一种抗BCMA的单域抗体及其应用的制作方法

本发明涉及肿瘤的细胞免疫治疗领域，尤其涉及一种抗BCMA的单域抗体及其应用。
背景技术：
：B细胞成熟抗原(B-cellmaturationantigen，BCMA)，包括184个氨基酸残基，BCMA属于缺少信号肽的I型跨膜信号蛋白，是肿瘤坏死因子受体家族(TNFR)的成员，能够与B细胞激活因子BAFF或增殖诱导配体(Aproliferationinducedligand，APRIL)两种配体相结合。在正常组织中，BCMA表达于成熟B细胞和浆细胞表面，在维持浆细胞的存活中起了重要的作用，其机制主要包括BCMA与BAFF蛋白结合，并上调抗凋亡基因Bcl-2，Mcl-1及Bclw等，维持细胞生长。BCMA普遍表达于多发性骨髓瘤细胞系，对骨髓瘤细胞的恶性增生起了重要的促进作用，提示BCMA可以作为CAR-T细胞的靶点之一用于多发性骨髓瘤的细胞免疫治疗。多发性骨髓瘤是一种恶性浆细胞疾病，表现为骨髓浆细胞恶性克隆性增生，分泌单克隆免疫球蛋白或其片段(M蛋白)，导致骨骼、肾脏等相关靶器官或组织损伤，常见临床表现为骨痛、贫血、肾功能不全、感染等。目前多发性骨髓瘤为血液系统第二大恶性肿瘤，占血液系统恶性肿瘤的10％，多发病于男性，其发病率随着年龄的增长逐年增高，近几年更是有年轻化的趋势。嵌合抗原受体T细胞(ChimericAntigenReceptor-Tcell，CAR-T)是指经基因修饰后，能以MHC非限制性方式识别特定目的抗原，并且持续活化扩增的T细胞。生物免疫细胞治疗已经成为手术、放疗、化疗外的第四种治疗肿瘤的手段，并将成为未来肿瘤治疗必选手段。CAR-T细胞回输治疗是当前肿瘤治疗中最明确有效的免疫治疗形式。大量研究表明，CAR-T细胞可以有效的识别肿瘤抗原，引起特异性的抗肿瘤免疫应答，显著改善患者的生存状况。因此，提供一种新型的抗BCMA的单域抗体并应用于制备嵌合抗原受体，在制备预防和/或治疗和/或诊断肿瘤的药物领域具有广阔的应用前景和巨大的市场价值。技术实现要素：针对现有技术的不足和市场的需求，本发明提供一种抗BCMA的单域抗体及其应用，本发明通过实验筛选得到七个单域抗体，制备得到靶向BCMA的嵌合抗原受体，其中抗体QL197G8对靶细胞杀伤效率高，细胞因子IFN-γ的释放量大，亲和力强，特异性好，具有广阔的应用前景和巨大的市场价值。为达此目的，本发明采用以下技术方案：一种抗BCMA的单域抗体或其抗原结合片段，其特征在于，所述抗BCMA单域抗体包含氨基酸序列为SEQIDNO.1-3、SEQIDNO.4-6、SEQIDNO.7-9、SEQIDNO.10-12、SEQIDNO.13-15、SEQIDNO.16-18或SEQIDNO.19-21中任意一组的CDR，或与SEQIDNO.1-3、SEQIDNO.4-6、SEQIDNO.7-9、SEQIDNO.10-12、SEQIDNO.13-15、SEQIDNO.16-18或SEQIDNO.19-21中任意一组具有至少90％同一性的CDR。本发明中，抗BCMA单域抗体包含氨基酸序列可以是与SEQIDNO.1-3、SEQIDNO.4-6、SEQIDNO.7-9、SEQIDNO.10-12、SEQIDNO.13-15、SEQIDNO.16-18或SEQIDNO.19-21中任意一组的CDR具有至少90％同一性的CDR，例如可以是90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％同一性，不改变所述CDR区结构和活性的同一性CDR均在本发明保护范围之内。优选地，其特征在于，所述抗BCMA的抗体的氨基酸序列如SEQIDNO.22-28所示。本发明中，发明人用BCMA抗原胞外区多肽免疫美洲驼(llama)，通过对大量BCMA抗体进行筛选分析，筛选得到七个单域抗体，其中抗体QL197G8亲和力强，特异性好，而且它们还可以发挥个头小的优势，识别肿瘤细胞表面更小、更隐蔽的结合位点。制备得到靶向BCMA的嵌合抗原受体，对靶细胞杀伤效率高，具有广阔的应用前景和巨大的市场价值。序列见表1和表2；表1抗体名称氨基酸序列QL197A11CDR1SEQIDNO.1RTVSTATMQL197A11CDR2SEQIDNO.2AALNWSGNKSYYADSQL197A11CDR3SEQIDNO.3AGPDLNYYTNYDARRYDHQL197C9CDR1SEQIDNO.4HTTAINAAQL197C9CDR2SEQIDNO.5LVGLIHNDGSTQYAQFAQL197C9CDR3SEQIDNO.6NIDSRGVGPVWAQL197D11CDR1SEQIDNO.7NIYSINRMAQL197D11CDR2SEQIDNO.8VVATSLVDGTTNYGDSVKDQL197D11CDR3SEQIDNO.9NVEGNRIDYAPGSRYPTHSYVELQL197E2CDR1SEQIDNO.10ASFNDYHMQL197E2CDR2SEQIDNO.11FVAQIARYGAATYYARAVQQL197E2CDR3SEQIDNO.12TADRSNYYIDNALPDQL197F4CDR1SEQIDNO.13RNFNSYAMQL197F4CDR2SEQIDNO.14FLATISRAAGSTYYADSAKQL197F4CDR3SEQIDNO.15AESWTPTTGWPPTKADEFDQL197F7CDR1SEQIDNO.16FNLDDYAIQL197F7CDR2SEQIDNO.17RVSCISSDGRTSHTGSAKQL197F7CDR3SEQIDNO.18ERTSRLCSFLSYDQL197G8CDR1SEQIDNO.19RTFSDHTLQL197G8CDR2SEQIDNO.20GAISWSGGSTYYADSVSQL197G8CDR3SEQIDNO.21ADDRYSDYR表2第二方面，本发明提供一种编码如第一方面所述抗BCMA的单域抗体或其抗原结合片段的核酸片段。所述核酸片段如SEQIDNO.29-35所示，具体如下表3；表3抗体亲和力成熟是抗体技术中的常用手段，可以通过单点完全随机突变结合热点组合途径(PM)及区段完全随机突变(BM)途径改变抗体的亲和力。前者通常用于小幅亲和力提高/最小程度序列改造，后者用于显著亲和力成熟。本发明要求保护7个纳米抗体的氨基酸，重点保护各抗体的CDR1，2，3序列，以及同上述CDR区序列同一性大于90％的序列。第三方面，本发明提供一种载体，所述载体包含至少一个拷贝的如第二方面所述的核酸片段。第四方面，本发明提供一种宿主细胞，所述宿主细胞包含第三方面所述的载体。第五方面，本发明提供一种嵌合抗原受体，所述胞外结构域为如第一方面所述抗BCMA的抗体。优选地，所述嵌合抗原受体的氨基酸序列如SEQIDNO.36-42所示。所述序列见表4；表4所述嵌合抗原受体的核酸序列如下表5所示；表5第六方面，本发明提供一种T细胞，采用如第五方面所述的嵌合抗原受体通过其编码的核酸序列转染到T细胞中表达。优选地，所述转染的方式为通过病毒载体和/或真核表达质粒转染到T细胞，优选为通过病毒载体转染到T细胞。本发明中，所述T细胞具有良好的靶向杀伤作用，同时能够释放低剂量免疫因子，具备低毒性高免疫杀伤反应性质。第七方面，本发明提供一种重组慢病毒，将包含如第三方面所述的载体与包装辅助质粒共转染哺乳细胞得到的重组慢病毒。优选地，所述哺乳细胞为293细胞、293T细胞或293F细胞中的任意一种或至少两种的组合。第八方面，本发明提供一种药物组合物，所述药物组合物包括如第一方面所述的单域抗体或其抗原结合片段、第二方面所述的核酸、如第三方面所述的载体、如第四方面所述的宿主细胞、如第五方面所述的嵌合抗原受体、如第六方面所述的T细胞或如第七方面所述的重组慢病毒中的任意一种或至少两种的组合。可选地，其还包括药学上可接受的载体和/或赋形剂。优选地，其还包含一种或者至少两种的化疗药物。第九方面，如第一方面所述的单域抗体或其抗原结合片段、如第二方面所述的核酸、如第三方面所述的载体、如第四方面所述的宿主细胞、如第五方面所述的嵌合抗原受体、如第六方面所述的T细胞、如第七方面所述的重组慢病毒或如第八方面所述的药物组合物在制备预防和/或治疗和/或诊断肿瘤的药物中的应用。根据本发明，所述肿瘤为与B细胞成熟抗原蛋白的表达相关的疾病，例如多发性骨髓瘤、霍奇金淋巴瘤、白血病或胶质母细胞瘤。与现有技术相比，本发明具有如下有益效果：本发明中，发明人用BCMA抗原胞外区多肽免疫美洲驼(llama)，通过对大量BCMA抗体进行筛选分析，筛选得到七个单域抗体，其中抗体QL197G8亲和力强，特异性好，而且它们还可以发挥个头小的优势，识别肿瘤细胞表面更小、更隐蔽的结合位点。制备得到靶向BCMA的嵌合抗原受体，对靶细胞杀伤效率高，细胞因子IFN-γ的释放量大，具有广阔的应用前景和巨大的市场价值。附图说明图1为本发明的CAR结构示意图；图2为本发明的本发明的pLVX-EF1-MCS载体图谱；图3为本发明的CAR-T细胞对靶细胞的杀伤效率图；图4为本发明的CAR-T细胞与靶细胞共孵育后细胞因子IFN-γ的释放情况；图5(A)-图5(B)为本发明的SDS-PAGE电泳结果图；图6(A)-图6(F)为本发明的anti-humanBCMA纳米抗体亲和力分析结果；图7为本发明7个抗BCMA纳米抗体与BCMA的结合能力的ELISA检测结果。具体实施方式为更进一步阐述本发明所采取的技术手段及其效果，以下结合附图并通过具体实施方式来进一步说明本发明的技术方案，但本发明并非局限在实施例范围内。实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购获得的常规产品。实施例1抗体筛选两个美洲驼(llama)用BCMA胞外区抗原肽免疫，1只用游离肽(QL197)免疫，另1只用血蓝蛋白(KLH)偶联肽(QL198)免疫。QL197llama产生高滴度抗体，获取其外周血单个核细胞(PBMC)用于VHH文库构建和抗体克隆分离，QL198羊驼没有产生良好的抗体而被终止；(1)QL197和QL198滴度由BCMA-freepeptide、BSA和protein测定：用ELISA法测定抗血清中抗体效价，包被抗原，血清稀释至1:10000-1:100万，免疫接种开始前的血清作为对照，用HRP偶联的山羊抗羊驼抗体(重链+轻链)和TMB底物检测捕获的羊驼抗体。由于滴度不好，终止了QL198的实验；(2)QL197VHH单域抗体文库的PBMC细胞数量：3.2×10e8；(3)总RNA纯化：采用苯酚/氯仿萃取法纯化PBMC总RNA，硅胶自旋柱法纯化；用无RNA酶的H2O洗脱总RNA；采用OD260/280比值(2.0)和琼脂糖凝胶电泳(非变性)评价RNA的质量；用1.0OD260＝40μg/mL的公式估计RNA浓度；(4)cDNA合成:按照美国QoolAbs公司的方案，使用llamaIgG重链特异的反向引物启动总RNA，采用重链特异性引物PCR检测cDNA的质量；(5)文库构建：从cDNA中扩增出预期大小的VHH产物；琼脂糖凝胶分离VH和VHH，进一步纯化VHH产物，用sfil位点修饰，克隆到pADL20c噬菌体显示载体中，结扎后的DNA转化为TG1细胞，该文库共获得1.3×10e9个独立克隆。注:由于框架4和铰链的高度多样性，VHH插入件分为两部分:一端在FR4上，产生～420bp产品；另一端在铰链后，产生～580bp产品。文库大小:1.3×10e9独立克隆；特异性克隆筛选:采用Qoolabs噬菌体筛选方案，在人BCMA涂布板上筛选噬菌体展示抗体文库，经过4轮筛选，每筛选95个克隆，随机选出阳性克隆个体。原代ELISA法检测R4克隆的培养裂解产物，ELISA包被抗原为人BCMA，见表6；表6验证标记克隆培养裂解产物的ELISA结果，ELISA包被抗原以BCMA游离肽、人BCMA、HSA为阴性对照，见表7；表7所有克隆均在平板上划线，每个克隆取2个单菌落进行ELISA验证，然后进行测序。