CN113493772B - 可分泌性表达免疫毒素的嵌合抗原受体修饰的免疫细胞 - Google Patents

可分泌性表达免疫毒素的嵌合抗原受体修饰的免疫细胞 Download PDF

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CN113493772B
CN113493772B CN202010271115.XA CN202010271115A CN113493772B CN 113493772 B CN113493772 B CN 113493772B CN 202010271115 A CN202010271115 A CN 202010271115A CN 113493772 B CN113493772 B CN 113493772B
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丰明乾
陈鑫
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Huazhong Agricultural University
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Abstract

本发明公开了一种可分泌性表达免疫毒素的嵌合抗原受体(CAR)表达系统，将嵌合抗原受体遗传修饰的免疫细胞(CAR‑免疫细胞)和免疫毒素相结合，构建了一种能够分泌性表达免疫毒素的新型CAR‑免疫细胞，用于肿瘤的免疫治疗。本发明的技术方案中的CAR和免疫毒素识别肿瘤细胞上两种不同的抗原或者同一个肿瘤抗原的两个不同的抗原表位。CAR基因和免疫毒素基因在免疫细胞中的表达可以是组成性地(持续性地)表达，也可以被表达调控元件调控(例如通过SynNotch开关，当所述CAR‑免疫细胞识别并结合其中一种肿瘤抗原(或抗原表位)后，激活靶向另一种肿瘤抗原(或抗原表位)的CAR和免疫毒素的表达)。

Description

可分泌性表达免疫毒素的嵌合抗原受体修饰的免疫细胞
技术领域
本发明属于生物技术领域。具体涉及一种新型的可分泌表达免疫毒素的嵌合抗原受体表达系统以及该系统在制备抗肿瘤药物中的应用。
背景技术
二十世纪以来人类的生活环境污染日趋加剧、生活环境不断恶化，人们与致癌因素密切接触，使得恶性肿瘤的发明率逐年递增，恶性肿瘤已经成为人类健康的头号敌人。据2018年全球癌症数据分析全球癌症新发病病例为1810万人，癌症死亡病例为960万人。肿瘤免疫很早就被提出，同时也是成为继肿瘤传统治疗方法(化学治疗、手术治疗及放射治疗)之后的第四种肿瘤疗法，其可激活特异性，重要的免疫细胞，直接靶向攻击肿瘤细胞，具有较高的疗效和安全性，是目前全球肿瘤治疗研究的热点。
肿瘤免疫治疗是激发或调动机体的免疫系统，增强肿瘤微环境抗肿瘤免疫力，从而控制和杀伤肿瘤细胞。主要包括免疫细胞治疗(主要是CAR-T细胞和CAR-NK细胞)、抗体治疗和溶瘤病毒。免疫细胞包括淋巴细胞和各种吞噬细胞等。淋巴细胞是免疫系统的基本成分，主要包括T淋巴细胞、B淋巴细胞、K淋巴细胞、NK淋巴细胞，T淋巴细胞在外周血占淋巴细胞总数的65％-75％，而且有很多T细胞亚群。免疫细胞治疗有CIK(cytokine inducedkiller cell)，将患者自身免疫细胞(主要是T细胞和NK细胞)进行分离，体外活化培养，然后再回输给患者，整个过程称为细胞过继治疗。抗体治疗又可细分为裸抗(抑制肿瘤的生长信号，抗血管生成)、抗体-毒素偶联物、免疫检查点抑制剂、双特异性抗体，抗体-细胞毒性蛋白融合(如pseudomonas exotoxin PE,ricin toxin,RNase等)、抗体-细胞因子融合蛋白(IL-2,IL-12,IFN等)。
从肿瘤免疫治疗活性上看，CAR-T、T细胞介导的双特异抗体和抗体-毒素偶联物要普遍强于其它治疗手段，是目前研究最多、最有希望的肿瘤免疫治疗手段。但是这三种免疫治疗手段都面临着各自的问题，除了需要进一步挖掘活性提升机制和解决抗药性外，剂量限制性毒副作用(dose-limited toxicities)严重制约了其治疗潜力的发挥。CAR-T细胞在制备的过程中就已经将T细胞进行了激活，因此在给药后即使未进入肿瘤部位也会产生大量的细胞因子(细胞因子风暴)，严重限制了CAR-T的临床治疗。双特异抗体和免疫毒素在理论上不存在预激活T细胞(pre-activated T cell)的情况,但是单抗原靶向性的双特异抗体和免疫毒素仍然存在“在靶脱瘤”(on-target off-tumor)的风险,因为几乎所有的肿瘤抗原都在其它正常组织有低水平的表达。目前只有针对血液肿瘤B淋巴瘤表面抗原CD19的CAR-T治疗在临床上取得了可喜的成绩，但是随着大量临床实验的进行发现靶向CD19的CAR-T并不是所向披靡，部分患者仍旧对其没有反应，还产生了耐受作用，归其原因发现肿瘤表面CD19的抗原丢失，表达量下调。此外除了血液肿瘤，免疫治疗在实体瘤治疗不尽如意，主要是实体瘤肿瘤微环境、存在大量负调控蛋白，加上免疫治疗在实体瘤治疗中伴随大量炎症因子释放等，使得免疫治疗在实体瘤中的治疗效果很差，远远低于血液肿瘤的免疫治疗。
免疫毒素(immunotoxin)是一类将识别肿瘤抗原的单克隆抗体与杀死肿瘤细胞的毒素蛋白进行基因重组所构建的融合蛋白，其中的单抗分子一般是采用单链scFv抗体或者单域VH抗体，毒素蛋白多采用铜绿假单胞菌外毒素(Pseudomonas exotoxin A,简称PE，如PE38、PE24,数字代表截短的毒素的分子量)和白喉毒素(Diphtheria toxin,简称DT，如DT390，数字代表N-端390个氨基酸)，其它毒素还有霍乱毒素(cholera toxin,CET40)、炭疽毒素(anthrax toxin)、植物性毒素(如蓖麻毒素(ricin)、相思豆毒蛋白，槲寄生凝集素、美洲商陆抗病毒蛋白、白树毒素(gelonin)、丝瓜毒素或皂草素(saporin))。PE、DT和CET40属于同一类型毒素。PE类的免疫毒素作用机制是抗体通过与细胞表面抗原蛋白结合，靶向肿瘤细胞，将免疫毒素蛋白带入到肿瘤细胞内，使肿瘤细胞的翻译延伸因子2(eEF2)进行不可逆地ADP化修饰失活，从而抑制肿瘤细胞的蛋白质合成。免疫毒素活性很强，但是免疫毒素攻击癌症细胞的同时也会产生非特异性毒性问题，也能导致正常细胞的死亡，同时免疫毒素由于分子量较小半衰期较短，需要持续给药，还有毒副作用非常大，常见血管渗透综合征。
针对以上的面临的问题，如何提高CAR-T的肿瘤杀伤能力是目前研究者们关注的热点。
发明内容
本发明针对现有肿瘤免疫治疗技术的不足，结合免疫毒素和嵌合抗原受体的细胞靶向性和肿瘤细胞杀伤力，构建了一种双效的、能分泌免疫毒素的嵌合抗原受体表达系统，利用该系统遗传改造的免疫细胞(CAR-免疫细胞)能同时表达嵌合抗原受体和免疫毒素，具有更强的抗肿瘤活性。本发明的技术方案中CAR基因和免疫毒素基因识别肿瘤细胞上两种不同的抗原或者同一个肿瘤抗原的两个不同的抗原表位。CAR基因和免疫毒素基因在免疫细胞中的表达可以是组成性的，也可以被表达调控元件调控，例如，通过SynNotch开关，CAR-免疫细胞靶向肿瘤组织并结合肿瘤细胞表面抗原后，免疫细胞启动嵌合抗原受体和免疫毒素的表达，使免疫毒素在肿瘤部位选择性地富集，避免了免疫毒素系统性给药所带来的副作用，充分发挥免疫毒素和免疫细胞治疗的双重联合杀伤机制。
本发明具体技术方案如下：
一种分泌性表达免疫毒素的嵌合抗原受体表达系统，包含嵌合抗原受体表达元件和免疫毒素表达元件，所述嵌合抗原受体和免疫毒素分别识别肿瘤细胞表面相同或不同的抗原或抗原表位。
本发明所述表达系统的核心元件由真核细胞启动子和嵌合抗原受体的编码基因、免疫毒素的编码基因构成。启动子包括但不限于经典的哺乳动物细胞组成性启动子CMV,SV40,EF2。所述嵌合抗原受体和免疫毒素分别识别肿瘤细胞表面不同的抗原或抗原表位或同一个抗原的不同表位。
本发明所述嵌合抗原受体包括但不限于经典的CAR结构，通常包括抗原结合区和功能区(铰链区、跨膜结构区、共刺激信号传导区和T细胞信号传导区)。也适用于各种升级型CAR结构，如一代、二代、三代CAR。
优选的，嵌合抗原受体的功能区氨基酸序列如SEQ ID No：1、3、5所示或SEQ IDNo：1、3、5的同效突变体(序列SEQ ID No：1、3、5对应的核苷酸序列分别为SEQ ID No：2、4、6)，所述的同效突变体是指突变体的序列和所述的序列有90％或以上的相似性、但是突变体的功能性质和所述序列相同。
一个优选示例，本发明所述嵌合抗原受体氨基酸序列如SEQ ID No：31所示。
本发明所述的免疫毒素包括抗原识别片段和活性片段，其中活性片段包括细菌性毒素(如假单胞菌外毒素A(pseudomonas exotoxin A，PE or ETA)、白喉毒素(diphtheriatoxins，DT)、霍乱毒素(Cholera toxin)、炭疽毒素(anthrax toxin))、植物性毒素(如蓖麻毒素、相思豆毒蛋白，槲寄生凝集素、美洲商陆抗病毒蛋白、白树毒素(gelonin)、丝瓜毒素或皂草素(saporin))和人源性蛋白毒素(如RNA酶和促凋亡蛋白，如RNA DFF40，通过裂解RNA起到杀伤细胞的作用。促凋亡蛋白主要包括Bcl-2家族蛋白DNA片段化因子40(DNAfragmentation factor 40，DFF40)及颗粒酶等。)优选地，所述杀伤肿瘤细胞的活性肽段选自：铜绿假单胞菌(Pseudomonas aeruginos)毒素PE24(氨基酸序列如SEQ ID No：17所示，核苷酸序列如SEQ ID No：18所示)、PE38(氨基酸序列如SEQ ID No：19所示，核苷酸序列如SEQ ID No：20所示)、白喉杆菌(Corynebacterium diphtheriae)毒素DT390(氨基酸序列如SEQ ID No：21所示，核苷酸序列如SEQ ID No：22所示)、蓖麻(Ricinus communis)毒素(氨基酸序列如SEQ ID No：23所示，核苷酸序列如SEQ ID No：24所示)、霍乱弧菌(Vibriocholerae)毒素CET40(氨基酸序列如SEQ ID No：25所示，核苷酸序列如SEQ ID No：26所示)、RNase(氨基酸序列如SEQ ID No：27所示，核苷酸序列如SEQ ID No：28所示)或上述序列的同效突变体，所述的同效突变体是指突变体的序列和所述的序列有90％或以上的相似性、但是突变体的功能性质和所述序列相同。
优选的，本发明所述嵌合抗原受体的抗原结合区和免疫毒素的抗原识别片段选自抗体、抗体的抗原结合片段或其任意组合。可以是完整的抗体、Fab、Fab'、(Fab')2、Fv、VH片段或者单链可变区片段scFv中的一种或几种。
优选的，本发明所述嵌合抗原受体抗原结合区以及免疫毒素的抗原识别片段能够结合肿瘤抗原。所述肿瘤为前列腺癌、肾细胞癌、霍奇金淋巴瘤、非霍奇金淋巴瘤、白血病(如慢性淋巴细胞性白血病、急性淋巴细胞细胞性白血病、小淋巴细胞性白血病、急性髓细胞性白血病)、多发性骨髓瘤、腺癌、大肠癌、乳腺癌、实体瘤、头颈部癌、胶质母细胞瘤、神经母细胞瘤、肉瘤、转移性癌、多形性胶质母细胞瘤等。
优选的，所述肿瘤抗原选自Glypican-3(GPC3)，B7-H3，Mesothelin，EGFRvIII中的一种或几种。
进一步的，本发明所述的嵌合抗原受体表达系统，嵌合抗原受体和免疫毒素的表达方式可以为组成性的表达方式，或者为受表达调控元件调控的条件诱导性的表达方式。所述的组成性的表达方式是指嵌合抗原受体和免疫毒素在免疫细胞中的表达不需要施加特殊的诱导条件。
优选的，所述表达调控元件选自synNotch系统、四环素介导的调控系统、雌激素受体(estrogen receptor,ER)介导的调控系统中的一种或几种。
本发明一个优选的示例中，所述表达调控元件为synNotch系统。
SynNotch是一种嵌合跨膜受体，是基于天然Notch蛋白的跨膜信号转导机制，将天然Notch蛋白的胞外域(配体识别域)用其它能够识别肿瘤细胞抗原的单链抗体(scFv)取代，将天然Notch蛋白的胞内域用其它转录激活域(Gal4-VP64、tetR-VP64等)取代，将synNotch基因导入免疫细胞后，SynNotch在免疫细胞表面表达，识别并结合肿瘤细胞表面的抗原后，SynNotch被激活并在跨膜区发生断裂，释放的胞内转录激活域进入细胞核激活其它基因的表达。
所述synNotch系统的胞外域与嵌合抗原受体和免疫毒素识别肿瘤细胞表面的抗原或抗原表位不同。
优选的synNotch系统的synNotch蛋白包括胞外抗原识别区和核心控制区，所述synNotch蛋白核心控制区的氨基酸序列如SEQ ID No：29所示(核苷酸序列如SEQ ID No：30所示)或SEQ ID No：29的同效突变体。
优选的，所述synNotch蛋白的胞外抗原识别区、嵌合抗原受体的胞外抗原识别区和免疫毒素抗原识别肽段相同或不同。
本发明一个优选的示例，所述synNotch系统的胞外域与嵌合抗原受体和免疫毒素识别的肿瘤细胞表面抗原选自Glypican-3(GPC3)、B7-H3、Mesothelin、EGFRvIII。优选的，抗原识别片段选自hYP7(氨基酸序列SEQ ID No：7，核苷酸序列SEQ ID No：8)、HN3(氨基酸序列SEQ ID No：9，核苷酸序列SEQ ID No：10)、anti-B7H3(氨基酸序列SEQ ID No：11，核苷酸序列SEQ ID No：12)、SS1(氨基酸序列SEQ ID No：13，核苷酸序列SEQ ID No：14)、YP158(氨基酸序列SEQ ID No：15，核苷酸序列SEQ ID No：16)中的任意一种或几种。
本发明公开了一种双靶点增效的嵌合抗原受体表达系统，包含synNotch系统和嵌合抗原受体表达元件和免疫毒素表达元件。优选地，synNotch系统靶向肿瘤细胞表面抗原后，其胞内转录因子激活嵌合抗原受体和免疫毒素的表达，synNotch的抗原识别片段和嵌合抗原受体以及免疫毒素分别识别并靶向肿瘤细胞不同的表面抗原或抗原表位，或者相同肿瘤抗原的不同抗原表位。
一个具体的示例中，所述synNotch系统氨基酸序列如SEQ ID No：29所示，嵌合抗原受体氨基酸序列如SEQ ID No：31所示，免疫毒素氨基酸序列如SEQ ID No：32-41所示。
本发明所述的嵌合抗原受体表达系统，可以为携带嵌合抗原受体和免疫毒素编码基因的重组质粒、表达框、病毒载体或细胞。
所述细胞选自自体的或异体的的T细胞、NK细胞、细胞毒性T淋巴细胞或调节T细胞、记忆性T细胞、双特异性T细胞、CIK细胞、NKT细胞、γδ-T细胞。
本发明还公开了本发明所述的嵌合抗原受体表达系统在制备抗肿瘤药物中的应用。所述的嵌合抗原受体和免疫毒素表达系统作为药物活性成分用于肿瘤免疫治疗。
优选的，所述肿瘤选自肝癌、肺癌、结直肠癌、胃癌、卵巢癌、胰腺癌、前列腺癌、肾细胞癌、霍奇金淋巴瘤、非霍奇金淋巴瘤、白血病(如慢性淋巴细胞性白血病、急性淋巴细胞细胞性白血病、小淋巴细胞性白血病、急性髓细胞性白血病)、多发性骨髓瘤、腺癌、乳腺癌、头颈部癌、胶质母细胞瘤、神经母细胞瘤、肉瘤、多形性胶质母细胞瘤等。
本发明另一目的在于公开一种对免疫细胞进行遗传修饰和基因编辑的方法，利用权利本发明所述的嵌合抗原受体和免疫毒素表达系统使免疫细胞共表达嵌合抗原受体和免疫毒素。
本发明优点：
(1)本发明以CAR-免疫细胞分泌表达免疫毒素，利用CAR的肿瘤抗原识别能力，提高免疫毒素的靶向性，实现免疫毒素在肿瘤部位的特异性富集，避免了免疫毒素全身性给药的毒副作用。同时通过免疫毒素对肿瘤细胞的杀伤作用，提高CAR-免疫细胞的治疗效果。
(2)由于双靶点蛋白在肿瘤组织共表达的概率要远远高于在正常组织。本发明采用双靶点的设计思路很好地解决免疫治疗单靶标抗原蛋白丢失的问题，提高了免疫治疗的靶向性。本发明以SynNotch系统作为靶向和调控表达元件，只有当SynNotch识别并靶向肿瘤细胞后，CAR和免疫毒素才能够分泌表达，降低了免疫毒素对正常细胞的毒性。
(3)本发明以SynNotch系统作为嵌合抗原受体和免疫毒素的表达开关，一方面使免疫细胞的靶向性更强，另一方面免疫毒素的表达变得更为可控，提高了治疗的安全性。
附图说明
图1为本发明实施例制备组成性和可控制性分泌免疫毒素的双靶向GPC3/B7H3CAR-T的质粒示意图。
图2为本发明实施例制备分泌免疫毒素的双靶向Mesothelin/B7H3 CAR-T的质粒示意图。
图3为本发明组成性和可控制性分泌免疫毒素的双靶向GPC3/B7H3 CAR-T对肝癌细胞Hep3B的体外杀伤结果。
图4为本发明组成性和可控制性分泌免疫毒素的双靶向Mesothelin/B7H3 CAR-T对肺癌细胞H226的体外杀伤结果。
图5为本发明组成性和可控制性分泌免疫毒素的CAR-T体内抗肿瘤活性结果。
图6为本发明组成性和可控制性分泌免疫毒素的CAR-T在体内的抑瘤活性对比。
图7为本发明分泌不同类型免疫毒素的双靶向GPC3/B7H3 CAR-T对肝癌细胞Hep3B的体外杀伤结果。
图8为本发明分泌不同类型免疫毒素的双靶向Mesothelin/B7H3 CAR-T对肺癌细胞H226的体外杀伤结果。
具体实施方式
以下通过实施例说明本发明的具体步骤，但不局限于实施例范围。在本发明中使用的术语，除非另有说明，一般具有本领域普通技术人员通常理解的含义。下面结合具体实施例并参照数据进一步详细描述本发明，应理解，这些实施例只是为了举例说明本发明，而非以任何方式限制本发明的范围。在以下实施例中，未详细描述的各种过程和方法是本领域中公知的常规方法。
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此。
实施例1组成性和可控制性分泌免疫毒素的双靶向GPC3/B7H3 CAR-T质粒构建双靶向GPC3/B7H3 CAR-T构建由2种慢病毒载体构成。载体1和载体2的图谱见附图1。载体1是CAR和免疫毒素的启动蛋白，携带一个表达框(c-Myc-hYP7 scFv)-SynNotch-(SV40 NLS-tTA)-T2A-[eEF2(G717R)]，hYP7-SynNotch-tTA和eEF2(G717R)共转录、共翻译，翻译后在T2A连接肽处断开，各自发挥功能。hYP7 scFv识别肿瘤细胞表面的GPC3，其N-末端融合的c-Myc标签可以用来指示病毒载体1的转导效率和SynNotch的表达情况。SynNotch是跨膜的信号转导结构域，tTA是转录激活域，在hYP7结合到GPC3上之后，SynNotch被激活，释放tTA，被释放的tTA进入细胞核启动CAR和免疫毒素的表达(载体2提供)。mEF2是突变型的延伸因子EF2(G717R)，是免疫毒素的抗性基因。hYP7-synNotch-tTA氨基酸序列如SEQ ID No:42所示。构建过程：对化学合成的两端带EcoRV和BstBI酶切位点的表达框进行EcoRV和BstBI双酶切，然后用T4连接酶(Takara)克隆到EcoRV/BstBI线性化的pLenti载体(ThermoFisher)。采用类似的方法，对化学合成的SynNotch-(SV40 NLS-tTA)-T2A-[eEF2(G717R)]表达框(仅-缺失hYP7识别抗体)用BspEI和BstBI双酶切，克隆到pLenti载体，得到不含hYP7的载体pLenti-SynNotch-tTA-mEF2。载体2携带tTA的启动子TRE3G pro和TRE3G pro驱动的CAR基因、免疫毒素基因。下游为转录框：CD8 leader-anti-B7H3-CD8hinge-TM-41BB-CD3z-T2A-IgGsignal-HN3-mPE24-IRES-mScarletI，其中的CAR基因由识别肿瘤细胞表面B7-H3的单抗m276 scFv和4-1BB激活域、CD3z激活域融合构成，氨基酸序列如SEQ ID No:31所示。免疫毒素为靶向GPC3的HN3-PE24，氨基酸序列如SEQ ID No:32所示，CAR基因和免疫毒素之间由T2A连接肽隔开，翻译后断开。mScarlet为红色荧光蛋白，共转录后通过IRES元件独立翻译。mScarlet可以用来指示病毒载体2的转导效率和CAR基因的表达情况。构建过程：对化学合成的两端带ClaI和BstBI酶切位点的表达框进行ClaI/BstBI双酶切，T4连接酶克隆到ClaI/BstBI线性化的pLVX载体(CloneTech)。载体3与载体2类似，只是将载体2中的TRE启动子替换为CMV启动子，CAR和免疫毒素的表达由SynNotch控制变为不受控制的组成性的表达。构建过程：将化学合成的两端含有ClaI和EcoRI酶切位点的CMV启动子取代载体2中的TRE启动子。
