CN115894704B - 一种特异性靶向肿瘤EpCAM抗原的抗体及应用 - Google Patents

一种特异性靶向肿瘤EpCAM抗原的抗体及应用 Download PDF

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CN115894704B
CN115894704B CN202111120042.5A CN202111120042A CN115894704B CN 115894704 B CN115894704 B CN 115894704B CN 202111120042 A CN202111120042 A CN 202111120042A CN 115894704 B CN115894704 B CN 115894704B
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王玮
魏于全
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Sichuan University
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Abstract

本发明属于基因工程和生物免疫治疗领域，提供了一种特异性靶向肿瘤EpCAM抗原的嵌合抗原受体，包括胞外抗原识别结构域、跨膜结构域和胞内信号结构域，所述胞外抗原识别结构域包括VHH抗体或其片段，所述VHH抗体或其片段具有特异性靶向肿瘤EpCAM抗原的互补决定区CDR，其能有效识别EpCAM表达阳性的肿瘤细胞，同时不会攻击正常的EpCAM低表达上皮细胞。

Description

一种特异性靶向肿瘤EpCAM抗原的抗体及应用
技术领域
.本发明属于基因工程和生物免疫治疗领域，具体涉及一种特异性靶向肿瘤EpCAM抗原的抗体及其应用。
背景技术
.在肿瘤免疫治疗过程中，诱导患者产生有效的抗肿瘤免疫是疾病治疗的关键。肿瘤细胞可以产生一系列的逃逸机制来避开免疫系统监视。包括下调主要组织相容性复合物(MHC)、下调自身抗原表达、下调免疫检查点分子配体，形成局部抑制微环境等，从而限制自身效应细胞对肿瘤细胞的识别和攻击1。
.目前已知的肿瘤抗原靶点主要分为两大类，一类是仅在肿瘤细胞中表达，而在正常细胞中不表达的抗原，具有肿瘤细胞特异性，被认为是肿瘤特异性抗原；另一类是在肿瘤细胞中高表达，在正常细胞中低表达或不表达的抗原，其表达具有肿瘤相关性，称为肿瘤相关抗原。EpCAM即为肿瘤相关抗原的一种，它在结直肠癌，卵巢癌等肿瘤细胞中高表达，在人的上皮来源细胞中也表达，虽然表达量相对较低，但是也缺乏肿瘤特异性。因此，选择EpCAM作为免疫治疗靶点需要考虑其免疫耐受。
.靶向EpCAM阳性肿瘤的治疗目前集中于抗体药物的开发和疗效评估。如Edrecolomab，Adecatumumab和Catumaxomab，以及抗体-药物偶联药(ADC)。EpCAM单抗药物Edrecolomab在III期结肠癌的辅助治疗中没有观察到其对患者总体生存率和无负荷生存期的改善19-21。Adecatumumab在转移性乳腺癌单药治疗临床研究中发现其具有剂量依赖性和靶抗原依赖性，但未观察到客观肿瘤消退22。Catumaxomab为靶向CD3和EpCAM的双特异性抗体药物，在上皮细胞癌继发的恶性腹水治疗中显示出明显临床获益，并且安全性可控23,24，已于2009年获EMA批准上市用于恶性腹水的治疗，令人遗憾的是该药物由于销售惨淡于2017年退市。此外，抗EpCAM的抗体-药物偶联药如Oportuzumab monatox25和Tucotuzumab26在临床研究中显示出良好的耐受，但是对患者疾病改善和生存期的延长需要更多的临床数据来加以证实。总体来说，靶向EpCAM的抗体药物取得了一定的进展，但仍需要开发新的免疫治疗药物/方法来补充现有医疗技术的不足。
.嵌合抗原受体修饰的T细胞(CAR-T)疗法被认为是一种有效的肿瘤免疫治疗手段。该疗法是在T细胞中表达称为嵌合抗原受体(CAR)的一类重组受体，该受体能将T细胞重定向到所选靶点的肿瘤细胞。CAR分子由多个结构原件组成，分别为胞外的抗原识别结构域，铰链区、跨膜域，和胞内的信号转导结构域。抗原识别结构域包括但不限于免疫球蛋白来源的单链抗体(Single-chain variable fragment,scFv)，该单链抗体由免疫球蛋白来源的重链和轻链的可变区通过G4S等结构连接组成；以及其它非抗体来源的分子，如自然杀伤细胞受体NKG2D能与靶细胞表面的配体NKG2DL结合而识别肿瘤抗原等2。铰链区则为CAR分子的胞外域和胞内域提供柔性连接作用，通常为免疫球蛋白重链的CH2和CH3结构域来源。跨膜区能够将CAR分子锚定在细胞膜上，其来源通常为CD4、CD8、或CD28、4-1BB等共刺激分子的跨膜区。胞内信号转导域由共刺激分子如CD28,4-1BB，ICOS等和胞内激活分子如CD3ζ或FcRγ等组成，能够将胞外域的抗原配体识别信号传导并放大，使T细胞或NK细胞等被激活。
.CAR分子通常是由胞外的抗原识别结构域提供特异性抗原识别功能，将T细胞或NK细胞重定向抗原表达的肿瘤细胞位置。靶抗原的识别能激活CAR分子下游的信号转导，启动T细胞或NK细胞的效应功能。激活的T细胞或NK细胞通过分泌IFN-γ、TNF-α、IL-2、颗粒酶、穿孔素等效应分子，直接作用于靶细胞或募集其它免疫效应细胞参与免疫反应，最终导致靶细胞裂解死亡。因此，目前对CAR分子结构的改造主要思路都是增强CAR-T细胞的功能和靶细胞毒性。
.综上所述，本发明将提供一种新型纳米抗体应用于CAR分子结构的设计中，使其能够特异性识别EpCAM阳性表达的肿瘤细胞，以补充现有医疗技术的不足。
发明内容
.有鉴于此，本发明旨在提供一种特异性靶向肿瘤EpCAM抗原的抗体及应用，具体技术方案如下。
.一种特异性靶向肿瘤EpCAM抗原的嵌合抗原受体，包括胞外抗原识别结构域、跨膜结构域和胞内信号结构域，所述胞外抗原识别结构域包括VHH抗体或其片段，所述VHH抗体或其片段具有特异性靶向肿瘤EpCAM抗原的互补决定区CDR。
.VHH全称为重链单域抗体，为羊驼来源的一类抗体，与传统的IgG不同，VHH只包含重链，其体积小，分子量更小(～15kDa)，因此又称为纳米抗体。与传统的单克隆抗体相比较，它具有独特优势，比如更容易进入抗原表位、免疫原性低、溶解性好、稳定性高，亲和力不逊于scFv等。
.VHH抗体与EpCAM靶点特异性识别的机制即抗体的重链可变区的CDR部位与肿瘤抗原识别结合的过程，其中，VHH的CDR结构决定其靶向EpCAM的特异性。
.进一步，所述VHH抗体或其片段可选地选自Seq ID NO.1-9中的任一氨基酸序列构成的多肽或表位。
.进一步，所述互补决定区CDR可选地选自Seq ID NO.10-18中氨基酸序列的一个或多个。
.现有研究已经证实，存在靶向多个靶点(一般是两个)的情况，这种情况下会在CAR中设计含有多个抗体的识别区，即有多个抗体的CDR区，这些CDR区可以是串联在一起的，也可以是整合在一个CAR结构中在细胞里表达后再分开识别抗原。
.进一步，所述跨膜结构域可选地包括CD3ζ、CD3ε、CD4、CD8α、CD28、CD5、CD16、CD9、CD22、CD33、CD27、CD37、CD45、CD64、CD80、CD86、CD127、CD137、CD134、CD152、CD154、PD-1或Dectin-1中的一个或多个。
.进一步，所述胞内信号结构域可选地包括CD3ζ、CD27、CD28、CD30、CD137、CD134、CD154、Dectin-1、FcRγ或ICOS中的一个或多个。
.进一步，所述VHH抗体或其片段的核苷酸可选地选自Seq ID NO.19-27中的任一序列。
.包含上述嵌合抗原受体的基因转移方式。
.进一步，所述基因转移方式可选地包含病毒载体、转座子系统、电穿孔法或CRISPR/Cas9基因编辑工具。
.一种CAR-T细胞，表达上述的嵌合抗原受体。
.进一步，表达的所述嵌合抗原受体的氨基酸序列如Seq ID NO.28-36任一项所示。
.进一步，表达的所述嵌合抗原受体的核苷酸序列如Seq ID NO.37-45任一项所示。
.一种CAR-NK细胞，表达上述的嵌合抗原受体。
.进一步，表达的所述嵌合抗原受体的氨基酸序列如Seq ID NO.28-36任一项所示。
.进一步，表达的所述嵌合抗原受体的核苷酸序列如Seq ID NO.37-45任一项所示。
.一种抗肿瘤药物，含有本发明所述的CAR-T细胞和药学上允许添加的辅料和/或助剂。
.进一步，所述肿瘤包括上皮细胞来源的实体肿瘤，且EpCAM表达为阳性。
.进一步，所述肿瘤包括上皮来源的恶性肿瘤和循环肿瘤细胞，以及肿瘤干细胞。
.进一步，所述肿瘤包括肠癌、肺癌、卵巢癌、肝癌或胃癌。
.EpCAM全称上皮细胞粘附分子(Epithelial cell adhesion molecule)，是由肿瘤相关钙信号转导1基因编码的一个40KD的单次跨膜糖蛋白，参与调节细胞黏附和迁移，调控增殖和分化以及介导信号转导14。它在1979年被确定为肿瘤相关抗原15，表达于肠癌，肺癌，前列腺癌，卵巢癌等大多数上皮来源的恶性肿瘤细胞16。
.本发明所述的嵌合抗原受体的合成方法，包括以下步骤：(1)构建Anti-EpCAM-VHH-CAR完整基因；(2)使用引物对Primer1和Primer2扩增所述Anti-EpCAM-VHH-CAR基因；(3)利用BamHI和EcoRI限制酶消化扩增出的基因序列并用病毒载体包装。
.进一步，所述病毒载体包括慢病毒载体、腺病毒载体或反转录病毒载体。
.进一步，所述引物Primer1的序列如Seq ID NO.46所示，引物Primer2的序列如Seq ID NO.47所示。
.一种特异性靶向肿瘤EpCAM抗原的VHH抗体或其片段在制备双特异性抗体和抗体-药物偶联药物ADC中的应用，所述VHH抗体或其片段具有特异性靶向肿瘤EpCAM抗原的互补决定区CDR，所述互补决定区CDR可选地选自Seq ID NO.10-18中氨基酸序列的一个或多个。
.一种特异性靶向肿瘤EpCAM抗原的VHH抗体或其片段在制备肿瘤诊断试剂盒的应用，所述VHH抗体或其片段具有特异性靶向肿瘤EpCAM抗原的互补决定区CDR，所述互补决定区CDR可选地选自Seq ID NO.10-18中氨基酸序列的一个或多个。
.进一步，所述肿瘤为上皮细胞来源的实体肿瘤，且EpCAM表达为阳性。
.有益效果
.本发明首先提供了一种特异性靶向肿瘤EpCAM抗原的抗体。如本领域技术人员所知，EpCAM是一种肿瘤相关性抗原，其在肿瘤细胞中高表达，但在正常上皮来源细胞中也表达，虽然表达量相对较低，但是该靶点缺乏肿瘤特异性。本发明团队在选择靶向EpCAM的抗体时，着重考虑了抗体的亲和力问题。当抗体亲和力太高时，虽然能识别EpCAM高表达的肿瘤细胞，但同时会导致CAR-T细胞识别并攻击EpCAM低表达的正常上皮来源细胞；而当抗体的亲和力太低时，则不能有效识别EpCAM表达的肿瘤细胞，导致治疗失败。所以本发明筛选的抗EpCAM的VHH抗体亲和力适中，能够保证有效识别到肿瘤细胞，同时保证其不会识别攻击正常的EpCAM低表达上皮细胞。
.本发明利用筛选出的亲和力适中的纳米级VHH抗体或其片段来构建第二代和第三代嵌合抗原受体分子核酸序列，所述嵌合抗原受体分子在T细胞、NK细胞等免疫效应细胞中表达能够赋予这些免疫细胞靶向识别并裂解表达EpCAM分子的肿瘤细胞的能力，达到治疗肿瘤疾病的目的。
.具体来说，本发明构建的Anti-EpCAM-VHH纳米抗体对EpCAM表达阳性细胞具有高亲和力，用Anti-EpCAM-VHH-CAR转导待治疗患者的T淋巴细胞，将转导后的T淋巴细胞回输给患者，能使成功转导的T细胞重定向到EpCAM阳性表达的靶细胞，产生免疫响应。该方法能增强T细胞对靶细胞的免疫应答。用Anti-EpCAM-VHH-CAR转导T细胞表达嵌合抗原受体的过程能够在体外完成，也能够在体内进行，最终转导成功的细胞都为Anti-EpCAM-VHH-CAR-T细胞。
.此外，本发明筛选出的该抗体或其片段也可用于双特异性结合抗体、抗体-药物偶联药物等靶向治疗产品的研究，同时也可应用于抗体类检测产品。
附图说明
.为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作一简单地介绍。显而易见地，下面描述中的附图是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其它的附图。
.图1为Anti-EpCAM-VHH hFc蛋白分子量鉴定：经HEK293细胞表达的VHH-hFc融合蛋白经二硫苏糖醇(DTT)还原，用SDS-PAGE电泳鉴定相对分子量大小位于43-55kD之间；
.图2为不同VHH hFc结合EpCAM-His的ELISA检测结果；
.图3为Anti-EpCAM-VHH-CAR结构：Anti-EpCAM-VHH-CAR基因装载在本发明自构的慢病毒载体上，利用EF1-α启动子启动CAR基因表达，胞外域为IL-2引导序列和EpCAM-VHH，胞内域为CD8a铰链区和跨膜区、CD28和4-1BB以及CD3ζ胞内段氨基酸残基；
.图4为Anti-EpCAM-VHH-CAR在细胞膜上的表达模式：Anti-EpCAM-VHH-CAR的EpCAM-VHH和CD8a铰链区位于细胞膜外侧，用于识别靶抗原，CD8a跨膜区镶嵌在细胞膜上，用于在膜上固定CAR分子，信号转导域位于膜内，用于传导放大细胞激活信号；
.图5为Anti-EpCAM-VHH-CAR在T细胞中表达检测：利用流式细胞术，使用异硫氰酸酯(FITC)标记的兔抗羊驼抗体能够检测到CAR分子的表达情况；
.图6为Anti-EpCAM-VHH-CAR-T靶向EpCAM表达阳性细胞释放细胞因子；
.图7为Anti-EpCAM-VHH-CAR-T细胞毒性检测。
具体实施方式
.为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述。显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
.需要说明的是，在本文中，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的过程、方法、物品或者装置不仅包括那些要素，而且还包括没有明确列出的其他要素，或者是还包括为这种过程、方法、物品或者装置所固有的要素。在没有更多限制的情况下，由语句“包括一个……”限定的要素，并不排除在包括该要素的过程、方法、物品或者装置中还存在另外的相同要素。