测序前验证阳性克隆培养裂解产物ELISA结果，ELISA包被抗原以BCMA游离肽、人BCMA、HSA为阴性对照，见表8；表8获得7个序列不同的阳性克隆，其DNA序列见SEQIDNO.29-35；实施例2：嵌合抗原受体的设计本实施例构建了抗BCMA的嵌合抗原受体，结构示意图见图1，嵌合抗原受体包括一段CD8α的信号肽序列(Leader)，与BCMA抗原特异性结合的单域抗体序列(Anti-BCMAVHH)，CD8α的铰链区(Hinge)和跨膜区序列(Transmembrane)，4-1BB共刺激域序列和CD3ζ信号传导域序列，具体各部分序列如下：CD8α信号肽(leader)的氨基酸序列(SEQIDNO.50)：MALPVTALLLPLALLLHAARP；CD8α信号肽(leader)的核苷酸序列(SEQIDNO.51)：ATGGCACTGCCAGTGACAGCCCTGCTGCTGCCACTGGCCCTGCTGCTGCACGCAGCACGCCCT；CD8α铰链区(hinge)的氨基酸序列(SEQIDNO.52)：TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD；CD8α铰链区(hinge)的核苷酸序列(SEQIDNO.53)：ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT；CD8α跨膜区(TM)的氨基酸序列(SEQIDNO.54)：IYIWAPLAGTCGVLLLSLVITLYC；CD8α跨膜区(TM)的核苷酸序列(SEQIDNO.55)：ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC；4-1BB胞内共刺激域(ICD)的氨基酸序列(SEQIDNO.56)：KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL；4-1BB胞内共刺激域(ICD)的核苷酸序列(SEQIDNO.57)：AAGAGAGGCAGGAAGAAGCTGCTGTACATCTTCAAGCAGCCCTTCATGCGCCCCGTGCAGACAACCCAGGAGGAGGACGGCTGCAGCTGTCGGTTCCCAGAGGAGGAGGAGGGAGGATGTGAGCTG；CD3ζ信号传导域的氨基酸序列(SEQIDNO.58)：RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR；CD3ζ信号传导域的核苷酸序列(SEQIDNO.59)：AGGGTGAAGTTTTCTCGGAGCGCCGATGCACCAGCATATCAGCAGGGACAGAATCAGCTGTACAACGAGCTGAATCTGGGCAGGCGCGAGGAGTACGACGTGCTGGATAAGCGGAGAGGCAGAGATCCCGAGATGGGAGGCAAGCCAAGGAGGAAGAACCCTCAGGAGGGCCTGTATAATGAGCTGCAGAAGGACAAGATGGCCGAGGCCTACTCTGAGATCGGCATGAAGGGAGAGCGGAGAAGGGGCAAGGGACACGATGGCCTGTATCAGGGCCTGAGCACAGCCACCAAGGACACCTACGATGCACTGCACATGCAGGCCCTGCCACCTAGG.实施例3：构建抗BCMA的嵌合抗原受体表达载体(1)根据CAR基因的核酸序列，在广州艾基生物技术有限公司进行全基因合成；(2)用EcoRI和BamHI双酶切全基因合成的CAR基因和空载体pLVX-EF1-MCS,于37℃水浴中酶切30min后，使用1.5％的琼脂糖凝胶进行DNA电泳，然后使用天根的琼脂糖凝胶试剂盒纯化回收处理；(3)pLVX-EF1-MCS载体与CAR基因片段的连接：连接体系如表9所示：表9组件添加量(μl)pLVX-EF1-MCS载体2(50ng)CAR基因10(150ng)T4DNA连接缓冲液2T4DNA连接酶(NEB)1ddH2O5总共20于22℃连接1h，连接产物直接转化Stbl3大肠杆菌感受态细胞，取200μl转化产物涂布氨苄抗性的LB平板，LB平板于37℃的培养箱中倒置培养过夜。次日早晨随机挑选3个单克隆进行菌落PCR鉴定，将阳性克隆送样测序。其中，抗BCMA的嵌合抗原受体慢病毒表达载体pLVX-EF1α-G8CAR-Km的载体图谱见图2。实施例4：慢病毒包装分别对实施例中的慢病毒表达载体进行慢病毒包装，采用四质粒系统，具体步骤如下：(1)四质粒系统分别表达慢病毒载体包装所需的gag/pol、Rev、VSV-G及本发明构建的CAR表达载体：将四质粒进行瞬时转染293T细胞，DNA含量为2μg/mL；(2)将上述质粒与PEI转染试剂混合，加入至一定体积的无血清的DMEM中，混匀后放置15分钟，将上述混合液加入至铺有293T细胞的细胞的T75培养瓶中，轻轻混匀，于37℃、5％CO2细胞培养箱培养6h；(3)6h后更换新鲜培养基，继续进行培养，并且加入10mM的丁酸钠溶液，72小时后收集慢病毒的培养上清进行纯化检测。实施例5：CAR-T细胞的扩增每位志愿者采30ml的全血，将外周血与生理盐水1:1进行稀释，在离心管内加入Ficoll，缓缓加入稀释后的外周血，1500rpm离心30min轻轻吸取PBMC层移入另一离心管内；用生理盐水洗涤PBMC多次，转入X-VIVO培养基(含50ng/mL的OKT3，300IU/mL的IL-2)中进行培养，PBMC分离后，需要用含50ng/mL的OKT3，300IU/ml的IL-2的X-VIVO进行激活，2日后将培养基更换成含300IU/mL的X-VIVO进行扩大培养，而后每两天进行一次计数并更换含300IU/mL的X-VIVO，并且将细胞浓度维持在0.5×106-1×106/mL，连续观察10天。实施例6：慢病毒感染T细胞利用RetroNectin提高慢病毒对T细胞的感染效率，将30μg的RetroNectin，包被于6孔板内，放于37℃细胞培养箱2h；吸取RetroNectin，利用含2.5％BSA的Hank’s溶液封闭包被后的6孔板，放于37℃细胞培养箱0.5h；吸取封闭液，利用含2％Hepes的Hank’s溶液洗涤6孔板，加入X-VIVO培养基，加入适量的慢病毒溶液，2000g，离心2h；弃上清，加入1×106的T细胞(CD3阳性 90％)，1000g，离心10min，于37℃、5％CO2和一定湿度的细胞培养箱内培养，第二日重复上述过程。实施例7：杀伤效率检测感染7-10天后进行细胞毒性检测实验，并用空白对照NC(未转染的T细胞)作为对照，具体步骤如下：利用LDH检测CAR-T细胞对K562和K562-BCMA(稳定表达BCMA的K562细胞)的毒性；将各种细胞离心后，用无血清无酚红的，RPMI1640培养基多次洗涤后计数，取1×106的K562和K562-BCMA细胞各50μL，铺板于96孔板内，作为靶细胞，按靶细胞：效应细胞＝1:1分别加入未转染的T细胞和各CAR-T细胞，于37℃、5％CO2和一定湿度的细胞培养箱内培养12h；加入裂解液作为阳性对照，而后250g离心5min，每孔取100μL培养上清，加入新的96孔板内，加入20μL反应液，放于暗室中反应20-30min。酶标仪590nm测定，根据公式计算溶解百分率：细胞毒性(％)＝[(实验孔－培养基背景孔)－(效应细胞自发LDH释放孔－培养基背景孔)－(靶细胞自发LDH释放孔－培养基背景孔)]/[(靶细胞最大LDH释放孔－体积校正孔)－(靶细胞自发LDH释放孔－培养基背景孔)]×100％。具体的步骤参照申请号为201510362935.5的专利《CD33特异性嵌合抗原受体及其应用》。CAR-T细胞对靶细胞的杀伤效率见图3；由图3可知，未转染CAR的T细胞(Tmock)对K562和K562-BCMA都无显著杀伤；各个CAR-T细胞都能不同程度地杀伤BCMA表达阳性的细胞K562-BCMA，而对BCMA阴性细胞K562几乎没有杀伤能力；其中G8CAR-T的杀伤率最高。实施例8：IFN-γ的释放检测分别将K562和K562-BCMA细胞按照5×105细胞/孔接种24孔板。按每孔5×105细胞分别加入CAR-T、未转染的T细胞(Tmock)细胞，补充培养液至1.5mL，于孵箱中共培养12小时后；采用人IFN-γELISA检测试剂盒(欣博盛生物)，对共培养上清进行检测(具体步骤见ELISA检测试剂盒说明书)。CAR-T细胞与靶细胞共孵育后细胞因子IFN-γ的释放情况见图4。由图4可知：未转染CAR的T细胞(Tmock)与K562和K562-BCMA共培养后几乎不分泌IFN-γ；各个CAR-T细胞与K562-BCMA共培养后，上清中都能检测到不同浓度的IFN-γ，而与BCMA阴性细胞K562共培养后很少有细胞因子IFN-γ的释放。实施例9亲和力测试将表2的7个羊驼抗体序列连同IgG引导肽序列(MAVVLAALLQSVQA)、llama的IgG2b的铰链区序列(epktpkpqpqpqpqpnptteskcpkcpapellggps)和His标签序列(HHHHHH)，进行密码子优化后构建至pcDNA4/HisB载体上，得到质粒。序列说明： QL197A11SEQIDNO.60MAVVLAALLQSVQAqvllvesggglvqaggslrlscavsgrtvstatmgwfrqapgkerefvaalnwsgnksyyadsvkgrfaisrdeakntvylqmnslkpedtavyhcaagpdlnyytnydarrydhwgqgtqvtvssepktpkpqpqpqpqpnptteskcpkcpapellggpsHHHHHH QL197C9SEQIDNO.61MAVVLAALLQSVQAqgqyvesgggsvqageslrlscigshttainaagwyrqtpgkqrelvglihndgstqyaqfakgrftisrddakdavylqmnslkvedtgvyycnidsrgvgpvwahwgqgtqvtvssepktpkpqpqpqpqpnptteskcpkcpapellggpshhhhhh QL197D11SEQIDNO.62MAVVLAALLQSVQAqaqlkesgggsvrtgeslrlsceasgniysinrmawyrqvsgmqrevvatslvdgttnygdsvkdrftvsrdnakkmvflqmnslepadtgvyycnvegnridyapgsrypthsyvelwgqgtqvtvssepktpkpqpqpqpqpnptteskcpkcpapellggpsHHHHHH QL197E2SEQIDNO.63MAVVLAALLQSVQAqvqlvenggglvqpggslrlscaasgasfndyhmgwfrqapgqerkfvaqiarygaatyyaravqgrftisvddakntvylqmngltpddtgvyyctadrsnyyidnalpdywgqgtqvtvssepktpkpqpqpqpqpnptteskcpkcpapellggpsHHHHHH QL197F4SEQIDNO.64MAVVLAALLQSVQAqvqliesggglvqagtsltlscassgrnfnsyamgwfrqapgkereflatisraagstyyadsakgrftisrdnrkefaylqihdlkpddtavyycaaeswtpttgwpptkadefdywgqgtqvtvssepktpkpqpqpqpqpnptteskcpkcpapellggpsHHHHHH QL197F7SEQIDNO.65MAVVLAALLQSVQAqdrlvesgggsvqaggslklscrtsgfnlddyaigwfrqapgkerervscissdgrtshtgsakgrftirsanarntvylqlnrltpedagvyfcaaertsrlcsflsydywgqgtqvtvssepktpkpqpqpqpqpnptteskcpkcpapellggpsHHHHHH QL197G8SEQIDNO.66MAVVLAALLQSVQAqvqlvesggglvqaggslrlscaasgrtfsdhtlgwfrqapgkerefvgaiswsggstyyadsvsgrftisrdkakntgylqmnslkpedtavyycaaaddrysdyrywgqgtqvtvssepktpkpqpqpqpqpnptteskcpkcpapellggpsHHHHHH对照抗体BBHL-Fab-VLSEQIDNO.67：DIVLTQSPASLAVSLGERATINCRASESVSVIGAHLIHWYQQKPGQPPKLLIYLASNLETGVPARFSGSGSGTDFTLTISSLQAEDAAIYYCLQSRIFPRTFGQGTKLEIK对照抗体BBHL-Fab-VHSEQIDNO.68：QVQLVQSGSELKKPGASVKVSCKASGYTFTDYSINWVRQAPGQGLEWMGWINTETREPAYAYDFRGRFVFSLDTSVSTAYLQISSLKAEDTAVYYCARDYSYAMDYWGQGTLVTVSS上述质粒测序正确后，进行293F瞬时转染，2天后收获细胞(可能存在降解的情况)，Ni-His亲和柱亲和纯化纳米抗体后，SDS-PAGE电泳检测，结果见图5(A)和图5(B)，其中图5(A)为15％还原性SDS-PAGE分析7个抗BCMA纳米抗体的分子量结果图，5(B)为15％非还原性SDS-PAGE分析7个抗BCMA纳米抗体的分子量结果图；由图5(A)和图5(B)可知，7个抗体分子都实现表达，C端存在降解情况。使用Anti-hFc固定传感器，loaddinghumanBCMA-hFc抗原，7种抗体蛋白初始浓度浓度设定为200nM，进行亲和力分析，结果见图6(A)-图6(B)和表10；由图6(A)、图6(B)和表10可知，7种抗体中QL197E2和QL197G8亲和力可以达到与对照BBHL-Fab相当的水平，但是还需要验证二者是否是与humanFc段的结合；表10使用Anti-hFc固定传感器，loaddinghumanBCMA-hFc抗原及无关抗原humanPD1-hFc，根据上面的亲和力分析结果，将QL197E2设定浓度为100nm，将QL197G8设定浓度为200nm，将BBHL-Fab(阳性对照抗体)设定浓度为50nm，分析三种蛋白是否是非特异结合，结果见图6(C)-图6(F)和表11；表11由图6(C)-图6(F)和表11可知，QL197E2是和humanFc段结合的抗体，而QL197G8不与humanFc段结合，可以进行后续的研究。我们进一步用ELISA检测7个抗BCMA纳米抗体与BCMA的结合能力(图7)，分别包被7种抗体及对照抗体BBHL-Fab，浓度为0.5ug/mL，将humanBCMA-hFc与无关带humanFc的抗原分别稀释浓度为20ng/ml及100ng/ml，加入Anti-humanHRP显色。由图7可知：QL197E2是和humanFc段结合的抗体，QL197G8是特异性结合，可作为后续研究。综上所述，本发明提供了一种抗BCMA的单域抗体及其应用，其中，QL197G8对靶细胞杀伤效率高，细胞因子IFN-γ的释放量大，且不与humanFc段结合，为B细胞相关疾病提供一个更有效的治疗选择。申请人声明，本发明通过上述实施例来说明本发明的详细方法，但本发明并不局限于上述详细方法，即不意味着本发明必须依赖上述详细方法才能实施。所属
