CN107082811B - 一种嵌合抗原受体并融合诱导性凋亡酶的复合蛋白 - Google Patents
一种嵌合抗原受体并融合诱导性凋亡酶的复合蛋白 Download PDFInfo
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Abstract
本发明公开了一种嵌合抗原受体并融合诱导性凋亡酶的复合蛋白，其编码序列和含有复合蛋白的修饰的T淋巴细胞。本发明还涉及上述复合蛋白和修饰的T淋巴细胞在预防和治疗乳腺癌中的用途。本发明构建的特异性嵌合抗原受体融合诱导性凋亡酶复合蛋白具有结合Her2受体，强化T淋巴细胞激活状态以及调控细胞凋亡功能；该修饰的T淋巴细胞能高效杀伤Her‑2阳性乳腺癌细胞，并且在药物AP1903的作用下能诱导修饰的T淋巴细胞凋亡，作为安全保障以终止其扩大性杀伤作用。
Description
技术领域
本发明涉及基因工程学和肿瘤学领域，尤其是一种嵌合抗原受体并融合诱导性凋亡酶的复合蛋白及其构建方法，本发明还涉及上述复合蛋白在制备治疗乳腺癌药物中的用途。
背景技术
我国乳腺癌的发病率呈现逐年上升的趋势，特别是晚期转移乳腺癌的病例增多。目前治疗乳腺癌仍然是以手术为主的综合治疗。自从提出了“乳腺癌是全身性疾病”的概念以后，全身治疗的方案越来越受到重视。但是，已经发生全身转移的乳腺癌仍然属于难治之症，这些病例往往对多种化疗和放疗方案都不敏感，各种治疗手段除了稍能延长寿命，并没有突破性进展。
研究发现，约25～30％的乳腺癌病人的癌组织细胞表达一种表皮细胞生长因子受体2(Human Epiderma lGrowth Factor Receptor 2，Her2)。虽然Her2在乳腺癌的表达率不算高，但是阳性的病例往往预后差、发展快。Her2在乳腺癌中的过量表达不仅与癌细胞的增殖率和发生转移的机会呈正相关，而且这些病例往往对多种化疗放疗不敏感。这主要由于Her2作为表皮生长因子受体，具有促癌细胞生长，扩散和肿瘤血管增生等功能，因此，Her2蛋白是乳腺癌恶化的分子标记，现在临床上已经采用检测癌组织中Her2表达来预测乳腺癌病人的预后，抗Her2治疗也已经通过了试验阶段，正式应用于临床实践。
2012年，我们课题组应用具有结合Her2受体和携带抗癌siRNA药物双向功能的融合蛋白作为载体成功识别Her2阳性乳腺癌细胞，并能在人源化小鼠荷瘤模型中成功输送功能性RNA序列抑制肿瘤细胞增殖迁移。因此Her-2抗体是有效结合Her-2分子的蛋白分子。
目前通过阻断Her2受体来治疗乳腺癌的途径主要有以下几类：
1.抗Her2单克隆抗体：
通过封闭乳腺癌细胞表面Her2受体，阻断其与各种生长因子的结合，从而抑制受体激活，同时诱导受体经胞吞作用进入细胞，减少细胞表面Her2的表达量。其中单抗Trastuzumab，又称herceptin，是第一个获美国FDA批准应用于晚期乳腺癌治疗的抗Her2药物。数年来多个中心大宗病例的临床应用结果证明，Herceptin单药治疗能使Her2阳性的晚期乳腺癌病例进入临床缓解期，并维持18个月之久。与化疗药物联合使用治疗已经发生全身转移的乳腺癌能增加肿瘤对化疗的敏感性，明显地延长病人的寿命。但是，距离真正有效地控制和治愈乳腺癌，Herceptin的疗效尚不够理想，这主要由于乳腺癌细胞Her2受体是在基因水平过量表达，而Herceptin只是通过阻断受体蛋白起作用，新的Her2蛋白仍在源源不断地合成并输送到细胞膜，补充丧失的受体。而且鉴于心脏毒性和过敏反应，单抗的用量不可能无限制地加大。另一方面，Herceptin只作用于Her2跨膜蛋白的细胞外部分，其胞内部分的酪氨酸激酶仍可被邻近未被阻断的表皮生长因子受体激活来传导增殖信号。
2.酪氨酸激酶抑制剂：
Her2刺激细胞增殖的信号主要通过酪氨酸激酶系统的级联活化来传导。通过抑制Her2受体蛋白胞内部分的酪氨酸激酶(如细胞外信号调节激酶Erk1抑制剂)，在细胞培养和动物实验中能抑制乳腺癌细胞增殖。但是与酪氨酸激酶抑制剂往往只针对级联网络系统的某一环节，而Her2信号还可以通过旁路途径传导。此外，酪氨酸激酶抑制剂不能直接抑制过量表达的Her2基因，更不能彻底地阻断Her2蛋白的合成。更重要的是，酪氨酸激酶也是正常细胞所必需，使用其抑制剂干扰了很多正常组织细胞的信号传导途径，引起多种毒副作用。
因此，要进一步扩展抗Her2治疗乳腺癌的临床成果，有必要发展毒性低，又能彻底抑制整个Her2跨膜蛋白表达的有效方法。
3.传统的反义基因方法：
包括特异性的反义寡核苷酸和核糖酶(ribozyme)均曾用于抑制乳腺癌细胞Her2基因表达的实验研究。但是由于这些传统的反义基因方法抑制基因表达的效果较弱，因此不能满足临床应用的需求。
4.CART细胞即过继性细胞免疫治疗是抗肿瘤治疗中的新型手段
CAR-T疗法，即Chimeric Antigen Receptor T Cel lImmunotherapy，嵌合抗原受体T细胞免疫疗法。它是过继性细胞免疫治疗的一种，这一概念较早就出现，是近几年才被改良使用到临床上，并在急性白血病和非霍奇金淋巴瘤等肿瘤的治疗上取得较显著的疗效的新型细胞疗法。被认为是最有前景的肿瘤治疗方式之一。CAR-T技术经过一系列漫长的演化过程中，逐渐走向成熟，并在抗肿瘤治疗上展现出日益优越的特点。
人体内正常免疫细胞在执行抗肿瘤功能时主要以识别并启动杀伤途径达到清除肿瘤细胞的作用，其中，T淋巴细胞是主要直接杀手。但由于肿瘤细胞的免疫逃逸以及表面抗原的多变性使得具有抗肿瘤细胞功能的免疫细胞不能及时识别肿瘤细胞造成肿瘤细胞不能被及早完全清除而形成肿瘤，进而危害人体健康。而具有特异抗原受体修饰的T淋巴细胞可以直接识别肿瘤抗原，并快速活化启动杀伤效应，可达到快速，特异，精准的效果。
细胞凋亡(apoptosis)，是生物体细胞的主动消亡过程，是多细胞有机体调控机体发育、控制细胞衰老、维持内环境稳定的重要机制。一般认为，细胞凋亡存在三条主要通路：线粒体通路、内质网通路和死亡受体通路，各通路间互相联系，共同调节细胞凋亡。其中，胱天蛋白酶(caspases)-9是线粒体通路上启动细胞凋亡的一个关键蛋白，caspase-9酶原的激活可以启动下游效应蛋白酶从而引起细胞凋亡。早在2001年已有科学家研究出一种诱导型的caspase-9(简称iCasp-9，与细胞内结合蛋白Fkbp12融合)可在药物AP1903的调控下自激活引起细胞凋亡，开启人为可调控的细胞凋亡，即通过药物可以及时让细胞“自杀”。这种设计随即在肿瘤细胞的研究上进行。后续的研究表明，AP1903对含有iCasp-9酶的细胞诱导凋亡快，体内代谢快，对机体其他部位影响小，是合适的调控药物。因此，iCasp-9可以作为调控细胞凋亡也即是控制CAR-T治疗的一个安全“按钮”。
目前，CART细胞疗法在白血病等非实体肿瘤的治疗中取得显著的进展，在国外已进入临床试验阶段。而在实体瘤的治疗上也进行了较多尝试，如黑色素癌，前列腺癌等等。CAR-T治疗应用的主要障碍在于特异识别特定的肿瘤细胞时，可能出现的细胞脱靶效应，以及由于重新输入大量激活型T淋巴细胞可能诱发的细胞炎症因子风暴对患者的机体伤害。当大量CAR-T细胞输入体内在快速地杀伤肿瘤细胞时，有可能会因为肿瘤细胞凋亡产生的碎片等，引起自身机体短时间内大量炎症因子的产生，或因为大量杀伤产生级联放大效应，损伤正常器官细胞。
发明内容
基于此，本发明的目的在于克服上述现有技术的不足之处而提供一种能避免诱发细胞炎症因子风暴，特异性识别并高效杀死肿瘤细胞并能够在淋巴细胞发生失常时及时诱导“自杀”的方法。
为实现上述目的，本发明采取的技术方案为：一种嵌合抗原受体并融合诱导性凋亡酶的复合蛋白，所述复合蛋白由SEQ ID NO：2所示的氨基酸序列组成。此处抗原指Her2单链中的一个片段。
在另一个方面，本发明还涉及一种分离的核酸，所述核酸编码上述复合蛋白。优选地，所述核酸的核苷酸序列如SEQ ID NO：1所示。