.如在本说明书中使用的，术语“大约”，典型地表示为所述值的+/-5％，更典型的是所述值的+/-4％，更典型的是所述值的+/-3％，更典型的是所述值的+/-2％，甚至更典型的是所述值的+/-1％，甚至更典型的是所述值的+/-0.5％。
.在本说明书中，某些实施方式可能以一种处于某个范围的格式公开。应该理解，这种“处于某个范围”的描述仅仅是为了方便和简洁，且不应该被解释为对所公开范围的僵化限制。因此，范围的描述应该被认为是已经具体地公开了所有可能的子范围以及在此范围内的独立数字值。例如，范围的描述应该被看作已经具体地公开了子范围如从1到3，从1到4，从1到5，从2到4，从2到6，从3到6等，以及此范围内的单独数字，例如1，2，3，4，5和6。无论该范围的广度如何，均适用以上规则。
.名词解释
.本发明所述的“特异性靶向肿瘤EpCAM抗原”是指本发明提供的VHH抗体或其片段能靶向肿瘤细胞上表达丰度较高的EpCAM，而对其他EpCAM低表达的上皮细胞识别能力较弱。
.本发明所述的“表位”是指抗原上被抗体识别、结合并与抗体相互作用的部位。
.在本发明的其中一个实施例中，Anti-EpCAM-VHH-CAR分子的胞外抗原识别域Anti-EpCAM-VHH能够识别并结合人EpCAM分子。
.在本发明的其中一个实施例中，铰链区和跨膜区来源于CD8α，也可以是CD8α以外的其它分子，如CD4，CD28等。共刺激信号转导域来源包括但不限于CD28,4-1BB,也可以来源于ICOS，OX40等，且共刺激分子数量可以为1个或多个。
.在本发明的其中一个实施例中，CD3ζ信号转导域为CD3ζ分子胞内段61-164氨基酸残基。CD3ζ的61-164氨基酸残基含3个受体酪氨酸激活基序(ITAM)，分别为氨基酸残基61-89，100-128，131-159，三个ITAM均能独立介导CD3ζ激活信号转导28。因而在CAR分子结构中使用CD3ζ胞内段、单独或任意组合使用CD3ζITAM构建的CAR分子均包含在本发明范围内。
.在本发明的其中一个实施例中，CD3ζ信号转导域也可以由其它信号转导结构替换，如FcRγ，数目可以为一个或多个。通过改变胞外抗原识别域以外的其它区域构建的嵌合抗原受体都在本发明范围内。
.在本发明的其中一个实施例中，通过在T淋巴细胞中表达Anti-EpCAM-VHH-CAR核酸序列，能够使在T细胞中产生Anti-EpCAM-VHH-CAR分子。此外，该核酸序列也能够在NK细胞中能够表达，因此构建的CAR-NK细胞，也在本发明范围内。
.在本发明的其中一个实施例中，Anti-EpCAM-VHH-CAR核酸分子是构建在慢病毒载体质粒上，利用EF1-α启动子启动CAR基因表达，除慢病毒载体外，使用其它的基因转移方式表达本发明的CAR，如逆转录病毒载体、转座子系统，电穿孔法、CRISPR/Cas9等基因编辑工具，也包含在本发明范围内。
.在本发明的其中一个实施例中，VHH抗体或其片段通过大肠杆菌表达蛋白免疫对羊驼定期免疫得到。
.在本发明的其中一个实施例中，VHH抗体或其片段经过转录和扩增后为单域抗体基因片段。
.以下通过具体实施例进行说明：
.实施例1
.特异性靶向人肿瘤EpCAM抗原的VHH抗体制备
.(1)羊驼免疫和cDNA文库构建。使用人EpCAM抗原1.5mg与佐剂1:1混合后多点注射到羊驼颈部淋巴结附近进行免疫，每周免疫两次，一共进行7次免疫。从第4次免疫开始每次采集外周血进行免疫评估并分离外周血淋巴细胞。用Trizol液裂解淋巴细胞后提取淋巴细胞总RNA，利用反转录试剂盒将提取到的RNA反转录成cDNA，再利用cDNA文库构建试剂盒构建大肠杆菌cDNA文库。
.(2)噬菌体文库制备。将大肠杆菌cDNA文库菌接种到含四环素和氨苄抗生素的2XYT培养基中，37℃震荡培养直到DO600＝0.5。向培养体系中加入辅助噬菌体M13KO7至终浓度1E12 cfu/ml，37℃培养一小时后加入卡那霉素，后30℃培养过夜。之后将培养液5000g离心10分钟取上清，加入PEG/NaCl溶液混匀后4℃静置20分钟。再将溶液4000g离心20分钟弃上清，向沉淀中加入PBS悬浮沉淀，后16000g离心10min即得到噬菌体文库。
.(3)噬菌体筛选VHH。将文库噬菌体和提前包被EpCAM-camFc(人EpCAM与羊驼Fc重组蛋白)的免疫管用3％BSA液室温封闭2h.将封闭后的免疫管用PBS清洗多次，然后将封闭后的噬菌体加入免疫管中，室温旋转孵育1h。用PBS-T液清洗免疫管多次后，加入100mM TEA洗脱，室温孵育10分钟，再加入1M Tris-HCl，所得的洗脱液即为筛选后的噬菌体。该筛选过程需重复2次以去除非特异性噬菌体。
.(4)噬菌体ELISA鉴定VHH。将步骤(3)得到的噬菌体稀释后接种到OD600＝0.5的SS320菌液中，37℃培养30分钟后取培养液涂布2X YT平板。37℃培养过夜后，挑取单克隆接种于含2X YT的48孔板中，37℃培养3-4小时，再向孔板中的加入卡那霉素和辅助噬菌体，30℃过夜培养，离心培养物获得上清液。用EpCAM-camFc提前包被96孔板，并用3％BSA封闭孔板，加入上清液室温孵育1小时，经PBS清洗后，用anti-M13 HRP结合，再用TMB显色，测定OD450值，根据OD450值大小判断不同VHH和EpCAM的亲和力。对比样本孔和阴性对照的OD450值，OD450显著升高的孔对应的蛋白样品进行测序，即获得不同VHH氨基酸序列。通过序列对比去除具有相同CDR区的序列和冗余序列后，得到9条单一氨基酸序列(ID NO.1-9)。利用IMGT数据库(国际免疫遗传学数据库)分析序列确定每条序列的CDR区。在下面列出的序列中标黑加粗的片段，从左到右依次为CDR1、CDR2、CDR3。不同的CDR区序列的组合有不同的VHH-EpCAM亲和力。
.NO.1:
.NO.2:
.NO.3:
.NO.4:
.NO.5:
.NO.6:
.NO.7:
.NO.8:
.NO.9:
.实施例2
.Anti-EpCAM-VHH抗体鉴定。为了在分子水平和细胞水平鉴定Anti-EpCAM-VHH，将筛选到的Anti-EpCAM-VHH融合到人源信号肽和humanFc(hFc，约29kD)的N端，转换到哺乳动物瞬转表达载体pQDFc上，进行HEK293细胞瞬转表达。经过亲和纯化，得到VHH-hFc融合蛋白。经HEK293细胞表达的VHH-hFc融合蛋白经二硫苏糖醇(DTT)还原，用SDS-PAGE电泳鉴定相对分子量大小在43-55kD之间(图1)。
.实施例3
.VHH-hFc结合能力鉴定。VHH-hFc结合能力鉴定用ELISA方法，鉴定VHH-hFc与EpCAM抗原的结合能力。将EpCAM-His蛋白1ng/ul包被在96孔板中，4度置放过夜，后用5倍梯度稀释的VHH-hFc蛋白(起始溶度为20ng/ul)与孔板中的EpCAM-His蛋白结合1小时，再用anti-hFc-HRP二抗结合VHH-hFc，显色后检测450nm波长下的OD值，根据OD450判断VHH-hFc与EpCAM-His蛋白的结合能力。
.实验结论：筛选到的9条Anti-EpCAM-VHH序列与EpCAM有不同程度的亲和力，其中1194亲和力最强，1201亲和力最弱，VHH 1191与EpCAM的亲和力处于中等水平(图2)。
.实施例4
.靶向EpCAM的嵌合抗原受体(Anti-EpCAM-VHH-CAR)构建及慢病毒载体构建。在Anti-EpCAM-VHH-CAR构建过程中，包含Anti-EpCAM-VHH的胞外段、CD8α跨膜区与胞内信号转导域(8α28BBζ)为合成的完整基因。使用Primer1：5‘-CGGGATCCATGTACCGGATGCAG-3’(SEQID NO.46)和Primer2：5‘-CGGAATTCTTAGCGAGGGGGC-3’(SEQ ID NO.47)扩增Anti-EpCAM-VHH-CAR基因。在引物序列ID NO.46和ID NO.47中分别添加了BamHI和EcoRI限制性内切酶位点，扩增得到的Anti-EpCAM-VHH-CAR核苷酸在5’段引入了BamHI位点，在3’端引入了EcoRI位点，利用BamHI和EcoRI限制酶消化Anti-EpCAM-VHH-CAR产物和自构载体PCLK质粒，能够在两种产物中获得相同的BamHI和EcoRI粘性末端，便于两种基因片段在相同的粘性末端利用T4连接酶连接成完整的DNA质粒环。
.实施例5
.人淋巴细胞培养及慢病毒转导。提取健康人的外周血单核细胞，培养在人淋巴细胞培养基X-vivo-15中，添加5％FBS和100IU/ml人IL-2。用CD3/CD28抗体包被的磁珠刺激细胞(磁珠：细胞＝3：1)24小时，使T细胞被激活。再在包被了纤维连接蛋白(50ug/ml)的孔板中加入慢病毒颗粒和激活后的T细胞，感染复数为2-4，1000xg离心2小时促进慢病毒感染T细胞。成功被慢病毒感染的T细胞表达Anti-EpCAM-VHH-CAR基因，即为Anti-EpCAM-VHH-CAR-T细胞。
.实施例6
.人淋巴细胞表达嵌合抗原受体检测在逆转录感染48-72小时进行。由于Anti-EpCAM-VHH-CAR胞外域的Anti-EpCAM-VHH为羊驼来源，使用兔抗羊驼VHH抗体(Genscript，南京)能够进行检测，检测方法为流式细胞术。
.实施例7
.靶向特异性细胞因子释放检测。由于Anti-EpCAM-VHH-CAR-T对EpCAM阳性表达的细胞具有特异性细胞毒作用，本发明选用了三种肿瘤细胞作为靶细胞，分别是高表达EpCAM的人结肠癌肿瘤细胞HT29，低表达EpCAM的人卵巢癌细胞SKOV3和不表达EpCAM的人宫颈癌细胞Hela。在细胞因子检测前，将1*105个(E:T＝10:1)或5*104(E:T＝5:1)个Anti-EpCAM-VHH-CAR-T细胞分别与三种肿瘤细胞在96孔板中共培养，肿瘤细胞数为1*104个/孔，共培养24小时候后取共培养上清检测IFN-γ、TNF-α和IL-2，检测方法为酶联免疫吸附试验。
.实验结论：Anti-EpCAM-VHH-CAR-T细胞在共培养体系中识别并结合靶细胞表面的EpCAM，促使CAR-T细胞效应功能被激活。释放大量的细胞因子，如IFN-γ，TNF-α等，通过对共培养上清中的细胞因子分泌量进行检测，能够评估CAR-T细胞的靶向特异性激活程度。图6中的CAR-T细胞能够被EpCAM阳性的HT29和SKOV3细胞刺激释放细胞因子，但是不能被EpCAM阴性的Hela细胞所激活，且CAR-T细胞的因子释放水平随着靶细胞抗原表达的增强而升高，具有抗原依赖性。
.实施例8
.细胞毒性检测。使用实时细胞毒性检测法测定Anti-EpCAM-VHH-CAR-T对肿瘤细胞毒性。具体方案为取HT29和Hela细胞铺实施标记检测板，细胞数为1*104个/孔，至于TRCA检测仪上记录细胞贴壁情况，铺板24小时细胞贴壁稳定后按照设定的效应细胞：靶细胞(E:T)比例添加Anti-EpCAM-CAR-T细胞，后置于TRCA检测仪上记录细胞贴壁情况。靶细胞被CAR-T细胞杀伤后将失去贴壁性。
.实验结论：通过实时检测靶细胞的贴壁性来评估CAR-T细胞的细胞毒性。图7显示在靶细胞HT29和Hela中加入T细胞后，ControlT细胞组HT29细胞数有所增加后缓慢减少，而CAR-T细胞组的HT29细胞迅速减少；在Hela细胞中加入Control T细胞和CAR-T细胞后，细胞均同等程度的减少，无显著变化。证实CAR-T细胞具有靶向EpCAM+细胞毒性。
.本发明涉及的序列如下表所示：
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.本发明涉及到的参考文献
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.上面结合附图对本发明的实施例进行了描述，但是本发明并不局限于上述的具体实施方式，上述的具体实施方式仅仅是示意性的，而不是限制性的，本领域的普通技术人员在本发明的启示下，在不脱离本发明宗旨和权利要求所保护的范围情况下，还可做出很多形式，这些均属于本发明的保护之内。
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Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Pro Thr Ser Ser Leu Arg
20 25 30
Thr Met Gly Trp Phe Arg Gln Ala Ser Gly Lys Glu Arg Glu Arg Val
35 40 45
Ala Val Ile Trp Asp Gly Arg Thr Thr Asp Tyr Asp Asp Ser Val Gln
50 55 60
Asp Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys Ser Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Thr Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala
85 90 95
Ala Ser Pro Arg Ile Val Pro Phe Ala Ser Thr Tyr Phe Gln His
100 105 110
<210> 16
<211> 116
<212> PRT
<213> 人工序列
<400> 16
His Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Gly Ser
20 25 30
Ile Phe Ser Arg Cys Gly Met Arg Trp Tyr Arg Gln Ala Pro Gly Lys
35 40 45
Glu Arg Glu Leu Val Ser Ser Thr Ser Lys Asp Gly Phe Thr Ser Tyr
50 55 60
Thr Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Asn
65 70 75 80
Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Lys Thr Glu Asp Thr Ala