技术领域：
的技术人员应该明了，对本发明的任何改进，对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。SEQUENCELISTING 110 广州百暨基因科技有限公司 120 一种抗BCMA的单域抗体及其应用 130 2019 160 68 170 PatentInversion3.3 210 1 211 8 212 PRT 213 人工合成序列 400 1ArgThrValSerThrAlaThrMet15 210 2 211 15 212 PRT 213 人工合成序列 400 2AlaAlaLeuAsnTrpSerGlyAsnLysSerTyrTyrAlaAspSer151015 210 3 211 18 212 PRT 213 人工合成序列 400 3AlaGlyProAspLeuAsnTyrTyrThrAsnTyrAspAlaArgArgTyr151015AspHis 210 4 211 8 212 PRT 213 人工合成序列 400 4HisThrThrAlaIleAsnAlaAla15 210 5 211 17 212 PRT 213 人工合成序列 400 5LeuValGlyLeuIleHisAsnAspGlySerThrGlnTyrAlaGlnPhe151015Ala 210 6 211 12 212 PRT 213 人工合成序列 400 6AsnIleAspSerArgGlyValGlyProValTrpAla1510 210 7 211 9 212 PRT 213 人工合成序列 400 7AsnIleTyrSerIleAsnArgMetAla15 210 8 211 19 212 PRT 213 人工合成序列 400 8ValValAlaThrSerLeuValAspGlyThrThrAsnTyrGlyAspSer151015ValLysAsp 210 9 211 23 212 PRT 213 人工合成序列 400 9AsnValGluGlyAsnArgIleAspTyrAlaProGlySerArgTyrPro151015ThrHisSerTyrValGluLeu20 210 10 211 8 212 PRT 213 人工合成序列 400 10AlaSerPheAsnAspTyrHisMet15 210 11 211 19 212 PRT 213 人工合成序列 400 11PheValAlaGlnIleAlaArgTyrGlyAlaAlaThrTyrTyrAlaArg151015AlaValGln 210 12 211 15 212 PRT 213 人工合成序列 400 12ThrAlaAspArgSerAsnTyrTyrIleAspAsnAlaLeuProAsp151015 210 13 211 8 212 PRT 213 人工合成序列 400 13ArgAsnPheAsnSerTyrAlaMet15 210 14 211 19 212 PRT 213 人工合成序列 400 14PheLeuAlaThrIleSerArgAlaAlaGlySerThrTyrTyrAlaAsp151015SerAlaLys 210 15 211 19 212 PRT 213 人工合成序列 400 15AlaGluSerTrpThrProThrThrGlyTrpProProThrLysAlaAsp151015GluPheAsp 210 16 211 8 212 PRT 213 人工合成序列 400 16PheAsnLeuAspAspTyrAlaIle15 210 17 211 18 212 PRT 213 人工合成序列 400 17ArgValSerCysIleSerSerAspGlyArgThrSerHisThrGlySer151015AlaLys 210 18 211 13 212 PRT 213 人工合成序列 400 18GluArgThrSerArgLeuCysSerPheLeuSerTyrAsp1510 210 19 211 8 212 PRT 213 人工合成序列 400 19ArgThrPheSerAspHisThrLeu15 210 20 211 17 212 PRT 213 人工合成序列 400 20GlyAlaIleSerTrpSerGlyGlySerThrTyrTyrAlaAspSerVal151015Ser 210 21 211 9 212 PRT 213 人工合成序列 400 21AlaAspAspArgTyrSerAspTyrArg15 210 22 211 126 212 PRT 213 人工合成序列 400 22GlnValLeuLeuValGluSerGlyGlyGlyLeuValGlnAlaGlyGly151015SerLeuArgLeuSerCysAlaValSerGlyArgThrValSerThrAla202530ThrMetGlyTrpPheArgGlnAlaProGlyLysGluArgGluPheVal354045AlaAlaLeuAsnTrpSerGlyAsnLysSerTyrTyrAlaAspSerVal505560LysGlyArgPheAlaIleSerArgAspGluAlaLysAsnThrValTyr65707580LeuGlnMetAsnSerLeuLysProGluAspThrAlaValTyrHisCys859095AlaAlaGlyProAspLeuAsnTyrTyrThrAsnTyrAspAlaArgArg100105110TyrAspHisTrpGlyGlnGlyThrGlnValThrValSerSer115120125 210 23 211 118 212 PRT 213 人工合成序列 400 23GlnGlyGlnTyrValGluSerGlyGlyGlySerValGlnAlaGlyGlu151015SerLeuArgLeuSerCysIleGlySerHisThrThrAlaIleAsnAla202530AlaGlyTrpTyrArgGlnThrProGlyLysGlnArgGluLeuValGly354045LeuIleHisAsnAspGlySerThrGlnTyrAlaGlnPheAlaLysGly505560ArgPheThrIleSerArgAspAspAlaLysAspAlaValTyrLeuGln65707580MetAsnSerLeuLysValGluAspThrGlyValTyrTyrCysAsnIle859095AspSerArgGlyValGlyProValTrpAlaHisTrpGlyGlnGlyThr100105110GlnValThrValSerSer115 210 24 211 129 212 PRT 213 人工合成序列 400 24GlnAlaGlnLeuLysGluSerGlyGlyGlySerValArgThrGlyGlu151015SerLeuArgLeuSerCysGluAlaSerGlyAsnIleTyrSerIleAsn202530ArgMetAlaTrpTyrArgGlnValSerGlyMetGlnArgGluValVal354045AlaThrSerLeuValAspGlyThrThrAsnTyrGlyAspSerValLys505560AspArgPheThrValSerArgAspAsnAlaLysLysMetValPheLeu65707580GlnMetAsnSerLeuGluProAlaAspThrGlyValTyrTyrCysAsn859095ValGluGlyAsnArgIleAspTyrAlaProGlySerArgTyrProThr100105110HisSerTyrValGluLeuTrpGlyGlnGlyThrGlnValThrValSer115120125Ser 210 25 211 123 212 PRT 213 人工合成序列 400 25GlnValGlnLeuValGluAsnGlyGlyGlyLeuValGlnProGlyGly151015SerLeuArgLeuSerCysAlaAlaSerGlyAlaSerPheAsnAspTyr202530HisMetGlyTrpPheArgGlnAlaProGlyGlnGluArgLysPheVal354045AlaGlnIleAlaArgTyrGlyAlaAlaThrTyrTyrAlaArgAlaVal505560GlnGlyArgPheThrIleSerValAspAspAlaLysAsnThrValTyr65707580LeuGlnMetAsnGlyLeuThrProAspAspThrGlyValTyrTyrCys859095ThrAlaAspArgSerAsnTyrTyrIleAspAsnAlaLeuProAspTyr100105110TrpGlyGlnGlyThrGlnValThrValSerSer115120 210 26 211 128 212 PRT 213 人工合成序列 400 26GlnValGlnLeuIleGluSerGlyGlyGlyLeuValGlnAlaGlyThr151015SerLeuThrLeuSerCysAlaSerSerGlyArgAsnPheAsnSerTyr202530AlaMetGlyTrpPheArgGlnAlaProGlyLysGluArgGluPheLeu354045AlaThrIleSerArgAlaAlaGlySerThrTyrTyrAlaAspSerAla505560LysGlyArgPheThrIleSerArgAspAsnArgLysGluPheAlaTyr65707580LeuGlnIleHisAspLeuLysProAspAspThrAlaValTyrTyrCys859095AlaAlaGluSerTrpThrProThrThrGlyTrpProProThrLysAla100105110AspGluPheAspTyrTrpGlyGlnGlyThrGlnValThrValSerSer115120125 210 27 211 122 212 PRT 213 人工合成序列 400 27GlnAspArgLeuValGluSerGlyGlyGlySerValGlnAlaGlyGly151015SerLeuLysLeuSerCysArgThrSerGlyPheAsnLeuAspAspTyr202530AlaIleGlyTrpPheArgGlnAlaProGlyLysGluArgGluArgVal354045SerCysIleSerSerAspGlyArgThrSerHisThrGlySerAlaLys505560GlyArgPheThrIleArgSerAlaAsnAlaArgAsnThrValTyrLeu65707580GlnLeuAsnArgLeuThrProGluAspAlaGlyValTyrPheCysAla859095AlaGluArgThrSerArgLeuCysSerPheLeuSerTyrAspTyrTrp100105110GlyGlnGlyThrGlnValThrValSerSer115120 210 28 211 119 212 PRT 213 人工合成序列 400 28GlnValGlnLeuValGluSerGlyGlyGlyLeuValGlnAlaGlyGly151015SerLeuArgLeuSerCysAlaAlaSerGlyArgThrPheSerAspHis202530ThrLeuGlyTrpPheArgGlnAlaProGlyLysGluArgGluPheVal354045GlyAlaIleSerTrpSerGlyGlySerThrTyrTyrAlaAspSerVal505560SerGlyArgPheThrIleSerArgAspLysAlaLysAsnThrGlyTyr65707580LeuGlnMetAsnSerLeuLysProGluAspThrAlaValTyrTyrCys859095AlaAlaAlaAspAspArgTyrSerAspTyrArgTyrTrpGlyGlnGly100105110ThrGlnValThrValSerSer115 210 29 211 378 212 PRT 213 人工合成序列 400 