CN110028588A - 抗原-Fc融合蛋白及其检测阳性CAR-T细胞的应用 - Google Patents

抗原-Fc融合蛋白及其检测阳性CAR-T细胞的应用 Download PDF

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CN110028588A
CN110028588A CN201810026940.6A CN201810026940A CN110028588A CN 110028588 A CN110028588 A CN 110028588A CN 201810026940 A CN201810026940 A CN 201810026940A CN 110028588 A CN110028588 A CN 110028588A
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金华君
杨欢
黄晨
刘祥箴
江芏青
何周
师传胤
钱其军
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Shanghai Cell Therapy Research Institute
Shanghai Cell Therapy Group Co Ltd
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Shanghai Cell Therapy Group Co Ltd
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Abstract

本发明提供了一种抗原‑Fc融合蛋白及其检测阳性CAR‑T细胞的应用。该抗原‑Fc融合蛋白含有突变的Fc片段，其氨基酸序列如SEQ ID NO:1所示。本发明还提供了带标记物的抗原‑Fc融合蛋白、其编码核酸、含有其的核酸构建体、其制备方法、含有其的试剂盒和阳性CAR‑T细胞的检测方法。带标记物的抗原‑Fc融合蛋白包含该抗原‑Fc融合蛋白和标记物。该阳性CAR‑T细胞的检测方法为将带标记物的抗原‑Fc融合蛋白与待检的CAR‑T细胞孵育、洗涤后检测。本发明带标记的抗原‑Fc融合蛋白不与T细胞γ‑2受体结合，可与阳性CAR‑T细胞表面受体有效结合，流式检测阳性率重复性好、准确性高、特异性强，操作简便。

Description

抗原-Fc融合蛋白及其检测阳性CAR-T细胞的应用
技术领域
本发明涉及生物技术领域，具体地涉及一种抗原-Fc融合蛋白及其检测阳性CAR-T细胞的应用。
背景技术
癌症是人类高发性恶性疾病，在全球范围内死亡率排名首位。癌症病人体内的肿瘤细胞能够通过“免疫逃逸”来对付T细胞的杀伤和清除作用，具体是由于肿瘤细胞表面MHC-I类分子的表达降低，使之不能与T细胞表面抗原形成复合物，进而不能被T细胞受体识别。T细胞激活过程的复杂调控和肿瘤细胞的免疫逃逸机制大大限制了T细胞杀伤肿瘤细胞的能力。
嵌合抗原受体T(CAR-T)细胞是对T细胞进行基因修饰改造，使其表达嵌合抗原受体(CAR)，进而识别肿瘤细胞进行精准杀伤的治疗手段。相对于目前临床使用的放疗、化疗而言，CAR-T细胞治疗具有毒副作用小、治疗效果佳的优点(Jaspers JE,等.PharmacolTher,2017,doi:10.1016/j.pharmthera.2017.03.012；Grupp SA,等.N Engl J Med,2013,368:1509-1518)。CAR-T细胞阳性率的检测是CAR-T细胞生产过程中的重要质控步骤，现有的可用于检测CAR-T细胞阳性率检测的方法有羊抗人IgG F(ab)2-FITC法、Protein L+链亲和素-PE法以及生物素标记的抗原-Fc融合蛋白法。与羊抗人IgG F(ab)2-FITC法和ProteinL+链亲和素-PE法相比，生物素标记的抗原-Fc融合蛋白法稳定性更高，重复性更好，但生物素标记的抗原-Fc融合蛋白容易与测试样品中T细胞上存在的少量γ-2受体产生非特异性结合，导致最终测定结果包含部分假阳性结果。
因此，目前本领域尚缺少一种能够稳定、重复性高同时又精准地测定CAR-T细胞阳性率的方法和试剂。
发明内容
本发明所要解决的技术问题是为了克服目前本领域已有的抗原-Fc融合蛋白检测阳性CAR-T细胞的方法中天然的Fc片段与T细胞中存在的少量γ-2受体产生非特异性结合，进而导致最终检测结果中包含部分假阳性结果的技术问题，提供了一种新的抗原-Fc融合蛋白。本发明的新的抗原-Fc融合蛋白中含有的Fc片段为包含CH2区缺失的Fc片段，稳定性好、不与T细胞上存在的γ-2受体产生非特异性结合，有效地减少了检测阳性CAR-T细胞过程中出现的假阳性与假阴性结果。同时本发明的新的抗原-Fc融合蛋白的突变Fc片段部分还带有标记物，使阳性CAR-T细胞能有效地被标记，进而可以进行后续的分析或分选操作。
具体地，本发明通过以下技术方案来解决本发明所要解决的技术问题。
本发明一方面提供了一种Fc片段突变体，其序列为SEQ ID NO:1所示序列。
本发明另一方面提供了前述Fc片段突变体的编码核酸序列及其互补序列。优选地，所述编码核酸序列为SEQ ID NO:2所示序列。
本发明另一方面提供了一种抗原-Fc融合蛋白，其包含抗原和含有突变的Fc片段。
在一个或多个实施方案种，所述抗原为全长蛋白分子或细胞跨膜蛋白的胞外结构域。
在一个或多个实施方案中，所述抗原为疾病相关的蛋白质抗原，针对所述疾病相关的蛋白质抗原具有特异性靶向的抗原结合分子。优选地，所述抗原结合分子为抗体。
在一个或多个实施方案中，所述疾病相关的蛋白质抗原为肿瘤相关抗原(TAA)。
在一个或多个实施方案种，所述肿瘤相关抗原任选地为选自ErbB2(HER2/neu)、癌胚抗原(CEA)、上皮细胞黏附分子(EpCAM)、表皮生长因子受体(EGFR、EGFR变体III(EGFRvIII))、CD19、CD20、CD30、CD40、双唾液酸神经节苷脂GD2、GD3、C型凝集素样分子-1(CLL-1)、导管上皮粘蛋白、gp36、TAG-72、鞘糖脂、胶质瘤相关抗原、β-人绒毛膜促性腺激素、甲胎蛋白(AFP)、凝集素反应性AFP、甲状腺球蛋白、RAGE-1、MN-CA IX、人端粒酶逆转录酶、RU1、RU2(AS)、肠羧基酯酶、mut hsp70-2、M-CSF、前列腺酶、前列腺酶特异性抗原(PSA)、PAP、NY-ESO-1、LAGA-1a、p53、前列腺蛋白、PSMA、存活蛋白和端粒酶、前列腺癌肿瘤抗原-1(PCTA-1)、MAGE、ELF2M、中性粒细胞弹性蛋白酶、肝配蛋白B2、CD22、胰岛素生长因子(IGF1)-I、IGF-II、IGFI受体、间皮素(MSLN)、呈递肿瘤特异性肽表位的主要组织相容性复合物(MHC)分子、5T4、ROR1、Nkp30、NKG2D、肿瘤基质抗原、纤连蛋白的额外结构域A(EDA)和额外结构域B(EDB)以及肌腱蛋白-C的A1结构域(TnC A1)以及成纤维细胞相关蛋白(fap)、LRP6、黑素瘤相关硫酸软骨素蛋白聚糖(MCSP)、CD38/CS1、MART1、WT1、MUC1、LMP2、独特型、NY-ESO-1、Ras突变体、gp100、蛋白酶3、bcr-abl、酪氨酸酶、hTERT、EphA2、ML-TAP、ERG、NA17、PAX3、ALK、雄激素受体；谱系特异性或组织特异性抗原如CD3、CD4、CD8、CD24、CD25、CD33、CD34、CD70、CD79、CD116、CD117、CD135、CD123、CD133、CD138、CTLA-4、B7-1(CD80)、B7-2(CD86)、内皮糖蛋白、主要组织相容性复合物(MHC)分子、BCMA(CD269、TNFRSF 17)和FLT-3中任一种。优选地为选自CD19、MSLN、Muc1和EGFR中的任一种。更优选地，为选自CD19的胞外结构域、MSLN的胞外结构域、Muc1的胞外结构域和EGFR的胞外结构域中任一种。
在一个或多个实施方案种，所述Fc片段为IgG1、IgG2、IgG3或IgG4上的Fc片段。优选地，为IgG4Fc片段。
在一个或多个实施方案种，所述突变是替换突变、缺失突变或插入突变。优选地，所述突变是IgG4Fc片段的CH2区缺失的突变。
在一个或多个实施方案中，所述含有突变的Fc片段的氨基酸序列为SEQ ID NO:1所示的序列。
在一个或多个实施方案中，所述抗原位于N端，所述含有突变的Fc片段位于C端。
在一个或多个实施方案中，所述抗原位于C端，所述含有突变的Fc片段位于N端。
在一个或多个实施方案中，所述抗原-Fc融合蛋白还包含接头，所述接头两端分别连接所述抗原和所述含有突变的Fc片段。优选地，所述接头为IgG分子铰链区。更优选地，所述接头为IgG4铰链区。
在一个或多个实施方案中，所述抗原位于所述接头的N端，所述含有突变的Fc片段位于所述接头的C端。
在一个或多个实施方案中，所述抗原位于所述接头的C端，所述含有突变的Fc片段位于所述接头的N端。
在一个或多个实施方案中，所述抗原-Fc融合蛋白还包含分泌型信号肽，所述分泌型信号肽位于位于所述抗原-Fc融合蛋白的N端。优选地，所述分泌型信号肽为轻链信号肽，更优选地，其序列如SEQ ID NO:3所示
在一个或多个实施方案中，所述抗原-Fc融合蛋白的氨基酸序列为选自SEQ IDNO:4、SEQ ID NO:6、SEQ ID NO：8和SEQ ID NO:10中的任一种。
在一个或多个实施方案中，所述抗原-Fc融合蛋白的N端或C端还含有一个或多个标签。优选地，所述标签为选自FLAG、HA、HA1、c-Myc、Poly-His、Poly-Arg、Strep-TagII、AU1、EE、T7、4A6、ε、B、gE和Ty1中的一种或多种。
本发明另一方面提供了编码前述抗原-Fc融合蛋白的分离的核酸，其编码所述抗原-Fc融合蛋白，其是DNA或RNA。
在一个或多个实施方案中，所述分离的核酸为单链DNA或双链DNA，优选地为双链DNA。
在一个或多个实施方案中，所述DNA的序列为SEQ ID NO:5、SEQ ID NO:7、SEQ IDNO:9或SEQ ID NO:11所示序列。
在一个或多个实施方案中，所述分离的核酸为未经修饰的RNA或经过化学修饰的RNA。
在一个或多个实施方案中，所述RNA的序列为SEQ ID NO:12、SEQ ID NO:13、SEQID NO:14或SEQ ID NO:15所示序列。
在一个或多个实施方案中，所述经过化学修饰的RNA上的化学修饰包括核糖环修饰，磷酸二酯键骨架修饰和碱基修饰。优选地，所述核糖环修饰修饰为2’-氧烷基修饰和2’-F修饰修饰。优选地，所述磷酸二酯键骨架修饰为锁核酸(LNA)修饰。优选地，所述碱基修饰为4-硫脲嘧啶修饰、2-硫脲嘧啶修饰和C-连接的伪尿嘧啶修饰。
在一个或多个实施方案中，所述经过化学修饰的RNA包含一种或多种上述化学修饰。
本发明另一方面提供了一种核酸构建体，其含有编码前述抗原-Fc融合蛋白的DNA序列和与所述含有编码前述抗原-Fc融合蛋白的DNA序列可操作地连接的调控序列。
在一个或多个实施方案中，所述调控序列是启动子，优选地，所述调控序列是CMV启动子。
在一个或多个实施方案中，所述调控序列还包括转录终止序列，所述转录终止序列与编码前述抗原-Fc融合蛋白的DNA序列的3’末端可操作地连接。优选地，所述转录终止序列为SV40-polyA转录终止序列。
在一个或多个实施方案中，所述调控序列还包括增强子。优选地，所述增强子位于所述启动子的上游。优选地所述增强子为选自mCMV增强子、hCMV增强子、EF1a启动子的CCEF增强子、腺病毒主要晚期蛋白增强子、CD3e增强子和SV40增强子中的任一种。更优选地，所述增强子为hCMV增强子或mCMV增强子。最优选地，所述增强子为hCMV增强子。
在一个或多个实施方案中，所述核酸构建体是载体。优选地，所述核酸构建体是表达载体。更优选地，所述核酸构建体是真核表达载体。
在一个或多个实施方案中，所述核酸构建体是真核表达载体，所述真核表达载体是含有编码前述抗原-Fc融合蛋白的DNA序列的pCDNA3.4载体。优选地，所述编码前述抗原-Fc融合蛋白的DNA序列为选自SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9和SEQ ID NO:11中任一种。更优选地，所述编码前述抗原-Fc融合蛋白的DNA序列为选自SEQ ID NO:21、SEQ IDNO:23、SEQ ID NO:25和SEQ ID NO:27中任一种。
本发明另一方面提供了前述抗原-Fc融合蛋白的制备方法，其包括化学合成前述抗原-Fc融合蛋白的氨基酸序列或用含有编码前述抗原-Fc融合蛋白的DNA的表达载体表达前述抗原-Fc融合蛋白。
在一个或多个实施方案中，所述制备方法为用含有编码前述抗原-Fc融合蛋白的DNA的表达载体表达前述抗原-Fc融合蛋白。优选地，所述表达载体为真核表达载体。更优选地，所述表达载体为pCDNA3.4载体。
在一个或多个实施方案中，编码所述抗原-Fc融合蛋白的DNA序列为编码肿瘤相关抗原-Fc融合蛋白的DNA序列。优选地，为编码肿瘤相关抗原胞外结构域-Fc融合蛋白的DNA序列。更优选地，为编码CD19胞外结构域-Fc、间皮素胞外结构域-Fc、Muc1胞外结构域-Fc或EGFR胞外结构域-Fc融合蛋白的DNA序列。
在一个或多个实施方案中，所述表达载体为pCDNA3.4载体，所述pCDNA3.4载体包含的编码所述抗原-Fc融合蛋白的DNA序列为选自SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9和SEQ ID NO:11中任一种。更优选地，所述pCDNA3.4载体包含的编码所述所述抗原-Fc融合蛋白的DNA序列为选自SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25和SEQ ID NO:27中任一种。
在一个或多个实施方案中，所述制备方法还包括纯化过程。优选地，所述纯化过程包括选自盐析、亲和层析、离子交换层析、分子筛层析和吸附层析中的一种或多种，任选地，所述纯化过程为亲和层析。
在一个或多个实施方案中，所述纯化为亲和层析。优选地，所述亲和层析为基于Fc与Protein A或Protein G之间的相互作用的亲和层析。
本发明另一方面提供了一种带标记物的抗原-Fc融合蛋白，其包含前述抗原-Fc融合蛋白和与其相连接的标记物。
在一个或多个实施方案中，所述标记物为荧光基团或化学发光剂。
在一个或多个实施方案中，所述荧光基团为选自FITC、TRITC、PE、FAM、RRX、TR、Cy2、Cy3、Cy5、ECD、PC5.5、PC7、APC、APC-A70、APC-A75、Pac-Blue、Alexa488、mBBr、5-IAF、E-118、DTAF、罗丹明绿和KrO中的任一种。任选地，所述荧光基团为PE荧光素。
在一个或多个实施方案中，所述标记物通过共价结合方式或非共价结合方式与前述抗原-Fc融合蛋白相连接。优选地，所述标记物通过非共价结合方式与前述抗原-Fc融合蛋白相连接。
在一个或多个实施方案中，所述非共价结合方式为含有配体与受体相互作用的非共价结合方式。优选地，所述标记物与配体共价相连接，前述抗原-Fc融合蛋白与受体共价相连接，所述标记物通过受体-配体间的非共价相互作用与前述抗原-Fc融合蛋白相连接。
在一个或多个实施方案中，所述标记物与链亲合素共价相连接，前述抗原-Fc融合蛋白与生物素共价相连接，所述标记物通过链亲合素-生物素间的非共价相互作用与前述抗原-Fc融合蛋白相连接。
在一个或多个实施方案中，所述标记物为与链亲合素共价相连接的PE荧光素，前述抗原-Fc融合蛋白上的赖氨酸与生物素共价相连接，所述PE荧光素通过链亲合素-生物素间的非共价相互作用与前述抗原-Fc融合蛋白相连接。
在一个或多个实施方案中，所述带标记物的抗原-Fc融合蛋白为选自带PE荧光素标记的CD19胞外结构域-Fc融合蛋白、带PE荧光素标记的MSLN胞外结构域-Fc融合蛋白、带PE荧光素标记的Muc1胞外结构域-Fc融合蛋白和带PE荧光素标记的CD19胞外结构域-Fc融合蛋白中的一种或多种。
在一个或多个实施方案中，所述带标记物的抗原-Fc融合蛋白为选自带PE荧光素标记的序列为SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8和SEQ ID NO:10蛋白中的一种或多种，所述PE荧光素与链亲合素共价地相连接，所述带标记物的抗原-Fc融合蛋白的肽链与生物素共价地相连接，所述PE荧光素与所述肽链通过链亲合素与生物素的相互作用非共价地相连接。
本发明另一方面提供了前述带标记物的抗原-Fc融合蛋白的制备方法，其包括将标记物与前述抗原-Fc融合蛋白相连接。
在一个或多个实施方案中，所述制备方法包括将所述标记物与前述抗原-Fc融合蛋白共价地相连接或将所述标记物与前述抗原-Fc融合蛋白非共价地相连接。
在一个或多个实施方案中，所述制备方法包括将所述标记物通过链亲合素-生物素间的非共价相互作用与前述抗原-Fc融合蛋白相连接。
在一个或多个实施方案中，所述制备方法包括将所述标记物与链亲合素共价相连接，将前述抗原-Fc融合蛋白与生物素共价相连接，通过链亲合素-生物素间的非共价相互作用将所述标记物与前述抗原-Fc融合蛋白非共价相连接。优选地，所述标记物为荧光基团或化学发光剂，更优选地为荧光基团，任选地位PE荧光素。优选地，前述抗原-Fc融合蛋白为选自CD19胞外结构域-Fc融合蛋白、MSLN胞外结构域-Fc融合蛋白、Muc1胞外结构域-Fc融合蛋白和CD19胞外结构域-Fc融合蛋白中的任一种，更优选地，前述抗原-Fc融合蛋白的氨基酸序列为选自SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8和SEQ ID NO:10中的任一种。
本发明另一方面提供了一种检测阳性CAR-T细胞的试剂盒，其含有前述抗原-Fc融合蛋白、前述编码所述抗原-Fc融合蛋白的分离的核酸、前述核酸构建体和前述带标记物的抗原-Fc融合蛋白中的一种或多种。
在一个或多个实施方案中，所述检测阳性CAR-T细胞的试剂盒包括前述带标记物的抗原-Fc融合蛋白。优选地，包括带PE荧光素标记的抗原-Fc融合蛋白。
在一个或多个实施方案中，所述检测阳性CAR-T细胞的试剂盒包括带PE荧光素标记的CD19胞外结构域-Fc融合蛋白、带PE荧光素标记的MSLN胞外结构域-Fc融合蛋白、带PE荧光素标记的Muc1胞外结构域-Fc融合蛋白和带PE荧光素标记的CD19胞外结构域-Fc融合蛋白中的一种或多种。优选地，包括带PE荧光素标记的序列为SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8和SEQ ID NO:10蛋白中的一种或多种。
在一个或多个实施方案中，所述检测阳性CAR-T细胞的试剂盒还包括标记物、配体和受体中的一种或多种，所述配体与所述标记物共价相连接，所述受体与所述抗原-Fc融合蛋白共价相连接，所述配体与所述受体非共价相连接。优选地，所述标记物为荧光基团或化学发光剂。更优选地，所述标记物为荧光基团，所述荧光基团包括选自FITC、TRITC、PE、FAM、RRX、TR、Cy2、Cy3、Cy5、ECD、PC5.5、PC7、APC、APC-A70、APC-A75、Pac-Blue、Alexa488、mBBr、5-IAF、E-118、DTAF、罗丹明绿和KrO中的一种或多种。
在一个或多个实施方案中，所述检测阳性CAR-T细胞的试剂盒还包括链亲合素、PE荧光素和生物素中的一种或多种。
在一个或多个实施方案中，所述检测阳性CAR-T细胞的试剂盒还包括生物素和/或与链亲合素共价地相连接的PE荧光素。
在一个或多个实施方案中，所述检测阳性CAR-T细胞的试剂盒还包括反应试剂，其将所述受体和前述抗原-Fc融合蛋白共价相连接。优选地，所述受体为生物素，所述反应试剂为N-羟基琥珀酰亚胺(NHS)和/或1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)。
本发明另一方面提供了一种阳性CAR-T细胞的检测方法，包括将前述带标记物的抗原-Fc融合蛋白与待检测的CAR-T细胞孵育，洗涤后进行检测。
在一个或多个实施方案中，前述带标记物的抗原-Fc融合蛋白为带PE荧光素标记的抗原-Fc融合蛋白，所述检测为流式细胞检测。优选地，所述带标记物的抗原-Fc融合蛋白为带PE荧光素标记的序列为SEQ ID NO:4、SEQ ID NO:6、SEQ ID NO:8和SEQ ID NO:10蛋白中的一种或多种。
在一个或多个实施方案中，所述孵育的温度为37℃，和/或，所述孵育的时间为1小时；和/或，所述孵育的CO2的浓度为5％。
在一个或多个实施方案中，所述洗涤为将细胞用洗涤缓冲液重悬、离心。优选地，所述离心的转速为1000转/分。优选地，所述离心的时间为3分钟。优选地，所述洗涤的次数为3次。优选地，所述洗涤缓冲液为PBS。
附图说明
以下结合附图说明本发明的特征和有益效果。
图1、PE荧光素标记抗原-Fc融合蛋白检测阳性CAR-T细胞的原理。
图2、PE荧光素标记的CD19、MSLN、Muc1和EGFR的抗原-Fc融合蛋白分别通过流式细胞计数法检测CD19-CAR-T、Meso-CAR-T、Muc1-CAR-T和EGFR-CAR-T细胞阳性率的结果。
图3、PE荧光素标记的抗原-Fc融合蛋白与羊抗人IgG F(ab)2-FITC法和ProteinL-链亲和素-PE法通过流式细胞计数法检测阳性CD19-CAR-T细胞的结果对比。
图4、PE荧光素标记的抗原-Fc融合蛋白与PE荧光素标记的抗原-野生型Fc融合蛋白检测CD19-CAR-T、Meso-CAR-T、Muc1-CAR-T和EGFR-CAR-T细胞阳性率的结果对比。
具体实施方式
下面对本发明涉及的部分术语进行定义。
除非另有注释，本申请依照常规用法使用技术术语。分子生物学中的常用术语的定义可以在Benjamin Lewin,Genes X,published by Jones&Bartlett Publishers,2009；和Meyers et al.(eds.),The Encyclopedia of Cell Biology and MolecularMedicine,published by Wiley-VCH in 16volumes,2008和其它类似参考文献中找到。
在本发明中，术语“抗原”表示在引入体内时，被免疫系统识别的任何物质。例如蛋白质或细胞跨膜蛋白的胞外结构域。
术语"抗体"是指免疫球蛋白分子和免疫球蛋白(Ig)分子的免疫活性部分，即，含特异性结合抗原(与抗原免疫反应)的抗原结合位点的分子。“特异性结合”或“与抗原免疫反应”意指抗体与所需抗原的一个或多个抗原决定簇反应和不与其它多肽反应。抗体包括但不限于多克隆抗体、单克隆抗体、嵌合抗体、dAb(结构域抗体)、单链抗体、Fab、Fab’和F(ab')2片段、scFv和Fab表达文库。
术语“Fc”或“Fc片段”是指抗体的可结晶段(fragment crystallizable,Fc)，指位于抗体分子"Y"结构的柄部末端，包含抗体重链恒定区CH2和CH3结构域的肽段,是抗体与效应分子或者细胞相互作用的部位。
术语“野生型”是指具有如在自然界中发现的呈“正常”(如与突变体、患病者、改变者等形成对比)状态或情形的结构和/或活性的实体。
术语“接头”是连接不同蛋白或多肽之间的多肽片段，其目的是使所连接的蛋白或多肽保持各自的空间构象，以维持蛋白或多肽的功能或活性。示例性的接头包括含有G和/或S的接头，以及IgG的铰链区，如IgG4铰链区。
术语“核酸构建体”是指可以被引入靶细胞或组织中的人工构建的核酸区段。典型地包括DNA构建体，指已被亚克隆进载体中的编码目的蛋白质的核苷酸序列的DNA插入。
术语“载体”是指用于将一个或多个核酸或一个或多个多核苷酸引入或转移到靶细胞或组织中的核酸构建体或多核苷酸构建体。典型地，载体用于将外源DNA引入另一个细胞或组织中。载体可以包含用于在细菌中生长的细菌抗性基因和用于在生物体中表达目的蛋白质的启动子。DNA可以通过PCR或任何其他本领域技术人员已知的一种或多种合适的技术在体外产生。
术语“表达载体”是指重组或合成产生的用于在靶细胞中表达目的核酸的核酸构建体或载体(例如外源核酸或转基因)。典型地，目的核酸表达目的蛋白质。
术语“表达框”是指表达一个基因所需的完整元件，包括启动子、基因编码序列、PolyA加尾信号序列。优选地还包括增强子元件。
术语“嵌合抗原受体”(CAR)是人工改造受体，能够将识别肿瘤细胞表面抗原的特异性分子(如抗体)锚定在免疫细胞(如T细胞)上，使免疫细胞识别肿瘤抗原或病毒抗原和杀死肿瘤细胞或病毒感染的细胞。CAR通常依次包含任选的信号肽、结合肿瘤细胞膜抗原的多肽如单链抗体、铰链区、跨膜区和胞内信号区。通常，结合肿瘤细胞膜抗原的多肽能够以中等亲和力结合肿瘤细胞广泛表达的膜抗原。结合肿瘤细胞膜抗原的多肽可以是天然多肽或人工合成多肽；优选地，人工合成多肽为单链抗体或Fab片段。
术语“阳性CAR-T细胞”是指在嵌合抗原受体(CAR)的表达能够被现有的常规检测手段所检测到的CAR-T细胞。
术语“可操作地连接”是指核酸序列与核苷酸的调节序列(例如启动子、增强子、转录和翻译终止位点以及其他信号序列)的功能关系并且表明两个或更多个DNA片段结合在一起，使得它们为了预期的目的而一致起作用。例如，核酸序列(通常为DNA)与调节序列或启动子区域的有效连接是指DNA与调节序列或启动子之间的物理和功能关系，使得通过特异性识别、结合和转录该DNA的RNA聚合酶从调节序列或启动子启动这种DNA的转录。为了优化表达和/或体外转录，可能需要修饰其所表达的细胞类型中核酸或DNA表达的调节序列。对这种修饰的期望或需要可以根据经验确定。
术语“标记物”是指可通过光谱、光化学、生物化学、免疫化学、化学或其它物理手段检测的物质。例如，包括荧光染料(荧光基团)、化学发光剂、高电子密度试剂、酶(ELISA中常用)、生物素、地高辛、32P、35S或其它同位素、半抗原、以及可被检测的蛋白质(例如通过将放射性标签整合至肽中或用于检测与肽特异性反应的抗体)。该术语包括单一标记试剂或标记试剂的组合，例如，提供独特可检测特征(例如，特定波长或波长组合下)的荧光团的组合。可以采用本领域已知的用于将标记物偶联到需要的试剂的任何方法。
术语“非特异性结合”是指一种分子以非特异的和瞬时的方式与另一种分子相互作用。例如具有总体正电荷的分子与具有总体负电荷的分子的相互作用，然而，缔合作用并不紧密，并且在温和的盐类存在的条件下很可能解离。
在目前现有的几种检测阳性CAR-T细胞的方法中，生物素标记抗原与野生型Fc融合蛋白的方法与其他几种方法相比具有稳定性更高、重复性更好的优点，野生型的Fc片段容易与测试样品中的T细胞上存在的少量γ-2受体产生非特异性结合而导致产生假阳性结果，降低了该方法检测的准确度。本发明人经过大量实验与筛选，获得了一种含有缺失突变的Fc片段，该含有缺失突变的Fc片段与抗原通过接头形成的融合蛋白稳定性好，其用于检测CAR-T细胞时能够消除Fc与T细胞上存在的少量γ-2受体之间形成的非特异性结合，极大地提高了检测的准确度，进而完成了本发明。
因此，本发明提供了一种Fc片段突变体，所述Fc片段突变体的序列为SEQ ID NO:1所示序列。
本发明还提供了前述Fc片段突变体的编码核酸序列或其互补序列。优选地，所述编码核酸序列为SEQ ID NO:2所示序列。
本发明还提供了一种抗原-Fc融合蛋白，其包含抗原和含有突变的Fc片段，不与T细胞上存在的γ-2受体产生非特异性结合。
在某些实施方案中，所述抗原可以是本领域常规所指的抗原，典型地为全长蛋白分子或细胞跨膜蛋白的胞外结构域。
在某些实施方案中，所述抗原为疾病相关的蛋白质抗原，针对所述疾病相关的蛋白质抗原具有特异性靶向的抗原结合分子，如抗体。所述疾病相关的蛋白质抗原优选地为肿瘤相关抗原(TAA)。所述肿瘤相关抗原任选地为选自ErbB2(HER2/neu)、癌胚抗原(CEA)、上皮细胞黏附分子(EpCAM)、表皮生长因子受体(EGFR、EGFR变体III(EGFRvIII))、CD19、CD20、CD30、CD40、双唾液酸神经节苷脂GD2、GD3、C型凝集素样分子-1(CLL-1)、导管上皮粘蛋白、gp36、TAG-72、鞘糖脂、胶质瘤相关抗原、β-人绒毛膜促性腺激素、甲胎蛋白(AFP)、凝集素反应性AFP、甲状腺球蛋白、RAGE-1、MN-CA IX、人端粒酶逆转录酶、RU1、RU2(AS)、肠羧基酯酶、mut hsp70-2、M-CSF、前列腺酶、前列腺酶特异性抗原(PSA)、PAP、NY-ESO-1、LAGA-1a、p53、前列腺蛋白、PSMA、存活蛋白和端粒酶、前列腺癌肿瘤抗原-1(PCTA-1)、MAGE、ELF2M、中性粒细胞弹性蛋白酶、肝配蛋白B2、CD22、胰岛素生长因子(IGF1)-I、IGF-II、IGFI受体、间皮素(MSLN)、呈递肿瘤特异性肽表位的主要组织相容性复合物(MHC)分子、5T4、ROR1、Nkp30、NKG2D、肿瘤基质抗原、纤连蛋白的额外结构域A(EDA)和额外结构域B(EDB)以及肌腱蛋白-C的A1结构域(TnC A1)以及成纤维细胞相关蛋白(fap)、LRP6、黑素瘤相关硫酸软骨素蛋白聚糖(MCSP)、CD38/CS1、MART1、WT1、MUC1、LMP2、独特型、NY-ESO-1、Ras突变体、gp100、蛋白酶3、bcr-abl、酪氨酸酶、hTERT、EphA2、ML-TAP、ERG、NA17、PAX3、ALK、雄激素受体；谱系特异性或组织特异性抗原如CD3、CD4、CD8、CD24、CD25、CD33、CD34、CD70、CD79、CD116、CD117、CD135、CD123、CD133、CD138、CTLA-4、B7-1(CD80)、B7-2(CD86)、内皮糖蛋白、主要组织相容性复合物(MHC)分子、BCMA(CD269、TNFRSF 17)和FLT-3中任一种，优选地为选自CD19、MSLN、Muc1和EGFR中的任一种，更优选地为选自CD19的胞外结构域、MSLN的胞外结构域、Muc1的胞外结构域和EGFR的胞外结构域中任一种。
在某些实施方案中，所述Fc片段可以为本领域常规的IgG上的Fc片段，任选地包括IgG1、IgG2、IgG3或IgG4上的Fc片段，优选地为IgG4上的Fc片段。所述突变任选地可以是替换突变、缺失突变或插入突变，只要此种突变能够使得所述Fc片段与T细胞上存在的少量γ-2受体之间产生的非特异性结合消除，同时又能起到稳定抗原胞外区片段的效果即可。优选地，所述突变是缺失突变，缺失Fc片段的CH2区。优选地，所述突变的Fc片段的氨基酸序列为SEQ ID NO:1所示序列。
在某些实施方案中，所述抗原-Fc融合蛋白的各组成部分在空间上可以是所述抗原位于N端，所述Fc片段位于C端，或所述Fc片段位于N端，所述抗原位于C端，两种中的任一种空间上的排列顺序都能达到本发明的技术效果。优选地，所述抗原位于N端，所述Fc片段位于C端。
在某些实施方案中，所述抗原-Fc融合蛋白还包含接头，所述接头两端分别连接所述抗原和所述含有突变的Fc片段。
在某些实施方案中，所述接头可以为本领域常规地连接两个各自独立形成结构域的接头，包括GS重复的序列和IgG分子铰链区。IgG分子铰链区指免疫球蛋白重链CH1和CH2功能区之间的区域，该区富含脯氨酸，不形成α螺旋，易发生伸展及一定程度扭曲，有利于抗原与Fc片段保持各自的空间构型，发挥各自的功能而不会相互影响。接头的长度可以是3～25个氨基酸残基，例如3～15、5～15、10～20个氨基酸残基。在某些实施方案中，接头序列是多甘氨酸接头序列。接头序列中甘氨酸的数量无特别限制，通常为2～20个，例如2～15、2～10、2～8个。除甘氨酸和丝氨酸来，接头中还可含有其它已知的氨基酸残基，例如丙氨酸(A)、亮氨酸(L)、苏氨酸(T)、谷氨酸(E)、苯丙氨酸(F)、精氨酸(R)、谷氨酰胺(Q)等。在某些实施例中，所述接头优选地为IgG分子铰链区。所述IgG分子铰链区可以为IgG1、IgG2、IgG3或IgG4的铰链区，优选地为IgG4铰链区。
在某些实施方案中，所述抗原-Fc融合蛋白的各组成部分在空间上可以是所述抗原位于所述接头的N端，所述含有突变的Fc片段位于所述接头的C端，或所述含有突变的Fc片段位于所述接头的N端，所述抗原位于所述接头的C端，任意一种空间上的排列顺序都能达到本发明的技术效果。优选地，所述抗原位于所述接头的N端，所述Fc片段位于所述接头的C端。
在某些实施方案中，所述抗原-Fc融合蛋白还可以包含分泌型信号肽，所述分泌型信号肽位于所述抗原-Fc融合蛋白的N端。
在某些实施方案中，所述分泌型信号肽可以为本领域常规的分泌型信号肽，能够提高与其相融合的蛋白分泌到细胞外的水平。优选地，所述分泌型信号肽为轻链信号肽，更优选地，其序列如SEQ ID NO:3所示。
在某些实施方案中，前述抗原-Fc融合蛋白的氨基酸序列为SEQ ID NO:4、SEQ IDNO:6、SEQ ID NO:8或SEQ ID NO:10所示序列。
应理解，在基因克隆操作中，常常需要设计合适的酶切位点，这势必在所表达的氨基酸序列末端引入了一个或多个不相干的残基，而这并不影响目的序列的活性。为了构建融合蛋白、促进重组蛋白的表达、获得自动分泌到宿主细胞外的重组蛋白、或利于重组蛋白的纯化，常常需要将一些氨基酸添加至重组蛋白的N-末端、C-末端或该蛋白内的其它合适区域内，例如，包括但不限于，适合的接头肽、信号肽、前导肽、末端延伸等。因此，本发明的抗原-Fc融合蛋白的氨基端或羧基端还可含有一个或多个标签。任何合适的标签都可以用于本发明。例如，所述的标签可以是FLAG、HA、HA1、c-Myc、Poly-His、Poly-Arg、Strep-TagII、AU1、EE、T7、4A6、ε、B、gE以及Ty1。这些标签可用于对蛋白进行纯化。
本发明还提供了编码前述抗原-Fc融合蛋白的核酸，所述核酸编码所述抗原-Fc融合蛋白，所述核酸是DNA或RNA。
在某些实施方案中，所述DNA包括cDNA或人工合成的DNA。DNA可以是单链DNA或双链DNA。本发明的抗原-Fc融合蛋白DNA形式的核酸通常可以用PCR扩增法获得。具体而言，可根据本发明所公开的核苷酸序列来设计引物，并用市售的cDNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板，扩增得到有关序列。在某些实施方案中，所述抗原-Fc融合蛋白的核酸的序列为SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9或SEQ ID NO:11所示序列。
在某些实施方案中，所述RNA可以是未经修饰的RNA或经过化学修饰的RNA，所述化学修饰的RNA较之未经修饰的RNA更加稳定。所述化学修饰可以为本领域常规的旨在提高RNA稳定性而对RNA进行的修饰，包括核糖环修饰，磷酸二酯键骨架修饰和碱基修饰。所述核糖环修饰包括但不限于2’-氧烷基修饰和2’-F修饰；所述磷酸二酯键骨架修饰包括但不限于锁核酸(LNA)修饰；所述碱基修饰包括但不限于4-硫脲嘧啶修饰、2-硫脲嘧啶修饰和C-连接的伪尿嘧啶修饰。所述化学修饰的RNA任选地含有上述几种化学修饰中的一种或多种。
在某些实施方案中，所述RNA的序列为SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14或SEQ ID NO:15所示序列。
本发明还提供了核酸构建体，其含有编码前述抗原-Fc融合蛋白的DNA序列以及与所述含有编码前述抗原-Fc融合蛋白的DNA序列可操作地连接的调控序列。
在某些实施方案中，所述调控序列可以是合适的启动子。启动子通常与待表达蛋白的编码序列可操作的连接。启动子可以是在所选择的宿主细胞中显示转录活性的任何核苷酸序列，包括突变的、截短的和杂合启动子，并且可以从编码与该宿主细胞同源或异源的胞外或胞内多肽的基因获得。
优选地，所述启动子是即时早期巨细胞病毒(CMV)启动子、PGK启动子、EEF启动子、延伸生长因子-1α(EF-1α)、类人猿病毒40(SV40)早期启动子、小鼠乳癌病毒(MMTV)、人免疫缺陷病毒(HIV)长末端重复(LTR)启动子、MoMuLV启动子、鸟类白血病病毒启动子、EB病毒即时早期启动子、鲁斯氏肉瘤病毒启动子、人基因启动子、beta-actin启动子、肌球蛋白启动子、血红素启动子或肌酸激酶启动子。
在某些实施方案中，所述调控序列可以是合适的转录终止序列，由宿主细胞识别以终止转录的序列。所述转录终止序列与编码该多肽的核苷酸序列的3’末端操作性连接。在选择的宿主细胞中有功能的任何终止序列都可用于本发明。优选地，所述转录终止序列为SV40-polyA转录终止序列。
在某些实施方案中，所述调控序列除启动子外还可以包括增强子，所述增强子可以为本领域常规的增强子，其位于所述启动子的上游或下游，能够提高所述启动子序列在所选择的宿主细胞中的转录效率。所述增强子包括但不限于mCMV增强子、hCMV增强子、EF1a启动子的CCEF增强子、腺病毒主要晚期蛋白增强子、CD3e增强子和SV40增强子，优选地为mCMV增强子、hCMV增强子或SV40增强子，更优选地为hCMV或mCMV增强子，最优选地为hCMV增强子。
在某些实施方案中，所述核酸构建体是载体。具体地，将编码前述抗原-Fc融合蛋白的DNA序列克隆进入多种类型地载体即可获得。这些载体的类型包括但不限于质粒、噬菌粒、噬菌体衍生物、动物病毒和粘粒。优选地，所述载体可以是表达载体。所述表达载体可以为原核表达载体或真核表达载体，包括但不限于pET系列载体、pCDNA系列表达载体、昆虫杆状病毒表达载体和酵母真核表达载体。优选地为pCDNA系列表达载体，如pCDNA3.4表达载体。优选地，所述编码前述抗原-Fc融合蛋白的DNA序列为选自SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9和SEQ ID NO:11中任一种。更优选地，所述编码前述抗原-Fc融合蛋白的DNA序列为选自SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25和SEQ ID NO:27中任一种。
本发明还提供了前述抗原-Fc融合蛋白的制备方法，其包括化学合成前述抗原-Fc融合蛋白的氨基酸序列或用含有编码所述抗原-Fc融合蛋白的DNA的表达载体表达所述抗原-Fc融合蛋白。
在某些实施方案中，所述制备方法优选地为用含有编码所述抗原-Fc融合蛋白的DNA的表达载体表达所述抗原-Fc融合蛋白。所述表达载体可以为原核表达载体或真核表达载体，包括但不限于质粒、噬菌粒、噬菌体衍生物、动物病毒和粘粒。优选地，所述载体可以是表达载体。所述表达载体可以为原核表达载体或真核表达载体，包括但不限于pET系列载体、pCDNA系列表达载体、昆虫杆状病毒表达载体和酵母真核表达载体。优选地为pCDNA系列表达载体，如pCDNA3.4表达载体。在某些实施方案中，编码所述抗原-Fc融合蛋白的DNA的序列为编码肿瘤相关抗原-Fc融合蛋白的DNA序列，优选地为编码肿瘤相关抗原胞外结构域-Fc融合蛋白的DNA序列，任选地为编码CD19胞外结构域-Fc融合蛋白、间皮素胞外结构域-Fc融合蛋白、Muc1胞外结构域-Fc融合蛋白或EGFR胞外结构域-Fc融合蛋白的DNA序列。在某些实施方案中，编码所述抗原-Fc融合蛋白的DNA序列为SEQ ID NO:5、SEQ ID NO:7、SEQ IDNO:9或SEQ ID NO:11。更优选地，所述pCDNA3.4载体包含的编码所述所述抗原-Fc融合蛋白的DNA序列为选自SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25和SEQ ID NO:27中任一种。
在某些实施方案中，所述制备方法优选地还包括纯化过程，将原核或真核表达体系中表达得到的前述抗原-Fc融合蛋白从细胞裂解液中分离。所述纯化过程可以为本领域常规的蛋白纯化过程，包括但不限于盐析、亲和层析、离子交换层析、分子筛层析和吸附层析，优选地为亲和层析，更优选地为基于Fc与Protein A或Fc与Protein G之间相互作用的亲和层析。
本发明还提供了带标记物的抗原-Fc融合蛋白，其包含前述抗原-Fc融合蛋白和与其相连接的标记物。
所述标记物可以为本领域常规的标记物，指可通过光谱、光化学、生物化学、免疫化学、化学或其它物理手段检测的物质。例如，包括荧光基团、化学发光剂、高电子密度试剂、酶(ELISA中常用)、生物素、地高辛、32P或其它同位素、半抗原、以及可被检测的蛋白质(例如通过将放射性标签整合至肽中或用于检测与肽特异性反应的抗体)，优选地为荧光基团或化学发光剂。在某些实施方案中，所述标记物优选地为荧光基团，所述荧光基团可以为本领域常规的荧光基团，包括但不限于FITC、TRITC、PE、FAM、RRX、TR、Cy2、Cy3、Cy5、ECD、PC5.5、PC7、APC、APC-A70、APC-A75、Pac-Blue、Alexa488、mBBr、5-IAF、E-118、DTAF、罗丹明绿和KrO，任选地为PE。
所述标记物可以通过共价结合方式或非共价结合方式与前述抗原-Fc融合蛋白相连接。所述共价结合方式通常是利用荧光基团上的特定基团与蛋白质肽链侧链上的基团发生化学反应从而形成共价键，例如FITC上的N＝C＝S基团与蛋白质肽链上游离的-NH2可以发生反应，又如与-SH反应的马来酰亚胺活化的荧光素，如荧光素-5-马来酰亚胺。所述非共价结合方式包括含有配体与受体相互作用的结合方式，即在待标记蛋白上引入受体，与所述受体的配体共价相连接的标记物通过配体-受体相互作用标记上带标记蛋白。例如六组氨酸标签-镍-荧光团络合物体系，在待标记的蛋白序列内引入6组氨酸标签，Cy3或Cy5荧光团通过两个连接臂经2个镍离子与组氨酸标签通过强配位键络合，实现标记；再例如将生物素与待标记蛋白发生反应，通过赖氨酸共价偶联，与链亲合素共价结合的标记物即可通过链亲合素-生物素间的强非共价相互作用标记待标记的蛋白。
在某些实施方案中，所述标记物通过非共价结合的方式与前述抗原-Fc融合蛋白相连接，优选地，所述标记物与配体共价相连接，前述抗原-Fc融合蛋白与受体共价相连接，所述标记物通过受体-配体间的非共价相互作用与前述抗原-Fc融合蛋白相连接。在某些实施方案中，所述标志物与链亲合素共价相连接，前述抗原-Fc融合蛋白上的赖氨酸与生物素共价相连接，所述标记物通过链亲合素-生物素间的非共价相互作用与前述抗原-Fc融合蛋白相连接。优选地，所述标记物为与链亲合素共价相连接的PE荧光素，前述抗原-Fc融合蛋白上的赖氨酸与生物素共价相连接，PE通过链亲合素-生物素间的非共价相互作用与前述抗原-Fc融合蛋白相连接。
在某些实施方案中，所述带标记物的抗原-Fc融合蛋白中的抗原为选自CD19的胞外结构域、MSLN的胞外结构域、Muc1的胞外结构域和EGFR的胞外结构域中的任一种，所述Fc的氨基酸序列如SEQ ID NO:1所示，所述抗原与所述Fc通过IgG4铰链区共价相连接。优选地，所述带标记物的抗原-Fc融合蛋白的氨基酸序列为选自SEQ ID NO:4、SEQ ID NO:6、SEQID NO:8和SEQ ID NO:10中的任一种，其与生物素共价相连接；所述标记物为PE荧光素，其与链亲合素共价相连接，与生物素共价相连接的氨基酸序列和与链亲合素共价相连接的PE荧光素通过生物素与链亲合素之间的非共价结合方式相连接。
本发明还提供了前述带标记物的抗原-Fc融合蛋白的制备方法，其包括将所述标记物与前述抗原-Fc融合蛋白相连接。
所述制备方法中，可以将所述标记物与前述抗原-Fc融合蛋白共价地或非共价地相连接，优选地，将两者非共价地连接。在某些实施方案中，将所述标记物通过链亲合素-生物素间的非共价相互作用与前述抗原-Fc融合蛋白相连接。优选地，将所述标记物与链亲合素共价相连，将前述抗原-Fc融合蛋白与生物素共价偶联，通过链亲合素-生物素间的非共价相互作用将标记物与前述抗原-Fc融合蛋白相连。生物素与蛋白分子共价偶联的方法可以通过本领域常规的缩合反应进行，在加入N-羟基琥珀酰亚胺(NHS)与1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)的条件下，生物素的戊酸侧链羧基可与蛋白的ε-氨基缩合形成酰胺键，进而与蛋白质分子肽链中的赖氨酸共价地相连接。应理解，由于在二十种常规氨基酸中只有赖氨酸具有ε-氨基，理论上肽链中的每个赖氨酸都有可能与生物素共价相连。在某些实施方案中，将PE荧光素与链亲合素共价相连，将前述抗原-Fc融合蛋白与生物素共价偶联，通过链亲合素-生物素间的非共价相互作用将PE荧光素与前述抗原-Fc融合蛋白相连。
