CN109265564A - 一种鼠抗cd3重组免疫毒素及其制备方法和应用 - Google Patents

一种鼠抗cd3重组免疫毒素及其制备方法和应用 Download PDF

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CN109265564A
CN109265564A CN201811129984.8A CN201811129984A CN109265564A CN 109265564 A CN109265564 A CN 109265564A CN 201811129984 A CN201811129984 A CN 201811129984A CN 109265564 A CN109265564 A CN 109265564A
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陈宏远
芮雯
陈鸿策
游思远
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Abstract

本发明公开一种鼠抗CD3重组免疫毒素及其制备方法和应用，属于生物技术领域。本发明采用基因工程技术与策略，通过改变单链抗体与DT390的结合方式以提高其亲和力，优化密码子系列得到以DT结构为毒性中心的三种重组免疫毒素，构建其原核表达系统，得到靶向T细胞受体CD3ε的抗小鼠单链抗体偶联免疫毒素以剔除外周免疫器官的T淋巴细胞，利用其进行移植免疫模型建立，免疫耐受和自身性免疫疾病的等的治疗研究。证实靶向鼠抗CD3ε重组免疫毒素剔除鼠外周血及淋巴结T细胞的有效性。在小鼠T淋巴细胞剔除、自身性免疫疾病、器官移植、免疫耐受及肿瘤治疗的研究方面具有应用意义，后续可能有临床前药学研究及转化的潜能。

Description

一种鼠抗CD3重组免疫毒素及其制备方法和应用
技术领域
本发明属于生物技术领域，特别涉及一类用于选择性破坏具有某一特异性标记细胞的融合蛋白，是将细胞选择性配体与毒素分子通过基因融合的方法组合而成的嵌合重组体及其制备方法和应用。
背景技术
免疫毒素(immunotoxin，IT)可作为一种靶向治疗药物，通常是将细胞选择性作用配体与毒素分子通过化学连接或基因融合的方法组合而成的嵌合体蛋白。作用机制由德国免疫学家Ehrilich在20世纪初提出。常用的靶向载体包括单克隆抗体如CD3、CDS、CD22、IL-2(白细胞介素2)、IL-3(白细胞介素3)GM-CSF(粒单集落刺激因子)和短肽激素等；毒素如蓖麻毒素(ricin)、假单胞菌外毒素(Pseudomonas Exotoxin，PE)、白喉毒素(diphtheriatoxin，DT)等。嵌合毒素的组成可以使单链的可变区片段免疫毒素(ScFvimmunotoxin)、双链的可变区片段免疫毒素(dsFv immunotoxin)和趋化因子或生长因子相关的免疫毒素等。嵌合毒素与野生型毒素相比具有稳定性强、渗透性好、较强的导向特异性，且制备时简洁稳定，可短期内大量复制。免疫毒素的毒性发挥部分还可以是小分子化合物毒素、放射性元素毒素和蛋白毒素(植物毒素和细菌毒素分子比较常见)。
DT是一种感染B棒状噬菌体的白喉杆菌的外毒素，完整的DT分子由535个氨基酸组成，分子量为58.4kD。根据其结构和功能特点，自N端向C端可分为3个结构区：催化区(C区)，由1～193氨基酸残基组成；跨膜区(T区)，由205～378氨基酸残基组成；受体结合区(R区)，由386～535氨基酸残基组成。R区能与真核细胞表面DT受体结合，T区在R区与受体结合后插入细胞膜中，使C区进入细胞内。C区含有ADP依赖的核糖体转移酶活性。能催化NAD+的ADP核糖基向延长因子EF-2转移，生成烟肽胺，使EF-2失活，阻断细胞蛋白质的合成。DT具有强烈的毒性，只需1个DT分子进入细胞内就可杀死1个真核细胞。利用DT分子的3个功能区在空间上相互独立的特点，可通过基因重组，将R区剔除，取而代之以特异性识别细胞表面受体分子的配体结构如上述生长因子、单克隆抗体等。
DT的免疫毒素大多保留了其C区和T区，以利用其ADP-核糖基转移酶活性和跨膜转运功能，而R区则被突变失活或被剔除，替之以与靶细胞特异性结合的导向部分(如抗体的Fab)，从而赋予其靶向细胞特异性。目前已进入临床前期研究的毒素效应部分常采用截短的天然DT(如DT390、DT388等)或DT突变体。基于DT的融合毒素包括：DT388-IL-3、DT385-VEGF、DT388等与uPA的融合。这些融合毒素中，DT388-IL3在临床试验中取得了较好的效果。目前DT389-IL2重组融合毒素(地尼白介素2)被美国FDA批准上市，用于治疗成人皮肤T细胞淋巴瘤。T细胞(抗原)受体(T cell receptor，TCR)为所有T细胞表面的特征性标志，以非共价键与CD3结合，形成TCR-CD3复合物。抗CD3免疫毒素通过单链抗体结构域靶向并作用于T淋巴细胞，通过细胞内化，结合DT390功能与来抑制蛋白质的合成，诱导细胞死亡。以化学偶联制备的是第一代免疫毒素，已有用此法合成的抗CD3的毒素可有效剔除猪的T淋巴细胞，降低造血干细胞移植过程中的移植物抗宿主反应(GVHD)。但是存在产量低，连接物的异源性，剂量限制及因其非特异性中和作用导致的剂量限制，这类免疫毒素制备复杂，在以化学方法制备的过程中易损失抗体的活性，且免疫毒素稳定性差，动物试验效果不好。鉴于DT化学毒性作用，选择合适的表达载体以及感受态细胞是当前制约基因工程方法生产免疫毒素的瓶颈之一。目前，CD3的单克隆抗体药物已在我国研发成功并上市，但未见其鼠源特异性强，半衰期长并具有药学活性的免疫毒素报道。
以基因工程融合法制备的是第二代免疫毒素，是依据分子生物学技术将导向与毒性效应两部分的基因串联，在大肠杆菌或者酵母等合适细胞体系中表达，得到免疫毒素的重组融合蛋白。这类免疫毒素制备相对简单，蛋白稳定、均一、分子量小、渗透性好。经FDA批准上市的ONTAK(DAB389-IL2)是第二代免疫毒素的成功例子。Zhirui Wang等报道了重组的抗人及抗猴的CD3T细胞免疫毒素，这类免疫毒素含有DT390，通过(G4S)3与其C末端共价偶联，然后融合单链抗体CD3+ScFv，但采用与后者不同抗CD3Fv区与DT390的连接方式，重组毒素的亲和力有提高30倍以上，且在外周血及淋巴循环中的半衰期也不同。纯化的免疫毒素可以使外周血T细胞降低80％，淋巴结T细胞降低74％，免疫毒素处理4天后，淋巴结T细胞仅保留了24％。因此，设计并生产高亲和力，具有较好的半衰期并符合药学特性CD3重组免疫毒素蛋白以用于小鼠体内剔除T淋巴细胞，将在小鼠T淋巴细胞剔除、自身性免疫疾病、器官移植、免疫耐受、及肿瘤治疗方面极具科研及药物开发价值。
发明内容
为了克服现有技术的缺点与不足，本发明的目的在于提供一种鼠抗CD3重组免疫毒素。
本发明基于仓鼠抗小鼠的CD3的Epsilon亚结构域基因与DT基因的嵌合重组开发免疫毒素蛋白。
本发明的另一目的在于提供编码上述鼠抗CD3重组免疫毒素的基因。
本发明的另一目的在于提供鼠抗CD3重组免疫毒素的制备方法。
本发明的再一目的在于提供上述鼠抗CD3重组免疫毒素的应用。
本发明采用基因工程技术与策略，通过改变单链抗体与DT390的结合方式以提高其亲和力，优化密码子系列得到以DT结构为毒性中心的三种重组免疫毒素：ScFv-CD3-DT390，Bi-CD3-DT390，Foldback-CD3-DT390，构建其原核表达系统，得到靶向T细胞受体CD3ε的抗小鼠单链抗体偶联免疫毒素以剔除外周免疫器官的T淋巴细胞，利用其进行移植免疫模型建立，免疫耐受和自身性免疫疾病的等的治疗研究。
单克隆抗体在机体内主要依赖宿主自身抗体依赖性介导的细胞毒性作用(ADCC)和补体依赖性细胞毒性作用(CDC)，来发挥其对靶细胞的杀灭作用。当机体免疫功能缺陷或免疫功能下降时，就需要研发一种其他的药物来代替或辅助抗体治疗法。因此，根据抗体的F化片段设计单链抗体(ScFv)，与白喉毒素(DT390)相结合构建重组蛋白。DT的免疫毒素大多保留了其C区和T区，以利用其ADP-核糖基转移酶活性和跨膜转运功能，而R区则被突变失活或被剔除，替之以与靶细胞特异性结合的导向部分(如抗体的Fab)从而赋予其靶向细胞特异性。
本发明的目的通过下述技术方案实现：
本发明提供一种鼠抗CD3重组免疫毒素，包括2部分：一部分为细菌毒素或植物毒素，另一部分为scFv、Bi-scFv结构域或Foldback-scFv，两部分通过由四个甘氨酸和丝氨酸残基(G4S)组成的接头进行连接得到。