在另一个方面，本发明还涉及一种载体，所述载体表达上述复合蛋白。可用于插入目的多核苷酸的载体是本领域公知的，包括但不限于克隆载体和表达载体，在一个实施方案中，载体可以是例如质粒，粘粒，噬菌体，柯斯质粒等等。
在另一个方面，本发明还涉及一种修饰的T淋巴细胞，所述修饰的T淋巴细胞表达上述复合蛋白。
在另一个方面，本发明还涉及一种药物组合物，所述药物组合物包含上述复合蛋白，或上述分离的核酸，或上述载体，或上述修饰的T淋巴细胞。应当说明的是，所述复合蛋白包括但不限于SEQ ID NO：2所示的氨基酸序列，还可以包括复合蛋白的变体。
在另一个方面，本发明还涉及上述复合蛋白，或分离的核酸，或载体，或修饰的T淋巴细胞在制备治疗乳腺癌的药物中的用途。
在另一个方面，本发明还涉及上述修饰的T淋巴细胞和AP1903的联合使用在制备治疗乳腺癌的药物中的用途。由此，AP1903能够在修饰的T淋巴细胞发生失常时及时诱导其“自杀”。此处“失常”是指，在修饰的T淋巴细胞快速地杀伤肿瘤细胞时，有可能会因为肿瘤细胞凋亡产生的碎片等，引起自身机体的短时间内大量炎症因子的产生，或因为大量杀伤产生级联放大效应损伤正常细胞。
下面对本发明中相关术语进行相应说明和解释：
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的细胞培养、分子遗传学、核酸化学、免疫学实验室操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
根据本发明，术语“变体”是指这样的蛋白，其氨基酸序列与本发明的复合蛋白(如SEQ ID NO：2所示的蛋白)的氨基酸序列具有一个或多个(例如1～3个或1～5个或1～10个)氨基酸不同或者具有至少60％，80％，85％，90％，95％，96％，97％，98％，或99％的同一性，并且其保留了所述复合蛋白的必要特性。此处术语“必要特性”是指：能够结合Her2受体；能强化激活免疫T淋巴细胞；能诱导肿瘤细胞凋亡；能够在真核表达系统中可溶性地表达；利用本发明所涉及的表达纯化方法能够获得高产率的纯化蛋白。术语“同一性”是对核苷酸序列或氨基酸序列的相似性的量度。通常将序列排列起来，以获得最大限度的匹配。“同一性”本身具有本领域公知的意义并且可用公开的算法(例如BLAST)来计算。
在本发明中，术语“真核表达系统”是指由293FT细胞与慢病毒质粒表达载体组成的表达系统，其中293FT细胞从市场上购买获得。
根据本发明，术语“载体(vector)”是指，可将多核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸所编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌体；柯斯质粒等等。
可用于本发明的方法中的缓冲液是本领域公知的，包括但不限于，Tris缓冲液，磷酸盐缓冲液，HEPES缓冲液，MOPS缓冲液等等。
综上所述，本发明的有益效果为：
1、基于体内自身免疫细胞识别杀伤肿瘤细胞的原理，通过基因工程的方法，使自身T淋巴细胞表达靶向Her-2表面分子的CAR(嵌合抗原受体)串联两种免疫激活信号分子片段(CD137与CD3ζ)得到本发明的复合蛋白，含有该复合蛋白的T淋巴细胞在药物AP1903的配合下，可实现肿瘤细胞被快速识别，并且被杀伤进而导致凋亡，同时，能避免以往CART疗法中出现的炎症因子风暴等严重不良反应出现；
2、本发明的复合蛋白将抗Her-2分子单链Fv区域与T细胞受体的细胞内T细胞信号区相结合，能将具有细胞毒性的T淋巴细胞靶向于表达Her-2的细胞(乳腺癌细胞等)，然后通过质粒转染技术，对细胞毒性T细胞进行修饰，并且该复合蛋白设有开启“自杀”的开关，具有高效精准安全的优点。
附图说明
图1为实施例3中pMSCV puro-Her2-scfv-CD8α-CD137-CD3ζ-iCasp9用EcoRI和XhoI的酶切鉴定后的电泳结果，其中图1(a)中，M为marker，2979bp处上方有相应的条带，与预期条带的位置相吻合；图1(b)为maeker的各DNA条带的电泳图；
图2为pMSCV puro-Her2-scfv-CD8α-CD137-CD3ζ-iCasp9结构图；
图3为实施例8修饰的CAR-T淋巴细胞活化鉴定结果图；
图4为实施例9中修饰的CAR-T淋巴细胞杀伤能力检测结果图；
图5为实施例9中修饰的CAR-T淋巴细胞的靶向特异性检测结果图；
图6为实施例9中修饰的CAR-T淋巴细胞在药物AP1903的调控下的结果图。
具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。除非特别指明，本发明中所使用的分子生物学实验方法和免疫检测法，基本上参照J.Sambrook等人，分子克隆：实验室手册，第2版，冷泉港实验室出版社，1989，以及F.M.Ausubel等人，精编分子生物学实验指南，第3版，JohnWiley&Sons，Inc.，1995中所述的方法进行；限制性内切酶的使用依照产品制造商推荐的条件。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。
本发明旨在构建具有结合Her2受体，同时能强化激活免疫T淋巴细胞，又能够调控细胞凋亡的特异性嵌合抗原受体并融合诱导性细胞凋亡酶的复合蛋白；以及开发CAR-T细胞在杀伤肿瘤细胞的效应能力以及调控能力，加速CAR-T技术应用于临床；并为治疗晚期乳腺癌提供有效的武器，发展新型的抗癌细胞疗法。
通过本发明的方法构建的特异性嵌合抗原受体并融合诱导性细胞凋亡酶的复合蛋白具有结合Her2受体，强化T淋巴细胞激活状态以及调控细胞凋亡的功能；其能高效杀伤Her-2阳性乳腺癌细胞，并且在药物AP1903的诱导下能发生细胞凋亡，进一步验证了CAR-T细胞的治疗效果和临床可行性；此复合蛋白的成功开发为发展嵌和抗原受体细胞免疫治疗法奠定了坚实的基础，将为治疗晚期乳腺癌提供又一个高质量且有力的手段。
为更好的说明本发明的目的、技术方案和优点，下面将结合附图和具体实施例对本发明作进一步说明。
实施例1构建具有结合Her2受体、同时强化激活免疫T淋巴细胞、又能够调控细胞凋亡的特异受体蛋白的质粒
1.1引物设计：从基因文库中查到Her2单链片段抗体，激活片段(CD8α链，CD137跨膜段，CD3ζ胞内段)，诱导片段(Fkbp12以及caspase-9的保守段)的序列后，用Primer软件分别设计相关引物以及连接引物。以上涉及片段全场序列均可从基因Bank中查找获得。
1.1.1.Her2-ScFv引物：
Forward：NcoI为内切酶
5’-GGGCCATGGCCCAGGTGCAGCTGTTGCAGTCTGGGGCAGAG-3’(SEQ ID NO：3)
Reverse：NotI为内切酶
5’-TTGCGGCCGCTCCGGAATTCACCTAGGACGGTCAGCTTGGTCCC-3’(SEQ ID NO：4)
1.1.2.激活片段的引物：
①CD8α链引物：
F：CCAGCGAAGCCCACCACGAC(SEQ ID NO：5)
R：AGGAGATGATATCACAGGCGAAGTCCAGCC(SEQ ID NO：6)
②CD137引物：
F：CGCCTGTGATATCATCTCCTTCTTTCTTGC(SEQ ID NO：7)
R：ACTTCACTCTTCACAGTTCACATCCTCCTT(SEQ ID NO：8)
③CD3ζ胞内段引物：
F：TGAACTGTGAAGAGTGAAGTTCAGCAGGAG(SEQ ID NO：9)
R：AGGGGGCAGGGCCTGCATG(SEQ ID NO：10)
1.1.3.诱导片段引物
F1：ACTAACAGGCAAGCAGCAAAGTTGT(SEQ ID NO：11)
F2：AGCGGCGGCGGCAGCACTAACAGG(SEQ ID NO：12)
R：TTATGATGTTTTAAAGAAAAGTTTTTTCCG(SEQ ID NO：13)
1.2 PCR克隆相关目的片段：
1.2.1.PCR反应体系1：50ul体系