在某些实施方案中，所述带标记物的抗原-Fc融合蛋白的抗原为选自CD19的胞外结构域、MSLN的胞外结构域、Muc1的胞外结构域和EGFR的胞外结构域中的任一种，所述Fc的氨基酸序列如SEQ ID NO:1所示，所述抗原与所述Fc通过IgG4铰链区共价相连接。优选地，所述带标记物的抗原-Fc融合蛋白的氨基酸序列为选自SEQ ID NO:4、SEQ ID NO:6、SEQ IDNO:8和SEQ ID NO:10中的任一种，其与生物素共价相连接；所述标记物为PE荧光素，其与链亲合素共价相连接。所述制备方法包括将前述抗原-Fc融合蛋白与所述标记物通过生物素-链亲合素非共价相互作用相连接。
本发明还提供了检测阳性CAR-T细胞的试剂盒，其含有前述抗原-Fc融合蛋白、前述核酸构建体和前述带标记物的抗原-Fc融合蛋白中的一种或多种。
在某些实施方案中，所述检测阳性CAR-T细胞的试剂盒还可以包括标记物、配体和受体中的一种或多种。所述标记物可以为本领域常规的标记物，包括荧光基团、化学发光剂、高电子密度试剂、酶(ELISA中常用)、生物素、地高辛、35S、32P或其它同位素、半抗原、以及可被检测的蛋白质(例如通过将放射性标签整合至肽中或用于检测与肽特异性反应的抗体)，优选地为荧光基团或化学发光剂。在某些实施方案中，所述标记物优选地为荧光基团，所述荧光基团可以为本领域常规的荧光基团，包括但不限于FITC、TRITC、PE、FAM、RRX、TR、Cy2、Cy3、Cy5、ECD、PC5.5、PC7、APC、APC-A70、APC-A75、Pac-Blue、Alexa488、mBBr、5-IAF、E-118、DTAF、罗丹明绿和KrO，任选地为PE。所述配体可以与所述标记物共价相连。优选地，所述配体为链亲合素。优选地，所述受体为生物素。
在某些实施方案中，所述检测阳性CAR-T细胞的试剂盒还包括与链亲合素共价地相连接的PE荧光素。
在某些实施方案中，所述检测阳性CAR-T细胞的试剂盒还可以包括将所述受体和前述抗原-Fc融合蛋白共价相连的反应试剂。所述受体可以在加入所述反应试剂的条件下与前述抗原-Fc融合蛋白发生化学反应进而与其共价相连。优选地，所述反应试剂为生物素与蛋白质肽链中的ε-氨基发生缩合反应所需要的试剂，如N-羟基琥珀酰亚胺(NHS)和/或1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)。
本发明还提供了阳性CAR-T细胞的检测方法，包括将前述带标记物的抗原-Fc融合蛋白与待检测的CAR-T细胞孵育，洗涤后进行检测。
所述检测可以为本领域常规的检测方法，与不同的标记物种类相适应。可以包括但不限于流式细胞检测、Western blot、ELISA和同位素标记的检测方法如X射线放射性自显影或磷屏感应。在某些实施方案中，前述带标记物的抗原-Fc融合蛋白的标记物为荧光基团，任选地为PE，所述检测为流式细胞检测。
在某些实施方案中，所述孵育可以为本领域常规的孵育操作，优选地，所述孵育的温度为37℃。优选地，所述孵育的时间为1小时。
在某些实施方案中，所述洗涤可以为本领域常规的洗涤操作，包括将细胞用洗涤缓冲液重悬、离心。所述洗涤旨在去除游离的、未与生物素结合的PE荧光素。优选地，所述离心的转速为1000rpm。优选地，所述离心的时间为3min。优选地，所述洗涤的次数为3次。优选地，所述洗涤缓冲液为PBS。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明的积极进步效果在于：本发明将抗原Fc段进行CH2区缺失突变，得到的生物素标记-突变抗原不与γ-2受体结合，但可以与CAR-T阳性细胞表面相应的CAR受体有效结合，流式检测CAR T细胞阳性率重复性好、准确性高、特异性强，操作也相对简便。
下面将结合实施案例对本发明所涉及的实施方案进行详细描述。本领域技术人员将会理解，下面的实施案例仅用于说明本发明，而不应视为限定本发明的范围。实施案例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或者按照产品说明书进行。
流式细胞仪购自Beckman-Coult，型号为FC500-MCL；
CD19羊抗人IgG F(ab)2–FITC多克隆抗体购自Jackson Immuno Research公司，货号为：109-095-006。
带PE荧光素标记的链亲合素：购自上海金斯瑞生物科技有限公司；
Lenti-XTM表达系统：购自Clontech公司，货号：631253；
T cell TransActTM试剂盒：购自Miltenyi生物，货号：130-111-160
ExpiCHOTM表达系统：购自Thermo Fisher公司，货号：A29133
MabSelect亲和层析树脂：购自GE Healthcare，货号：17519902
DNA序列合成：上海捷瑞生物工程有限公司
以下实施例中所使用的CAR-T细胞按下述方法制得：
1)采用Ficoll差速离心法从四个不同供体的外周血PBMC中分离得到四类不同的人初始T细胞，分别命名为T1、T2、T3和T4；
2)合成分别靶向CD19、MSLN、Muc1和EGFR的嵌合抗原受体(CAR)的DNA编码序列，分别如SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18和SEQ ID NO:19所示，在上述序列的5’端和3’端分别连接EcoRI与XbaI酶切位点连接头，连接至Lenti-XTM表达系统中的pLVX-EF1a-IRES-Puro载体上，得到表达靶向CD19、MSLN、Muc1和EGFR的CAR的慢病毒表达载体；
3)按Lenti-XTM表达系统说明书操作，包装获得表达靶向CD19、MSLN、Muc1和EGFR的CAR的慢病毒；使用pLVX-EF1a-IRES-Puro空载，包装获得负对照慢病毒；按Naldini L,Blomer U,Gallay P,Ory D,Mulligan R,Gage FH,Verma IM,Trono D:In vivo genedelivery and stable transduction of nondividing cells by a lentiviralvector.Science 1996,272:263–267一文的方法纯化、浓缩所获得慢病毒；
4)按MOI＝50-100，用3)中获得的分别表达靶向CD19、MSLN、Muc1和EGFR的CAR的慢病毒分别转导1)中得到的T1、T2、T3和T4细胞，制成CAR-T细胞；用3)中获得负对照慢病毒分别转导1)中得到的T1、T2、T3和T4细胞，制成各CAR-T细胞各自的对照T细胞(Control T1、Control T2、Control T3、Control T4)；
5)对步骤4)中获得的各CAR-T细胞及各自的对照T细胞使用T cell TransActTM试剂盒(Miltenyi生物)根据说明书的操作步骤进行刺激扩增、培养。
最终获得的靶向CD19、MSLN、Muc1和EGFR的CAR-T细胞以下分别命名为CD19-CAR-T、Meso-CAR-T、Muc1-CAR-T和EGFR-CAR-T。
以下实施例中，各缩略词组所代表的意思如下所定义：
Ag-muFc：抗原与CH2区缺失的IgG4Fc片段融合蛋白的概括表述。具体的可以为CD19-muFc、MSLN-muFc、Muc1-muFc或EGFR-muFc；
Ag-Fc：抗原与野生型IgG4Fc片段融合蛋白的概括表述。具体的可以为CD19-wtFc、MSLN-wtFc、Muc1-wtFc或EGFR-wtFc；
EGFR-IFc：不含有任何Fc片段的EGFR胞外结构域。
PE-Ag-muFc：PE标记的Ag-muFc；
PE-Ag-Fc：PE标记的Ag-Fc；
PE-EGFR-IFc：PE标记的EGFR-IFc。
所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1抗原-Fc融合蛋白及对照蛋白的制备
1、选取CD19、MSLN、Muc1与EGFR四个基因表达的蛋白的胞外区与IgG4铰链区及含有CH2区域缺失的IgG4Fc片段构建形成融合蛋白。具体地，合成序列为SEQ ID NO:21、SEQID NO:23、SEQ ID NO:25和SEQ ID NO:27所示的DNA片段及其反向互补片段，复性形成双链DNA，上述4段DNA序列分别在序列SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9和SEQ ID NO:11的5’端增加了轻链信号肽的编码DNA序列，表达氨基酸序列分别为SEQ ID NO:20、SEQ IDNO:22、SEQ ID NO:24和SEQ ID NO:26所示的N端含有轻链信号肽的四种抗原-Fc融合蛋白，旨在使得这四种融合蛋白能够被有效分泌到真核细胞外，方便后续纯化。用末端转移酶(TdT)在序列为SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25和SEQ ID NO:27的双链DNA的两条链的5’端加上A后，连接到pCDNA3.4载体上(pCDNA3.4载体购自Thermo Fisher，货号：A14697)，构建得到过表达CD19、MSLN、Muc1与EGFR胞外结构域与CH2区缺失的IgG4Fc片段融合蛋白的表达载体。根据ExpiCHOTM表达系统说明书，使用ExpiCHOTM表达系统对上述融合蛋白进行过表达后，使用GE Healthcare的MabSelect亲和层析树脂按说明书操作步骤对表达产物进行纯化。所制得的四种蛋白的胞外区与缺失CH2的IgG4Fc片段所形成的抗原-Fc融合蛋白分别命名为CD19-muFc、MSLN-muFc、Muc1-muFc与EGFR-muFc；
2、合成序列为分别为SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30和SEQ ID NO:31所示的DNA序列，分别编码CD19、MSLN、Muc1与EGFR胞外结构域与野生型IgG4Fc片段融合蛋白，按1中的方法制备得到CD19、MSLN、Muc1与EGFR胞外结构域与野生型IgG4Fc片段融合蛋白，分别命名为CD19-wtFc、MSLN-wtFc、Muc1-wtFc与EGFR-wtFc；
3、合成序列为SEQ ID NO:32所示的DNA序列，其编码EGFR胞外结构域，按1中的方法制备得到EGFR胞外结构域蛋白，命名为EGFR-IFc。
实施例2PE荧光素标记的抗原-Fc融合蛋白的制备
1、蛋白与生物素的偶联
取生物素0.2mg，分别与实施例1中制得的四种抗原-Fc融合蛋白(CD19-muFc、MSLN-muFc、Muc1-muFc与EGFR-muFc)和五种对照蛋白(CD19-wtFc、MSLN-wtFc、Muc1-wtFc、EGFR-wtFc与EGFR-IFc)各50.0mg一起加入10mL水中，搅拌混合15min直至混匀，在避光条件下加入N-羟基琥珀酰亚胺(NHS)与1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)各0.1mg，继续避光搅拌3h，反应完毕后将产物装入透析袋内(截留分子量1000Da)，纯水透析72h，冻干即得到各蛋白与生物素的偶联物；
2、荧光标记抗原-Fc融合蛋白-生物素偶联物与对照蛋白-生物素偶联物
将1中得到的四种抗原-Fc融合蛋白(CD19-muFc、MSLN-muFc、Muc1-muFc与EGFR-muFc)的生物素偶联物与五种对照蛋白(CD19-wtFc、MSLN-wtFc、Muc1-wtFc、EGFR-wtFc与EGFR-IFc)的生物素偶联物各取25mg加入到5mL PBS中，涡旋振荡15s后加入5.0mg带PE荧光素标记的链亲合素，再次涡旋振荡15s，室温静置30min后，3000rpm离心3min，弃去上清，沉淀加入新鲜PBS重悬，重复此操作三次，得到各PE荧光素标记的蛋白(PE-CD19-muFc、PE-MSLN-muFc、PE-Muc1-muFc、PE-EGFR-muFc、PE-CD19-wtFc、PE-MSLN-wtFc、PE-Muc1-wtFc、PE-EGFR-wtFc与PE-EGFR-IFc)的沉淀，冻干备用。
PE荧光素标记的抗原-Fc融合蛋白的结构与工作原理如图1所示。带PE荧光素标记的链亲合素与蛋白分子上共价偶联的生物所形成强非共价相互作用，使抗原-Fc融合蛋白被PE荧光素标记。
实施例3PE荧光素标记的抗原-Fc融合蛋白检测阳性CAR-T细胞
1、将实施例2制得的PE荧光素标记的各抗原-Fc融合蛋白溶于PBS中配置成浓度为10.0mg/mL的100×贮备液，使用前用PBS稀释100倍即得PE荧光素标记的抗原-Fc融合蛋白稀释液
2、分别取靶向CD19、MSLN、Muc1和EGFR的CAR-T细胞及各自的对照T(Control T1、Control T2、Control T3和Control T4)细胞各1×106个，1000rpm离心3min，弃去上层培养基，用1mL新鲜培养基重悬，向5种CAR-T细胞中分别加入1、2、5μL步骤1的PE荧光素标记的抗原-Fc融合蛋白稀释液，37℃、5％CO2孵育1h；
3、用冷PBS洗涤步骤2孵育后的5种CAR-T细胞三次，每次洗涤用mL冷PBS重悬CAR-T细胞，1000rpm离心3min。三次以后用流式细胞仪检测细胞的荧光强度，分析各CAR-T细胞的阳性率。
结果如图2和表1所示。图2显示了PE荧光素标记的抗原-Fc融合蛋白稀释液的用量分别为1、2、5μL时Muc1-CAR-T细胞、Meso-CAR-T细胞与各自对应的Control T细胞的阳性率结果。结果显示Muc1-CAR-T和Meso-CAR-T各自三种标记剂量下检测的阳性率都较为接近，Muc1-CAR-T细胞的阳性率三种标记剂量均在66％以上，Meso-CAR-T细胞的阳性率三种标记剂量均在87％以上，且Control T3与Control T2的阳性率均非常低。
同时图2结果也表明PE荧光素标记的抗原-Fc融合蛋白稀释液的用量在1、2、5μL时的检测结果基本一致。这表明在1μL用量的条件下PE荧光素标记的抗原-Fc融合蛋白相对于1×106个各种CAR-T细胞已相对过量，与各种CAR-T细胞表面受体的结合达到饱和，提高浓度也不会使结合效果进一步增强。后续实验种的用量均为每1×106个CAR-T细胞使用1μLPE荧光素标记的抗原-Fc融合蛋白稀释液。
表1
第一次 第二次 第三次
CD19-CAR-T 69.83％ 69.42％ 68.50％
Meso-CAR-T 88.92％ 89.60％ 89.74％
Muc1-CAR-T 67.87％ 66.49％ 67.21％
EGFR-CAR-T 90.15％ 89.42％ 90.03％
表1为PE荧光素标记的抗原-Fc融合蛋白检测CD19-CAR-T、Meso-CAR-T、Muc1-CAR-T和EGFR-CAR-T细胞阳性率重复性测试结果。由表1可知使用PE荧光素标记的抗原-Fc融合蛋白检测各种CAR-T细胞的结果重复性好，准确度高，表明本发明方法符合CAR-T细胞阳性率的检测要求。
对比例1PE荧光素标记的抗原-Fc融合蛋白与羊抗人IgG F(ab)2-FITC法和Protein L-链亲和素-PE法检测阳性CD19-CAR-T细胞的比较
PE荧光素标记的抗原-Fc融合蛋白检测CD19-CAR-T细胞的步骤同实施例3中所述，不再重复；
CD19羊抗人IgG F(ab)2–FITC多克隆抗体按Jackson Immuno Research公司的抗体说明书使用，流式检测FITC荧光强度，分析CD19-CAR-T细胞阳性率；
Protein L+链亲和素-PE按文献Zheng Z,Chinnasamy N,Morgan RA Protein L:anovel reagent for the detection of chimeric antigen receptor(CAR)expressionby flow cytometry.J Transl Med.2012Feb 13；10:29所述方法使用，流式检测PE荧光强度，分析CD19-CAR-T细胞阳性率。
结果如图3所示，CD19羊抗人IgG F(ab)2–FITC法检测Control T细胞阳性率三次结果均大于25％，其中一次甚至达57.43％，存在严重的假阳性结果，使同期测试的CD19-CAR-T细胞阳性率检测结果偏高；并且结果不稳定，阳性率数值分布从71％到91％。
Protein L+链亲和素-PE法检测的结果不稳定，阳性率数值从23％附近到接近69％都有分布，重复性较差。
本发明的PE荧光素标记的抗原-Fc融合蛋白检测CD19-CAR-T细胞阳性率的结果表明，本发明的方法假阳性率低，且三次重复试验的结果稳定，可重复性好，与前两种检测方法相比具有显著的优势。
对比例2本发明PE荧光素标记的抗原-Fc融合蛋白(PE-Ag-muFc)与PE荧光素标记的抗原-野生型Fc融合蛋白(PE-Ag-wtFc)以及PE荧光素标记的未融合Fc片段的EGFR胞外结构域(PE-EGFR-IFc)检测CAR-T细胞阳性率结果对比
CD19、MSLN、Muc1、EGFR四种蛋白的PE-Ag-wtFc与PE-EGFR-IFc的制备过程见实施例1和实施例2，浓度均调整为为50μg/mL。
本发明PE荧光素标记的抗原-Fc融合蛋白(PE-Ag-muFc)、PE荧光素标记的抗原-野生型Fc融合蛋白(PE-Ag-wtFc)与PE荧光素标记的未融合Fc片段的EGFR胞外结构域(PE-EGFR-IFc)检测CD19-CAR-T、MSLN-CAR-T、Muc1-CAR-T与EGFR-CAR-T细胞阳性率的方法同实施例3。CD19、MSLN、Muc1与EGFR的PE-Ag-wtFc及EGFR的PE-Ag-IFc的用量均为2μL。
结果如图4和表2所示。图4显示，四种PE-Ag-wtFc检测各自的Control T细胞时普遍存在10-20％的假阳性结果，导致CAR-T细胞的阳性率检测结果普遍偏高。相比之下，本发明的四种PE-Ag-muFc检测各自的Control T细胞时阳性率均小于0.2％，几乎不包含假阳性结果。因此与PE荧光素标记的抗原-野生型Fc融合蛋白相比，本发明的PE荧光素标记的抗原-Fc融合蛋白检测阳性CAR-T细胞的准确率有大幅度地提高，检测结果更为准确可信。
表2
表2显示，虽然两组结果的Control T阳性率均较低，但PE-EGFR-IFc由于缺失IgG4Fc融合蛋白，稳定性较差，进而导致与EGFR-CAR-T细胞胞外的抗原结合域的结合能力较差，测得的阳性率普遍偏低，因而存在假阴性结果。而PE-EGFR-muFc组测得的EGFR-CAR-T细胞阳性率正常，不存在假阴性结果。
应理解，在阅读了本发明的上述内容之后，本领域技术人员可以对本发明的相关条件作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
序列表
<110> 上海细胞治疗研究院
上海细胞治疗工程技术研究中心集团有限公司
<120> 抗原-Fc融合蛋白及其检测阳性CAR-T细胞的应用
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Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu
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Gly Leu Gly Cys Gly Leu Lys Asn Arg Ser Ser Glu Gly Pro Ser Ser
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Pro Ser Gly Lys Leu Met Ser Pro Lys Leu Tyr Val Trp Ala Lys Asp
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Ser Leu Asn Gln Ser Leu Ser Gln Asp Leu Thr Met Ala Pro Gly Ser
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Thr Ala Arg Pro Val Leu Trp His Trp Leu Leu Arg Thr Gly Gly Trp
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Met Pro Glu Glu Pro Leu Val Val Lys Val Glu Glu Gly Asp Asn Ala
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Gly Leu Gly Cys Gly Leu Lys Asn Arg Ser Ser Glu Gly Pro Ser Ser
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130 135 140
cgc cct gag atc tgg gag gga gag cct ccg tgt ctc cca ccg agg gac 480
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145 150 155 160
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Ser Leu Asn Gln Ser Leu Ser Gln Asp Leu Thr Met Ala Pro Gly Ser
165 170 175
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Thr Leu Trp Leu Ser Cys Gly Val Pro Pro Asp Ser Val Ser Arg Gly
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ccc agc gac atc gcc gtg gag tgg gag agc aat ggg cag ccg gag aac 1008
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
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aac tac aag acc acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc 1056
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370 375 380
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Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
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<213> 人工序列(Artificial Sequence)
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Pro Asn Gly Tyr Leu Val Leu Asp Leu Ser Met Gln Glu Ala Leu Ser
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Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
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Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
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Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
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Leu Ser Leu Ser Leu Gly Lys
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<210> 7
<211> 1272
<212> DNA
<213> 人工序列(Artificial Sequence)
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<222> (1)..(1269)
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Met Glu Val Glu Lys Thr Ala Cys Pro Ser Gly Lys Lys Ala Arg Glu
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Pro Phe Thr Tyr Glu Gln Leu Asp Val Leu Lys His Lys Leu Asp Glu
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65 70 75 80
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Leu Phe Leu Lys Met Ser Pro Glu Asp Ile Arg Lys Trp Asn Val Thr
85 90 95
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Ser Leu Glu Thr Leu Lys Ala Leu Leu Glu Val Asn Lys Gly His Glu
100 105 110
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Met Ser Pro Gln Val Ala Thr Leu Ile Asp Arg Phe Val Lys Gly Arg
115 120 125
ggc cag cta gac aaa gac acc cta gac acc ctg acc gcc ttc tac cct 432
Gly Gln Leu Asp Lys Asp Thr Leu Asp Thr Leu Thr Ala Phe Tyr Pro
130 135 140
ggg tac ctg tgc tcc ctc agc ccc gag gag ctg agc tcc gtg ccc ccc 480
Gly Tyr Leu Cys Ser Leu Ser Pro Glu Glu Leu Ser Ser Val Pro Pro
145 150 155 160
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Ser Ser Ile Trp Ala Val Arg Pro Gln Asp Leu Asp Thr Cys Asp Pro
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agg cag ctg gac gtc ctc tat ccc aag gcc cgc ctt gct ttc cag aac 576
Arg Gln Leu Asp Val Leu Tyr Pro Lys Ala Arg Leu Ala Phe Gln Asn
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Met Asn Gly Ser Glu Tyr Phe Val Lys Ile Gln Ser Phe Leu Gly Gly
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Ala Pro Thr Glu Asp Leu Lys Ala Leu Ser Gln Gln Asn Val Ser Met
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Asp Leu Ala Thr Phe Met Lys Leu Arg Thr Asp Ala Val Leu Pro Leu
225 230 235 240
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Thr Val Ala Glu Val Gln Lys Leu Leu Gly Pro His Val Glu Gly Leu
245 250 255
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Lys Ala Glu Glu Arg His Arg Pro Val Arg Asp Trp Ile Leu Arg Gln
260 265 270
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Arg Gln Asp Asp Leu Asp Thr Leu Gly Leu Gly Leu Gln Gly Gly Ile
275 280 285
ccc aac ggc tac ctg gtc cta gac ctc agc atg caa gag gcc ctc tcg 912
Pro Asn Gly Tyr Leu Val Leu Asp Leu Ser Met Gln Glu Ala Leu Ser
290 295 300
gag tcc aaa tat ggt ccc cca tgc cca cca tgc cca ggg cag ccc cga 960
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gln Pro Arg
305 310 315 320
gag cca cag gtg tac acc ctg ccc cca tcc cag gag gag atg acc aag 1008
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
325 330 335
aac cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tac ccc agc gac 1056
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
340 345 350
atc gcc gtg gag tgg gag agc aat ggg cag ccg gag aac aac tac aag 1104
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
355 360 365
acc acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc 1152
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
370 375 380
agg cta acc gtg gac aag agc agg tgg cag gag ggg aat gtc ttc tca 1200
Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
385 390 395 400
tgc tcc gtg atg cat gag gct ctg cac aac cac tac aca cag aag agc 1248
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
405 410 415
ctc tcc ctg tct ctg ggt aaa tga 1272
Leu Ser Leu Ser Leu Gly Lys
420
<210> 8
<211> 550
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<221> PEPTIDE
<222> (1)..(550)
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Met Ser Gly His Ala Ser Ser Thr Pro Gly Gly Glu Lys Glu Thr Ser
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20 25 30
Ser Met Thr Ser Ser Val Leu Ser Ser His Ser Pro Gly Ser Gly Ser
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Ser Thr Thr Gln Gly Gln Asp Val Thr Leu Ala Pro Ala Thr Glu Pro
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Val Thr Arg Pro Ala Leu Gly Ser Thr Thr Pro Pro Ala His Asp Val
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Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro
130 135 140
Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro
145 150 155 160
Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val
165 170 175
Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro
180 185 190
Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser
195 200 205
Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Asn Arg Pro
210 215 220
Ala Leu Gly Ser Thr Ala Pro Pro Val His Asn Val Thr Ser Ala Ser
225 230 235 240
Gly Ser Ala Ser Gly Ser Ala Ser Thr Leu Val His Asn Gly Thr Ser
245 250 255
Ala Arg Ala Thr Thr Thr Pro Ala Ser Lys Ser Thr Pro Phe Ser Ile
260 265 270
Pro Ser His His Ser Asp Thr Pro Thr Thr Leu Ala Ser His Ser Thr
275 280 285
Lys Thr Asp Ala Ser Ser Thr His His Ser Ser Val Pro Pro Leu Thr
290 295 300
Ser Ser Asn His Ser Thr Ser Pro Gln Leu Ser Thr Gly Val Ser Phe
305 310 315 320
Phe Phe Leu Ser Phe His Ile Ser Asn Leu Gln Phe Asn Ser Ser Leu
325 330 335
Glu Asp Pro Ser Thr Asp Tyr Tyr Gln Glu Leu Gln Arg Asp Ile Ser
340 345 350
Glu Met Phe Leu Gln Ile Tyr Lys Gln Gly Gly Phe Leu Gly Leu Ser
355 360 365
Asn Ile Lys Phe Arg Pro Gly Ser Val Val Val Gln Leu Thr Leu Ala
370 375 380
Phe Arg Glu Gly Thr Ile Asn Val His Asp Val Glu Thr Gln Phe Asn
385 390 395 400
Gln Tyr Lys Thr Glu Ala Ala Ser Arg Tyr Asn Leu Thr Ile Ser Asp
405 410 415
Val Ser Val Ser Asp Val Pro Phe Pro Phe Ser Ala Gln Ser Gly Glu
420 425 430
Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gln Pro Arg Glu
435 440 445
Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn
450 455 460
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
465 470 475 480
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
485 490 495
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg
500 505 510
Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys
515 520 525
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
530 535 540
Ser Leu Ser Leu Gly Lys
545 550
<210> 9
<211> 1653
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> CDS
<222> (1)..(1653)
<223> Muc1-铰链-IgG4Fc
<400> 9
atg tct ggt cat gca agc tct acc cca ggt gga gaa aag gag act tcg 48
Met Ser Gly His Ala Ser Ser Thr Pro Gly Gly Glu Lys Glu Thr Ser
1 5 10 15
gct acc cag aga agt tca gtg ccc agc tct act gag aag aat gct gtg 96
Ala Thr Gln Arg Ser Ser Val Pro Ser Ser Thr Glu Lys Asn Ala Val
20 25 30
agt atg acc agc agc gta ctc tcc agc cac agc ccc ggt tca ggc tcc 144
Ser Met Thr Ser Ser Val Leu Ser Ser His Ser Pro Gly Ser Gly Ser
35 40 45
tcc acc act cag gga cag gat gtc act ctg gcc ccg gcc acg gaa cca 192
Ser Thr Thr Gln Gly Gln Asp Val Thr Leu Ala Pro Ala Thr Glu Pro
50 55 60
gct tca ggt tca gct gcc ctg tgg gga cag gat gtc acc tcg gtc cca 240
Ala Ser Gly Ser Ala Ala Leu Trp Gly Gln Asp Val Thr Ser Val Pro
65 70 75 80
gtc acc agg cca gcc ctg ggc tcc acc acc ccg cca gcc cac gat gtc 288
Val Thr Arg Pro Ala Leu Gly Ser Thr Thr Pro Pro Ala His Asp Val
85 90 95
acc tca gcc ccg gac aac aag cca gcc ccg ggc tcc acc gcc ccc cca 336
Thr Ser Ala Pro Asp Asn Lys Pro Ala Pro Gly Ser Thr Ala Pro Pro
100 105 110
gcc cac ggt gtc acc tcg gcc ccg gac acc agg ccg gcc ccg ggc tcc 384
Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser
115 120 125
acc gcc ccc cca gcc cac ggt gtc acc tcg gcc ccg gac acc agg ccg 432
Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro
130 135 140
gcc ccg ggc tcc acc gcc ccc cca gcc cac ggt gtc acc tcg gcc ccg 480
Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro
145 150 155 160
gac acc agg ccg gcc ccg ggc tcc acc gcc ccc cca gcc cac ggt gtc 528
Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val
165 170 175
acc tcg gcc ccg gac acc agg ccg gcc ccg ggc tcc acc gcc ccc cca 576
Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro
180 185 190
gcc cac ggt gtc acc tcg gcc ccg gac acc agg ccg gcc ccg ggc tcc 624
Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser
195 200 205
acc gcc ccc cca gcc cat ggt gtc acc tcg gcc ccg gac aac agg ccc 672
Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Asn Arg Pro
210 215 220
gcc ttg ggc tcc acc gcc cct cca gtc cac aat gtc acc tcg gcc tca 720
Ala Leu Gly Ser Thr Ala Pro Pro Val His Asn Val Thr Ser Ala Ser
225 230 235 240
ggc tct gca tca ggc tca gct tct act ctg gtg cac aac ggc acc tct 768
Gly Ser Ala Ser Gly Ser Ala Ser Thr Leu Val His Asn Gly Thr Ser
245 250 255
gcc agg gct acc aca acc cca gcc agc aag agc act cca ttc tca att 816
Ala Arg Ala Thr Thr Thr Pro Ala Ser Lys Ser Thr Pro Phe Ser Ile
260 265 270