优选的，所述的细菌毒素为白喉毒素、绿脓杆菌(Pseudomonas aeruginosa)外毒素等；所述的植物毒素为蓖麻毒素、相思豆毒素、白素毒素等。
更优选的，所述的细菌毒素为DT390，得到的鼠抗CD3重组免疫毒素分别命名为ScFv-CD3-DT390，Bi-CD3-DT390，Foldback-CD3-DT390；
所述的DT390的氨基酸序列如SEQ ID NO：1所示；
所述的scFv的氨基酸序列如SEQ ID NO：2所示；
所述的Bi-scFv的氨基酸序列如SEQ ID NO：3所示；
所述的Foldback-scFv的氨基酸序列如SEQ ID NO：4所示；
所述的ScFv-CD3-DT390的氨基酸序列如SEQ ID NO：5所示；
所述的Bi-CD3-DT390的氨基酸序列如SEQ ID NO：6所示；
所述的Foldback-CD3-DT390的氨基酸序列如SEQ ID NO：7所示；
本发明还提供一种编码鼠抗CD3重组免疫毒素的基因。
其中，
所述的编码重组免疫毒素ScFv-CD3-DT390的基因如SEQ ID NO：8所示。
所述的重组免疫毒素Bi-CD3-DT390的基因如SEQ ID NO：9所示。
所述的重组免疫毒素Foldback-CD3-DT390的基因如SEQ ID NO：10所示。
采用基因工程技术与策略，通过改变单链抗体与DT390的结合方式以提高其亲和力，优化密码子系列得到以DT结构为毒性中心的三种重组免疫毒素：ScFv-CD3-DT390，Bi-CD3-DT390，Foldback-CD3-DT390，构建其原核表达系统。
本发明还提供了含有上述基因的载体。
本发明还提供了含有上述载体的宿主细胞。
本发明还提供一种鼠抗CD3重组免疫毒素的制备方法，包括如下步骤：以DT390为例：
(1)对上述鼠抗CD3重组免疫毒素的氨基酸序列分别进行优化，设计了如SEQ IDNO：8～SEQ ID NO：10所述的基因，另外在5′端引入KpnI酶切位点以及引入EK识别位点-(Asp)4Lys编码序列并设计上起始密码子ATG，其中EK识别位点-(Asp)4Lys编码序列以便切除最终表达的融合蛋白的His-Tag；在3′端设计上6个组氨酸的密码子CGTCGTCGTCGTCGTCGT、终止密码子TAATGA并引入XhoI酶切位点，使每个构建体易于蛋白质纯化与检测，同时去除编码序列3′端的终止密码子；
(2)目的基因采用pET-30a(+)表达载体进行构建重组表达载体；
(3)将重组表达载体转化宿主菌后诱导表达、纯化，获得鼠抗CD3重组免疫毒素。
本发明还提供了鼠抗CD3重组免疫毒素、基因、载体或宿主细胞将在小鼠T淋巴细胞剔除、自身性免疫疾病、器官移植、免疫耐受及肿瘤治疗等领域具有广泛的应用。
本发明相对于现有技术具有如下的优点及效果：
(1)通过优化目的基因的密码子及筛选单链抗体与DT390的融合方式，三种单链抗体与DT连接，提高重组免疫毒素的亲和力。
(2)证实靶向鼠抗CD3ε重组免疫毒素剔除鼠外周血及淋巴结T细胞的有效性。
(3)此类免疫毒素具备开发成基因工程药物，前期可作为实验动物的工具药，在小鼠T淋巴细胞剔除、自身性免疫疾病、器官移植、免疫耐受及肿瘤治疗的研究方面具有应用意义。后续可能有临床前药学研究及转化的潜能。
附图说明
图1是表达并纯化后的重组免疫毒素的SDS-PAGE分析结果；其中，M1：SDS-PAGEMarker；Lane 1：BSA；A为DT390；B为ScFv-CD3-DT390；C为Bi-CD3-DT390；D为Foldback-CD3-DT390。
图2是表达并纯化后的重组免疫毒素的Western Blotting结果；其中，M2：WesternBlot Marker；A为DT390；B为ScFv-CD3-DT390；C为Bi-CD3-DT390；D为Foldback-CD3-DT390。
图3是表达并纯化后的重组免疫毒素的流式细胞术分析结果；其中，A为DT390；B为ScFv-CD3-DT390；C为Bi-CD3-DT390；D为Foldback-CD3-DT390。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。
下列实施例中未注明具体实验条件的试验方法，通常按照常规实验条件或按照制造厂所建议的实验条件。所使用的材料、试剂等，如无特别说明，均为从商业途径得到的试剂和材料。
实施例1
(1)鼠抗CD3重组免疫毒素的表达载体构建：
重组免疫毒素：ScFv-CD3-DT390，Bi-CD3-DT390，Foldback-CD3-DT390，构建其原核表达系统。
设计优化的鼠抗CD3重组免疫毒素的密码子核苷酸序列，见SEQ ID NO：8、SEQ IDNO：9、SEQ ID NO：10；分别在5′端引入KpnI酶切位点以及引入EK识别位点-(Asp)4Lys编码序列并设计上起始密码子ATG，其中EK识别位点-(Asp)4Lys编码序列以便切除最终表达的融合蛋白的His-Tag；在3′端设计上6个组氨酸的密码子CGTCGTCGTCGTCGTCGT、终止密码子TAATGA并引入XhoI酶切位点，使每个构建体易于蛋白质纯化与检测，同时去除编码序列3′端的终止密码子。
核苷酸序列由公司合成，其后采用PCR技术扩增，常规方法进行分子克隆，目的基因采用pET-30a(+)表达载体进行表达。
(2)表达载体转化及诱导表达：取100μL冰上融化的大肠杆菌BL21(DE3)感受态细胞，将构建好的含有重组蛋白基因的质粒转化到大肠杆菌BL21(DE3)感受态细胞中，冰上静止25min。然后均匀涂布到LB平板上(含50μg/mL的硫酸卡那霉素)，之后倒置于37℃培养箱过夜。从转化的平板中挑选单克隆，接种到4mL的TB培养基中(含50μg/mL的硫酸卡那霉素)，待培养至OD600为0.5～0.8，向试管培养液中加入终浓度0.5mM的IPTG，之后分别置于15℃、28℃、37℃诱导表达。同样诱导含有空载体的大肠杆菌BL21(DE3)感受态细胞，以此作对照。
(3)SDS-PAGE分析鉴定诱导表达结果：取诱导后的培养液12000rpm离心5min，去除上清液，加入PBS液重悬沉淀，最后加入SDS-PAGE上样缓冲液于100℃下加热样品10min，然后离心取上清电泳。电泳前10min时，100V稳压电泳，待溴酚蓝指示剂进入分离胶后200V稳压电泳至溴酚蓝带迁移至离凝胶底部1cm，取出凝胶用考马斯亮蓝染色液染色，随后转入脱色液中，脱色至背景清晰。
(4)重组蛋白放大培养：培养3L的表达菌，生长至OD600＝0.8时，加入终浓度为0.5mM IPTG，37℃诱导16h后收集菌体。
(5)重组蛋白纯化(整个纯化过程在低温下操作)，及SDS-PAGE分析：
1)上清通过亲和层析纯化重组蛋白：全菌采用50mM Tris(pH8.0)，150mM NaCl含1％Triton X-100，1μg/mL Pepstatin A，1μg/mL Leupeptin，1mM DTT超声裂解，同时以50mM Tris(pH8.0)，150mM NaCl缓冲液平衡Ni-IDA柱，之后用不同浓度咪唑的平衡缓冲液洗脱目标蛋白，并收集每个洗脱组分进行SDS-PAGE分析检测。经Ni-IDA亲和层析纯化分析，重组蛋白蛋白表达在包涵体中。
2)包涵体通过亲和层析纯化重组蛋白蛋白：包涵体采用50mM Tris(pH8.0)、150mMNaCl含1％Triton X-100、5mM EDTA、2mM DTT洗涤后，以50mM Tris(pH8.0)，150mM NaCl，8MUrea缓冲液溶解包涵体同时平衡Ni-IDA柱，最后用不同浓度咪唑的平衡缓冲液洗脱目标蛋白，并收集每个洗脱组分进行SDS-PAGE分析检测。结果如图1所示，表明纯化后的3种重组免疫毒素蛋白用SDS-PAGE凝胶电泳检测，结果显示免疫毒素ScFv-CD3-DT390，Bi-CD3-DT390和Foldback-CD3-DT390蛋白大小符合预期，蛋白的条带清晰且没有其他杂带，说明经过纯化后，蛋白纯度较高。
(6)Western-blotting分析：SDS-PAGE凝胶电泳后，进行电转。用PBS冲洗NC膜3次，然后将膜浸泡于含10％脱脂奶粉的TBST中，37℃摇动封闭2h；封闭结束后PBST清洗NC膜3次，再加入1：200稀释的6×His tag antibody[GT359](GTX628914，GeneTex，USA)，37℃轻摇孵育1h；取出后用PBST清洗3次，每次15min；洗完后加入1：2 000稀释的辣根过氧化物酶(HRP)标记的山羊抗鼠IgG二抗，37℃轻摇孵育1h，TBST清洗3次，每次15min。将膜取出后进行显色。结果如图2所示，表明：经纯化后的重组免疫毒素蛋白用抗His标签抗体进行Western-blotting检测，结果显示融合6个His的嵌合免疫毒素ScFv-CD3-DT390，Bi-CD3-DT390和Foldback-CD3-DT390His标签均为阳性，且蛋白大小符合预期，表明目标蛋白在原核载体表达系统得以成功表达。