1.2.2.PCR反应循环
1.3.PCR产物酶切
1.3.1.Her2ScFv的PCR产物用XhoI和NotI内切酶切开
50ul体系
Her2ScFv的PCR产物酶体系在37℃水浴4小时。
1.3.2.pMSCV puro用XhoI和EcoRI内切酶切开
50ul体系：
pMSCV puro的酶切体系在37℃水浴4小时。
1.4将上述三个PCR产物进行切胶回收目的片段
1.4.1将酶切鉴定片段大小正确的PCR产物全部上样1％Agarose胶电泳；
1.4.2将目的DNA片段用干净的手术刀切下，放入1.5ml离心管，放入回收胶前离心管称重；
1.4.3称重后，在1.5ml离心管中加入以每毫克胶加入1微升体积的bindingbuffer；
1.4.4 60℃水浴中放置10min(至胶完全溶解)，每2～3分钟混匀一次(溶胶液应与member binding buffer颜色相同)；
1.4.5加入1倍胶体积的异丙醇然后充分混匀，静置片刻；
1.4.6将柱子放样品转移入柱子中(柱子的最大容量为800ul)，室温放置1min，然后16000g×1min离心；
1.4.7取下柱子，弃流出液，将柱子放回原收集管中；
1.4.8加入700u lwashing buffer至收集管，室温放置1min，然后16000g×1min离心；
1.4.9加入500u lwashing buffer至收集管，然后16000g×1min离心；
1.4.10取下收集管，弃流出液，再次16000g×1min离心；
1.4.11将收集管放置在一支新的1.5ml离心管中，加入50u lElubte buffer(或无菌H2O)至膜中央，静置片刻，然后16000g×1min离心，收集回收胶里的PCR产物。
1.5将切胶回收的目的片段进行连接
1.6连接产物转化感受态细胞
1.6.1 CaCl2制备感受态细胞DH5α
1.6.1.1在15ml试管中加入3mlLB培养基，从平板上挑取一单克隆接种，37℃，250rpm，培养过夜，约16小时。
1.6.1.2取2ml过夜菌接入200ml新鲜的LB培养基，37℃，260rpm，培养，直至OD 600＝0.4～0.5，然后将菌液冰浴30～60分钟。
1.6.1.3 4℃，5000rpm×5min离心收集细菌，弃上清。
1.6.1.4用10ml0.1M的CaCl2轻轻吹打，充分悬浮，冰浴10min。
1.6.1.5 4℃，5000rpm×5min离心，弃上清。
1.6.1.6加入2ml0.1M的CaCl2轻轻吹打，充分悬浮，冰浴30min即感受态制备完成。
1.6.2转化感受态细胞
1.6.2.1准备1.5ml的离心管2支，冰浴预冷；
1.6.2.2分别取100ulDH5α感受态细胞，各加入10ul连接产物，混合均匀；
1.6.2.3将上述混合物放置冰上30min；
1.6.2.4将混合物转移至42℃水浴中温育90s；
1.6.2.5将混合物转移至冰上放置2min后，加入800ul的LB培养液，在摇床上37℃，150rpm培养60min；
1.6.2.6 4000rpm×5min离心；
1.6.2.7弃800ul的上清液，剩余100ul混匀后涂LA板；
1.6.2.8先取20ul IPTG和70ul Xgal混合后涂布在LA板上进行蓝白斑筛选，然后把上步骤剩余的100ul菌液混匀后涂LA板；
1.6.2.9 LA板置37℃培养箱过夜培养；
1.6.2.10在过夜培养的LA板上挑取10个长得比较饱满的白色克隆，分别加入6mlLA培养基中，在摇床上37℃，250rpm培养16h。
实施例2 pMSCV puro-Her2-scfv-CD8α-CD137-CD3ζ-iCasp9质粒小量抽提
2.1收菌：以12000g×1min离心收菌。(一般抽提一份用3～4ml菌液)；
2.2弃上清；
2.3加250u l冰浴Buffer S1(含Rnase A)，涡旋振荡，使菌体充分悬浮；
2.4加250ulBuffer S2，温和混匀6次(此过程不超过5min)；加350u lBuffer S3，温和混匀6次；
2.5以12000g×10min离心；
2.6取上清，过DNA制备管，以12000g×1min，弃滤液；
2.7 DNA制备管内加700ulBuffer W1，12000g×1min，弃滤液；
2.8 DNA制备管内加500ulBufferW2，12000g×1min，弃滤液；
2.9再次以12000g×1min离心；
2.10加40ulddH2O(或EB buffer)于DNA制备管的膜中央，室温静置1min(50℃预热EB buffer可能洗脱效果更好)；
2.11 12000g×1min离心并收集洗脱液，即质粒DNA。
实施例3酶切鉴定嵌合受体融合诱导凋亡酶复合蛋白构建的质粒pMSCVpuro-Her2-scfv-CD8α-CD137-CD3ζ-iCasp9
用序列分析软件BioEdit对pMSCV puro-Her2-scfv-CD8α-CD137-CD3ζ-iCasp9质粒进行分析，正确定位各酶切位点的位置。
EcoRI(G＾AATTC)的酶切位点处于：4537；XhoI(C＾TCGAG)的酶切位点处于：1417；酶切后的条带大小为：3120bp(由于酶切的特性，限制性内切酶会从酶切位点前后的3个碱基处切下，因此，切下条带大小会比实际插入序列多6个碱基)，位置准确，如图1所示。
实施例4 pMSCV puro-Her2-scfv-CD8α-CD137-CD3ζ-iCasp9质粒大量抽提与测序
4.1进行质粒大量抽提以获得较高纯度的质粒，为其在T淋巴细胞表达做准备。
4.1.1吸取0.5ml上述菌液加入500mlLA培养基中在摇床上37℃，250rpm培养16h，菌液密度大约为3～4×109细胞/ml。
4.1.2将菌液收集在200ml离心管中，在4℃条件下，以6000g的速度离心15min。以同样的条件重复3次，把细菌收集在同一个离心管中。
4.1.3将细菌沉淀重悬在20mlBuffer P1(使用前加入RNase A和LyseBluereagent)中。
4.1.4向离心管中加入20ml Buffer P2，并上下颠倒4～6次混匀，在室温(15～25℃)下放置5min，此时混合液变为蓝色。
4.1.5向离心管中加入20ml Buffer P3，并上下颠倒4～6次混匀，在冰上放置30min，此时混合液蓝色褪去，变为无色。
4.1.6将上述液体在4℃20，000g离心30min，并迅速收集含有质粒DNA的上清液。
4.1.7将上一步骤获得的上清液4℃20，000g离心15min，并迅速收集含有质粒DNA的上清液。
4.1.8向上清液中加入42ml异丙醇以沉淀其中的质粒DNA，并4℃20，000g离心15min，小心弃去上清液。
4.1.9向质粒DNA沉淀中加入500μlTE buffer(pH 8.0)，并加入Buffer Q BT至总容量5ml。
4.1.10使用QIAGEN-tip 100进行质粒抽提，取4mlBuffer QBT加入QIAGEN-tip100柱子进行平衡，并让液体在重力的作用下缓慢流尽。
4.1.11将步骤9所获得的含有质粒DNA的液体加入柱子，让其在重力的作用下缓慢进入交换树脂。
4.1.12用Buffer QC洗柱，2×10ml。
4.1.13用5ml Buffer QF过柱进行洗脱，用一个干净的离心管接洗脱液。
4.1.14于洗脱液中加3.5ml异丙醇以沉淀质粒DNA，并4℃15，000g离心30min。离心后将上清液小心弃去。
4.1.15室温下用2ml 70％的酒精洗质粒DNA，然后在4℃15，000g离心10min，离心后小心弃去上清液。
4.1.16沉淀物风干10min后加入250μl的TE buffer(pH 8.0)。
4.2抽提质粒浓度测定
应用紫外分光光度计测定质粒大量抽提后获得的pMSCVpuro-Her2-scfv-CD8α-CD137-CD3ζ-iCasp9质粒的浓度为：
0.025×1000×50＝1.25ug/ul
4.3 Her2-scfv-CD8α-CD137-CD3ζ-iCasp9的测序