实施例2组成性和可控制性分泌免疫毒素的双靶向Mesothelin/B7H3 CAR-T质粒构建
质粒图谱如图2所示。在载体1的基础上用anti-Mesothelin抗体YP158替换hYP7，得到载体4(pLenti-YP158-SynNotch-tTA-eEF2)。构建方法：对化学合成的两端带EcoRV和BspEI酶切位点的anti-Mesothelin抗体YP158进行EcoRV和BspEI双酶切，然后用T4连接酶(Takara)克隆到实施例1中的载体1(替换hYP7)。YP158-SynNotch-tTA氨基酸序列如SEQ IDNo:43所示。载体5(pLVX-TRE3G-CAR-B7H3-SS1-PE38-mScarlet)表达识别B7H3的CAR(同实施例1)和识别mesothelin的免疫毒素(SS1-PE38)。构建方法：对化学合成的两端带NheI和XhoI酶切位点的免疫毒素片段SS1-PE38进行NheI/XhoI双酶切，替换载体2中的HN3-PE24。SS1-PE38氨基酸序列如SEQ ID No:37所示。载体6为组成性地表达CAR(识别B7H3，同实施例1)和免疫毒素(SS1-PE38)。构建方法：对化学合成的两端带NheI和XhoI酶切位点的免疫毒素片段SS1-PE38进行NheI/XhoI双酶切，替换载体3中的HN3-PE24。
实施例3组成性和可控制性分泌免疫毒素的CAR-T质粒逆转录病毒包装。
1.提前将293T细胞接种到T25中，等293T细胞长到50％左右进行转染，准备要包装的质粒复合物5μg，质粒复合物的比例为pMission-Gagpol：表达质粒：pMission-VSVG＝3：1：1，质粒复合物和转染试剂PEI(15μg)分别加到500μl的Opti-MEM培养基，在2ml离心管里混匀，加到养293T细胞中。37度培养8h，去除培养基，用PBS清洗一遍，加入新的不含血清的DMEM培养基(含1％双抗)。
2.等48h后收集病毒，用0.45μm的针头滤器过滤，分装存放保存于-80度，待用。
实施例4逆转录病毒感染人的PBMC细胞
采用Ficoll分离液(天津灏洋)密度梯度离心法分离PBMC，分离的PBMC用含100IU/ml IL-2(北京同立海源)的RPMI-1640培养基培养，并加入
Figure BDA0002443189960000081
Human T-ActivatorCD3/CD28(ThermoFisher)磁珠激活2天。将激活的PBMC与逆转录病毒液共孵育48小时后，更换新鲜的培养基(含100IU/ml IL-2)，T细胞活化培养48小时，感染后每天计数T细胞的密度，在T细胞快速增殖的对数期，收获T细胞，进行后期的试验。
实施例5组成性和可控制性分泌免疫毒素的双靶向GPC3/B7H3 CAR-T对肝癌细胞的体外杀伤。
将构建的CAR-T细胞和Luciferase标记的肝癌细胞Hep3B(GPC3阳性)按照0.5:1、1:1、5:1、10:1的比例混合，接种到96-孔细胞培养板(肿瘤细胞固定在10,000/孔)，培养基补充到200μl，37℃共孵育24小时，然后用Spark多功能全波长酶标仪测定Luciferase的活性，计算肿瘤细胞存活率。活性测试试验设置对照组及实验组分别为：(1)hYP7-SynNotch-tTA-T(只有抗原识别蛋白hYP7修饰的T细胞，缺失CAR和免疫毒素)，(2)SynNotch-tTA//CAR-B7H3-HN3-PE24(缺失第一靶向基团hYP7，CAR和免疫毒素都无法有效表达)，(3)hYP7-SynNotch-tTA//CAR-B7H3(只表达CAR)，(4)hYP7-SynNotch-tTA//HN3-PE24-T(只表达免疫毒素)，(5)为(3)和(4)的混合，(6)CAR-B7H3-HN3-PE24-T(CAR和免疫毒素在CMV启动子驱动下的组成性表达)，(7)hYP7-SynNotch-tTA//CAR-B7H3-HN3-PE24-T(受SynNotch控制的，同时表达CAR和免疫毒素)。结果如图3所示，结果表明：1)仅仅hYP7遗传修饰的T细胞、缺失靶向基团hYP7的SynNotch-tTA//CAR-B7H3-HN3-PE24 T细胞对肿瘤细胞的杀伤活性都很弱；2)受SynNotch控制的只表达CAR的T细胞和受SynNotch控制的只表达免疫毒素的T细胞对Hep3B均有一定程度的杀伤活性，但是两者混合的增效效果并不明显；3)同时表达CAR和免疫毒素的T细胞对肿瘤细胞的杀伤活性显著强于受SynNotch控制的只表达CAR的T细胞和受SynNotch控制的只表达免疫毒素的T细胞的混合，受SynNotch控制的同时表达CAR和免疫毒素的T细胞对肿瘤细胞的杀伤活性最强。
实施例6组成性和可控制性分泌免疫毒素的双靶向Mesothelin/B7H3 CAR-T对肺癌细胞的体外杀伤活性。
组成性和可控制性分泌免疫毒素的双靶向Mesothelin/B7H3 CAR-T参考实例5来设置实验方案，肿瘤细胞用肺癌H226(Mesothelin阳性)细胞，实验结果如图4，与实施例5的杀伤效果一致，都显示出共表达CAR和免疫毒素的T细胞以及受SynNotch控制的共表达CAR和免疫毒素的T细胞具有优越的H226肿瘤细胞的杀伤活性。
实施例7组成性和可控制性分泌免疫毒素的CAR-T体内抗肿瘤活性。
体内活性测定采用皮下移植瘤模型。体内实验组设置实施例5中的设置一致。将5×106Hep3B细胞接种到NSG小鼠后臀部的皮下，当42天时肿瘤组织长到约100mm3，将1×107CAR-T(或修饰的T)细胞尾静脉注射给小鼠。肿瘤的生长曲线采用游标卡尺测量，每2天测量一次，直到试验测到68天结束。实验结果如图5所示，结果显示不表达CAR和免疫毒素的T细胞(hYP7-SynNotch-tTA-T组和缺失靶向基团hYP7的SynNotch-tTA//CAR-B7H3-HN3-PE24-T)几乎没有抑瘤活性，可控制性分泌免疫毒素的CAR-T细胞(hYP7-SynNotch-tTA//CAR-B7H3-HN3-PE24-T)能显著地抑制肿瘤的生长，只表达CAR(hYP7-SynNotch-tTA//CAR-B7H3-T)或只表达免疫毒素的T细胞(hYP7-SynNotch-tTA//HN3-PE24-T)，抑瘤活性大大降低，只表达CAR和只表达免疫毒素的T细胞外在联合抑瘤活性稍弱。
为了比较组成性地共表达CAR和免疫毒素(CAR-B7H3-HN3-PE24-T)、SynNotch控制性地共表达CAR和免疫毒素(hYP7-SynNotch-tTA//CAR-B7H3-HN3-PE24-T)两种方式在体内抑瘤活性上的差异，本实施例还考察了CAR-B7H3-T、单独给予免疫毒素HN3-PE24、CAR-B7H3-T+HN3-PE24混合、共表达CAR-B7H3-HN3-PE24-T以及hYP7-SynNotch-tTA//CAR-B7H3-HN3-PE24-T对Hep3B移植瘤模型活性。结果如图6所示，结果显示，1mg/kg的重组免疫毒素HN3-PE24单独给药(尾静脉注射，每周3次，共6次)、常规的单靶向CAR-B7H3-T、以及免疫毒素HN3-PE24和单靶向CAR-B7H3-T的联合给药虽然都具有一定程度的抑瘤活性，但是共表达CAR和免疫毒素T细胞的抑瘤效果明显优于上述实验组，并且SynNotch控制性地共表达CAR和免疫毒素效果最为显著。
实施例8分泌不同类型免疫毒素的CAR-T的活性考察
参考实施例1-4的方法将靶向GPC3的HN3单抗和靶向Mesothelin的SS1单抗分别与以下毒素进行融合：白喉毒素(DT390)、蓖麻毒素(RICIN)、霍乱毒素(CET40)、人源性蛋白毒素核糖核酸酶(RNase)，构建分泌不同类型免疫毒素的CAR-T细胞CAR-B7H3-NH3-DT390-T、CAR-B7H3-NH3-RICIN-T、CAR-B7H3-NH3-CET40-T、CAR-B7H3-NH3-Rnase-T、CAR-B7H3-NH3-PE24-T以及CAR-B7H3-SS1-DT390-T、CAR-B7H3-SS1-RICIN-T、CAR-B7H3-SS1-CET40-T、CAR-B7H3-SS1-Rnase-T、CAR-B7H3-SS1-PE38-T。构建过程：化学合成两端带有NheI和XhoI酶切位点的免疫毒素片段HN3-DT390、HN3-RICIN、HN3-CET40、HN3-RNase酶切替换实施例1中的HN3-PE24，HN3-DT390序列如SEQ ID No:33所示，HN3-RICIN序列如SEQ ID No:35所示，HN3-CET40序列如SEQ ID No:34所示，HN3-Rnase序列如SEQ ID No:36所示，化学合成两端带有NheI和XhoI酶切位点的免疫毒素片段SS1-DT390、SS1-RICIN、SS1-CET40、SS1-Rnase酶切替换实施例2中的SS1-PE38，SS1-DT390序列如SEQ ID No:38所示，SS1-RICIN序列如SEQ IDNo:40所示，SS1-CET40序列如SEQ ID No:39所示，SS1-Rnase序列如SEQ ID No:41所示。各种毒素的序列：DT390氨基酸序列如SEQ ID No:21所示，DT390核苷酸序列如SEQ ID No:22所示；CET40氨基酸序列如SEQ ID No:25所示，CET40核苷酸序列如SEQ ID No:26所示；RICIN氨基酸序列如SEQ ID No:23所示，RICIN核苷酸序列如SEQ ID No:24所示；RNase氨基酸序列如SEQ ID No:27所示，RNase核苷酸序列如SEQ ID No:28所示。以肝癌Hep3B和肺癌H226为测试细胞株的体外杀伤实验显示，所有共表达HN3免疫毒素的CAR-B7H3都对Hep3B细胞显示了很强的肿瘤杀伤活性(图7)，所有共表达SS1免疫毒素的CAR-B7H3都对H226细胞显示了很强的肿瘤杀伤活性(图8)，在两种测试细胞中，共表达PE免疫毒素的CAR-T活性最强，共表达DT390免疫毒素的CAR-T活性次之。
序列表
<110> 华中农业大学
<120> 可分泌性表达免疫毒素的嵌合抗原受体修饰的免疫细胞
<160> 43
<170> SIPOSequenceListing 1.0
<210> 1
<211> 224
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
1 5 10 15
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
20 25 30
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile
35 40 45
Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val
50 55 60
Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe
65 70 75 80
Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly
85 90 95
Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg
100 105 110
Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln
115 120 125
Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp
130 135 140
Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro
145 150 155 160
Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys
165 170 175
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
180 185 190
Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala
195 200 205
Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
210 215 220
<210> 2
<211> 672
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
accacaaccc cagcacctag accacctaca ccagcaccaa ccatcgcatc ccagccactg 60
tctctgcgcc ccgaggcatg caggcctgca gcaggcggcg ccgtgcacac caggggcctg 120
gacttcgcct gcgacatcta catctgggca ccactggcag gaacatgtgg cgtgctgctg 180
ctgtccctgg tcatcaccct gtactgcaag agaggcagga agaagctgct gtatatcttc 240
aagcagccct tcatgagacc cgtgcagaca acccaggagg aggacggctg ctcttgtagg 300
ttccctgagg aggaggaggg cggctgtgag ctgagagtga agtttagcag gtccgccgat 360
gcaccagcat accagcaggg acagaaccag ctgtataacg agctgaatct gggccggaga 420
gaggagtacg acgtgctgga taagaggagg ggacgcgacc cagagatggg aggcaagcca 480
cagaggagaa agaaccctca ggagggcctg tacaatgagc tgcagaagga caagatggcc 540
gaggcctata gcgagatcgg catgaaggga gagaggcgcc ggggcaaggg acacgatggc 600
ctgtaccagg gcctgtccac agccaccaag gacacatatg atgccctgca catgcaggcc 660
ctgccaccaa gg 672
<210> 3
<211> 219
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn
1 5 10 15
Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu
20 25 30
Phe Pro Gly Pro Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly
35 40 45
Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe
50 55 60
Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn
65 70 75 80
Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr
85 90 95
Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser
100 105 110
Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr
115 120 125
Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys
130 135 140
Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn
145 150 155 160
Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu
165 170 175
Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly
180 185 190
His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr
195 200 205
Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
210 215
<210> 4
<211> 657
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
attgaagtta tgtatcctcc tccttaccta gacaatgaga agagcaatgg aaccattatc 60
catgtgaaag ggaaacacct ttgtccaagt cccctatttc ccggaccttc taagcccttt 120
tgggtgctgg tggtggttgg gggagtcctg gcttgctata gcttgctagt aacagtggcc 180
tttattattt tctgggtgag gagtaagagg agcaggctcc tgcacagtga ctacatgaac 240
atgactcccc gccgccccgg gcccacccgc aagcattacc agccctatgc cccaccacgc 300
gacttcgcag cctatcgctc cagagtgaag ttcagcagga gcgcagacgc ccccgcgtac 360
cagcagggcc agaaccagct ctataacgag ctcaatctag gacgaagaga ggagtacgat 420
gttttggaca agagacgtgg ccgggaccct gagatggggg gaaagccgag aaggaagaac 480