实施例2
体外细胞模型分析鼠抗CD3重组免疫毒素生物学特性及对小鼠CD3表型剔除作用
1.体外细胞模型分析鼠抗CD3重组免疫毒素对小鼠CD3结合及表型剔除
采用健康小鼠外周血细胞分析鼠抗CD3重组免疫毒素在体外结合和杀死鼠外周血单个核细胞。
(1)准备足够的小鼠外周血淋巴细胞，用细胞培养液稀释为2.0×106个细胞/mL，每孔500μL加入24孔板中。
(2)将纯化的鼠抗CD3重组免疫毒素用淋巴细胞培养液分别稀释为1.0×10-6M，1.0×10-7M，1.0×10-8M及1.0×10-9M。
(3)取50μL稀释的鼠抗CD3重组免疫毒素或阴性对照加入对应的孔内，37℃温箱中培养48h。
(4)将细胞收集至5mL离心管中，从24孔板中收集细胞时，要轻轻吹打几次，尽可能收集所有的细胞。
(5)用2mL FACS Buffer洗涤细胞两次，弃去上清液。流式细胞仪分析鼠抗CD3重组免疫毒素体外结合鼠外周血CD3单个核细胞，鼠外周血单个核细胞与生物素标记的鼠抗CD3重组免疫毒素为主色，PE标记的链霉素亲和素作为第二色。
(6)为了进一步排除免疫毒素的脱靶效应，还分析其他的外周血单个核细胞用单链折叠重组免疫毒素(Foldback-CD3-DT390)孵育48h后的数量。B淋巴细胞、NK细胞和单核细胞未见明显变化，表明三种免疫毒素在选择性作用于小鼠T细胞方面具有较强的靶向性。
2.体内细胞模型分析免疫毒素对小鼠CD3结合及表型剔除作用
(1)选择6-7周龄BALB/C小鼠，实验随机分4组，鼠抗CD3重组免疫毒素的剂量按照步骤(2)进行，每组8只，考虑DT390的毒性作用，2天内给药完毕。
(2)实验组静脉推注隔日总剂量分别设置为0.2，0.4，0.6，0.8，1.2，1.6及2μg/只小鼠。
(3)28日内定时在淋巴结和胸腺的T细胞将通过流式细胞仪测定外周血T细胞耗竭水平，并计算其血液中T细胞的绝对数量。结果如图3所示，表明三种重组免疫毒素使用小鼠外周血T淋巴细胞分别降低了80％，83％和90％。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
序列表
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Ala Lys Tyr Glu Tyr Met Ala Gln Ala Cys Ala Gly Asn Arg Val Arg
180 185 190
Arg Ser Val Gly Ser Ser Leu Ser Cys Ile Asn Leu Asp Trp Asp Val
195 200 205
Ile Arg Asp Lys Thr Lys Thr Lys Ile Glu Ser Leu Lys Glu His Gly
210 215 220
Pro Ile Lys Asn Lys Met Ser Glu Ser Pro Asn Lys Thr Val Ser Glu
225 230 235 240
Glu Lys Ala Lys Gln Tyr Leu Glu Glu Phe His Gln Thr Ala Leu Glu
245 250 255
His Pro Glu Leu Ser Glu Leu Lys Thr Val Thr Gly Thr Asn Pro Val
260 265 270
Phe Ala Gly Ala Asn Tyr Ala Ala Trp Ala Val Asn Val Ala Gln Val
275 280 285
Ile Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys Thr Thr Ala Ala Leu
290 295 300
Ser Ile Leu Pro Gly Ile Gly Ser Val Met Gly Ile Ala Asp Gly Ala
305 310 315 320
Val His His Asn Thr Glu Glu Ile Val Ala Gln Ser Ile Ala Leu Ser
325 330 335
Ser Leu Met Val Ala Gln Ala Ile Pro Leu Val Gly Glu Leu Val Asp
340 345 350
Ile Gly Phe Ala Ala Tyr Asn Phe Val Glu Ser Ile Ile Asn Leu Phe
355 360 365
Gln Val Val His Asn Ser Tyr Asn Arg Pro Ala Tyr Ser Pro Gly His
370 375 380
Lys Thr Gln Pro Phe Leu Gly Gly Gly Gly Ser Asp Ile Gln Met Thr
385 390 395 400
Gln Ser Pro Ser Ser Leu Pro Ala Ser Leu Gly Asp Arg Val Thr Ile
405 410 415
Asn Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn Trp Tyr Gln
420 425 430
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Asn Lys
435 440 445
Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Arg
450 455 460
Asp Ser Ser Phe Thr Ile Ser Ser Leu Glu Ser Glu Asp Ile Gly Ser
465 470 475 480
Tyr Tyr Cys Gln Gln Tyr Tyr Asn Tyr Pro Trp Thr Phe Gly Pro Gly
485 490 495
Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
500 505 510
Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
515 520 525
Val Gln Pro Gly Lys Ser Leu Lys Leu Ser Cys Glu Ala Ser Gly Phe
530 535 540
Thr Phe Ser Gly Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Arg
545 550 555 560
Gly Leu Glu Ser Val Ala Tyr Ile Thr Ser Ser Ser Ile Asn Ile Lys
565 570 575
Tyr Ala Asp Ala Val Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala
580 585 590
Lys Asn Leu Leu Phe Leu Gln Met Asn Ile Leu Lys Ser Glu Asp Thr
595 600 605
Ala Met Tyr Tyr Cys Ala Arg Phe Asp Trp Asp Lys Asn Tyr Trp Gly
610 615 620
Gln Gly Thr Met Val Thr Val Ser Ser
625 630
<210> 6
<211> 886
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> Bi-CD3-DT390的氨基酸序列
<400> 6
Gly Ala Asp Asp Val Val Asp Ser Ser Lys Ser Phe Val Met Glu Asn
1 5 10 15
Phe Ser Ser Tyr His Gly Thr Lys Pro Gly Tyr Val Asp Ser Ile Gln
20 25 30
Lys Gly Ile Gln Lys Pro Lys Ser Gly Thr Gln Gly Asn Tyr Asp Asp
35 40 45
