本发明涉及生物
技术领域：
，具体涉及一种cart-cd123及其制备与应用。
背景技术：
：急性髓系白血病(acutemyeloidleukemia，aml)是失去正常定向分化功能的非成熟的白血病细胞在体内堆积而产生异常造血的一种克隆性疾病。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。fda批准用于治疗aml的药物主要集中在砷剂、环磷酰胺、长春花碱、阿糖胞苷类，这些药物在临床上的收益比较低副作用较强，患者五年生存率很低，急切需求新的疗法。嵌合抗原受体(chimericantigenreceptor,car)是通过胞外配体识别区域(一般为单链抗体或单链受体)对目标抗原进行特异性识别，并激活胞内信号区域，使细胞因该种信号而发生定向的变化。将t细胞用特异性嵌合抗原受体修饰，胞内增加共刺激信号，构建成嵌合抗原受体t淋巴细胞(car-t)，其特点为car-t细胞的活化，可不依赖于tcr，仅依赖于car与抗原间识别作用，这就极大降低了由于肿瘤细胞表面低水平表达mhc分子而引起的细胞免疫逃逸。在多种恶性肿瘤治疗，尤其是all及cll中，car-t疗法展现出了惊人的疗效，例如ctl019等，并获得fda突破性药物认证。2007年irapastan等人利用抗cd123抗体26292(stemlinetherapeutics)偶联pe38毒素，合成出免疫毒素(immunotoxin)在体外证实抗cd123抗体可以特异性抑制aml细胞系的增殖。现今，美国国家癌症研究所(nationalcancerinstitute)与德克萨斯大学安德森肿瘤研究中心(mdandersoncancercenter)、美国希望之城医学中心(hopeofcitymedicalcenter)以及宾夕法尼亚大学正在开展以cd123为靶点的嵌合抗原受体t细胞疗法的临床实验；临床试验中所应用的抗cd123的单链抗体为26292。由于抗体的亲和力对抗体对免疫治疗的活性有显著影响。因此，本领域技术人员致力于开发亲和力更高的抗体及其应用。技术实现要素：鉴于现有技术的情况，本发明的目的在于提供一种cart-cd123及其制备与应用。为了实现上述目的及其他相关目的，本发明采用如下技术方案：本发明的第一方面，提供一种针对cd123的单链抗体，包括重链可变区和轻链可变区，所述重链可变区包括重链可变区互补决定区1cdr-h1、重链可变区互补决定区2cdr-h2和重链可变区互补决定区3cdr-h3；所述轻链包括轻链可变区，所述轻链包括轻链可变区互补决定区1cdr-l1、轻链可变区互补决定区2cdr-l2和轻链可变区互补决定区3cdr-l3。优选地，所述重链可变区互补决定区1cdr-h1的氨基酸序列如seqidno.1所示。优选地，所述重链可变区互补决定区2cdr-h2的氨基酸序列如seqidno.2所示。优选地，所述重链可变区互补决定区3cdr-h3的氨基酸序列如seqidno.3所示。优选地，所述轻链可变区互补决定区1cdr-l1的氨基酸序列如seqidno.4所示。优选地，所述轻链可变区互补决定区2cdr-l2的氨基酸序列如seqidno.5所示。优选地，所述轻链可变区互补决定区3cdr-l3的氨基酸序列如seqidno.6所示。优选地，所述重链可变区的氨基酸序列如seqidno.7所示。优选地，所述轻链可变区的氨基酸序列如seqidno.8所示。优选地，所述重链可变区和轻链可变区之间通过连接肽连接。进一步地，所述连接肽的氨基酸序列如seqidno.9所示。优选地，所述针对cd123的单链抗体的氨基酸序列如seqidno.10所示。本发明的第二方面，提供前述针对cd123的单链抗体在制备靶向于cd123的嵌合抗原受体中的用途。本发明的第三方面，提供一种靶向于cd123的嵌合抗原受体，所述嵌合抗原受体包括依次连接的cd8信号肽、前述针对cd123的单链抗体、cd8铰链及跨膜区、胞内4-1bb以及胞内cd3ζ。优选地，所述cd8信号肽的氨基酸序列如seqidno.28所示。优选地，所述cd8铰链及跨膜区的氨基酸序列如seqidno.29所示。优选地，所述胞内4-1bb(亦即cd137)的氨基酸序列如seqidno.30所示。优选地，所述胞内cd3ζ的氨基酸序列如seqidno.31所示。进一步地，所述靶向于cd123的嵌合抗原受体的氨基酸序列如seqidno.27所示。本发明第四方面提供了一种多核苷酸，其编码所述靶向于cd123的嵌合抗原受体。本发明第五方面提供了一种表达载体，其含有所述多核苷酸。本发明第六方面提供了一种宿主细胞，其被所述表达载体所转化。本发明第七方面，提供一种制备所述靶向于cd123的嵌合抗原受体的方法，包括步骤：构建含有靶向于cd123的嵌合抗原受体编码多核苷酸序列的表达载体，然后将所述表达载体转化至宿主细胞中诱导表达，从表达产物中分离获得所述靶向于cd123的嵌合抗原受体。本发明的第八方面，提供所述靶向于cd123的嵌合抗原受体在制备嵌合抗原受体t细胞及肿瘤治疗药物中的用途。本发明的第九方面，提供一种嵌合抗原受体t细胞，其表达所述靶向于cd123的嵌合抗原受体。本发明的第十方面，提供一种肿瘤治疗药物，其含有所述嵌合抗原受体t细胞。本发明的第十一方面，提供一种肿瘤治疗方法，包括步骤：将所述嵌合抗原受体t细胞施用于肿瘤患者。优选地，所述肿瘤为cd123高表达的肿瘤。例如：血液癌(白血病)、胃癌、肝癌、肾脏肿瘤、肺癌、小肠癌、骨癌、前列腺癌、结直肠癌、乳腺癌、大肠癌、前列腺癌、宫颈癌、肾上腺肿瘤、或膀胱肿瘤。。与现有技术相比，本发明的有益效果为：(1)本发明的抗cd123蛋白的抗体，亲和力显著提高；(2)基于抗cd123抗体改造成的嵌合抗原受体t细胞可以增强对表达cd123肿瘤细胞的杀伤能力。附图说明图1：cd123-scfv的elisa实验结果。图2：二代car结构示意图。图3：cart-cd123及cart-对照组制备流程图。图4a：cart-对照组gfp表达结果。图4b：cart-cd123gfp表达结果。图5：cart-cd123细胞分群检测结果。图6：cart-对照组细胞分群检测结果。图7a：流式细胞仪检测k562-cd123上cd123表达结果。图7b：流式细胞仪检测k562上cd123表达结果。图8：cart-cd123及cart-对照组细胞分泌。图9：cart-cd123、cart-对照组对靶细胞及非靶细胞的杀伤作用。具体实施方式car-tcar-t，全称是chimericantigenreceptort-cellimmunotherapy，嵌合抗原受体t细胞免疫疗法。嵌合抗原受体chimericantigenreceptor(car)。以下通过特定的具体实例说明本发明的实施方式，本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用，本说明书中的各项细节也可以基于不同观点与应用，在没有背离本发明的精神下进行各种修饰或改变。在进一步描述本发明具体实施方式之前，应理解，本发明的保护范围不局限于下述特定的具体实施方案；还应当理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围；在本发明说明书和权利要求书中，除非文中另外明确指出，单数形式“一个”、“一”和“这个”包括复数形式。当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术语与本
技术领域：
技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本
技术领域：
的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。除非另外说明，本发明中所公开的实验方法、检测方法、制备方法均采用本
技术领域：
常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组dna技术及相关领域的常规技术。这些技术在现有文献中已有完善说明，具体可参见sambrook等molecularcloning：alaboratorymanual，secondedition，coldspringharborlaboratorypress，1989andthirdedition，2001；ausubel等，currentprotocolsinmolecularbiology，johnwiley&sons，newyork，1987andperiodicupdates；theseriesmethodsinenzymology，academicpress，sandiego；wolffe，chromatinstructureandfunction，thirdedition，academicpress，sandiego，1998；methodsinenzymology，vol.304，chromatin(p.m.wassarmananda.p.wolffe，eds.)，academicpress，sandiego，1999；和methodsinmolecularbiology，vol.119，chromatinprotocols(p.b.becker，ed.)humanapress，totowa，1999等。实施例1cd123-scfv抗原序列噬菌体展示筛选cd123-scfv抗原序列噬菌体展示筛选，包括如下步骤：1、突变库的构建以pcan-scfv26292质粒(购自ge公司)为模板，利用随机引物pcr引入突变，引物如下表1所示：表1获得的scfv26292的重链(h)及轻链(l)的cdr1、cdr2、cdr3突变库pcr产物分别命名为h1、h2、h3、l1、l2和l3。用sfii和noti对pcr产物及pcantab5e酶切回收后，经t4dna连接酶16℃连接过夜。连接产物电转至tg1，2yt重悬并于37℃复苏1h后，取菌液梯度稀释进行平板计数，得到各个突变库库容至少108，其余菌液全部涂布2yt(ga)平板。从上述突变库中分别随机挑取20个单克隆送测序，多样性均100％。2、噬菌体抗体库的淘选加入20nmcd123-his-biotin抗原(cd123抗原购自sinobiological；biotin标记试剂购自sigma)与噬菌体抗体库室温孵育2h，再将混合物转入链霉亲和素磁珠中(购自lifetechnology)室温共孵育15min。pbst-pbs洗去未结合的噬菌体，再加入胰酶37℃作用30min，从而洗脱下结合的噬菌体。将胰酶消化洗脱下的噬菌体感染4ml对数期的tg1菌体，37℃静置30min，取部分菌液梯度稀释进行平板计数，其余菌液全部涂布2yt(ga：5％葡萄糖，100ug/ml青霉素)平板，用于下一轮的包装。包装后的噬菌体可用于下一轮的淘选，共进行4轮淘选富集，每轮淘选10倍稀释梯度降低抗原浓度，并逐轮增加pbst-pbs洗涤次数。3、scfv的筛选和鉴定对淘选出克隆的表达上清中的可溶性scfv进行elisa筛选，从而获得cd123抗原的特异性抗体。