委托英潍捷基贸易有限公司进行测序，测序结果如下：
CTCGAGATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCC GATGGCCCAGGTGCAGCTGGTGCAGTCTGGGGCAGAGGTGAAAAAGCCCGGGGAGTCTCTGAAGATCTCCTGTAAG GGTTCTGGATACAGCTTTACCAGCTACTGGATCGCCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTACATGG GGCTCATCTATCCTGGTGACTCTGACACCAAATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGTCGACAA GTCCGTCAGCACTGCCTACTTGCAATGGAGCAGTCTGAAGCCCTCGGACAGCGCCGTGTATTTTTGTGCGAGACAT GACGTGGGATATTGCAGTAGTTCCAACTGCGCAAAGTGGCCTGAATACTTCCAGCATTGGGGCCAGGGCACCCTGG TCACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGCAGTCTGTGTTGACGCA GCCGCCCTCAGTGTCTGCGGCCCCAGGACAGAAGGTCACCATCTCCTGCTCTGGAAGCAGCTCCAACATTGGGAAT AATTATGTATCCTGGTACCAGCAGCTCCCAGGAACAGCCCCCAAACTCCTCATCTATGATCACACCAATCGGCCCG CAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGTTCCGGTCCGA GGATGAGGCTGATTATTACTGTGCCTCCTGGGACTACACCCTCTCGGGCTGGGGTTCGGCGGAGGAACCAAGCTGA CCGTCCTAGGTGCGGCCGCCGGCGGAGGAGGATCTCCAGCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAAC ACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTG CACACGAGGGGGCTGGACTTCGCCTGTGATATCATCTCCTTCTTTCTTGCGCTGACGTCGACTGCGTTGCTCTTCC TGCTGTTCTTCCTCACGCTCCGTTTCTCTGTTGTTAAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACC ATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGA TGTGAACTGAGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACG GCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAG CCGCAGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTG AGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAA GGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGCCATCATCACCACCACCATCAGTGCACCAACTAT GCGCTGCTGAAACTGGCGGGCGATGTGGAAAGCAACCCGGGCCCGATGGGAGTGCAGGTGGAAACCATCTCCCCAG GAGACGGGCGCACCTTCCCCAAGCGCGGCCAGACCTGCGTGGTGCACTACACCGGGATGCTTGAAGATGGAAAGAA AGTTGATTCCTCCCGGGACAGAAACAAGCCCTTTAAGTTTATGCTAGGCAAGCAGGAGGTGATCCGAGGCTGGGAA GAAGGGGTTGCCCAGATGAGTGTGGGTCAGAGAGCCAAACTGACTATATCTCCAGATTATGCCTATGGTGCCACTG GGCACCCAGGCATCATCCCACCACATGCCACTCTCGTCTTCGATGTGGAGCTTCTAAAACTGGAAAGCGGCGGCGG CAGCACTAACAGGCAAGCAGCAAAGTTGTCGAAGCCAACCCTAGAAAACCTTACCCCAGTGGTGCTCAGACCAGAG ATTCGCAAACCAGAGGTTCTCAGACCGGAAACACCCAGACCAGTGGACATTGGTTCTGGAGGATTTGGTGATGTCG GTGCTCTTGAGAGTTTGAGGGGAAATGCAGATTTGGCTTACATCCTGAGCATGGAGCCCTGTGGCCACTGCCTCAT TATCAACAATGTGAACTTCTGCCGTGAGTCCGGGCTCCGCACCCGCACTGGCTCCAACATCGACTGTGAGAAGTTG CGGCGTCGCTTCTCCTCGCTGCATTTCATGGTGGAGGTGAAGGGCGACCTGACTGCCAAGAAAATGGTGCTGGCTT TGCTGGAGCTGGCGCAGCAGGACCACGGTGCTCTGACTGCTGCGTGGTGGTCATTCTCTCTCACGGCTGTCAGGCC AGCCACCTGCAGTTCCCAGGGGCTGTCTACGGCACAGATGGATGCCCTGTGTCGGTCGAGAAGATTGTGAACATCT TCAATGGGACCAGCTGCCCCAGCCTGGGAGGGAAGCCCAAGCTCTTTTTCATCCAGGCCTGTGGTGGGGAGCAGAA AGACCATGGGTTTGAGGTGGCCTCCACTTCCCCTGAAGACGAGTCCCCTGGCAGTAACCCCGAGCCAGATGCCACC CCGTTCCAGGAAGGTTTGAGGACCTTCGACCAGCTGGACGCCATATCTAGTTTGCCCACACCCAGTGACATCTTTG TGTCCTACTCTACTTTCCCAGGTTTTGTTTCCTGGAGGGACCCCAAGAGTGGCTCCTGGTACGTTGAGACCCTGGA CGACATCTTTGAGCAGTGGGCTCACTCTGAAGACCTGCAGTCCCTCCTGCTTAGGGTCGCTAATGCTGTTTCGGTG AAAGGGATTTATAAACAGATGCCTGGTTGCTTTAATTTCCTCCGGAAAAAACTTTTCTTTAAAACATCACATCATC ACCACCACCATTAAGA-3’
结果显示上述序列(下划线部分)与Her2-scfv-CD8α-CD137-CD3ζ-iCasp9的理论序列(即SEQ ID NO：1所示的DNA序列)完全一致。
实施例5质粒图
所构建质粒pMSCV puro-Her2-scfv-CD8α-CD137-CD3ζ-iCasp9的绘制质粒图如图2所示。这个质粒图阐明了质粒的英文全称为pMSCV puro-Her2-scfv-CD8α-CD137-CD3ζ-iCasp9，序列的总长度为9225bp，以及目标序列Her2-scfv-CD8α-CD137-CD3ζ-iCasp9(即SEQ ID NO：1所示的DNA序列)的插入位置，并说明相应的酶切位点，质粒表达嘌呤霉素抗性等质粒信息。
实施例6 pMSCV慢病毒表达载体的包装及纯化
6.1pMSCV puro表达质粒慢病毒的构建