Asp Trp Lys Gly Phe Tyr Ser Thr Asp Asn Lys His Asp Ala Ala Gly
50 55 60
Tyr Ser Val Asp Asn Glu Asn Pro Leu Ser Gly Lys Ala Gly Gly Val
65 70 75 80
Val Lys Val Thr Tyr Pro Gly Leu Thr Lys Val Leu Ala Leu Lys Val
85 90 95
Asp Asn Ala Glu Thr Ile Lys Lys Glu Leu Gly Leu Ser Leu Thr Glu
100 105 110
Pro Leu Met Glu Gln Val Gly Thr Glu Glu Phe Ile Lys Arg Phe Gly
115 120 125
Asp Gly Ala Ser Arg Val Val Leu Ser Leu Pro Phe Ala Glu Gly Ser
130 135 140
Ser Ser Val Glu Tyr Ile Asn Asn Trp Glu Gln Ala Lys Ala Leu Ser
145 150 155 160
Val Glu Leu Glu Ile Asn Phe Glu Thr Arg Gly Lys Arg Gly Gln Asp
165 170 175
Ala Lys Tyr Glu Tyr Met Ala Gln Ala Cys Ala Gly Asn Arg Val Arg
180 185 190
Arg Ser Val Gly Ser Ser Leu Ser Cys Ile Asn Leu Asp Trp Asp Val
195 200 205
Ile Arg Asp Lys Thr Lys Thr Lys Ile Glu Ser Leu Lys Glu His Gly
210 215 220
Pro Ile Lys Asn Lys Met Ser Glu Ser Pro Asn Lys Thr Val Ser Glu
225 230 235 240
Glu Lys Ala Lys Gln Tyr Leu Glu Glu Phe His Gln Thr Ala Leu Glu
245 250 255
His Pro Glu Leu Ser Glu Leu Lys Thr Val Thr Gly Thr Asn Pro Val
260 265 270
Phe Ala Gly Ala Asn Tyr Ala Ala Trp Ala Val Asn Val Ala Gln Val
275 280 285
Ile Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys Thr Thr Ala Ala Leu
290 295 300
Ser Ile Leu Pro Gly Ile Gly Ser Val Met Gly Ile Ala Asp Gly Ala
305 310 315 320
Val His His Asn Thr Glu Glu Ile Val Ala Gln Ser Ile Ala Leu Ser
325 330 335
Ser Leu Met Val Ala Gln Ala Ile Pro Leu Val Gly Glu Leu Val Asp
340 345 350
Ile Gly Phe Ala Ala Tyr Asn Phe Val Glu Ser Ile Ile Asn Leu Phe
355 360 365
Gln Val Val His Asn Ser Tyr Asn Arg Pro Ala Tyr Ser Pro Gly His
370 375 380
Lys Thr Gln Pro Phe Leu Gly Gly Gly Gly Ser Asp Ile Gln Met Thr
385 390 395 400
Gln Ser Pro Ser Ser Leu Pro Ala Ser Leu Gly Asp Arg Val Thr Ile
405 410 415
Asn Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn Trp Tyr Gln
420 425 430
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Asn Lys
435 440 445
Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Arg
450 455 460
Asp Ser Ser Phe Thr Ile Ser Ser Leu Glu Ser Glu Asp Ile Gly Ser
465 470 475 480
Tyr Tyr Cys Gln Gln Tyr Tyr Asn Tyr Pro Trp Thr Phe Gly Pro Gly
485 490 495
Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
500 505 510
Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
515 520 525
Val Gln Pro Gly Lys Ser Leu Lys Leu Ser Cys Glu Ala Ser Gly Phe
530 535 540
Thr Phe Ser Gly Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Arg
545 550 555 560
Gly Leu Glu Ser Val Ala Tyr Ile Thr Ser Ser Ser Ile Asn Ile Lys
565 570 575
Tyr Ala Asp Ala Val Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala
580 585 590
Lys Asn Leu Leu Phe Leu Gln Met Asn Ile Leu Lys Ser Glu Asp Thr
595 600 605
Ala Met Tyr Tyr Cys Ala Arg Phe Asp Trp Asp Lys Asn Tyr Trp Gly
610 615 620
Gln Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly
625 630 635 640
Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro
645 650 655
Ser Ser Leu Pro Ala Ser Leu Gly Asp Arg Val Thr Ile Asn Cys Gln
660 665 670
Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro
675 680 685
Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Asn Lys Leu Ala Asp
690 695 700
Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Arg Asp Ser Ser
705 710 715 720
Phe Thr Ile Ser Ser Leu Glu Ser Glu Asp Ile Gly Ser Tyr Tyr Cys
725 730 735
Gln Gln Tyr Tyr Asn Tyr Pro Trp Thr Phe Gly Pro Gly Thr Lys Leu
740 745 750
Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
755 760 765
Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
770 775 780
Gly Lys Ser Leu Lys Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe Ser
785 790 795 800
Gly Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Arg Gly Leu Glu