elisa初步筛选后，挑取阳性信号至少2倍大于阴性信号的克隆送测序，分析测序结果，提取出富集较多的cdr区域对应的克隆。试验流程：(1).从第3轮和第4轮淘选后的h和l突变库各随机挑取单克隆于96孔板中，37℃摇床培养过夜；(2).将过夜培养的单克隆培养液转接于新的96孔板中，37℃摇床培养至对数期后加iptg至终浓度1mm，30℃摇床培养过夜；(3).往包被有亲和素的96孔板中加入100μlcd123-his-biotin(1μg/ml)，室温孵育1h；(4).pbst洗板3次；(5).先往洗后的96孔板中加50μl稀释液(pbs/0.05％tween20/1％bsa)，再每孔加入相应的50μl昨日诱导过夜后的菌液上清，其中孔h11和h12分别为阴性及阳性对照；(6).室温孵育1h；(7).pbst洗板3次；(8).每孔加入100μlanti-myc-hrp(1:10,000稀释)，其中孔h12加入100μlanti-his-hrp(1:10,000稀释)，室温孵育1h；(9).pbst洗板3次；(10).加入100μltmb，避光、室温孵育10min；(11).加入100μl2mh2so4终止反应，并读取a450nm。elisa实验结果如下表2及图1所示：表2如上表2及图1中，negativecontrol具体是指没有插入scfv的噬菌体。positivecontrol具体是指将cd123抗原包被在板子上，用抗myc标签抗体检测，此处阳性特指cd123抗原成功包被在板子上。通过上表2及图1中的结果可知，噬菌体上的scfv可以结合cd123抗原。得到cd123-scfv，包括重链可变区和轻链可变区，重链可变区和轻链可变区之间通过连接肽连接，所述重链可变区包括重链可变区互补决定区1cdr-h1、重链可变区互补决定区2cdr-h2和重链可变区互补决定区3cdr-h3；所述轻链包括轻链可变区，所述轻链包括轻链可变区互补决定区1cdr-l1、轻链可变区互补决定区2cdr-l2和轻链可变区互补决定区3cdr-l3。所述重链可变区互补决定区1cdr-h1的氨基酸序列如seqidno.1所示，具体为：gytftqywmn。所述重链可变区互补决定区2cdr-h2的氨基酸序列如seqidno.2所示，具体为：ridpydsethynqkfkd。所述重链可变区互补决定区3cdr-h3的氨基酸序列如seqidno.3所示，具体为：gnwddy。所述轻链可变区互补决定区1cdr-l1的氨基酸序列如seqidno.4所示，具体为：rasksiskdla。所述轻链可变区互补决定区2cdr-l2的氨基酸序列如seqidno.5所示，具体为：sgstlqs。所述轻链可变区互补决定区3cdr-l3的氨基酸序列如seqidno.6所示，具体为：qqhnkypyt。所述重链可变区的氨基酸序列如seqidno.7所示，具体为：qvqlqqpgaelvrpgasvklsckasgytftqywmnwvkqrpdqglewigridpydsethynqkfkdkailtvdkssstaymqlssltsedsavyycargnwddywgqgttltvss。所述轻链可变区的氨基酸序列如seqidno.8所示，具体为：dvqitqspsylaaspgetitincrasksiskdlawyqekpgktnklliysgstlqsgipsrfsgsgsgtdftltisslepedfamyycqqhnkypytfgggtkleik。所述连接肽的氨基酸序列如seqidno.9所示，具体为：ggggsggggsgggs。所述cd123-scfv的氨基酸序列如seqidno.10所示，具体为：qvqlqqpgaelvrpgasvklsckasgytftqywmnwvkqrpdqglewigridpydsethynqkfkdkailtvdkssstaymqlssltsedsavyycargnwddywgqgttltvssggggsggggsgggsdvqitqspsylaaspgetitincrasksiskdlawyqekpgktnklliysgstlqsgipsrfsgsgsgtdftltisslepedfamyycqqhnkypytfgggtkleik。对应地，所述重链可变区互补决定区1cdr-h1的核苷酸序列如seqidno.11所示，具体为：ggctacaccttcacccagtactggatgaac。所述重链可变区互补决定区2cdr-h2的核苷酸序列如seqidno.12所示，具体为：cgcatcgacccctacgacagcgagacccactacaaccagaagttcaaggac。所述重链可变区互补决定区3cdr-h3的核苷酸序列如seqidno.13所示，具体为：ggcaactgggacgactac。所述轻链可变区互补决定区1cdr-l1的核苷酸序列如seqidno.14所示，具体为：cgcgccagcaagagcatcagcaaggacctggcc。所述轻链可变区互补决定区2cdr-l2的核苷酸序列如seqidno.15所示，具体为：agcggcagcaccctgcagagc。所述轻链可变区互补决定区3cdr-l3的核苷酸序列如seqidno.16所示，具体为：cagcagcacaacaagtacccctacacc。所述重链可变区的核苷酸序列如seqidno.17所示，具体为：caggtgcagctgcagcagcccggcgccgagctggtgcgccccggcgccagcgtgaagctgagctgcaaggccagcggctacaccttcacccagtactggatgaactgggtgaagcagcgccccgaccgggcctggagtggatcggccgcatcgacccctacgacagcgagacccactacaaccagaagttcaaggacaaggccatcctgaccgtggacaagagcagcagcaccgcctacatgcagctgagcagctgaccagcgaggacagcgccgtgtactactgcgcccgcggcaactgggacgactactggggccagggcaccaccctgaccgtgagcagc。所述轻链可变区的核苷酸序列如seqidno.18所示，具体为：gacgtgcagatcacccagagccccagctacctggccgccagccccggcgagaccatcaccatcaactgccgcgccagcaagagcatcagcaaggacctggcctggtaccaggagaagcccgcaagaccaacaagctgctgatctacagcggcagcaccctgcagagcggcatccccagccgcttcagcggcagcggcagcggcaccgacttcaccctgaccatcagcagcctggagcccgaggacttcgccatgtactactgccagcagcacaacaagtacccctacaccttcggcggcgcaccaagctggagatcaag。所述连接肽的核苷酸序列如seqidno.19所示，具体为：ggtggaggcggttcaggcggaggtggctctggcggtggggatcg。所述cd123-scfv的核苷酸序列如seqidno.20所示，具体为：caggtgcagctgcagcagcccggcgccgagctggtgcgccccggcgccagcgtgaagctgagctgcaaggccagcggctacaccttcacccagtactggatgaactgggtgaagcagcgccccgaccgggcctggagtggatcggccgcatcgacccctacgacagcgagacccactacaaccagaagttcaaggacaaggccatcctgaccgtggacaagagcagcagcaccgcctacatgcagctgagcagctgaccagcgaggacagcgccgtgtactactgcgcccgcggcaactgggacgactactggggccagggcaccaccctgaccgtgagcagcggtggaggcggttcaggcggaggtggctctggcggtggggatcggacgtgcagatcacccagagccccagctacctggccgccagccccggcgagaccatcaccatcaactgccgcgccagcaagagcatcagcaaggacctggcctggtaccaggagaagcccgcaagaccaacaagctgctgatctacagcggcagcaccctgcagagcggcatccccagccgcttcagcggcagcggcagcggcaccgacttcaccctgaccatcagcagcctggagcccgaggacttcgccatgtactactgccagcagcacaacaagtacccctacaccttcggcggcgcaccaagctggagatcaag。实施例2car-cd123的设计与构建2.1car的设计说明本发明选用二代car框结构，其结构如图2所示。2.2car的结构及构建本发明中，靶向于cd123的嵌合抗原受体可简称为car-cd123。本发明中，car-cd123由依次连接的cd8信号肽、cd123-scfv、cd8铰链及跨膜区、胞内4-1bb(亦即cd137)、胞内cd3ζ融合而成。所述cd8信号肽的核苷酸序列如seqidno.21所示，具体为：atgtacaggatgcaactcctgtcttgcattgcactaagtcttgcacttgtcacgaactcggccg。所述cd123-scfv的核苷酸序列如seqidno.20所示。所述cd8铰链及跨膜区的核苷酸序列如seqidno.22所示，具体为：accacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgc。所述胞内4-1bb的核苷酸序列如seqidno.23所示，具体为：aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactg。所述胞内cd3ζ的核苷酸序列如seqidno.24所示，具体为：agagtgaagttcagcaggagcgcagacgcccccgcgtacaagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc。所述car-cd123的核苷酸序列如seqidno.25所示，具体为：atgtacaggatgcaactcctgtcttgcattgcactaagtcttgcacttgtcacgaactcggccgcaggtgcagctgcagcagcccggcgccgagctggtgcgccccggcgccagcgtgaagctgagctgcaaggccagcggctacaccttcacccagtactggatgaactgggtgaagcagcgccccgaccagggcctggagtggatcggccgcatcgacccctacgacagcgagacccactacaaccagaagttcaaggacaaggccatcctgaccgtggacaagagcagcagcaccgcctacatgcagctgagcagcctgaccagcgaggacagcgccgtgtactactgcgcccgcggcaactgggacgactactggggccagggcaccaccctgaccgtgagcagcggtggaggcggttcaggcggaggtggctctggcggtggcggatcggacgtgcagatcacccagagccccagctacctggccgccagccccggcgagaccatcaccatcaactgccgcgccagcaagagcatcagcaaggacctggcctggtaccaggagaagcccggcaagaccaacaagctgctgatctacagcggcagcaccctgcagagcggcatccccagccgcttcagcggcagcggcagcggcaccgacttcaccctgaccatcagcagcctggagcccgaggacttcgccatgtactactgccagcagcacaacaagtacccctacaccttcggcggcggcaccaagctggagatcaagaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccccgcgtacaagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgc。本发明还设置了car-对照组，所述car-对照组由依次连接的cd8信号肽、cd123-scfv、cd8铰链及跨膜区、cd8-δcd3ζ融合而成，所述car-对照组的核苷酸序列如seqidno.26所示，具体为：atgtacaggatgcaactcctgtcttgcattgcactaagtcttgcacttgtcacgaactcggccgcaggtgcagctgcagcagcccggcgccgagctggtgcgccccggcgccagcgtgaagctgagctgcaaggccagcggctacaccttcacccagtactggatgaactgggtgaagcagcgccccgaccagggcctggagtggatcggccgcatcgacccctacgacagcgagacccactacaaccagaagttcaaggacaaggccatcctgaccgtggacaagagcagcagcaccgcctacatgcagctgagcagcctgaccagcgaggacagcgccgtgtactactgcgcccgcggcaactgggacgactactggggccagggcaccaccctgaccgtgagcagcggtggaggcggttcaggcggaggtggctctggcggtggcggatcggacgtgcagatcacccagagccccagctacctggccgccagccccggcgagaccatcaccatcaactgccgcgccagcaagagcatcagcaaggacctggcctggtaccaggagaagcccggcaagaccaacaagctgctgatctacagcggcagcaccctgcagagcggcatccccagccgcttcagcggcagcggcagcggcaccgacttcaccctgaccatcagcagcctggagcccgaggacttcgccatgtactactgccagcagcacaacaagtacccctacaccttcggcggcggcaccaagctggagatcaagaccacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgcagagtgaagttcagcaggagcgcagacgcccccgcgtactaa。合成所述car-cd123以及car-对照组的整个表达框，插入表达载体(购自上海吉凯基因化学技术有限公司)，经测序正确后，使用qiagen公司的质粒纯化试剂盒提取并纯化质粒，获得重组表达载体。经测序可知，car-cd123以及car-对照组的全长基因序列正确，均与预期相符。具体地，car-cd123的核苷酸序列如seqidno.25所示。所述car-对照组的核苷酸序列如seqidno.26所示。可得知，car-cd123的氨基酸序列如seqidno.27所示，具体为：myrmqllscialslalvtnsaqvqlqqpgaelvrpgasvklsckasgytftqywmnwvkqrpdqglewigridpydsethynqkfkdkailtvdkssstaymqlssltsedsavyycargnwddywgqgttltvssggggsggggsggggsdvqitqspsylaaspgetitincrasksiskdlawyqekpgktnklliysgstlqsgipsrfsgsgsgtdftltisslepedfamyycqqhnkypytfgggtkleikrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalpprtttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlyckrgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcelrvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr。其中，cd8信号肽的氨基酸序列如seqidno.28所示，具体为：myrmqllscialslalvtnsa。所述cd123-scfv的氨基酸序列如seqidno.10所示。所述cd8铰链及跨膜区的氨基酸序列如seqidno.29所示，具体为：prrqrrdhqhrrppsrrspcpcaqrragqrrgaqctrggwtspvistsgrpwpglvgsfschwlspft。所述胞内4-1bb(亦即cd137)的氨基酸序列如seqidno.30所示，具体为：krgrkkllyifkqpfmrpvqttqeedgcscrfpeeeeggcel。所述胞内cd3ζ的氨基酸序列如seqidno.31所示，具体为：rvkfsrsadapaykqgqnqlynelnlgrreeydvldkrrgrdpemggkprrknpqeglynelqkdkmaeayseigmkgerrrgkghdglyqglstatkdtydalhmqalppr。所述car-对照组的氨基酸序列如seqidno.32所示，myrmqllscialslalvtnsaqvqlqqpgaelvrpgasvklsckasgytftqywmnwvkqrpdqglewigridpydsethynqkfkdkailtvdkssstaymqlssltsedsavyycargnwddywgqgttltvssggggsggggsggggsdvqitqspsylaaspgetitincrasksiskdlawyqekpgktnklliysgstlqsgipsrfsgsgsgtdftltisslepedfamyycqqhnkypytfgggtkleiktttpaprpptpaptiasqplslrpeacrpaaggavhtrgldfacdiyiwaplagtcgvlllslvitlycrvkfsrsadapay。实施例3car-cd123修饰的t细胞(cart-cd123)的制备与表型验证3.1cart-cd123及cart-对照组的制备(1)实验目的制备cart-cd123和cart-对照组。(2)实验设计cart-cd123及cart-对照组制备流程如图3所示。具体的试验方法包括如下步骤：1.pbmc分离a.将新鲜血液转移到50ml离心管中。用生理盐水按照1:1(体积比)进行稀释，轻轻上下吹打3-5次混匀。b.将6mlficoll加入到15ml离心管中，竖直放置到水平台面上静止备用。将稀释好的血液(总体积8ml)用移液管缓慢均匀加入到竖直放置在台面的含ficoll溶液的离心管中。c.将加好血液与ficoll的离心管轻轻转移至离心机中。在转移离心管过程尽量减少晃动，保持离心管竖直，以避免打破液面分层。室温400g离心30分钟。d.离心结束后，轻轻转移离心管至超净台中。用移液器吸取中间的白膜层(即淋巴细胞层)转移至新的50ml离心管中。尽量避免吸取到下层或上层的细胞，避免造成污染。e.加入30ml生理盐水至上述转移出来的淋巴细胞中，轻轻吹打混匀，60-100g室温离心10分钟。重复洗涤淋巴细胞，去除上清，加入一定量的完全培养基重悬细胞。f.计数调整密度为5*105细胞/ml，预先处理过的12孔板中每孔加入1ml。将培养板放置于37℃，5％co2培养箱培养48小时。2.慢病毒感染g.收集上述pbmc，室温300g离心4min，调整密度为1*106细胞/ml，新的6孔板中每孔加入1ml细胞，实验组加入10.5ulcd123二代car的慢病毒(病毒滴度为2*108/ml，moi＝3)；对照组加入105ul对照病毒(病毒滴度为2*107/ml，moi＝3)，每孔加入一定的transb感染试剂。h.将细胞培养板置于37℃，5％co2培养箱培养。i.第三天每孔加入1ml新鲜的培养基，继续培养。j.第七天计数计算扩增倍数。(3)实验结果及分析本次制备，初始pbmc细胞量均为1e+6，第七天时对照组pbmc细胞量为1.02e+07，其中99.9％为cd3+t细胞，见图6，细胞扩增倍数约为10倍；实验组pbmc细胞量为9.40e+06，其中99.8％为cd3+t细胞，见图5，细胞扩增倍数约为9.4倍，见表3。表3cart-cd123及cart-对照组细胞增殖3.2cart-cd123表型检测(1)实验目的确定cart-cd123及cart-对照组的感染效率。(2)实验设计流式细胞仪检测所制备细胞gfp的表达。具体的试验方法包括如下步骤：a.计数每个细胞取出1.0x106个，1000rpm，离心3min，加入100ulfacsbuffer重悬细胞，加入15ulapcanti-humancd8、15ulpeanti-humancd4、15ulpercy5.5anti-humancd3。b.4℃避光孵育30min，1000rpm，离心3min，去掉上清加入facsbuffer再洗两遍。c.加入150ulfacsbuffer重悬细胞，facs检测荧光。(3)实验结果及分析cart-对照组gfp表达结果如图4a所示，cart-对照组感染效率约为90.7％。cart-cd123gfp表达结果如图4b所示，cart-cd123感染效率约为92.92％。3.3cart的细胞分群检测(1)实验目的确定cart-cd123的细胞中t细胞的亚群分布特征。(2)实验设计流式细胞仪检测所制备细胞表面标志物，检测标的及意义如下表4所示。具体的试验方法包括如下步骤：a.计数每个细胞取出1.0x106个，1000rpm，离心3min，加入100ulfacsbuffer重悬细胞，加入15ulapcanti-humancd8、15ulpeanti-humancd4、15ulpercy5.5anti-humancd3。