6.1接种1×106个293FT细胞于100mm组织培养皿中，初始细胞密度应在50～70％铺满培养过夜。
6.2除去培养基，加入4ml优化培养基opti-MEM以及1ml完全培养基(含10％胎牛血清的DMEM培养基)。确认培养皿的所有区域被培养基覆盖，以防止细胞干燥死亡。
6.3于一无菌15ml离心管中混合制备慢病毒的三个质粒：psPAX2 4μg，Pmd2.G1.4ug和pMSCV puro-Her2-scfv-CD8α-CD137-CD3ζ-iCasp9表达质粒5.6ug，加入opti-MEM，总体积1500ul。Lipofectamine3000 25ul，加入到无血清无抗生素opti-MEM，总体积1500ul，室温静置5min。
混合上述溶液，室温静置30min。
6.4将上述混合液逐滴加入组织培养皿，每加入2到3滴便温和地晃动培养皿以使起其与转染混合液混匀。
6.5 37度培养10～14小时。
6.6约12小时以后，除去转染溶液，并加入20ml完全培养基。37℃培养约48～60小时。
6.7约48小时后，收集上清，再加入10ml培养基，继续培养约24小时后收集含病毒的培养液。
6.8将两次收集的病毒原液合并后于4℃条件下3000r/min离心5min，去除包装细胞，然后通过0.22um的一次性滤器过滤，即得病毒原液。再将之置于超速离心管内25000r/min，2小时。弃上清，用100ulPBS重悬沉淀，分装到无菌的EP管，-80℃保存。
实施例7 Her2-scfv-CD8α-CD137-CD3ζ-iCasp9复合蛋白在人CD8T淋巴细胞上的表达
7.1人CD8T淋巴细胞的分离。
7.1.1人外周血单个核细胞的分离。
7.1.1.1将健康人外周血约30ml全血用约30ml生理盐水稀释。
7.1.1.2取两支50ml离心管加入约15mlFicoll溶液，再将稀释的血液轻轻倾斜加入离心管中，注意避免两种溶液混合在一起，每管加入30ml血液。
7.1.1.3室温，450g离心25分钟，降速加速度设置为0，得到分层四层的液体，从上到下分别是血浆层，单核细胞层，Ficoll层，红细胞和中性粒细胞层。
7.1.1.4用巴氏管将白色的单核细胞层吸取置于另一管中，加入生理盐水约30～40ml混匀，450g，5min室温离心弃去上清。重复加入生理盐水，300g，5min室温离心，弃上清。再重复一次，加入20ml生理盐水后先计数单核细胞的个数，然后再300g，8min离心弃上清。
7.1.2人CD8T淋巴细胞的筛选
7.1.2.1对于每107个细胞，加入40ul缓冲液与10ul一抗溶液，轻柔重悬，4℃静置5min。
7.1.2.2对于每107个细胞，加入30ul缓冲液与20ul二抗溶液，轻柔混匀，4℃静置10min，然后加入500ul缓冲液每107个细胞。
7.1.2.3将分离柱放置于磁力架上，先用5ml缓冲液润管，整个筛选过程尽量少泡。
7.1.2.4待缓冲液流完，在柱子下放置新的离心管，在柱子中加入上述的细胞悬液，轻柔，用3ml缓冲液冲洗之前有细胞悬液的离心管，然后再吸取过柱。再用3ml缓冲液加入柱子。滤下的细胞即CD8T淋巴细胞。弃去柱子。将滤过的淋巴细胞300g、5min室温离心，用含有10％血清的1640培养基重悬并37℃培养。
7.2 Her2-scfv-CD8α-CD137-CD3ζ-iCasp9复合蛋白在CD8T淋巴细胞的表达
7.2.1计数3＊107个CD8细胞，用约400ul含5％血清培养基重悬，(培养基预先加入polybrene，终浓度为8mg/L)，加入之前浓缩的病毒1ul，轻柔混匀后置于12孔培养板中，37℃培养箱培养12小时，离心弃上清，用1ml 10％血清培养基重悬培养箱培养12小时。再重复转染一次。
7.2.2再次转染48小时后加入嘌呤霉素筛选3～4天，浓度为5mg/L。由于表达质粒为嘌呤霉素抗性，因此筛选后的细胞为转染成功的CAR-T淋巴细胞。
实施例8修饰的CAR-T淋巴细胞活化鉴定。
8.1取Her2高表达乳腺癌细胞株BT474细胞104个，分为两组，每组分别加入105个筛选出的T淋巴细胞和CAR-T细胞，37℃培养箱共培养2小时。
8.2取出细胞，离心去上清，生理盐水重悬洗一遍，再离心弃上清，用100ul生理盐水重悬，加入1ul人CD69荧光抗体，4℃孵育30min，洗去多余的抗体，用流式分析仪分析CD69表达情况。
如图3所示，对于未转染的T淋巴细胞，在2小时时即可检测到CAR-T细胞CD69有较高的表达，说明转染了复合蛋白的T细胞具有高效的活化效应。
实施例9修饰的CAR-T淋巴细胞功能鉴定
9.1修饰的CAR-T淋巴细胞杀伤能力检测
9.1.1取Her2高表达乳腺癌细胞株BT474细胞8×104个，平均分7组，组①直接接种于12孔细胞板中。后6组细胞一起用500ul生理盐水重悬，加入1ul celltracker染料，避光，37℃15min后，加入500ul含血清的培养基，1000rpm离心3min，弃上清。后续实验也在避光条件下进行。
9.1.2将后6组细胞用2ml含血清培养基重悬，各250ul分别加入12孔培养板中另外6个孔，即为②③④⑤⑥⑦组，每组约为104个细胞。
9.1.3按照肿瘤：淋巴细胞个数＝1/1，1/5，1/10的比例，在②③④组中分别加入转染的CAR-T细胞，在⑤⑥⑦组中分别加入筛选出来的T淋巴细胞。每孔的体系都为1.2ml。置于37℃培养箱培养6～8小时。
9.1.4 6～8小时后，将每孔的细胞吹下吸取到1.5ml EP管中，300g，4min室温离心，弃上清。再用200ul生理盐水重悬，加入流式抗体PI，在流式分析仪上分析肿瘤细胞被杀伤的情况。第一组作为未染色对照。
如图4所示，对于未转染的T淋巴细胞，CAR-T细胞对Her2阳性乳腺癌细胞具有较高的杀伤能力，并且，随着T淋巴细胞数量逐步调整为肿瘤细胞的1倍、5倍、10倍，该杀伤能力也逐渐增高。
9.2修饰的CAR-T淋巴细胞的靶向特异性
9.2.1取HER-2表达阴性细胞MDA-MB-231细胞约3×104个，平均分为3组，接种于新的12孔板中。
9.2.2按照肿瘤：淋巴细胞个数＝1/1，1/5，1/10的比例，在肿瘤细胞中分别加入转染的CAR-T细胞。每孔的体系都为1.2ml。置于37℃培养箱培养6～8小时。
9.2.3 6-8小时后，将每孔的细胞吹下吸取到1.5mlEP管中，300g，4min室温离心，弃上清。再用200ul生理盐水重悬，加入流式抗体PI，在流式分析仪上分析肿瘤细胞被杀伤的情况。
如图5所示，对于Her 2阴性的乳腺癌细胞，CAR-T细胞的杀伤能力和普通T淋巴细胞相似，说明CAR-T细胞的杀伤肿瘤细胞能力是有靶向特异性的。
9.3修饰的CAR-T淋巴细胞在药物AP1903的调控下能够快速凋亡
9.3.1各取105个CAR-T淋巴细胞和筛选的CD8T淋巴细胞分别各接种于3个12孔培养板中，一一对应为三组，每孔都加入AP1903，终浓度为100nM。
9.3.2分别于0，0.5小时的时候吸出相对的一组细胞，300g，4min，室温离心弃上清。用300ul预冷的生理盐水重悬，加入5ul的Annexin V-APC混匀后，避光，室温孵育15分钟。300g、4min离心弃上清。
9.3.3用预冷的200ul生理盐水重悬，加入流式抗体PI，在流式分析仪上分析淋巴细胞凋亡的情况。另取未作处理的淋巴细胞作为未染色对照。
如图6所示，在加入诱导药物的情况下，CAR-T细胞可迅速达到早期凋亡状态，而普通T细胞则不受影响，说明药物AP1903是有效安全的诱导药物，CAR-T细胞安全调控的设计成功。
综上，我们用流式等分析实验发现，修饰的CAR-T淋巴细胞具有高于普通CD8T淋巴细胞对Her2乳腺癌细胞的杀伤能力，同时，在药物AP1903的调控下，CAR-T细胞能够在短时间内快速诱导自身凋亡，当大量CAR-T细胞输入体内，在其快速地杀伤肿瘤细胞时，有可能会因为肿瘤细胞凋亡产生的碎片等，引起自身机体的短时间内大量炎症因子的产生，或因为大量杀伤产生级联放大效应损伤正常细胞时，可以应用这一“安全按钮”(即药物AP1903)清除异常的CART细胞而保障机体的稳定安全。