29CysAlaGlyGlyThrGlyCysThrGlyCysThrGlyGlyThrAlaGly151015AlaGlyThrCysThrGlyGlyGlyGlyGlyAlaGlyGlyAlaThrThr202530GlyGlyThrGlyCysAlaGlyGlyCysThrGlyGlyGlyGlyGlyCys354045ThrCysThrCysThrGlyAlaGlyAlaCysThrCysThrCysCysThr505560GlyThrGlyCysAlaGlyThrGlyThrCysThrGlyGlyAlaCysGly65707580CysAlaCysCysGlyThrCysAlaGlyThrAlaCysCysGlyCysAla859095AlaCysCysAlaThrGlyGlyGlyCysThrGlyGlyThrThrCysCys100105110GlyCysCysAlaGlyGlyCysThrCysCysAlaGlyGlyGlyAlaAla115120125GlyGlyAlaGlyCysGlyThrGlyAlaGlyThrThrThrGlyThrCys130135140GlyCysAlaGlyCysGlyCysThrThrAlaAlaCysThrGlyGlyAla145150155160GlyThrGlyGlyCysAlaAlaThrAlaAlaAlaThrCysAlaThrAla165170175CysThrAlaThrGlyCysThrGlyAlaThrThrCysCysGlyThrGly180185190AlaAlaGlyGlyGlyCysCysGlyAlaThrThrCysGlyCysCysAla195200205ThrCysThrCysCysAlaGlyAlaGlyAlaCysGlyAlaAlaGlyCys210215220CysAlaAlaGlyAlaAlaCysAlaCysGlyGlyThrGlyThrAlaThr225230235240CysThrAlaCysAlaGlyAlaThrGlyAlaAlaCysAlaGlyCysCys245250255ThrGlyAlaAlaAlaCysCysThrGlyAlaGlyGlyAlaCysAlaCys260265270GlyGlyCysCysGlyThrThrThrAlaThrCysAlaCysThrGlyThr275280285GlyCysAlaGlyCysAlaGlyGlyAlaCysCysGlyGlyAlaThrThr290295300ThrAlaAlaAlaThrThrAlaCysThrAlaThrAlaCysCysAlaAla305310315320ThrThrAlaThrGlyAlaCysGlyCysCysCysGlyAlaCysGlyGly325330335ThrAlaThrGlyAlaCysCysAlaThrThrGlyGlyGlyGlyGlyCys340345350AlaGlyGlyGlyGlyAlaCysCysCysAlaGlyGlyThrCysAlaCys355360365CysGlyThrCysThrCysCysThrCysAla370375 210 30 211 354 212 DNA 213 人工合成序列 400 30caggggcagtacgtggagtctgggggcggatcggtccaggctggggagtctctaagactc60tcgtgcatcgggtctcacactactgccatcaacgccgcgggctggtatcgtcagactcca120gggaagcagcgcgaactggtcggcctgattcacaatgatggcagtacgcaatatgcccaa180ttcgcgaagggccgattcactatttccagggacgacgccaaggacgcggtgtacctgcaa240atgaacagcctaaaagttgaagacacgggcgtctattattgcaatatcgactcgcgcgga300gtggggccggtctgggcccactggggcccggggactcaggtcaccgtctcctca354 210 31 211 387 212 DNA 213 人工合成序列 400 31caggcgcagctgaaggagtctgggggaggctcggtgcggactggggagtctctgcggctc60tcctgtgaagcctccggaaatatctacagtatcaatagaatggcctggtaccgccaggtt120tcagggatgcagcgcgaggtggtcgcgaccagtctcgttgatggtacaacgaactatgga180gactccgtgaaggaccgattcaccgtttccagagacaacgccaaaaaaatggtgtttctg240cagatgaatagcctagaacccgcggacacgggcgtctattattgtaatgtcgagggaaat300cggatcgactatgcacctggtagtcgctacccgacacacagttatgtcgaactttggggc360caggggctcgcggtcactgtctcttca387 210 32 211 369 212 DNA 213 人工合成序列 400 32caggtgcaactggtagaaaatgggggaggattggtgcagcctgggggctctctgagactc60tcctgtgcagcctctggtgcctccttcaatgactatcacatgggctggttccgccaggct120ccggggcaggagcgaaagtttgtcgctcagattgcacggtatggtgctgccacgtattat180gcgcgggccgttcagggccggttcaccatctccgtcgacgacgccaagaatacggtgtat240ctgcaaatgaacggtttgacacctgacgacacgggcgtttattactgtacagcagatcgc300agcaattattatatcgacaatgccctgcctgattattggggccaggggacccaggtgacc360gtctcctca369 210 33 211 384 212 DNA 213 人工合成序列 400 33caggtgcagttgatcgagtctggaggaggattggtgcaggcgggaacgtctctgacactc60tcctgtgcgtcctccggacgcaacttcaatagttacgccatgggctggtttcgccaggct120ccaggaaaggagcgtgaatttctggctactattagccgggctgctggcagcacatattat180gctgactccgcgaagggccgattcaccatttctcgagacaaccgcaaggaattcgcgtat240ctacaaatacacgacctgaaacctgacgacacggccgtttattactgtgcagcagagtca300tggaccccgactacgggctggcctccgacgaaggcagatgagtttgactactggggccag360ggtacccaggtcaccgtctcctca384 210 34 211 366 212 DNA 213 人工合成序列 400 34caggaccggctcgtggaatccgggggaggctcggtgcaggctggggggtccctgaaactc60tcctgtcgaacctctggattcaatctcgatgattatgccatcggctggttccgccaggcc120ccaggaaaggagcgtgaaagggtctcttgtattagtagtgatggccggacgtcacatacc180ggctccgcgaagggccgatttaccatcaggagcgccaacgcccggaatacggtgtatctc240caactgaatcgcctgacccctgaagacgcgggcgtttatttttgtgctgcagagcggacg300agtcgattgtgttccttcctatcgtatgattactggggccaggggacccaggtcaccgtc360tcctca366 210 35 211 357 212 DNA 213 人工合成序列 400 35caggtgcagctggtagagtctgggggaggattggtgcaggctgggggctctctgagactc60tcctgtgcagcctctggacgtaccttcagtgaccataccctgggctggttccgccaggct120cccgggaaggagcgtgagtttgtaggagctatttcctggagtggtggtagcacatactat180gcagactccgtgagcggccgattcaccatctctcgagacaaggccaagaacacgggctat240ctgcaaatgaacagcctgaaacctgaggacacggccgtttattactgtgcagcagccgac300gatcgctatagtgactatcgctactggggccaggggacccaggtcaccgtctcctca357 210 36 211 370 212 PRT 213 人工合成序列 400 36MetAlaLeuProValThrAlaLeuLeuLeuProLeuAlaLeuLeuLeu151015HisAlaAlaArgProGlnValLeuLeuValGluSerGlyGlyGlyLeu202530ValGlnAlaGlyGlySerLeuArgLeuSerCysAlaValSerGlyArg354045ThrValSerThrAlaThrMetGlyTrpPheArgGlnAlaProGlyLys505560GluArgGluPheValAlaAlaLeuAsnTrpSerGlyAsnLysSerTyr65707580TyrAlaAspSerValLysGlyArgPheAlaIleSerArgAspGluAla859095LysAsnThrValTyrLeuGlnMetAsnSerLeuLysProGluAspThr100105110AlaValTyrHisCysAlaAlaGlyProAspLeuAsnTyrTyrThrAsn115120125TyrAspAlaArgArgTyrAspHisTrpGlyGlnGlyThrGlnValThr130135140ValSerSerThrThrThrProAlaProArgProProThrProAlaPro145150155160ThrIleAlaSerGlnProLeuSerLeuArgProGluAlaCysArgPro165170175AlaAlaGlyGlyAlaValHisThrArgGlyLeuAspPheAlaCysAsp180185190IleTyrIleTrpAlaProLeuAlaGlyThrCysGlyValLeuLeuLeu195200205SerLeuValIleThrLeuTyrCysLysArgGlyArgLysLysLeuLeu210215220TyrIlePheLysGlnProPheMetArgProValGlnThrThrGlnGlu225230235240GluAspGlyCysSerCysArgPheProGluGluGluGluGlyGlyCys245250255GluLeuArgValLysPheSerArgSerAlaAspAlaProAlaTyrGln260265270GlnGlyGlnAsnGlnLeuTyrAsnGluLeuAsnLeuGlyArgArgGlu275280285GluTyrAspValLeuAspLysArgArgGlyArgAspProGluMetGly290295300GlyLysProArgArgLysAsnProGlnGluGlyLeuTyrAsnGluLeu305310315320GlnLysAspLysMetAlaGluAlaTyrSerGluIleGlyMetLysGly325330335GluArgArgArgGlyLysGlyHisAspGlyLeuTyrGlnGlyLeuSer340345350ThrAlaThrLysAspThrTyrAspAlaLeuHisMetGlnAlaLeuPro355360365ProArg370 210 37 211 362 212 PRT 213 人工合成序列 400 37MetAlaLeuProValThrAlaLeuLeuLeuProLeuAlaLeuLeuLeu151015HisAlaAlaArgProGlnGlyGlnTyrValGluSerGlyGlyGlySer202530ValGlnAlaGlyGluSerLeuArgLeuSerCysIleGlySerHisThr354045ThrAlaIleAsnAlaAlaGlyTrpTyrArgGlnThrProGlyLysGln505560ArgGluLeuValGlyLeuIleHisAsnAspGlySerThrGlnTyrAla65707580GlnPheAlaLysGlyArgPheThrIleSerArgAspAspAlaLysAsp859095AlaValTyrLeuGlnMetAsnSerLeuLysValGluAspThrGlyVal100105110TyrTyrCysAsnIleAspSerArgGlyValGlyProValTrpAlaHis115120125TrpGlyGlnGlyThrGlnValThrValSerSerThrThrThrProAla130135140ProArgProProThrProAlaProThrIleAlaSerGlnProLeuSer145150155160LeuArgProGluAlaCysArgProAlaAlaGlyGlyAlaValHisThr165170175ArgGlyLeuAspPheAlaCysAspIleTyrIleTrpAlaProLeuAla180185190GlyThrCysGlyValLeuLeuLeuSerLeuValIleThrLeuTyrCys195200205LysArgGlyArgLysLysLeuLeuTyrIlePheLysGlnProPheMet210215220ArgProValGlnThrThrGlnGluGluAspGlyCysSerCysArgPhe225230235240ProGluGluGluGluGlyGlyCysGluLeuArgValLysPheSerArg245250255SerAlaAspAlaProAlaTyrGlnGlnGlyGlnAsnGlnLeuTyrAsn260265270GluLeuAsnLeuGlyArgArgGluGluTyrAspValLeuAspLysArg275280285ArgGlyArgAspProGluMetGlyGlyLysProArgArgLysAsnPro290295300GlnGluGlyLeuTyrAsnGluLeuGlnLysAspLysMetAlaGluAla305310315320TyrSerGluIleGlyMetLysGlyGluArgArgArgGlyLysGlyHis325330335AspGlyLeuTyrGlnGlyLeuSerThrAlaThrLysAspThrTyrAsp340345350AlaLeuHisMetGlnAlaLeuProProArg355360 