cctcaggaag gcctgtacaa tgaactgcag aaagataaga tggcggaggc ctacagtgag 540
attgggatga aaggcgagcg ccggaggggc aaggggcacg atggccttta ccagggtctc 600
agtacagcca ccaaggacac ctacgacgcc cttcacatgc aggccctgcc ccctcgc 657
<210> 5
<211> 283
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro
1 5 10 15
Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu
20 25 30
Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg
35 40 45
Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly
50 55 60
Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn
65 70 75 80
His Arg Asn Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met
85 90 95
Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro
100 105 110
Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Phe Ser Val
115 120 125
Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe
130 135 140
Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg
145 150 155 160
Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser
165 170 175
Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr
180 185 190
Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys
195 200 205
Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn
210 215 220
Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu
225 230 235 240
Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly
245 250 255
His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr
260 265 270
Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
275 280
<210> 6
<211> 849
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
ttcgtgccgg tcttcctgcc agcgaagccc accacgacgc cagcgccgcg accaccaaca 60
ccggcgccca ccatcgcgtc gcagcccctg tccctgcgcc cagaggcgtg ccggccagcg 120
gcggggggcg cagtgcacac gagggggctg gacttcgcct gtgacatcta catctgggcg 180
cccttggccg ggacttgtgg ggtccttctc ctgtcactgg ttatcaccct ttactgcaac 240
cacaggaaca ggagtaagag gagcaggctc ctgcacagtg actacatgaa catgactccc 300
cgccgccccg ggcccacccg caagcattac cagccctatg ccccaccacg cgacttcgca 360
gcctatcgct cccgtttctc tgttgttaaa cggggcagaa agaaactcct gtatatattc 420
aaacaaccat ttatgagacc agtacaaact actcaagagg aagatggctg tagctgccga 480
tttccagaag aagaagaagg aggatgtgaa ctgagagtga agttcagcag gagcgcagac 540
gcccccgcgt accagcaggg ccagaaccag ctctataacg agctcaatct aggacgaaga 600
gaggagtacg atgttttgga caagagacgt ggccgggacc ctgagatggg gggaaagccg 660
agaaggaaga accctcagga aggcctgtac aatgaactgc agaaagataa gatggcggag 720
gcctacagtg agattgggat gaaaggcgag cgccggaggg gcaaggggca cgatggcctt 780
taccagggtc tcagtacagc caccaaggac acctacgacg cccttcacat gcaggccctg 840
ccccctcgc 849
<210> 7
<211> 266
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
20 25 30
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
35 40 45
Thr Phe Asn Lys Asn Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys
50 55 60
Gly Leu Glu Trp Val Gly Arg Ile Arg Asn Lys Thr Asn Asn Tyr Ala
65 70 75 80
Thr Tyr Tyr Ala Asp Ser Val Lys Ala Arg Phe Thr Ile Ser Arg Asp
85 90 95
Asp Ser Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu
100 105 110
Asp Thr Ala Val Tyr Tyr Cys Val Ala Gly Asn Ser Phe Ala Tyr Trp
115 120 125
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly
130 135 140
Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser
145 150 155 160
Pro Asp Ser Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys
165 170 175
Lys Ser Ser Gln Ser Leu Leu Tyr Ser Ser Asn Gln Lys Asn Tyr Leu
180 185 190
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr
195 200 205
Trp Ala Ser Ser Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser
210 215 220
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu
225 230 235 240
Asp Val Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Asn Tyr Pro Leu Thr
245 250 255
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
260 265
<210> 8
<211> 798
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccggaggtgc agcttgttga gtctggtgga ggattggtgc agcctggagg gtcattgaga 120
ctctcatgtg cagcctctgg attcaccttc aataagaatg ccatgaattg ggtccgccag 180
gctccaggaa agggtttgga atgggttggc cgcataagaa ataaaactaa taattatgca 240
acatattatg ccgattcagt gaaagccagg tttaccatct ccagagatga ttcaaagaac 300
tcactctatc tgcaaatgaa cagcttgaaa accgaggaca cagccgtgta ctattgtgtg 360
gctggtaact cgtttgctta ctggggccaa gggactctgg tcactgtgag ctcaggtggc 420
ggtggctctg gcggtggtgg cagcggcggt ggcggttcag acattgtgat gacccagtct 480
ccagactccc tagctgtgtc actgggagag agggccacta tcaactgcaa gtccagtcag 540
agccttttat atagcagcaa tcaaaagaac tacttggcct ggtaccaaca gaaaccaggg 600
cagcctccta aactgctgat ttactgggca tccagtaggg aatctggggt ccctgatcgc 660
ttcagtggca gtggatctgg gacagatttc actctcacca tcagcagtct gcaggctgaa 720
gacgtggcag tttattactg tcagcaatat tataactatc cgctcacgtt cggtcagggg 780
accaagttgg agatcaaa 798
<210> 9
<211> 135
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly
1 5 10 15
Val His Ser Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln
20 25 30
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Tyr Phe Asp Phe
35 40 45
Asp Ser Tyr Glu Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
50 55 60
Glu Trp Ile Gly Ser Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Pro
65 70 75 80
Ser Leu Lys Ser Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
85 90 95
Leu Tyr Leu Gln Met Asn Thr Leu Arg Ala Glu Asp Thr Ala Thr Tyr
100 105 110
Tyr Cys Ala Arg Val Asn Met Asp Arg Phe Asp Tyr Trp Gly Gln Gly
115 120 125
Thr Leu Val Thr Val Ser Ser
130 135
<210> 10
<211> 405
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
atggagtggt cctgggtgtt cctgttcttt ctgtccgtca caaccggcgt gcactcccag 60
gtgcagctgg tgcagtctgg gggaggcttg gtacagcctg gagggtccct gagactctcc 120
tgtgcagcct cttatttcga tttcgattct tatgaaatga gctgggtccg ccaggctcca 180
gggaagggcc tagagtggat tgggagtatc tatcatagtg ggagcaccta ctacaacccg 240
tccctcaaga gtcgagtcac catctccaga gacaattcca agaacacgct gtatctgcaa 300
atgaacaccc tgagagccga ggacacagcc acgtattact gtgcgagagt aaatatggac 360
cgatttgact actggggcca gggaaccctg gtcaccgtct cctca 405
<210> 11
<211> 264
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val
20 25 30
Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly
35 40 45
Thr Phe Ser Ser Tyr Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln
50 55 60
Gly Leu Glu Trp Met Gly Gly Ile Ile Pro Ile Leu Gly Ile Ala Asn
65 70 75 80
Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Gln Ser
85 90 95
Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
100 105 110
Ala Val Tyr Tyr Cys Ala Arg Gly Gly Ser Gly Ser Tyr His Met Asp
115 120 125
Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly
130 135 140
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr
145 150 155 160
Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu
165 170 175
Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala Trp Tyr Gln
180 185 190
Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser Asn
195 200 205
Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr
210 215 220
Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val
225 230 235 240
Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro Arg Ile Thr Phe Gly
245 250 255
Gln Gly Thr Arg Leu Glu Ile Lys
260
<210> 12
<211> 792
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
atggccctgc ccgtgaccgc cctgctgctg cccctggccc tgctgctgca cgccgcccgg 60
ccccaggtgc agctgcagca gagcggcgcc gaggtgaaga agcccggcag cagcgtgaag 120
gtgagctgca aggccagcgg cggcaccttc agcagctacg ccatcagctg ggtgcggcag 180
gcccccggcc agggcctgga gtggatgggc ggcatcatcc ccatcctggg catcgccaaa 240
tacgcccaga agttccaggg ccgggtgacc atcaccgccg accagagcac cagcaccgcc 300
tacatggagc tgagcagcct gcggagcgag gacaccgccg tgtactactg cgcccggggc 360
ggcagcggca gctaccacat ggacgtgtgg ggcaagggca ccaccgtgac cgtgagcagc 420
ggcggcggcg gcagcggcgg cggcggcagc ggcggcggcg gcagcgagat cgtgctgacc 480
cagagccccg ccaccctgag cctgagcccc ggcgagcggg ccaccctgag ctgccgggcc 540
agccagagcg tgagcagcta cctggcctgg taccagcaga agcccggcca ggccccccgg 600
ctgctgatct acgacgccag caaacgggcc accggcatcc ccgcccggtt cagcggcagc 660
ggcagcggca ccgacttcac cctgaccatc agcagcctgg agcccgagga cttcgccgtg 720
tactactgcc agcagcggag caaatggccc ccccggatca ccttcggcca gggcacccgg 780
ctggagatca ag 792
<210> 13
<211> 259
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly
1 5 10 15
Val His Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Glu Lys
20 25 30
Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe
35 40 45
Thr Gly Tyr Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu
50 55 60
Glu Trp Ile Gly Leu Ile Thr Pro Tyr Asn Gly Ala Ser Ser Tyr Asn