85 90 95
Val Tyr Ser Cys Ala Ala Ile Cys Ala Val Gly Gly Tyr Ser Leu Ser
100 105 110
Thr Tyr Thr Tyr
115
<210> 17
<211> 113
<212> PRT
<213> 人工序列
<400> 17
Glu Val Gln Leu Val Glu Ser Gly Gly Asp Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Ser Pro Gly Ser Tyr
20 25 30
Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Arg Val
35 40 45
Ala Ile Ile Glu Ser Arg Gly Thr Val Thr Tyr Val Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys Ala
85 90 95
Ala Ser Arg Pro Trp Ser Gly Val Arg Cys Leu His Asp Lys Tyr Asp
100 105 110
Tyr
<210> 18
<211> 118
<212> PRT
<213> 人工序列
<400> 18
His Val Gln Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ser Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr Ala Tyr Ser Ser Leu
20 25 30
Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val Ala Ala
35 40 45
Leu Leu Thr Ala Ile Gly Gly Pro Thr Arg Thr Thr Tyr Ala Asp Ser
50 55 60
Val Lys Gly Arg Leu Ala Ile Ser Gln Asp His Ala Lys Asn Thr Leu
65 70 75 80
Tyr Leu Gln Met Ser Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr
85 90 95
Cys Ala Ala Gly Arg Pro Ala Gly Thr Pro Arg Trp Leu Leu Leu Ala
100 105 110
Pro Arg Asp Tyr Asn Tyr
115
<210> 19
<211> 387
<212> DNA
<213> 人工序列
<400> 19
gacgtgcagc tggtggagag cggcggcggc agcgtgcaga gcggcggcag cctgaggctg 60
agctgcgccg ccagcggcta cacctacagg aggtactaca tgggctggtt caggcaggcc 120
cccggcgagc agagggaggg cgtggccgtg atcaacaacg acggcaggac caactacgcc 180
gacagcgtga agggcaggtt caggatcagc agggacaacg ccgagaacac cctgcacctg 240
gagatgaaca gcctgaagcc cgaggacacc gccatgtact actgcgccgc caccggcaac 300
atcctgcccc ccatgaccgc cgtgcccccc ctgggcaggc agtggtaccc ctactggggc 360
aggggcaccc tggtgaccgt gagcagc 387
<210> 20
<211> 390
<212> DNA
<213> 人工序列
<400> 20
cacgtgcagc tggtggagag cggcggcggc agcgtgcaga gcggcggcag cctgaggctg 60
agctgcgccg ccagcggcta cgccgtgaag aactgcatgg gctggttcag gcaggccccc 120
ggcaaggaga gggagggcgt ggccgtgatc aacaggaacg gcatcaccac ctacgccgac 180
agcgtgaagg gcaggttcac catcagccag gacaaggaca agaacaccct ggacctgcag 240
atgaacagcc tgaagcccga ggacaccgcc atgtactact gcgccgccac ccccaccctg 300
ctgaccatcc ccgccaggtt cctgtgcgac gtgaggaacc ccagcggctt caccgactgg 360
ggccagggca ccctggtgac cgtgagcagc 390
<210> 21
<211> 384
<212> DNA
<213> 人工序列
<400> 21
caggtgcagc tggtggagag cggcggcggc agcgtgcagg ccggcggcag cctgaggctg 60
agctgcgtgg tgagcgccta cagcgcctac acctacaaga ccatgtgcat gggctggttc 120
aggcaggccc ccggcaagga gagggagggc gtggccgcca tctacagggg cggcctgaac 180
acctactacg ccgacagcgt gaagggcagg ttcatcatca gcagggacaa cgccgagagc 240
accatgtacc tgcagatgaa cagcctgaag cccgaggaca ccgccatgta ctactgcgcc 300
gccgactggc tgaggggcga cgactgcaac atcggcgcca acttcgacta ctggggccag 360
ggcacccagg tgaccgtgag cagc 384
<210> 22
<211> 396
<212> DNA
<213> 人工序列
<400> 22
caggtgcagc tggtggagag cggcggcggc agcgtgcagg ccggcggcag cctgaggctg 60
agctgcgtgg ccaccggctt caccatcagc aggaagtgca tgggctggtt cagggaggcc 120
cccggcaaga agagggaggt gatcgccacc atcaacaccg gcagcagcag cccctactac 180
gccgacggcg tgaagggcag gttcaccatc agccaggaca acgccaagaa caccgtgtac 240
ctgcagatga acagcctgaa gcccgaggac accgccatgt actactgcgc cgccaccaag 300
ggcgtggtgg tgggcaccgg ctactgcggc ggcccctacg tggagaggcc caacagcgcc 360
tactggggcc agggcaccca ggtgaccgtg agcagc 396
<210> 23
<211> 378
<212> DNA
<213> 人工序列
<400> 23
gacgtgcagc tggtggagag cggcggcggc agcgtgcagg ccggcaggag cctgaggctg 60
agctgcgagc tgagcgacta cacctggagc accgtgtgca tgggctggtt caggcaggcc 120
cccggcaagg agagggaggg cgtggccgtg atctacacca ggagcggcgg caccacctac 180
gccgacagcg ccaagggcag gttcaccatc agcagggaca acgccaagga caccctgtac 240
ctgcagatgg acagcctgaa gcccgaggac accgccatgt actactgcgc cgccggcccc 300
ctgtacgacg gcaggtgcac ctacaggagc cccgccttcc actactgggg ccagggcacc 360
caggtgaccg tgagcagc 378
<210> 24
<211> 366
<212> DNA
<213> 人工序列
<400> 24
gacgtgcagc tggtggaaag cggaggagga agcgcccagg ccggaggaag cctgagactg 60
agctgcgccg ccagcggacc cacctcttca ctgagaacaa tgggatggtt cagacaggcc 120
tctggcaaag aaagagagag ggtggccgtc atttgggatg gcagaacaac cgactacgac 180
gactccgtgc aggacagatt caccatcagc caggacaacg ccaagagcac agtctatctg 240
cagatgaaca cactgaagcc cgaagatacc gccatgtact actgcgcagc cagccccaga 300
atcgtgccct tcgccagcac ctacttccag cactggggac agggaaccca ggtgaccgtc 360
agctcc 366
<210> 25
<211> 381
<212> DNA
<213> 人工序列
<400> 25
cacgtgcagc tggtggagag cggcggcggc agcgtgcagg ccggcggcag cctgaagctg 60
agctgcgccg ccagcggcag catcttcagc ggcagcatct tcagcaggtg cggcatgagg 120
tggtacaggc aggcccccgg caaggagagg gagctggtga gcagcaccag caaggacggc 180
ttcaccagct acaccgacag cgtgaagggc aggttcacca tcagccagga caacgccaac 240
aacaccctgt acctgcagat gagcagcctg aagaccgagg acaccgccgt gtacagctgc 300
gccgccatct gcgccgtggg cggctacagc ctgagcacct acacctactg gggccagggc 360
acccaggtga ccgtgagcag c 381
<210> 26
<211> 372
<212> DNA
<213> 人工序列
<400> 26
gaggtgcagc tggtggagag cggcggcgac agcgtgcagg ccggcggcag cctgaggctg 60
agctgcgccg ccagcggcta cagccccggc agctactgca tgggctggtt caggcaggcc 120
cccggcaagg agagggagag ggtggccatc atcgagagca ggggcaccgt gacctacgtg 180
gacagcgtga agggcaggtt caccatcagc aaggacaacg ccaagaacac cctgtacctg 240
cagatgaaca gcctgaagcc cgaggacacc gccatgtact actgcgccgc cagcaggccc 300
tggagcggcg tgaggtgcct gcacgacaag tacgactact ggggccaggg cacccaggtg 360
accgtgagca gc 372
<210> 27
<211> 387
<212> DNA
<213> 人工序列
<400> 27
cacgtgcagc tggtggagag cggcggcggc agcgtgcaga gcggcggcag cctgaggctg 60
agctgcgccg tgagcggcta cgcctacagc agcctggcct ggttcaggca ggcccccggc 120
aaggagaggg agggcgtggc cgccctgctg accgccatcg gcggccccac caggaccacc 180
tacgccgaca gcgtgaaggg caggctggcc atcagccagg accacgccaa gaacaccctg 240
tacctgcaga tgagcagcct gaagcccgag gacaccgcca tgtactactg cgccgccggc 300
aggcccgccg gcacccccag gtggctgctg ctggccccca gggactacaa ctactggggc 360
cagggcaccc aggtgaccgt gagcagc 387
<210> 28
<211> 403
<212> PRT
<213> 人工序列
<400> 28
Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu
1 5 10 15
Val Thr Asn Ser Ala Asp Val Gln Leu Val Glu Ser Gly Gly Gly Ser
20 25 30
Val Gln Ser Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr
35 40 45
Thr Tyr Arg Arg Tyr Tyr Met Gly Trp Phe Arg Gln Ala Pro Gly Glu
50 55 60
Gln Arg Glu Gly Val Ala Val Ile Asn Asn Asp Gly Arg Thr Asn Tyr
65 70 75 80
Ala Asp Ser Val Lys Gly Arg Phe Arg Ile Ser Arg Asp Asn Ala Glu
85 90 95
Asn Thr Leu His Leu Glu Met Asn Ser Leu Lys Pro Glu Asp Thr Ala
100 105 110