210 38 211 373 212 PRT 213 人工合成序列 400 38MetAlaLeuProValThrAlaLeuLeuLeuProLeuAlaLeuLeuLeu151015HisAlaAlaArgProGlnAlaGlnLeuLysGluSerGlyGlyGlySer202530ValArgThrGlyGluSerLeuArgLeuSerCysGluAlaSerGlyAsn354045IleTyrSerIleAsnArgMetAlaTrpTyrArgGlnValSerGlyMet505560GlnArgGluValValAlaThrSerLeuValAspGlyThrThrAsnTyr65707580GlyAspSerValLysAspArgPheThrValSerArgAspAsnAlaLys859095LysMetValPheLeuGlnMetAsnSerLeuGluProAlaAspThrGly100105110ValTyrTyrCysAsnValGluGlyAsnArgIleAspTyrAlaProGly115120125SerArgTyrProThrHisSerTyrValGluLeuTrpGlyGlnGlyThr130135140GlnValThrValSerSerThrThrThrProAlaProArgProProThr145150155160ProAlaProThrIleAlaSerGlnProLeuSerLeuArgProGluAla165170175CysArgProAlaAlaGlyGlyAlaValHisThrArgGlyLeuAspPhe180185190AlaCysAspIleTyrIleTrpAlaProLeuAlaGlyThrCysGlyVal195200205LeuLeuLeuSerLeuValIleThrLeuTyrCysLysArgGlyArgLys210215220LysLeuLeuTyrIlePheLysGlnProPheMetArgProValGlnThr225230235240ThrGlnGluGluAspGlyCysSerCysArgPheProGluGluGluGlu245250255GlyGlyCysGluLeuArgValLysPheSerArgSerAlaAspAlaPro260265270AlaTyrGlnGlnGlyGlnAsnGlnLeuTyrAsnGluLeuAsnLeuGly275280285ArgArgGluGluTyrAspValLeuAspLysArgArgGlyArgAspPro290295300GluMetGlyGlyLysProArgArgLysAsnProGlnGluGlyLeuTyr305310315320AsnGluLeuGlnLysAspLysMetAlaGluAlaTyrSerGluIleGly325330335MetLysGlyGluArgArgArgGlyLysGlyHisAspGlyLeuTyrGln340345350GlyLeuSerThrAlaThrLysAspThrTyrAspAlaLeuHisMetGln355360365AlaLeuProProArg370 210 39 211 367 212 PRT 213 人工合成序列 400 39MetAlaLeuProValThrAlaLeuLeuLeuProLeuAlaLeuLeuLeu151015HisAlaAlaArgProGlnValGlnLeuValGluAsnGlyGlyGlyLeu202530ValGlnProGlyGlySerLeuArgLeuSerCysAlaAlaSerGlyAla354045SerPheAsnAspTyrHisMetGlyTrpPheArgGlnAlaProGlyGln505560GluArgLysPheValAlaGlnIleAlaArgTyrGlyAlaAlaThrTyr65707580TyrAlaArgAlaValGlnGlyArgPheThrIleSerValAspAspAla859095LysAsnThrValTyrLeuGlnMetAsnGlyLeuThrProAspAspThr100105110GlyValTyrTyrCysThrAlaAspArgSerAsnTyrTyrIleAspAsn115120125AlaLeuProAspTyrTrpGlyGlnGlyThrGlnValThrValSerSer130135140ThrThrThrProAlaProArgProProThrProAlaProThrIleAla145150155160SerGlnProLeuSerLeuArgProGluAlaCysArgProAlaAlaGly165170175GlyAlaValHisThrArgGlyLeuAspPheAlaCysAspIleTyrIle180185190TrpAlaProLeuAlaGlyThrCysGlyValLeuLeuLeuSerLeuVal195200205IleThrLeuTyrCysLysArgGlyArgLysLysLeuLeuTyrIlePhe210215220LysGlnProPheMetArgProValGlnThrThrGlnGluGluAspGly225230235240CysSerCysArgPheProGluGluGluGluGlyGlyCysGluLeuArg245250255ValLysPheSerArgSerAlaAspAlaProAlaTyrGlnGlnGlyGln260265270AsnGlnLeuTyrAsnGluLeuAsnLeuGlyArgArgGluGluTyrAsp275280285ValLeuAspLysArgArgGlyArgAspProGluMetGlyGlyLysPro290295300ArgArgLysAsnProGlnGluGlyLeuTyrAsnGluLeuGlnLysAsp305310315320LysMetAlaGluAlaTyrSerGluIleGlyMetLysGlyGluArgArg325330335ArgGlyLysGlyHisAspGlyLeuTyrGlnGlyLeuSerThrAlaThr340345350LysAspThrTyrAspAlaLeuHisMetGlnAlaLeuProProArg355360365 210 40 211 372 212 PRT 213 人工合成序列 400 40MetAlaLeuProValThrAlaLeuLeuLeuProLeuAlaLeuLeuLeu151015HisAlaAlaArgProGlnValGlnLeuIleGluSerGlyGlyGlyLeu202530ValGlnAlaGlyThrSerLeuThrLeuSerCysAlaSerSerGlyArg354045AsnPheAsnSerTyrAlaMetGlyTrpPheArgGlnAlaProGlyLys505560GluArgGluPheLeuAlaThrIleSerArgAlaAlaGlySerThrTyr65707580TyrAlaAspSerAlaLysGlyArgPheThrIleSerArgAspAsnArg859095LysGluPheAlaTyrLeuGlnIleHisAspLeuLysProAspAspThr100105110AlaValTyrTyrCysAlaAlaGluSerTrpThrProThrThrGlyTrp115120125ProProThrLysAlaAspGluPheAspTyrTrpGlyGlnGlyThrGln130135140ValThrValSerSerThrThrThrProAlaProArgProProThrPro145150155160AlaProThrIleAlaSerGlnProLeuSerLeuArgProGluAlaCys165170175ArgProAlaAlaGlyGlyAlaValHisThrArgGlyLeuAspPheAla180185190CysAspIleTyrIleTrpAlaProLeuAlaGlyThrCysGlyValLeu195200205LeuLeuSerLeuValIleThrLeuTyrCysLysArgGlyArgLysLys210215220LeuLeuTyrIlePheLysGlnProPheMetArgProValGlnThrThr225230235240GlnGluGluAspGlyCysSerCysArgPheProGluGluGluGluGly245250255GlyCysGluLeuArgValLysPheSerArgSerAlaAspAlaProAla260265270TyrGlnGlnGlyGlnAsnGlnLeuTyrAsnGluLeuAsnLeuGlyArg275280285ArgGluGluTyrAspValLeuAspLysArgArgGlyArgAspProGlu290295300MetGlyGlyLysProArgArgLysAsnProGlnGluGlyLeuTyrAsn305310315320GluLeuGlnLysAspLysMetAlaGluAlaTyrSerGluIleGlyMet325330335LysGlyGluArgArgArgGlyLysGlyHisAspGlyLeuTyrGlnGly340345350LeuSerThrAlaThrLysAspThrTyrAspAlaLeuHisMetGlnAla355360365LeuProProArg370 210 41 211 366 212 PRT 213 人工合成序列 400 41MetAlaLeuProValThrAlaLeuLeuLeuProLeuAlaLeuLeuLeu151015HisAlaAlaArgProGlnAspArgLeuValGluSerGlyGlyGlySer202530ValGlnAlaGlyGlySerLeuLysLeuSerCysArgThrSerGlyPhe354045AsnLeuAspAspTyrAlaIleGlyTrpPheArgGlnAlaProGlyLys505560GluArgGluArgValSerCysIleSerSerAspGlyArgThrSerHis65707580ThrGlySerAlaLysGlyArgPheThrIleArgSerAlaAsnAlaArg859095AsnThrValTyrLeuGlnLeuAsnArgLeuThrProGluAspAlaGly100105110ValTyrPheCysAlaAlaGluArgThrSerArgLeuCysSerPheLeu115120125SerTyrAspTyrTrpGlyGlnGlyThrGlnValThrValSerSerThr130135140ThrThrProAlaProArgProProThrProAlaProThrIleAlaSer145150155160GlnProLeuSerLeuArgProGluAlaCysArgProAlaAlaGlyGly165170175AlaValHisThrArgGlyLeuAspPheAlaCysAspIleTyrIleTrp180185190AlaProLeuAlaGlyThrCysGlyValLeuLeuLeuSerLeuValIle195200205ThrLeuTyrCysLysArgGlyArgLysLysLeuLeuTyrIlePheLys210215220GlnProPheMetArgProValGlnThrThrGlnGluGluAspGlyCys225230235240SerCysArgPheProGluGluGluGluGlyGlyCysGluLeuArgVal245250255LysPheSerArgSerAlaAspAlaProAlaTyrGlnGlnGlyGlnAsn260265270GlnLeuTyrAsnGluLeuAsnLeuGlyArgArgGluGluTyrAspVal275280285LeuAspLysArgArgGlyArgAspProGluMetGlyGlyLysProArg290295300ArgLysAsnProGlnGluGlyLeuTyrAsnGluLeuGlnLysAspLys305310315320MetAlaGluAlaTyrSerGluIleGlyMetLysGlyGluArgArgArg325330335GlyLysGlyHisAspGlyLeuTyrGlnGlyLeuSerThrAlaThrLys340345350AspThrTyrAspAlaLeuHisMetGlnAlaLeuProProArg355360365 