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CN110452294B - 五种铰链区及其嵌合抗原受体和免疫细胞 - Google Patents

五种铰链区及其嵌合抗原受体和免疫细胞 Download PDF

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CN110452294B
CN110452294B CN201910720495.8A CN201910720495A CN110452294B CN 110452294 B CN110452294 B CN 110452294B CN 201910720495 A CN201910720495 A CN 201910720495A CN 110452294 B CN110452294 B CN 110452294B
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储以微
王月弟
骆菲菲
高一源
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Fudan University
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Abstract

本发明提供了五种铰链区及其嵌合抗原受体和免疫细胞，属于生物医药技术领域，所述五种铰链区分别为铰链区V‑5、铰链区V‑4‑5、铰链区V‑3‑4‑5、铰链区V‑2‑3‑4‑5和铰链区V‑1‑2‑3‑4‑5。利用本发明铰链区的CAR结构，相较于应用传统铰链区的CAR结构，能够显著提升CAR‑T细胞的抗原特异性免疫反应，增强CAR‑T细胞的敏感性，并抵抗耗竭，从而增强CAR‑T细胞的治疗效果，提升抗肿瘤治疗疗效。同时，包含本发明铰链区的CAR适用于对T细胞、NK细胞等免疫细胞的转染，能够广谱应用于肿瘤治疗。

Description

五种铰链区及其嵌合抗原受体和免疫细胞
技术领域
本发明涉及生物医药技术领域，尤其涉及五种铰链区及其嵌合抗原受体和免疫细胞。
背景技术
嵌合抗原受体(Chimeric Antigen Receptor，CAR)制备技术是一种利用基因工程编辑的方法将非MHC限制识别目标抗原的单链抗体(scFv)与铰链区(hinge)、跨膜基序(TM)和T细胞活化相关基序串联，通过借助电穿孔转染或病毒感染等方式修饰T或NK等免疫细胞，赋予它们识别、杀伤肿瘤细胞的能力。以CAR修饰的T细胞在血液肿瘤治疗中取得了良好的疗效，2017年有两款靶向CD19肿瘤抗原的CAR-T细胞产品在美国获批上市，用于治疗复发或难治性儿童和年轻成人急性淋巴细胞白血病。但是，CAR制备技术发展相对缓慢，自1993年出现第一个应用CAR修饰T细胞杀伤卵巢肿瘤，20多年来，从第一代CAR技术发展至第三代，以及近年来一系列基于第二、三代CAR的技术优化，整体疗效提升有限。在非实体肿瘤治疗方面，仍面临治疗抵抗或反复复发等困境；在针对实体瘤治疗方面，更是难见显著有效报道。因此，迫切需要开发新一代CAR制备技术以提升肿瘤治疗整体疗效。
目前广泛应用的CAR结构主要由四个部分组成：1.最外端的单克隆单链抗体序列，主要识别抗原，决定CAR-T细胞的靶向性；2.铰链区，连接单克隆抗体序列和跨膜序列；3.跨膜基序，使得CAR可以锚定在免疫细胞胞膜上；4.胞内信号传导序列，主要为一个或者多个共刺激分子胞内段以及CD3ζ链串联，为CAR-T细胞提供活化所需的第一、二信号。因此，在综合分析了上述四部分结构和功能后，近年来的研究都将目光投注于单链抗体段和胞内信号段的序列优化，以期增强CAR-T细胞体内杀伤功能。但是，迄今尚未有CAR-T细胞疗效显著提升的报道。而铰链区虽为CAR结构中的重要组成，但因传统认为其仅发挥连接作用，致使铰链区是否参与细胞功能发挥的相关研究在国内外基本空白。
发明内容
本发明的目的在于提供五种铰链区及其嵌合抗原受体和免疫细胞，以增强免疫细胞的抗原特异性免疫应答。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种铰链区V-5，所述铰链区V-5的核苷酸序列如SEQ ID NO.1所示。
本发明提供了一种铰链区V-4-5，所述铰链区V-4-5的核苷酸序列如SEQ ID NO.2所示。
本发明提供了一种铰链区V-3-4-5，所述铰链区V-3-4-5的核苷酸序列如SEQ IDNO.3所示。
本发明提供了一种铰链区V-2-3-4-5，所述铰链区V-2-3-4-5的核苷酸序列如SEQID NO.4所示。
本发明提供了一种铰链区V-1-2-3-4-5，所述铰链区V-1-2-3-4-5的核苷酸序列如SEQ ID NO.5所示。
本发明还提供了一种嵌合抗原受体，所述嵌合抗原受体的铰链区为铰链区V-5、铰链区V-4-5、铰链区V-3-4-5、铰链区V-2-3-4-5和铰链区V-1-2-3-4-5中的任意一种。
本发明还提供了一种包含上述方案所述嵌合抗原受体的免疫细胞。
优选的，所述免疫细胞为T细胞或NK细胞。
本发明还提供了上述方案任意一种所述铰链区在制备提高免疫细胞的抗原特异性免疫应答的药物中的应用。
本发明还提供了上述方案任意一种所述铰链区在制备抗肿瘤药物中的应用。
本发明的有益效果：本发明提供了五种铰链区，分别为铰链区V-5、铰链区V-4-5、铰链区V-3-4-5、铰链区V-2-3-4-5和铰链区V-1-2-3-4-5；所述铰链区V-5、铰链区V-4-5、铰链区V-3-4-5、铰链区V-2-3-4-5和铰链区V-1-2-3-4-5的核苷酸序列分别如SEQ ID NO.1～SEQ ID NO.5所示。利用本发明铰链区的CAR结构，相较于应用传统铰链区的CAR结构，能够显著提升CAR-T细胞的抗原特异性免疫反应，增强CAR-T细胞的敏感性，并抵抗耗竭，从而增强CAR-T细胞的治疗效果，提升抗肿瘤治疗疗效。同时，包含本发明铰链区的CAR适用于对T细胞、NK细胞等免疫细胞的转染，能够广谱应用于肿瘤治疗。
附图说明
图1表示4-1BB V(5)的结构示意图；
图2表示4-1BB V(4-5)的结构示意图；
图3表示4-1BB V(3-4-5)的结构示意图；
图4表示4-1BB V(2-3-4-5)的结构示意图；
图5表示4-1BB V(1-2-3-4-5)的结构示意图；
图6表示No-H的结构示意图；
图7表示CD8a-H的结构式意图；
图8表示CD28-H的结构式意图；
图9表示IgG4-H的结构示意图；
图10表示靶向HER2-CAR电转后T细胞活力；
图11表示靶向HER2-CAR电转后T细胞大小；
图12表示靶向HER2-CAR电转后T细胞数目；
图13表示靶向HER2-CAR电转后的表达水平；
图14表示HER2-CAR-T细胞CD107a膜转位水平；
图15表示HER2-CAR-T细胞IFN-γ的分泌情况；
图16表示HER2-CAR-T细胞IL-2的分泌情况；
图17表示GPC3-CAR-T细胞的CD107a膜转位；
图18表示GPC3-CAR-T细胞的IFN-γ的分泌；
图19表示GPC3-CAR-T细胞的IL-2的分泌；
图20表示CD19-CAR-T细胞的CD107a膜转位；
图21表示CD19-CAR-T细胞的IFN-γ的分泌；
图22表示CD19-CAR-T细胞的IL-2的分泌；
图23表示CAR-T细胞在不同浓度抗原刺激下细胞因子TNF-α分泌；
图24表示CAR-T细胞在不同浓度抗原刺激下细胞因子IFN-γ分泌；
图25表示CAR-T细胞在不同浓度抗原刺激下活化水平的差异(CD25表达)；
图26表示CAR-T细胞活化后下游信号传导(pSTAT1)；
图27表示CAR-T细胞活化后下游信号传导(pSTAT5)；
图28表示CAR-T细胞表面耗竭性标志(PD-1、TIM3)的表达水平。
具体实施方式
本发明提供了一种铰链区V-5，所述铰链区V-5的核苷酸序列如SEQ ID NO.1所示。
本发明提供了一种铰链区V-4-5，所述铰链区V-4-5的核苷酸序列如SEQ ID NO.2所示。
本发明提供了一种铰链区V-3-4-5，所述铰链区V-3-4-5的核苷酸序列如SEQ IDNO.3所示。
本发明提供了一种铰链区V-2-3-4-5，所述铰链区V-2-3-4-5的核苷酸序列如SEQID NO.4所示。
本发明提供了一种铰链区V-1-2-3-4-5，所述铰链区V-1-2-3-4-5的核苷酸序列如SEQ ID NO.5所示。
本发明还提供了一种嵌合抗原受体，所述嵌合抗原受体的铰链区为铰链区V-5、铰链区V-4-5、铰链区V-3-4-5、铰链区V-2-3-4-5和铰链区V-1-2-3-4-5中的任意一种；所述嵌合抗原受体由非MHC限制识别目标抗原的单链抗体(scFv)与铰链区(hinge)、跨膜基序(TM)和T细胞活化相关基序顺次串联得到。
本发明中，所述铰链区V-5、铰链区V-4-5、铰链区V-3-4-5、铰链区V-2-3-4-5和铰链区V-1-2-3-4-5均含有一段共同的核苷酸序列，如SEQ ID NO.1所示。
本发明具体实施过程中，包含铰链区V-5的嵌合抗原受体为嵌合抗原受体4-1BB V(5)，结构示意图如图1所示；所述嵌合抗原受体4-1BB V(5)的核苷酸序列如SEQ ID NO.6所示；所述嵌合抗原受体4-1BB V(5)编码的蛋白质的氨基酸序列如SEQ ID NO.7所示；所述嵌合抗原受体4-1BB V(5)优选的由上海生工有限公司合成。
本发明具体实施过程中，包含铰链区V-4-5的嵌合抗原受体为嵌合抗原受体4-1BBV(4-5)，结构示意图如图2所示；所述嵌合抗原受体4-1BB V(4-5)的核苷酸序列如SEQ IDNO.8所示；所述嵌合抗原受体4-1BB V(4-5)编码的蛋白质的氨基酸序列如SEQ ID NO.9所示；所述嵌合抗原受体4-1BB V(4-5)优选的由上海生工有限公司合成。
本发明具体实施过程中，包含铰链区V-3-4-5的嵌合抗原受体为嵌合抗原受体4-1BB V(3-4-5)，结构示意图如图3所示；所述嵌合抗原受体4-1BB V(3-4-5)的核苷酸序列如SEQ ID NO.10所示；所述嵌合抗原受体4-1BB V(3-4-5)编码的蛋白质的氨基酸序列如SEQID NO.11所示；所述嵌合抗原受体4-1BB V(3-4-5)优选的由上海生工有限公司合成。
本发明具体实施过程中，包含铰链区V-2-3-4-5的嵌合抗原受体为嵌合抗原受体4-1BB V(2-3-4-5)，结构示意图如图4所示；所述嵌合抗原受体4-1BB V(2-3-4-5)的核苷酸序列如SEQ ID NO.12所示；所述嵌合抗原受体4-1BB V(2-3-4-5)编码的蛋白质的氨基酸序列如SEQ ID NO.13所示；所述嵌合抗原受体4-1BB V(2-3-4-5)优选的由上海生工有限公司合成。
本发明具体实施过程中，包含铰链区V-1-2-3-4-5的嵌合抗原受体为嵌合抗原受体4-1BB V(1-2-3-4-5)，结构示意图如图5所示；所述嵌合抗原受体4-1BB V(1-2-3-4-5)的核苷酸序列如SEQ ID NO.14所示；所述嵌合抗原受体4-1BB V(1-2-3-4-5)编码的蛋白质的氨基酸序列如SEQ ID NO.15所示；所述嵌合抗原受体4-1BB V(1-2-3-4-5)优选的由上海生工有限公司合成。
本发明中，去除铰链区的嵌合抗原受体No-H的结构示意图如图6所示；所述No-H的核苷酸序列如SEQ ID NO.16所示；所述No-H编码No-H的蛋白质的氨基酸序列如SEQ IDNO.17所示。
本发明还提供了一种包含上述方案所述嵌合抗原受体的免疫细胞；所述免疫细胞优选为T细胞或NK细胞。
本发明还提供了上述方案任意一种所述铰链区在制备提高免疫细胞的抗原特异性免疫应答的药物中的应用。
本发明还提供了上述方案任意一种所述铰链区在制备抗肿瘤药物中的应用。
本发明中，利用本发明铰链区的CAR结构，相较于应用传统铰链区的CAR结构，能够显著提升CAR-T细胞的抗原特异性免疫反应，增强CAR-T细胞的敏感性，并抵抗耗竭，从而增强CAR-T细胞的治疗效果，提升抗肿瘤治疗疗效。
下面结合实施例对本发明提供的技术方案进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
实施例1
1.委托上海生工有限公司根据核酸序列合成相应的目的基因，其中目的基因包括4-1BB V(1-2-3-4-5)、4-1BB V(2-3-4-5)、4-1BB V(3-4-5)、4-1BB V(4-5)、4-1BB V(5)、No-H、CD8a-H、CD28-H和IgG4-H，其中CD8a-H的结构式意图如图7所示，CD8a-H的核苷酸序列如SEQ ID NO.18所示，CD8a-H编码的氨基酸序列如SEQ ID NO.19所示；CD28-H的结构式意图如图8所示，CD28-H的核苷酸序列如SEQ ID NO.20所示，CD28-H编码的氨基酸序列如SEQID NO.21所示；IgG4-H的结构示意图如图9所示，IgG4-H的核苷酸序列如SEQ ID NO.22所示，IgG4-H编码的氨基酸序列如SEQ ID NO.23所示。通过基因克隆的手段将目的基因连接到载体pGEM4Z/GFP/A64中，我们利用neb的Not I和Hind III两个酶切掉GFP基因，然后将上述目的基因插入。
2.制备mRNA：将质粒利用neb的SpeI酶切过夜，利用QIAGEN的QIAquick PCRPurification Kit试剂盒纯化酶切产物，使用CELLSCRIPTTM的T7 mScriptTMStandard mRNAProduction System试剂盒制备RNA。
3.T细胞制备：健康人外周血分离的单个核细胞，调整浓度为1×106/mL，铺于24孔板中，1mL/孔。培养条件5ug/mL anti-hCD3(pre-coated)，2ug/mL anti-hCD28。五天后去除anti-hCD3/28刺激，给予40ng/mL(100 U/mL)rmIL-2维持量，每2天根据细胞浓度补充培养基，待9～11天后T细胞体积缩小为8um以下时，细胞冻存备用。
4.RNA电转T细胞：首先用冷Opti-MEM洗细胞三次，重悬细胞于合适浓度(20～300×106/mL)；然后取100L细胞悬液与5～10ug RNA混匀，加入0.2M电转杯；电转条件：500uv700us(刺激后T细胞)，500uv 800us(静息T细胞)，500uv 500us(肿瘤细胞)；电转后迅速转入预热的1640完全培养基中。
5.电转RNA后，分别进行功能评价。
5.1电转HER2-CAR的24h后，使用thermo fisher公司的细胞计数仪，测量T细胞活力、大小和数目。测量结果参见图10～图12，其中图10表示靶向HER2-CAR电转后T细胞活力；图11表示靶向HER2-CAR电转后T细胞大小；图12表示靶向HER2-CAR电转后T细胞数目。由图10～图12可以看出，靶向HER2-CAR电转后不影响T细胞活力、直径和数目。
5.2电转HER2-CAR的24h后，使用荧光标记抗体进行染色，后在流式细胞仪上测量各组T细胞表面HER2-CAR的表达情况。测量结果参见图13。由图13可以看出不同的铰链区会影响CAR的表达情况，其中4-1BB V(3-4-5)表达最高。
5.3电转HER2-CAR RNA的24h后，各组T细胞分别与K562(HER2阴性)肿瘤细胞和N87(HER2阳性)肿瘤细胞按比例(1:4)共孵育4h，检测T细胞表面CD107a膜转位情况。检测结果参见图14。结果发现，与K562(Her2阴性)肿瘤细胞共孵育时，除4-1BB V(1-2-3-4-5)组外，各组T细胞表面均只检测到较低水平的CD107a膜转位；但是当与N87(HER2阳性)肿瘤细胞共孵育时，与未转RNA组T细胞相比，电转HER2 CAR RNA组(No-H组除外)的T细胞表面均可检测到高水平的CD107a膜转位，且No-H组类似没有电转的T细胞组。