ccc agc cac cac tct gat act cct acc acc ctt gcc agc cat agc acc 864
Pro Ser His His Ser Asp Thr Pro Thr Thr Leu Ala Ser His Ser Thr
275 280 285
aag act gat gcc agt agc act cac cat agc tcg gta cct cct ctc acc 912
Lys Thr Asp Ala Ser Ser Thr His His Ser Ser Val Pro Pro Leu Thr
290 295 300
tcc tcc aat cac agc act tct ccc cag ttg tct act ggg gtc tct ttc 960
Ser Ser Asn His Ser Thr Ser Pro Gln Leu Ser Thr Gly Val Ser Phe
305 310 315 320
ttt ttc ctg tct ttt cac att tca aac ctc cag ttt aat tcc tct ctg 1008
Phe Phe Leu Ser Phe His Ile Ser Asn Leu Gln Phe Asn Ser Ser Leu
325 330 335
gaa gat ccc agc acc gac tac tac caa gag ctg cag aga gac att tct 1056
Glu Asp Pro Ser Thr Asp Tyr Tyr Gln Glu Leu Gln Arg Asp Ile Ser
340 345 350
gaa atg ttt ttg cag att tat aaa caa ggg ggt ttt ctg ggc ctc tcc 1104
Glu Met Phe Leu Gln Ile Tyr Lys Gln Gly Gly Phe Leu Gly Leu Ser
355 360 365
aat att aag ttc agg cca gga tct gtg gtg gta caa ttg act ctg gcc 1152
Asn Ile Lys Phe Arg Pro Gly Ser Val Val Val Gln Leu Thr Leu Ala
370 375 380
ttc cga gaa ggt acc atc aat gtc cac gac gtg gag aca cag ttc aat 1200
Phe Arg Glu Gly Thr Ile Asn Val His Asp Val Glu Thr Gln Phe Asn
385 390 395 400
cag tat aaa acg gaa gca gcc tct cga tat aac ctg acg atc tca gac 1248
Gln Tyr Lys Thr Glu Ala Ala Ser Arg Tyr Asn Leu Thr Ile Ser Asp
405 410 415
gtc agc gtg agt gat gtg cca ttt cct ttc tct gcc cag tct ggg gag 1296
Val Ser Val Ser Asp Val Pro Phe Pro Phe Ser Ala Gln Ser Gly Glu
420 425 430
tcc aaa tat ggt ccc cca tgc cca cca tgc cca ggg cag ccc cga gag 1344
Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gln Pro Arg Glu
435 440 445
cca cag gtg tac acc ctg ccc cca tcc cag gag gag atg acc aag aac 1392
Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn
450 455 460
cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tac ccc agc gac atc 1440
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
465 470 475 480
gcc gtg gag tgg gag agc aat ggg cag ccg gag aac aac tac aag acc 1488
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
485 490 495
acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc agg 1536
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg
500 505 510
cta acc gtg gac aag agc agg tgg cag gag ggg aat gtc ttc tca tgc 1584
Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys
515 520 525
tcc gtg atg cat gag gct ctg cac aac cac tac aca cag aag agc ctc 1632
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
530 535 540
tcc ctg tct ctg ggt aaa tga 1653
Ser Leu Ser Leu Gly Lys
545 550
<210> 10
<211> 740
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(740)
<223> EGFR-铰链-IgG4Fc
<400> 10
Met Leu Glu Glu Lys Lys Val Cys Gln Gly Thr Ser Asn Lys Leu Thr
1 5 10 15
Gln Leu Gly Thr Phe Glu Asp His Phe Leu Ser Leu Gln Arg Met Phe
20 25 30
Asn Asn Cys Glu Val Val Leu Gly Asn Leu Glu Ile Thr Tyr Val Gln
35 40 45
Arg Asn Tyr Asp Leu Ser Phe Leu Lys Thr Ile Gln Glu Val Ala Gly
50 55 60
Tyr Val Leu Ile Ala Leu Asn Thr Val Glu Arg Ile Pro Leu Glu Asn
65 70 75 80
Leu Gln Ile Ile Arg Gly Asn Met Tyr Tyr Glu Asn Ser Tyr Ala Leu
85 90 95
Ala Val Leu Ser Asn Tyr Asp Ala Asn Lys Thr Gly Leu Lys Glu Leu
100 105 110
Pro Met Arg Asn Leu Gln Glu Ile Leu His Gly Ala Val Arg Phe Ser
115 120 125
Asn Asn Pro Ala Leu Cys Asn Val Glu Ser Ile Gln Trp Arg Asp Ile
130 135 140
Val Ser Ser Asp Phe Leu Ser Asn Met Ser Met Asp Phe Gln Asn His
145 150 155 160
Leu Gly Ser Cys Gln Lys Cys Asp Pro Ser Cys Pro Asn Gly Ser Cys
165 170 175
Trp Gly Ala Gly Glu Glu Asn Cys Gln Lys Leu Thr Lys Ile Ile Cys
180 185 190
Ala Gln Gln Cys Ser Gly Arg Cys Arg Gly Lys Ser Pro Ser Asp Cys
195 200 205
Cys His Asn Gln Cys Ala Ala Gly Cys Thr Gly Pro Arg Glu Ser Asp
210 215 220
Cys Leu Val Cys Arg Lys Phe Arg Asp Glu Ala Thr Cys Lys Asp Thr
225 230 235 240
Cys Pro Pro Leu Met Leu Tyr Asn Pro Thr Thr Tyr Gln Met Asp Val
245 250 255
Asn Pro Glu Gly Lys Tyr Ser Phe Gly Ala Thr Cys Val Lys Lys Cys
260 265 270
Pro Arg Asn Tyr Val Val Thr Asp His Gly Ser Cys Val Arg Ala Cys
275 280 285
Gly Ala Asp Ser Tyr Glu Met Glu Glu Asp Gly Val Arg Lys Cys Lys
290 295 300
Lys Cys Glu Gly Pro Cys Arg Lys Val Cys Asn Gly Ile Gly Ile Gly
305 310 315 320
Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe
325 330 335
Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala
340 345 350
Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu
355 360 365
Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile
370 375 380
Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu
385 390 395 400
Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala
405 410 415
Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu
420 425 430
Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr
435 440 445
Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys
450 455 460
Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly
465 470 475 480
Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu
485 490 495
Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys
500 505 510
Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu
515 520 525
Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met
530 535 540
Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala
545 550 555 560
His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val
565 570 575
Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His
580 585 590
Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro
595 600 605
Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro Glu Ser Lys
610 615 620
Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gln Pro Arg Glu Pro Gln
625 630 635 640
Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
645 650 655
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
660 665 670
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
675 680 685
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr
690 695 700
Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
705 710 715 720
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
725 730 735
Ser Leu Gly Lys
740
<210> 11
<211> 2223
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> CDS
<222> (1)..(2223)
<223> EGFR-铰链-IgG4Fc
<400> 11
atg ctg gag gaa aag aaa gtt tgc caa ggc acg agt aac aag ctc acg 48
Met Leu Glu Glu Lys Lys Val Cys Gln Gly Thr Ser Asn Lys Leu Thr
1 5 10 15
cag ttg ggc act ttt gaa gat cat ttt ctc agc ctc cag agg atg ttc 96
Gln Leu Gly Thr Phe Glu Asp His Phe Leu Ser Leu Gln Arg Met Phe
20 25 30
aat aac tgt gag gtg gtc ctt ggg aat ttg gaa att acc tat gtg cag 144
Asn Asn Cys Glu Val Val Leu Gly Asn Leu Glu Ile Thr Tyr Val Gln
35 40 45
agg aat tat gat ctt tcc ttc tta aag acc atc cag gag gtg gct ggt 192
Arg Asn Tyr Asp Leu Ser Phe Leu Lys Thr Ile Gln Glu Val Ala Gly
50 55 60
tat gtc ctc att gcc ctc aac aca gtg gag cga att cct ttg gaa aac 240
Tyr Val Leu Ile Ala Leu Asn Thr Val Glu Arg Ile Pro Leu Glu Asn
65 70 75 80
ctg cag atc atc aga gga aat atg tac tac gaa aat tcc tat gcc tta 288
Leu Gln Ile Ile Arg Gly Asn Met Tyr Tyr Glu Asn Ser Tyr Ala Leu
85 90 95
gca gtc tta tct aac tat gat gca aat aaa acc gga ctg aag gag ctg 336
Ala Val Leu Ser Asn Tyr Asp Ala Asn Lys Thr Gly Leu Lys Glu Leu
100 105 110
ccc atg aga aat tta cag gaa atc ctg cat ggc gcc gtg cgg ttc agc 384
Pro Met Arg Asn Leu Gln Glu Ile Leu His Gly Ala Val Arg Phe Ser
115 120 125
aac aac cct gcc ctg tgc aac gtg gag agc atc cag tgg cgg gac ata 432
Asn Asn Pro Ala Leu Cys Asn Val Glu Ser Ile Gln Trp Arg Asp Ile
130 135 140
gtc agc agt gac ttt ctc agc aac atg tcg atg gac ttc cag aac cac 480
Val Ser Ser Asp Phe Leu Ser Asn Met Ser Met Asp Phe Gln Asn His
145 150 155 160
ctg ggc agc tgc caa aag tgt gat cca agc tgt ccc aat ggg agc tgc 528
Leu Gly Ser Cys Gln Lys Cys Asp Pro Ser Cys Pro Asn Gly Ser Cys
165 170 175
tgg ggt gca gga gag gag aac tgc cag aaa ctg acc aaa atc atc tgt 576
Trp Gly Ala Gly Glu Glu Asn Cys Gln Lys Leu Thr Lys Ile Ile Cys
180 185 190
gcc cag cag tgc tcc ggg cgc tgc cgt ggc aag tcc ccc agt gac tgc 624
Ala Gln Gln Cys Ser Gly Arg Cys Arg Gly Lys Ser Pro Ser Asp Cys
195 200 205
tgc cac aac cag tgt gct gca ggc tgc aca ggc ccc cgg gag agc gac 672
Cys His Asn Gln Cys Ala Ala Gly Cys Thr Gly Pro Arg Glu Ser Asp
210 215 220
tgc ctg gtc tgc cgc aaa ttc cga gac gaa gcc acg tgc aag gac acc 720
Cys Leu Val Cys Arg Lys Phe Arg Asp Glu Ala Thr Cys Lys Asp Thr
225 230 235 240
tgc ccc cca ctc atg ctc tac aac ccc acc acg tac cag atg gat gtg 768
Cys Pro Pro Leu Met Leu Tyr Asn Pro Thr Thr Tyr Gln Met Asp Val
245 250 255
aac ccc gag ggc aaa tac agc ttt ggt gcc acc tgc gtg aag aag tgt 816
Asn Pro Glu Gly Lys Tyr Ser Phe Gly Ala Thr Cys Val Lys Lys Cys
260 265 270
ccc cgt aat tat gtg gtg aca gat cac ggc tcg tgc gtc cga gcc tgt 864
Pro Arg Asn Tyr Val Val Thr Asp His Gly Ser Cys Val Arg Ala Cys
275 280 285
ggg gcc gac agc tat gag atg gag gaa gac ggc gtc cgc aag tgt aag 912
Gly Ala Asp Ser Tyr Glu Met Glu Glu Asp Gly Val Arg Lys Cys Lys
290 295 300
aag tgc gaa ggg cct tgc cgc aaa gtg tgt aac gga ata ggt att ggt 960
Lys Cys Glu Gly Pro Cys Arg Lys Val Cys Asn Gly Ile Gly Ile Gly
305 310 315 320
gaa ttt aaa gac tca ctc tcc ata aat gct acg aat att aaa cac ttc 1008
Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe
325 330 335
aaa aac tgc acc tcc atc agt ggc gat ctc cac atc ctg ccg gtg gca 1056
Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala
340 345 350
ttt agg ggt gac tcc ttc aca cat act cct cct ctg gat cca cag gaa 1104
Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu
355 360 365
ctg gat att ctg aaa acc gta aag gaa atc aca ggg ttt ttg ctg att 1152
Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile
370 375 380
cag gct tgg cct gaa aac agg acg gac ctc cat gcc ttt gag aac cta 1200
Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu
385 390 395 400
gaa atc ata cgc ggc agg acc aag caa cat ggt cag ttt tct ctt gca 1248
Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala
405 410 415
gtc gtc agc ctg aac ata aca tcc ttg gga tta cgc tcc ctc aag gag 1296
Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu
420 425 430
ata agt gat gga gat gtg ata att tca gga aac aaa aat ttg tgc tat 1344
Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr
435 440 445
gca aat aca ata aac tgg aaa aaa ctg ttt ggg acc tcc ggt cag aaa 1392
Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys
450 455 460
acc aaa att ata agc aac aga ggt gaa aac agc tgc aag gcc aca ggc 1440
Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly
465 470 475 480
cag gtc tgc cat gcc ttg tgc tcc ccc gag ggc tgc tgg ggc ccg gag 1488
Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu
485 490 495
ccc agg gac tgc gtc tct tgc cgg aat gtc agc cga ggc agg gaa tgc 1536
Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys
500 505 510
gtg gac aag tgc aac ctt ctg gag ggt gag cca agg gag ttt gtg gag 1584
Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu
515 520 525
aac tct gag tgc ata cag tgc cac cca gag tgc ctg cct cag gcc atg 1632
Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met
530 535 540
aac atc acc tgc aca gga cgg gga cca gac aac tgt atc cag tgt gcc 1680
Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala
545 550 555 560
cac tac att gac ggc ccc cac tgc gtc aag acc tgc ccg gca gga gtc 1728
His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val
565 570 575
atg gga gaa aac aac acc ctg gtc tgg aag tac gca gac gcc ggc cat 1776
Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His
580 585 590
gtg tgc cac ctg tgc cat cca aac tgc acc tac gga tgc act ggg cca 1824
Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro
595 600 605
ggt ctt gaa ggc tgt cca acg aat ggg cct aag atc ccg gag tcc aaa 1872
Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro Glu Ser Lys
610 615 620
tat ggt ccc cca tgc cca cca tgc cca ggg cag ccc cga gag cca cag 1920
Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gln Pro Arg Glu Pro Gln
625 630 635 640
gtg tac acc ctg ccc cca tcc cag gag gag atg acc aag aac cag gtc 1968
Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
645 650 655
agc ctg acc tgc ctg gtc aaa ggc ttc tac ccc agc gac atc gcc gtg 2016
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
660 665 670
gag tgg gag agc aat ggg cag ccg gag aac aac tac aag acc acg cct 2064
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
675 680 685
ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc agg cta acc 2112
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr
690 695 700
gtg gac aag agc agg tgg cag gag ggg aat gtc ttc tca tgc tcc gtg 2160
Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
705 710 715 720
atg cat gag gct ctg cac aac cac tac aca cag aag agc ctc tcc ctg 2208
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
725 730 735
tct ctg ggt aaa tga 2223
Ser Leu Gly Lys
740
<210> 12
<211> 1182
<212> RNA
<213> 人工序列(Artificial Sequence)
<220>
<221> CDS
<222> (1)..(1182)
<223> CD19-铰链-IgG4Fc
<400> 12
aug ccc gag gaa ccu cua gug gug aag gug gaa gag gga gau aac gcu 48
Met Pro Glu Glu Pro Leu Val Val Lys Val Glu Glu Gly Asp Asn Ala
1 5 10 15
gug cug cag ugc cuc aag ggg acc uca gau ggc ccc acu cag cag cug 96
Val Leu Gln Cys Leu Lys Gly Thr Ser Asp Gly Pro Thr Gln Gln Leu
20 25 30
acc ugg ucu cgg gag ucc ccg cuu aaa ccc uuc uua aaa cuc agc cug 144
Thr Trp Ser Arg Glu Ser Pro Leu Lys Pro Phe Leu Lys Leu Ser Leu
35 40 45
ggg cug cca ggc cug gga auc cac aug agg ccc cug gcc auc ugg cuu 192
Gly Leu Pro Gly Leu Gly Ile His Met Arg Pro Leu Ala Ile Trp Leu
50 55 60
uuc auc uuc aac guc ucu caa cag aug ggg ggc uuc uac cug ugc cag 240
Phe Ile Phe Asn Val Ser Gln Gln Met Gly Gly Phe Tyr Leu Cys Gln
65 70 75 80
ccg ggg ccc ccc ucu gag aag gcc ugg cag ccu ggc ugg aca guc aau 288
Pro Gly Pro Pro Ser Glu Lys Ala Trp Gln Pro Gly Trp Thr Val Asn
85 90 95
gug gag ggc agc ggg gag cug uuc cgg ugg aau guu ucg gac cua ggu 336
Val Glu Gly Ser Gly Glu Leu Phe Arg Trp Asn Val Ser Asp Leu Gly
100 105 110
ggc cug ggc ugu ggc cug aag aac agg ucc uca gag ggc ccc agc ucc 384
Gly Leu Gly Cys Gly Leu Lys Asn Arg Ser Ser Glu Gly Pro Ser Ser
115 120 125
ccu ucc ggg aag cuc aug agc ccc aag cug uau gug ugg gcc aaa gac 432
Pro Ser Gly Lys Leu Met Ser Pro Lys Leu Tyr Val Trp Ala Lys Asp
130 135 140
cgc ccu gag auc ugg gag gga gag ccu ccg ugu cuc cca ccg agg gac 480
Arg Pro Glu Ile Trp Glu Gly Glu Pro Pro Cys Leu Pro Pro Arg Asp
145 150 155 160
agc cug aac cag agc cuc agc cag gac cuc acc aug gcc ccu ggc ucc 528
Ser Leu Asn Gln Ser Leu Ser Gln Asp Leu Thr Met Ala Pro Gly Ser
165 170 175
aca cuc ugg cug ucc ugu ggg gua ccc ccu gac ucu gug ucc agg ggc 576
Thr Leu Trp Leu Ser Cys Gly Val Pro Pro Asp Ser Val Ser Arg Gly
180 185 190
ccc cuc ucc ugg acc cau gug cac ccc aag ggg ccu aag uca uug cug 624
Pro Leu Ser Trp Thr His Val His Pro Lys Gly Pro Lys Ser Leu Leu
195 200 205
agc cua gag cug aag gac gau cgc ccg gcc aga gau aug ugg gua aug 672
Ser Leu Glu Leu Lys Asp Asp Arg Pro Ala Arg Asp Met Trp Val Met
210 215 220
gag acg ggu cug uug uug ccc cgg gcc aca gcu caa gac gcu gga aag 720
Glu Thr Gly Leu Leu Leu Pro Arg Ala Thr Ala Gln Asp Ala Gly Lys
225 230 235 240
uau uau ugu cac cgu ggc aac cug acc aug uca uuc cac cug gag auc 768
Tyr Tyr Cys His Arg Gly Asn Leu Thr Met Ser Phe His Leu Glu Ile
245 250 255
acu gcu cgg cca gua cua ugg cac ugg cug cug agg acu ggu ggc ugg 816
Thr Ala Arg Pro Val Leu Trp His Trp Leu Leu Arg Thr Gly Gly Trp
260 265 270
aag guc uca gag ucc aaa uau ggu ccc cca ugc cca cca ugc cca ggg 864
Lys Val Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly
275 280 285
cag ccc cga gag cca cag gug uac acc cug ccc cca ucc cag gag gag 912
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu
290 295 300
aug acc aag aac cag guc agc cug acc ugc cug guc aaa ggc uuc uac 960
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
305 310 315 320
ccc agc gac auc gcc gug gag ugg gag agc aau ggg cag ccg gag aac 1008
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
325 330 335
aac uac aag acc acg ccu ccc gug cug gac ucc gac ggc ucc uuc uuc 1056
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
340 345 350
cuc uac agc agg cua acc gug gac aag agc agg ugg cag gag ggg aau 1104
Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn
355 360 365
guc uuc uca ugc ucc gug aug cau gag gcu cug cac aac cac uac aca 1152
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
370 375 380
cag aag agc cuc ucc cug ucu cug ggu aaa 1182
Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
385 390
<210> 13
<211> 1269
<212> RNA
<213> 人工序列(Artificial Sequence)
<220>
<221> CDS
<222> (1)..(1269)
<223> MSLN-铰链-IgG4Fc
<400> 13
aug gaa gug gag aag aca gcc ugu ccu uca ggc aag aag gcc cgc gag 48
Met Glu Val Glu Lys Thr Ala Cys Pro Ser Gly Lys Lys Ala Arg Glu
1 5 10 15
aua gac gag agc cuc auc uuc uac aag aag ugg gag cug gaa gcc ugc 96
Ile Asp Glu Ser Leu Ile Phe Tyr Lys Lys Trp Glu Leu Glu Ala Cys
20 25 30
gug gau gcg gcc cug cug gcc acc cag aug gac cgc gug aac gcc auc 144
Val Asp Ala Ala Leu Leu Ala Thr Gln Met Asp Arg Val Asn Ala Ile
35 40 45
ccc uuc acc uac gag cag cug gac guc cua aag cau aaa cug gau gag 192
Pro Phe Thr Tyr Glu Gln Leu Asp Val Leu Lys His Lys Leu Asp Glu
50 55 60
cuc uac cca caa ggu uac ccc gag ucu gug auc cag cac cug ggc uac 240
Leu Tyr Pro Gln Gly Tyr Pro Glu Ser Val Ile Gln His Leu Gly Tyr
65 70 75 80
cuc uuc cuc aag aug agc ccu gag gac auu cgc aag ugg aau gug acg 288
Leu Phe Leu Lys Met Ser Pro Glu Asp Ile Arg Lys Trp Asn Val Thr
85 90 95
ucc cug gag acc cug aag gcu uug cuu gaa guc aac aaa ggg cac gaa 336
Ser Leu Glu Thr Leu Lys Ala Leu Leu Glu Val Asn Lys Gly His Glu
100 105 110
aug agu ccu cag gug gcc acc cug auc gac cgc uuu gug aag gga agg 384
Met Ser Pro Gln Val Ala Thr Leu Ile Asp Arg Phe Val Lys Gly Arg
115 120 125
ggc cag cua gac aaa gac acc cua gac acc cug acc gcc uuc uac ccu 432
Gly Gln Leu Asp Lys Asp Thr Leu Asp Thr Leu Thr Ala Phe Tyr Pro
130 135 140
ggg uac cug ugc ucc cuc agc ccc gag gag cug agc ucc gug ccc ccc 480
Gly Tyr Leu Cys Ser Leu Ser Pro Glu Glu Leu Ser Ser Val Pro Pro
145 150 155 160
agc agc auc ugg gcg guc agg ccc cag gac cug gac acg ugu gac cca 528
Ser Ser Ile Trp Ala Val Arg Pro Gln Asp Leu Asp Thr Cys Asp Pro
165 170 175
agg cag cug gac guc cuc uau ccc aag gcc cgc cuu gcu uuc cag aac 576
Arg Gln Leu Asp Val Leu Tyr Pro Lys Ala Arg Leu Ala Phe Gln Asn
180 185 190
aug aac ggg ucc gaa uac uuc gug aag auc cag ucc uuc cug ggu ggg 624
Met Asn Gly Ser Glu Tyr Phe Val Lys Ile Gln Ser Phe Leu Gly Gly
195 200 205
gcc ccc acg gag gau uug aag gcg cuc agu cag cag aau gug agc aug 672
Ala Pro Thr Glu Asp Leu Lys Ala Leu Ser Gln Gln Asn Val Ser Met
210 215 220
gac uug gcc acg uuc aug aag cug cgg acg gau gcg gug cug ccg uug 720
Asp Leu Ala Thr Phe Met Lys Leu Arg Thr Asp Ala Val Leu Pro Leu
225 230 235 240
acu gug gcu gag gug cag aaa cuu cug gga ccc cac gug gag ggc cug 768
Thr Val Ala Glu Val Gln Lys Leu Leu Gly Pro His Val Glu Gly Leu
245 250 255
aag gcg gag gag cgg cac cgc ccg gug cgg gac ugg auc cua cgg cag 816
Lys Ala Glu Glu Arg His Arg Pro Val Arg Asp Trp Ile Leu Arg Gln
260 265 270
cgg cag gac gac cug gac acg cug ggg cug ggg cua cag ggc ggc auc 864
Arg Gln Asp Asp Leu Asp Thr Leu Gly Leu Gly Leu Gln Gly Gly Ile
275 280 285
ccc aac ggc uac cug guc cua gac cuc agc aug caa gag gcc cuc ucg 912
Pro Asn Gly Tyr Leu Val Leu Asp Leu Ser Met Gln Glu Ala Leu Ser