Met Tyr Tyr Cys Ala Ala Thr Gly Asn Ile Leu Pro Pro Met Thr Ala
115 120 125
Val Pro Pro Leu Gly Arg Gln Trp Tyr Pro Tyr Trp Gly Arg Gly Thr
130 135 140
Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr
145 150 155 160
Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala
165 170 175
Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe
180 185 190
Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val
195 200 205
Leu Leu Leu Ser Leu Val Ile Thr Arg Ser Lys Arg Ser Arg Leu Leu
210 215 220
His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg
225 230 235 240
Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg
245 250 255
Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe
260 265 270
Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg
275 280 285
Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Ala Pro Ala Tyr Gln
290 295 300
Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu
305 310 315 320
Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly
325 330 335
Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu
340 345 350
Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys
355 360 365
Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu
370 375 380
Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu
385 390 395 400
Pro Pro Arg
<210> 29
<211> 404
<212> PRT
<213> 人工序列
<400> 29
Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu
1 5 10 15
Val Thr Asn Ser Ala His Val Gln Leu Val Glu Ser Gly Gly Gly Ser
20 25 30
Val Gln Ser Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr
35 40 45
Ala Val Lys Asn Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu
50 55 60
Arg Glu Gly Val Ala Val Ile Asn Arg Asn Gly Ile Thr Thr Tyr Ala
65 70 75 80
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Lys Asp Lys Asn
85 90 95
Thr Leu Asp Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met
100 105 110
Tyr Tyr Cys Ala Ala Thr Pro Thr Leu Leu Thr Ile Pro Ala Arg Phe
115 120 125
Leu Cys Asp Val Arg Asn Pro Ser Gly Phe Thr Asp Trp Gly Gln Gly
130 135 140
Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro
145 150 155 160
Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu
165 170 175
Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp
180 185 190
Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly
195 200 205
Val Leu Leu Leu Ser Leu Val Ile Thr Arg Ser Lys Arg Ser Arg Leu
210 215 220
Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr
225 230 235 240
Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr
245 250 255
Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro
260 265 270
Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys
275 280 285
Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Ala Pro Ala Tyr
290 295 300
Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg
305 310 315 320
Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met
325 330 335
Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn
340 345 350
Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met
355 360 365
Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly
370 375 380
Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala
385 390 395 400
Leu Pro Pro Arg
<210> 30
<211> 396
<212> PRT
<213> 人工序列
<400> 30
Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu
1 5 10 15
Val Thr Asn Ser Ala His Val Gln Leu Val Glu Ser Gly Gly Gly Ser
20 25 30
Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ala
35 40 45
Thr Arg Arg Thr Thr Cys Val Ser Trp Phe Arg Gln Ala Pro Gly Lys
50 55 60
Glu Arg Glu Arg Leu Ala Thr Ile Leu Thr Gly Thr Ser Tyr Thr Asn
65 70 75 80
Tyr Ala Asp Ser Val Lys Gly Arg Phe Ile Ile Ser Gln Asp Asn Ala
85 90 95
Lys Asn Thr Val Tyr Leu Gln Met Ser Ser Leu Lys Pro Glu Asp Thr
100 105 110
Ala Met Tyr Tyr Cys Ala Ala Asn Leu Gly Gly Leu Cys Pro Pro Ala
115 120 125
Glu Tyr Ala Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Thr
130 135 140
Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser
145 150 155 160
Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly
165 170 175
Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp
180 185 190
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
195 200 205
Thr Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met
210 215 220
Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala
225 230 235 240
Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys
245 250 255
Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr
260 265 270
Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly
275 280 285
Gly Cys Glu Leu Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr
290 295 300
Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys
305 310 315 320
Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys
325 330 335
Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala
340 345 350
Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys
355 360 365
Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr
370 375 380
Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
385 390 395
<210> 31
<211> 402
<212> PRT
<213> 人工序列
<400> 31
Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu
1 5 10 15
Val Thr Asn Ser Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser
20 25 30
Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Val Val Ser Ala Tyr
35 40 45
Ser Ala Tyr Thr Tyr Lys Thr Met Cys Met Gly Trp Phe Arg Gln Ala
50 55 60
Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Ile Tyr Arg Gly Gly Leu
65 70 75 80
Asn Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Ile Ile Ser Arg
85 90 95
Asp Asn Ala Glu Ser Thr Met Tyr Leu Gln Met Asn Ser Leu Lys Pro
100 105 110
Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Asp Trp Leu Arg Gly Asp
115 120 125
Asp Cys Asn Ile Gly Ala Asn Phe Asp Tyr Trp Gly Gln Gly Thr Gln
130 135 140
Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro
145 150 155 160
Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys
165 170 175
Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala
180 185 190
Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu
195 200 205
Leu Leu Ser Leu Val Ile Thr Arg Ser Lys Arg Ser Arg Leu Leu His
210 215 220
Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys
225 230 235 240