5.4电转HER2-CAR RNA的24h后，各组T细胞分别与K562(HER2阴性)肿瘤细胞和N87(HER2阳性)肿瘤细胞按比例(1:1)共孵育24h，检测细胞因子IFN-以及IL-2的分泌。检测结果参见图15和图16，其中图15表示HER2-CAR-T细胞IFN-γ的分泌情况；图16表示HER2-CAR-T细胞IL-2的分泌情况。结果发现，与K562(HER2阴性)组对比，电转HER2 CAR RNA组(No-H组除外)的T细胞分泌较高的IFN-，4-1BB V(3-4-5)组分泌较高水平的IL-2，No-H组类似没有电转的T细胞组。
5.5电转GPC3-CAR RNA(其中GPC3 scFv的核苷酸序列如SEQ ID NO.24所示，GPC3scFv编码的氨基酸序列如SEQ ID NO.25所示)的24h后，检测CD107a膜转位以及细胞因子的分泌，GPC3阳性细胞为Huh7，GPC3阴性细胞为K562。结果参见图17～图19，其中图17表示GPC3-CAR-T细胞的CD107a膜转位；图18表示GPC3-CAR-T细胞的IFN-γ的分泌；图19表示GPC3-CAR-T细胞的IL-2的分泌。由图17～图19可以看出，所有的GPC3-CAR-T均具有良好的特异性，识别GPC3阳性的肿瘤Huh7，且包含铰链区V-3-4-5的GPC3-CAR-T活化水平更高，表现出细胞因子(IL-2，IFN-γ)的高分泌，以及最优的杀伤效应(CD107a膜转位)。
5.6电转CD19-CAR RNA(其中CD19 scFv的核苷酸序列如SEQ ID NO.26所示，CD19scFv编码的氨基酸序列如SEQ ID NO.27所示)的24h后，检测CD107a膜转位以及细胞因子的分泌，CD19阳性细胞为Ramos，CD19阴性细胞为K562。结果参见图20～图22，其中图20表示CD19-CAR-T细胞的CD107a膜转位；图21表示CD19-CAR-T细胞的IFN-γ的分泌；图22表示CD19-CAR-T细胞的IL-2的分泌。由图20～图22可以看出，与HER2-CAR,GPC3-CAR结果一致，铰链区V-3-4-5赋予CD19-CAR-T细胞更高的活化水平和杀伤效应。
5.7电转HER2-CAR RNA后，使用纯化的HER2抗原按照不同剂量刺激，检测该优化的铰链区(4-1BB V(3-4-5))对抗原刺激的敏感性，并设置最广泛使用的传统铰链区(CD8-H)和去除铰链区作为对照，实验结果参见图23，图24和图25，其中图23表示CAR-T细胞在不同浓度抗原(3μg/mL,1μg/mL和0μg/mL)刺激下细胞因子TNF-α的分泌；图24表示CAR-T细胞在不同浓度抗原刺激下IFN-γ的分泌；图25表示CAR-T细胞在不同抗原刺激下活化水平(CD25)的差异。结果证实4-1BB V(3-4-5)作为铰链区组在低剂量的抗原刺激下仍保持高水平的免疫反应性，提示该新型铰链区显著增强CAR-T细胞对抗原反应的敏感性。
5.8电转HER2-CAR RNA后，与抗原阳性的肿瘤细胞共同培养48h，后检测CAR-T活化后下游信号传导(pSTAT1、pSTAT5)以及表面耗竭性标志(PD-1、TIM3)，比较不同铰链区对CAR-T细胞的耗竭的影响，结果参见图26～图28，其中图26表示CAR-T细胞活化后下游信号传导(pSTAT1)；图27表示CAR-T细胞活化后下游信号传导(pSTAT5)；图28表示CAR-T细胞表面耗竭性标志(PD-1、TIM3)的表达水平。结果证实铰链区4-1BB V(3-4-5)在有效提升CAR-T细胞的活化外，还可以维持其持久性，表现较低水平的耗竭标志的表达。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 复旦大学
<120> 五种铰链区及其嵌合抗原受体和免疫细胞
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Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
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Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
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Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys
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His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
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Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val
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Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser
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Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val
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Asp Gly Phe Tyr Ala Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr
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Val Ser Ser Pro Ser Pro Ala Asp Leu Ser Pro Gly Ala Ser Ser Val
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Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val
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Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu
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Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr
325 330 335
Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr
340 345 350
Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro
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Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys
370 375 380
Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe
385 390 395 400
Ser Arg Ser Ala Glu Pro Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu
405 410 415
Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp
420 425 430
Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys
435 440 445
Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala
450 455 460
Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys
465 470 475 480
Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr
485 490 495
Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
500 505
<210> 8
<211> 1650
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
atggattttc aggtgcagat tttcagcttc ctgctaatca gtgcctcagt cataatgtcc 60
agaggagata tccagatgac ccagtccccg agctccctgt ccgcctctgt gggcgatagg 120
gtcaccatca cctgccgtgc cagtcaggat gtgaatactg ctgtagcctg gtatcaacag 180
aaaccaggaa aagctccgaa actactgatt tactcggcat ccttccttta ttctggagtc 240
ccttctcgct tctctggatc tagatctggg acggatttca ctctgaccat cagcagtctg 300
cagccggaag acttcgcaac ttattactgt cagcaacatt atactactcc tcccacgttc 360
ggacagggta ccaaggtgga gatcaaaggt ggtggtggtt ctggcggcgg cggctccgag 420
gttcagctgg tggagtctgg cggtggcctg gtgcagccag ggggctcact ccgtttgtcc 480
tgtgcagctt ctggcttcaa cattaaagac acctatatac actgggtgcg tcaggccccg 540
ggtaagggcc tggaatgggt tgcaaggatt tatcctacga atggttatac tagatatgcc 600
gatagcgtca agggccgttt cactataagc gcagacacat ccaaaaacac agcctacctg 660
cagatgaaca gcctgcgtgc tgaggacact gccgtctatt attgttctag atggggaggg 720
gacggcttct atgctatgga cgtgtggggt caaggaaccc tggtcaccgt ctcctcggac 780
tgttgctttg ggacatttaa cgatcagaaa cgtggcatct gtcgaccctg gacaaactgt 840
tctttggatg gaaagtctgt gcttgtgaat gggacgaagg agagggacgt ggtctgtgga 900
ccatctccag ccgacctctc tccgggagca tcctctgtga ccccgcctgc ccctgcgaga 960
gagccaggac actctccgca gttttgggtg ctggtggtgg ttggtggagt cctggcttgc 1020
tatagcttgc tagtaacagt ggcctttatt attttctggg tgaggagtaa gaggagcagg 1080
ctcctgcaca gtgactacat gaacatgact ccccgccgcc ccgggcccac ccgcaagcat 1140
taccagccct atgccccacc acgcgacttc gcagcctatc gctccaaacg gggcagaaag 1200
aaactcctgt atatattcaa acaaccattt atgagaccag tacaaactac tcaagaggaa 1260
gatggctgta gctgccgatt tccagaagaa gaagaaggag gatgtgaact gagagtgaag 1320
ttcagcagga gcgcagagcc ccccgcgtac cagcagggcc agaaccagct ctataacgag 1380
ctcaatctag gacgaagaga ggagtacgat gttttggaca agagacgtgg ccgggaccct 1440
gagatggggg gaaagccgag aaggaagaac cctcaggaag gcctgtacaa tgaactgcag 1500
aaagataaga tggcggaggc ctacagtgag attgggatga aaggcgagcg ccggaggggc 1560
aaggggcacg atggccttta ccagggtctc agtacagcca ccaaggacac ctacgacgcc 1620
cttcacatgc aggccctgcc ccctcgctaa 1650
<210> 9
<211> 549
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
20 25 30
Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
35 40 45
Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys
50 55 60
Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val
65 70 75 80
Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr
85 90 95
Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
100 105 110
His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
115 120 125
Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val
130 135 140
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser
145 150 155 160
Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val
165 170 175
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Tyr Pro
180 185 190
Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
195 200 205
Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser
210 215 220