b.4℃避光孵育30min，1000rpm，离心3min，去掉上清加入facsbuffer再洗两遍。c.加入150ulfacsbuffer重悬细胞，facs检测荧光。表4t细胞亚群分布检测标的意义cd3鉴定t淋巴细胞占总细胞群体的比例cd3/cd8/cd4鉴定t效应淋巴细胞/t辅助淋巴细胞在总细胞群体的比例(3)实验结果及分析cart-cd123细胞分群检测结果如图5所示。cart-对照组细胞分群检测结果如图6所示。由上述结果可知，所制备的cart-cd123细胞分群如表5：表5cart-cd123细胞亚群分布比例所制备的cart-对照组细胞分群如表6：表6cart-对照组细胞亚群分布比例t淋巴细胞(cd3+)占总细胞群体的比例99.9％t效应淋巴细胞(cd3+cd8+)占t细胞的比例29％t辅助淋巴细胞(cd3+cd4+)占t细胞的比例66.2％4.cart-cd123的功能验证4.1靶细胞的验证(1)实验目的检测靶细胞和非靶细胞上cd123的表达。(2)实验设计使用cd123的流式抗体对靶细胞(k562-cd123)及对照细胞(k562)进行检测。具体的试验方法包括如下步骤：1.细胞按照下表8中所列培养基培养在37℃、5％co2培养箱中。表82.实验时，吸掉培养基，用5ml0.25％的胰酶冲洗细胞层，然后吸掉胰酶，将细胞放在培养箱中消化3～5分钟，加入新鲜培养基重悬细胞。3.计数每个细胞取出1.0x105个，1000rpm，离心3min，加入100ulfacsbuffer重悬细胞，加入2ulpe-cy5anti-humancd123。4.4℃避光孵育30min，1000rpm，离心3min，去掉上清加入facsbuffer再洗两遍。5.加入150ulfacsbuffer重悬细胞，facs检测荧光。(3)实验结果与分析流式细胞仪检测k562-cd123上cd123表达结果如图7a所示，靶细胞k562-cd123中cd123表达较高。流式细胞仪检测k562上cd123表达结果如图7b，结果显示阴性对照k562中cd123表达较低。4.2cart-cd123的体外功能验证4.2.1细胞因子分泌实验(1)实验目的通过检测cart-cd123细胞与靶细胞共同孵育后，细胞因子分泌是否有上升判断cart是否有功能。(2)实验设计分别对比cart-cd123、cart-对照组与靶细胞和非靶细胞共同孵育后，培养基上清中细胞因子的分泌情况。具体的试验方法包括如下步骤：(一)细胞因子分泌a.收集靶细胞，用稀释缓冲液将细胞洗涤一次，1000rpm3min离心，弃上清，以含2％fbs的1640重悬细胞，计数，最终将细胞稀释到1*106个/ml浓度；b.收集cart细胞(效应细胞)，用稀释缓冲液将细胞洗涤一次，1000rpm3min离心，弃上清，2％fbs的1640重悬细胞，计数，最终将细胞稀释到1*106个/ml浓度；c.将100ul靶细胞和100ulcart细胞1:1混合加入96孔板中，37℃、5％co2共同培养20～24小时；d.1000rpm5min离心，收集上清检测细胞因子。(二)细胞因子检测1.标准品制备e.准备15ml离心管，在瓶盖上标注1，将标准品转移至ep管内，并向其中加入标准品稀释液2ml，轻柔颠倒混匀，室温防止15min-20min平衡。f.取9支1.5mlep管，并向每管中加入300ul标准品稀释液，瓶盖上标注编号2-9，在1管中取300ul至2管内混匀；在2管内取出300ul至3管内，依次类推进行2倍梯度稀释，另取一支1.5mlep管，加入300ul标准品稀释液作为阴性对照。2.准备捕获珠g.确定好需要检测的细胞因子及样品个数。h.取出待检测的细胞因子捕获珠子瓶，用力混匀。每种珠子取出(待检测样品数+标准品数+阴性对照数)*10ul的捕获珠子量。并将每种珠子进行涡轮震荡混匀。3.样品准备及检测i.用标准品稀释液对样品进行稀释(稀释倍数可自行决定，如1：10、1：100等)j.取n个1.5mlep管(n＝样品数+标准品数+对照数)k.向每管(标准品、样品、阴性对照)中加入50ul混合珠子；并向各管中加入相应待测试剂(标准品、样品、对照)50ul以及pedetectionreagent50ul，充分混合后，避光室温孵育3小时。l.向每管中加入1mlwashbuffer，轻柔吹匀后200g离心5分钟。m.小心吸去上清，并向每管中加入300ulwashbuffer，并重悬沉淀。n.将样品进行流式细胞仪检测。(3)实验结果与分析本次实验分别检测了il-2、il-4、il-5、il-10、tnf-α、ifn-γ的分泌，结果如图8所示，cart-cd123在存在靶细胞时，il-2、tnf-α、ifn-γ的分泌高于cart-对照组结果显示cart-cd123具有一定的功能活性。4.2.2细胞杀伤能力检测(1)实验目的检测cart-cd123及cart-对照组的杀伤能力。(2)实验设计通过ldh释放实验，判断cart细胞对靶细胞的特异杀伤。具体的试验方法包括如下步骤：。1.ldh释放a.收集靶细胞，用稀释缓冲液将细胞洗涤一次，1000rpm3min离心，弃上清，以含2％fbs的1640重悬细胞、计数，最终将细胞密度调整为1*105个/ml，备用；b.收集cart(效应细胞)，用稀释缓冲液将细胞洗涤一次，1000rpm3min离心，弃上清，以含2％fbs的1640重悬细胞、计数，将细胞密度分别调整为2*106个/ml、1*106个/ml、0.5*106个/ml、0.25*106个/ml、0.125*106个/ml；c.将上述细胞分别加入u型底96孔板中(200ul体系)，分组如下表7，e：t分别为20:1、15:1、10:1、5:1、1:1；表7d.细胞放入含5％co2的37℃培养箱中进行培养4-6小时；e.实验终点前45min，在靶细胞最大释放组中加入20ul10*lysis(试剂盒自带)；f.培养终点1500rpm5min离心培养板，每孔取50μl上清液转移到新的96孔板中，每孔加入50ul混合后的底物，室温避光孵育30min，加入50ul终止液，490nm检测吸收值。2.靶细胞存在下的cart增殖a.收集靶细胞，用稀释缓冲液将细胞洗涤一次，1000rpm3min离心，弃上清，以含2％fbs的1640重悬细胞、计数，最终将细胞密度调整为1*105个/ml，备用；b.收集cart(效应细胞)，用稀释缓冲液将细胞洗涤一次，1000rpm3min离心，弃上清，以含2％fbs的1640重悬细胞、计数，将细胞密度分别调整为2*105个/ml；c.上述效应细胞和靶细胞各1ml加入12孔板中，37℃、5％co2培养箱中培养72小时。d.facs检测gfp阳性细胞。(3)实验结果与分析cart-cd123和cart-对照组对靶细胞及非靶细胞的裂解效应如图9所示。与cart-对照组相比，cart-cd123对靶细胞具有一定杀伤活性，在cart-cd123：靶细胞比例为20:1共培养4hr后，靶细胞裂解率为29.09％。对比例本发明在研发过程中，还尝试了其他序列的cd123-scfv，结果效果都没有实施例1中的cd123-scfv效果好。具体地，当cd123-scfv的氨基酸序列如seqidno.33所示时，采用实施例1中相同的elisa实验方法，所获得的elisa实验结果为2.57。qvqlqqpgaelvrpgasvklsckasgytmqsywmnwvkqrpdqglewigridpydsethynqkfkdkailtvdkssstaymqlssltsedsavyycargnwddywgqgttltvssggggsggggsggggsdvqitqspsylaaspgetitincrasksiskdlawyqekpgktnklliysgstlqsgipsrfsgsgsgtdftltisslepedfamyycqqhnkypytfgggtkleik(seqidno.33)。当cd123-scfv的氨基酸序列如seqidno.34所示时，采用实施例1中相同的elisa实验方法，所获得的elisa实验结果为1.56。qvqlqqpgaelvrpgasvklsckasgytftsywmnwvkqrpdqglewigridpwdskthynqkfkdkailtvdkssstaymqlssltsedsavyycargnwddywgqgttltvssggggsggggsggggsdvqitqspsylaaspgetitincrasksiskdlawyqekpgktnklliysgstlqsgipsrfsgsgsgtdftltisslepedfamyycqqhnkypytfgggtkleik(seqidno.34)。当cd123-scfv的氨基酸序列如seqidno.35所示时，采用实施例1中相同的elisa实验方法，所获得的elisa实验结果为1.46。qvqlqqpgaelvrpgasvklsckasgytftsywmnwvkqrpdqglewigridpydsethynqkfkdkailtvdkssstaymqlssltsedsavyycarsqvsdywgqgttltvssggggsggggsggggsdvqitqspsylaaspgetitincrasksiskdlawyqekpgktnklliysgstlqsgipsrfsgsgsgtdftltisslepedfamyycqqhnkypytfgggtkleik(seqidno.35)。当cd123-scfv的氨基酸序列如seqidno.36所示时，采用实施例1中相同的elisa实验方法，所获得的elisa实验结果为1.30。qvqlqqpgaelvrpgasvklsckasgytftsywmnwvkqrpdqglewigridpydsethynqkfkdkailtvdkssstaymqlssltsedsavyycarfqvmtfwgqgttltvssggggsggggsggggsdvqitqspsylaaspgetitincrasksiskdlawyqekpgktnklliysgstlqsgipsrfsgsgsgtdftltisslepedfamyycqqhnkypytfgggtkleik(seqidno.36)。当cd123-scfv的氨基酸序列如seqidno.37所示时，采用实施例1中相同的elisa实验方法，所获得的elisa实验结果为1.90。qvqlqqpgaelvrpgasvklsckasgytftsywmnwvkqrpdqglewigridpydsethynqkfkdkailtvdkssstaymqlssltsedsavyycargnwddywgqgttltvssggggsggggsggggsdvqitqspsylaaspgetitincrasksiskdlawyqekpgktnklliysdshyqsgipsrfsgsgsgtdftltisslepedfamyycqqhnkypytfgggtkleik(seqidno.37)。当cd123-scfv的氨基酸序列如seqidno.38所示时，采用实施例1中相同的elisa实验方法，所获得的elisa实验结果为1.47。