最后所应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明作了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。
SEQUENCE LISTING
<110> 中山大学
<120> 一种嵌合抗原受体并融合诱导性凋亡酶的复合蛋白
<130> 2017
<160> 13
<170> PatentIn version 3.5
<210> 1
<211> 3114
<212> DNA
<213> 人工序列
<400> 1
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccgatggccc aggtgcagct ggtgcagtct ggggcagagg tgaaaaagcc cggggagtct 120
ctgaagatct cctgtaaggg ttctggatac agctttacca gctactggat cgcctgggtg 180
cgccagatgc ccgggaaagg cctggagtac atggggctca tctatcctgg tgactctgac 240
accaaataca gcccgtcctt ccaaggccag gtcaccatct cagtcgacaa gtccgtcagc 300
actgcctact tgcaatggag cagtctgaag ccctcggaca gcgccgtgta tttttgtgcg 360
agacatgacg tgggatattg cagtagttcc aactgcgcaa agtggcctga atacttccag 420
cattggggcc agggcaccct ggtcaccgtc tcctcaggtg gaggcggttc aggcggaggt 480
ggctctggcg gtggcggatc gcagtctgtg ttgacgcagc cgccctcagt gtctgcggcc 540
ccaggacaga aggtcaccat ctcctgctct ggaagcagct ccaacattgg gaataattat 600
gtatcctggt accagcagct cccaggaaca gcccccaaac tcctcatcta tgatcacacc 660
aatcggcccg caggggtccc tgaccgattc tctggctcca agtctggcac ctcagcctcc 720
ctggccatca gtgggttccg gtccgaggat gaggctgatt attactgtgc ctcctgggac 780
tacaccctct cgggctgggt gttcggcgga ggaaccaagc tgaccgtcct aggtgcggcc 840
gccggcggag gaggatctcc agcgaagccc accacgacgc cagcgccgcg accaccaaca 900
ccggcgccca ccatcgcgtc gcagcccctg tccctgcgcc cagaggcgtg ccggccagcg 960
gcggggggcg cagtgcacac gagggggctg gacttcgcct gtgatatcat ctccttcttt 1020
cttgcgctga cgtcgactgc gttgctcttc ctgctgttct tcctcacgct ccgtttctct 1080
gttgttaaac ggggcagaaa gaaactcctg tatatattca aacaaccatt tatgagacca 1140
gtacaaacta ctcaagagga agatggctgt agctgccgat ttccagaaga agaagaagga 1200
ggatgtgaac tgccagcgaa gcccaccacg acgccagcgc cgcgaccacc aacaccggcg 1260
cccaccatcg cgtcgcagcc cctgtccctg cgcccagagg cgtgccggcc agcggcgggg 1320
ggcgcagtgc acacgagggg gctggacttc gcctgtgata gagtgaagtt cagcaggagc 1380
gcagacgccc ccgcgtacca gcagggccag aaccagctct ataacgagct caatctagga 1440
cgaagagagg agtacgatgt tttggacaag agacgtggcc gggaccctga gatgggggga 1500
aagccgcaga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1560
atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 1620
gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1680
caggccctgc cccctcgcca tcatcaccac caccatcagt gcaccaacta tgcgctgctg 1740
aaactggcgg gcgatgtgga aagcaacccg ggcccgatgg gagtgcaggt ggaaaccatc 1800
tccccaggag acgggcgcac cttccccaag cgcggccaga cctgcgtggt gcactacacc 1860
gggatgcttg aagatggaaa gaaagttgat tcctcccggg acagaaacaa gccctttaag 1920
tttatgctag gcaagcagga ggtgatccga ggctgggaag aaggggttgc ccagatgagt 1980
gtgggtcaga gagccaaact gactatatct ccagattatg cctatggtgc cactgggcac 2040
ccaggcatca tcccaccaca tgccactctc gtcttcgatg tggagcttct aaaactggaa 2100
agcggcggcg gcagcactaa caggcaagca gcaaagttgt cgaagccaac cctagaaaac 2160
cttaccccag tggtgctcag accagagatt cgcaaaccag aggttctcag accggaaaca 2220
cccagaccag tggacattgg ttctggagga tttggtgatg tcggtgctct tgagagtttg 2280
aggggaaatg cagatttggc ttacatcctg agcatggagc cctgtggcca ctgcctcatt 2340
atcaacaatg tgaacttctg ccgtgagtcc gggctccgca cccgcactgg ctccaacatc 2400
gactgtgaga agttgcggcg tcgcttctcc tcgctgcatt tcatggtgga ggtgaagggc 2460
gacctgactg ccaagaaaat ggtgctggct ttgctggagc tggcgcagca ggaccacggt 2520
gctctggact gctgcgtggt ggtcattctc tctcacggct gtcaggccag ccacctgcag 2580
ttcccagggg ctgtctacgg cacagatgga tgccctgtgt cggtcgagaa gattgtgaac 2640
atcttcaatg ggaccagctg ccccagcctg ggagggaagc ccaagctctt tttcatccag 2700