His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser
245 250 255
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
260 265 270
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
275 280 285
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Ala Pro Ala Tyr Gln Gln
290 295 300
Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu
305 310 315 320
Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly
325 330 335
Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu
340 345 350
Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly
355 360 365
Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser
370 375 380
Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro
385 390 395 400
Pro Arg
<210> 32
<211> 406
<212> PRT
<213> 人工序列
<400> 32
Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu
1 5 10 15
Val Thr Asn Ser Ala Gln Val Gln Leu Val Glu Ser Gly Gly Gly Ser
20 25 30
Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Val Ala Thr Gly Phe
35 40 45
Thr Ile Ser Arg Lys Cys Met Gly Trp Phe Arg Glu Ala Pro Gly Lys
50 55 60
Lys Arg Glu Val Ile Ala Thr Ile Asn Thr Gly Ser Ser Ser Pro Tyr
65 70 75 80
Tyr Ala Asp Gly Val Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala
85 90 95
Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr
100 105 110
Ala Met Tyr Tyr Cys Ala Ala Thr Lys Gly Val Val Val Gly Thr Gly
115 120 125
Tyr Cys Gly Gly Pro Tyr Val Glu Arg Pro Asn Ser Ala Tyr Trp Gly
130 135 140
Gln Gly Thr Gln Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg
145 150 155 160
Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg
165 170 175
Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly
180 185 190
Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr
195 200 205
Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Arg Ser Lys Arg Ser
210 215 220
Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly
225 230 235 240
Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala
245 250 255
Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys
260 265 270
Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys
275 280 285
Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Ala Pro
290 295 300
Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly
305 310 315 320
Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro
325 330 335
Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu
340 345 350
Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile
355 360 365
Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr
370 375 380
Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met
385 390 395 400
Gln Ala Leu Pro Pro Arg
405
<210> 33
<211> 400
<212> PRT
<213> 人工序列
<400> 33
Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu
1 5 10 15
Val Thr Asn Ser Ala Asp Val Gln Leu Val Glu Ser Gly Gly Gly Ser
20 25 30
Val Gln Ala Gly Arg Ser Leu Arg Leu Ser Cys Glu Leu Ser Asp Tyr
35 40 45
Thr Trp Ser Thr Val Cys Met Gly Trp Phe Arg Gln Ala Pro Gly Lys
50 55 60
Glu Arg Glu Gly Val Ala Val Ile Tyr Thr Arg Ser Gly Gly Thr Thr
65 70 75 80
Tyr Ala Asp Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
85 90 95
Lys Asp Thr Leu Tyr Leu Gln Met Asp Ser Leu Lys Pro Glu Asp Thr
100 105 110
Ala Met Tyr Tyr Cys Ala Ala Gly Pro Leu Tyr Asp Gly Arg Cys Thr
115 120 125
Tyr Arg Ser Pro Ala Phe His Tyr Trp Gly Gln Gly Thr Gln Val Thr
130 135 140
Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro
145 150 155 160
Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro
165 170 175
Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
180 185 190
Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu
195 200 205
Ser Leu Val Ile Thr Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp
210 215 220
Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr
225 230 235 240
Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Lys Arg
245 250 255
Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro
260 265 270
Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu
275 280 285
Glu Glu Glu Gly Gly Cys Glu Leu Ala Pro Ala Tyr Gln Gln Gly Gln
290 295 300
Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp
305 310 315 320
Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro
325 330 335
Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
340 345 350
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
355 360 365
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
370 375 380
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
385 390 395 400
<210> 34
<211> 396
<212> PRT
<213> 人工序列
<400> 34
Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu
1 5 10 15
Val Thr Asn Ser Ala Asp Val Gln Leu Val Glu Ser Gly Gly Gly Ser
20 25 30
Ala Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Pro
35 40 45
Thr Ser Ser Leu Arg Thr Met Gly Trp Phe Arg Gln Ala Ser Gly Lys
50 55 60
Glu Arg Glu Arg Val Ala Val Ile Trp Asp Gly Arg Thr Thr Asp Tyr
65 70 75 80
Asp Asp Ser Val Gln Asp Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys
85 90 95
Ser Thr Val Tyr Leu Gln Met Asn Thr Leu Lys Pro Glu Asp Thr Ala
100 105 110
Met Tyr Tyr Cys Ala Ala Ser Pro Arg Ile Val Pro Phe Ala Ser Thr
115 120 125
Tyr Phe Gln His Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Thr
130 135 140
Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser
145 150 155 160
Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly
165 170 175
Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp
180 185 190
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
195 200 205
Thr Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met
210 215 220
Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala
225 230 235 240
Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys
245 250 255
Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr
260 265 270
Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly
275 280 285
Gly Cys Glu Leu Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr
290 295 300
Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys
305 310 315 320
Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys
325 330 335
Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala
340 345 350
Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys
355 360 365
Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr
370 375 380
Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
385 390 395
<210> 35
<211> 401
<212> PRT
<213> 人工序列
<400> 35
Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu
1 5 10 15
Val Thr Asn Ser Ala His Val Gln Leu Val Glu Ser Gly Gly Gly Ser
20 25 30
Val Gln Ala Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Ser
35 40 45
Ile Phe Ser Gly Ser Ile Phe Ser Arg Cys Gly Met Arg Trp Tyr Arg
50 55 60
Gln Ala Pro Gly Lys Glu Arg Glu Leu Val Ser Ser Thr Ser Lys Asp
65 70 75 80
Gly Phe Thr Ser Tyr Thr Asp Ser Val Lys Gly Arg Phe Thr Ile Ser
85 90 95
Gln Asp Asn Ala Asn Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Lys
100 105 110
Thr Glu Asp Thr Ala Val Tyr Ser Cys Ala Ala Ile Cys Ala Val Gly
115 120 125
Gly Tyr Ser Leu Ser Thr Tyr Thr Tyr Trp Gly Gln Gly Thr Gln Val
130 135 140
Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala
145 150 155 160
Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg
165 170 175
Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys
180 185 190
Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu
195 200 205
Leu Ser Leu Val Ile Thr Arg Ser Lys Arg Ser Arg Leu Leu His Ser
210 215 220
Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His
225 230 235 240
Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Lys
245 250 255
Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg
260 265 270
Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro
275 280 285
Glu Glu Glu Glu Gly Gly Cys Glu Leu Ala Pro Ala Tyr Gln Gln Gly
290 295 300
Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr
305 310 315 320
Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
325 330 335
Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln
340 345 350
Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu
355 360 365
Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr
370 375 380
Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro
385 390 395 400
Arg
<210> 36
<211> 403
<212> PRT
<213> 人工序列
<400> 36
Met Tyr Arg Met Gln Leu Leu Ser Cys Ile Ala Leu Ser Leu Ala Leu
1 5 10 15
Val Thr Asn Ser Ala His Val Gln Leu Val Glu Ser Gly Gly Gly Ser
20 25 30
Val Gln Ser Gly Gly Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Tyr
35 40 45
Ala Tyr Ser Ser Leu Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg
50 55 60
Glu Gly Val Ala Ala Leu Leu Thr Ala Ile Gly Gly Pro Thr Arg Thr
65 70 75 80
Thr Tyr Ala Asp Ser Val Lys Gly Arg Leu Ala Ile Ser Gln Asp His
85 90 95
Ala Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Lys Pro Glu Asp
100 105 110
Thr Ala Met Tyr Tyr Cys Ala Ala Gly Arg Pro Ala Gly Thr Pro Arg
115 120 125
Trp Leu Leu Leu Ala Pro Arg Asp Tyr Asn Tyr Trp Gly Gln Gly Thr
130 135 140
Gln Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr
145 150 155 160
Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala
165 170 175
Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe
180 185 190
Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val
195 200 205
Leu Leu Leu Ser Leu Val Ile Thr Arg Ser Lys Arg Ser Arg Leu Leu
210 215 220
His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg
225 230 235 240
Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg
245 250 255
Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe
260 265 270
Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg
275 280 285
Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Ala Pro Ala Tyr Gln
290 295 300
Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu
305 310 315 320
Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly
325 330 335
Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu
340 345 350
Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys
355 360 365
Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu
370 375 380
Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu
385 390 395 400
Pro Pro Arg
<210> 37
<211> 1220
<212> DNA
<213> 人工序列
<400> 37
atgtaccgga tgcagctgct gagctgtatc gccctgagcc tggccctggt gaccaacagc 60
gccgacgtgc agctggtgga gagcggcggc ggcagcgtgc agagcggcgg cagcctgagg 120
ctgagctgcg ccgccagcgg ctacacctac aggaggtact acatgggctg gttcaggcag 180
gcccccggcg agcagaggga gggcgtggcc gtgatcaaca acgacggcag gaccaactac 240
gccgacagcg tgaagggcag gttcaggatc agcagggaca acgccgagaa caccctgcac 300
ctggagatga acagcctgaa gcccgaggac accgccatgt actactgcgc cgccaccggc 360
aacatcctgc cccccatgac cgccgtgccc cccctgggca ggcagtggta cccctactgg 420
ggcaggggca ccctggtgac cgtgagcagc accacgacgc cagcgccgcg accaccaaca 480
ccggcgccca ccatcgcgtc gcagcccctg tccctgcgcc cagaggcgtg ccggccagcg 540
gcggggggcg cagtgcacac gagggggctg gacttcgcct gtgatatcta catctgggcg 600
cccctggccg ggacttgtgg ggtccttctc ctgtcactgg ttatcaccag gagtaagagg 660
agcaggctcc tgcacagtga ctacatgaac atgactcccc gccgccccgg gcccacccgc 720
aagcattacc agccctatgc cccaccacgc gacttcgcag cctatcgctc caaacggggc 780
agaaagaaac tcctgtatat attcaaacaa ccatttatga gaccagtaca aactactcaa 840
gaggaagatg gctgtagctg ccgatttcca gaagaagaag aaggaggatg tgaactggcc 900
cccgcgtacc agcagggcca gaaccagctc tataacgagc tcaatctagg acgaagagag 960
gagtacgatg ttttggacaa gagacgtggc cgggaccctg agatgggggg aaagccgcag 1020
agaaggaaga accctcagga aggcctgtac aatgaactgc agaaagataa gatggcggag 1080
gcctacagtg agattgggat gaaaggcgag cgccggaggg gcaaggggca cgatggcctt 1140
taccagggtc tcagtacagc caccaaggac acctacgacg cccttcacat gcaggccctg 1200
ccccctcgct aagaattccg 1220
<210> 38
<211> 1223
<212> DNA
<213> 人工序列
<400> 38
atgtaccgga tgcagctgct gagctgtatc gccctgagcc tggccctggt gaccaacagc 60
gcccacgtgc agctggtgga gagcggcggc ggcagcgtgc agagcggcgg cagcctgagg 120
ctgagctgcg ccgccagcgg ctacgccgtg aagaactgca tgggctggtt caggcaggcc 180
cccggcaagg agagggaggg cgtggccgtg atcaacagga acggcatcac cacctacgcc 240
gacagcgtga agggcaggtt caccatcagc caggacaagg acaagaacac cctggacctg 300
cagatgaaca gcctgaagcc cgaggacacc gccatgtact actgcgccgc cacccccacc 360
ctgctgacca tccccgccag gttcctgtgc gacgtgagga accccagcgg cttcaccgac 420
tggggccagg gcaccctggt gaccgtgagc agcaccacga cgccagcgcc gcgaccacca 480
acaccggcgc ccaccatcgc gtcgcagccc ctgtccctgc gcccagaggc gtgccggcca 540
gcggcggggg gcgcagtgca cacgaggggg ctggacttcg cctgtgatat ctacatctgg 600
gcgcccctgg ccgggacttg tggggtcctt ctcctgtcac tggttatcac caggagtaag 660
aggagcaggc tcctgcacag tgactacatg aacatgactc cccgccgccc cgggcccacc 720