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser Arg Trp Gly Gly
225 230 235 240
Asp Gly Phe Tyr Ala Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr
245 250 255
Val Ser Ser Asp Cys Cys Phe Gly Thr Phe Asn Asp Gln Lys Arg Gly
260 265 270
Ile Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Lys Ser Val Leu
275 280 285
Val Asn Gly Thr Lys Glu Arg Asp Val Val Cys Gly Pro Ser Pro Ala
290 295 300
Asp Leu Ser Pro Gly Ala Ser Ser Val Thr Pro Pro Ala Pro Ala Arg
305 310 315 320
Glu Pro Gly His Ser Pro Gln Phe Trp Val Leu Val Val Val Gly Gly
325 330 335
Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe
340 345 350
Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn
355 360 365
Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr
370 375 380
Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys
385 390 395 400
Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr
405 410 415
Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu
420 425 430
Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Glu Pro Pro
435 440 445
Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly
450 455 460
Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro
465 470 475 480
Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr
485 490 495
Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly
500 505 510
Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln
515 520 525
Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln
530 535 540
Ala Leu Pro Pro Arg
545
<210> 10
<211> 1746
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
atggattttc aggtgcagat tttcagcttc ctgctaatca gtgcctcagt cataatgtcc 60
agaggagata tccagatgac ccagtccccg agctccctgt ccgcctctgt gggcgatagg 120
gtcaccatca cctgccgtgc cagtcaggat gtgaatactg ctgtagcctg gtatcaacag 180
aaaccaggaa aagctccgaa actactgatt tactcggcat ccttccttta ttctggagtc 240
ccttctcgct tctctggatc tagatctggg acggatttca ctctgaccat cagcagtctg 300
cagccggaag acttcgcaac ttattactgt cagcaacatt atactactcc tcccacgttc 360
ggacagggta ccaaggtgga gatcaaaggt ggtggtggtt ctggcggcgg cggctccgag 420
gttcagctgg tggagtctgg cggtggcctg gtgcagccag ggggctcact ccgtttgtcc 480
tgtgcagctt ctggcttcaa cattaaagac acctatatac actgggtgcg tcaggccccg 540
ggtaagggcc tggaatgggt tgcaaggatt tatcctacga atggttatac tagatatgcc 600
gatagcgtca agggccgttt cactataagc gcagacacat ccaaaaacac agcctacctg 660
cagatgaaca gcctgcgtgc tgaggacact gccgtctatt attgttctag atggggaggg 720
gacggcttct atgctatgga cgtgtggggt caaggaaccc tggtcaccgt ctcctcggac 780
tgcactccag ggtttcactg cctgggggca ggatgcagca tgtgtgaaca ggattgtaaa 840
caaggtcaag aactgacaaa aaaaggttgt aaagactgtt gctttgggac atttaacgat 900
cagaaacgtg gcatctgtcg accctggaca aactgttctt tggatggaaa gtctgtgctt 960
gtgaatggga cgaaggagag ggacgtggtc tgtggaccat ctccagccga cctctctccg 1020
ggagcatcct ctgtgacccc gcctgcccct gcgagagagc caggacactc tccgcagttt 1080
tgggtgctgg tggtggttgg tggagtcctg gcttgctata gcttgctagt aacagtggcc 1140
tttattattt tctgggtgag gagtaagagg agcaggctcc tgcacagtga ctacatgaac 1200
atgactcccc gccgccccgg gcccacccgc aagcattacc agccctatgc cccaccacgc 1260
gacttcgcag cctatcgctc caaacggggc agaaagaaac tcctgtatat attcaaacaa 1320
ccatttatga gaccagtaca aactactcaa gaggaagatg gctgtagctg ccgatttcca 1380
gaagaagaag aaggaggatg tgaactgaga gtgaagttca gcaggagcgc agagcccccc 1440
gcgtaccagc agggccagaa ccagctctat aacgagctca atctaggacg aagagaggag 1500
tacgatgttt tggacaagag acgtggccgg gaccctgaga tggggggaaa gccgagaagg 1560
aagaaccctc aggaaggcct gtacaatgaa ctgcagaaag ataagatggc ggaggcctac 1620
agtgagattg ggatgaaagg cgagcgccgg aggggcaagg ggcacgatgg cctttaccag 1680
ggtctcagta cagccaccaa ggacacctac gacgcccttc acatgcaggc cctgccccct 1740
cgctaa 1746
<210> 11
<211> 581
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
20 25 30
Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
35 40 45
Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys
50 55 60
Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val
65 70 75 80
Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr
85 90 95
Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
100 105 110
His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
115 120 125
Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val
130 135 140
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser
145 150 155 160
Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val
165 170 175
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Tyr Pro
180 185 190
Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
195 200 205
Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser
210 215 220
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser Arg Trp Gly Gly
225 230 235 240
Asp Gly Phe Tyr Ala Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr
245 250 255
Val Ser Ser Asp Cys Thr Pro Gly Phe His Cys Leu Gly Ala Gly Cys
260 265 270
Ser Met Cys Glu Gln Asp Cys Lys Gln Gly Gln Glu Leu Thr Lys Lys
275 280 285
Gly Cys Lys Asp Cys Cys Phe Gly Thr Phe Asn Asp Gln Lys Arg Gly
290 295 300
Ile Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Lys Ser Val Leu
305 310 315 320
Val Asn Gly Thr Lys Glu Arg Asp Val Val Cys Gly Pro Ser Pro Ala
325 330 335
Asp Leu Ser Pro Gly Ala Ser Ser Val Thr Pro Pro Ala Pro Ala Arg
340 345 350
Glu Pro Gly His Ser Pro Gln Phe Trp Val Leu Val Val Val Gly Gly
355 360 365
Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe
370 375 380
Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn
385 390 395 400
Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr
405 410 415
Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys
420 425 430
Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr
435 440 445
Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu
450 455 460
Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Glu Pro Pro
465 470 475 480
Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly
485 490 495
Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro
500 505 510
Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr
515 520 525
Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly
530 535 540
Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln
545 550 555 560
Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln
565 570 575
Ala Leu Pro Pro Arg
580
<210> 12
<211> 1869
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
atggattttc aggtgcagat tttcagcttc ctgctaatca gtgcctcagt cataatgtcc 60
agaggagata tccagatgac ccagtccccg agctccctgt ccgcctctgt gggcgatagg 120
gtcaccatca cctgccgtgc cagtcaggat gtgaatactg ctgtagcctg gtatcaacag 180
aaaccaggaa aagctccgaa actactgatt tactcggcat ccttccttta ttctggagtc 240
ccttctcgct tctctggatc tagatctggg acggatttca ctctgaccat cagcagtctg 300
cagccggaag acttcgcaac ttattactgt cagcaacatt atactactcc tcccacgttc 360
ggacagggta ccaaggtgga gatcaaaggt ggtggtggtt ctggcggcgg cggctccgag 420
gttcagctgg tggagtctgg cggtggcctg gtgcagccag ggggctcact ccgtttgtcc 480
tgtgcagctt ctggcttcaa cattaaagac acctatatac actgggtgcg tcaggccccg 540
ggtaagggcc tggaatgggt tgcaaggatt tatcctacga atggttatac tagatatgcc 600
gatagcgtca agggccgttt cactataagc gcagacacat ccaaaaacac agcctacctg 660
cagatgaaca gcctgcgtgc tgaggacact gccgtctatt attgttctag atggggaggg 720
gacggcttct atgctatgga cgtgtggggt caaggaaccc tggtcaccgt ctcctcgagt 780
ccctgtcctc caaatagttt ctccagcgca ggtggacaaa ggacctgtga catatgcagg 840
cagtgtaaag gtgttttcag gaccaggaag gagtgttcct ccaccagcaa tgcagagtgt 900
gactgcactc cagggtttca ctgcctgggg gcaggatgca gcatgtgtga acaggattgt 960
aaacaaggtc aagaactgac aaaaaaaggt tgtaaagact gttgctttgg gacatttaac 1020
gatcagaaac gtggcatctg tcgaccctgg acaaactgtt ctttggatgg aaagtctgtg 1080
cttgtgaatg ggacgaagga gagggacgtg gtctgtggac catctccagc cgacctctct 1140
ccgggagcat cctctgtgac cccgcctgcc cctgcgagag agccaggaca ctctccgcag 1200
ttttgggtgc tggtggtggt tggtggagtc ctggcttgct atagcttgct agtaacagtg 1260
gcctttatta ttttctgggt gaggagtaag aggagcaggc tcctgcacag tgactacatg 1320
aacatgactc cccgccgccc cgggcccacc cgcaagcatt accagcccta tgccccacca 1380
cgcgacttcg cagcctatcg ctccaaacgg ggcagaaaga aactcctgta tatattcaaa 1440
caaccattta tgagaccagt acaaactact caagaggaag atggctgtag ctgccgattt 1500
ccagaagaag aagaaggagg atgtgaactg agagtgaagt tcagcaggag cgcagagccc 1560
cccgcgtacc agcagggcca gaaccagctc tataacgagc tcaatctagg acgaagagag 1620
gagtacgatg ttttggacaa gagacgtggc cgggaccctg agatgggggg aaagccgaga 1680
aggaagaacc ctcaggaagg cctgtacaat gaactgcaga aagataagat ggcggaggcc 1740
tacagtgaga ttgggatgaa aggcgagcgc cggaggggca aggggcacga tggcctttac 1800
cagggtctca gtacagccac caaggacacc tacgacgccc ttcacatgca ggccctgccc 1860
cctcgctaa 1869
<210> 13
<211> 622
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
20 25 30
Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
35 40 45
Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys
50 55 60
Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val
65 70 75 80
Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr
85 90 95
Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
100 105 110
His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
115 120 125
Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val
130 135 140
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser
145 150 155 160
Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val
165 170 175
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Tyr Pro
180 185 190
Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
195 200 205
Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser
210 215 220
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser Arg Trp Gly Gly
225 230 235 240
Asp Gly Phe Tyr Ala Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr
245 250 255
Val Ser Ser Ser Pro Cys Pro Pro Asn Ser Phe Ser Ser Ala Gly Gly
260 265 270
Gln Arg Thr Cys Asp Ile Cys Arg Gln Cys Lys Gly Val Phe Arg Thr
275 280 285
Arg Lys Glu Cys Ser Ser Thr Ser Asn Ala Glu Cys Asp Cys Thr Pro
290 295 300
Gly Phe His Cys Leu Gly Ala Gly Cys Ser Met Cys Glu Gln Asp Cys
305 310 315 320
Lys Gln Gly Gln Glu Leu Thr Lys Lys Gly Cys Lys Asp Cys Cys Phe
325 330 335
Gly Thr Phe Asn Asp Gln Lys Arg Gly Ile Cys Arg Pro Trp Thr Asn
340 345 350
Cys Ser Leu Asp Gly Lys Ser Val Leu Val Asn Gly Thr Lys Glu Arg
355 360 365
Asp Val Val Cys Gly Pro Ser Pro Ala Asp Leu Ser Pro Gly Ala Ser
370 375 380
Ser Val Thr Pro Pro Ala Pro Ala Arg Glu Pro Gly His Ser Pro Gln
385 390 395 400
Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu
405 410 415
Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser
420 425 430
Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly
435 440 445
Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala
450 455 460
Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys
465 470 475 480
Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys
485 490 495
Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val
500 505 510
Lys Phe Ser Arg Ser Ala Glu Pro Pro Ala Tyr Gln Gln Gly Gln Asn
515 520 525
Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val
530 535 540
Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg
545 550 555 560
Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys
565 570 575
Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg
580 585 590
Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys
595 600 605
Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
610 615 620
<210> 14
<211> 1935
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
atggattttc aggtgcagat tttcagcttc ctgctaatca gtgcctcagt cataatgtcc 60
agaggagata tccagatgac ccagtccccg agctccctgt ccgcctctgt gggcgatagg 120
gtcaccatca cctgccgtgc cagtcaggat gtgaatactg ctgtagcctg gtatcaacag 180
aaaccaggaa aagctccgaa actactgatt tactcggcat ccttccttta ttctggagtc 240
ccttctcgct tctctggatc tagatctggg acggatttca ctctgaccat cagcagtctg 300
cagccggaag acttcgcaac ttattactgt cagcaacatt atactactcc tcccacgttc 360
ggacagggta ccaaggtgga gatcaaaggt ggtggtggtt ctggcggcgg cggctccgag 420
gttcagctgg tggagtctgg cggtggcctg gtgcagccag ggggctcact ccgtttgtcc 480
tgtgcagctt ctggcttcaa cattaaagac acctatatac actgggtgcg tcaggccccg 540
ggtaagggcc tggaatgggt tgcaaggatt tatcctacga atggttatac tagatatgcc 600
gatagcgtca agggccgttt cactataagc gcagacacat ccaaaaacac agcctacctg 660
cagatgaaca gcctgcgtgc tgaggacact gccgtctatt attgttctag atggggaggg 720
gacggcttct atgctatgga cgtgtggggt caaggaaccc tggtcaccgt ctcctcgttg 780
caggatcctt gtagtaactg cccagctggt acattctgtg ataataacag gaatcagatt 840
tgcagtccct gtcctccaaa tagtttctcc agcgcaggtg gacaaaggac ctgtgacata 900
tgcaggcagt gtaaaggtgt tttcaggacc aggaaggagt gttcctccac cagcaatgca 960
gagtgtgact gcactccagg gtttcactgc ctgggggcag gatgcagcat gtgtgaacag 1020
gattgtaaac aaggtcaaga actgacaaaa aaaggttgta aagactgttg ctttgggaca 1080
tttaacgatc agaaacgtgg catctgtcga ccctggacaa actgttcttt ggatggaaag 1140
tctgtgcttg tgaatgggac gaaggagagg gacgtggtct gtggaccatc tccagccgac 1200
ctctctccgg gagcatcctc tgtgaccccg cctgcccctg cgagagagcc aggacactct 1260
ccgcagtttt gggtgctggt ggtggttggt ggagtcctgg cttgctatag cttgctagta 1320
acagtggcct ttattatttt ctgggtgagg agtaagagga gcaggctcct gcacagtgac 1380
tacatgaaca tgactccccg ccgccccggg cccacccgca agcattacca gccctatgcc 1440
ccaccacgcg acttcgcagc ctatcgctcc aaacggggca gaaagaaact cctgtatata 1500
ttcaaacaac catttatgag accagtacaa actactcaag aggaagatgg ctgtagctgc 1560
cgatttccag aagaagaaga aggaggatgt gaactgagag tgaagttcag caggagcgca 1620
gagccccccg cgtaccagca gggccagaac cagctctata acgagctcaa tctaggacga 1680
agagaggagt acgatgtttt ggacaagaga cgtggccggg accctgagat ggggggaaag 1740
ccgagaagga agaaccctca ggaaggcctg tacaatgaac tgcagaaaga taagatggcg 1800
gaggcctaca gtgagattgg gatgaaaggc gagcgccgga ggggcaaggg gcacgatggc 1860
ctttaccagg gtctcagtac agccaccaag gacacctacg acgcccttca catgcaggcc 1920
ctgccccctc gctaa 1935
<210> 15
<211> 644
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 15