65 70 75 80
Gln Lys Phe Arg Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser
85 90 95
Thr Ala Tyr Met Asp Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val
100 105 110
Tyr Phe Cys Ala Arg Gly Gly Tyr Asp Gly Arg Gly Phe Asp Tyr Trp
115 120 125
Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Val Gly Gly Ser Gly
130 135 140
Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Glu Leu Thr Gln Ser
145 150 155 160
Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys
165 170 175
Ser Ala Ser Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Ser
180 185 190
Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser
195 200 205
Gly Val Pro Gly Arg Phe Ser Gly Ser Gly Ser Gly Asn Ser Tyr Ser
210 215 220
Leu Thr Ile Ser Ser Val Glu Ala Glu Asp Asp Ala Thr Tyr Tyr Cys
225 230 235 240
Gln Gln Trp Ser Gly Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu
245 250 255
Glu Ile Lys
<210> 14
<211> 777
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
atggagtgga gctgggtgtt cctgttcttc ctgagcgtga ccaccggcgt gcacagccag 60
gtgcagctgc agcagagcgg ccccgagctg gagaagcccg gcgccagcgt gaagatcagc 120
tgcaaggcca gcggctacag cttcaccggc tacaccatga aatgggtgaa gcagagccac 180
ggcaagagcc tggagtggat cggcctgatc accccctaca aaggcgccag cagctacaaa 240
cagaagttcc ggggcaaggc caccctgacc gtggacaaga gcagcagcac cgcctacatg 300
gacctgctga gcctgaccag cgaggacagc gccgtgtact tctgcgcccg gggcggctac 360
gacggccggg gcttcgacta ctggggccag ggcaccaccg tgaccgtgag cagcggcgtg 420
ggcggcagcg gcggcggcgg cagcggcggc ggcggcagcg acatcgagct gacccagagc 480
cccgccatca tgagcgccag ccccggcgag aaggtgacca tgacctgcag cgccagcagc 540
agcgtgagct acatgcactg gtaccagcag aagagcggca ccagccccaa gcggtggatc 600
tacgacacca gcaagctggc cagcggcgtg cccggccggt tcagcggcag cggcagcggc 660
aaaagctaca gcctgaccat cagcagcgtg gaggccgagg acgacgccac ctactactgc 720
cagcagtgga gcggctaccc cctgaccttc ggcgccggca ccaagctgga gatcaag 777
<210> 15
<211> 268
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 15
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gln Ser Leu Glu Glu Ser Gly Gly Asp Leu Val
20 25 30
Lys Pro Gly Ala Ser Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser
35 40 45
Phe Ser Gly Asp Tyr Tyr Met Cys Trp Val Arg Gln Ala Pro Gly Lys
50 55 60
Gly Leu Glu Trp Ile Ala Cys Ile Gly Gly Gly Ser Asn Thr Ala Thr
65 70 75 80
Tyr Tyr Ala Thr Trp Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser
85 90 95
Ser Thr Thr Val Thr Leu Gln Met Thr Ser Leu Thr Ala Ala Asp Thr
100 105 110
Ala Thr Tyr Phe Cys Ala Arg Asp Leu Gly Phe Val Asp Tyr Ala Leu
115 120 125
Glu Leu Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly
130 135 140
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Val Met
145 150 155 160
Thr Gln Thr Pro Ala Ser Val Glu Val Ala Val Gly Gly Thr Val Thr
165 170 175
Ile Lys Cys Gln Ala Ser Glu Asn Met Tyr Asn Ser Leu Ala Trp Tyr
180 185 190
Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Arg Ala Ser
195 200 205
Thr Leu Glu Ser Gly Val Pro Ser Arg Phe Lys Gly Ser Gly Ser Gly
210 215 220
Thr Glu Tyr Thr Leu Thr Ile Ser Asp Leu Glu Cys Ala Asp Ala Ala
225 230 235 240
Thr Tyr Tyr Cys Gln Cys Thr Phe Tyr Ser His Asn Asn Asn Tyr Gly
245 250 255
Gly Ala Phe Gly Gly Gly Thr Glu Val Val Val Lys
260 265
<210> 16
<211> 804
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
atggccctgc ccgtgaccgc cctgctgctg cccctggccc tgctgctgca cgccgcccgg 60
ccccagagcc tggaggagag cggcggcgac ctggtgaagc ccggcgccag cctgaccctg 120
acctgcaccg ccagcggctt cagcttcagc ggcgactact acatgtgctg ggtgcggcag 180
gcccccggca agggcctgga gtggatcgcc tgcatcggcg gcggcagcaa aaccgccacc 240
tactacgcca cctgggccaa gggccggttc accatcagca agaccagcag caccaccgtg 300
accctgcaga tgaccagcct gaccgccgcc gacaccgcca cctacttctg cgcccgggac 360
ctgggcttcg tggactacgc cctggagctg tggggccccg gcaccctggt gaccgtgagc 420
agcggcggcg gcggcagcgg cggcggcggc agcggcggcg gcggcagcga cgtggtgatg 480
acccagaccc ccgccagcgt ggaggtggcc gtgggcggca ccgtgaccat caagtgccag 540
gccagcgaga aaatgtacaa aagcctggcc tggtaccagc agaagcccgg ccagcccccc 600
aagctgctga tctaccgggc cagcaccctg gagagcggcg tgcccagccg gttcaagggc 660
agcggcagcg gcaccgagta caccctgacc atcagcgacc tggagtgcgc cgacgccgcc 720
acctactact gccagtgcac cttctacagc cacaaaaaaa aatacggcgg cgccttcggc 780
ggcggcaccg aggtggtggt gaag 804
<210> 17
<211> 219
<212> PRT
<213> 铜绿假单胞菌(Pseudomonas aeruginosa)
<400> 17
Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val Ser Phe Ser
1 5 10 15
Thr Arg Gly Thr Gln Asn Trp Thr Val Glu Arg Leu Leu Gln Ala His
20 25 30
Arg Gln Leu Glu Glu Arg Gly Tyr Val Phe Val Gly Tyr His Gly Thr
35 40 45
Phe Leu Glu Ala Ala Gln Ser Ile Val Phe Gly Gly Val Arg Ala Arg
50 55 60
Ser Gln Asp Leu Asp Ala Ile Trp Arg Gly Phe Tyr Ile Ala Gly Asp
65 70 75 80
Pro Ala Leu Ala Tyr Gly Tyr Ala Gln Asp Gln Glu Pro Asp Ala Arg
85 90 95
Gly Arg Ile Arg Asn Gly Ala Leu Leu Arg Val Tyr Val Pro Arg Ser
100 105 110
Ser Leu Pro Gly Phe Tyr Arg Thr Ser Leu Thr Leu Ala Ala Pro Glu
115 120 125
Ala Ala Gly Glu Val Glu Arg Leu Ile Gly His Pro Leu Pro Leu Arg
130 135 140
Leu Asp Ala Ile Thr Gly Pro Glu Glu Glu Gly Gly Arg Leu Glu Thr
145 150 155 160
Ile Leu Gly Trp Pro Leu Ala Glu Arg Thr Val Val Ile Pro Ser Ala
165 170 175
Ile Pro Thr Asp Pro Arg Asn Val Gly Gly Asp Leu Asp Pro Ser Ser
180 185 190
Ile Pro Asp Lys Glu Gln Ala Ile Ser Ala Leu Pro Asp Tyr Ala Ser
195 200 205
Gln Pro Gly Lys Pro Pro Arg Glu Asp Leu Lys
210 215
<210> 18
<211> 657
<212> DNA
<213> 铜绿假单胞菌(Pseudomonas aeruginosa)
<400> 18
cccaccggcg ccgagttcct gggcgacggc ggcgacgtga gcttcagcac ccggggcacc 60
cagaaatgga ccgtggagcg gctgctgcag gcccaccggc agctggagga gcggggctac 120
gtgttcgtgg gctaccacgg caccttcctg gaggccgccc agagcatcgt gttcggcggc 180
gtgcgggccc ggagccagga cctggacgcc atctggcggg gcttctacat cgccggcgac 240
cccgccctgg cctacggcta cgcccaggac caggagcccg acgcccgggg ccggatccgg 300
aaaggcgccc tgctgcgggt gtacgtgccc cggagcagcc tgcccggctt ctaccggacc 360
agcctgaccc tggccgcccc cgaggccgcc ggcgaggtgg agcggctgat cggccacccc 420
ctgcccctgc ggctggacgc catcaccggc cccgaggagg agggcggccg gctggagacc 480
atcctgggct ggcccctggc cgagcggacc gtggtgatcc ccagcgccat ccccaccgac 540
ccccggaaag tgggcggcga cctggacccc agcagcatcc ccgacaagga gcaggccatc 600
agcgccctgc ccgactacgc cagccagccc ggcaagcccc cccgggagga cctgaag 657
<210> 19
<211> 329
<212> PRT
<213> 铜绿假单胞菌(Pseudomonas aeruginosa)
<400> 19
Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu Tyr Leu Ala
1 5 10 15
Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg Asn Ala Leu
20 25 30
Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile Arg Glu Gln
35 40 45
Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala Glu Ser Glu
50 55 60
Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala Asn
65 70 75 80
Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala Ala Gly Glu Cys Ala
85 90 95
Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn Tyr Pro Thr
100 105 110
Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val Ser Phe Ser Thr Arg
115 120 125
Gly Thr Gln Asn Trp Thr Val Glu Arg Leu Leu Gln Ala His Arg Gln
130 135 140
Leu Glu Glu Arg Gly Tyr Val Phe Val Gly Tyr His Gly Thr Phe Leu
145 150 155 160
Glu Ala Ala Gln Ser Ile Val Phe Gly Gly Val Arg Ala Arg Ser Gln
165 170 175
Asp Leu Asp Ala Ile Trp Arg Gly Phe Tyr Ile Ala Gly Asp Pro Ala
180 185 190
Leu Ala Tyr Gly Tyr Ala Gln Asp Gln Glu Pro Asp Ala Arg Gly Arg
195 200 205
Ile Arg Asn Gly Ala Leu Leu Arg Val Tyr Val Pro Arg Ser Ser Leu
210 215 220
Pro Gly Phe Tyr Arg Thr Ser Leu Thr Leu Ala Ala Pro Glu Ala Ala
225 230 235 240
Gly Glu Val Glu Arg Leu Ile Gly His Pro Leu Pro Leu Arg Leu Asp
245 250 255
Ala Ile Thr Gly Pro Glu Glu Glu Gly Gly Arg Leu Glu Thr Ile Leu
260 265 270
Gly Trp Pro Leu Ala Glu Arg Thr Val Val Ile Pro Ser Ala Ile Pro
275 280 285
Thr Asp Pro Arg Asn Val Gly Gly Asp Leu Asp Pro Ser Ser Ile Pro
290 295 300
Asp Lys Glu Gln Ala Ile Ser Ala Leu Pro Asp Tyr Ala Ser Gln Pro
305 310 315 320
Gly Lys Pro Pro Arg Glu Asp Leu Lys
325
<210> 20
<211> 987
<212> DNA
<213> 铜绿假单胞菌(Pseudomonas aeruginosa)
<400> 20
gagcagtgcg gctaccccgt gcagcggctg gtggccctgt acctggccgc ccggctgagc 60
tggaaacagg tggaccaggt gatccggaaa gccctggcca gccccggcag cggcggcgac 120
ctgggcgagg ccatccggga gcagcccgag caggcccggc tggccctgac cctggccgcc 180
gccgagagcg agcggttcgt gcggcagggc accggcaaag acgaggccgg cgccgccaaa 240
gccgacgtgg tgagcctgac ctgccccgtg gccgccggcg agtgcgccgg ccccgccgac 300
agcggcgacg ccctgctgga gcggaaatac cccaccggcg ccgagttcct gggcgacggc 360