cgcaagcatt accagcccta tgccccacca cgcgacttcg cagcctatcg ctccaaacgg 780
ggcagaaaga aactcctgta tatattcaaa caaccattta tgagaccagt acaaactact 840
caagaggaag atggctgtag ctgccgattt ccagaagaag aagaaggagg atgtgaactg 900
gcccccgcgt accagcaggg ccagaaccag ctctataacg agctcaatct aggacgaaga 960
gaggagtacg atgttttgga caagagacgt ggccgggacc ctgagatggg gggaaagccg 1020
cagagaagga agaaccctca ggaaggcctg tacaatgaac tgcagaaaga taagatggcg 1080
gaggcctaca gtgagattgg gatgaaaggc gagcgccgga ggggcaaggg gcacgatggc 1140
ctttaccagg gtctcagtac agccaccaag gacacctacg acgcccttca catgcaggcc 1200
ctgccccctc gctaagaatt ccg 1223
<210> 39
<211> 1199
<212> DNA
<213> 人工序列
<400> 39
atgtaccgga tgcagctgct gagctgtatc gccctgagcc tggccctggt gaccaacagc 60
gcccacgtgc agctggtgga gagcggcggc ggcagcgtgc aggccggcgg cagcctgagg 120
ctgagctgcg ccgccagcgg cgccaccagg aggaccacct gcgtgagctg gttcaggcag 180
gcccccggca aggagaggga gaggctggcc accatcctga ccggcaccag ctacaccaac 240
tacgccgaca gcgtgaaggg caggttcatc atcagccagg acaacgccaa gaacaccgtg 300
tacctgcaga tgagcagcct gaagcccgag gacaccgcca tgtactactg cgccgccaac 360
ctgggcggcc tgtgcccccc cgccgagtac gcctactggg gccagggcac ccaggtgacc 420
gtgagcagca ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg 480
cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg 540
agggggctgg acttcgcctg tgatatctac atctgggcgc ccctggccgg gacttgtggg 600
gtccttctcc tgtcactggt tatcaccagg agtaagagga gcaggctcct gcacagtgac 660
tacatgaaca tgactccccg ccgccccggg cccacccgca agcattacca gccctatgcc 720
ccaccacgcg acttcgcagc ctatcgctcc aaacggggca gaaagaaact cctgtatata 780
ttcaaacaac catttatgag accagtacaa actactcaag aggaagatgg ctgtagctgc 840
cgatttccag aagaagaaga aggaggatgt gaactggccc ccgcgtacca gcagggccag 900
aaccagctct ataacgagct caatctagga cgaagagagg agtacgatgt tttggacaag 960
agacgtggcc gggaccctga gatgggggga aagccgcaga gaaggaagaa ccctcaggaa 1020
ggcctgtaca atgaactgca gaaagataag atggcggagg cctacagtga gattgggatg 1080
aaaggcgagc gccggagggg caaggggcac gatggccttt accagggtct cagtacagcc 1140
accaaggaca cctacgacgc ccttcacatg caggccctgc cccctcgcta agaattccg 1199
<210> 40
<211> 1217
<212> DNA
<213> 人工序列
<400> 40
atgtaccgga tgcagctgct gagctgtatc gccctgagcc tggccctggt gaccaacagc 60
gcccaggtgc agctggtgga gagcggcggc ggcagcgtgc aggccggcgg cagcctgagg 120
ctgagctgcg tggtgagcgc ctacagcgcc tacacctaca agaccatgtg catgggctgg 180
ttcaggcagg cccccggcaa ggagagggag ggcgtggccg ccatctacag gggcggcctg 240
aacacctact acgccgacag cgtgaagggc aggttcatca tcagcaggga caacgccgag 300
agcaccatgt acctgcagat gaacagcctg aagcccgagg acaccgccat gtactactgc 360
gccgccgact ggctgagggg cgacgactgc aacatcggcg ccaacttcga ctactggggc 420
cagggcaccc aggtgaccgt gagcagcacc acgacgccag cgccgcgacc accaacaccg 480
gcgcccacca tcgcgtcgca gcccctgtcc ctgcgcccag aggcgtgccg gccagcggcg 540
gggggcgcag tgcacacgag ggggctggac ttcgcctgtg atatctacat ctgggcgccc 600
ctggccggga cttgtggggt ccttctcctg tcactggtta tcaccaggag taagaggagc 660
aggctcctgc acagtgacta catgaacatg actccccgcc gccccgggcc cacccgcaag 720
cattaccagc cctatgcccc accacgcgac ttcgcagcct atcgctccaa acggggcaga 780
aagaaactcc tgtatatatt caaacaacca tttatgagac cagtacaaac tactcaagag 840
gaagatggct gtagctgccg atttccagaa gaagaagaag gaggatgtga actggccccc 900
gcgtaccagc agggccagaa ccagctctat aacgagctca atctaggacg aagagaggag 960
tacgatgttt tggacaagag acgtggccgg gaccctgaga tggggggaaa gccgcagaga 1020
aggaagaacc ctcaggaagg cctgtacaat gaactgcaga aagataagat ggcggaggcc 1080
tacagtgaga ttgggatgaa aggcgagcgc cggaggggca aggggcacga tggcctttac 1140
cagggtctca gtacagccac caaggacacc tacgacgccc ttcacatgca ggccctgccc 1200
cctcgctaag aattccg 1217
<210> 41
<211> 1229
<212> DNA
<213> 人工序列
<400> 41
atgtaccgga tgcagctgct gagctgtatc gccctgagcc tggccctggt gaccaacagc 60
gcccaggtgc agctggtgga gagcggcggc ggcagcgtgc aggccggcgg cagcctgagg 120
ctgagctgcg tggccaccgg cttcaccatc agcaggaagt gcatgggctg gttcagggag 180
gcccccggca agaagaggga ggtgatcgcc accatcaaca ccggcagcag cagcccctac 240
tacgccgacg gcgtgaaggg caggttcacc atcagccagg acaacgccaa gaacaccgtg 300
tacctgcaga tgaacagcct gaagcccgag gacaccgcca tgtactactg cgccgccacc 360
aagggcgtgg tggtgggcac cggctactgc ggcggcccct acgtggagag gcccaacagc 420
gcctactggg gccagggcac ccaggtgacc gtgagcagca ccacgacgcc agcgccgcga 480
ccaccaacac cggcgcccac catcgcgtcg cagcccctgt ccctgcgccc agaggcgtgc 540
cggccagcgg cggggggcgc agtgcacacg agggggctgg acttcgcctg tgatatctac 600
atctgggcgc ccctggccgg gacttgtggg gtccttctcc tgtcactggt tatcaccagg 660
agtaagagga gcaggctcct gcacagtgac tacatgaaca tgactccccg ccgccccggg 720
cccacccgca agcattacca gccctatgcc ccaccacgcg acttcgcagc ctatcgctcc 780
aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 840
actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 900
gaactggccc ccgcgtacca gcagggccag aaccagctct ataacgagct caatctagga 960
cgaagagagg agtacgatgt tttggacaag agacgtggcc gggaccctga gatgggggga 1020
aagccgcaga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1080
atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 1140
gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1200
caggccctgc cccctcgcta agaattccg 1229
<210> 42
<211> 1211
<212> DNA
<213> 人工序列
<400> 42
atgtaccgga tgcagctgct gagctgtatc gccctgagcc tggccctggt gaccaacagc 60
gccgacgtgc agctggtgga gagcggcggc ggcagcgtgc aggccggcag gagcctgagg 120
ctgagctgcg agctgagcga ctacacctgg agcaccgtgt gcatgggctg gttcaggcag 180
gcccccggca aggagaggga gggcgtggcc gtgatctaca ccaggagcgg cggcaccacc 240
tacgccgaca gcgccaaggg caggttcacc atcagcaggg acaacgccaa ggacaccctg 300
tacctgcaga tggacagcct gaagcccgag gacaccgcca tgtactactg cgccgccggc 360
cccctgtacg acggcaggtg cacctacagg agccccgcct tccactactg gggccagggc 420
acccaggtga ccgtgagcag caccacgacg ccagcgccgc gaccaccaac accggcgccc 480
accatcgcgt cgcagcccct gtccctgcgc ccagaggcgt gccggccagc ggcggggggc 540
gcagtgcaca cgagggggct ggacttcgcc tgtgatatct acatctgggc gcccctggcc 600
gggacttgtg gggtccttct cctgtcactg gttatcacca ggagtaagag gagcaggctc 660
ctgcacagtg actacatgaa catgactccc cgccgccccg ggcccacccg caagcattac 720
cagccctatg ccccaccacg cgacttcgca gcctatcgct ccaaacgggg cagaaagaaa 780
ctcctgtata tattcaaaca accatttatg agaccagtac aaactactca agaggaagat 840
ggctgtagct gccgatttcc agaagaagaa gaaggaggat gtgaactggc ccccgcgtac 900
cagcagggcc agaaccagct ctataacgag ctcaatctag gacgaagaga ggagtacgat 960
gttttggaca agagacgtgg ccgggaccct gagatggggg gaaagccgca gagaaggaag 1020