gcctgtggtg gggagcagaa agaccatggg tttgaggtgg cctccacttc ccctgaagac 2760
gagtcccctg gcagtaaccc cgagccagat gccaccccgt tccaggaagg tttgaggacc 2820
ttcgaccagc tggacgccat atctagtttg cccacaccca gtgacatctt tgtgtcctac 2880
tctactttcc caggttttgt ttcctggagg gaccccaaga gtggctcctg gtacgttgag 2940
accctggacg acatctttga gcagtgggct cactctgaag acctgcagtc cctcctgctt 3000
agggtcgcta atgctgtttc ggtgaaaggg atttataaac agatgcctgg ttgctttaat 3060
ttcctccgga aaaaactttt ctttaaaaca tcacatcatc accaccacca ttaa 3114
<210> 2
<211> 1037
<212> PRT
<213> 人工序列
<223> 一种嵌合抗原受体并融合诱导性凋亡酶的复合蛋白
<400> 2
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Met Ala Gln Val Gln Leu Val Gln Ser Gly Ala
20 25 30
Glu Val Lys Lys Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser
35 40 45
Gly Tyr Ser Phe Thr Ser Tyr Trp Ile Ala Trp Val Arg Gln Met Pro
50 55 60
Gly Lys Gly Leu Glu Tyr Met Gly Leu Ile Tyr Pro Gly Asp Ser Asp
65 70 75 80
Thr Lys Tyr Ser Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Val Asp
85 90 95
Lys Ser Val Ser Thr Ala Tyr Leu Gln Trp Ser Ser Leu Lys Pro Ser
100 105 110
Asp Ser Ala Val Tyr Phe Cys Ala Arg His Asp Val Gly Tyr Cys Ser
115 120 125
Ser Ser Asn Cys Ala Lys Trp Pro Glu Tyr Phe Gln His Trp Gly Gln
130 135 140
Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
145 150 155 160
Gly Ser Gly Gly Gly Gly Ser Gln Ser Val Leu Thr Gln Pro Pro Ser
165 170 175
Val Ser Ala Ala Pro Gly Gln Lys Val Thr Ile Ser Cys Ser Gly Ser
180 185 190
Ser Ser Asn Ile Gly Asn Asn Tyr Val Ser Trp Tyr Gln Gln Leu Pro
195 200 205
Gly Thr Ala Pro Lys Leu Leu Ile Tyr Asp His Thr Asn Arg Pro Ala
210 215 220
Gly Val Pro Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser
225 230 235 240
Leu Ala Ile Ser Gly Phe Arg Ser Glu Asp Glu Ala Asp Tyr Tyr Cys
245 250 255
Ala Ser Trp Asp Tyr Thr Leu Ser Gly Trp Val Phe Gly Gly Gly Thr
260 265 270
Lys Leu Thr Val Leu Gly Ala Ala Ala Gly Gly Gly Gly Ser Pro Ala
275 280 285
Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr
290 295 300
Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala
305 310 315 320
Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile
325 330 335
Ile Ser Phe Phe Leu Ala Leu Thr Ser Thr Ala Leu Leu Phe Leu Leu
340 345 350
Phe Phe Leu Thr Leu Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys
355 360 365
Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr
370 375 380
Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly
385 390 395 400
Gly Cys Glu Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro
405 410 415
Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro
420 425 430
Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu
435 440 445
Asp Phe Ala Cys Asp Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro
450 455 460
Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly
465 470 475 480
Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro
485 490 495
Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu
500 505 510
Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile
515 520 525
Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr
530 535 540
Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met
545 550 555 560
Gln Ala Leu Pro Pro Arg His His His His His His Gln Cys Thr Asn
565 570 575
Tyr Ala Leu Leu Lys Leu Ala Gly Asp Val Glu Ser Asn Pro Gly Pro
580 585 590
Met Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe
595 600 605
Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu
610 615 620
Asp Gly Lys Lys Val Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys
625 630 635 640
Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val
645 650 655
Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp
660 665 670
Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala
675 680 685
Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Ser Gly Gly Gly
690 695 700
Ser Thr Asn Arg Gln Ala Ala Lys Leu Ser Lys Pro Thr Leu Glu Asn
705 710 715 720
Leu Thr Pro Val Val Leu Arg Pro Glu Ile Arg Lys Pro Glu Val Leu
725 730 735
Arg Pro Glu Thr Pro Arg Pro Val Asp Ile Gly Ser Gly Gly Phe Gly
740 745 750
Asp Val Gly Ala Leu Glu Ser Leu Arg Gly Asn Ala Asp Leu Ala Tyr
755 760 765
Ile Leu Ser Met Glu Pro Cys Gly His Cys Leu Ile Ile Asn Asn Val
770 775 780
Asn Phe Cys Arg Glu Ser Gly Leu Arg Thr Arg Thr Gly Ser Asn Ile
785 790 795 800
Asp Cys Glu Lys Leu Arg Arg Arg Phe Ser Ser Leu His Phe Met Val
805 810 815
Glu Val Lys Gly Asp Leu Thr Ala Lys Lys Met Val Leu Ala Leu Leu
820 825 830
Glu Leu Ala Gln Gln Asp His Gly Ala Leu Asp Cys Cys Val Val Val
835 840 845
Ile Leu Ser His Gly Cys Gln Ala Ser His Leu Gln Phe Pro Gly Ala
850 855 860
Val Tyr Gly Thr Asp Gly Cys Pro Val Ser Val Glu Lys Ile Val Asn
865 870 875 880
Ile Phe Asn Gly Thr Ser Cys Pro Ser Leu Gly Gly Lys Pro Lys Leu
885 890 895
Phe Phe Ile Gln Ala Cys Gly Gly Glu Gln Lys Asp His Gly Phe Glu
900 905 910
Val Ala Ser Thr Ser Pro Glu Asp Glu Ser Pro Gly Ser Asn Pro Glu
915 920 925
Pro Asp Ala Thr Pro Phe Gln Glu Gly Leu Arg Thr Phe Asp Gln Leu
930 935 940
Asp Ala Ile Ser Ser Leu Pro Thr Pro Ser Asp Ile Phe Val Ser Tyr
945 950 955 960
Ser Thr Phe Pro Gly Phe Val Ser Trp Arg Asp Pro Lys Ser Gly Ser
965 970 975
Trp Tyr Val Glu Thr Leu Asp Asp Ile Phe Glu Gln Trp Ala His Ser
980 985 990
Glu Asp Leu Gln Ser Leu Leu Leu Arg Val Ala Asn Ala Val Ser Val
995 1000 1005
Lys Gly Ile Tyr Lys Gln Met Pro Gly Cys Phe Asn Phe Leu Arg
1010 1015 1020
Lys Lys Leu Phe Phe Lys Thr Ser His His His His His His
1025 1030 1035
<210> 3
<211> 41
<212> DNA
<213> 人工序列
<400> 3
gggccatggc ccaggtgcag ctgttgcagt ctggggcaga g 41
<210> 4
<211> 44
<212> DNA
<213> 人工序列
<400> 4
ttgcggccgc tccggaattc acctaggacg gtcagcttgg tccc 44
<210> 5
<211> 20
<212> DNA
<213> 人工序列
<400> 5
ccagcgaagc ccaccacgac 20
<210> 6
<211> 30
<212> DNA
<213> 人工序列
<400> 6
aggagatgat atcacaggcg aagtccagcc 30
<210> 7
<211> 30
<212> DNA
<213> 人工序列
<400> 7
cgcctgtgat atcatctcct tctttcttgc 30
<210> 8
<211> 30
<212> DNA
<213> 人工序列
<400> 8
acttcactct tcacagttca catcctcctt 30
<210> 9
<211> 30
<212> DNA
<213> 人工序列
<400> 9
tgaactgtga agagtgaagt tcagcaggag 30
<210> 10
<211> 19
<212> DNA
<213> 人工序列
<400> 10
agggggcagg gcctgcatg 19
<210> 11
<211> 25
<212> DNA
<213> 人工序列
<400> 11
actaacaggc aagcagcaaa gttgt 25
<210> 12
<211> 24
<212> DNA
<213> 人工序列
<400> 12
agcggcggcg gcagcactaa cagg 24
<210> 13
<211> 30
<212> DNA
<213> 人工序列
<400> 13
ttatgatgtt ttaaagaaaa gttttttccg 30
Claims (8)
1.一种嵌合抗原受体并融合诱导性凋亡酶的复合蛋白，其特征在于，所述复合蛋白由SEQ ID NO：2所示的氨基酸序列组成。
2.一种分离的核酸，其特征在于，所述核酸编码如权利要求1所述的复合蛋白。
3.如权利要求2所述的核酸，其特征在于，所述核酸的核苷酸序列如SEQ ID NO：1所示。
4.一种载体，其特征在于，所述载体表达如权利要求1所述的复合蛋白。
5.一种修饰的T淋巴细胞，其特征在于，所述修饰的T淋巴细胞表达如权利要求1所述的复合蛋白。
6.一种药物组合物，其特征在于，所述药物组合物包含如权利要求1所述的复合蛋白，或如权利要求2所述的分离的核酸，或如权利要求4所述的载体，或如权利要求5所述的修饰的T淋巴细胞。
7.如权利要求1所述的复合蛋白，或如权利要求2所述的分离的核酸，或如权利要求4所述的载体，或如权利要求5所述的修饰的T淋巴细胞在制备治疗乳腺癌的药物中的用途。
8.如权利要求5所述的修饰的T淋巴细胞和AP1903的联合使用在制备治疗乳腺癌的药物中的用途。
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