ggcgacgtga gcttcagcac ccggggcacc cagaaatgga ccgtggagcg gctgctgcag 420
gcccaccggc agctggagga gcggggctac gtgttcgtgg gctaccacgg caccttcctg 480
gaggccgccc agagcatcgt gttcggcggc gtgcgggccc ggagccagga cctggacgcc 540
atctggcggg gcttctacat cgccggcgac cccgccctgg cctacggcta cgcccaggac 600
caggagcccg acgcccgggg ccggatccgg aaaggcgccc tgctgcgggt gtacgtgccc 660
cggagcagcc tgcccggctt ctaccggacc agcctgaccc tggccgcccc cgaggccgcc 720
ggcgaggtgg agcggctgat cggccacccc ctgcccctgc ggctggacgc catcaccggc 780
cccgaggagg agggcggccg gctggagacc atcctgggct ggcccctggc cgagcggacc 840
gtggtgatcc ccagcgccat ccccaccgac ccccggaaag tgggcggcga cctggacccc 900
agcagcatcc ccgacaagga gcaggccatc agcgccctgc ccgactacgc cagccagccc 960
ggcaagcccc cccgggagga cctgaag 987
<210> 21
<211> 390
<212> PRT
<213> 白喉杆菌(Corynebacterium diphtheriae)
<400> 21
Gly Ala Asp Asp Val Val Asp Ser Ser Lys Ser Phe Val Met Glu Asn
1 5 10 15
Phe Ser Ser Tyr His Gly Thr Lys Pro Gly Tyr Val Asp Ser Ile Gln
20 25 30
Lys Gly Ile Gln Lys Pro Lys Ser Gly Thr Gln Gly Asn Tyr Asp Asp
35 40 45
Asp Trp Lys Gly Phe Tyr Ser Thr Asp Asn Lys Tyr Asp Ala Ala Gly
50 55 60
Tyr Ser Val Asp Asn Glu Asn Pro Leu Ser Gly Lys Ala Gly Gly Val
65 70 75 80
Val Lys Val Thr Tyr Pro Gly Leu Thr Lys Val Leu Ala Leu Lys Val
85 90 95
Asp Asn Ala Glu Thr Ile Lys Lys Glu Leu Gly Leu Ser Leu Thr Glu
100 105 110
Pro Leu Met Glu Gln Val Gly Thr Glu Glu Phe Ile Lys Arg Phe Gly
115 120 125
Asp Gly Ala Ser Arg Val Val Leu Ser Leu Pro Phe Ala Glu Gly Ser
130 135 140
Ser Ser Val Glu Tyr Ile Asn Asn Trp Glu Gln Ala Lys Ala Leu Ser
145 150 155 160
Val Glu Leu Glu Ile Asn Phe Glu Thr Arg Gly Lys Arg Gly Gln Asp
165 170 175
Ala Met Tyr Glu Tyr Met Ala Gln Ala Cys Ala Gly Asn Arg Val Arg
180 185 190
Arg Ser Val Gly Ser Ser Leu Ser Cys Ile Asn Leu Asp Trp Asp Val
195 200 205
Ile Arg Asp Lys Thr Lys Thr Lys Ile Glu Ser Leu Lys Glu His Gly
210 215 220
Pro Ile Lys Asn Lys Met Ser Glu Ser Pro Asn Lys Thr Val Ser Glu
225 230 235 240
Glu Lys Ala Lys Gln Tyr Leu Glu Glu Phe His Gln Thr Ala Leu Glu
245 250 255
His Pro Glu Leu Ser Glu Leu Lys Thr Val Thr Gly Thr Asn Pro Val
260 265 270
Phe Ala Gly Ala Asn Tyr Ala Ala Trp Ala Val Asn Val Ala Gln Val
275 280 285
Ile Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys Thr Thr Ala Ala Leu
290 295 300
Ser Ile Leu Pro Gly Ile Gly Ser Val Met Gly Ile Ala Asp Gly Ala
305 310 315 320
Val His His Asn Thr Glu Glu Ile Val Ala Gln Ser Ile Ala Leu Ser
325 330 335
Ser Leu Met Val Ala Gln Ala Ile Pro Leu Val Gly Glu Leu Val Asp
340 345 350
Ile Gly Phe Ala Ala Tyr Asn Phe Val Glu Ser Ile Ile Asn Leu Phe
355 360 365
Gln Val Val His Asn Ser Tyr Asn Arg Pro Ala Tyr Ser Pro Gly His
370 375 380
Lys Thr Gln Pro Phe Leu
385 390
<210> 22
<211> 1170
<212> DNA
<213> 白喉杆菌(Corynebacterium diphtheriae)
<400> 22
ggcgccgacg acgtggtgga cagcagcaag agcttcgtga tggagaaatt cagcagctac 60
cacggcacca agcccggcta cgtggacagc atccagaagg gcatccagaa gcccaagagc 120
ggcacccagg gcaaatacga cgacgactgg aagggcttct acagcaccga caaaaagtac 180
gacgccgccg gctacagcgt ggacaaagag aaacccctga gcggcaaggc cggcggcgtg 240
gtgaaggtga cctaccccgg cctgaccaag gtgctggccc tgaaggtgga caaagccgag 300
accatcaaga aggagctggg cctgagcctg accgagcccc tgatggagca ggtgggcacc 360
gaggagttca tcaagcggtt cggcgacggc gccagccggg tggtgctgag cctgcccttc 420
gccgagggca gcagcagcgt ggagtacatc aaaaaatggg agcaggccaa ggccctgagc 480
gtggagctgg agatcaaatt cgagacccgg ggcaagcggg gccaggacgc catgtacgag 540
tacatggccc aggcctgcgc cggcaaacgg gtgcggcgga gcgtgggcag cagcctgagc 600
tgcatcaaac tggactggga cgtgatccgg gacaagacca agaccaagat cgagagcctg 660
aaggagcacg gccccatcaa gaaaaagatg agcgagagcc ccaaaaagac cgtgagcgag 720
gagaaggcca agcagtacct ggaggagttc caccagaccg ccctggagca ccccgagctg 780
agcgagctga agaccgtgac cggcaccaaa cccgtgttcg ccggcgccaa atacgccgcc 840
tgggccgtga aagtggccca ggtgatcgac agcgagaccg ccgacaaact ggagaagacc 900
accgccgccc tgagcatcct gcccggcatc ggcagcgtga tgggcatcgc cgacggcgcc 960
gtgcaccaca aaaccgagga gatcgtggcc cagagcatcg ccctgagcag cctgatggtg 1020
gcccaggcca tccccctggt gggcgagctg gtggacatcg gcttcgccgc ctacaaattc 1080
gtggagagca tcatcaaact gttccaggtg gtgcacaaaa gctacaaacg gcccgcctac 1140
agccccggcc acaagaccca gcccttcctg 1170
<210> 23
<211> 267
<212> PRT
<213> 蓖麻(Ricinus communis)
<400> 23
Ile Phe Pro Lys Gln Tyr Pro Ile Ile Asn Phe Thr Thr Ala Gly Ala
1 5 10 15
Thr Val Gln Ser Tyr Thr Asn Phe Ile Arg Ala Val Arg Gly Arg Leu
20 25 30
Thr Thr Gly Ala Asp Val Arg His Glu Ile Pro Val Leu Pro Asn Arg
35 40 45
Val Gly Leu Pro Ile Asn Gln Arg Phe Ile Leu Val Glu Leu Ser Asn
50 55 60
His Ala Glu Leu Ser Val Thr Leu Ala Leu Asp Val Thr Asn Ala Tyr
65 70 75 80
Val Val Gly Tyr Arg Ala Gly Asn Ser Ala Tyr Phe Phe His Pro Asp
85 90 95
Asn Gln Glu Asp Ala Glu Ala Ile Thr His Leu Phe Thr Asp Val Gln
100 105 110
Asn Arg Tyr Thr Phe Ala Phe Gly Gly Asn Tyr Asp Arg Leu Glu Gln
115 120 125
Leu Ala Gly Asn Leu Arg Glu Asn Ile Glu Leu Gly Asn Gly Pro Leu
130 135 140
Glu Glu Ala Ile Ser Ala Leu Tyr Tyr Tyr Ser Thr Gly Gly Thr Gln
145 150 155 160
Leu Pro Thr Leu Ala Arg Ser Phe Ile Ile Cys Ile Gln Met Ile Ser
165 170 175
Glu Ala Ala Arg Phe Gln Tyr Ile Glu Gly Glu Met Arg Thr Arg Ile
180 185 190
Arg Tyr Asn Arg Arg Ser Ala Pro Asp Pro Ser Val Ile Thr Leu Glu
195 200 205
Asn Ser Trp Gly Arg Leu Ser Thr Ala Ile Gln Glu Ser Asn Gln Gly
210 215 220
Ala Phe Ala Ser Pro Ile Gln Leu Gln Arg Arg Asn Gly Ser Lys Phe
225 230 235 240
Ser Val Tyr Asp Val Ser Ile Leu Ile Pro Ile Ile Ala Leu Met Val
245 250 255
Tyr Arg Cys Ala Pro Pro Pro Ser Ser Gln Phe
260 265
<210> 24
<211> 801
<212> DNA
<213> 蓖麻(Ricinus communis)
<400> 24
atcttcccca agcagtaccc catcatcaaa ttcaccaccg ccggcgccac cgtgcagagc 60
tacaccaaat tcatccgggc cgtgcggggc cggctgacca ccggcgccga cgtgcggcac 120
gagatccccg tgctgcccaa acgggtgggc ctgcccatca aacagcggtt catcctggtg 180
gagctgagca aacacgccga gctgagcgtg accctggccc tggacgtgac caaagcctac 240
gtggtgggct accgggccgg caaaagcgcc tacttcttcc accccgacaa acaggaggac 300
gccgaggcca tcacccacct gttcaccgac gtgcagaaac ggtacacctt cgccttcggc 360
ggcaaatacg accggctgga gcagctggcc ggcaaactgc gggagaaaat cgagctgggc 420
aaaggccccc tggaggaggc catcagcgcc ctgtactact acagcaccgg cggcacccag 480
ctgcccaccc tggcccggag cttcatcatc tgcatccaga tgatcagcga ggccgcccgg 540
ttccagtaca tcgagggcga gatgcggacc cggatccggt acaaacggcg gagcgccccc 600
gaccccagcg tgatcaccct ggagaaaagc tggggccggc tgagcaccgc catccaggag 660
agcaaacagg gcgccttcgc cagccccatc cagctgcagc ggcggaaagg cagcaagttc 720
agcgtgtacg acgtgagcat cctgatcccc atcatcgccc tgatggtgta ccggtgcgcc 780
ccccccccca gcagccagtt c 801
<210> 25
<211> 364
<212> PRT
<213> 霍乱弧菌(Vibrio cholerae)
<400> 25
Ala Leu Ala Ala His Arg Val Cys Gly Val Pro Leu Glu Thr Leu Ala
1 5 10 15
Arg Ser Arg Lys Pro Arg Asp Leu Pro Asp Asp Leu Ser Cys Ala Tyr
20 25 30
Gln Ala Gln Asn Ile Val Ser Leu Phe Val Ala Thr Arg Ile Leu Phe
35 40 45
Ser His Leu Asp Ser Val Phe Thr Leu Asn Leu Asp Glu Gln Glu Pro
50 55 60
Glu Val Ala Glu Arg Leu Ser Ala Leu Arg Gln Ile Asn Glu Asn Asn
65 70 75 80
Pro Gly Met Val Thr Gln Val Leu Thr Val Ala Arg Gln Ile Tyr Asn
85 90 95
Asp Tyr Val Thr His His Pro Gly Leu Ile Pro Glu Gln Thr Ser Ala
100 105 110
Gly Ala Gln Ala Ala Asp Ile Leu Ser Leu Phe Cys Pro Asp Ala Asp
115 120 125
Lys Pro Cys Val Ala Ser Asn Asn Asp Gln Ala Asn Ile Asn Ile Glu
130 135 140
Ser Arg Ser Gly Arg Ser Tyr Leu Pro Glu Asn Arg Ala Val Ile Thr
145 150 155 160
Pro Gln Gly Val Thr Asn Trp Thr Tyr Gln Glu Leu Glu Ala Thr His
165 170 175
Gln Ala Leu Thr Arg Glu Gly Tyr Val Phe Val Gly Tyr His Gly Thr
180 185 190
Asn His Val Ala Ala Gln Thr Ile Val Asn Arg Ile Ala Pro Val Pro
195 200 205
Arg Gly Asn Asn Thr Glu Asn Glu Glu Lys Trp Gly Gly Leu Tyr Val
210 215 220
Ala Thr His Ala Glu Val Ala His Gly Tyr Ala Arg Ile Lys Glu Gly
225 230 235 240
Thr Gly Glu Tyr Gly Leu Pro Thr Arg Ala Glu Arg Glu Ala Arg Gly
245 250 255
Val Met Leu Arg Val Tyr Ile Pro Arg Ala Ser Leu Glu Arg Phe Tyr
260 265 270
Arg Thr Asn Thr Pro Leu Glu Asn Ala Glu Arg His Ile Thr Gln Val
275 280 285
Ile Gly His Ser Leu Pro Leu Arg Asn Glu Ala Phe Thr Gly Pro Glu
290 295 300
Ser Ala Gly Gly Glu Asp Glu Thr Val Ile Gly Trp Asp Met Ala Ile
305 310 315 320
His Ala Val Ala Ile Pro Ser Thr Ile Pro Gly Asn Ala Tyr Glu Glu
325 330 335
Leu Ala Ile Asp Glu Glu Ala Val Ala Lys Glu Gln Ser Ile Ser Ala
340 345 350
Lys Pro Pro Tyr Lys Glu Gln Lys Asp Glu Leu Lys
355 360
<210> 26
<211> 1092
<212> DNA
<213> 霍乱弧菌(Vibrio cholerae)
<400> 26