210 42 211 363 212 PRT 213 人工合成序列 400 42MetAlaLeuProValThrAlaLeuLeuLeuProLeuAlaLeuLeuLeu151015HisAlaAlaArgProGlnValGlnLeuValGluSerGlyGlyGlyLeu202530ValGlnAlaGlyGlySerLeuArgLeuSerCysAlaAlaSerGlyArg354045ThrPheSerAspHisThrLeuGlyTrpPheArgGlnAlaProGlyLys505560GluArgGluPheValGlyAlaIleSerTrpSerGlyGlySerThrTyr65707580TyrAlaAspSerValSerGlyArgPheThrIleSerArgAspLysAla859095LysAsnThrGlyTyrLeuGlnMetAsnSerLeuLysProGluAspThr100105110AlaValTyrTyrCysAlaAlaAlaAspAspArgTyrSerAspTyrArg115120125TyrTrpGlyGlnGlyThrGlnValThrValSerSerThrThrThrPro130135140AlaProArgProProThrProAlaProThrIleAlaSerGlnProLeu145150155160SerLeuArgProGluAlaCysArgProAlaAlaGlyGlyAlaValHis165170175ThrArgGlyLeuAspPheAlaCysAspIleTyrIleTrpAlaProLeu180185190AlaGlyThrCysGlyValLeuLeuLeuSerLeuValIleThrLeuTyr195200205CysLysArgGlyArgLysLysLeuLeuTyrIlePheLysGlnProPhe210215220MetArgProValGlnThrThrGlnGluGluAspGlyCysSerCysArg225230235240PheProGluGluGluGluGlyGlyCysGluLeuArgValLysPheSer245250255ArgSerAlaAspAlaProAlaTyrGlnGlnGlyGlnAsnGlnLeuTyr260265270AsnGluLeuAsnLeuGlyArgArgGluGluTyrAspValLeuAspLys275280285ArgArgGlyArgAspProGluMetGlyGlyLysProArgArgLysAsn290295300ProGlnGluGlyLeuTyrAsnGluLeuGlnLysAspLysMetAlaGlu305310315320AlaTyrSerGluIleGlyMetLysGlyGluArgArgArgGlyLysGly325330335HisAspGlyLeuTyrGlnGlyLeuSerThrAlaThrLysAspThrTyr340345350AspAlaLeuHisMetGlnAlaLeuProProArg355360 210 43 211 1086 212 DNA 213 人工合成序列 400 43atggcactgccagtgacagccctgctgctgccactggccctgctgctgcacgcagcacgc60cctcaggtgctgctggtagagtctgggggaggattggtgcaggctgggggctctctgaga120ctctcctgtgcagtgtctggacgcaccgtcagtaccgcaaccatgggctggttccgccag180gctccagggaaggagcgtgagtttgtcgcagcgcttaactggagtggcaataaatcatac240tatgctgattccgtgaagggccgattcgccatctccagagacgaagccaagaacacggtg300tatctacagatgaacagcctgaaacctgaggacacggccgtttatcactgtgcagcagga360ccggatttaaattactataccaattatgacgcccgacggtatgaccattgggggcagggg420acccaggtcaccgtctcctcactgaaacctgaggacacggccgtttattactgtgcagca480gccgacgatcgctatagtgactatcgctactggggccaggggacccaggtcaccgtctcc540tcaaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagccc600ctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgaggggg660ctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtcctt720ctcctgtcactggttatcaccctttactgcagggtgaagttttctcggagcgccgatgca780ccagcatatcagcagggacagaatcagctgtacaacgagctgaatctgggcaggcgcgag840gagtacgacgtgctggataagcggagaggcagagatcccgagatgggaggcaagccaagg900aggaagaaccctcaggagggcctgtataatgagctgcagaaggacaagatggccgaggcc960tactctgagatcggcatgaagggagagcggagaaggggcaagggacacgatggcctgtat1020cagggcctgagcacagccaccaaggacacctacgatgcactgcacatgcaggccctgcca1080cctagg1086 210 44 211 1062 212 DNA 213 人工合成序列 400 44atggcactgccagtgacagccctgctgctgccactggccctgctgctgcacgcagcacgc60cctcaggggcagtacgtggagtctgggggcggatcggtccaggctggggagtctctaaga120ctctcgtgcatcgggtctcacactactgccatcaacgccgcgggctggtatcgtcagact180ccagggaagcagcgcgaactggtcggcctgattcacaatgatggcagtacgcaatatgcc240caattcgcgaagggccgattcactatttccagggacgacgccaaggacgcggtgtacctg300caaatgaacagcctaaaagttgaagacacgggcgtctattattgcaatatcgactcgcgc360ggagtggggccggtctgggcccactggggcccggggactcaggtcaccgtctcctcactg420aaacctgaggacacggccgtttattactgtgcagcagccgacgatcgctatagtgactat480cgctactggggccaggggacccaggtcaccgtctcctcaaccacgacgccagcgccgcga540ccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgc600cggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctac660atctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctt720tactgcagggtgaagttttctcggagcgccgatgcaccagcatatcagcagggacagaat780cagctgtacaacgagctgaatctgggcaggcgcgaggagtacgacgtgctggataagcgg840agaggcagagatcccgagatgggaggcaagccaaggaggaagaaccctcaggagggcctg900tataatgagctgcagaaggacaagatggccgaggcctactctgagatcggcatgaaggga960gagcggagaaggggcaagggacacgatggcctgtatcagggcctgagcacagccaccaag1020gacacctacgatgcactgcacatgcaggccctgccacctagg1062 210 45 211 1095 212 DNA 213 人工合成序列 400 45atggcactgccagtgacagccctgctgctgccactggccctgctgctgcacgcagcacgc60cctcaggcgcagctgaaggagtctgggggaggctcggtgcggactggggagtctctgcgg120ctctcctgtgaagcctccggaaatatctacagtatcaatagaatggcctggtaccgccag180gtttcagggatgcagcgcgaggtggtcgcgaccagtctcgttgatggtacaacgaactat240ggagactccgtgaaggaccgattcaccgtttccagagacaacgccaaaaaaatggtgttt300ctgcagatgaatagcctagaacccgcggacacgggcgtctattattgtaatgtcgaggga360aatcggatcgactatgcacctggtagtcgctacccgacacacagttatgtcgaactttgg420ggccaggggctcgcggtcactgtctcttcactgaaacctgaggacacggccgtttattac480tgtgcagcagccgacgatcgctatagtgactatcgctactggggccaggggacccaggtc540accgtctcctcaaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcg600tcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcac660acgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgt720ggggtccttctcctgtcactggttatcaccctttactgcagggtgaagttttctcggagc780gccgatgcaccagcatatcagcagggacagaatcagctgtacaacgagctgaatctgggc840aggcgcgaggagtacgacgtgctggataagcggagaggcagagatcccgagatgggaggc900aagccaaggaggaagaaccctcaggagggcctgtataatgagctgcagaaggacaagatg960gccgaggcctactctgagatcggcatgaagggagagcggagaaggggcaagggacacgat1020ggcctgtatcagggcctgagcacagccaccaaggacacctacgatgcactgcacatgcag1080gccctgccacctagg1095 210 46 211 1077 212 DNA 213 人工合成序列 400 46atggcactgccagtgacagccctgctgctgccactggccctgctgctgcacgcagcacgc60cctcaggtgcaactggtagaaaatgggggaggattggtgcagcctgggggctctctgaga120ctctcctgtgcagcctctggtgcctccttcaatgactatcacatgggctggttccgccag180gctccggggcaggagcgaaagtttgtcgctcagattgcacggtatggtgctgccacgtat240tatgcgcgggccgttcagggccggttcaccatctccgtcgacgacgccaagaatacggtg300tatctgcaaatgaacggtttgacacctgacgacacgggcgtttattactgtacagcagat360cgcagcaattattatatcgacaatgccctgcctgattattggggccaggggacccaggtg420accgtctcctcactgaaacctgaggacacggccgtttattactgtgcagcagccgacgat480cgctatagtgactatcgctactggggccaggggacccaggtcaccgtctcctcaaccacg540acgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctg600cgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttc660gcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtca720ctggttatcaccctttactgcagggtgaagttttctcggagcgccgatgcaccagcatat780cagcagggacagaatcagctgtacaacgagctgaatctgggcaggcgcgaggagtacgac840gtgctggataagcggagaggcagagatcccgagatgggaggcaagccaaggaggaagaac900cctcaggagggcctgtataatgagctgcagaaggacaagatggccgaggcctactctgag960atcggcatgaagggagagcggagaaggggcaagggacacgatggcctgtatcagggcctg1020agcacagccaccaaggacacctacgatgcactgcacatgcaggccctgccacctagg1077 