis15<210>146<211>16<212>prt<213>artificialsequence<220><223>vhcdr2-1<400>146tyrilesertyrserglyserthrsertyrasnproserleuthrser151015<210>147<211>16<212>prt<213>artificialsequence<220><223>vhcdr2-2<400>147valiletrpalaaspglyserthrasntyrasnseralaleumetser151015<210>148<211>16<212>prt<213>artificialsequence<220><223>vhcdr2-3<400>148tyrileserpheglyglythrthrsertyrasnproserleulysser151015<210>149<211>16<212>prt<213>artificialsequence<220><223>vhcdr2-4<400>149valiletrpalaaspglyserthrhistyrasnserserleumetser151015<210>150<211>16<212>prt<213>artificialsequence<220><223>vhcdr2-5<400>150valiletrpalaaspglythrthrasntyrasnseralaleumetser151015<210>151<211>16<212>prt<213>artificialsequence<220><223>vhcdr2-6<400>151valiletrpalaaspglyserthrhistyrasnseralaleumetser151015<210>152<211>16<212>prt<213>artificialsequence<220><223>vhcdr2-7<400>152tyrilesertyrserglyvalthrsertyrasnproserleulysser151015<210>153<211>17<212>prt<213>artificialsequence<220><223>vhcdr2-8<400>153metileaspproseraspsergluthrargleuasnglnlysphelys151015asp<210>154<211>16<212>prt<213>artificialsequence<220><223>vhcdr2-9<400>154valiletrpalaaspglyserargasntyrasnseralaleumetser151015<210>155<211>16<212>prt<213>artificialsequence<220><223>vhcdr2-10<400>155tyrilesertyrserglyserthrsertyrasnproserleulysser151015<210>156<211>4<212>prt<213>artificialsequence<220><223>vhcdr3-1<400>156glythrasptyr1<210>157<211>10<212>prt<213>artificialsequence<220><223>vhcdr3-2<400>157gluglypheaspthrthrtrppheasptyr1510<210>158<211>4<212>prt<213>artificialsequence<220><223>vhcdr3-3<400>158glyleuasptyr1<210>159<211>10<212>prt<213>artificialsequence<220><223>vhcdr3-4<400>159gluglytyraspthrthrtrppheasptyr1510<210>160<211>10<212>prt<213>artificialsequence<220><223>vhcdr3-5<400>160gluglypheaspthrthrtrpphealatyr1510<210>161<211>7<212>prt<213>artificialsequence<220><223>vhcdr3-6<400>161argasptyrserleuasptyr15<210>162<211>9<212>prt<213>artificialsequence<220><223>vlcdr3-5<400>162glnglnasnserglytyrprophethr15<210>163<211>15<212>prt<213>artificialsequence<220><223>32716克隆vl-cdr1<400>163argalasergluservalaspasntyrglyasnthrphemethis151015<210>164<211>7<212>prt<213>artificialsequence<220><223>32716克隆vl-cdr2<400>164argalaserasnleugluser15<210>165<211>9<212>prt<213>artificialsequence<220><223>32716克隆vl-cdr3<400>165glnglnserasngluaspproprothr15<210>166<211>7<212>prt<213>artificialsequence<220><223>32716克隆vh-cdr1<400>166pheileasntyrglymetasn15<210>167<211>17<212>prt<213>artificialsequence<220><223>32716克隆vh-cdr2<400>167trpileasnthrtyrthrglygluserthrtyrseralaaspphelys151015gly<210>168<211>9<212>prt<213>artificialsequence<220><223>32716克隆vh-cdr3<400>168serglyglytyraspprometasptyr15<210>169<211>111<212>prt<213>artificialsequence<220><223>0033-car-vl<400>169aspilevalleuthrglnserproalaserleualavalserleugly151015glnargalathrilesercysargalasergluservalaspasntyr202530glyasnthrphemethistrptyrglnglnlysproglyglnpropro354045lysleuleuiletyrargalaserasnleugluserglyileproala505560argpheserglyserglyserargthraspphethrleuthrileasn65707580provalglualaaspaspvalalathrtyrtyrcysglnglnserasn859095gluaspproprothrpheglyalaglythrlysleugluleulys100105110<210>170<211>118<212>prt<213>artificialsequence<220><223>0033-car-vh<400>170glnileglnleuvalglnserglyprogluleulyslysproglyglu151015thrvallysilesercyslysalaserglytyrilepheileasntyr202530glymetasntrpvallysglnalaproglylysserphelystrpmet354045glytrpileasnthrtyrthrglygluserthrtyrseralaaspphe505560lysglyargphealapheserleugluthrseralaserthralatyr65707580leuhisileasnaspleulysasngluaspthralathrtyrphecys859095alaargserglyglytyraspprometasptyrtrpglyglnglythr100105110servalthrvalserser115当前第1页12