805 810 815
Ser Val Ala Tyr Ile Thr Ser Ser Ser Ile Asn Ile Lys Tyr Ala Asp
820 825 830
Ala Val Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Leu
835 840 845
Leu Phe Leu Gln Met Asn Ile Leu Lys Ser Glu Asp Thr Ala Met Tyr
850 855 860
Tyr Cys Ala Arg Phe Asp Trp Asp Lys Asn Tyr Trp Gly Gln Gly Thr
865 870 875 880
Met Val Thr Val Ser Ser
885
<210> 7
<211> 866
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> Foldback-CD3-DT390的氨基酸序列
<400> 7
Gly Ala Asp Asp Val Val Asp Ser Ser Lys Ser Phe Val Met Glu Asn
1 5 10 15
Phe Ser Ser Tyr His Gly Thr Lys Pro Gly Tyr Val Asp Ser Ile Gln
20 25 30
Lys Gly Ile Gln Lys Pro Lys Ser Gly Thr Gln Gly Asn Tyr Asp Asp
35 40 45
Asp Trp Lys Gly Phe Tyr Ser Thr Asp Asn Lys His Asp Ala Ala Gly
50 55 60
Tyr Ser Val Asp Asn Glu Asn Pro Leu Ser Gly Lys Ala Gly Gly Val
65 70 75 80
Val Lys Val Thr Tyr Pro Gly Leu Thr Lys Val Leu Ala Leu Lys Val
85 90 95
Asp Asn Ala Glu Thr Ile Lys Lys Glu Leu Gly Leu Ser Leu Thr Glu
100 105 110
Pro Leu Met Glu Gln Val Gly Thr Glu Glu Phe Ile Lys Arg Phe Gly
115 120 125
Asp Gly Ala Ser Arg Val Val Leu Ser Leu Pro Phe Ala Glu Gly Ser
130 135 140
Ser Ser Val Glu Tyr Ile Asn Asn Trp Glu Gln Ala Lys Ala Leu Ser
145 150 155 160
Val Glu Leu Glu Ile Asn Phe Glu Thr Arg Gly Lys Arg Gly Gln Asp
165 170 175
Ala Lys Tyr Glu Tyr Met Ala Gln Ala Cys Ala Gly Asn Arg Val Arg
180 185 190
Arg Ser Val Gly Ser Ser Leu Ser Cys Ile Asn Leu Asp Trp Asp Val
195 200 205
Ile Arg Asp Lys Thr Lys Thr Lys Ile Glu Ser Leu Lys Glu His Gly
210 215 220
Pro Ile Lys Asn Lys Met Ser Glu Ser Pro Asn Lys Thr Val Ser Glu
225 230 235 240
Glu Lys Ala Lys Gln Tyr Leu Glu Glu Phe His Gln Thr Ala Leu Glu
245 250 255
His Pro Glu Leu Ser Glu Leu Lys Thr Val Thr Gly Thr Asn Pro Val
260 265 270
Phe Ala Gly Ala Asn Tyr Ala Ala Trp Ala Val Asn Val Ala Gln Val
275 280 285
Ile Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys Thr Thr Ala Ala Leu
290 295 300
Ser Ile Leu Pro Gly Ile Gly Ser Val Met Gly Ile Ala Asp Gly Ala
305 310 315 320
Val His His Asn Thr Glu Glu Ile Val Ala Gln Ser Ile Ala Leu Ser
325 330 335
Ser Leu Met Val Ala Gln Ala Ile Pro Leu Val Gly Glu Leu Val Asp
340 345 350
Ile Gly Phe Ala Ala Tyr Asn Phe Val Glu Ser Ile Ile Asn Leu Phe
355 360 365
Gln Val Val His Asn Ser Tyr Asn Arg Pro Ala Tyr Ser Pro Gly His
370 375 380
Lys Thr Gln Pro Phe Leu Gly Gly Gly Gly Ser Asp Ile Gln Met Thr
385 390 395 400
Gln Ser Pro Ser Ser Leu Pro Ala Ser Leu Gly Asp Arg Val Thr Ile
405 410 415
Asn Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn Trp Tyr Gln
420 425 430
Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Thr Asn Lys
435 440 445
Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Arg
450 455 460
Asp Ser Ser Phe Thr Ile Ser Ser Leu Glu Ser Glu Asp Ile Gly Ser
465 470 475 480
Tyr Tyr Cys Gln Gln Tyr Tyr Asn Tyr Pro Trp Thr Phe Gly Pro Gly
485 490 495
Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Glu Val Gln Leu Val
500 505 510
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Lys Ser Leu Lys Leu Ser
515 520 525
Cys Glu Ala Ser Gly Phe Thr Phe Ser Gly Tyr Gly Met His Trp Val
530 535 540
Arg Gln Ala Pro Gly Arg Gly Leu Glu Ser Val Ala Tyr Ile Thr Ser
545 550 555 560
Ser Ser Ile Asn Ile Lys Tyr Ala Asp Ala Val Lys Gly Arg Phe Thr
565 570 575
Val Ser Arg Asp Asn Ala Lys Asn Leu Leu Phe Leu Gln Met Asn Ile
580 585 590
Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala Arg Phe Asp Trp
595 600 605
Asp Lys Asn Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Gly
610 615 620
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile
625 630 635 640
Gln Met Thr Gln Ser Pro Ser Ser Leu Pro Ala Ser Leu Gly Asp Arg
645 650 655
Val Thr Ile Asn Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn
660 665 670
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile Tyr Tyr
675 680 685
Thr Asn Lys Leu Ala Asp Gly Val Pro Ser Arg Phe Ser Gly Ser Gly
690 695 700
Ser Gly Arg Asp Ser Ser Phe Thr Ile Ser Ser Leu Glu Ser Glu Asp
705 710 715 720
Ile Gly Ser Tyr Tyr Cys Gln Gln Tyr Tyr Asn Tyr Pro Trp Thr Phe
725 730 735
Gly Pro Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Glu Val
740 745 750
Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Lys Ser Leu
755 760 765
Lys Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe Ser Gly Tyr Gly Met
770 775 780
His Trp Val Arg Gln Ala Pro Gly Arg Gly Leu Glu Ser Val Ala Tyr
785 790 795 800
Ile Thr Ser Ser Ser Ile Asn Ile Lys Tyr Ala Asp Ala Val Lys Gly
805 810 815
Arg Phe Thr Val Ser Arg Asp Asn Ala Lys Asn Leu Leu Phe Leu Gln
820 825 830
Met Asn Ile Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala Arg
835 840 845
Phe Asp Trp Asp Lys Asn Tyr Trp Gly Gln Gly Thr Met Val Thr Val
850 855 860
Ser Ser
865
<210> 8
<211> 1899
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> ScFv-CD3-DT390核酸编码序列
<400> 8
ggtgcggacg acgttgttga ctcttctaaa tctttcgtta tggaaaactt ctcttcttac 60
cacggtacca aaccgggtta cgttgactct atccagaaag gtatccagaa accgaaatct 120
ggtacccagg gtaactacga cgacgactgg aaaggtttct actctaccga caacaaacac 180
gacgcggcgg gttactctgt tgacaacgaa aacccgctgt ctggtaaagc gggtggtgtt 240
gttaaagtta cctacccggg tctgaccaaa gttctggcgc tgaaagttga caacgcggaa 300
accatcaaaa aagaactggg tctgtctctg accgaaccgc tgatggaaca ggttggtacc 360
gaagaattca tcaaacgttt cggtgacggt gcgtctcgtg ttgttctgtc tctgccgttc 420
gcggaaggtt cttcttctgt tgaatacatc aacaactggg aacaggcgaa agcgctgtct 480
gttgaactgg aaatcaactt cgaaacccgt ggtaaacgtg gtcaggacgc gaaatacgaa 540
tacatggcgc aggcgtgcgc gggtaaccgt gttcgtcgtt ctgttggttc ttctctgtct 600
tgcatcaacc tggactggga cgttatccgt gacaaaacca aaaccaaaat cgaatctctg 660
aaagaacacg gtccgatcaa aaacaaaatg tctgaatctc cgaacaaaac cgtttctgaa 720
gaaaaagcga aacagtacct ggaagaattc caccagaccg cgctggaaca cccggaactg 780
tctgaactga aaaccgttac cggtaccaac ccggttttcg cgggtgcgaa ctacgcggcg 840
tgggcggtta acgttgcgca ggttatcgac tctgaaaccg cggacaacct ggaaaaaacc 900
accgcggcgc tgtctatcct gccgggtatc ggttctgtta tgggtatcgc ggacggtgcg 960
gttcaccaca acaccgaaga aatcgttgcg cagtctatcg cgctgtcttc tctgatggtt 1020
gcgcaggcga tcccgctggt tggtgaactg gttgacatcg gtttcgcggc gtacaacttc 1080
gttgaatcta tcatcaacct gttccaggtt gttcacaact cttacaaccg tccggcgtac 1140
tctccgggtc acaaaaccca gccgttcctg ggtggtggtg gttctgacat ccagatgacc 1200
cagtctccgt cttctctgcc ggcgtctctg ggtgaccgtg ttaccatcaa ctgccaggcg 1260
tctcaggaca tctctaacta cctgaactgg taccagcaga aaccgggtaa agcgccgaaa 1320
ctgctgatct actacaccaa caaactggcg gacggtgttc cgtctcgttt ctctggttct 1380
ggttctggtc gtgactcttc tttcaccatc tcttctctgg aatctgaaga catcggttct 1440
tactactgcc agcagtacta caactacccg tggaccttcg gtccgggtac caaactggaa 1500
atcaaaggtg gtggtggttc tggtggtggt ggttctggtg gtggtggttc tgaagttcag 1560
ctggttgaat ctggtggtgg tctggttcag ccgggtaaat ctctgaaact gtcttgcgaa 1620
gcgtctggtt tcaccttctc tggttacggt atgcactggg ttcgtcaggc gccgggtcgt 1680
ggtctggaat ctgttgcgta catcacctct tcttctatca acatcaaata cgcggacgcg 1740
gttaaaggtc gtttcaccgt ttctcgtgac aacgcgaaaa acctgctgtt cctgcagatg 1800
aacatcctga aatctgaaga caccgcgatg tactactgcg cgcgtttcga ctgggacaaa 1860
aactactggg gtcagggtac catggttacc gtttcttct 1899
<210> 9
<211> 2658
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> Bi-CD3-DT390核酸编码序列序列
<400> 9
ggtgcggacg acgttgttga ctcttctaaa tctttcgtta tggaaaactt ctcttcttac 60
cacggtacca aaccgggtta cgttgactct atccagaaag gtatccagaa accgaaatct 120
ggtacccagg gtaactacga cgacgactgg aaaggtttct actctaccga caacaaacac 180