210 47 211 1092 212 DNA 213 人工合成序列 400 47atggcactgccagtgacagccctgctgctgccactggccctgctgctgcacgcagcacgc60cctcaggtgcagttgatcgagtctggaggaggattggtgcaggcgggaacgtctctgaca120ctctcctgtgcgtcctccggacgcaacttcaatagttacgccatgggctggtttcgccag180gctccaggaaaggagcgtgaatttctggctactattagccgggctgctggcagcacatat240tatgctgactccgcgaagggccgattcaccatttctcgagacaaccgcaaggaattcgcg300tatctacaaatacacgacctgaaacctgacgacacggccgtttattactgtgcagcagag360tcatggaccccgactacgggctggcctccgacgaaggcagatgagtttgactactggggc420cagggtacccaggtcaccgtctcctcactgaaacctgaggacacggccgtttattactgt480gcagcagccgacgatcgctatagtgactatcgctactggggccaggggacccaggtcacc540gtctcctcaaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcg600cagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacg660agggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggg720gtccttctcctgtcactggttatcaccctttactgcagggtgaagttttctcggagcgcc780gatgcaccagcatatcagcagggacagaatcagctgtacaacgagctgaatctgggcagg840cgcgaggagtacgacgtgctggataagcggagaggcagagatcccgagatgggaggcaag900ccaaggaggaagaaccctcaggagggcctgtataatgagctgcagaaggacaagatggcc960gaggcctactctgagatcggcatgaagggagagcggagaaggggcaagggacacgatggc1020ctgtatcagggcctgagcacagccaccaaggacacctacgatgcactgcacatgcaggcc1080ctgccacctagg1092 210 48 211 1074 212 DNA 213 人工合成序列 400 48atggcactgccagtgacagccctgctgctgccactggccctgctgctgcacgcagcacgc60cctcaggaccggctcgtggaatccgggggaggctcggtgcaggctggggggtccctgaaa120ctctcctgtcgaacctctggattcaatctcgatgattatgccatcggctggttccgccag180gccccaggaaaggagcgtgaaagggtctcttgtattagtagtgatggccggacgtcacat240accggctccgcgaagggccgatttaccatcaggagcgccaacgcccggaatacggtgtat300ctccaactgaatcgcctgacccctgaagacgcgggcgtttatttttgtgctgcagagcgg360acgagtcgattgtgttccttcctatcgtatgattactggggccaggggacccaggtcacc420gtctcctcactgaaacctgaggacacggccgtttattactgtgcagcagccgacgatcgc480tatagtgactatcgctactggggccaggggacccaggtcaccgtctcctcaaccacgacg540ccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgc600ccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcc660tgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactg720gttatcaccctttactgcagggtgaagttttctcggagcgccgatgcaccagcatatcag780cagggacagaatcagctgtacaacgagctgaatctgggcaggcgcgaggagtacgacgtg840ctggataagcggagaggcagagatcccgagatgggaggcaagccaaggaggaagaaccct900caggagggcctgtataatgagctgcagaaggacaagatggccgaggcctactctgagatc960ggcatgaagggagagcggagaaggggcaagggacacgatggcctgtatcagggcctgagc1020acagccaccaaggacacctacgatgcactgcacatgcaggccctgccacctagg1074 210 49 211 1065 212 DNA 213 人工合成序列 400 49atggcactgccagtgacagccctgctgctgccactggccctgctgctgcacgcagcacgc60cctcaggtgcagctggtagagtctgggggaggattggtgcaggctgggggctctctgaga120ctctcctgtgcagcctctggacgtaccttcagtgaccataccctgggctggttccgccag180gctcccgggaaggagcgtgagtttgtaggagctatttcctggagtggtggtagcacatac240tatgcagactccgtgagcggccgattcaccatctctcgagacaaggccaagaacacgggc300tatctgcaaatgaacagcctgaaacctgaggacacggccgtttattactgtgcagcagcc360gacgatcgctatagtgactatcgctactggggccaggggacccaggtcaccgtctcctca420ctgaaacctgaggacacggccgtttattactgtgcagcagccgacgatcgctatagtgac480tatcgctactggggccaggggacccaggtcaccgtctcctcaaccacgacgccagcgccg540cgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcg600tgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatc660tacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcacc720ctttactgcagggtgaagttttctcggagcgccgatgcaccagcatatcagcagggacag780aatcagctgtacaacgagctgaatctgggcaggcgcgaggagtacgacgtgctggataag840cggagaggcagagatcccgagatgggaggcaagccaaggaggaagaaccctcaggagggc900ctgtataatgagctgcagaaggacaagatggccgaggcctactctgagatcggcatgaag960ggagagcggagaaggggcaagggacacgatggcctgtatcagggcctgagcacagccacc1020aaggacacctacgatgcactgcacatgcaggccctgccacctagg1065 210 50 211 21 212 PRT 213 人工合成序列 400 50MetAlaLeuProValThrAlaLeuLeuLeuProLeuAlaLeuLeuLeu151015HisAlaAlaArgPro20 210 51 211 63 212 DNA 213 人工合成序列 400 51atggcactgccagtgacagccctgctgctgccactggccctgctgctgcacgcagcacgc60cct63 210 52 211 45 212 PRT 213 人工合成序列 400 52ThrThrThrProAlaProArgProProThrProAlaProThrIleAla151015SerGlnProLeuSerLeuArgProGluAlaCysArgProAlaAlaGly202530GlyAlaValHisThrArgGlyLeuAspPheAlaCysAsp354045 210 53 211 135 212 DNA 213 人工合成序列 400 53accacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctg60tccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctg120gacttcgcctgtgat135 210 54 211 24 212 PRT 213 人工合成序列 400 54IleTyrIleTrpAlaProLeuAlaGlyThrCysGlyValLeuLeuLeu151015SerLeuValIleThrLeuTyrCys20 210 55 211 72 212 DNA 213 人工合成序列 400 55atctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatc60accctttactgc72 210 56 211 42 212 PRT 213 人工合成序列 400 56LysArgGlyArgLysLysLeuLeuTyrIlePheLysGlnProPheMet151015ArgProValGlnThrThrGlnGluGluAspGlyCysSerCysArgPhe202530ProGluGluGluGluGlyGlyCysGluLeu3540 210 57 211 126 212 DNA 213 人工合成序列 400 57aagagaggcaggaagaagctgctgtacatcttcaagcagcccttcatgcgccccgtgcag60acaacccaggaggaggacggctgcagctgtcggttcccagaggaggaggagggaggatgt120gagctg126 210 58 211 112 212 PRT 213 人工合成序列 400 58ArgValLysPheSerArgSerAlaAspAlaProAlaTyrGlnGlnGly151015GlnAsnGlnLeuTyrAsnGluLeuAsnLeuGlyArgArgGluGluTyr202530AspValLeuAspLysArgArgGlyArgAspProGluMetGlyGlyLys354045ProArgArgLysAsnProGlnGluGlyLeuTyrAsnGluLeuGlnLys505560AspLysMetAlaGluAlaTyrSerGluIleGlyMetLysGlyGluArg65707580ArgArgGlyLysGlyHisAspGlyLeuTyrGlnGlyLeuSerThrAla859095ThrLysAspThrTyrAspAlaLeuHisMetGlnAlaLeuProProArg100105110 210 59 211 336 212 DNA 213 人工合成序列 400 