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
20 25 30
Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
35 40 45
Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys
50 55 60
Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val
65 70 75 80
Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr
85 90 95
Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
100 105 110
His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
115 120 125
Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val
130 135 140
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser
145 150 155 160
Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val
165 170 175
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Tyr Pro
180 185 190
Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
195 200 205
Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser
210 215 220
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser Arg Trp Gly Gly
225 230 235 240
Asp Gly Phe Tyr Ala Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr
245 250 255
Val Ser Ser Leu Gln Asp Pro Cys Ser Asn Cys Pro Ala Gly Thr Phe
260 265 270
Cys Asp Asn Asn Arg Asn Gln Ile Cys Ser Pro Cys Pro Pro Asn Ser
275 280 285
Phe Ser Ser Ala Gly Gly Gln Arg Thr Cys Asp Ile Cys Arg Gln Cys
290 295 300
Lys Gly Val Phe Arg Thr Arg Lys Glu Cys Ser Ser Thr Ser Asn Ala
305 310 315 320
Glu Cys Asp Cys Thr Pro Gly Phe His Cys Leu Gly Ala Gly Cys Ser
325 330 335
Met Cys Glu Gln Asp Cys Lys Gln Gly Gln Glu Leu Thr Lys Lys Gly
340 345 350
Cys Lys Asp Cys Cys Phe Gly Thr Phe Asn Asp Gln Lys Arg Gly Ile
355 360 365
Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Lys Ser Val Leu Val
370 375 380
Asn Gly Thr Lys Glu Arg Asp Val Val Cys Gly Pro Ser Pro Ala Asp
385 390 395 400
Leu Ser Pro Gly Ala Ser Ser Val Thr Pro Pro Ala Pro Ala Arg Glu
405 410 415
Pro Gly His Ser Pro Gln Phe Trp Val Leu Val Val Val Gly Gly Val
420 425 430
Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp
435 440 445
Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met
450 455 460
Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala
465 470 475 480
Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys
485 490 495
Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr
500 505 510
Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly
515 520 525
Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Glu Pro Pro Ala
530 535 540
Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg
545 550 555 560
Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu
565 570 575
Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn
580 585 590
Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met
595 600 605
Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly
610 615 620
Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala
625 630 635 640
Leu Pro Pro Arg
<210> 16
<211> 1446
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
atggattttc aggtgcagat tttcagcttc ctgctaatca gtgcctcagt cataatgtcc 60
agaggagata tccagatgac ccagtccccg agctccctgt ccgcctctgt gggcgatagg 120
gtcaccatca cctgccgtgc cagtcaggat gtgaatactg ctgtagcctg gtatcaacag 180
aaaccaggaa aagctccgaa actactgatt tactcggcat ccttccttta ttctggagtc 240
ccttctcgct tctctggatc tagatctggg acggatttca ctctgaccat cagcagtctg 300
cagccggaag acttcgcaac ttattactgt cagcaacatt atactactcc tcccacgttc 360
ggacagggta ccaaggtgga gatcaaaggt ggtggtggtt ctggcggcgg cggctccgag 420
gttcagctgg tggagtctgg cggtggcctg gtgcagccag ggggctcact ccgtttgtcc 480
tgtgcagctt ctggcttcaa cattaaagac acctatatac actgggtgcg tcaggccccg 540
ggtaagggcc tggaatgggt tgcaaggatt tatcctacga atggttatac tagatatgcc 600
gatagcgtca agggccgttt cactataagc gcagacacat ccaaaaacac agcctacctg 660
cagatgaaca gcctgcgtgc tgaggacact gccgtctatt attgttctag atggggaggg 720
gacggcttct atgctatgga cgtgtggggt caaggaaccc tggtcaccgt ctcctcgttt 780
tgggtgctgg tggtggttgg tggagtcctg gcttgctata gcttgctagt aacagtggcc 840
tttattattt tctgggtgag gagtaagagg agcaggctcc tgcacagtga ctacatgaac 900
atgactcccc gccgccccgg gcccacccgc aagcattacc agccctatgc cccaccacgc 960
gacttcgcag cctatcgctc caaacggggc agaaagaaac tcctgtatat attcaaacaa 1020
ccatttatga gaccagtaca aactactcaa gaggaagatg gctgtagctg ccgatttcca 1080
gaagaagaag aaggaggatg tgaactgaga gtgaagttca gcaggagcgc agagcccccc 1140
gcgtaccagc agggccagaa ccagctctat aacgagctca atctaggacg aagagaggag 1200
tacgatgttt tggacaagag acgtggccgg gaccctgaga tggggggaaa gccgagaagg 1260
aagaaccctc aggaaggcct gtacaatgaa ctgcagaaag ataagatggc ggaggcctac 1320
agtgagattg ggatgaaagg cgagcgccgg aggggcaagg ggcacgatgg cctttaccag 1380
ggtctcagta cagccaccaa ggacacctac gacgcccttc acatgcaggc cctgccccct 1440
cgctaa 1446
<210> 17
<211> 481
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 17
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
20 25 30
Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
35 40 45
Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys
50 55 60
Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val
65 70 75 80
Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr
85 90 95
Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
100 105 110
His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
115 120 125
Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val
130 135 140
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser
145 150 155 160
Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val
165 170 175
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Tyr Pro
180 185 190
Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
195 200 205
Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser
210 215 220
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser Arg Trp Gly Gly
225 230 235 240
Asp Gly Phe Tyr Ala Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr
245 250 255
Val Ser Ser Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys
260 265 270
Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser
275 280 285
Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg
290 295 300
Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg
305 310 315 320
Asp Phe Ala Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr
325 330 335
Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu
340 345 350
Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu
355 360 365
Leu Arg Val Lys Phe Ser Arg Ser Ala Glu Pro Pro Ala Tyr Gln Gln
370 375 380
Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu
385 390 395 400
Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly
405 410 415
Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln
420 425 430
Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu
435 440 445
Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr
450 455 460
Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro
465 470 475 480
Arg
<210> 18
<211> 1581
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