qvqlqqpgaelvrpgasvklsckasgytftsywmnwvkqrpdqglewigridpydsethynqkfkdkailtvdkssstaymqlssltsedsavyycargnwddywgqgttltvssggggsggggsggggsdvqitqspsylaaspgetitincrasksiskdlawyqekpgktnklliysdsqaisgipsrfsgsgsgtdftltisslepedfamyycqqhnkypytfgggtkleik(seqidno.38)。当cd123-scfv的氨基酸序列如seqidno.39所示时，采用实施例1中相同的elisa实验方法，所获得的elisa实验结果为1.94。qvqlqqpgaelvrpgasvklsckasgytftsywmnwvkqrpdqglewigridpydsethynqkfkdkailtvdkssstaymqlssltsedsavyycargnwddywgqgttltvssggggsggggsggggsdvqitqspsylaaspgetitincrasksiskdlawyqekpgktnklliyrvsrdqsgipsrfsgsgsgtdftltisslepedfamyycqqhnkypytfgggtkleik(seqidno.39)。当cd123-scfv的氨基酸序列如seqidno.40所示时，采用实施例1中相同的elisa实验方法，所获得的elisa实验结果为2.24。qvqlqqpgaelvrpgasvklsckasgytftsywmnwvkqrpdqglewigridpydsethynqkfkdkailtvdkssstaymqlssltsedsavyycargnwddywgqgttltvssggggsggggsggggsdvqitqspsylaaspgetitincrasksiskdlawyqekpgktnklliysgstlqsgipsrfsgsgsgtdftltisslepedfamyycqqghkypytfgggtkleik(seqidno.40)。当cd123-scfv的氨基酸序列如seqidno.41所示时，采用实施例1中相同的elisa实验方法，所获得的elisa实验结果为1.58。qvqlqqpgaelvrpgasvklsckasgytftsywmnwvkqrpdqglewigridpydsethynqkfkdkailtvdkssstaymqlssltsedsavyycargnwddywgqgttltvssggggsggggsggggsdvqitqspsylaaspgetitincrasksiskdlawyqekpgktnklliysgstlqsgipsrfsgsgsgtdftltisslepedfamyycqqhnkypytfgggtkleik(seqidno.41)。以上的实施例是为了说明本发明公开的实施方案，并不能理解为对本发明的限制。此外，本文所列出的各种修改以及发明中方法、组合物的变化，在不脱离本发明的范围和精神的前提下对本领域内的技术人员来说是显而易见的。虽然已结合本发明的多种具体优选实施例对本发明进行了具体的描述，但应当理解，本发明不应仅限于这些具体实施例。事实上，各种如上所述的对本领域内的技术人员来说显而易见的修改来获取发明都应包括在本发明的范围内。序列表<110>刘爽张青<120>cart-cd123及其制备与应用<130>181359<160>55<170>siposequencelisting1.0<210>1<211>10<212>prt<213>人工序列(artificialsequence)<400>1glytyrthrphethrglntyrtrpmetasn1510<210>2<211>17<212>prt<213>人工序列(artificialsequence)<400>2argileaspprotyraspsergluthrhistyrasnglnlysphelys151015asp<210>3<211>6<212>prt<213>人工序列(artificialsequence)<400>3glyasntrpaspasptyr15<210>4<211>11<212>prt<213>人工序列(artificialsequence)<400>4argalaserlysserileserlysaspleuala1510<210>5<211>7<212>prt<213>人工序列(artificialsequence)<400>5serglyserthrleuglnser15<210>6<211>9<212>prt<213>人工序列(artificialsequence)<400>6glnglnhisasnlystyrprotyrthr15<210>7<211>115<212>prt<213>人工序列(artificialsequence)<400>7glnvalglnleuglnglnproglyalagluleuvalargproglyala151015servallysleusercyslysalaserglytyrthrphethrglntyr202530trpmetasntrpvallysglnargproaspglnglyleuglutrpile354045glyargileaspprotyraspsergluthrhistyrasnglnlysphe505560lysasplysalaileleuthrvalasplysserserserthralatyr65707580metglnleuserserleuthrsergluaspseralavaltyrtyrcys859095alaargglyasntrpaspasptyrtrpglyglnglythrthrleuthr100105110valserser115<210>8<211>107<212>prt<213>人工序列(artificialsequence)<400>8aspvalglnilethrglnserprosertyrleualaalaserprogly151015gluthrilethrileasncysargalaserlysserileserlysasp202530leualatrptyrglnglulysproglylysthrasnlysleuleuile354045tyrserglyserthrleuglnserglyileproserargphesergly505560serglyserglythraspphethrleuthrileserserleuglupro65707580gluaspphealamettyrtyrcysglnglnhisasnlystyrprotyr859095thrpheglyglyglythrlysleugluilelys100105<210>9<211>14<212>prt<213>人工序列(artificialsequence)<400>9glyglyglyglyserglyglyglyglyserglyglyglyser1510<210>10<211>236<212>prt<213>人工序列(artificialsequence)<400>10glnvalglnleuglnglnproglyalagluleuvalargproglyala151015servallysleusercyslysalaserglytyrthrphethrglntyr202530trpmetasntrpvallysglnargproaspglnglyleuglutrpile354045glyargileaspprotyraspsergluthrhistyrasnglnlysphe505560lysasplysalaileleuthrvalasplysserserserthralatyr65707580metglnleuserserleuthrsergluaspseralavaltyrtyrcys859095alaargglyasntrpaspasptyrtrpglyglnglythrthrleuthr100105110valserserglyglyglyglyserglyglyglyglyserglyglygly115120125seraspvalglnilethrglnserprosertyrleualaalaserpro130135140glygluthrilethrileasncysargalaserlysserileserlys145150155160aspleualatrptyrglnglulysproglylysthrasnlysleuleu165170175iletyrserglyserthrleuglnserglyileproserargpheser180185190glyserglyserglythraspphethrleuthrileserserleuglu195200205progluaspphealamettyrtyrcysglnglnhisasnlystyrpro210215220tyrthrpheglyglyglythrlysleugluilelys225230235<210>11<211>30<212>dna<213>人工序列(artificialsequence)<400>11ggctacaccttcacccagtactggatgaac30<210>12<211>51<212>dna<213>人工序列(artificialsequence)<400>12cgcatcgacccctacgacagcgagacccactacaaccagaagttcaaggac51<210>13<211>18<212>dna<213>人工序列(artificialsequence)<400>13ggcaactgggacgactac18<210>14<211>33<212>dna<213>人工序列(artificialsequence)<400>14cgcgccagcaagagcatcagcaaggacctggcc33<210>15<211>21<212>dna<213>人工序列(artificialsequence)<400>15agcggcagcaccctgcagagc21<210>16<211>27<212>dna<213>人工序列(artificialsequence)<400>16cagcagcacaacaagtacccctacacc27<210>17<211>343<212>dna<213>人工序列(artificialsequence)<400>17caggtgcagctgcagcagcccggcgccgagctggtgcgccccggcgccagcgtgaagctg60agctgcaaggccagcggctacaccttcacccagtactggatgaactgggtgaagcagcgc120cccgaccgggcctggagtggatcggccgcatcgacccctacgacagcgagacccactaca180accagaagttcaaggacaaggccatcctgaccgtggacaagagcagcagcaccgcctaca240tgcagctgagcagctgaccagcgaggacagcgccgtgtactactgcgcccgcggcaactg300ggacgactactggggccagggcaccaccctgaccgtgagcagc343<210>18<211>319<212>dna<213>人工序列(artificialsequence)<400>18gacgtgcagatcacccagagccccagctacctggccgccagccccggcgagaccatcacc60atcaactgccgcgccagcaagagcatcagcaaggacctggcctggtaccaggagaagccc120gcaagaccaacaagctgctgatctacagcggcagcaccctgcagagcggcatccccagcc180gcttcagcggcagcggcagcggcaccgacttcaccctgaccatcagcagcctggagcccg240aggacttcgccatgtactactgccagcagcacaacaagtacccctacaccttcggcggcg300caccaagctggagatcaag319<210>19<211>44<212>dna<213>人工序列(artificialsequence)<400>19ggtggaggcggttcaggcggaggtggctctggcggtggggatcg44<210>20<211>706<212>dna<213>人工序列(artificialsequence)<400>20caggtgcagctgcagcagcccggcgccgagctggtgcgccccggcgccagcgtgaagctg60agctgcaaggccagcggctacaccttcacccagtactggatgaactgggtgaagcagcgc120cccgaccgggcctggagtggatcggccgcatcgacccctacgacagcgagacccactaca180accagaagttcaaggacaaggccatcctgaccgtggacaagagcagcagcaccgcctaca240tgcagctgagcagctgaccagcgaggacagcgccgtgtactactgcgcccgcggcaactg300ggacgactactggggccagggcaccaccctgaccgtgagcagcggtggaggcggttcagg360cggaggtggctctggcggtggggatcggacgtgcagatcacccagagccccagctacctg420gccgccagccccggcgagaccatcaccatcaactgccgcgccagcaagagcatcagcaag480gacctggcctggtaccaggagaagcccgcaagaccaacaagctgctgatctacagcggca540gcaccctgcagagcggcatccccagccgcttcagcggcagcggcagcggcaccgacttca600ccctgaccatcagcagcctggagcccgaggacttcgccatgtactactgccagcagcaca660acaagtacccctacaccttcggcggcgcaccaagctggagatcaag706<210>21<211>64<212>dna<213>人工序列(artificialsequence)<400>21atgtacaggatgcaactcctgtcttgcattgcactaagtcttgcacttgtcacgaactcg60gccg64<210>22<211>207<212>dna<213>人工序列(artificialsequence)<400>22accacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctg60tccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctg120gacttcgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctc180ctgtcactggttatcaccctttactgc207<210>23<211>126<212>dna<213>人工序列(artificialsequence)<400>23aaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaa60actactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgt120gaactg126<210>24<211>336<212>dna<213>人工序列(artificialsequence)<400>24agagtgaagttcagcaggagcgcagacgcccccgcgtacaagcagggccagaaccagctc60tataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggc120cgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaat180gaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgc240cggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacc300tacgacgcccttcacatgcaggccctgccccctcgc336<210>25<211>1444<212>dna<213>人工序列(artificialsequence)<400>25atgtacaggatgcaactcctgtcttgcattgcactaagtcttgcacttgtcacgaactcg60gccgcaggtgcagctgcagcagcccggcgccgagctggtgcgccccggcgccagcgtgaa120gctgagctgcaaggccagcggctacaccttcacccagtactggatgaactgggtgaagca180gcgccccgaccagggcctggagtggatcggccgcatcgacccctacgacagcgagaccca240ctacaaccagaagttcaaggacaaggccatcctgaccgtggacaagagcagcagcaccgc300ctacatgcagctgagcagcctgaccagcgaggacagcgccgtgtactactgcgcccgcgg360caactgggacgactactggggccagggcaccaccctgaccgtgagcagcggtggaggcgg420ttcaggcggaggtggctctggcggtggcggatcggacgtgcagatcacccagagccccag480ctacctggccgccagccccggcgagaccatcaccatcaactgccgcgccagcaagagcat540cagcaaggacctggcctggtaccaggagaagcccggcaagaccaacaagctgctgatcta600cagcggcagcaccctgcagagcggcatccccagccgcttcagcggcagcggcagcggcac660cgacttcaccctgaccatcagcagcctggagcccgaggacttcgccatgtactactgcca720gcagcacaacaagtacccctacaccttcggcggcggcaccaagctggagatcaagaccac780gacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccct840gcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggactt900cgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtc960actggttatcaccctttactgcaaacggggcagaaagaaactcctgtatatattcaaaca1020accatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttcc1080agaagaagaagaaggaggatgtgaactgagagtgaagttcagcaggagcgcagacgcccc1140cgcgtacaagcagggccagaaccagctctataacgagctcaatctaggacgaagagagga1200gtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaag1260gaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggccta1320cagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttacca1380gggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccc1440tcgc1444<210>26<211>1024<212>dna<213>人工序列(artificialsequence)<400>26atgtacaggatgcaactcctgtcttgcattgcactaagtcttgcacttgtcacgaactcg60gccgcaggtgcagctgcagcagcccggcgccgagctggtgcgccccggcgccagcgtgaa120gctgagctgcaaggccagcggctacaccttcacccagtactggatgaactgggtgaagca180gcgccccgaccagggcctggagtggatcggccgcatcgacccctacgacagcgagaccca240ctacaaccagaagttcaaggacaaggccatcctgaccgtggacaagagcagcagcaccgc300ctacatgcagctgagcagcctgaccagcgaggacagcgccgtgtactactgcgcccgcgg360caactgggacgactactggggccagggcaccaccctgaccgtgagcagcggtggaggcgg420ttcaggcggaggtggctctggcggtggcggatcggacgtgcagatcacccagagccccag480ctacctggccgccagccccggcgagaccatcaccatcaactgccgcgccagcaagagcat540cagcaaggacctggcctggtaccaggagaagcccggcaagaccaacaagctgctgatcta600cagcggcagcaccctgcagagcggcatccccagccgcttcagcggcagcggcagcggcac660cgacttcaccctgaccatcagcagcctggagcccgaggacttcgccatgtactactgcca720gcagcacaacaagtacccctacaccttcggcggcggcaccaagctggagatcaagaccac780gacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccct840gcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggactt900cgcctgtgatatctacatctgggcgcccttggccgggacttgtggggtccttctcctgtc960actggttatcaccctttactgcagagtgaagttcagcaggagcgcagacgcccccgcgta1020ctaa1024<210>27<211>593<212>prt<213>人工序列(artificialsequence)<400>27mettyrargmetglnleuleusercysilealaleuserleualaleu151015valthrasnseralaglnvalglnleuglnglnproglyalagluleu202530valargproglyalaservallysleusercyslysalaserglytyr354045thrphethrglntyrtrpmetasntrpvallysglnargproaspgln505560glyleuglutrpileglyargileaspprotyraspsergluthrhis65707580tyrasnglnlysphelysasplysalaileleuthrvalasplysser859095serserthralatyrmetglnleuserserleuthrsergluaspser100105110alavaltyrtyrcysalaargglyasntrpaspasptyrtrpglygln115120125glythrthrleuthrvalserserglyglyglyglyserglyglygly130135140glyserglyglyglyglyseraspvalglnilethrglnserproser145150155160tyrleualaalaserproglygluthrilethrileasncysargala165170175serlysserileserlysaspleualatrptyrglnglulysprogly180185190lysthrasnlysleuleuiletyrserglyserthrleuglnsergly195200205ileproserargpheserglyserglyserglythraspphethrleu210215220thrileserserleugluprogluaspphealamettyrtyrcysgln225230235240glnhisasnlystyrprotyrthrpheglyglyglythrlysleuglu245250255ilelysargvallyspheserargseralaaspalaproalatyrlys260265270glnglyglnasnglnleutyrasngluleuasnleuglyargargglu275280285glutyraspvalleuasplysargargglyargaspproglumetgly290295300glylysproargarglysasnproglngluglyleutyrasngluleu305310315320glnlysasplysmetalaglualatyrsergluileglymetlysgly325330335gluargargargglylysglyhisaspglyleutyrglnglyleuser340345350thralathrlysaspthrtyraspalaleuhismetglnalaleupro355360365proargthrthrthrproalaproargproprothrproalaprothr370375380ilealaserglnproleuserleuargproglualacysargproala385390395400alaglyglyalavalhisthrargglyleuaspphealacysaspile405410415tyriletrpalaproleualaglythrcysglyvalleuleuleuser420425430leuvalilethrleutyrcyslysargglyarglyslysleuleutyr435440445ilephelysglnprophemetargprovalglnthrthrglngluglu450455460aspglycyssercysargpheproglugluglugluglyglycysglu465470475480leuargvallyspheserargseralaaspalaproalatyrlysgln485490495glyglnasnglnleutyrasngluleuasnleuglyargarggluglu500505510tyraspvalleuasplysargargglyargaspproglumetglygly515520525lysproargarglysasnproglngluglyleutyrasngluleugln530535540lysasplysmetalaglualatyrsergluileglymetlysglyglu545550555560argargargglylysglyhisaspglyleutyrglnglyleuserthr565570575alathrlysaspthrtyraspalaleuhismetglnalaleupropro580585590arg<210>28<211>21<212>prt<213>人工序列(artificialsequence)<400>28mettyrargmetglnleuleusercysilealaleuserleualaleu151015valthrasnserala20<210>29<211>68<212>prt<213>人工序列(artificialsequence)<400>29proargargglnargargasphisglnhisargargproproserarg151015argserprocysprocysalaglnargargalaglyglnargarggly202530alaglncysthrargglyglytrpthrserprovalileserthrser354045glyargprotrpproglyleuvalglyserphesercyshistrpleu505560serprophethr65<210>30<211>42<212>prt<213>人工序列(artificialsequence)<400>30lysargglyarglyslysleuleutyrilephelysglnprophemet151015argprovalglnthrthrglnglugluaspglycyssercysargphe202530proglugluglugluglyglycysgluleu3540<210>31<211>112<212>prt<213>人工序列(artificialsequence)<400>31argvallyspheserargseralaaspalaproalatyrlysglngly151015glnasnglnleutyrasngluleuasnleuglyargarggluglutyr202530aspvalleuasplysargargglyargaspproglumetglyglylys354045proargarglysasnproglngluglyleutyrasngluleuglnlys505560asplysmetalaglualatyrsergluileglymetlysglygluarg65707580argargglylysglyhisaspglyleutyrglnglyleuserthrala859095thrlysaspthrtyraspalaleuhismetglnalaleuproproarg100105110<210>32<211>340<212>prt<213>人工序列(artificialsequence)<400>32mettyrargmetglnleuleusercysilealaleuserleualaleu151015valthrasnseralaglnvalglnleuglnglnproglyalagluleu202530valargproglyalaservallysleusercyslysalaserglytyr354045thrphethrglntyrtrpmetasntrpvallysglnargproaspgln505560glyleuglutrpileglyargileaspprotyraspsergluthrhis65707580tyrasnglnlysphelysasplysalaileleuthrvalasplysser859095serserthralatyrmetglnleuserserleuthrsergluaspser100105110alavaltyrtyrcysalaargglyasntrpaspasptyrtrpglygln115120125glythrthrleuthrvalserserglyglyglyglyserglyglygly130135140glyserglyglyglyglyseraspvalglnilethrglnserproser145150155160tyrleualaalaserproglygluthrilethrileasncysargala165170175serlysserileserlysaspleualatrptyrglnglulysprogly180185190lysthrasnlysleuleuiletyrserglyserthrleuglnsergly195200205ileproserargpheserglyserglyserglythraspphethrleu210215220thrileserserleugluprogluaspphealamettyrtyrcysgln225230235240glnhisasnlystyrprotyrthrpheglyglyglythrlysleuglu245250255ilelysthrthrthrproalaproargproprothrproalaprothr260265270ilealaserglnproleuserleuargproglualacysargproala275280285alaglyglyalavalhisthrargglyleuaspphealacysaspile290295300tyriletrpalaproleualaglythrcysglyvalleuleuleuser305310315320leuvalilethrleutyrcysargvallyspheserargseralaasp325330335alaproalatyr340<210>33<211>237<212>prt<213>人工序列(artificialsequence)<400>33glnvalglnleuglnglnproglyalagluleuvalargproglyala151015servallysleusercyslysalaserglytyrthrmetglnsertyr202530trpmetasntrpvallysglnargproaspglnglyleuglutrpile354045glyargileaspprotyraspsergluthrhistyrasnglnlysphe505560lysasplysalaileleuthrvalasplysserserserthralatyr65707580metglnleuserserleuthrsergluaspseralavaltyrtyrcys859095alaargglyasntrpaspasptyrtrpglyglnglythrthrleuthr100105110valserserglyglyglyglyserglyglyglyglyserglyglygly115120125glyseraspvalglnilethrglnserprosertyrleualaalaser130135140proglygluthrilethrileasncysargalaserlysserileser145150155160lysaspleualatrptyrglnglulysproglylysthrasnlysleu165170175leuiletyrserglyserthrleuglnserglyileproserargphe180185190serglyserglyserglythraspphethrleuthrileserserleu195200205gluprogluaspphealamettyrtyrcysglnglnhisasnlystyr210215220protyrthrpheglyglyglythrlysleugluilelys225230235<210>34<211>237<212>prt<213>人工序列(artificialsequence)<400>34glnvalglnleuglnglnproglyalagluleuvalargproglyala151015servallysleusercyslysalaserglytyrthrphethrsertyr202530trpmetasntrpvallysglnargproaspglnglyleuglutrpile354045glyargileaspprotrpaspserlysthrhistyrasnglnlysphe505560lysasplysalaileleuthrvalasplysserserserthralatyr65707580metglnleuserserleuthrsergluaspseralavaltyrtyrcys859095alaargglyasntrpaspasptyrtrpglyglnglythrthrleuthr100105110valserserglyglyglyglyserglyglyglyglyserglyglygly115120125glyseraspvalglnilethrglnserprosertyrleualaalaser130135140proglygluthrilethrileasncysargalaserlysserileser145150155160lysaspleualatrptyrglnglulysproglylysthrasnlysleu165170175leuiletyrserglyserthrleuglnserglyileproserargphe180185190serglyserglyserglythraspphethrleuthrileserserleu195200205gluprogluaspphealamettyrtyrcysglnglnhisasnlystyr210215220protyrthrpheglyglyglythrlysleugluilelys225230235<210>35<211>237<212>prt<213>人工序列(artificialsequence)<400>35glnvalglnleuglnglnproglyalagluleuvalargproglyala151015servallysleusercyslysalaserglytyrthrphethrsertyr202530trpmetasntrpvallysglnargproaspglnglyleuglutrpile354045glyargileaspprotyraspsergluthrhistyrasnglnlysphe505560lysasplysalaileleuthrvalasplysserserserthralatyr65707580metglnleuserserleuthrsergluaspseralavaltyrtyrcys859095alaargserglnvalserasptyrtrpglyglnglythrthrleuthr100105110valserserglyglyglyglyserglyglyglyglyserglyglygly115120125glyseraspvalglnilethrglnserprosertyrleualaalaser130135140proglygluthrilethrileasncysargalaserlysserileser145150155160lysaspleualatrptyrglnglulysproglylysthrasnlysleu165170175leuiletyrserglyserthrleuglnserglyileproserargphe180185190serglyserglyserglythraspphethrleuthrileserserleu195200205gluprogluaspphealamettyrtyrcysglnglnhisasnlystyr210215220protyrthrpheglyglyglythrlysleugluilelys225230235<210>36<211>237<212>prt<213>人工序列(artificialsequence)<400>36glnvalglnleuglnglnproglyalagluleuvalargproglyala151015servallysleusercyslysalaserglytyrthrphethrsertyr202530trpmetasntrpvallysglnargproaspglnglyleuglutrpile354045glyargileaspprotyraspsergluthrhistyrasnglnlysphe505560lysasplysalaileleuthrvalasplysserserserthralatyr65707580metglnleuserserleuthrsergluaspseralavaltyrtyrcys859095alaargpheglnvalmetthrphetrpglyglnglythrthrleuthr100105110valserserglyglyglyglyserglyglyglyglyserglyglygly115120125glyseraspvalglnilethrglnserprosertyrleualaalaser130135140proglygluthrilethrileasncysargalaserlysserileser145150155160lysaspleualatrptyrglnglulysproglylysthrasnlysleu165170175leuiletyrserglyserthrleuglnserglyileproserargphe180185190serglyserglyserglythraspphethrleuthrileserserleu195200205gluprogluaspphealamettyrtyrcysglnglnhisasnlystyr210215220protyrthrpheglyglyglythrlysleugluilelys225230235<210>37<211>237<212>prt<213>人工序列(artificialsequence)<400>37glnvalglnleuglnglnproglyalagluleuvalargproglyala151015servallysleusercyslysalaserglytyrthrphethrsertyr202530trpmetasntrpvallysglnargproaspglnglyleuglutrpile354045glyargileaspprotyraspsergluthrhistyrasnglnlysphe505560lysasplysalaileleuthrvalasplysserserserthralatyr6570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>人工序列(artificialsequence)<400>49agcggataacaatttcacacagga24<210>50<211>21<212>dna<213>人工序列(artificialsequence)<400>50gccgctggccttgcagctcag21<210>51<211>20<212>dna<213>人工序列(artificialsequence)<400>51gatgcggccgatccactcca20<210>52<211>20<212>dna<213>人工序列(artificialsequence)<400>52gcgggcgcagtagtacacgg20<210>53<211>20<212>dna<213>人工序列(artificialsequence)<400>53gctggcgcggcagttgatgg20<210>54<211>20<212>dna<213>人工序列(artificialsequence)<400>54gtagatcagcagcttgttgg20<210>55<211>22<212>dna<213>人工序列(artificialsequence)<400>55ctggcagtagtacatggcgaag22当前第1页12