59agggtgaagttttctcggagcgccgatgcaccagcatatcagcagggacagaatcagctg60tacaacgagctgaatctgggcaggcgcgaggagtacgacgtgctggataagcggagaggc120agagatcccgagatgggaggcaagccaaggaggaagaaccctcaggagggcctgtataat180gagctgcagaaggacaagatggccgaggcctactctgagatcggcatgaagggagagcgg240agaaggggcaagggacacgatggcctgtatcagggcctgagcacagccaccaaggacacc300tacgatgcactgcacatgcaggccctgccacctagg336 210 60 211 182 212 PRT 213 人工合成序列 400 60MetAlaValValLeuAlaAlaLeuLeuGlnSerValGlnAlaGlnVal151015LeuLeuValGluSerGlyGlyGlyLeuValGlnAlaGlyGlySerLeu202530ArgLeuSerCysAlaValSerGlyArgThrValSerThrAlaThrMet354045GlyTrpPheArgGlnAlaProGlyLysGluArgGluPheValAlaAla505560LeuAsnTrpSerGlyAsnLysSerTyrTyrAlaAspSerValLysGly65707580ArgPheAlaIleSerArgAspGluAlaLysAsnThrValTyrLeuGln859095MetAsnSerLeuLysProGluAspThrAlaValTyrHisCysAlaAla100105110GlyProAspLeuAsnTyrTyrThrAsnTyrAspAlaArgArgTyrAsp115120125HisTrpGlyGlnGlyThrGlnValThrValSerSerGluProLysThr130135140ProLysProGlnProGlnProGlnProGlnProAsnProThrThrGlu145150155160SerLysCysProLysCysProAlaProGluLeuLeuGlyGlyProSer165170175HisHisHisHisHisHis180 210 61 211 174 212 PRT 213 人工合成序列 400 61MetAlaValValLeuAlaAlaLeuLeuGlnSerValGlnAlaGlnGly151015GlnTyrValGluSerGlyGlyGlySerValGlnAlaGlyGluSerLeu202530ArgLeuSerCysIleGlySerHisThrThrAlaIleAsnAlaAlaGly354045TrpTyrArgGlnThrProGlyLysGlnArgGluLeuValGlyLeuIle505560HisAsnAspGlySerThrGlnTyrAlaGlnPheAlaLysGlyArgPhe65707580ThrIleSerArgAspAspAlaLysAspAlaValTyrLeuGlnMetAsn859095SerLeuLysValGluAspThrGlyValTyrTyrCysAsnIleAspSer100105110ArgGlyValGlyProValTrpAlaHisTrpGlyGlnGlyThrGlnVal115120125ThrValSerSerGluProLysThrProLysProGlnProGlnProGln130135140ProGlnProAsnProThrThrGluSerLysCysProLysCysProAla145150155160ProGluLeuLeuGlyGlyProSerHisHisHisHisHisHis165170 210 62 211 185 212 PRT 213 人工合成序列 400 62MetAlaValValLeuAlaAlaLeuLeuGlnSerValGlnAlaGlnAla151015GlnLeuLysGluSerGlyGlyGlySerValArgThrGlyGluSerLeu202530ArgLeuSerCysGluAlaSerGlyAsnIleTyrSerIleAsnArgMet354045AlaTrpTyrArgGlnValSerGlyMetGlnArgGluValValAlaThr505560SerLeuValAspGlyThrThrAsnTyrGlyAspSerValLysAspArg65707580PheThrValSerArgAspAsnAlaLysLysMetValPheLeuGlnMet859095AsnSerLeuGluProAlaAspThrGlyValTyrTyrCysAsnValGlu100105110GlyAsnArgIleAspTyrAlaProGlySerArgTyrProThrHisSer115120125TyrValGluLeuTrpGlyGlnGlyThrGlnValThrValSerSerGlu130135140ProLysThrProLysProGlnProGlnProGlnProGlnProAsnPro145150155160ThrThrGluSerLysCysProLysCysProAlaProGluLeuLeuGly165170175GlyProSerHisHisHisHisHisHis180185 210 63 211 179 212 PRT 213 人工合成序列 400 63MetAlaValValLeuAlaAlaLeuLeuGlnSerValGlnAlaGlnVal151015GlnLeuValGluAsnGlyGlyGlyLeuValGlnProGlyGlySerLeu202530ArgLeuSerCysAlaAlaSerGlyAlaSerPheAsnAspTyrHisMet354045GlyTrpPheArgGlnAlaProGlyGlnGluArgLysPheValAlaGln505560IleAlaArgTyrGlyAlaAlaThrTyrTyrAlaArgAlaValGlnGly65707580ArgPheThrIleSerValAspAspAlaLysAsnThrValTyrLeuGln859095MetAsnGlyLeuThrProAspAspThrGlyValTyrTyrCysThrAla100105110AspArgSerAsnTyrTyrIleAspAsnAlaLeuProAspTyrTrpGly115120125GlnGlyThrGlnValThrValSerSerGluProLysThrProLysPro130135140GlnProGlnProGlnProGlnProAsnProThrThrGluSerLysCys145150155160ProLysCysProAlaProGluLeuLeuGlyGlyProSerHisHisHis165170175HisHisHis 210 64 211 184 212 PRT 213 人工合成序列 400 64MetAlaValValLeuAlaAlaLeuLeuGlnSerValGlnAlaGlnVal151015GlnLeuIleGluSerGlyGlyGlyLeuValGlnAlaGlyThrSerLeu202530ThrLeuSerCysAlaSerSerGlyArgAsnPheAsnSerTyrAlaMet354045GlyTrpPheArgGlnAlaProGlyLysGluArgGluPheLeuAlaThr505560IleSerArgAlaAlaGlySerThrTyrTyrAlaAspSerAlaLysGly65707580ArgPheThrIleSerArgAspAsnArgLysGluPheAlaTyrLeuGln859095IleHisAspLeuLysProAspAspThrAlaValTyrTyrCysAlaAla100105110GluSerTrpThrProThrThrGlyTrpProProThrLysAlaAspGlu115120125PheAspTyrTrpGlyGlnGlyThrGlnValThrValSerSerGluPro130135140LysThrProLysProGlnProGlnProGlnProGlnProAsnProThr145150155160ThrGluSerLysCysProLysCysProAlaProGluLeuLeuGlyGly165170175ProSerHisHisHisHisHisHis180 210 65 211 178 212 PRT 213 人工合成序列 400 65MetAlaValValLeuAlaAlaLeuLeuGlnSerValGlnAlaGlnAsp151015ArgLeuValGluSerGlyGlyGlySerValGlnAlaGlyGlySerLeu202530LysLeuSerCysArgThrSerGlyPheAsnLeuAspAspTyrAlaIle354045GlyTrpPheArgGlnAlaProGlyLysGluArgGluArgValSerCys505560IleSerSerAspGlyArgThrSerHisThrGlySerAlaLysGlyArg65707580PheThrIleArgSerAlaAsnAlaArgAsnThrValTyrLeuGlnLeu859095AsnArgLeuThrProGluAspAlaGlyValTyrPheCysAlaAlaGlu100105110ArgThrSerArgLeuCysSerPheLeuSerTyrAspTyrTrpGlyGln115120125GlyThrGlnValThrValSerSerGluProLysThrProLysProGln130135140ProGlnProGlnProGlnProAsnProThrThrGluSerLysCysPro145150155160LysCysProAlaProGluLeuLeuGlyGlyProSerHisHisHisHis165170175HisHis 210 66 211 175 212 PRT 213 人工合成序列 400 66MetAlaValValLeuAlaAlaLeuLeuGlnSerValGlnAlaGlnVal151015GlnLeuValGluSerGlyGlyGlyLeuValGlnAlaGlyGlySerLeu202530ArgLeuSerCysAlaAlaSerGlyArgThrPheSerAspHisThrLeu354045GlyTrpPheArgGlnAlaProGlyLysGluArgGluPheValGlyAla505560IleSerTrpSerGlyGlySerThrTyrTyrAlaAspSerValSerGly65707580ArgPheThrIleSerArgAspLysAlaLysAsnThrGlyTyrLeuGln859095MetAsnSerLeuLysProGluAspThrAlaValTyrTyrCysAlaAla100105110AlaAspAspArgTyrSerAspTyrArgTyrTrpGlyGlnGlyThrGln115120125ValThrValSerSerGluProLysThrProLysProGlnProGlnPro130135140GlnProGlnProAsnProThrThrGluSerLysCysProLysCysPro145150155160AlaProGluLeuLeuGlyGlyProSerHisHisHisHisHisHis165170175 210 67 211 111 212 PRT 213 人工合成序列 400 67AspIleValLeuThrGlnSerProAlaSerLeuAlaValSerLeuGly151015GluArgAlaThrIleAsnCysArgAlaSerGluSerValSerValIle202530GlyAlaHisLeuIleHisTrpTyrGlnGlnLysProGlyGlnProPro354045LysLeuLeuIleTyrLeuAlaSerAsnLeuGluThrGlyValProAla505560ArgPheSerGlySerGlySerGlyThrAspPheThrLeuThrIleSer65707580SerLeuGlnAlaGluAspAlaAlaIleTyrTyrCysLeuGlnSerArg859095IlePheProArgThrPheGlyGlnGlyThrLysLeuGluIleLys100105110 210 68 211 117 212 PRT 213 人工合成序列 400 68GlnValGlnLeuValGlnSerGlySerGluLeuLysLysProGlyAla151015SerValLysValSerCysLysAlaSerGlyTyrThrPheThrAspTyr202530SerIleAsnTrpValArgGlnAlaProGlyGlnGlyLeuGluTrpMet354045GlyTrpIleAsnThrGluThrArgGluProAlaTyrAlaTyrAspPhe505560ArgGlyArgPheValPheSerLeuAspThrSerValSerThrAlaTyr65707580LeuGlnIleSerSerLeuLysAlaGluAspThrAlaValTyrTyrCys859095AlaArgAspTyrSerTyrAlaMetAspTyrTrpGlyGlnGlyThrLeu100105110ValThrValSerSer115当前第1页1 2 3