atggattttc aggtgcagat tttcagcttc ctgctaatca gtgcctcagt cataatgtcc 60
agaggagata tccagatgac ccagtccccg agctccctgt ccgcctctgt gggcgatagg 120
gtcaccatca cctgccgtgc cagtcaggat gtgaatactg ctgtagcctg gtatcaacag 180
aaaccaggaa aagctccgaa actactgatt tactcggcat ccttccttta ttctggagtc 240
ccttctcgct tctctggatc tagatctggg acggatttca ctctgaccat cagcagtctg 300
cagccggaag acttcgcaac ttattactgt cagcaacatt atactactcc tcccacgttc 360
ggacagggta ccaaggtgga gatcaaaggt ggtggtggtt ctggcggcgg cggctccgag 420
gttcagctgg tggagtctgg cggtggcctg gtgcagccag ggggctcact ccgtttgtcc 480
tgtgcagctt ctggcttcaa cattaaagac acctatatac actgggtgcg tcaggccccg 540
ggtaagggcc tggaatgggt tgcaaggatt tatcctacga atggttatac tagatatgcc 600
gatagcgtca agggccgttt cactataagc gcagacacat ccaaaaacac agcctacctg 660
cagatgaaca gcctgcgtgc tgaggacact gccgtctatt attgttctag atggggaggg 720
gacggcttct atgctatgga cgtgtggggt caaggaaccc tggtcaccgt ctcctcgacc 780
acgacgccag cgccgcgacc accaacaccg gcgcccacca tcgcgtcgca gcccctgtcc 840
ctgcgcccag aggcgtgccg gccagcggcg gggggcgcag tgcacacgag ggggctggac 900
ttcgcctgtg atttttgggt gctggtggtg gttggtggag tcctggcttg ctatagcttg 960
ctagtaacag tggcctttat tattttctgg gtgaggagta agaggagcag gctcctgcac 1020
agtgactaca tgaacatgac tccccgccgc cccgggccca cccgcaagca ttaccagccc 1080
tatgccccac cacgcgactt cgcagcctat cgctccaaac ggggcagaaa gaaactcctg 1140
tatatattca aacaaccatt tatgagacca gtacaaacta ctcaagagga agatggctgt 1200
agctgccgat ttccagaaga agaagaagga ggatgtgaac tgagagtgaa gttcagcagg 1260
agcgcagagc cccccgcgta ccagcagggc cagaaccagc tctataacga gctcaatcta 1320
ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1380
ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1440
atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 1500
gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1560
caggccctgc cccctcgcta a 1581
<210> 19
<211> 525
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 19
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
20 25 30
Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
35 40 45
Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys
50 55 60
Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val
65 70 75 80
Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr
85 90 95
Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
100 105 110
His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
115 120 125
Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val
130 135 140
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser
145 150 155 160
Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val
165 170 175
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Tyr Pro
180 185 190
Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
195 200 205
Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser
210 215 220
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser Arg Trp Gly Gly
225 230 235 240
Asp Gly Phe Tyr Ala Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr
245 250 255
Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro
260 265 270
Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro
275 280 285
Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
290 295 300
Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu
305 310 315 320
Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser
325 330 335
Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly
340 345 350
Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala
355 360 365
Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys
370 375 380
Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys
385 390 395 400
Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val
405 410 415
Lys Phe Ser Arg Ser Ala Glu Pro Pro Ala Tyr Gln Gln Gly Gln Asn
420 425 430
Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val
435 440 445
Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg
450 455 460
Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys
465 470 475 480
Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg
485 490 495
Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys
500 505 510
Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro
515 520 525
<210> 20
<211> 1581
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
atggattttc aggtgcagat tttcagcttc ctgctaatca gtgcctcagt cataatgtcc 60
agaggagata tccagatgac ccagtccccg agctccctgt ccgcctctgt gggcgatagg 120
gtcaccatca cctgccgtgc cagtcaggat gtgaatactg ctgtagcctg gtatcaacag 180
aaaccaggaa aagctccgaa actactgatt tactcggcat ccttccttta ttctggagtc 240
ccttctcgct tctctggatc tagatctggg acggatttca ctctgaccat cagcagtctg 300
cagccggaag acttcgcaac ttattactgt cagcaacatt atactactcc tcccacgttc 360
ggacagggta ccaaggtgga gatcaaaggt ggtggtggtt ctggcggcgg cggctccgag 420
gttcagctgg tggagtctgg cggtggcctg gtgcagccag ggggctcact ccgtttgtcc 480
tgtgcagctt ctggcttcaa cattaaagac acctatatac actgggtgcg tcaggccccg 540
ggtaagggcc tggaatgggt tgcaaggatt tatcctacga atggttatac tagatatgcc 600
gatagcgtca agggccgttt cactataagc gcagacacat ccaaaaacac agcctacctg 660
cagatgaaca gcctgcgtgc tgaggacact gccgtctatt attgttctag atggggaggg 720
gacggcttct atgctatgga cgtgtggggt caaggaaccc tggtcaccgt ctcctcggat 780
atttacttct gcaaaattga agttatgtat cctcctcctt acctagacaa tgagaagagc 840
aatggaacca ttatccatgt gaaagggaaa cacctttgtc caagtcccct atttcccgga 900
ccttctaagc ccttttgggt gctggtggtg gttggtggag tcctggcttg ctatagcttg 960
ctagtaacag tggcctttat tattttctgg gtgaggagta agaggagcag gctcctgcac 1020
agtgactaca tgaacatgac tccccgccgc cccgggccca cccgcaagca ttaccagccc 1080
tatgccccac cacgcgactt cgcagcctat cgctccaaac ggggcagaaa gaaactcctg 1140
tatatattca aacaaccatt tatgagacca gtacaaacta ctcaagagga agatggctgt 1200
agctgccgat ttccagaaga agaagaagga ggatgtgaac tgagagtgaa gttcagcagg 1260
agcgcagagc cccccgcgta ccagcagggc cagaaccagc tctataacga gctcaatcta 1320
ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1380
ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1440
atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 1500
gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1560
caggccctgc cccctcgcta a 1581
<210> 21
<211> 526
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 21
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
20 25 30
Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
35 40 45
Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys
50 55 60
Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val
65 70 75 80
Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr
85 90 95
Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
100 105 110
His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
115 120 125
Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val
130 135 140
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser
145 150 155 160
Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val
165 170 175
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Tyr Pro