gacgcggcgg gttactctgt tgacaacgaa aacccgctgt ctggtaaagc gggtggtgtt 240
gttaaagtta cctacccggg tctgaccaaa gttctggcgc tgaaagttga caacgcggaa 300
accatcaaaa aagaactggg tctgtctctg accgaaccgc tgatggaaca ggttggtacc 360
gaagaattca tcaaacgttt cggtgacggt gcgtctcgtg ttgttctgtc tctgccgttc 420
gcggaaggtt cttcttctgt tgaatacatc aacaactggg aacaggcgaa agcgctgtct 480
gttgaactgg aaatcaactt cgaaacccgt ggtaaacgtg gtcaggacgc gaaatacgaa 540
tacatggcgc aggcgtgcgc gggtaaccgt gttcgtcgtt ctgttggttc ttctctgtct 600
tgcatcaacc tggactggga cgttatccgt gacaaaacca aaaccaaaat cgaatctctg 660
aaagaacacg gtccgatcaa aaacaaaatg tctgaatctc cgaacaaaac cgtttctgaa 720
gaaaaagcga aacagtacct ggaagaattc caccagaccg cgctggaaca cccggaactg 780
tctgaactga aaaccgttac cggtaccaac ccggttttcg cgggtgcgaa ctacgcggcg 840
tgggcggtta acgttgcgca ggttatcgac tctgaaaccg cggacaacct ggaaaaaacc 900
accgcggcgc tgtctatcct gccgggtatc ggttctgtta tgggtatcgc ggacggtgcg 960
gttcaccaca acaccgaaga aatcgttgcg cagtctatcg cgctgtcttc tctgatggtt 1020
gcgcaggcga tcccgctggt tggtgaactg gttgacatcg gtttcgcggc gtacaacttc 1080
gttgaatcta tcatcaacct gttccaggtt gttcacaact cttacaaccg tccggcgtac 1140
tctccgggtc acaaaaccca gccgttcctg ggtggtggtg gttctgacat ccagatgacc 1200
cagtctccgt cttctctgcc ggcgtctctg ggtgaccgtg ttaccatcaa ctgccaggcg 1260
tctcaggaca tctctaacta cctgaactgg taccagcaga aaccgggtaa agcgccgaaa 1320
ctgctgatct actacaccaa caaactggcg gacggtgttc cgtctcgttt ctctggttct 1380
ggttctggtc gtgactcttc tttcaccatc tcttctctgg aatctgaaga catcggttct 1440
tactactgcc agcagtacta caactacccg tggaccttcg gtccgggtac caaactggaa 1500
atcaaaggtg gtggtggttc tggtggtggt ggttctggtg gtggtggttc tgaagttcag 1560
ctggttgaat ctggtggtgg tctggttcag ccgggtaaat ctctgaaact gtcttgcgaa 1620
gcgtctggtt tcaccttctc tggttacggt atgcactggg ttcgtcaggc gccgggtcgt 1680
ggtctggaat ctgttgcgta catcacctct tcttctatca acatcaaata cgcggacgcg 1740
gttaaaggtc gtttcaccgt ttctcgtgac aacgcgaaaa acctgctgtt cctgcagatg 1800
aacatcctga aatctgaaga caccgcgatg tactactgcg cgcgtttcga ctgggacaaa 1860
aactactggg gtcagggtac catggttacc gtttcttctg gtggtggtgg ttctggtggt 1920
ggtggttctg gtggtggtgg ttctgacatc cagatgaccc agtctccgtc ttctctgccg 1980
gcgtctctgg gtgaccgtgt taccatcaac tgccaggcgt ctcaggacat ctctaactac 2040
ctgaactggt accagcagaa accgggtaaa gcgccgaaac tgctgatcta ctacaccaac 2100
aaactggcgg acggtgttcc gtctcgtttc tctggttctg gttctggtcg tgactcttct 2160
ttcaccatct cttctctgga atctgaagac atcggttctt actactgcca gcagtactac 2220
aactacccgt ggaccttcgg tccgggtacc aaactggaaa tcaaaggtgg tggtggttct 2280
ggtggtggtg gttctggtgg tggtggttct gaagttcagc tggttgaatc tggtggtggt 2340
ctggttcagc cgggtaaatc tctgaaactg tcttgcgaag cgtctggttt caccttctct 2400
ggttacggta tgcactgggt tcgtcaggcg ccgggtcgtg gtctggaatc tgttgcgtac 2460
atcacctctt cttctatcaa catcaaatac gcggacgcgg ttaaaggtcg tttcaccgtt 2520
tctcgtgaca acgcgaaaaa cctgctgttc ctgcagatga acatcctgaa atctgaagac 2580
accgcgatgt actactgcgc gcgtttcgac tgggacaaaa actactgggg tcagggtacc 2640
atggttaccg tttcttct 2658
<210> 10
<211> 2598
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> Foldback-CD3-DT390核酸编码序列
<400> 10
ggtgcggacg acgttgttga ctcttctaaa tctttcgtta tggaaaactt ctcttcttac 60
cacggtacca aaccgggtta cgttgactct atccagaaag gtatccagaa accgaaatct 120
ggtacccagg gtaactacga cgacgactgg aaaggtttct actctaccga caacaaacac 180
gacgcggcgg gttactctgt tgacaacgaa aacccgctgt ctggtaaagc gggtggtgtt 240
gttaaagtta cctacccggg tctgaccaaa gttctggcgc tgaaagttga caacgcggaa 300
accatcaaaa aagaactggg tctgtctctg accgaaccgc tgatggaaca ggttggtacc 360
gaagaattca tcaaacgttt cggtgacggt gcgtctcgtg ttgttctgtc tctgccgttc 420
gcggaaggtt cttcttctgt tgaatacatc aacaactggg aacaggcgaa agcgctgtct 480
gttgaactgg aaatcaactt cgaaacccgt ggtaaacgtg gtcaggacgc gaaatacgaa 540