290 295 300
gag ucc aaa uau ggu ccc cca ugc cca cca ugc cca ggg cag ccc cga 960
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gln Pro Arg
305 310 315 320
gag cca cag gug uac acc cug ccc cca ucc cag gag gag aug acc aag 1008
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
325 330 335
aac cag guc agc cug acc ugc cug guc aaa ggc uuc uac ccc agc gac 1056
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
340 345 350
auc gcc gug gag ugg gag agc aau ggg cag ccg gag aac aac uac aag 1104
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
355 360 365
acc acg ccu ccc gug cug gac ucc gac ggc ucc uuc uuc cuc uac agc 1152
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
370 375 380
agg cua acc gug gac aag agc agg ugg cag gag ggg aau guc uuc uca 1200
Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
385 390 395 400
ugc ucc gug aug cau gag gcu cug cac aac cac uac aca cag aag agc 1248
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
405 410 415
cuc ucc cug ucu cug ggu aaa 1269
Leu Ser Leu Ser Leu Gly Lys
420
<210> 14
<211> 1650
<212> RNA
<213> 人工序列(Artificial Sequence)
<220>
<221> CDS
<222> (1)..(1650)
<223> Muc1-铰链-IgG4Fc
<400> 14
aug ucu ggu cau gca agc ucu acc cca ggu gga gaa aag gag acu ucg 48
Met Ser Gly His Ala Ser Ser Thr Pro Gly Gly Glu Lys Glu Thr Ser
1 5 10 15
gcu acc cag aga agu uca gug ccc agc ucu acu gag aag aau gcu gug 96
Ala Thr Gln Arg Ser Ser Val Pro Ser Ser Thr Glu Lys Asn Ala Val
20 25 30
agu aug acc agc agc gua cuc ucc agc cac agc ccc ggu uca ggc ucc 144
Ser Met Thr Ser Ser Val Leu Ser Ser His Ser Pro Gly Ser Gly Ser
35 40 45
ucc acc acu cag gga cag gau guc acu cug gcc ccg gcc acg gaa cca 192
Ser Thr Thr Gln Gly Gln Asp Val Thr Leu Ala Pro Ala Thr Glu Pro
50 55 60
gcu uca ggu uca gcu gcc cug ugg gga cag gau guc acc ucg guc cca 240
Ala Ser Gly Ser Ala Ala Leu Trp Gly Gln Asp Val Thr Ser Val Pro
65 70 75 80
guc acc agg cca gcc cug ggc ucc acc acc ccg cca gcc cac gau guc 288
Val Thr Arg Pro Ala Leu Gly Ser Thr Thr Pro Pro Ala His Asp Val
85 90 95
acc uca gcc ccg gac aac aag cca gcc ccg ggc ucc acc gcc ccc cca 336
Thr Ser Ala Pro Asp Asn Lys Pro Ala Pro Gly Ser Thr Ala Pro Pro
100 105 110
gcc cac ggu guc acc ucg gcc ccg gac acc agg ccg gcc ccg ggc ucc 384
Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser
115 120 125
acc gcc ccc cca gcc cac ggu guc acc ucg gcc ccg gac acc agg ccg 432
Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro
130 135 140
gcc ccg ggc ucc acc gcc ccc cca gcc cac ggu guc acc ucg gcc ccg 480
Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro
145 150 155 160
gac acc agg ccg gcc ccg ggc ucc acc gcc ccc cca gcc cac ggu guc 528
Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val
165 170 175
acc ucg gcc ccg gac acc agg ccg gcc ccg ggc ucc acc gcc ccc cca 576
Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro
180 185 190
gcc cac ggu guc acc ucg gcc ccg gac acc agg ccg gcc ccg ggc ucc 624
Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser
195 200 205
acc gcc ccc cca gcc cau ggu guc acc ucg gcc ccg gac aac agg ccc 672
Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Asn Arg Pro
210 215 220
gcc uug ggc ucc acc gcc ccu cca guc cac aau guc acc ucg gcc uca 720
Ala Leu Gly Ser Thr Ala Pro Pro Val His Asn Val Thr Ser Ala Ser
225 230 235 240
ggc ucu gca uca ggc uca gcu ucu acu cug gug cac aac ggc acc ucu 768
Gly Ser Ala Ser Gly Ser Ala Ser Thr Leu Val His Asn Gly Thr Ser
245 250 255
gcc agg gcu acc aca acc cca gcc agc aag agc acu cca uuc uca auu 816
Ala Arg Ala Thr Thr Thr Pro Ala Ser Lys Ser Thr Pro Phe Ser Ile
260 265 270
ccc agc cac cac ucu gau acu ccu acc acc cuu gcc agc cau agc acc 864
Pro Ser His His Ser Asp Thr Pro Thr Thr Leu Ala Ser His Ser Thr
275 280 285
aag acu gau gcc agu agc acu cac cau agc ucg gua ccu ccu cuc acc 912
Lys Thr Asp Ala Ser Ser Thr His His Ser Ser Val Pro Pro Leu Thr
290 295 300
ucc ucc aau cac agc acu ucu ccc cag uug ucu acu ggg guc ucu uuc 960
Ser Ser Asn His Ser Thr Ser Pro Gln Leu Ser Thr Gly Val Ser Phe
305 310 315 320
uuu uuc cug ucu uuu cac auu uca aac cuc cag uuu aau ucc ucu cug 1008
Phe Phe Leu Ser Phe His Ile Ser Asn Leu Gln Phe Asn Ser Ser Leu
325 330 335
gaa gau ccc agc acc gac uac uac caa gag cug cag aga gac auu ucu 1056
Glu Asp Pro Ser Thr Asp Tyr Tyr Gln Glu Leu Gln Arg Asp Ile Ser
340 345 350
gaa aug uuu uug cag auu uau aaa caa ggg ggu uuu cug ggc cuc ucc 1104
Glu Met Phe Leu Gln Ile Tyr Lys Gln Gly Gly Phe Leu Gly Leu Ser
355 360 365
aau auu aag uuc agg cca gga ucu gug gug gua caa uug acu cug gcc 1152
Asn Ile Lys Phe Arg Pro Gly Ser Val Val Val Gln Leu Thr Leu Ala
370 375 380
uuc cga gaa ggu acc auc aau guc cac gac gug gag aca cag uuc aau 1200
Phe Arg Glu Gly Thr Ile Asn Val His Asp Val Glu Thr Gln Phe Asn
385 390 395 400
cag uau aaa acg gaa gca gcc ucu cga uau aac cug acg auc uca gac 1248
Gln Tyr Lys Thr Glu Ala Ala Ser Arg Tyr Asn Leu Thr Ile Ser Asp
405 410 415
guc agc gug agu gau gug cca uuu ccu uuc ucu gcc cag ucu ggg gag 1296
Val Ser Val Ser Asp Val Pro Phe Pro Phe Ser Ala Gln Ser Gly Glu
420 425 430
ucc aaa uau ggu ccc cca ugc cca cca ugc cca ggg cag ccc cga gag 1344
Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gln Pro Arg Glu
435 440 445
cca cag gug uac acc cug ccc cca ucc cag gag gag aug acc aag aac 1392
Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn
450 455 460
cag guc agc cug acc ugc cug guc aaa ggc uuc uac ccc agc gac auc 1440
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
465 470 475 480
gcc gug gag ugg gag agc aau ggg cag ccg gag aac aac uac aag acc 1488
Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr
485 490 495
acg ccu ccc gug cug gac ucc gac ggc ucc uuc uuc cuc uac agc agg 1536
Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg
500 505 510
cua acc gug gac aag agc agg ugg cag gag ggg aau guc uuc uca ugc 1584
Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys
515 520 525
ucc gug aug cau gag gcu cug cac aac cac uac aca cag aag agc cuc 1632
Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
530 535 540
ucc cug ucu cug ggu aaa 1650
Ser Leu Ser Leu Gly Lys
545
<210> 15
<211> 2220
<212> RNA
<213> 人工序列(Artificial Sequence)
<220>
<221> CDS
<222> (1)..(2220)
<223> EGFR-铰链-IgG4Fc
<400> 15
aug cug gag gaa aag aaa guu ugc caa ggc acg agu aac aag cuc acg 48
Met Leu Glu Glu Lys Lys Val Cys Gln Gly Thr Ser Asn Lys Leu Thr
1 5 10 15
cag uug ggc acu uuu gaa gau cau uuu cuc agc cuc cag agg aug uuc 96
Gln Leu Gly Thr Phe Glu Asp His Phe Leu Ser Leu Gln Arg Met Phe
20 25 30
aau aac ugu gag gug guc cuu ggg aau uug gaa auu acc uau gug cag 144
Asn Asn Cys Glu Val Val Leu Gly Asn Leu Glu Ile Thr Tyr Val Gln
35 40 45
agg aau uau gau cuu ucc uuc uua aag acc auc cag gag gug gcu ggu 192
Arg Asn Tyr Asp Leu Ser Phe Leu Lys Thr Ile Gln Glu Val Ala Gly
50 55 60
uau guc cuc auu gcc cuc aac aca gug gag cga auu ccu uug gaa aac 240
Tyr Val Leu Ile Ala Leu Asn Thr Val Glu Arg Ile Pro Leu Glu Asn
65 70 75 80
cug cag auc auc aga gga aau aug uac uac gaa aau ucc uau gcc uua 288
Leu Gln Ile Ile Arg Gly Asn Met Tyr Tyr Glu Asn Ser Tyr Ala Leu
85 90 95
gca guc uua ucu aac uau gau gca aau aaa acc gga cug aag gag cug 336
Ala Val Leu Ser Asn Tyr Asp Ala Asn Lys Thr Gly Leu Lys Glu Leu
100 105 110
ccc aug aga aau uua cag gaa auc cug cau ggc gcc gug cgg uuc agc 384
Pro Met Arg Asn Leu Gln Glu Ile Leu His Gly Ala Val Arg Phe Ser
115 120 125
aac aac ccu gcc cug ugc aac gug gag agc auc cag ugg cgg gac aua 432
Asn Asn Pro Ala Leu Cys Asn Val Glu Ser Ile Gln Trp Arg Asp Ile
130 135 140
guc agc agu gac uuu cuc agc aac aug ucg aug gac uuc cag aac cac 480
Val Ser Ser Asp Phe Leu Ser Asn Met Ser Met Asp Phe Gln Asn His
145 150 155 160
cug ggc agc ugc caa aag ugu gau cca agc ugu ccc aau ggg agc ugc 528
Leu Gly Ser Cys Gln Lys Cys Asp Pro Ser Cys Pro Asn Gly Ser Cys
165 170 175
ugg ggu gca gga gag gag aac ugc cag aaa cug acc aaa auc auc ugu 576
Trp Gly Ala Gly Glu Glu Asn Cys Gln Lys Leu Thr Lys Ile Ile Cys
180 185 190
gcc cag cag ugc ucc ggg cgc ugc cgu ggc aag ucc ccc agu gac ugc 624
Ala Gln Gln Cys Ser Gly Arg Cys Arg Gly Lys Ser Pro Ser Asp Cys
195 200 205
ugc cac aac cag ugu gcu gca ggc ugc aca ggc ccc cgg gag agc gac 672
Cys His Asn Gln Cys Ala Ala Gly Cys Thr Gly Pro Arg Glu Ser Asp
210 215 220
ugc cug guc ugc cgc aaa uuc cga gac gaa gcc acg ugc aag gac acc 720
Cys Leu Val Cys Arg Lys Phe Arg Asp Glu Ala Thr Cys Lys Asp Thr
225 230 235 240
ugc ccc cca cuc aug cuc uac aac ccc acc acg uac cag aug gau gug 768
Cys Pro Pro Leu Met Leu Tyr Asn Pro Thr Thr Tyr Gln Met Asp Val
245 250 255
aac ccc gag ggc aaa uac agc uuu ggu gcc acc ugc gug aag aag ugu 816
Asn Pro Glu Gly Lys Tyr Ser Phe Gly Ala Thr Cys Val Lys Lys Cys
260 265 270
ccc cgu aau uau gug gug aca gau cac ggc ucg ugc guc cga gcc ugu 864
Pro Arg Asn Tyr Val Val Thr Asp His Gly Ser Cys Val Arg Ala Cys
275 280 285
ggg gcc gac agc uau gag aug gag gaa gac ggc guc cgc aag ugu aag 912
Gly Ala Asp Ser Tyr Glu Met Glu Glu Asp Gly Val Arg Lys Cys Lys
290 295 300
aag ugc gaa ggg ccu ugc cgc aaa gug ugu aac gga aua ggu auu ggu 960
Lys Cys Glu Gly Pro Cys Arg Lys Val Cys Asn Gly Ile Gly Ile Gly
305 310 315 320
gaa uuu aaa gac uca cuc ucc aua aau gcu acg aau auu aaa cac uuc 1008
Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe
325 330 335
aaa aac ugc acc ucc auc agu ggc gau cuc cac auc cug ccg gug gca 1056
Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala
340 345 350
uuu agg ggu gac ucc uuc aca cau acu ccu ccu cug gau cca cag gaa 1104
Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu
355 360 365
cug gau auu cug aaa acc gua aag gaa auc aca ggg uuu uug cug auu 1152
Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile
370 375 380
cag gcu ugg ccu gaa aac agg acg gac cuc cau gcc uuu gag aac cua 1200
Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu
385 390 395 400
gaa auc aua cgc ggc agg acc aag caa cau ggu cag uuu ucu cuu gca 1248
Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala
405 410 415
guc guc agc cug aac aua aca ucc uug gga uua cgc ucc cuc aag gag 1296
Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu
420 425 430
aua agu gau gga gau gug aua auu uca gga aac aaa aau uug ugc uau 1344
Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr
435 440 445
gca aau aca aua aac ugg aaa aaa cug uuu ggg acc ucc ggu cag aaa 1392
Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys
450 455 460
acc aaa auu aua agc aac aga ggu gaa aac agc ugc aag gcc aca ggc 1440
Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly
465 470 475 480
cag guc ugc cau gcc uug ugc ucc ccc gag ggc ugc ugg ggc ccg gag 1488
Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu
485 490 495
ccc agg gac ugc guc ucu ugc cgg aau guc agc cga ggc agg gaa ugc 1536
Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys
500 505 510
gug gac aag ugc aac cuu cug gag ggu gag cca agg gag uuu gug gag 1584
Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu
515 520 525
aac ucu gag ugc aua cag ugc cac cca gag ugc cug ccu cag gcc aug 1632
Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met
530 535 540
aac auc acc ugc aca gga cgg gga cca gac aac ugu auc cag ugu gcc 1680
Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala
545 550 555 560
cac uac auu gac ggc ccc cac ugc guc aag acc ugc ccg gca gga guc 1728
His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val
565 570 575
aug gga gaa aac aac acc cug guc ugg aag uac gca gac gcc ggc cau 1776
Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His
580 585 590
gug ugc cac cug ugc cau cca aac ugc acc uac gga ugc acu ggg cca 1824
Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro
595 600 605
ggu cuu gaa ggc ugu cca acg aau ggg ccu aag auc ccg gag ucc aaa 1872
Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro Glu Ser Lys
610 615 620
uau ggu ccc cca ugc cca cca ugc cca ggg cag ccc cga gag cca cag 1920
Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gln Pro Arg Glu Pro Gln
625 630 635 640
gug uac acc cug ccc cca ucc cag gag gag aug acc aag aac cag guc 1968
Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
645 650 655
agc cug acc ugc cug guc aaa ggc uuc uac ccc agc gac auc gcc gug 2016
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
660 665 670
gag ugg gag agc aau ggg cag ccg gag aac aac uac aag acc acg ccu 2064
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
675 680 685
ccc gug cug gac ucc gac ggc ucc uuc uuc cuc uac agc agg cua acc 2112
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr
690 695 700
gug gac aag agc agg ugg cag gag ggg aau guc uuc uca ugc ucc gug 2160
Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val
705 710 715 720
aug cau gag gcu cug cac aac cac uac aca cag aag agc cuc ucc cug 2208
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
725 730 735
ucu cug ggu aaa 2220
Ser Leu Gly Lys
<210> 16
<211> 1461
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> CDS
<222> (1)..(1458)
<223> CD19-CAR
<400> 16
atg gcc tta cca gtg acc gcc ttg ctc ctg ccg ctg gcc ttg ctg ctc 48
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
cac gcc gcc agg ccg gaa att gtg atg acc cag tca ccc gcc act ctt 96
His Ala Ala Arg Pro Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu
20 25 30
agc ctt tca ccc ggt gag cgc gca acc ctg tct tgc aga gcc tcc caa 144
Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln
35 40 45
gac atc tca aaa tac ctt aat tgg tat caa cag aag ccc gga cag gct 192
Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Gln Ala
50 55 60
cct cgc ctt ctg atc tac cac acc agc cgg ctc cat tct gga atc cct 240
Pro Arg Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Ile Pro
65 70 75 80
gcc agg ttc agc ggt agc gga tct ggg acc gac tac acc ctc act atc 288
Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile
85 90 95
agc tca ctg cag cca gag gac ttc gct gtc tat ttc tgt cag caa ggg 336
Ser Ser Leu Gln Pro Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Gly
100 105 110
aac acc ctg ccc tac acc ttt gga cag ggc acc aag ctc gag att aaa 384
Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
115 120 125
ggt gga ggt ggc agc gga gga ggt ggg tcc ggc ggt gga gga agc cag 432
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
130 135 140
gtc caa ctc caa gaa agc gga ccg ggt ctt gtg aag cca tca gaa act 480
Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr
145 150 155 160
ctt tca ctg act tgt act gtg agc gga gtg tct ctc ccc gat tac ggg 528
Leu Ser Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly
165 170 175
gtg tct tgg atc aga cag cca ccg ggg aag ggt ctg gaa tgg att gga 576
Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile Gly
180 185 190
gtg att tgg ggc tct gag act act tac tac tct tca tcc ctc aag tca 624
Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Ser Ser Ser Leu Lys Ser
195 200 205
cgc gtc acc atc tca aag gac aac tct aag aat cag gtg tca ctg aaa 672
Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Asn Gln Val Ser Leu Lys
210 215 220
ctg tca tct gtg acc gca gcc gac acc gcc gtg tac tat tgc gct aag 720
Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Lys
225 230 235 240
cat tac tat tat ggc ggg agc tac gca atg gat tac tgg gga cag ggt 768
His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly
245 250 255
act ctg gtc acc gtg tcc agc acc acg acg cca gcg ccg cga cca cca 816
Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro
260 265 270
aca ccg gcg ccc acc atc gcg tcg cag ccc ctg tcc ctg cgc cca gag 864
Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu
275 280 285
gcg tgc cgg cca gcg gcg ggg ggc gca gtg cac acg agg ggg ctg gac 912
Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp
290 295 300
ttc gcc tgt gat atc tac atc tgg gcg ccc ctg gcc ggg act tgt ggg 960
Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly
305 310 315 320
gtc ctt ctc ctg tca ctg gtt atc acc ctt tac tgc aaa cgg ggc aga 1008
Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg
325 330 335
aag aag ctc ctg tat ata ttc aaa caa cca ttt atg aga cca gta caa 1056
Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln
340 345 350
act act caa gag gaa gat ggc tgt agc tgc cga ttt cca gaa gaa gaa 1104
Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu
355 360 365
gaa gga gga tgt gaa ctg aga gtg aag ttc agc agg agc gca gac gcc 1152
Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala
370 375 380
ccc gcg tac cag cag ggc cag aac cag ctc tat aac gag ctc aat cta 1200
Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu
385 390 395 400
gga cga aga gag gag tac gat gtt ttg gac aag aga cgt ggc cgg gac 1248
Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp
405 410 415
cct gag atg ggg gga aag ccg aga agg aag aac cct cag gaa ggc ctg 1296
Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu
420 425 430
tac aat gaa ctg cag aaa gat aag atg gcg gag gcc tac agt gag att 1344
Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile
435 440 445
ggg atg aaa ggc gag cgc cgg agg ggc aag ggg cac gat ggc ctt tac 1392
Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr
450 455 460
cag ggt ctc agt aca gcc acc aag gac acc tac gac gcc ctt cac atg 1440
Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met
465 470 475 480
cag gcc ctg ccc cct cgc tga 1461
Gln Ala Leu Pro Pro Arg
485
<210> 17
<211> 1479
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> CDS
<222> (1)..(1479)
<223> MSLN-CAR
<400> 17
atg gcc tta cca gtg acc gcc ttg ctc ctg ccg ctg gcc ttg ctg ctc 48
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
cac gcc gcc agg ccg gac gtg gtg atg acc cag acc ccc gcc agc gtg 96
His Ala Ala Arg Pro Asp Val Val Met Thr Gln Thr Pro Ala Ser Val
20 25 30
agc gag ccc gtg ggc ggc acc gtg acc atc aag tgc cag gcc agc cag 144
Ser Glu Pro Val Gly Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Gln
35 40 45
agg atc agc agc tac ctg agc tgg tac cag cag aag ccc ggc cag agg 192
Arg Ile Ser Ser Tyr Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Arg
50 55 60
ccc aag ctg ctg atc ttc ggc gcc agc acc ctg gcc agc ggc gtg ccc 240
Pro Lys Leu Leu Ile Phe Gly Ala Ser Thr Leu Ala Ser Gly Val Pro
65 70 75 80
agc agg ttc aag ggc agc ggc agc ggc acc gag tac acc ctg acc atc 288
Ser Arg Phe Lys Gly Ser Gly Ser Gly Thr Glu Tyr Thr Leu Thr Ile
85 90 95
agc gac ctg gag tgc gcc gac gcc gcc acc tac tac tgc cag agc tac 336
Ser Asp Leu Glu Cys Ala Asp Ala Ala Thr Tyr Tyr Cys Gln Ser Tyr
100 105 110
gcc tac ttc gac agc aac aac tgg cac gcc ttc ggc ggc ggc acc gag 384
Ala Tyr Phe Asp Ser Asn Asn Trp His Ala Phe Gly Gly Gly Thr Glu
115 120 125
gtg gtg gtg ggc ggc ggc ggc agc ggc ggc ggc ggc agc ggc ggc ggc 432
Val Val Val Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
130 135 140
ggc agc cag cag cag ctg gag gag agc ggc ggc ggc ctg gtg aag ccc 480
Gly Ser Gln Gln Gln Leu Glu Glu Ser Gly Gly Gly Leu Val Lys Pro
145 150 155 160
gag ggc agc ctg acc ctg acc tgc aag gcc agc ggc ttc gac ctg ggc 528
Glu Gly Ser Leu Thr Leu Thr Cys Lys Ala Ser Gly Phe Asp Leu Gly
165 170 175
ttc tac ttc tac gcc tgc tgg gtg agg cag gcc ccc ggc aag ggc ctg 576
Phe Tyr Phe Tyr Ala Cys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
180 185 190
gag tgg atc gcc tgc atc tac acc gcc ggc agc ggc agc acc tac tac 624
Glu Trp Ile Ala Cys Ile Tyr Thr Ala Gly Ser Gly Ser Thr Tyr Tyr
195 200 205