180 185 190
Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
195 200 205
Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser
210 215 220
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser Arg Trp Gly Gly
225 230 235 240
Asp Gly Phe Tyr Ala Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr
245 250 255
Val Ser Ser Asp Ile Tyr Phe Cys Lys Ile Glu Val Met Tyr Pro Pro
260 265 270
Pro Tyr Leu Asp Asn Glu Lys Ser Asn Gly Thr Ile Ile His Val Lys
275 280 285
Gly Lys His Leu Cys Pro Ser Pro Leu Phe Pro Gly Pro Ser Lys Pro
290 295 300
Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu
305 310 315 320
Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser
325 330 335
Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly
340 345 350
Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala
355 360 365
Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys
370 375 380
Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys
385 390 395 400
Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val
405 410 415
Lys Phe Ser Arg Ser Ala Glu Pro Pro Ala Tyr Gln Gln Gly Gln Asn
420 425 430
Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val
435 440 445
Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg
450 455 460
Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys
465 470 475 480
Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg
485 490 495
Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys
500 505 510
Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
515 520 525
<210> 22
<211> 1761
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
atggattttc aggtgcagat tttcagcttc ctgctaatca gtgcctcagt cataatgtcc 60
agaggagata tccagatgac ccagtccccg agctccctgt ccgcctctgt gggcgatagg 120
gtcaccatca cctgccgtgc cagtcaggat gtgaatactg ctgtagcctg gtatcaacag 180
aaaccaggaa aagctccgaa actactgatt tactcggcat ccttccttta ttctggagtc 240
ccttctcgct tctctggatc tagatctggg acggatttca ctctgaccat cagcagtctg 300
cagccggaag acttcgcaac ttattactgt cagcaacatt atactactcc tcccacgttc 360
ggacagggta ccaaggtgga gatcaaaggt ggtggtggtt ctggcggcgg cggctccgag 420
gttcagctgg tggagtctgg cggtggcctg gtgcagccag ggggctcact ccgtttgtcc 480
tgtgcagctt ctggcttcaa cattaaagac acctatatac actgggtgcg tcaggccccg 540
ggtaagggcc tggaatgggt tgcaaggatt tatcctacga atggttatac tagatatgcc 600
gatagcgtca agggccgttt cactataagc gcagacacat ccaaaaacac agcctacctg 660
cagatgaaca gcctgcgtgc tgaggacact gccgtctatt attgttctag atggggaggg 720
gacggcttct atgctatgga cgtgtggggt caaggaaccc tggtcaccgt ctcctcgaaa 780
gccaaagggc agccccgaga accacaggtg tacaccctgc ccccatcccg ggatgagctg 840
accaagaacc aggtcagcct gacctgcctg gtcaaaggct tctatcccag cgacatcgcc 900
gtggagtggg agagcaatgg gcagccggag aacaactaca agaccacgcc tcccgtgctg 960
gactccgacg gctccttctt cctctacagc aagctcaccg tggacaagag caggtggcag 1020
caggggaacg tcttctcatg ctccgtgatg catgaggctc tgcacaacca ctacacgcag 1080
aagagcctct ccttttgggt gctggtggtg gttggtggag tcctggcttg ctatagcttg 1140
ctagtaacag tggcctttat tattttctgg gtgaggagta agaggagcag gctcctgcac 1200
agtgactaca tgaacatgac tccccgccgc cccgggccca cccgcaagca ttaccagccc 1260
tatgccccac cacgcgactt cgcagcctat cgctccaaac ggggcagaaa gaaactcctg 1320
tatatattca aacaaccatt tatgagacca gtacaaacta ctcaagagga agatggctgt 1380
agctgccgat ttccagaaga agaagaagga ggatgtgaac tgagagtgaa gttcagcagg 1440
agcgcagagc cccccgcgta ccagcagggc cagaaccagc tctataacga gctcaatcta 1500
ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 1560
ggaaagccga gaaggaagaa ccctcaggaa ggcctgtaca atgaactgca gaaagataag 1620
atggcggagg cctacagtga gattgggatg aaaggcgagc gccggagggg caaggggcac 1680
gatggccttt accagggtct cagtacagcc accaaggaca cctacgacgc ccttcacatg 1740
caggccctgc cccctcgcta a 1761
<210> 23
<211> 586
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 23
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Asp Ile Gln Met Thr Gln Ser Pro Ser Ser
20 25 30
Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
35 40 45
Gln Asp Val Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys
50 55 60
Ala Pro Lys Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val
65 70 75 80
Pro Ser Arg Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr
85 90 95
Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
100 105 110
His Tyr Thr Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
115 120 125
Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val
130 135 140
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser
145 150 155 160
Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr Tyr Ile His Trp Val
165 170 175
Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Arg Ile Tyr Pro
180 185 190
Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
195 200 205
Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr Leu Gln Met Asn Ser
210 215 220
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ser Arg Trp Gly Gly
225 230 235 240
Asp Gly Phe Tyr Ala Met Asp Val Trp Gly Gln Gly Thr Leu Val Thr
245 250 255
Val Ser Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
260 265 270
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
275 280 285
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
290 295 300
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
305 310 315 320
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys
325 330 335
Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu
340 345 350
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Phe Trp Val Leu
355 360 365
Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val
370 375 380
Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His
385 390 395 400
Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys
405 410 415
His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser
420 425 430
Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met
435 440 445
Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe
450 455 460
Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg
465 470 475 480
Ser Ala Glu Pro Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn
485 490 495
Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg
500 505 510
Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro
515 520 525
Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala
530 535 540
Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His
545 550 555 560
Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp
565 570 575
Ala Leu His Met Gln Ala Leu Pro Pro Arg
580 585
<210> 24
<211> 778
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
atggattttc aggtgcagat tttcagcttc ctgctaatca gtgcctcagt cataatgtcc 60
agaggagatg ttgtgatgac tcagtctcca ctctccctgc ccgtcacccc tggagagccg 120
gcctcctctc ctgcagatct agtcagagcc ttgtacacag taatgccaac acctatttac 180
attggtacct gcagaagcca gggcagtctc cacagctcct gatctataaa gtttccaacc 240
gattttctgg ggtccctgac aggttcagtg gcagtggatc aggcacagat tttacactga 300