gccctggccg cccaccgggt gtgcggcgtg cccctggaga ccctggcccg gagccggaag 60
ccccgggacc tgcccgacga cctgagctgc gcctaccagg cccagaaaat cgtgagcctg 120
ttcgtggcca cccggatcct gttcagccac ctggacagcg tgttcaccct gaaactggac 180
gagcaggagc ccgaggtggc cgagcggctg agcgccctgc ggcagatcaa agagaaaaaa 240
cccggcatgg tgacccaggt gctgaccgtg gcccggcaga tctacaaaga ctacgtgacc 300
caccaccccg gcctgatccc cgagcagacc agcgccggcg cccaggccgc cgacatcctg 360
agcctgttct gccccgacgc cgacaagccc tgcgtggcca gcaaaaaaga ccaggccaaa 420
atcaaaatcg agagccggag cggccggagc tacctgcccg agaaacgggc cgtgatcacc 480
ccccagggcg tgaccaaatg gacctaccag gagctggagg ccacccacca ggccctgacc 540
cgggagggct acgtgttcgt gggctaccac ggcaccaaac acgtggccgc ccagaccatc 600
gtgaaacgga tcgcccccgt gccccggggc aaaaaaaccg agaaagagga gaagtggggc 660
ggcctgtacg tggccaccca cgccgaggtg gcccacggct acgcccggat caaggagggc 720
accggcgagt acggcctgcc cacccgggcc gagcgggagg cccggggcgt gatgctgcgg 780
gtgtacatcc cccgggccag cctggagcgg ttctaccgga ccaaaacccc cctggagaaa 840
gccgagcggc acatcaccca ggtgatcggc cacagcctgc ccctgcggaa agaggccttc 900
accggccccg agagcgccgg cggcgaggac gagaccgtga tcggctggga catggccatc 960
cacgccgtgg ccatccccag caccatcccc ggcaaagcct acgaggagct ggccatcgac 1020
gaggaggccg tggccaagga gcagagcatc agcgccaagc ccccctacaa ggagcagaag 1080
gacgagctga ag 1092
<210> 27
<211> 124
<212> PRT
<213> 人(human)
<400> 27
Lys Glu Ser Arg Ala Lys Lys Phe Gln Arg Gln His Met Asp Ser Asp
1 5 10 15
Ser Ser Pro Ser Ser Ser Ser Thr Tyr Cys Asn Gln Met Met Arg Arg
20 25 30
Arg Asn Met Thr Gln Gly Arg Cys Lys Pro Val Asn Thr Phe Val His
35 40 45
Glu Pro Leu Val Asp Val Gln Asn Val Cys Phe Gln Glu Lys Val Thr
50 55 60
Cys Lys Asn Gly Gln Gly Asn Cys Tyr Lys Ser Asn Ser Ser Met His
65 70 75 80
Ile Thr Asp Cys Arg Leu Thr Asn Gly Ser Arg Tyr Pro Asn Cys Ala
85 90 95
Tyr Arg Thr Ser Pro Lys Glu Arg His Ile Ile Val Ala Cys Glu Gly
100 105 110
Ser Pro Tyr Val Pro Val His Phe Asp Ala Ser Val
115 120
<210> 28
<211> 372
<212> DNA
<213> 人(human)
<400> 28
aaggagagcc gggccaagaa gttccagcgg cagcacatgg acagcgacag cagccccagc 60
agcagcagca cctactgcaa acagatgatg cggcggcgga aaatgaccca gggccggtgc 120
aagcccgtga aaaccttcgt gcacgagccc ctggtggacg tgcagaaagt gtgcttccag 180
gagaaggtga cctgcaagaa aggccagggc aaatgctaca agagcaaaag cagcatgcac 240
atcaccgact gccggctgac caaaggcagc cggtacccca aatgcgccta ccggaccagc 300
cccaaggagc ggcacatcat cgtggcctgc gagggcagcc cctacgtgcc cgtgcacttc 360
gacgccagcg tg 372
<210> 29
<211> 582
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 29
Ile Leu Asp Tyr Ser Phe Thr Gly Gly Ala Gly Arg Asp Ile Pro Pro
1 5 10 15
Pro Gln Ile Glu Glu Ala Cys Glu Leu Pro Glu Cys Gln Val Asp Ala
20 25 30
Gly Asn Lys Val Cys Asn Leu Gln Cys Asn Asn His Ala Cys Gly Trp
35 40 45
Asp Gly Gly Asp Cys Ser Leu Asn Phe Asn Asp Pro Trp Lys Asn Cys
50 55 60
Thr Gln Ser Leu Gln Cys Trp Lys Tyr Phe Ser Asp Gly His Cys Asp
65 70 75 80
Ser Gln Cys Asn Ser Ala Gly Cys Leu Phe Asp Gly Phe Asp Cys Gln
85 90 95
Leu Thr Glu Gly Gln Cys Asn Pro Leu Tyr Asp Gln Tyr Cys Lys Asp
100 105 110
His Phe Ser Asp Gly His Cys Asp Gln Gly Cys Asn Ser Ala Glu Cys
115 120 125
Glu Trp Asp Gly Leu Asp Cys Ala Glu His Val Pro Glu Arg Leu Ala
130 135 140
Ala Gly Thr Leu Val Leu Val Val Leu Leu Pro Pro Asp Gln Leu Arg
145 150 155 160
Asn Asn Ser Phe His Phe Leu Arg Glu Leu Ser His Val Leu His Thr
165 170 175
Asn Val Val Phe Lys Arg Asp Ala Gln Gly Gln Gln Met Ile Phe Pro
180 185 190
Tyr Tyr Gly His Glu Glu Glu Leu Arg Lys His Pro Ile Lys Arg Ser
195 200 205
Thr Val Gly Trp Ala Thr Ser Ser Leu Leu Pro Gly Thr Ser Gly Gly
210 215 220
Arg Gln Arg Arg Glu Leu Asp Pro Met Asp Ile Arg Gly Ser Ile Val
225 230 235 240
Tyr Leu Glu Ile Asp Asn Arg Gln Cys Val Gln Ser Ser Ser Gln Cys
245 250 255
Phe Gln Ser Ala Thr Asp Val Ala Ala Phe Leu Gly Ala Leu Ala Ser
260 265 270
Leu Gly Ser Leu Asn Ile Pro Tyr Lys Ile Glu Ala Val Lys Ser Glu
275 280 285
Pro Val Glu Pro Pro Leu Pro Ser Gln Leu His Leu Met Tyr Val Ala
290 295 300
Ala Ala Ala Phe Val Leu Leu Phe Phe Val Gly Cys Gly Val Leu Leu
305 310 315 320
Ser Arg Lys Arg Arg Arg Met Ala Pro Lys Lys Lys Arg Lys Val Ser
325 330 335
Arg Leu Asp Lys Ser Lys Val Ile Asn Ser Ala Leu Glu Leu Leu Asn
340 345 350
Glu Val Gly Ile Glu Gly Leu Thr Thr Arg Lys Leu Ala Gln Lys Leu
355 360 365
Gly Val Glu Gln Pro Thr Leu Tyr Trp His Val Lys Asn Lys Arg Ala
370 375 380
Leu Leu Asp Ala Leu Ala Ile Glu Met Leu Asp Arg His His Thr His
385 390 395 400
Phe Cys Pro Leu Glu Gly Glu Ser Trp Gln Asp Phe Leu Arg Asn Asn
405 410 415
Ala Lys Ser Phe Arg Cys Ala Leu Leu Ser His Arg Asp Gly Ala Lys
420 425 430
Val His Leu Gly Thr Arg Pro Thr Glu Lys Gln Tyr Glu Thr Leu Glu
435 440 445
Asn Gln Leu Ala Phe Leu Cys Gln Gln Gly Phe Ser Leu Glu Asn Ala
450 455 460
Leu Tyr Ala Leu Ser Ala Val Gly His Phe Thr Leu Gly Cys Val Leu
465 470 475 480
Glu Asp Gln Glu His Gln Val Ala Lys Glu Glu Arg Glu Thr Pro Thr
485 490 495
Thr Asp Ser Met Pro Pro Leu Leu Arg Gln Ala Ile Glu Leu Phe Asp
500 505 510
His Gln Gly Ala Glu Pro Ala Phe Leu Phe Gly Leu Glu Leu Ile Ile
515 520 525
Cys Gly Leu Glu Lys Gln Leu Lys Cys Glu Ser Gly Gly Pro Ala Asp
530 535 540
Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Pro Ala Asp Ala Leu Asp
545 550 555 560
Asp Phe Asp Leu Asp Met Leu Pro Ala Asp Ala Leu Asp Asp Phe Asp
565 570 575
Leu Asp Met Leu Pro Gly
580
<210> 30
<211> 1746
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
atcctggact acagcttcac aggtggcgct gggcgcgaca ttcccccacc gcagattgag 60
gaggcctgtg agctgcctga gtgccaggtg gatgcaggca ataaggtctg caacctgcag 120
tgtaataatc acgcatgtgg ctgggatggt ggcgactgct ccctcaactt caatgacccc 180
tggaagaact gcacgcagtc tctacagtgc tggaagtatt ttagcgacgg ccactgtgac 240
agccagtgca actcggccgg ctgcctcttt gatggcttcg actgccagct caccgaggga 300
cagtgcaacc ccctgtatga ccagtactgc aaggaccact tcagtgatgg ccactgcgac 360
cagggctgta acagtgccga atgtgagtgg gatggcctag actgtgctga gcatgtaccc 420
gagcggctgg cagccggcac cctggtgctg gtggtgctgc ttccacccga ccagctacgg 480
aacaactcct tccactttct gcgggagctc agccacgtgc tgcacaccaa cgtggtcttc 540
aagcgtgatg cgcaaggcca gcagatgatc ttcccgtact atggccacga ggaagagctg 600
cgcaagcacc caatcaagcg ctctacagtg ggttgggcca cctcttcact gcttcctggt 660
accagtggtg ggcgccagcg cagggagctg gaccccatgg acatccgtgg ctccattgtc 720
tacctggaga tcgacaaccg gcaatgtgtg cagtcatcct cgcagtgctt ccagagtgcc 780
accgatgtgg ctgccttcct aggtgctctt gcgtcacttg gcagcctcaa tattccttac 840
aagattgagg ccgtgaagag tgagccggtg gagcctccgc tgccctcgca gctgcacctc 900
atgtacgtgg cagcggccgc cttcgtgctc ctgttctttg tgggctgtgg ggtgctgctg 960
tcccgcaagc gccggcggat ggctcctaag aagaagagga aggtgtctag actggacaag 1020
agcaaagtca taaactctgc tctggaatta ctcaatgaag tcggtatcga aggcctgacg 1080
acaaggaaac tcgctcaaaa gctgggagtt gagcagccta ccctgtactg gcacgtgaag 1140
aacaagcggg ccctgctcga tgccctggca atcgagatgc tggacaggca tcatacccac 1200
ttctgccccc tggaaggcga gtcatggcaa gactttctgc ggaacaacgc caagtcattc 1260
cgctgtgctc tcctctcaca tcgcgacggg gctaaagtgc atctcggcac ccgcccaaca 1320
gagaaacagt acgaaaccct ggaaaatcag ctcgcgttcc tgtgtcagca aggcttctcc 1380
ctggagaacg cactgtacgc tctgtccgcc gtgggccact ttacactggg ctgcgtattg 1440
gaggatcagg agcatcaagt agcaaaagag gaaagagaga cacctaccac cgattctatg 1500
cccccacttc tgagacaagc aattgagctg ttcgaccatc agggagccga acctgccttc 1560
cttttcggcc tggaactaat catatgtggc ctggagaaac agctaaagtg cgaaagcggc 1620
gggccggccg acgcccttga cgattttgac ttagacatgc tcccagccga tgcccttgac 1680
gactttgacc ttgatatgct gcctgctgac gctcttgacg attttgacct tgacatgctc 1740
cccggg 1746
<210> 31
<211> 488
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 31
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val
20 25 30
Lys Lys Pro Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly
35 40 45
Thr Phe Ser Ser Tyr Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln
50 55 60
Gly Leu Glu Trp Met Gly Gly Ile Ile Pro Ile Leu Gly Ile Ala Asn
65 70 75 80
Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Ile Thr Ala Asp Gln Ser
85 90 95
Thr Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
100 105 110
Ala Val Tyr Tyr Cys Ala Arg Gly Gly Ser Gly Ser Tyr His Met Asp
115 120 125
Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly
130 135 140
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ile Val Leu Thr
145 150 155 160
Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu
165 170 175
Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala Trp Tyr Gln
180 185 190
Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Asp Ala Ser Asn