gcc agc tgg gcc aag ggc agg ttc acc atc agc aag gcc agc agc acc 672
Ala Ser Trp Ala Lys Gly Arg Phe Thr Ile Ser Lys Ala Ser Ser Thr
210 215 220
acc gtg acc ctg cag atg acc agc ctg gcc gcc gcc gac acc gcc acc 720
Thr Val Thr Leu Gln Met Thr Ser Leu Ala Ala Ala Asp Thr Ala Thr
225 230 235 240
tac ttc tgc gcc agg agc acc gcc aac acc agg agc acc tac tac ctg 768
Tyr Phe Cys Ala Arg Ser Thr Ala Asn Thr Arg Ser Thr Tyr Tyr Leu
245 250 255
aac ctg tgg ggc ccc ggc acc ctg gtg acc gtg agc agc acc acg acg 816
Asn Leu Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr
260 265 270
cca gcg ccg cga cca cca aca ccg gcg ccc acc atc gcg tcg cag ccc 864
Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro
275 280 285
ctg tcc ctg cgc cca gag gcg tgc cgg cca gcg gcg ggg ggc gca gtg 912
Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val
290 295 300
cac acg agg ggg ctg gac ttc gcc tgt gat atc tac atc tgg gcg ccc 960
His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro
305 310 315 320
ctg gcc ggg act tgt ggg gtc ctt ctc ctg tca ctg gtt atc acc ctt 1008
Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu
325 330 335
tac tgc aaa cgg ggc aga aag aag ctc ctg tat ata ttc aaa caa cca 1056
Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro
340 345 350
ttt atg aga cca gta caa act act caa gag gaa gat ggc tgt agc tgc 1104
Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys
355 360 365
cga ttt cca gaa gaa gaa gaa gga gga tgt gaa ctg aga gtg aag ttc 1152
Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe
370 375 380
agc agg agc gca gac gcc ccc gcg tac cag cag ggc cag aac cag ctc 1200
Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu
385 390 395 400
tat aac gag ctc aat cta gga cga aga gag gag tac gat gtt ttg gac 1248
Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp
405 410 415
aag aga cgt ggc cgg gac cct gag atg ggg gga aag ccg aga agg aag 1296
Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys
420 425 430
aac cct cag gaa ggc ctg tac aat gaa ctg cag aaa gat aag atg gcg 1344
Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala
435 440 445
gag gcc tac agt gag att ggg atg aaa ggc gag cgc cgg agg ggc aag 1392
Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys
450 455 460
ggg cac gat ggc ctt tac cag ggt ctc agt aca gcc acc aag gac acc 1440
Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr
465 470 475 480
tac gac gcc ctt cac atg cag gcc ctg ccc cct cgc tga 1479
Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
485 490
<210> 18
<211> 1461
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> CDS
<222> (1)..(1458)
<223> Muc1-CAR
<400> 18
atg gcc tta cca gtg acc gcc ttg ctc ctg ccg ctg gcc ttg ctg ctc 48
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
cac gcc gcc agg ccg cag agc gtg ctg acc cag ccc ccc agc gtg agc 96
His Ala Ala Arg Pro Gln Ser Val Leu Thr Gln Pro Pro Ser Val Ser
20 25 30
gtg gcc ccc ggc aag acc gcc agg atc acc tgc ggc ggc aac aac atc 144
Val Ala Pro Gly Lys Thr Ala Arg Ile Thr Cys Gly Gly Asn Asn Ile
35 40 45
ggc agc aag agc gtg cac tgg tac cag cag aag ccc ggc cag gcc ccc 192
Gly Ser Lys Ser Val His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
50 55 60
gtg ctg gtg atc tac tac gac agc gac agg ccc agc ggc atc ccc gag 240
Val Leu Val Ile Tyr Tyr Asp Ser Asp Arg Pro Ser Gly Ile Pro Glu
65 70 75 80
agg ttc agc ggc agc aac agc ggc aac acc gcc acc ctg acc atc agc 288
Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser
85 90 95
agg gtg gag gcc ggc gac gag gcc gac tac tac tgc cag gtg tgg gac 336
Arg Val Glu Ala Gly Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp
100 105 110
agc agc agc gac tgg gtg ttc ggc ggc ggc acc aag ctg acc gtg ctg 384
Ser Ser Ser Asp Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu
115 120 125
ggc ggc ggc ggc agc ggc ggc ggc ggc agc ggc ggc ggc ggc agc cag 432
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
130 135 140
atg cag ctg gtg cag agc ggc gcc gag gtg aag aag ccc ggc gcc agc 480
Met Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala Ser
145 150 155 160
gtg aag gtg agc tgc aag gcc agc ggc tac acc ttc acc ggc cac tac 528
Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Gly His Tyr
165 170 175
atg cac tgg gtg agg cag gcc ccc ggc cag ggc ctg gag tgg atg ggc 576
Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met Gly
180 185 190
tgg atc gac ccc gtg acc ggc ggc acc aag tac gcc cag aac ttc cag 624
Trp Ile Asp Pro Val Thr Gly Gly Thr Lys Tyr Ala Gln Asn Phe Gln
195 200 205
ggc tgg gtg acc atg acc agg gac acc agc atc agg acc gcc tac atg 672
Gly Trp Val Thr Met Thr Arg Asp Thr Ser Ile Arg Thr Ala Tyr Met
210 215 220
gag ctg agc agg ctg agg agc gac gac acc gcc atg tac tac tgc gcc 720
Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Met Tyr Tyr Cys Ala
225 230 235 240
agg gag gtg acc ggc gac agg ggc cag ttc gac aag tgg ggc cag ggc 768
Arg Glu Val Thr Gly Asp Arg Gly Gln Phe Asp Lys Trp Gly Gln Gly
245 250 255
acc ctg gtg acc gtg gcc agc acc acg acg cca gcg ccg cga cca cca 816
Thr Leu Val Thr Val Ala Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro
260 265 270
aca ccg gcg ccc acc atc gcg tcg cag ccc ctg tcc ctg cgc cca gag 864
Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu
275 280 285
gcg tgc cgg cca gcg gcg ggg ggc gca gtg cac acg agg ggg ctg gac 912
Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp
290 295 300
ttc gcc tgt gat atc tac atc tgg gcg ccc ctg gcc ggg act tgt ggg 960
Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly
305 310 315 320
gtc ctt ctc ctg tca ctg gtt atc acc ctt tac tgc aaa cgg ggc aga 1008
Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg
325 330 335
aag aag ctc ctg tat ata ttc aaa caa cca ttt atg aga cca gta caa 1056
Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln
340 345 350
act act caa gag gaa gat ggc tgt agc tgc cga ttt cca gaa gaa gaa 1104
Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu
355 360 365
gaa gga gga tgt gaa ctg aga gtg aag ttc agc agg agc gca gac gcc 1152
Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala
370 375 380
ccc gcg tac cag cag ggc cag aac cag ctc tat aac gag ctc aat cta 1200
Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu
385 390 395 400
gga cga aga gag gag tac gat gtt ttg gac aag aga cgt ggc cgg gac 1248
Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp
405 410 415
cct gag atg ggg gga aag ccg aga agg aag aac cct cag gaa ggc ctg 1296
Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu
420 425 430
tac aat gaa ctg cag aaa gat aag atg gcg gag gcc tac agt gag att 1344
Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile
435 440 445
ggg atg aaa ggc gag cgc cgg agg ggc aag ggg cac gat ggc ctt tac 1392
Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr
450 455 460
cag ggt ctc agt aca gcc acc aag gac acc tac gac gcc ctt cac atg 1440
Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met
465 470 475 480
cag gcc ctg ccc cct cgc tga 1461
Gln Ala Leu Pro Pro Arg
485
<210> 19
<211> 1473
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> CDS
<222> (1)..(1470)
<223> EGFR-CAR
<400> 19
atg gcc tta cca gtg acc gcc ttg ctc ctg ccg ctg gcc ttg ctg ctc 48
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
cac gcc gcc agg ccg gaa atc cag ctg gtc caa tcg gga gct gag gtc 96
His Ala Ala Arg Pro Glu Ile Gln Leu Val Gln Ser Gly Ala Glu Val
20 25 30
aag aag ccg gga gcc acc gtc aag atc tca tgc aag ggg tcg gga ttc 144
Lys Lys Pro Gly Ala Thr Val Lys Ile Ser Cys Lys Gly Ser Gly Phe
35 40 45
aac atc gag gac tac tac att cac tgg gtg cag caa gct ccg gga aaa 192
Asn Ile Glu Asp Tyr Tyr Ile His Trp Val Gln Gln Ala Pro Gly Lys
50 55 60
ggc ctg gaa tgg atg ggc aga atc gac cca gaa aac gac gaa act aag 240
Gly Leu Glu Trp Met Gly Arg Ile Asp Pro Glu Asn Asp Glu Thr Lys
65 70 75 80
tac gga ccg att ttc caa gga aga gtg act atc acc gcc gat act tca 288
Tyr Gly Pro Ile Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser
85 90 95
acc aat acc gtc tac atg gaa ctg agc tcg ctc cgg tcc gaa gat act 336
Thr Asn Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
100 105 110
gca gtg tat tac tgt gcc ttt cgc gga ggg gtg tac tgg ggc caa gga 384
Ala Val Tyr Tyr Cys Ala Phe Arg Gly Gly Val Tyr Trp Gly Gln Gly
115 120 125
act act gtc act gtc tcg tca gga ggc gga ggg tcg gga gga ggc ggg 432
Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
130 135 140
agc gga ggc ggt ggc tcg ggt ggc gga gga agc gac gtg gtg atg acc 480
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr
145 150 155 160
cag tcc ccg gac tcc ctc gcc gtg agc ctc gga gag agg gcg act atc 528
Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile
165 170 175
aat tgc aag tcg tcc cag tca ctt ctg gat tcc gat ggt aaa acg tac 576
Asn Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser Asp Gly Lys Thr Tyr
180 185 190
ctc aac tgg ctg cag caa aag cca ggg cag cca ccc aaa cgg ttg atc 624
Leu Asn Trp Leu Gln Gln Lys Pro Gly Gln Pro Pro Lys Arg Leu Ile
195 200 205
tcc ctt gtg tcc aaa ctg gat agc gga gtg cct gac cgc ttc tcg ggt 672
Ser Leu Val Ser Lys Leu Asp Ser Gly Val Pro Asp Arg Phe Ser Gly
210 215 220
tcc ggt agc ggg acc gac ttc acc ctg acg atc agc tca ctg cag gcg 720
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala
225 230 235 240
gag gac gtg gca gtg tac tac tgc tgg cag gga acc cac ttc cct ggc 768
Glu Asp Val Ala Val Tyr Tyr Cys Trp Gln Gly Thr His Phe Pro Gly
245 250 255
acc ttt gga ggt ggc acc aag gtg gag atc aag acc acg acg cca gcg 816
Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Thr Thr Thr Pro Ala
260 265 270
ccg cga cca cca aca ccg gcg ccc acc atc gcg tcg cag ccc ctg tcc 864
Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser
275 280 285
ctg cgc cca gag gcg tgc cgg cca gcg gcg ggg ggc gca gtg cac acg 912
Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr
290 295 300
agg ggg ctg gac ttc gcc tgt gat atc tac atc tgg gcg ccc ctg gcc 960
Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala
305 310 315 320
ggg act tgt ggg gtc ctt ctc ctg tca ctg gtt atc acc ctt tac tgc 1008
Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys
325 330 335
aaa cgg ggc aga aag aag ctc ctg tat ata ttc aaa caa cca ttt atg 1056
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
340 345 350
aga cca gta caa act act caa gag gaa gat ggc tgt agc tgc cga ttt 1104
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
355 360 365
cca gaa gaa gaa gaa gga gga tgt gaa ctg aga gtg aag ttc agc agg 1152
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg
370 375 380
agc gca gac gcc ccc gcg tac cag cag ggc cag aac cag ctc tat aac 1200
Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn
385 390 395 400
gag ctc aat cta gga cga aga gag gag tac gat gtt ttg gac aag aga 1248
Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg
405 410 415
cgt ggc cgg gac cct gag atg ggg gga aag ccg aga agg aag aac cct 1296
Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro
420 425 430
cag gaa ggc ctg tac aat gaa ctg cag aaa gat aag atg gcg gag gcc 1344
Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala
435 440 445
tac agt gag att ggg atg aaa ggc gag cgc cgg agg ggc aag ggg cac 1392
Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His
450 455 460
gat ggc ctt tac cag ggt ctc agt aca gcc acc aag gac acc tac gac 1440
Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp
465 470 475 480
gcc ctt cac atg cag gcc ctg ccc cct cgc tga 1473
Ala Leu His Met Gln Ala Leu Pro Pro Arg
485 490
<210> 20
<211> 413
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(413)
<223> SP-CD19-铰链-IgG4Fc
<400> 20
Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro
1 5 10 15
Asp Thr Thr Gly Pro Glu Glu Pro Leu Val Val Lys Val Glu Glu Gly
20 25 30
Asp Asn Ala Val Leu Gln Cys Leu Lys Gly Thr Ser Asp Gly Pro Thr
35 40 45
Gln Gln Leu Thr Trp Ser Arg Glu Ser Pro Leu Lys Pro Phe Leu Lys
50 55 60
Leu Ser Leu Gly Leu Pro Gly Leu Gly Ile His Met Arg Pro Leu Ala
65 70 75 80
Ile Trp Leu Phe Ile Phe Asn Val Ser Gln Gln Met Gly Gly Phe Tyr
85 90 95
Leu Cys Gln Pro Gly Pro Pro Ser Glu Lys Ala Trp Gln Pro Gly Trp
100 105 110
Thr Val Asn Val Glu Gly Ser Gly Glu Leu Phe Arg Trp Asn Val Ser
115 120 125
Asp Leu Gly Gly Leu Gly Cys Gly Leu Lys Asn Arg Ser Ser Glu Gly
130 135 140
Pro Ser Ser Pro Ser Gly Lys Leu Met Ser Pro Lys Leu Tyr Val Trp
145 150 155 160
Ala Lys Asp Arg Pro Glu Ile Trp Glu Gly Glu Pro Pro Cys Leu Pro
165 170 175
Pro Arg Asp Ser Leu Asn Gln Ser Leu Ser Gln Asp Leu Thr Met Ala
180 185 190
Pro Gly Ser Thr Leu Trp Leu Ser Cys Gly Val Pro Pro Asp Ser Val
195 200 205
Ser Arg Gly Pro Leu Ser Trp Thr His Val His Pro Lys Gly Pro Lys
210 215 220
Ser Leu Leu Ser Leu Glu Leu Lys Asp Asp Arg Pro Ala Arg Asp Met
225 230 235 240
Trp Val Met Glu Thr Gly Leu Leu Leu Pro Arg Ala Thr Ala Gln Asp
245 250 255
Ala Gly Lys Tyr Tyr Cys His Arg Gly Asn Leu Thr Met Ser Phe His
260 265 270
Leu Glu Ile Thr Ala Arg Pro Val Leu Trp His Trp Leu Leu Arg Thr
275 280 285
Gly Gly Trp Lys Val Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro
290 295 300
Cys Pro Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
305 310 315 320
Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
325 330 335
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
340 345 350
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
355 360 365
Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln
370 375 380
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
385 390 395 400
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
405 410
<210> 21
<211> 1242
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> CDS
<222> (1)..(1239)
<223> SP-CD19-铰链-IgG4Fc
<400> 21
atg gaa gcc cca gct cag ctt ctc ttc ctc ctg cta ctc tgg ctc cca 48
Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro
1 5 10 15
gat acc acc gga ccc gag gaa cct cta gtg gtg aag gtg gaa gag gga 96
Asp Thr Thr Gly Pro Glu Glu Pro Leu Val Val Lys Val Glu Glu Gly
20 25 30
gat aac gct gtg ctg cag tgc ctc aag ggg acc tca gat ggc ccc act 144
Asp Asn Ala Val Leu Gln Cys Leu Lys Gly Thr Ser Asp Gly Pro Thr
35 40 45
cag cag ctg acc tgg tct cgg gag tcc ccg ctt aaa ccc ttc tta aaa 192
Gln Gln Leu Thr Trp Ser Arg Glu Ser Pro Leu Lys Pro Phe Leu Lys
50 55 60
ctc agc ctg ggg ctg cca ggc ctg gga atc cac atg agg ccc ctg gcc 240
Leu Ser Leu Gly Leu Pro Gly Leu Gly Ile His Met Arg Pro Leu Ala
65 70 75 80
atc tgg ctt ttc atc ttc aac gtc tct caa cag atg ggg ggc ttc tac 288
Ile Trp Leu Phe Ile Phe Asn Val Ser Gln Gln Met Gly Gly Phe Tyr
85 90 95
ctg tgc cag ccg ggg ccc ccc tct gag aag gcc tgg cag cct ggc tgg 336
Leu Cys Gln Pro Gly Pro Pro Ser Glu Lys Ala Trp Gln Pro Gly Trp
100 105 110
aca gtc aat gtg gag ggc agc ggg gag ctg ttc cgg tgg aat gtt tcg 384
Thr Val Asn Val Glu Gly Ser Gly Glu Leu Phe Arg Trp Asn Val Ser
115 120 125
gac cta ggt ggc ctg ggc tgt ggc ctg aag aac agg tcc tca gag ggc 432
Asp Leu Gly Gly Leu Gly Cys Gly Leu Lys Asn Arg Ser Ser Glu Gly
130 135 140
ccc agc tcc cct tcc ggg aag ctc atg agc ccc aag ctg tat gtg tgg 480
Pro Ser Ser Pro Ser Gly Lys Leu Met Ser Pro Lys Leu Tyr Val Trp
145 150 155 160
gcc aaa gac cgc cct gag atc tgg gag gga gag cct ccg tgt ctc cca 528
Ala Lys Asp Arg Pro Glu Ile Trp Glu Gly Glu Pro Pro Cys Leu Pro
165 170 175
ccg agg gac agc ctg aac cag agc ctc agc cag gac ctc acc atg gcc 576
Pro Arg Asp Ser Leu Asn Gln Ser Leu Ser Gln Asp Leu Thr Met Ala
180 185 190
cct ggc tcc aca ctc tgg ctg tcc tgt ggg gta ccc cct gac tct gtg 624
Pro Gly Ser Thr Leu Trp Leu Ser Cys Gly Val Pro Pro Asp Ser Val
195 200 205
tcc agg ggc ccc ctc tcc tgg acc cat gtg cac ccc aag ggg cct aag 672
Ser Arg Gly Pro Leu Ser Trp Thr His Val His Pro Lys Gly Pro Lys
210 215 220
tca ttg ctg agc cta gag ctg aag gac gat cgc ccg gcc aga gat atg 720
Ser Leu Leu Ser Leu Glu Leu Lys Asp Asp Arg Pro Ala Arg Asp Met
225 230 235 240
tgg gta atg gag acg ggt ctg ttg ttg ccc cgg gcc aca gct caa gac 768
Trp Val Met Glu Thr Gly Leu Leu Leu Pro Arg Ala Thr Ala Gln Asp
245 250 255
gct gga aag tat tat tgt cac cgt ggc aac ctg acc atg tca ttc cac 816
Ala Gly Lys Tyr Tyr Cys His Arg Gly Asn Leu Thr Met Ser Phe His
260 265 270
ctg gag atc act gct cgg cca gta cta tgg cac tgg ctg ctg agg act 864
Leu Glu Ile Thr Ala Arg Pro Val Leu Trp His Trp Leu Leu Arg Thr
275 280 285
ggt ggc tgg aag gtc tca gag tcc aaa tat ggt ccc cca tgc cca cca 912
Gly Gly Trp Lys Val Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro
290 295 300
tgc cca ggg cag ccc cga gag cca cag gtg tac acc ctg ccc cca tcc 960
Cys Pro Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
305 310 315 320
cag gag gag atg acc aag aac cag gtc agc ctg acc tgc ctg gtc aaa 1008
Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
325 330 335
ggc ttc tac ccc agc gac atc gcc gtg gag tgg gag agc aat ggg cag 1056
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
340 345 350
ccg gag aac aac tac aag acc acg cct ccc gtg ctg gac tcc gac ggc 1104
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
355 360 365
tcc ttc ttc ctc tac agc agg cta acc gtg gac aag agc agg tgg cag 1152
Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln
370 375 380
gag ggg aat gtc ttc tca tgc tcc gtg atg cat gag gct ctg cac aac 1200
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn
385 390 395 400
cac tac aca cag aag agc ctc tcc ctg tct ctg ggt aaa tga 1242
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
405 410
<210> 22
<211> 442
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(442)
<223> SP-MSLN-铰链-IgG4Fc
<400> 22
Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro
1 5 10 15
Asp Thr Thr Gly Glu Val Glu Lys Thr Ala Cys Pro Ser Gly Lys Lys
20 25 30
Ala Arg Glu Ile Asp Glu Ser Leu Ile Phe Tyr Lys Lys Trp Glu Leu
35 40 45
Glu Ala Cys Val Asp Ala Ala Leu Leu Ala Thr Gln Met Asp Arg Val
50 55 60
Asn Ala Ile Pro Phe Thr Tyr Glu Gln Leu Asp Val Leu Lys His Lys
65 70 75 80
Leu Asp Glu Leu Tyr Pro Gln Gly Tyr Pro Glu Ser Val Ile Gln His
85 90 95
Leu Gly Tyr Leu Phe Leu Lys Met Ser Pro Glu Asp Ile Arg Lys Trp
100 105 110
Asn Val Thr Ser Leu Glu Thr Leu Lys Ala Leu Leu Glu Val Asn Lys
115 120 125
Gly His Glu Met Ser Pro Gln Val Ala Thr Leu Ile Asp Arg Phe Val
130 135 140
Lys Gly Arg Gly Gln Leu Asp Lys Asp Thr Leu Asp Thr Leu Thr Ala
145 150 155 160
Phe Tyr Pro Gly Tyr Leu Cys Ser Leu Ser Pro Glu Glu Leu Ser Ser
165 170 175
Val Pro Pro Ser Ser Ile Trp Ala Val Arg Pro Gln Asp Leu Asp Thr
180 185 190
Cys Asp Pro Arg Gln Leu Asp Val Leu Tyr Pro Lys Ala Arg Leu Ala
195 200 205
Phe Gln Asn Met Asn Gly Ser Glu Tyr Phe Val Lys Ile Gln Ser Phe
210 215 220
Leu Gly Gly Ala Pro Thr Glu Asp Leu Lys Ala Leu Ser Gln Gln Asn
225 230 235 240
Val Ser Met Asp Leu Ala Thr Phe Met Lys Leu Arg Thr Asp Ala Val
245 250 255
Leu Pro Leu Thr Val Ala Glu Val Gln Lys Leu Leu Gly Pro His Val
260 265 270
Glu Gly Leu Lys Ala Glu Glu Arg His Arg Pro Val Arg Asp Trp Ile
275 280 285
Leu Arg Gln Arg Gln Asp Asp Leu Asp Thr Leu Gly Leu Gly Leu Gln
290 295 300
Gly Gly Ile Pro Asn Gly Tyr Leu Val Leu Asp Leu Ser Met Gln Glu
305 310 315 320
Ala Leu Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly
325 330 335
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu
340 345 350
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
355 360 365
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
370 375 380