aaccctcagg aaggcctgta caatgaactg cagaaagata agatggcgga ggcctacagt 1080
gagattggga tgaaaggcga gcgccggagg ggcaaggggc acgatggcct ttaccagggt 1140
ctcagtacag ccaccaagga cacctacgac gcccttcaca tgcaggccct gccccctcgc 1200
taagaattcc g 1211
<210> 43
<211> 1199
<212> DNA
<213> 人工序列
<400> 43
atgtaccgga tgcagctgct gagctgtatc gccctgagcc tggccctggt gaccaacagc 60
gccgacgtgc agctggtgga aagcggagga ggaagcgccc aggccggagg aagcctgaga 120
ctgagctgcg ccgccagcgg acccacctct tcactgagaa caatgggatg gttcagacag 180
gcctctggca aagaaagaga gagggtggcc gtcatttggg atggcagaac aaccgactac 240
gacgactccg tgcaggacag attcaccatc agccaggaca acgccaagag cacagtctat 300
ctgcagatga acacactgaa gcccgaagat accgccatgt actactgcgc agccagcccc 360
agaatcgtgc ccttcgccag cacctacttc cagcactggg gacagggaac ccaggtgacc 420
gtcagctcca ccacgacgcc agcgccgcga ccaccaacac cggcgcccac catcgcgtcg 480
cagcccctgt ccctgcgccc agaggcgtgc cggccagcgg cggggggcgc agtgcacacg 540
agggggctgg acttcgcctg tgatatctac atctgggcgc ccctggccgg gacttgtggg 600
gtccttctcc tgtcactggt tatcaccagg agtaagagga gcaggctcct gcacagtgac 660
tacatgaaca tgactccccg ccgccccggg cccacccgca agcattacca gccctatgcc 720
ccaccacgcg acttcgcagc ctatcgctcc aaacggggca gaaagaaact cctgtatata 780
ttcaaacaac catttatgag accagtacaa actactcaag aggaagatgg ctgtagctgc 840
cgatttccag aagaagaaga aggaggatgt gaactggccc ccgcgtacca gcagggccag 900
aaccagctct ataacgagct caatctagga cgaagagagg agtacgatgt tttggacaag 960
agacgtggcc gggaccctga gatgggggga aagccgcaga gaaggaagaa ccctcaggaa 1020
ggcctgtaca atgaactgca gaaagataag atggcggagg cctacagtga gattgggatg 1080
aaaggcgagc gccggagggg caaggggcac gatggccttt accagggtct cagtacagcc 1140
accaaggaca cctacgacgc ccttcacatg caggccctgc cccctcgcta agaattccg 1199
<210> 44
<211> 1214
<212> DNA
<213> 人工序列
<400> 44
atgtaccgga tgcagctgct gagctgtatc gccctgagcc tggccctggt gaccaacagc 60
gcccacgtgc agctggtgga gagcggcggc ggcagcgtgc aggccggcgg cagcctgaag 120
ctgagctgcg ccgccagcgg cagcatcttc agcggcagca tcttcagcag gtgcggcatg 180
aggtggtaca ggcaggcccc cggcaaggag agggagctgg tgagcagcac cagcaaggac 240
ggcttcacca gctacaccga cagcgtgaag ggcaggttca ccatcagcca ggacaacgcc 300
aacaacaccc tgtacctgca gatgagcagc ctgaagaccg aggacaccgc cgtgtacagc 360
tgcgccgcca tctgcgccgt gggcggctac agcctgagca cctacaccta ctggggccag 420
ggcacccagg tgaccgtgag cagcaccacg acgccagcgc cgcgaccacc aacaccggcg 480
cccaccatcg cgtcgcagcc cctgtccctg cgcccagagg cgtgccggcc agcggcgggg 540
ggcgcagtgc acacgagggg gctggacttc gcctgtgata tctacatctg ggcgcccctg 600
gccgggactt gtggggtcct tctcctgtca ctggttatca ccaggagtaa gaggagcagg 660
ctcctgcaca gtgactacat gaacatgact ccccgccgcc ccgggcccac ccgcaagcat 720
taccagccct atgccccacc acgcgacttc gcagcctatc gctccaaacg gggcagaaag 780
aaactcctgt atatattcaa acaaccattt atgagaccag tacaaactac tcaagaggaa 840
gatggctgta gctgccgatt tccagaagaa gaagaaggag gatgtgaact ggcccccgcg 900
taccagcagg gccagaacca gctctataac gagctcaatc taggacgaag agaggagtac 960
gatgttttgg acaagagacg tggccgggac cctgagatgg ggggaaagcc gcagagaagg 1020
aagaaccctc aggaaggcct gtacaatgaa ctgcagaaag ataagatggc ggaggcctac 1080
agtgagattg ggatgaaagg cgagcgccgg aggggcaagg ggcacgatgg cctttaccag 1140
ggtctcagta cagccaccaa ggacacctac gacgcccttc acatgcaggc cctgccccct 1200
cgctaagaat tccg 1214
<210> 45
<211> 1220
<212> DNA
<213> 人工序列
<400> 45
atgtaccgga tgcagctgct gagctgtatc gccctgagcc tggccctggt gaccaacagc 60
gcccacgtgc agctggtgga gagcggcggc ggcagcgtgc agagcggcgg cagcctgagg 120
ctgagctgcg ccgtgagcgg ctacgcctac agcagcctgg cctggttcag gcaggccccc 180
ggcaaggaga gggagggcgt ggccgccctg ctgaccgcca tcggcggccc caccaggacc 240
acctacgccg acagcgtgaa gggcaggctg gccatcagcc aggaccacgc caagaacacc 300
ctgtacctgc agatgagcag cctgaagccc gaggacaccg ccatgtacta ctgcgccgcc 360
ggcaggcccg ccggcacccc caggtggctg ctgctggccc ccagggacta caactactgg 420
ggccagggca cccaggtgac cgtgagcagc accacgacgc cagcgccgcg accaccaaca 480
ccggcgccca ccatcgcgtc gcagcccctg tccctgcgcc cagaggcgtg ccggccagcg 540
gcggggggcg cagtgcacac gagggggctg gacttcgcct gtgatatcta catctgggcg 600
cccctggccg ggacttgtgg ggtccttctc ctgtcactgg ttatcaccag gagtaagagg 660
agcaggctcc tgcacagtga ctacatgaac atgactcccc gccgccccgg gcccacccgc 720
aagcattacc agccctatgc cccaccacgc gacttcgcag cctatcgctc caaacggggc 780
agaaagaaac tcctgtatat attcaaacaa ccatttatga gaccagtaca aactactcaa 840
gaggaagatg gctgtagctg ccgatttcca gaagaagaag aaggaggatg tgaactggcc 900
cccgcgtacc agcagggcca gaaccagctc tataacgagc tcaatctagg acgaagagag 960
gagtacgatg ttttggacaa gagacgtggc cgggaccctg agatgggggg aaagccgcag 1020
agaaggaaga accctcagga aggcctgtac aatgaactgc agaaagataa gatggcggag 1080
gcctacagtg agattgggat gaaaggcgag cgccggaggg gcaaggggca cgatggcctt 1140
taccagggtc tcagtacagc caccaaggac acctacgacg cccttcacat gcaggccctg 1200
ccccctcgct aagaattccg 1220
<210> 46
<211> 23
<212> DNA
<213> 人工序列
<400> 46
cgggatccat gtaccggatg cag 23
<210> 47
<211> 21
<212> DNA
<213> 人工序列
<400> 47
cggaattctt agcgaggggg c 21

Claims (14)

1.一种特异性靶向肿瘤EpCAM抗原的嵌合抗原受体，包括胞外抗原识别结构域、跨膜结构域和胞内信号结构域，其特征在于，所述胞外抗原识别结构域包括VHH抗体，所述VHH抗体具有特异性靶向肿瘤EpCAM抗原的互补决定区CDR；所述VHH抗体为Seq ID NO.6所示的氨基酸序列。
2.如权利要求1所述的嵌合抗原受体，其特征在于，所述跨膜结构域包括CD3ζ、CD3ε、CD4、CD8α、CD28、CD5、CD16、CD9、CD22、CD33、CD27、CD37、CD45、CD64、CD80、CD86、CD127、CD137、CD134、CD152、CD154、PD-1或Dectin-1中的一个或多个。
3.如权利要求1所述的嵌合抗原受体，其特征在于，所述胞内信号结构域包括CD3ζ、CD27、CD28、CD30、CD137、CD134、CD154、Dectin-1、FcRγ或ICOS中的一个或多个。
4.如权利要求1所述的嵌合抗原受体，其特征在于，所述VHH抗体的核苷酸为Seq IDNO.24所示的序列。
5.一种CAR-T细胞，其特征在于，表达权利要求1-4任一项所述的嵌合抗原受体。
6.如权利要求5所述的CAR-T细胞，其特征在于，所述嵌合抗原受体的氨基酸序列如SeqID NO.34所示；或者，所述嵌合抗原受体的核苷酸序列如Seq ID NO.43所示。
7.一种CAR-NK细胞，其特征在于，表达权利要求1-4任一项所述的嵌合抗原受体。
8.如权利要求7所述的CAR-NK细胞，其特征在于，所述嵌合抗原受体的氨基酸序列如Seq ID NO.34所示；或者，所述嵌合抗原受体的核苷酸序列如Seq ID NO.43所示。
9.一种抗肿瘤药物，其特征在于，含有权利要求5所述的CAR-T细胞和药学上允许添加的辅料和/或助剂；所述肿瘤为结肠癌和卵巢癌。
10.权利要求1-4任一项所述的嵌合抗原受体的合成方法，其特征在于，包括以下步骤：（1）构建Anti-EpCAM-VHH-CAR完整基因；（2）使用引物对Primer1和Primer2扩增所述Anti-EpCAM-VHH-CAR基因；（3）利用BamHI和EcoRI限制酶消化扩增出的基因序列并用病毒载体包装。
11.如权利要求10所述的合成方法，其特征在于，所述病毒载体包括慢病毒载体、腺病毒载体或反转录病毒载体。
12.如权利要求10所述的合成方法，其特征在于，所述引物Primer1的序列如Seq IDNO.46所示，引物Primer2的序列如Seq ID NO.47所示。
13.一种特异性靶向肿瘤EpCAM抗原的VHH抗体在制备双特异性抗体和抗体-药物偶联药物ADC中的应用，其特征在于，所述VHH抗体具有特异性靶向肿瘤EpCAM抗原的互补决定区CDR，所述VHH抗体为Seq ID NO.6所示的氨基酸序列。
14.一种特异性靶向肿瘤EpCAM抗原的VHH抗体在制备肿瘤诊断试剂盒的应用，其特征在于，所述VHH抗体具有特异性靶向肿瘤EpCAM抗原的互补决定区CDR，所述VHH抗体为SeqID NO.6所示的氨基酸序列；所述肿瘤为结肠癌和卵巢癌。
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