195 200 205
Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr
210 215 220
Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val
225 230 235 240
Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Pro Arg Ile Thr Phe Gly
245 250 255
Gln Gly Thr Arg Leu Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro
260 265 270
Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro
275 280 285
Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu
290 295 300
Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys
305 310 315 320
Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly
325 330 335
Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val
340 345 350
Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu
355 360 365
Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp
370 375 380
Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn
385 390 395 400
Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg
405 410 415
Asp Pro Glu Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu
420 425 430
Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser
435 440 445
Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly
450 455 460
Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu
465 470 475 480
His Met Gln Ala Leu Pro Pro Arg
485
<210> 32
<211> 354
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 32
Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly
1 5 10 15
Val His Ser Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln
20 25 30
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Tyr Phe Asp Phe
35 40 45
Asp Ser Tyr Glu Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
50 55 60
Glu Trp Ile Gly Ser Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Pro
65 70 75 80
Ser Leu Lys Ser Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
85 90 95
Leu Tyr Leu Gln Met Asn Thr Leu Arg Ala Glu Asp Thr Ala Thr Tyr
100 105 110
Tyr Cys Ala Arg Val Asn Met Asp Arg Phe Asp Tyr Trp Gly Gln Gly
115 120 125
Thr Leu Val Thr Val Ser Ser Pro Thr Gly Ala Glu Phe Leu Gly Asp
130 135 140
Gly Gly Asp Val Ser Phe Ser Thr Arg Gly Thr Gln Asn Trp Thr Val
145 150 155 160
Glu Arg Leu Leu Gln Ala His Arg Gln Leu Glu Glu Arg Gly Tyr Val
165 170 175
Phe Val Gly Tyr His Gly Thr Phe Leu Glu Ala Ala Gln Ser Ile Val
180 185 190
Phe Gly Gly Val Arg Ala Arg Ser Gln Asp Leu Asp Ala Ile Trp Arg
195 200 205
Gly Phe Tyr Ile Ala Gly Asp Pro Ala Leu Ala Tyr Gly Tyr Ala Gln
210 215 220
Asp Gln Glu Pro Asp Ala Arg Gly Arg Ile Arg Asn Gly Ala Leu Leu
225 230 235 240
Arg Val Tyr Val Pro Arg Ser Ser Leu Pro Gly Phe Tyr Arg Thr Ser
245 250 255
Leu Thr Leu Ala Ala Pro Glu Ala Ala Gly Glu Val Glu Arg Leu Ile
260 265 270
Gly His Pro Leu Pro Leu Arg Leu Asp Ala Ile Thr Gly Pro Glu Glu
275 280 285
Glu Gly Gly Arg Leu Glu Thr Ile Leu Gly Trp Pro Leu Ala Glu Arg
290 295 300
Thr Val Val Ile Pro Ser Ala Ile Pro Thr Asp Pro Arg Asn Val Gly
305 310 315 320
Gly Asp Leu Asp Pro Ser Ser Ile Pro Asp Lys Glu Gln Ala Ile Ser
325 330 335
Ala Leu Pro Asp Tyr Ala Ser Gln Pro Gly Lys Pro Pro Arg Glu Asp
340 345 350
Leu Lys
<210> 33
<211> 525
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 33
Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly
1 5 10 15
Val His Ser Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln
20 25 30
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Tyr Phe Asp Phe
35 40 45
Asp Ser Tyr Glu Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
50 55 60
Glu Trp Ile Gly Ser Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Pro
65 70 75 80
Ser Leu Lys Ser Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
85 90 95
Leu Tyr Leu Gln Met Asn Thr Leu Arg Ala Glu Asp Thr Ala Thr Tyr
100 105 110
Tyr Cys Ala Arg Val Asn Met Asp Arg Phe Asp Tyr Trp Gly Gln Gly
115 120 125
Thr Leu Val Thr Val Ser Ser Gly Ala Asp Asp Val Val Asp Ser Ser
130 135 140
Lys Ser Phe Val Met Glu Asn Phe Ser Ser Tyr His Gly Thr Lys Pro
145 150 155 160
Gly Tyr Val Asp Ser Ile Gln Lys Gly Ile Gln Lys Pro Lys Ser Gly
165 170 175
Thr Gln Gly Asn Tyr Asp Asp Asp Trp Lys Gly Phe Tyr Ser Thr Asp
180 185 190
Asn Lys Tyr Asp Ala Ala Gly Tyr Ser Val Asp Asn Glu Asn Pro Leu
195 200 205
Ser Gly Lys Ala Gly Gly Val Val Lys Val Thr Tyr Pro Gly Leu Thr
210 215 220
Lys Val Leu Ala Leu Lys Val Asp Asn Ala Glu Thr Ile Lys Lys Glu
225 230 235 240
Leu Gly Leu Ser Leu Thr Glu Pro Leu Met Glu Gln Val Gly Thr Glu
245 250 255
Glu Phe Ile Lys Arg Phe Gly Asp Gly Ala Ser Arg Val Val Leu Ser
260 265 270
Leu Pro Phe Ala Glu Gly Ser Ser Ser Val Glu Tyr Ile Asn Asn Trp
275 280 285
Glu Gln Ala Lys Ala Leu Ser Val Glu Leu Glu Ile Asn Phe Glu Thr
290 295 300
Arg Gly Lys Arg Gly Gln Asp Ala Met Tyr Glu Tyr Met Ala Gln Ala
305 310 315 320
Cys Ala Gly Asn Arg Val Arg Arg Ser Val Gly Ser Ser Leu Ser Cys
325 330 335
Ile Asn Leu Asp Trp Asp Val Ile Arg Asp Lys Thr Lys Thr Lys Ile
340 345 350
Glu Ser Leu Lys Glu His Gly Pro Ile Lys Asn Lys Met Ser Glu Ser
355 360 365
Pro Asn Lys Thr Val Ser Glu Glu Lys Ala Lys Gln Tyr Leu Glu Glu
370 375 380
Phe His Gln Thr Ala Leu Glu His Pro Glu Leu Ser Glu Leu Lys Thr
385 390 395 400
Val Thr Gly Thr Asn Pro Val Phe Ala Gly Ala Asn Tyr Ala Ala Trp
405 410 415
Ala Val Asn Val Ala Gln Val Ile Asp Ser Glu Thr Ala Asp Asn Leu
420 425 430
Glu Lys Thr Thr Ala Ala Leu Ser Ile Leu Pro Gly Ile Gly Ser Val
435 440 445
Met Gly Ile Ala Asp Gly Ala Val His His Asn Thr Glu Glu Ile Val
450 455 460
Ala Gln Ser Ile Ala Leu Ser Ser Leu Met Val Ala Gln Ala Ile Pro
465 470 475 480
Leu Val Gly Glu Leu Val Asp Ile Gly Phe Ala Ala Tyr Asn Phe Val
485 490 495
Glu Ser Ile Ile Asn Leu Phe Gln Val Val His Asn Ser Tyr Asn Arg
500 505 510
Pro Ala Tyr Ser Pro Gly His Lys Thr Gln Pro Phe Leu
515 520 525
<210> 34
<211> 499
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 34
Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly
1 5 10 15
Val His Ser Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln
20 25 30
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Tyr Phe Asp Phe
35 40 45
Asp Ser Tyr Glu Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
50 55 60
Glu Trp Ile Gly Ser Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Pro
65 70 75 80
Ser Leu Lys Ser Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
85 90 95
Leu Tyr Leu Gln Met Asn Thr Leu Arg Ala Glu Asp Thr Ala Thr Tyr
100 105 110
Tyr Cys Ala Arg Val Asn Met Asp Arg Phe Asp Tyr Trp Gly Gln Gly
115 120 125
Thr Leu Val Thr Val Ser Ser Ala Leu Ala Ala His Arg Val Cys Gly
130 135 140
Val Pro Leu Glu Thr Leu Ala Arg Ser Arg Lys Pro Arg Asp Leu Pro
145 150 155 160
Asp Asp Leu Ser Cys Ala Tyr Gln Ala Gln Asn Ile Val Ser Leu Phe
165 170 175
Val Ala Thr Arg Ile Leu Phe Ser His Leu Asp Ser Val Phe Thr Leu
180 185 190
Asn Leu Asp Glu Gln Glu Pro Glu Val Ala Glu Arg Leu Ser Ala Leu
195 200 205
Arg Gln Ile Asn Glu Asn Asn Pro Gly Met Val Thr Gln Val Leu Thr
210 215 220
Val Ala Arg Gln Ile Tyr Asn Asp Tyr Val Thr His His Pro Gly Leu
225 230 235 240
Ile Pro Glu Gln Thr Ser Ala Gly Ala Gln Ala Ala Asp Ile Leu Ser
245 250 255
Leu Phe Cys Pro Asp Ala Asp Lys Pro Cys Val Ala Ser Asn Asn Asp
260 265 270
Gln Ala Asn Ile Asn Ile Glu Ser Arg Ser Gly Arg Ser Tyr Leu Pro
275 280 285
Glu Asn Arg Ala Val Ile Thr Pro Gln Gly Val Thr Asn Trp Thr Tyr
290 295 300
Gln Glu Leu Glu Ala Thr His Gln Ala Leu Thr Arg Glu Gly Tyr Val
305 310 315 320
Phe Val Gly Tyr His Gly Thr Asn His Val Ala Ala Gln Thr Ile Val
325 330 335
Asn Arg Ile Ala Pro Val Pro Arg Gly Asn Asn Thr Glu Asn Glu Glu
340 345 350
Lys Trp Gly Gly Leu Tyr Val Ala Thr His Ala Glu Val Ala His Gly
355 360 365
Tyr Ala Arg Ile Lys Glu Gly Thr Gly Glu Tyr Gly Leu Pro Thr Arg
370 375 380
Ala Glu Arg Glu Ala Arg Gly Val Met Leu Arg Val Tyr Ile Pro Arg
385 390 395 400
Ala Ser Leu Glu Arg Phe Tyr Arg Thr Asn Thr Pro Leu Glu Asn Ala
405 410 415
Glu Arg His Ile Thr Gln Val Ile Gly His Ser Leu Pro Leu Arg Asn
420 425 430
Glu Ala Phe Thr Gly Pro Glu Ser Ala Gly Gly Glu Asp Glu Thr Val
435 440 445
Ile Gly Trp Asp Met Ala Ile His Ala Val Ala Ile Pro Ser Thr Ile
450 455 460
Pro Gly Asn Ala Tyr Glu Glu Leu Ala Ile Asp Glu Glu Ala Val Ala
465 470 475 480
Lys Glu Gln Ser Ile Ser Ala Lys Pro Pro Tyr Lys Glu Gln Lys Asp
485 490 495
Glu Leu Lys
<210> 35
<211> 402
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 35
Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly
1 5 10 15
Val His Ser Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln
20 25 30