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
385 390 395 400
Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn
405 410 415
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
420 425 430
Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
435 440
<210> 23
<211> 1329
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> CDS
<222> (1)..(1326)
<223> SP-MSLN-铰链-IgG4Fc
<400> 23
atg gaa gcc cca gct cag ctt ctc ttc ctc ctg cta ctc tgg ctc cca 48
Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro
1 5 10 15
gat acc acc gga gaa gtg gag aag aca gcc tgt cct tca ggc aag aag 96
Asp Thr Thr Gly Glu Val Glu Lys Thr Ala Cys Pro Ser Gly Lys Lys
20 25 30
gcc cgc gag ata gac gag agc ctc atc ttc tac aag aag tgg gag ctg 144
Ala Arg Glu Ile Asp Glu Ser Leu Ile Phe Tyr Lys Lys Trp Glu Leu
35 40 45
gaa gcc tgc gtg gat gcg gcc ctg ctg gcc acc cag atg gac cgc gtg 192
Glu Ala Cys Val Asp Ala Ala Leu Leu Ala Thr Gln Met Asp Arg Val
50 55 60
aac gcc atc ccc ttc acc tac gag cag ctg gac gtc cta aag cat aaa 240
Asn Ala Ile Pro Phe Thr Tyr Glu Gln Leu Asp Val Leu Lys His Lys
65 70 75 80
ctg gat gag ctc tac cca caa ggt tac ccc gag tct gtg atc cag cac 288
Leu Asp Glu Leu Tyr Pro Gln Gly Tyr Pro Glu Ser Val Ile Gln His
85 90 95
ctg ggc tac ctc ttc ctc aag atg agc cct gag gac att cgc aag tgg 336
Leu Gly Tyr Leu Phe Leu Lys Met Ser Pro Glu Asp Ile Arg Lys Trp
100 105 110
aat gtg acg tcc ctg gag acc ctg aag gct ttg ctt gaa gtc aac aaa 384
Asn Val Thr Ser Leu Glu Thr Leu Lys Ala Leu Leu Glu Val Asn Lys
115 120 125
ggg cac gaa atg agt cct cag gtg gcc acc ctg atc gac cgc ttt gtg 432
Gly His Glu Met Ser Pro Gln Val Ala Thr Leu Ile Asp Arg Phe Val
130 135 140
aag gga agg ggc cag cta gac aaa gac acc cta gac acc ctg acc gcc 480
Lys Gly Arg Gly Gln Leu Asp Lys Asp Thr Leu Asp Thr Leu Thr Ala
145 150 155 160
ttc tac cct ggg tac ctg tgc tcc ctc agc ccc gag gag ctg agc tcc 528
Phe Tyr Pro Gly Tyr Leu Cys Ser Leu Ser Pro Glu Glu Leu Ser Ser
165 170 175
gtg ccc ccc agc agc atc tgg gcg gtc agg ccc cag gac ctg gac acg 576
Val Pro Pro Ser Ser Ile Trp Ala Val Arg Pro Gln Asp Leu Asp Thr
180 185 190
tgt gac cca agg cag ctg gac gtc ctc tat ccc aag gcc cgc ctt gct 624
Cys Asp Pro Arg Gln Leu Asp Val Leu Tyr Pro Lys Ala Arg Leu Ala
195 200 205
ttc cag aac atg aac ggg tcc gaa tac ttc gtg aag atc cag tcc ttc 672
Phe Gln Asn Met Asn Gly Ser Glu Tyr Phe Val Lys Ile Gln Ser Phe
210 215 220
ctg ggt ggg gcc ccc acg gag gat ttg aag gcg ctc agt cag cag aat 720
Leu Gly Gly Ala Pro Thr Glu Asp Leu Lys Ala Leu Ser Gln Gln Asn
225 230 235 240
gtg agc atg gac ttg gcc acg ttc atg aag ctg cgg acg gat gcg gtg 768
Val Ser Met Asp Leu Ala Thr Phe Met Lys Leu Arg Thr Asp Ala Val
245 250 255
ctg ccg ttg act gtg gct gag gtg cag aaa ctt ctg gga ccc cac gtg 816
Leu Pro Leu Thr Val Ala Glu Val Gln Lys Leu Leu Gly Pro His Val
260 265 270
gag ggc ctg aag gcg gag gag cgg cac cgc ccg gtg cgg gac tgg atc 864
Glu Gly Leu Lys Ala Glu Glu Arg His Arg Pro Val Arg Asp Trp Ile
275 280 285
cta cgg cag cgg cag gac gac ctg gac acg ctg ggg ctg ggg cta cag 912
Leu Arg Gln Arg Gln Asp Asp Leu Asp Thr Leu Gly Leu Gly Leu Gln
290 295 300
ggc ggc atc ccc aac ggc tac ctg gtc cta gac ctc agc atg caa gag 960
Gly Gly Ile Pro Asn Gly Tyr Leu Val Leu Asp Leu Ser Met Gln Glu
305 310 315 320
gcc ctc tcg gag tcc aaa tat ggt ccc cca tgc cca cca tgc cca ggg 1008
Ala Leu Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly
325 330 335
cag ccc cga gag cca cag gtg tac acc ctg ccc cca tcc cag gag gag 1056
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu
340 345 350
atg acc aag aac cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tac 1104
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
355 360 365
ccc agc gac atc gcc gtg gag tgg gag agc aat ggg cag ccg gag aac 1152
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
370 375 380
aac tac aag acc acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc 1200
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
385 390 395 400
ctc tac agc agg cta acc gtg gac aag agc agg tgg cag gag ggg aat 1248
Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn
405 410 415
gtc ttc tca tgc tcc gtg atg cat gag gct ctg cac aac cac tac aca 1296
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
420 425 430
cag aag agc ctc tcc ctg tct ctg ggt aaa tga 1329
Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
435 440
<210> 24
<211> 569
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(569)
<223> SP-Muc1-铰链-IgG4Fc
<400> 24
Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro
1 5 10 15
Asp Thr Thr Gly Ser Gly His Ala Ser Ser Thr Pro Gly Gly Glu Lys
20 25 30
Glu Thr Ser Ala Thr Gln Arg Ser Ser Val Pro Ser Ser Thr Glu Lys
35 40 45
Asn Ala Val Ser Met Thr Ser Ser Val Leu Ser Ser His Ser Pro Gly
50 55 60
Ser Gly Ser Ser Thr Thr Gln Gly Gln Asp Val Thr Leu Ala Pro Ala
65 70 75 80
Thr Glu Pro Ala Ser Gly Ser Ala Ala Leu Trp Gly Gln Asp Val Thr
85 90 95
Ser Val Pro Val Thr Arg Pro Ala Leu Gly Ser Thr Thr Pro Pro Ala
100 105 110
His Asp Val Thr Ser Ala Pro Asp Asn Lys Pro Ala Pro Gly Ser Thr
115 120 125
Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala
130 135 140
Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp
145 150 155 160
Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr
165 170 175
Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala
180 185 190
His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr
195 200 205
Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala
210 215 220
Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp
225 230 235 240
Asn Arg Pro Ala Leu Gly Ser Thr Ala Pro Pro Val His Asn Val Thr
245 250 255
Ser Ala Ser Gly Ser Ala Ser Gly Ser Ala Ser Thr Leu Val His Asn
260 265 270
Gly Thr Ser Ala Arg Ala Thr Thr Thr Pro Ala Ser Lys Ser Thr Pro
275 280 285
Phe Ser Ile Pro Ser His His Ser Asp Thr Pro Thr Thr Leu Ala Ser
290 295 300
His Ser Thr Lys Thr Asp Ala Ser Ser Thr His His Ser Ser Val Pro
305 310 315 320
Pro Leu Thr Ser Ser Asn His Ser Thr Ser Pro Gln Leu Ser Thr Gly
325 330 335
Val Ser Phe Phe Phe Leu Ser Phe His Ile Ser Asn Leu Gln Phe Asn
340 345 350
Ser Ser Leu Glu Asp Pro Ser Thr Asp Tyr Tyr Gln Glu Leu Gln Arg
355 360 365
Asp Ile Ser Glu Met Phe Leu Gln Ile Tyr Lys Gln Gly Gly Phe Leu
370 375 380
Gly Leu Ser Asn Ile Lys Phe Arg Pro Gly Ser Val Val Val Gln Leu
385 390 395 400
Thr Leu Ala Phe Arg Glu Gly Thr Ile Asn Val His Asp Val Glu Thr
405 410 415
Gln Phe Asn Gln Tyr Lys Thr Glu Ala Ala Ser Arg Tyr Asn Leu Thr
420 425 430
Ile Ser Asp Val Ser Val Ser Asp Val Pro Phe Pro Phe Ser Ala Gln
435 440 445
Ser Gly Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gln
450 455 460
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met
465 470 475 480
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
485 490 495
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
500 505 510
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
515 520 525
Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val
530 535 540
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
545 550 555 560
Lys Ser Leu Ser Leu Ser Leu Gly Lys
565
<210> 25
<211> 1710
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> CDS
<222> (1)..(1707)
<223> SP-Muc1-铰链-IgG4Fc
<400> 25
atg gaa gcc cca gct cag ctt ctc ttc ctc ctg cta ctc tgg ctc cca 48
Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro
1 5 10 15
gat acc acc gga tct ggt cat gca agc tct acc cca ggt gga gaa aag 96
Asp Thr Thr Gly Ser Gly His Ala Ser Ser Thr Pro Gly Gly Glu Lys
20 25 30
gag act tcg gct acc cag aga agt tca gtg ccc agc tct act gag aag 144
Glu Thr Ser Ala Thr Gln Arg Ser Ser Val Pro Ser Ser Thr Glu Lys
35 40 45
aat gct gtg agt atg acc agc agc gta ctc tcc agc cac agc ccc ggt 192
Asn Ala Val Ser Met Thr Ser Ser Val Leu Ser Ser His Ser Pro Gly
50 55 60
tca ggc tcc tcc acc act cag gga cag gat gtc act ctg gcc ccg gcc 240
Ser Gly Ser Ser Thr Thr Gln Gly Gln Asp Val Thr Leu Ala Pro Ala
65 70 75 80
acg gaa cca gct tca ggt tca gct gcc ctg tgg gga cag gat gtc acc 288
Thr Glu Pro Ala Ser Gly Ser Ala Ala Leu Trp Gly Gln Asp Val Thr
85 90 95
tcg gtc cca gtc acc agg cca gcc ctg ggc tcc acc acc ccg cca gcc 336
Ser Val Pro Val Thr Arg Pro Ala Leu Gly Ser Thr Thr Pro Pro Ala
100 105 110
cac gat gtc acc tca gcc ccg gac aac aag cca gcc ccg ggc tcc acc 384
His Asp Val Thr Ser Ala Pro Asp Asn Lys Pro Ala Pro Gly Ser Thr
115 120 125
gcc ccc cca gcc cac ggt gtc acc tcg gcc ccg gac acc agg ccg gcc 432
Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala
130 135 140
ccg ggc tcc acc gcc ccc cca gcc cac ggt gtc acc tcg gcc ccg gac 480
Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp
145 150 155 160
acc agg ccg gcc ccg ggc tcc acc gcc ccc cca gcc cac ggt gtc acc 528
Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr
165 170 175
tcg gcc ccg gac acc agg ccg gcc ccg ggc tcc acc gcc ccc cca gcc 576
Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala
180 185 190
cac ggt gtc acc tcg gcc ccg gac acc agg ccg gcc ccg ggc tcc acc 624
His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr
195 200 205
gcc ccc cca gcc cac ggt gtc acc tcg gcc ccg gac acc agg ccg gcc 672
Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala
210 215 220
ccg ggc tcc acc gcc ccc cca gcc cat ggt gtc acc tcg gcc ccg gac 720
Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp
225 230 235 240
aac agg ccc gcc ttg ggc tcc acc gcc cct cca gtc cac aat gtc acc 768
Asn Arg Pro Ala Leu Gly Ser Thr Ala Pro Pro Val His Asn Val Thr
245 250 255
tcg gcc tca ggc tct gca tca ggc tca gct tct act ctg gtg cac aac 816
Ser Ala Ser Gly Ser Ala Ser Gly Ser Ala Ser Thr Leu Val His Asn
260 265 270
ggc acc tct gcc agg gct acc aca acc cca gcc agc aag agc act cca 864
Gly Thr Ser Ala Arg Ala Thr Thr Thr Pro Ala Ser Lys Ser Thr Pro
275 280 285
ttc tca att ccc agc cac cac tct gat act cct acc acc ctt gcc agc 912
Phe Ser Ile Pro Ser His His Ser Asp Thr Pro Thr Thr Leu Ala Ser
290 295 300
cat agc acc aag act gat gcc agt agc act cac cat agc tcg gta cct 960
His Ser Thr Lys Thr Asp Ala Ser Ser Thr His His Ser Ser Val Pro
305 310 315 320
cct ctc acc tcc tcc aat cac agc act tct ccc cag ttg tct act ggg 1008
Pro Leu Thr Ser Ser Asn His Ser Thr Ser Pro Gln Leu Ser Thr Gly
325 330 335
gtc tct ttc ttt ttc ctg tct ttt cac att tca aac ctc cag ttt aat 1056
Val Ser Phe Phe Phe Leu Ser Phe His Ile Ser Asn Leu Gln Phe Asn
340 345 350
tcc tct ctg gaa gat ccc agc acc gac tac tac caa gag ctg cag aga 1104
Ser Ser Leu Glu Asp Pro Ser Thr Asp Tyr Tyr Gln Glu Leu Gln Arg
355 360 365
gac att tct gaa atg ttt ttg cag att tat aaa caa ggg ggt ttt ctg 1152
Asp Ile Ser Glu Met Phe Leu Gln Ile Tyr Lys Gln Gly Gly Phe Leu
370 375 380
ggc ctc tcc aat att aag ttc agg cca gga tct gtg gtg gta caa ttg 1200
Gly Leu Ser Asn Ile Lys Phe Arg Pro Gly Ser Val Val Val Gln Leu
385 390 395 400
act ctg gcc ttc cga gaa ggt acc atc aat gtc cac gac gtg gag aca 1248
Thr Leu Ala Phe Arg Glu Gly Thr Ile Asn Val His Asp Val Glu Thr
405 410 415
cag ttc aat cag tat aaa acg gaa gca gcc tct cga tat aac ctg acg 1296
Gln Phe Asn Gln Tyr Lys Thr Glu Ala Ala Ser Arg Tyr Asn Leu Thr
420 425 430
atc tca gac gtc agc gtg agt gat gtg cca ttt cct ttc tct gcc cag 1344
Ile Ser Asp Val Ser Val Ser Asp Val Pro Phe Pro Phe Ser Ala Gln
435 440 445
tct ggg gag tcc aaa tat ggt ccc cca tgc cca cca tgc cca ggg cag 1392
Ser Gly Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gln
450 455 460
ccc cga gag cca cag gtg tac acc ctg ccc cca tcc cag gag gag atg 1440
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met
465 470 475 480
acc aag aac cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tac ccc 1488
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
485 490 495
agc gac atc gcc gtg gag tgg gag agc aat ggg cag ccg gag aac aac 1536
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
500 505 510
tac aag acc acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc 1584
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
515 520 525
tac agc agg cta acc gtg gac aag agc agg tgg cag gag ggg aat gtc 1632
Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val
530 535 540
ttc tca tgc tcc gtg atg cat gag gct ctg cac aac cac tac aca cag 1680
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
545 550 555 560
aag agc ctc tcc ctg tct ctg ggt aaa tga 1710
Lys Ser Leu Ser Leu Ser Leu Gly Lys
565
<210> 26
<211> 759
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<221> PEPTIDE
<222> (1)..(759)
<223> SP-EGFR-铰链-IgG4Fc
<400> 26
Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro
1 5 10 15
Asp Thr Thr Gly Leu Glu Glu Lys Lys Val Cys Gln Gly Thr Ser Asn
20 25 30
Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe Leu Ser Leu Gln
35 40 45
Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn Leu Glu Ile Thr
50 55 60
Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys Thr Ile Gln Glu
65 70 75 80
Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val Glu Arg Ile Pro
85 90 95
Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr Tyr Glu Asn Ser
100 105 110
Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn Lys Thr Gly Leu
115 120 125
Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu His Gly Ala Val
130 135 140
Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu Ser Ile Gln Trp
145 150 155 160
Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met Ser Met Asp Phe
165 170 175
Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro Ser Cys Pro Asn
180 185 190
Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln Lys Leu Thr Lys
195 200 205
Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg Gly Lys Ser Pro
210 215 220
Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys Thr Gly Pro Arg
225 230 235 240
Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp Glu Ala Thr Cys
245 250 255
Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro Thr Thr Tyr Gln
260 265 270
Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly Ala Thr Cys Val
275 280 285
Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His Gly Ser Cys Val
290 295 300
Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu Asp Gly Val Arg
305 310 315 320
Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val Cys Asn Gly Ile
325 330 335
Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile
340 345 350
Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu
355 360 365
Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp
370 375 380
Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe
385 390 395 400
Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe
405 410 415
Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe
420 425 430
Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser
435 440 445
Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn
450 455 460
Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser
465 470 475 480
Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys
485 490 495
Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp
500 505 510
Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly
515 520 525
Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu
530 535 540
Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro
545 550 555 560
Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile
565 570 575
Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro
580 585 590
Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp
595 600 605
Ala Gly His Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys
610 615 620
Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro
625 630 635 640
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gln Pro Arg
645 650 655
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
660 665 670
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
675 680 685
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
690 695 700
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
705 710 715 720
Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
725 730 735
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
740 745 750
Leu Ser Leu Ser Leu Gly Lys
755
<210> 27
<211> 2280
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> CDS
<222> (1)..(2277)
<223> SP-EGFR-铰链-IgG4Fc
<400> 27
atg gaa gcc cca gct cag ctt ctc ttc ctc ctg cta ctc tgg ctc cca 48
Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro
1 5 10 15
gat acc acc gga ctg gag gaa aag aaa gtt tgc caa ggc acg agt aac 96
Asp Thr Thr Gly Leu Glu Glu Lys Lys Val Cys Gln Gly Thr Ser Asn
20 25 30
aag ctc acg cag ttg ggc act ttt gaa gat cat ttt ctc agc ctc cag 144
Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe Leu Ser Leu Gln
35 40 45
agg atg ttc aat aac tgt gag gtg gtc ctt ggg aat ttg gaa att acc 192
Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn Leu Glu Ile Thr
50 55 60
tat gtg cag agg aat tat gat ctt tcc ttc tta aag acc atc cag gag 240
Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys Thr Ile Gln Glu
65 70 75 80
gtg gct ggt tat gtc ctc att gcc ctc aac aca gtg gag cga att cct 288
Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val Glu Arg Ile Pro
85 90 95
ttg gaa aac ctg cag atc atc aga gga aat atg tac tac gaa aat tcc 336
Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr Tyr Glu Asn Ser
100 105 110
tat gcc tta gca gtc tta tct aac tat gat gca aat aaa acc gga ctg 384
Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn Lys Thr Gly Leu
115 120 125
aag gag ctg ccc atg aga aat tta cag gaa atc ctg cat ggc gcc gtg 432
Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu His Gly Ala Val
130 135 140
cgg ttc agc aac aac cct gcc ctg tgc aac gtg gag agc atc cag tgg 480
Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu Ser Ile Gln Trp
145 150 155 160
cgg gac ata gtc agc agt gac ttt ctc agc aac atg tcg atg gac ttc 528
Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met Ser Met Asp Phe
165 170 175
cag aac cac ctg ggc agc tgc caa aag tgt gat cca agc tgt ccc aat 576
Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro Ser Cys Pro Asn
180 185 190
ggg agc tgc tgg ggt gca gga gag gag aac tgc cag aaa ctg acc aaa 624
Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln Lys Leu Thr Lys
195 200 205
atc atc tgt gcc cag cag tgc tcc ggg cgc tgc cgt ggc aag tcc ccc 672
Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg Gly Lys Ser Pro
210 215 220
agt gac tgc tgc cac aac cag tgt gct gca ggc tgc aca ggc ccc cgg 720
Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys Thr Gly Pro Arg
225 230 235 240
gag agc gac tgc ctg gtc tgc cgc aaa ttc cga gac gaa gcc acg tgc 768
Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp Glu Ala Thr Cys
245 250 255
aag gac acc tgc ccc cca ctc atg ctc tac aac ccc acc acg tac cag 816
Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro Thr Thr Tyr Gln
260 265 270
atg gat gtg aac ccc gag ggc aaa tac agc ttt ggt gcc acc tgc gtg 864
Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly Ala Thr Cys Val
275 280 285
aag aag tgt ccc cgt aat tat gtg gtg aca gat cac ggc tcg tgc gtc 912
Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His Gly Ser Cys Val
290 295 300
cga gcc tgt ggg gcc gac agc tat gag atg gag gaa gac ggc gtc cgc 960
Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu Asp Gly Val Arg
305 310 315 320
aag tgt aag aag tgc gaa ggg cct tgc cgc aaa gtg tgt aac gga ata 1008
Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val Cys Asn Gly Ile
325 330 335
ggt att ggt gaa ttt aaa gac tca ctc tcc ata aat gct acg aat att 1056
Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile
340 345 350
aaa cac ttc aaa aac tgc acc tcc atc agt ggc gat ctc cac atc ctg 1104
Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu
355 360 365
ccg gtg gca ttt agg ggt gac tcc ttc aca cat act cct cct ctg gat 1152
Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp
370 375 380
cca cag gaa ctg gat att ctg aaa acc gta aag gaa atc aca ggg ttt 1200
Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe
385 390 395 400
ttg ctg att cag gct tgg cct gaa aac agg acg gac ctc cat gcc ttt 1248
Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe
405 410 415
gag aac cta gaa atc ata cgc ggc agg acc aag caa cat ggt cag ttt 1296
Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe
420 425 430
tct ctt gca gtc gtc agc ctg aac ata aca tcc ttg gga tta cgc tcc 1344
Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser
435 440 445
ctc aag gag ata agt gat gga gat gtg ata att tca gga aac aaa aat 1392
Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn
450 455 460
ttg tgc tat gca aat aca ata aac tgg aaa aaa ctg ttt ggg acc tcc 1440
Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser
465 470 475 480
ggt cag aaa acc aaa att ata agc aac aga ggt gaa aac agc tgc aag 1488
Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys
485 490 495
gcc aca ggc cag gtc tgc cat gcc ttg tgc tcc ccc gag ggc tgc tgg 1536
Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp
500 505 510
ggc ccg gag ccc agg gac tgc gtc tct tgc cgg aat gtc agc cga ggc 1584
Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly
515 520 525
agg gaa tgc gtg gac aag tgc aac ctt ctg gag ggt gag cca agg gag 1632
Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu
530 535 540
ttt gtg gag aac tct gag tgc ata cag tgc cac cca gag tgc ctg cct 1680
Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro
545 550 555 560
cag gcc atg aac atc acc tgc aca gga cgg gga cca gac aac tgt atc 1728
Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile
565 570 575
cag tgt gcc cac tac att gac ggc ccc cac tgc gtc aag acc tgc ccg 1776
Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro
580 585 590
gca gga gtc atg gga gaa aac aac acc ctg gtc tgg aag tac gca gac 1824
Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp
595 600 605
gcc ggc cat gtg tgc cac ctg tgc cat cca aac tgc acc tac gga tgc 1872
Ala Gly His Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys
610 615 620
act ggg cca ggt ctt gaa ggc tgt cca acg aat ggg cct aag atc ccg 1920
Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro
625 630 635 640
gag tcc aaa tat ggt ccc cca tgc cca cca tgc cca ggg cag ccc cga 1968
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Gly Gln Pro Arg
645 650 655
gag cca cag gtg tac acc ctg ccc cca tcc cag gag gag atg acc aag 2016
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
660 665 670
aac cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tac ccc agc gac 2064
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
675 680 685
atc gcc gtg gag tgg gag agc aat ggg cag ccg gag aac aac tac aag 2112
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
690 695 700
acc acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc 2160
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
705 710 715 720
agg cta acc gtg gac aag agc agg tgg cag gag ggg aat gtc ttc tca 2208
Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
725 730 735
tgc tcc gtg atg cat gag gct ctg cac aac cac tac aca cag aag agc 2256
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
740 745 750
ctc tcc ctg tct ctg ggt aaa tga 2280
Leu Ser Leu Ser Leu Gly Lys
755
<210> 28
<211> 1572
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> CDS
<222> (1)..(1569)
<223> SP-CD19-铰链-wtFc
<400> 28
atg gaa gcc cca gct cag ctt ctc ttc ctc ctg cta ctc tgg ctc cca 48
Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro
1 5 10 15
gat acc acc gga ccc gag gaa cct cta gtg gtg aag gtg gaa gag gga 96
Asp Thr Thr Gly Pro Glu Glu Pro Leu Val Val Lys Val Glu Glu Gly
20 25 30
gat aac gct gtg ctg cag tgc ctc aag ggg acc tca gat ggc ccc act 144
Asp Asn Ala Val Leu Gln Cys Leu Lys Gly Thr Ser Asp Gly Pro Thr
35 40 45
cag cag ctg acc tgg tct cgg gag tcc ccg ctt aaa ccc ttc tta aaa 192
Gln Gln Leu Thr Trp Ser Arg Glu Ser Pro Leu Lys Pro Phe Leu Lys
50 55 60
ctc agc ctg ggg ctg cca ggc ctg gga atc cac atg agg ccc ctg gcc 240
Leu Ser Leu Gly Leu Pro Gly Leu Gly Ile His Met Arg Pro Leu Ala
65 70 75 80
atc tgg ctt ttc atc ttc aac gtc tct caa cag atg ggg ggc ttc tac 288
Ile Trp Leu Phe Ile Phe Asn Val Ser Gln Gln Met Gly Gly Phe Tyr
85 90 95
ctg tgc cag ccg ggg ccc ccc tct gag aag gcc tgg cag cct ggc tgg 336
Leu Cys Gln Pro Gly Pro Pro Ser Glu Lys Ala Trp Gln Pro Gly Trp
100 105 110
aca gtc aat gtg gag ggc agc ggg gag ctg ttc cgg tgg aat gtt tcg 384
Thr Val Asn Val Glu Gly Ser Gly Glu Leu Phe Arg Trp Asn Val Ser
115 120 125
gac cta ggt ggc ctg ggc tgt ggc ctg aag aac agg tcc tca gag ggc 432
Asp Leu Gly Gly Leu Gly Cys Gly Leu Lys Asn Arg Ser Ser Glu Gly
130 135 140
ccc agc tcc cct tcc ggg aag ctc atg agc ccc aag ctg tat gtg tgg 480
Pro Ser Ser Pro Ser Gly Lys Leu Met Ser Pro Lys Leu Tyr Val Trp
145 150 155 160
gcc aaa gac cgc cct gag atc tgg gag gga gag cct ccg tgt ctc cca 528
Ala Lys Asp Arg Pro Glu Ile Trp Glu Gly Glu Pro Pro Cys Leu Pro
165 170 175
ccg agg gac agc ctg aac cag agc ctc agc cag gac ctc acc atg gcc 576
Pro Arg Asp Ser Leu Asn Gln Ser Leu Ser Gln Asp Leu Thr Met Ala
180 185 190
cct ggc tcc aca ctc tgg ctg tcc tgt ggg gta ccc cct gac tct gtg 624
Pro Gly Ser Thr Leu Trp Leu Ser Cys Gly Val Pro Pro Asp Ser Val
195 200 205
tcc agg ggc ccc ctc tcc tgg acc cat gtg cac ccc aag ggg cct aag 672
Ser Arg Gly Pro Leu Ser Trp Thr His Val His Pro Lys Gly Pro Lys
210 215 220
tca ttg ctg agc cta gag ctg aag gac gat cgc ccg gcc aga gat atg 720
Ser Leu Leu Ser Leu Glu Leu Lys Asp Asp Arg Pro Ala Arg Asp Met
225 230 235 240
tgg gta atg gag acg ggt ctg ttg ttg ccc cgg gcc aca gct caa gac 768
Trp Val Met Glu Thr Gly Leu Leu Leu Pro Arg Ala Thr Ala Gln Asp
245 250 255
gct gga aag tat tat tgt cac cgt ggc aac ctg acc atg tca ttc cac 816
Ala Gly Lys Tyr Tyr Cys His Arg Gly Asn Leu Thr Met Ser Phe His
260 265 270
ctg gag atc act gct cgg cca gta cta tgg cac tgg ctg ctg agg act 864
Leu Glu Ile Thr Ala Arg Pro Val Leu Trp His Trp Leu Leu Arg Thr
275 280 285
ggt ggc tgg aag gtc tca gag tcc aaa tat ggt ccc cca tgc cca cca 912
Gly Gly Trp Lys Val Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro
290 295 300
tgc cca gca cct gag ttc ctg ggg gga cca tca gtc ttc ctg ttc ccc 960
Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro
305 310 315 320
cca aaa ccc aag gac act ctc atg atc tcc cgg acc cct gag gtc acg 1008
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
325 330 335
tgc gtg gtg gtg gac gtg agc cag gaa gac ccc gag gtc cag ttc aac 1056
Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn
340 345 350
tgg tac gtg gat ggc gtg gag gtg cat aat gcc aag aca aag ccg cgg 1104
Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
355 360 365
gag gag cag ttc aac agc acg tac cgt gtg gtc agc gtc ctc acc gtc 1152
Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val
370 375 380
ctg cac cag gac tgg ctg aac ggc aag gag tac aag tgc aag gtc tcc 1200
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser
385 390 395 400
aac aaa ggc ctc ccg tcc tcc atc gag aaa acc atc tcc aaa gcc aaa 1248
Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys
405 410 415
ggg cag ccc cga gag cca cag gtg tac acc ctg ccc cca tcc cag gag 1296
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu
420 425 430
gag atg acc aag aac cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc 1344
Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
435 440 445
tac ccc agc gac atc gcc gtg gag tgg gag agc aat ggg cag ccg gag 1392
Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
450 455 460
aac aac tac aag acc acg cct ccc gtg ctg gac tcc gac ggc tcc ttc 1440
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
465 470 475 480
ttc ctc tac agc agg cta acc gtg gac aag agc agg tgg cag gag ggg 1488
Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly
485 490 495
aat gtc ttc tca tgc tcc gtg atg cat gag gct ctg cac aac cac tac 1536
Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
500 505 510
aca cag aag agc ctc tcc ctg tct ctg ggt aaa tga 1572
Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys
515 520
<210> 29
<211> 1659
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> CDS
<222> (1)..(1656)
<223> SP-MSLN-铰链-wtFc
<400> 29
atg gaa gcc cca gct cag ctt ctc ttc ctc ctg cta ctc tgg ctc cca 48
Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro
1 5 10 15
gat acc acc gga gaa gtg gag aag aca gcc tgt cct tca ggc aag aag 96
Asp Thr Thr Gly Glu Val Glu Lys Thr Ala Cys Pro Ser Gly Lys Lys
20 25 30
gcc cgc gag ata gac gag agc ctc atc ttc tac aag aag tgg gag ctg 144
Ala Arg Glu Ile Asp Glu Ser Leu Ile Phe Tyr Lys Lys Trp Glu Leu
35 40 45
gaa gcc tgc gtg gat gcg gcc ctg ctg gcc acc cag atg gac cgc gtg 192
Glu Ala Cys Val Asp Ala Ala Leu Leu Ala Thr Gln Met Asp Arg Val
50 55 60
aac gcc atc ccc ttc acc tac gag cag ctg gac gtc cta aag cat aaa 240
Asn Ala Ile Pro Phe Thr Tyr Glu Gln Leu Asp Val Leu Lys His Lys
65 70 75 80
ctg gat gag ctc tac cca caa ggt tac ccc gag tct gtg atc cag cac 288
Leu Asp Glu Leu Tyr Pro Gln Gly Tyr Pro Glu Ser Val Ile Gln His
85 90 95
ctg ggc tac ctc ttc ctc aag atg agc cct gag gac att cgc aag tgg 336
Leu Gly Tyr Leu Phe Leu Lys Met Ser Pro Glu Asp Ile Arg Lys Trp
100 105 110
aat gtg acg tcc ctg gag acc ctg aag gct ttg ctt gaa gtc aac aaa 384
Asn Val Thr Ser Leu Glu Thr Leu Lys Ala Leu Leu Glu Val Asn Lys
115 120 125
ggg cac gaa atg agt cct cag gtg gcc acc ctg atc gac cgc ttt gtg 432
Gly His Glu Met Ser Pro Gln Val Ala Thr Leu Ile Asp Arg Phe Val
130 135 140
aag gga agg ggc cag cta gac aaa gac acc cta gac acc ctg acc gcc 480
Lys Gly Arg Gly Gln Leu Asp Lys Asp Thr Leu Asp Thr Leu Thr Ala
145 150 155 160
ttc tac cct ggg tac ctg tgc tcc ctc agc ccc gag gag ctg agc tcc 528
Phe Tyr Pro Gly Tyr Leu Cys Ser Leu Ser Pro Glu Glu Leu Ser Ser
165 170 175
gtg ccc ccc agc agc atc tgg gcg gtc agg ccc cag gac ctg gac acg 576
Val Pro Pro Ser Ser Ile Trp Ala Val Arg Pro Gln Asp Leu Asp Thr
180 185 190
tgt gac cca agg cag ctg gac gtc ctc tat ccc aag gcc cgc ctt gct 624
Cys Asp Pro Arg Gln Leu Asp Val Leu Tyr Pro Lys Ala Arg Leu Ala
195 200 205
ttc cag aac atg aac ggg tcc gaa tac ttc gtg aag atc cag tcc ttc 672
Phe Gln Asn Met Asn Gly Ser Glu Tyr Phe Val Lys Ile Gln Ser Phe
210 215 220
ctg ggt ggg gcc ccc acg gag gat ttg aag gcg ctc agt cag cag aat 720
Leu Gly Gly Ala Pro Thr Glu Asp Leu Lys Ala Leu Ser Gln Gln Asn
225 230 235 240
gtg agc atg gac ttg gcc acg ttc atg aag ctg cgg acg gat gcg gtg 768
Val Ser Met Asp Leu Ala Thr Phe Met Lys Leu Arg Thr Asp Ala Val
245 250 255
ctg ccg ttg act gtg gct gag gtg cag aaa ctt ctg gga ccc cac gtg 816
Leu Pro Leu Thr Val Ala Glu Val Gln Lys Leu Leu Gly Pro His Val
260 265 270
gag ggc ctg aag gcg gag gag cgg cac cgc ccg gtg cgg gac tgg atc 864
Glu Gly Leu Lys Ala Glu Glu Arg His Arg Pro Val Arg Asp Trp Ile
275 280 285
cta cgg cag cgg cag gac gac ctg gac acg ctg ggg ctg ggg cta cag 912
Leu Arg Gln Arg Gln Asp Asp Leu Asp Thr Leu Gly Leu Gly Leu Gln
290 295 300
ggc ggc atc ccc aac ggc tac ctg gtc cta gac ctc agc atg caa gag 960
Gly Gly Ile Pro Asn Gly Tyr Leu Val Leu Asp Leu Ser Met Gln Glu
305 310 315 320
gcc ctc tcg gag tcc aaa tat ggt ccc cca tgc cca cca tgc cca gca 1008
Ala Leu Ser Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala
325 330 335
cct gag ttc ctg ggg gga cca tca gtc ttc ctg ttc ccc cca aaa ccc 1056
Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
340 345 350
aag gac act ctc atg atc tcc cgg acc cct gag gtc acg tgc gtg gtg 1104
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
355 360 365
gtg gac gtg agc cag gaa gac ccc gag gtc cag ttc aac tgg tac gtg 1152
Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val
370 375 380
gat ggc gtg gag gtg cat aat gcc aag aca aag ccg cgg gag gag cag 1200
Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
385 390 395 400
ttc aac agc acg tac cgt gtg gtc agc gtc ctc acc gtc ctg cac cag 1248
Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln
405 410 415
gac tgg ctg aac ggc aag gag tac aag tgc aag gtc tcc aac aaa ggc 1296
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly
420 425 430
ctc ccg tcc tcc atc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc 1344
Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
435 440 445
cga gag cca cag gtg tac acc ctg ccc cca tcc cag gag gag atg acc 1392
Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr
450 455 460
aag aac cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tac ccc agc 1440
Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
465 470 475 480
gac atc gcc gtg gag tgg gag agc aat ggg cag ccg gag aac aac tac 1488
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
485 490 495
aag acc acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac 1536
Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr
500 505 510
agc agg cta acc gtg gac aag agc agg tgg cag gag ggg aat gtc ttc 1584
Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe
515 520 525
tca tgc tcc gtg atg cat gag gct ctg cac aac cac tac aca cag aag 1632
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
530 535 540
agc ctc tcc ctg tct ctg ggt aaa tga 1659
Ser Leu Ser Leu Ser Leu Gly Lys
545 550
<210> 30
<211> 2040
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> CDS
<222> (1)..(2037)
<223> SP-Muc1-铰链-wtFc
<400> 30
atg gaa gcc cca gct cag ctt ctc ttc ctc ctg cta ctc tgg ctc cca 48
Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro
1 5 10 15
gat acc acc gga tct ggt cat gca agc tct acc cca ggt gga gaa aag 96
Asp Thr Thr Gly Ser Gly His Ala Ser Ser Thr Pro Gly Gly Glu Lys
20 25 30
gag act tcg gct acc cag aga agt tca gtg ccc agc tct act gag aag 144
Glu Thr Ser Ala Thr Gln Arg Ser Ser Val Pro Ser Ser Thr Glu Lys
35 40 45
aat gct gtg agt atg acc agc agc gta ctc tcc agc cac agc ccc ggt 192
Asn Ala Val Ser Met Thr Ser Ser Val Leu Ser Ser His Ser Pro Gly
50 55 60
tca ggc tcc tcc acc act cag gga cag gat gtc act ctg gcc ccg gcc 240
Ser Gly Ser Ser Thr Thr Gln Gly Gln Asp Val Thr Leu Ala Pro Ala
65 70 75 80
acg gaa cca gct tca ggt tca gct gcc ctg tgg gga cag gat gtc acc 288
Thr Glu Pro Ala Ser Gly Ser Ala Ala Leu Trp Gly Gln Asp Val Thr
85 90 95
tcg gtc cca gtc acc agg cca gcc ctg ggc tcc acc acc ccg cca gcc 336
Ser Val Pro Val Thr Arg Pro Ala Leu Gly Ser Thr Thr Pro Pro Ala
100 105 110
cac gat gtc acc tca gcc ccg gac aac aag cca gcc ccg ggc tcc acc 384
His Asp Val Thr Ser Ala Pro Asp Asn Lys Pro Ala Pro Gly Ser Thr
115 120 125
gcc ccc cca gcc cac ggt gtc acc tcg gcc ccg gac acc agg ccg gcc 432
Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala
130 135 140
ccg ggc tcc acc gcc ccc cca gcc cac ggt gtc acc tcg gcc ccg gac 480
Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp
145 150 155 160
acc agg ccg gcc ccg ggc tcc acc gcc ccc cca gcc cac ggt gtc acc 528
Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr
165 170 175
tcg gcc ccg gac acc agg ccg gcc ccg ggc tcc acc gcc ccc cca gcc 576
Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr Ala Pro Pro Ala
180 185 190
cac ggt gtc acc tcg gcc ccg gac acc agg ccg gcc ccg ggc tcc acc 624
His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala Pro Gly Ser Thr
195 200 205
gcc ccc cca gcc cac ggt gtc acc tcg gcc ccg gac acc agg ccg gcc 672
Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp Thr Arg Pro Ala
210 215 220
ccg ggc tcc acc gcc ccc cca gcc cat ggt gtc acc tcg gcc ccg gac 720
Pro Gly Ser Thr Ala Pro Pro Ala His Gly Val Thr Ser Ala Pro Asp
225 230 235 240
aac agg ccc gcc ttg ggc tcc acc gcc cct cca gtc cac aat gtc acc 768
Asn Arg Pro Ala Leu Gly Ser Thr Ala Pro Pro Val His Asn Val Thr
245 250 255
tcg gcc tca ggc tct gca tca ggc tca gct tct act ctg gtg cac aac 816
Ser Ala Ser Gly Ser Ala Ser Gly Ser Ala Ser Thr Leu Val His Asn
260 265 270
ggc acc tct gcc agg gct acc aca acc cca gcc agc aag agc act cca 864
Gly Thr Ser Ala Arg Ala Thr Thr Thr Pro Ala Ser Lys Ser Thr Pro
275 280 285
ttc tca att ccc agc cac cac tct gat act cct acc acc ctt gcc agc 912
Phe Ser Ile Pro Ser His His Ser Asp Thr Pro Thr Thr Leu Ala Ser
290 295 300
cat agc acc aag act gat gcc agt agc act cac cat agc tcg gta cct 960
His Ser Thr Lys Thr Asp Ala Ser Ser Thr His His Ser Ser Val Pro
305 310 315 320
cct ctc acc tcc tcc aat cac agc act tct ccc cag ttg tct act ggg 1008
Pro Leu Thr Ser Ser Asn His Ser Thr Ser Pro Gln Leu Ser Thr Gly
325 330 335
gtc tct ttc ttt ttc ctg tct ttt cac att tca aac ctc cag ttt aat 1056
Val Ser Phe Phe Phe Leu Ser Phe His Ile Ser Asn Leu Gln Phe Asn
340 345 350
tcc tct ctg gaa gat ccc agc acc gac tac tac caa gag ctg cag aga 1104
Ser Ser Leu Glu Asp Pro Ser Thr Asp Tyr Tyr Gln Glu Leu Gln Arg
355 360 365
gac att tct gaa atg ttt ttg cag att tat aaa caa ggg ggt ttt ctg 1152
Asp Ile Ser Glu Met Phe Leu Gln Ile Tyr Lys Gln Gly Gly Phe Leu
370 375 380
ggc ctc tcc aat att aag ttc agg cca gga tct gtg gtg gta caa ttg 1200
Gly Leu Ser Asn Ile Lys Phe Arg Pro Gly Ser Val Val Val Gln Leu
385 390 395 400
act ctg gcc ttc cga gaa ggt acc atc aat gtc cac gac gtg gag aca 1248
Thr Leu Ala Phe Arg Glu Gly Thr Ile Asn Val His Asp Val Glu Thr
405 410 415
cag ttc aat cag tat aaa acg gaa gca gcc tct cga tat aac ctg acg 1296
Gln Phe Asn Gln Tyr Lys Thr Glu Ala Ala Ser Arg Tyr Asn Leu Thr
420 425 430
atc tca gac gtc agc gtg agt gat gtg cca ttt cct ttc tct gcc cag 1344
Ile Ser Asp Val Ser Val Ser Asp Val Pro Phe Pro Phe Ser Ala Gln
435 440 445
tct ggg gag tcc aaa tat ggt ccc cca tgc cca cca tgc cca gca cct 1392
Ser Gly Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro
450 455 460
gag ttc ctg ggg gga cca tca gtc ttc ctg ttc ccc cca aaa ccc aag 1440
Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
465 470 475 480
gac act ctc atg atc tcc cgg acc cct gag gtc acg tgc gtg gtg gtg 1488
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
485 490 495
gac gtg agc cag gaa gac ccc gag gtc cag ttc aac tgg tac gtg gat 1536
Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp
500 505 510
ggc gtg gag gtg cat aat gcc aag aca aag ccg cgg gag gag cag ttc 1584
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe
515 520 525
aac agc acg tac cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac 1632
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
530 535 540
tgg ctg aac ggc aag gag tac aag tgc aag gtc tcc aac aaa ggc ctc 1680
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu
545 550 555 560
ccg tcc tcc atc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga 1728
Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
565 570 575
gag cca cag gtg tac acc ctg ccc cca tcc cag gag gag atg acc aag 1776
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys
580 585 590
aac cag gtc agc ctg acc tgc ctg gtc aaa ggc ttc tac ccc agc gac 1824