tacatggcgc aggcgtgcgc gggtaaccgt gttcgtcgtt ctgttggttc ttctctgtct 600
tgcatcaacc tggactggga cgttatccgt gacaaaacca aaaccaaaat cgaatctctg 660
aaagaacacg gtccgatcaa aaacaaaatg tctgaatctc cgaacaaaac cgtttctgaa 720
gaaaaagcga aacagtacct ggaagaattc caccagaccg cgctggaaca cccggaactg 780
tctgaactga aaaccgttac cggtaccaac ccggttttcg cgggtgcgaa ctacgcggcg 840
tgggcggtta acgttgcgca ggttatcgac tctgaaaccg cggacaacct ggaaaaaacc 900
accgcggcgc tgtctatcct gccgggtatc ggttctgtta tgggtatcgc ggacggtgcg 960
gttcaccaca acaccgaaga aatcgttgcg cagtctatcg cgctgtcttc tctgatggtt 1020
gcgcaggcga tcccgctggt tggtgaactg gttgacatcg gtttcgcggc gtacaacttc 1080
gttgaatcta tcatcaacct gttccaggtt gttcacaact cttacaaccg tccggcgtac 1140
tctccgggtc acaaaaccca gccgttcctg ggtggtggtg gttctgacat ccagatgacc 1200
cagtctccgt cttctctgcc ggcgtctctg ggtgaccgtg ttaccatcaa ctgccaggcg 1260
tctcaggaca tctctaacta cctgaactgg taccagcaga aaccgggtaa agcgccgaaa 1320
ctgctgatct actacaccaa caaactggcg gacggtgttc cgtctcgttt ctctggttct 1380
ggttctggtc gtgactcttc tttcaccatc tcttctctgg aatctgaaga catcggttct 1440
tactactgcc agcagtacta caactacccg tggaccttcg gtccgggtac caaactggaa 1500
atcaaaggtg gtggtggttc tgaagttcag ctggttgaat ctggtggtgg tctggttcag 1560
ccgggtaaat ctctgaaact gtcttgcgaa gcgtctggtt tcaccttctc tggttacggt 1620
atgcactggg ttcgtcaggc gccgggtcgt ggtctggaat ctgttgcgta catcacctct 1680
tcttctatca acatcaaata cgcggacgcg gttaaaggtc gtttcaccgt ttctcgtgac 1740
aacgcgaaaa acctgctgtt cctgcagatg aacatcctga aatctgaaga caccgcgatg 1800
tactactgcg cgcgtttcga ctgggacaaa aactactggg gtcagggtac catggttacc 1860
gtttcttctg gtggtggtgg ttctggtggt ggtggttctg gtggtggtgg ttctgacatc 1920
cagatgaccc agtctccgtc ttctctgccg gcgtctctgg gtgaccgtgt taccatcaac 1980
tgccaggcgt ctcaggacat ctctaactac ctgaactggt accagcagaa accgggtaaa 2040
gcgccgaaac tgctgatcta ctacaccaac aaactggcgg acggtgttcc gtctcgtttc 2100
tctggttctg gttctggtcg tgactcttct ttcaccatct cttctctgga atctgaagac 2160
atcggttctt actactgcca gcagtactac aactacccgt ggaccttcgg tccgggtacc 2220
aaactggaaa tcaaaggtgg tggtggttct gaagttcagc tggttgaatc tggtggtggt 2280
ctggttcagc cgggtaaatc tctgaaactg tcttgcgaag cgtctggttt caccttctct 2340
ggttacggta tgcactgggt tcgtcaggcg ccgggtcgtg gtctggaatc tgttgcgtac 2400
atcacctctt cttctatcaa catcaaatac gcggacgcgg ttaaaggtcg tttcaccgtt 2460
tctcgtgaca acgcgaaaaa cctgctgttc ctgcagatga acatcctgaa atctgaagac 2520
accgcgatgt actactgcgc gcgtttcgac tgggacaaaa actactgggg tcagggtacc 2580
atggttaccg tttcttct 2598

Claims (10)

1.一种鼠抗CD3重组免疫毒素，其特征在于：所述重组免疫毒素包括2部分：一部分为细菌毒素或植物毒素，另一部分为scFv、Bi-scFv结构域或Foldback-scFv，两部分通过由四个甘氨酸和丝氨酸残基组成的接头进行连接得到；
所述的scFv的氨基酸序列如SEQ ID NO：2所示；
所述的Bi-scFv的氨基酸序列如SEQ ID NO：3所示；
所述的Foldback-scFv的氨基酸序列如SEQ ID NO：4所示。
2.根据权利要求1所述的鼠抗CD3重组免疫毒素，其特征在于：
所述的细菌毒素为白喉毒素、绿脓杆菌外毒素；所述的植物毒素为蓖麻毒素、相思豆毒素、白素毒素。
3.根据权利要求2所述的鼠抗CD3重组免疫毒素，其特征在于：
所述的细菌毒素为DT390，得到的鼠抗CD3重组免疫毒素分别命名为ScFv-CD3-DT390，Bi-CD3-DT390，Foldback-CD3-DT390；
所述的ScFv-CD3-DT390的氨基酸序列如SEQ ID NO：5所示；
所述的Bi-CD3-DT390的氨基酸序列如SEQ ID NO：6所示；
所述的Foldback-CD3-DT390的氨基酸序列如SEQ ID NO：7所示。
4.编码权利要求3所述的鼠抗CD3重组免疫毒素的基因，其特征在于：
所述的编码重组免疫毒素ScFv-CD3-DT390的基因如SEQ ID NO：8所示。
5.编码权利要求3所述的鼠抗CD3重组免疫毒素的基因，其特征在于：
所述的重组免疫毒素Bi-CD3-DT390的基因如SEQ ID NO：9所示。
6.编码权利要求3所述的鼠抗CD3重组免疫毒素的基因，其特征在于：
所述的重组免疫毒素Foldback-CD3-DT390的基因如SEQ ID NO：10所示。
7.含权利要求4、5或6所述的基因的载体。
8.含有权利要求7所述的载体的宿主细胞。
9.权利要求3所述的鼠抗CD3重组免疫毒素的制备方法，其特征在于包括如下步骤：
(1)对权利要求3所述的鼠抗CD3重组免疫毒素的氨基酸序列分别进行优化，设计了如SEQ ID NO：8～SEQ ID NO：10所述的基因，另外在5′端引入KpnI酶切位点以及引入EK识别位点-(Asp)4Lys编码序列并设计上起始密码子ATG；在3′端设计上6个组氨酸的密码子CGTCGTCGTCGTCGTCGT、终止密码子TAATGA并引入XhoI酶切位点；
(2)目的基因采用pET-30a(+)表达载体进行构建重组表达载体；
(3)将重组表达载体转化宿主菌后诱导表达、纯化，获得鼠抗CD3重组免疫毒素。
10.权利要求1、2或3所述的鼠抗CD3重组免疫毒素、权利要求4、5或6所述的基因、权利要求7所述的载体、权利要求8所述的宿主细胞在制备小鼠T淋巴细胞剔除、自身性免疫疾病、器官移植、免疫耐受及肿瘤治疗的药物中的应用。
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