aaatcagcag agtggaggct gaggatgttg gggtttatta ctgctctcaa aatacacatg 360
ttcctcctac gtttggccag gggaccaagc tggagatcaa acgtgtggag gcggttcagg 420
cggaggtggc tctcaggtgc agctggtgca gtctggagct gaggtgaaga agcctggggc 480
ctcagtgaag gtctcctgca aggcttctgg atacaccttc accgactatg aaatgcactg 540
ggtgcgacag gcccctggac aagggcttga gtggatggga gctcttgatc ctaaaactgg 600
tgatactgcc tacagtcaga agttcaaggg cagagtcacg ctgaccgcgg acgaatccac 660
gagcacagcc tacatggagc tgagcagcct gagatctgag gacacggccg tgtattactg 720
tacaagattc tactcctata cttactgggg ccagggaacc ctggtcaccg tctcctca 778
<210> 25
<211> 260
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 25
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Asp Val Val Met Thr Gln Ser Pro Leu Ser
20 25 30
Leu Pro Val Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser
35 40 45
Gln Ser Leu Val His Ser Asn Ala Asn Thr Tyr Leu His Trp Tyr Leu
50 55 60
Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn
65 70 75 80
Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr
85 90 95
Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val
100 105 110
Tyr Tyr Cys Ser Gln Asn Thr His Val Pro Pro Thr Phe Gly Gln Gly
115 120 125
Thr Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly
130 135 140
Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly
145 150 155 160
Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp
165 170 175
Tyr Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp
180 185 190
Met Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys
195 200 205
Phe Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala
210 215 220
Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
225 230 235 240
Cys Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val
245 250 255
Thr Val Ser Ser
260
<210> 26
<211> 792
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
atggattttc aggtgcagat tttcagcttc ctgctaatca gtgcctcagt cataatgtcc 60
agaggagaca tccagatgac acagactaca tcctccctgt ctgcctctct gggagacaga 120
gtcaccatca gttgcagggc aagtcaggac attagtaaat atttaaattg gtatcagcag 180
aaaccagatg gaactgttaa actcctgatc taccatacat caagattaca ctcaggagtc 240
ccatcaaggt tcagtggcag tgggtctgga acagattatt ctctcaccat tagcaacctg 300
gagcaagaag atattgccac ttacttttgc caacagggta atacgcttcc gtacacgttc 360
ggagggggga ccaagctgga gatcacaggt ggcggtggct cgggcggtgg tgggtcgggt 420
ggcggcggat ctgaggtgaa actgcaggag tcaggacctg gcctggtggc gccctcacag 480
agcctgtccg tcacatgcac tgtctcaggg gtctcattac ccgactatgg tgtaagctgg 540
attcgccagc ctccacgaaa gggtctggag tggctgggag taatatgggg tagtgaaacc 600
acatactata attcagctct caaatccaga ctgaccatca tcaaggacaa ctccaagagc 660
caagttttct taaaaatgaa cagtctgcaa actgatgaca cagccattta ctactgtgcc 720
aaacattatt actacggtgg tagctatgct atggactact ggggccaagg aacctcagtc 780
accgtctcct ca 792
<210> 27
<211> 264
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 27
Met Asp Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser
1 5 10 15
Val Ile Met Ser Arg Gly Asp Ile Gln Met Thr Gln Thr Thr Ser Ser
20 25 30
Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser
35 40 45
Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly
50 55 60
Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val
65 70 75 80
Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr
85 90 95
Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln
100 105 110
Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile
115 120 125
Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln
145 150 155 160
Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr
165 170 175
Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu
180 185 190
Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys
195 200 205
Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
210 215 220
Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala
225 230 235 240
Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln
245 250 255
Gly Thr Ser Val Thr Val Ser Ser
260

Claims (5)

1.一种嵌合抗原受体，所述嵌合抗原受体为HER2-CAR；所述HER2-CAR中的铰链区为铰链区V-3-4-5、铰链区V-2-3-4-5或铰链区V-1-2-3-4-5；
所述铰链区V-3-4-5的核苷酸序列如SEQ ID NO.3所示；
所述铰链区V-2-3-4-5的核苷酸序列如SEQ ID NO.4所示；
所述铰链区V-1-2-3-4-5的核苷酸序列如SEQ ID NO.5所示；
包含铰链区V-3-4-5的嵌合抗原受体为嵌合抗原受体4-1BB V(3-4-5)；所述嵌合抗原受体4-1BB V(3-4-5)的氨基酸序列如SEQ ID NO.11所示；
包含铰链区V-2-3-4-5的嵌合抗原受体为嵌合抗原受体4-1BB V(2-3-4-5)；所述嵌合抗原受体4-1BB V(2-3-4-5)的氨基酸序列如SEQ ID NO.13所示；
包含铰链区V-1-2-3-4-5的嵌合抗原受体为嵌合抗原受体4-1BB V(1-2-3-4-5)；所述嵌合抗原受体4-1BB V(1-2-3-4-5)的氨基酸序列如SEQ ID NO.15所示。
2.一种包含权利要求1所述嵌合抗原受体的免疫细胞。
3.根据权利要求2所述的免疫细胞，其特征在于，所述免疫细胞为T细胞或者NK细胞。
4.权利要求1所述嵌合抗原受体在制备提高免疫细胞的抗原特异性免疫应答的药物中的应用。
5.权利要求1所述嵌合抗原受体在制备抗肿瘤药物中的应用。
CN201910720495.8A 2019-08-06 2019-08-06 五种铰链区及其嵌合抗原受体和免疫细胞 Active CN110452294B (zh)

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Publication number Priority date Publication date Assignee Title
CN111097043B (zh) * 2020-01-13 2023-07-04 广东昭泰体内生物医药科技有限公司 一种胃癌药物组合物及其应用
CN117924518B (zh) * 2024-01-05 2025-08-22 苏州艾凯利元生物科技有限公司 用于nk细胞的嵌合抗原受体及工程化的nk细胞

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106459206A (zh) * 2014-04-07 2017-02-22 中外制药株式会社 免疫活化抗原结合分子
CN108271377A (zh) * 2015-10-07 2018-07-10 豪夫迈·罗氏有限公司 具有针对共刺激性tnf受体的四价的双特异性抗体
CN108602887A (zh) * 2015-10-02 2018-09-28 豪夫迈·罗氏有限公司 对共刺激性tnf受体特异性的双特异性抗体
WO2018178074A1 (en) * 2017-03-29 2018-10-04 F. Hoffmann-La Roche Ag Trimeric antigen binding molecules specific for a costimulatory tnf receptor

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TWI476001B (zh) * 2011-12-26 2015-03-11 Ind Tech Res Inst 三倍體Ｆｃ融合蛋白及其用途
CN107849112B (zh) * 2015-06-25 2022-04-01 美商生物细胞基因治疗有限公司 嵌合抗原受体(car)、组合物及其使用方法
CA3029197A1 (en) * 2016-06-24 2017-12-28 Icell Gene Therapeutics Llc Chimeric antigen receptors (cars), compositions and methods thereof
KR102865130B1 (ko) * 2017-12-29 2025-09-29 메모리얼 슬로안 케터링 캔서 센터 향상된 키메라 항원 수용체 및 그의 용도
US20210054086A1 (en) * 2018-01-10 2021-02-25 The General Hospital Corporation Immune cells expressing a chimeric antigen receptor
CN108017717B (zh) * 2018-01-24 2019-08-16 首都医科大学宣武医院 一种用于体外高效定向扩增的嵌合抗原受体及其应用
CN108239144B (zh) 2018-01-26 2021-05-25 重庆精准生物技术有限公司 改造的铰链及其在构建car骨架中的应用
CN110078817B (zh) 2018-01-26 2021-02-19 重庆精准生物技术有限公司 一种改良的铰链区及其在构建car骨架中的应用

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106459206A (zh) * 2014-04-07 2017-02-22 中外制药株式会社 免疫活化抗原结合分子
CN108602887A (zh) * 2015-10-02 2018-09-28 豪夫迈·罗氏有限公司 对共刺激性tnf受体特异性的双特异性抗体
CN108271377A (zh) * 2015-10-07 2018-07-10 豪夫迈·罗氏有限公司 具有针对共刺激性tnf受体的四价的双特异性抗体
WO2018178074A1 (en) * 2017-03-29 2018-10-04 F. Hoffmann-La Roche Ag Trimeric antigen binding molecules specific for a costimulatory tnf receptor

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Differential responsiveness of tumour necrosis factor receptors (TNFR) type 1 and 2 : the critical roleof the TNFR stalk region;Christine Richter等;《Newcastle University》;Newcastle University;20111231;第9页，表3 *

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