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
595 600 605
atc gcc gtg gag tgg gag agc aat ggg cag ccg gag aac aac tac aag 1872
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
610 615 620
acc acg cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc 1920
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
625 630 635 640
agg cta acc gtg gac aag agc agg tgg cag gag ggg aat gtc ttc tca 1968
Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser
645 650 655
tgc tcc gtg atg cat gag gct ctg cac aac cac tac aca cag aag agc 2016
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
660 665 670
ctc tcc ctg tct ctg ggt aaa tga 2040
Leu Ser Leu Ser Leu Gly Lys
675
<210> 31
<211> 2610
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> CDS
<222> (1)..(2607)
<223> SP-EGFR-铰链-wtFc
<400> 31
atg gaa gcc cca gct cag ctt ctc ttc ctc ctg cta ctc tgg ctc cca 48
Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro
1 5 10 15
gat acc acc gga ctg gag gaa aag aaa gtt tgc caa ggc acg agt aac 96
Asp Thr Thr Gly Leu Glu Glu Lys Lys Val Cys Gln Gly Thr Ser Asn
20 25 30
aag ctc acg cag ttg ggc act ttt gaa gat cat ttt ctc agc ctc cag 144
Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe Leu Ser Leu Gln
35 40 45
agg atg ttc aat aac tgt gag gtg gtc ctt ggg aat ttg gaa att acc 192
Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn Leu Glu Ile Thr
50 55 60
tat gtg cag agg aat tat gat ctt tcc ttc tta aag acc atc cag gag 240
Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys Thr Ile Gln Glu
65 70 75 80
gtg gct ggt tat gtc ctc att gcc ctc aac aca gtg gag cga att cct 288
Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val Glu Arg Ile Pro
85 90 95
ttg gaa aac ctg cag atc atc aga gga aat atg tac tac gaa aat tcc 336
Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr Tyr Glu Asn Ser
100 105 110
tat gcc tta gca gtc tta tct aac tat gat gca aat aaa acc gga ctg 384
Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn Lys Thr Gly Leu
115 120 125
aag gag ctg ccc atg aga aat tta cag gaa atc ctg cat ggc gcc gtg 432
Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu His Gly Ala Val
130 135 140
cgg ttc agc aac aac cct gcc ctg tgc aac gtg gag agc atc cag tgg 480
Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu Ser Ile Gln Trp
145 150 155 160
cgg gac ata gtc agc agt gac ttt ctc agc aac atg tcg atg gac ttc 528
Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met Ser Met Asp Phe
165 170 175
cag aac cac ctg ggc agc tgc caa aag tgt gat cca agc tgt ccc aat 576
Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro Ser Cys Pro Asn
180 185 190
ggg agc tgc tgg ggt gca gga gag gag aac tgc cag aaa ctg acc aaa 624
Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln Lys Leu Thr Lys
195 200 205
atc atc tgt gcc cag cag tgc tcc ggg cgc tgc cgt ggc aag tcc ccc 672
Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg Gly Lys Ser Pro
210 215 220
agt gac tgc tgc cac aac cag tgt gct gca ggc tgc aca ggc ccc cgg 720
Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys Thr Gly Pro Arg
225 230 235 240
gag agc gac tgc ctg gtc tgc cgc aaa ttc cga gac gaa gcc acg tgc 768
Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp Glu Ala Thr Cys
245 250 255
aag gac acc tgc ccc cca ctc atg ctc tac aac ccc acc acg tac cag 816
Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro Thr Thr Tyr Gln
260 265 270
atg gat gtg aac ccc gag ggc aaa tac agc ttt ggt gcc acc tgc gtg 864
Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly Ala Thr Cys Val
275 280 285
aag aag tgt ccc cgt aat tat gtg gtg aca gat cac ggc tcg tgc gtc 912
Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His Gly Ser Cys Val
290 295 300
cga gcc tgt ggg gcc gac agc tat gag atg gag gaa gac ggc gtc cgc 960
Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu Asp Gly Val Arg
305 310 315 320
aag tgt aag aag tgc gaa ggg cct tgc cgc aaa gtg tgt aac gga ata 1008
Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val Cys Asn Gly Ile
325 330 335
ggt att ggt gaa ttt aaa gac tca ctc tcc ata aat gct acg aat att 1056
Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile
340 345 350
aaa cac ttc aaa aac tgc acc tcc atc agt ggc gat ctc cac atc ctg 1104
Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu
355 360 365
ccg gtg gca ttt agg ggt gac tcc ttc aca cat act cct cct ctg gat 1152
Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp
370 375 380
cca cag gaa ctg gat att ctg aaa acc gta aag gaa atc aca ggg ttt 1200
Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe
385 390 395 400
ttg ctg att cag gct tgg cct gaa aac agg acg gac ctc cat gcc ttt 1248
Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe
405 410 415
gag aac cta gaa atc ata cgc ggc agg acc aag caa cat ggt cag ttt 1296
Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe
420 425 430
tct ctt gca gtc gtc agc ctg aac ata aca tcc ttg gga tta cgc tcc 1344
Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser
435 440 445
ctc aag gag ata agt gat gga gat gtg ata att tca gga aac aaa aat 1392
Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn
450 455 460
ttg tgc tat gca aat aca ata aac tgg aaa aaa ctg ttt ggg acc tcc 1440
Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser
465 470 475 480
ggt cag aaa acc aaa att ata agc aac aga ggt gaa aac agc tgc aag 1488
Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys
485 490 495
gcc aca ggc cag gtc tgc cat gcc ttg tgc tcc ccc gag ggc tgc tgg 1536
Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp
500 505 510
ggc ccg gag ccc agg gac tgc gtc tct tgc cgg aat gtc agc cga ggc 1584
Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly
515 520 525
agg gaa tgc gtg gac aag tgc aac ctt ctg gag ggt gag cca agg gag 1632
Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu
530 535 540
ttt gtg gag aac tct gag tgc ata cag tgc cac cca gag tgc ctg cct 1680
Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro
545 550 555 560
cag gcc atg aac atc acc tgc aca gga cgg gga cca gac aac tgt atc 1728
Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile
565 570 575
cag tgt gcc cac tac att gac ggc ccc cac tgc gtc aag acc tgc ccg 1776
Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro
580 585 590
gca gga gtc atg gga gaa aac aac acc ctg gtc tgg aag tac gca gac 1824
Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp
595 600 605
gcc ggc cat gtg tgc cac ctg tgc cat cca aac tgc acc tac gga tgc 1872
Ala Gly His Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys
610 615 620
act ggg cca ggt ctt gaa ggc tgt cca acg aat ggg cct aag atc ccg 1920
Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro
625 630 635 640
gag tcc aaa tat ggt ccc cca tgc cca cca tgc cca gca cct gag ttc 1968
Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe
645 650 655
ctg ggg gga cca tca gtc ttc ctg ttc ccc cca aaa ccc aag gac act 2016
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
660 665 670
ctc atg atc tcc cgg acc cct gag gtc acg tgc gtg gtg gtg gac gtg 2064
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
675 680 685
agc cag gaa gac ccc gag gtc cag ttc aac tgg tac gtg gat ggc gtg 2112
Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val
690 695 700
gag gtg cat aat gcc aag aca aag ccg cgg gag gag cag ttc aac agc 2160
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
705 710 715 720
acg tac cgt gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg ctg 2208
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
725 730 735
aac ggc aag gag tac aag tgc aag gtc tcc aac aaa ggc ctc ccg tcc 2256
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser
740 745 750
tcc atc gag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga gag cca 2304
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
755 760 765
cag gtg tac acc ctg ccc cca tcc cag gag gag atg acc aag aac cag 2352
Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
770 775 780
gtc agc ctg acc tgc ctg gtc aaa ggc ttc tac ccc agc gac atc gcc 2400
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
785 790 795 800
gtg gag tgg gag agc aat ggg cag ccg gag aac aac tac aag acc acg 2448
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
805 810 815
cct ccc gtg ctg gac tcc gac ggc tcc ttc ttc ctc tac agc agg cta 2496
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu
820 825 830
acc gtg gac aag agc agg tgg cag gag ggg aat gtc ttc tca tgc tcc 2544
Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
835 840 845
gtg atg cat gag gct ctg cac aac cac tac aca cag aag agc ctc tcc 2592
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
850 855 860
ctg tct ctg ggt aaa tga 2610
Leu Ser Leu Gly Lys
865
<210> 32
<211> 1923
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> CDS
<222> (1)..(1920)
<223> SP-EGFR-IFc
<400> 32
atg gaa gcc cca gct cag ctt ctc ttc ctc ctg cta ctc tgg ctc cca 48
Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro
1 5 10 15
gat acc acc gga ctg gag gaa aag aaa gtt tgc caa ggc acg agt aac 96
Asp Thr Thr Gly Leu Glu Glu Lys Lys Val Cys Gln Gly Thr Ser Asn
20 25 30
aag ctc acg cag ttg ggc act ttt gaa gat cat ttt ctc agc ctc cag 144
Lys Leu Thr Gln Leu Gly Thr Phe Glu Asp His Phe Leu Ser Leu Gln
35 40 45
agg atg ttc aat aac tgt gag gtg gtc ctt ggg aat ttg gaa att acc 192
Arg Met Phe Asn Asn Cys Glu Val Val Leu Gly Asn Leu Glu Ile Thr
50 55 60
tat gtg cag agg aat tat gat ctt tcc ttc tta aag acc atc cag gag 240
Tyr Val Gln Arg Asn Tyr Asp Leu Ser Phe Leu Lys Thr Ile Gln Glu
65 70 75 80
gtg gct ggt tat gtc ctc att gcc ctc aac aca gtg gag cga att cct 288
Val Ala Gly Tyr Val Leu Ile Ala Leu Asn Thr Val Glu Arg Ile Pro
85 90 95
ttg gaa aac ctg cag atc atc aga gga aat atg tac tac gaa aat tcc 336
Leu Glu Asn Leu Gln Ile Ile Arg Gly Asn Met Tyr Tyr Glu Asn Ser
100 105 110
tat gcc tta gca gtc tta tct aac tat gat gca aat aaa acc gga ctg 384
Tyr Ala Leu Ala Val Leu Ser Asn Tyr Asp Ala Asn Lys Thr Gly Leu
115 120 125
aag gag ctg ccc atg aga aat tta cag gaa atc ctg cat ggc gcc gtg 432
Lys Glu Leu Pro Met Arg Asn Leu Gln Glu Ile Leu His Gly Ala Val
130 135 140
cgg ttc agc aac aac cct gcc ctg tgc aac gtg gag agc atc cag tgg 480
Arg Phe Ser Asn Asn Pro Ala Leu Cys Asn Val Glu Ser Ile Gln Trp
145 150 155 160
cgg gac ata gtc agc agt gac ttt ctc agc aac atg tcg atg gac ttc 528
Arg Asp Ile Val Ser Ser Asp Phe Leu Ser Asn Met Ser Met Asp Phe
165 170 175
cag aac cac ctg ggc agc tgc caa aag tgt gat cca agc tgt ccc aat 576
Gln Asn His Leu Gly Ser Cys Gln Lys Cys Asp Pro Ser Cys Pro Asn
180 185 190
ggg agc tgc tgg ggt gca gga gag gag aac tgc cag aaa ctg acc aaa 624
Gly Ser Cys Trp Gly Ala Gly Glu Glu Asn Cys Gln Lys Leu Thr Lys
195 200 205
atc atc tgt gcc cag cag tgc tcc ggg cgc tgc cgt ggc aag tcc ccc 672
Ile Ile Cys Ala Gln Gln Cys Ser Gly Arg Cys Arg Gly Lys Ser Pro
210 215 220
agt gac tgc tgc cac aac cag tgt gct gca ggc tgc aca ggc ccc cgg 720
Ser Asp Cys Cys His Asn Gln Cys Ala Ala Gly Cys Thr Gly Pro Arg
225 230 235 240
gag agc gac tgc ctg gtc tgc cgc aaa ttc cga gac gaa gcc acg tgc 768
Glu Ser Asp Cys Leu Val Cys Arg Lys Phe Arg Asp Glu Ala Thr Cys
245 250 255
aag gac acc tgc ccc cca ctc atg ctc tac aac ccc acc acg tac cag 816
Lys Asp Thr Cys Pro Pro Leu Met Leu Tyr Asn Pro Thr Thr Tyr Gln
260 265 270
atg gat gtg aac ccc gag ggc aaa tac agc ttt ggt gcc acc tgc gtg 864
Met Asp Val Asn Pro Glu Gly Lys Tyr Ser Phe Gly Ala Thr Cys Val
275 280 285
aag aag tgt ccc cgt aat tat gtg gtg aca gat cac ggc tcg tgc gtc 912
Lys Lys Cys Pro Arg Asn Tyr Val Val Thr Asp His Gly Ser Cys Val
290 295 300
cga gcc tgt ggg gcc gac agc tat gag atg gag gaa gac ggc gtc cgc 960
Arg Ala Cys Gly Ala Asp Ser Tyr Glu Met Glu Glu Asp Gly Val Arg
305 310 315 320
aag tgt aag aag tgc gaa ggg cct tgc cgc aaa gtg tgt aac gga ata 1008
Lys Cys Lys Lys Cys Glu Gly Pro Cys Arg Lys Val Cys Asn Gly Ile
325 330 335
ggt att ggt gaa ttt aaa gac tca ctc tcc ata aat gct acg aat att 1056
Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile
340 345 350
aaa cac ttc aaa aac tgc acc tcc atc agt ggc gat ctc cac atc ctg 1104
Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu
355 360 365
ccg gtg gca ttt agg ggt gac tcc ttc aca cat act cct cct ctg gat 1152
Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp
370 375 380
cca cag gaa ctg gat att ctg aaa acc gta aag gaa atc aca ggg ttt 1200
Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe
385 390 395 400
ttg ctg att cag gct tgg cct gaa aac agg acg gac ctc cat gcc ttt 1248
Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe
405 410 415
gag aac cta gaa atc ata cgc ggc agg acc aag caa cat ggt cag ttt 1296
Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe
420 425 430
tct ctt gca gtc gtc agc ctg aac ata aca tcc ttg gga tta cgc tcc 1344
Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser
435 440 445
ctc aag gag ata agt gat gga gat gtg ata att tca gga aac aaa aat 1392
Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn
450 455 460
ttg tgc tat gca aat aca ata aac tgg aaa aaa ctg ttt ggg acc tcc 1440
Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser
465 470 475 480
ggt cag aaa acc aaa att ata agc aac aga ggt gaa aac agc tgc aag 1488
Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys
485 490 495
gcc aca ggc cag gtc tgc cat gcc ttg tgc tcc ccc gag ggc tgc tgg 1536
Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp
500 505 510
ggc ccg gag ccc agg gac tgc gtc tct tgc cgg aat gtc agc cga ggc 1584
Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly
515 520 525
agg gaa tgc gtg gac aag tgc aac ctt ctg gag ggt gag cca agg gag 1632
Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu
530 535 540
ttt gtg gag aac tct gag tgc ata cag tgc cac cca gag tgc ctg cct 1680
Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro
545 550 555 560
cag gcc atg aac atc acc tgc aca gga cgg gga cca gac aac tgt atc 1728
Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile
565 570 575
cag tgt gcc cac tac att gac ggc ccc cac tgc gtc aag acc tgc ccg 1776
Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro
580 585 590
gca gga gtc atg gga gaa aac aac acc ctg gtc tgg aag tac gca gac 1824
Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp
595 600 605
gcc ggc cat gtg tgc cac ctg tgc cat cca aac tgc acc tac gga tgc 1872
Ala Gly His Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys
610 615 620
act ggg cca ggt ctt gaa ggc tgt cca acg aat ggg cct aag atc ccg tga 1923
Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro
625 630 635 640

Claims (32)

1.一种抗原-Fc融合蛋白，其特征在于，其包含抗原和含有突变的Fc片段，所述突变的Fc片段的氨基酸序列为SEQ ID NO:1所示的序列。
2.根据权利要求1所述抗原-Fc融合蛋白，其特征在于，所述抗原为全长蛋白分子或细胞跨膜蛋白的胞外结构域。
3.根据权利要求1所述抗原-Fc融合蛋白，其特征在于，所述抗原为疾病相关的蛋白质抗原，优选地为肿瘤相关抗原；所述肿瘤相关抗原任选地为选自ErbB2(HER2/neu)、癌胚抗原(CEA)、上皮细胞黏附分子(EpCAM)、表皮生长因子受体(EGFR、EGFR变体III(EGFRvIII))、CD19、CD20、CD30、CD40、双唾液酸神经节苷脂GD2、GD3、C型凝集素样分子-1(CLL-1)、导管上皮粘蛋白、gp36、TAG-72、鞘糖脂、胶质瘤相关抗原、β-人绒毛膜促性腺激素、甲胎蛋白(AFP)、凝集素反应性AFP、甲状腺球蛋白、RAGE-1、MN-CA IX、人端粒酶逆转录酶、RU1、RU2(AS)、肠羧基酯酶、mut hsp70-2、M-CSF、前列腺酶、前列腺酶特异性抗原(PSA)、PAP、NY-ESO-1、LAGA-1a、p53、前列腺蛋白、PSMA、存活蛋白和端粒酶、前列腺癌肿瘤抗原-1(PCTA-1)、MAGE、ELF2M、中性粒细胞弹性蛋白酶、肝配蛋白B2、CD22、胰岛素生长因子(IGF1)-I、IGF-II、IGFI受体、间皮素(MSLN)、呈递肿瘤特异性肽表位的主要组织相容性复合物(MHC)分子、5T4、ROR1、Nkp30、NKG2D、肿瘤基质抗原、纤连蛋白的额外结构域A(EDA)和额外结构域B(EDB)以及肌腱蛋白-C的A1结构域(TnC A1)以及成纤维细胞相关蛋白(fap)、LRP6、黑素瘤相关硫酸软骨素蛋白聚糖(MCSP)、CD38/CS1、MART1、WT1、MUC1、LMP2、独特型、NY-ESO-1、Ras突变体、gp100、蛋白酶3、bcr-abl、酪氨酸酶、hTERT、EphA2、ML-TAP、ERG、NA17、PAX3、ALK、雄激素受体；谱系特异性或组织特异性抗原如CD3、CD4、CD8、CD24、CD25、CD33、CD34、CD70、CD79、CD116、CD117、CD135、CD123、CD133、CD138、CTLA-4、B7-1(CD80)、B7-2(CD86)、内皮糖蛋白、主要组织相容性复合物(MHC)分子、BCMA(CD269、TNFRSF 17)和FLT-3中任一种，优选地为选自CD19、MSLN、Muc1和EGFR中的任一种，更优选地为选自CD19的胞外结构域、MSLN的胞外结构域、Muc1的胞外结构域和EGFR的胞外结构域中的任一种。
4.根据权利要求1-3中任一项所述抗原-Fc融合蛋白，其特征在于，其还包含接头，所述接头两端分别连接所述抗原和所述含有突变的Fc片段；优选地，所述接头为IgG分子铰链区，更优选地，所述接头为IgG4铰链区。
5.根据权利要求4所述抗原-Fc融合蛋白，其特征在于，所述抗原位于所述接头的N端，所述含有突变的Fc片段位于所述接头的C端；或所述抗原位于所述接头的C端，所述含有突变的Fc片段位于所述接头的N端。
6.根据权利要求5所述抗原-Fc融合蛋白，其特征在于，其N端或C端还含有一个或多个标签，优选地，所述标签为选自FLAG、HA、HA1、c-Myc、Poly-His、Poly-Arg、Strep-TagII、AU1、EE、T7、4A6、ε、B、gE和Ty1中的一种或多种。
7.根据权利要求5所述抗原-Fc融合蛋白，其特征在于，其氨基酸序列为选自SEQ IDNO:4、SEQ ID NO:6、SEQ ID NO:8和SEQ ID NO:10中任一种。
8.根据权利要求5-7种任一项所述抗原-Fc融合蛋白，其特征在于，其还包含分泌型信号肽，所述分泌型信号肽位于其N端；优选地，所述分泌型信号肽为轻链信号肽；更优选地，所述分泌型信号肽的序列如SEQ ID NO:3所示，更优选地，所述抗原-Fc融合蛋白的氨基酸序列为选自SEQ ID NO:20、SEQ ID NO:22、SEQ ID NO:24和SEQ ID NO:26中任一种。
9.编码如权利要求1-8中任一项所述抗原-Fc融合蛋白的分离的核酸，其是DNA或RNA。
10.根据权利要求9所述分离的核酸，其特征在于，其为单链DNA或双链DNA；和/或，其序列为SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9或SEQ ID NO:11所示序列。
11.根据权利要求9所述分离的核酸，其特征在于，其为未经修饰的RNA或经过化学修饰的RNA；和/或，其序列为SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14或SEQ ID NO:15所示序列。
12.根据权利要求11所述分离的核酸，其特征在于，所述经过化学修饰的RNA上的化学修饰包括核糖环修饰，磷酸二酯键骨架修饰和碱基修饰；优选地，所述核糖环修饰修饰为2’-氧烷基修饰或2’-F修饰修饰；优选地，所述磷酸二酯键骨架修饰为锁核酸(LNA)修饰；优选地，所述碱基修饰为4-硫脲嘧啶修饰、2-硫脲嘧啶修饰或C-连接的伪尿嘧啶修饰；优选地，所述经过化学修饰的RNA包含一种或多种所述化学修饰。
13.一种核酸构建体，其特征在于，其含有根据权利要求9或10所述DNA序列和与所述DNA序列可操作地连接的调控序列。
14.根据权利要求13所述核酸构建体，其特征在于，所述调控序列是启动子，优选地，所述调控序列是CMV启动子。
15.根据权利要求14所述核酸构建体，其特征在于，所述调控序列还包括转录终止序列，所述转录终止序列与所述DNA序列的3’末端可操作地连接，优选地，所述转录终止序列为SV40-polyA转录终止序列。
16.根据权利要求14或15所述核酸构建体，其特征在于，所述调控序列还包括增强子，优选地，所述增强子位于所述启动子的上游；优选地，所述增强子为选自mCMV增强子、hCMV增强子、EF1a启动子的CCEF增强子、腺病毒主要晚期蛋白增强子、CD3e增强子和SV40增强子中的任一种，更优选地，所述增强子为hCMV增强子或mCMV增强子，最优选地，所述增强子为hCMV增强子。
17.根据权利要求16所述核酸构建体，其特征在于，其是载体，优选地，其是表达载体，更优选地，其是真核表达载体，最优选地，其是pCDNA3.4载体。
18.一种根据权利要求1-8中任一项所述抗原-Fc融合蛋白的制备方法，其特征在于，其包括以下步骤：化学合成根据权利要求1-8中任一项所述抗原-Fc融合蛋白的氨基酸序列或使用根据权利要求17所述表达载体表达所述抗原-Fc融合蛋白。
19.根据权利要求18所述制备方法，其特征在于，所述表达载体为真核表达载体，优选地，所述表达载体为pCDNA3.4，所述表达载体包含的编码所述抗原-Fc融合蛋白的DNA序列为选自SEQ ID NO:5、SEQ ID NO:7、SEQ ID NO:9和SEQ ID NO:11中任一种。
20.根据权利要求18或19所述制备方法，其特征在于，其还包括纯化过程；优选地，所述纯化过程包括选自盐析、亲和层析、离子交换层析、分子筛层析和吸附层析中的一种或多种；
任选地，所述纯化过程为亲和层析，优选地，所述亲和层析为基于Fc与Protein A或Protein G之间的相互作用的亲和层析。
21.一种带标记物的抗原-Fc融合蛋白，其特征在于，其包含根据权利要求1-8中任一项所述抗原-Fc融合蛋白和与其相连接的标记物。
22.根据权利要求21所述带标记物的抗原-Fc融合蛋白，其特征在于，所述标记物为荧光基团或化学发光剂。
23.根据权利要求22所述带标记物的抗原-Fc融合蛋白，其特征在于，所述标记物为荧光基团，所述荧光基团为选自FITC、TRITC、PE、FAM、RRX、TR、Cy2、Cy3、Cy5、ECD、PC5.5、PC7、APC、APC-A70、APC-A75、Pac-Blue、Alexa488、mBBr、5-IAF、E-118、DTAF、罗丹明绿和KrO中的任一种，优选地，所述荧光基团为PE荧光素。
24.根据权利要求21-23中任一项所述带标记物的抗原-Fc融合蛋白，其特征在于，所述标记物通过共价结合方式或非共价结合方式与所述抗原-Fc融合蛋白相连接。
25.根据权利要求24所述带标记物的抗原-Fc融合蛋白，其特征在于，所述标记物与链亲合素共价相连接，所述抗原-Fc融合蛋白与生物素共价相连接，所述标记物通过链亲合素与生物素间的非共价相互作用与前述抗原-Fc融合蛋白相连接；
优选地，所述标记物为PE荧光素；
优选地，所述抗原-Fc融合蛋白为根据权利要求4所述抗原-Fc融合蛋白，更优选地，所述抗原-Fc融合蛋白为根据权利要求7所述抗原-Fc融合蛋白。
26.一种根据权利要求21-25中任一项所述带标记物的抗原-Fc融合蛋白的制备方法，其特征在于，其包括以下步骤：将所述标记物与根据权利要求1-8中任一项所述抗原-Fc融合蛋白相连接。
27.根据权利要求26所述制备方法，其特征在于，其包括以下步骤：将所述标记物与链亲合素共价相连接，将生物素与所述抗原-Fc融合蛋白共价相连接，通过生物素与链亲合素间的非共价相互作用将所述标记物与所述抗原-Fc融合蛋白非共价相连接；
优选地，所述标记物为PE荧光素；
优选地，所述抗原-Fc融合蛋白为根据权利要求4所述抗原-Fc融合蛋白，更优选地，所述抗原-Fc融合蛋白为根据权利要求7所述抗原-Fc融合蛋白。
28.一种检测阳性CAR-T细胞的试剂盒，其特征在于，其含有根据权利要求1-8中任一项所述抗原-Fc融合蛋白、根据权利要求9-12中任一项所述分离的核酸、根据权利要求13-17中任一项所述核酸构建体和根据权利要求21-25中任一项所述带标记物的抗原-Fc融合蛋白中的一种或多种。
29.根据权利要求28所述试剂盒，其特征在于，其还包括标记物、配体和受体中的一种或多种，所述配体能够与所述标记物共价相连接，所述受体能够与所述抗原-Fc融合蛋白共价相连接，所述配体与所述受体之间能够非共价相连接；优选地，所述标记物为PE荧光素；优选地，所述配体为链亲合素，所述受体为生物素。
30.根据权利要求29所述试剂盒，其特征在于，其还包括与链亲合素共价相连接的PE荧光素和/或反应试剂，所述反应试剂为N-羟基琥珀酰亚胺和/或1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐。
31.一种阳性CAR-T细胞的检测方法，其特征在于，其包括以下步骤：将根据权利要求21-25中任一项所述带标记物的抗原-Fc融合蛋白与待检测的CAR-T细胞孵育、洗涤后进行检测。
32.根据权利要求31所述检测方法，其特征在于，所述检测为流式细胞检测；
和/或，所述孵育的温度为37℃；
和/或，所述孵育的时间为1小时；
和/或，所述孵育的CO2浓度为5％；
和/或，所述洗涤为将细胞用洗涤缓冲液重悬、离心；
和/或，所述洗涤的次数为3次。
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Application publication date: 20190719