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Tyr Phe Asp Phe
35 40 45
Asp Ser Tyr Glu Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
50 55 60
Glu Trp Ile Gly Ser Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Pro
65 70 75 80
Ser Leu Lys Ser Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
85 90 95
Leu Tyr Leu Gln Met Asn Thr Leu Arg Ala Glu Asp Thr Ala Thr Tyr
100 105 110
Tyr Cys Ala Arg Val Asn Met Asp Arg Phe Asp Tyr Trp Gly Gln Gly
115 120 125
Thr Leu Val Thr Val Ser Ser Ile Phe Pro Lys Gln Tyr Pro Ile Ile
130 135 140
Asn Phe Thr Thr Ala Gly Ala Thr Val Gln Ser Tyr Thr Asn Phe Ile
145 150 155 160
Arg Ala Val Arg Gly Arg Leu Thr Thr Gly Ala Asp Val Arg His Glu
165 170 175
Ile Pro Val Leu Pro Asn Arg Val Gly Leu Pro Ile Asn Gln Arg Phe
180 185 190
Ile Leu Val Glu Leu Ser Asn His Ala Glu Leu Ser Val Thr Leu Ala
195 200 205
Leu Asp Val Thr Asn Ala Tyr Val Val Gly Tyr Arg Ala Gly Asn Ser
210 215 220
Ala Tyr Phe Phe His Pro Asp Asn Gln Glu Asp Ala Glu Ala Ile Thr
225 230 235 240
His Leu Phe Thr Asp Val Gln Asn Arg Tyr Thr Phe Ala Phe Gly Gly
245 250 255
Asn Tyr Asp Arg Leu Glu Gln Leu Ala Gly Asn Leu Arg Glu Asn Ile
260 265 270
Glu Leu Gly Asn Gly Pro Leu Glu Glu Ala Ile Ser Ala Leu Tyr Tyr
275 280 285
Tyr Ser Thr Gly Gly Thr Gln Leu Pro Thr Leu Ala Arg Ser Phe Ile
290 295 300
Ile Cys Ile Gln Met Ile Ser Glu Ala Ala Arg Phe Gln Tyr Ile Glu
305 310 315 320
Gly Glu Met Arg Thr Arg Ile Arg Tyr Asn Arg Arg Ser Ala Pro Asp
325 330 335
Pro Ser Val Ile Thr Leu Glu Asn Ser Trp Gly Arg Leu Ser Thr Ala
340 345 350
Ile Gln Glu Ser Asn Gln Gly Ala Phe Ala Ser Pro Ile Gln Leu Gln
355 360 365
Arg Arg Asn Gly Ser Lys Phe Ser Val Tyr Asp Val Ser Ile Leu Ile
370 375 380
Pro Ile Ile Ala Leu Met Val Tyr Arg Cys Ala Pro Pro Pro Ser Ser
385 390 395 400
Gln Phe
<210> 36
<211> 259
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 36
Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly
1 5 10 15
Val His Ser Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln
20 25 30
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Tyr Phe Asp Phe
35 40 45
Asp Ser Tyr Glu Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
50 55 60
Glu Trp Ile Gly Ser Ile Tyr His Ser Gly Ser Thr Tyr Tyr Asn Pro
65 70 75 80
Ser Leu Lys Ser Arg Val Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
85 90 95
Leu Tyr Leu Gln Met Asn Thr Leu Arg Ala Glu Asp Thr Ala Thr Tyr
100 105 110
Tyr Cys Ala Arg Val Asn Met Asp Arg Phe Asp Tyr Trp Gly Gln Gly
115 120 125
Thr Leu Val Thr Val Ser Ser Lys Glu Ser Arg Ala Lys Lys Phe Gln
130 135 140
Arg Gln His Met Asp Ser Asp Ser Ser Pro Ser Ser Ser Ser Thr Tyr
145 150 155 160
Cys Asn Gln Met Met Arg Arg Arg Asn Met Thr Gln Gly Arg Cys Lys
165 170 175
Pro Val Asn Thr Phe Val His Glu Pro Leu Val Asp Val Gln Asn Val
180 185 190
Cys Phe Gln Glu Lys Val Thr Cys Lys Asn Gly Gln Gly Asn Cys Tyr
195 200 205
Lys Ser Asn Ser Ser Met His Ile Thr Asp Cys Arg Leu Thr Asn Gly
210 215 220
Ser Arg Tyr Pro Asn Cys Ala Tyr Arg Thr Ser Pro Lys Glu Arg His
225 230 235 240
Ile Ile Val Ala Cys Glu Gly Ser Pro Tyr Val Pro Val His Phe Asp
245 250 255
Ala Ser Val
<210> 37
<211> 588
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 37
Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly
1 5 10 15
Val His Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Glu Lys
20 25 30
Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe
35 40 45
Thr Gly Tyr Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu
50 55 60
Glu Trp Ile Gly Leu Ile Thr Pro Tyr Asn Gly Ala Ser Ser Tyr Asn
65 70 75 80
Gln Lys Phe Arg Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser
85 90 95
Thr Ala Tyr Met Asp Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val
100 105 110
Tyr Phe Cys Ala Arg Gly Gly Tyr Asp Gly Arg Gly Phe Asp Tyr Trp
115 120 125
Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Val Gly Gly Ser Gly
130 135 140
Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Glu Leu Thr Gln Ser
145 150 155 160
Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys
165 170 175
Ser Ala Ser Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Ser
180 185 190
Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser
195 200 205
Gly Val Pro Gly Arg Phe Ser Gly Ser Gly Ser Gly Asn Ser Tyr Ser
210 215 220
Leu Thr Ile Ser Ser Val Glu Ala Glu Asp Asp Ala Thr Tyr Tyr Cys
225 230 235 240
Gln Gln Trp Ser Gly Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu
245 250 255
Glu Ile Lys Glu Gln Cys Gly Tyr Pro Val Gln Arg Leu Val Ala Leu
260 265 270
Tyr Leu Ala Ala Arg Leu Ser Trp Asn Gln Val Asp Gln Val Ile Arg
275 280 285
Asn Ala Leu Ala Ser Pro Gly Ser Gly Gly Asp Leu Gly Glu Ala Ile
290 295 300
Arg Glu Gln Pro Glu Gln Ala Arg Leu Ala Leu Thr Leu Ala Ala Ala
305 310 315 320
Glu Ser Glu Arg Phe Val Arg Gln Gly Thr Gly Asn Asp Glu Ala Gly
325 330 335
Ala Ala Asn Ala Asp Val Val Ser Leu Thr Cys Pro Val Ala Ala Gly
340 345 350
Glu Cys Ala Gly Pro Ala Asp Ser Gly Asp Ala Leu Leu Glu Arg Asn
355 360 365
Tyr Pro Thr Gly Ala Glu Phe Leu Gly Asp Gly Gly Asp Val Ser Phe
370 375 380
Ser Thr Arg Gly Thr Gln Asn Trp Thr Val Glu Arg Leu Leu Gln Ala
385 390 395 400
His Arg Gln Leu Glu Glu Arg Gly Tyr Val Phe Val Gly Tyr His Gly
405 410 415
Thr Phe Leu Glu Ala Ala Gln Ser Ile Val Phe Gly Gly Val Arg Ala
420 425 430
Arg Ser Gln Asp Leu Asp Ala Ile Trp Arg Gly Phe Tyr Ile Ala Gly
435 440 445
Asp Pro Ala Leu Ala Tyr Gly Tyr Ala Gln Asp Gln Glu Pro Asp Ala
450 455 460
Arg Gly Arg Ile Arg Asn Gly Ala Leu Leu Arg Val Tyr Val Pro Arg
465 470 475 480
Ser Ser Leu Pro Gly Phe Tyr Arg Thr Ser Leu Thr Leu Ala Ala Pro
485 490 495
Glu Ala Ala Gly Glu Val Glu Arg Leu Ile Gly His Pro Leu Pro Leu
500 505 510
Arg Leu Asp Ala Ile Thr Gly Pro Glu Glu Glu Gly Gly Arg Leu Glu
515 520 525
Thr Ile Leu Gly Trp Pro Leu Ala Glu Arg Thr Val Val Ile Pro Ser
530 535 540
Ala Ile Pro Thr Asp Pro Arg Asn Val Gly Gly Asp Leu Asp Pro Ser
545 550 555 560
Ser Ile Pro Asp Lys Glu Gln Ala Ile Ser Ala Leu Pro Asp Tyr Ala
565 570 575
Ser Gln Pro Gly Lys Pro Pro Arg Glu Asp Leu Lys
580 585
<210> 38
<211> 649
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 38
Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly
1 5 10 15
Val His Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Glu Lys
20 25 30
Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe
35 40 45
Thr Gly Tyr Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu
50 55 60
Glu Trp Ile Gly Leu Ile Thr Pro Tyr Asn Gly Ala Ser Ser Tyr Asn
65 70 75 80
Gln Lys Phe Arg Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser
85 90 95
Thr Ala Tyr Met Asp Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val
100 105 110
Tyr Phe Cys Ala Arg Gly Gly Tyr Asp Gly Arg Gly Phe Asp Tyr Trp
115 120 125
Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Val Gly Gly Ser Gly
130 135 140
Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Glu Leu Thr Gln Ser
145 150 155 160
Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys
165 170 175
Ser Ala Ser Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Ser
180 185 190
Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser
195 200 205
Gly Val Pro Gly Arg Phe Ser Gly Ser Gly Ser Gly Asn Ser Tyr Ser
210 215 220
Leu Thr Ile Ser Ser Val Glu Ala Glu Asp Asp Ala Thr Tyr Tyr Cys
225 230 235 240
Gln Gln Trp Ser Gly Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu
245 250 255
Glu Ile Lys Gly Ala Asp Asp Val Val Asp Ser Ser Lys Ser Phe Val
260 265 270
Met Glu Asn Phe Ser Ser Tyr His Gly Thr Lys Pro Gly Tyr Val Asp
275 280 285
Ser Ile Gln Lys Gly Ile Gln Lys Pro Lys Ser Gly Thr Gln Gly Asn
290 295 300
Tyr Asp Asp Asp Trp Lys Gly Phe Tyr Ser Thr Asp Asn Lys Tyr Asp
305 310 315 320
Ala Ala Gly Tyr Ser Val Asp Asn Glu Asn Pro Leu Ser Gly Lys Ala
325 330 335
Gly Gly Val Val Lys Val Thr Tyr Pro Gly Leu Thr Lys Val Leu Ala
340 345 350
Leu Lys Val Asp Asn Ala Glu Thr Ile Lys Lys Glu Leu Gly Leu Ser
355 360 365
Leu Thr Glu Pro Leu Met Glu Gln Val Gly Thr Glu Glu Phe Ile Lys
370 375 380
Arg Phe Gly Asp Gly Ala Ser Arg Val Val Leu Ser Leu Pro Phe Ala
385 390 395 400
Glu Gly Ser Ser Ser Val Glu Tyr Ile Asn Asn Trp Glu Gln Ala Lys
405 410 415
Ala Leu Ser Val Glu Leu Glu Ile Asn Phe Glu Thr Arg Gly Lys Arg
420 425 430
Gly Gln Asp Ala Met Tyr Glu Tyr Met Ala Gln Ala Cys Ala Gly Asn
435 440 445
Arg Val Arg Arg Ser Val Gly Ser Ser Leu Ser Cys Ile Asn Leu Asp
450 455 460
Trp Asp Val Ile Arg Asp Lys Thr Lys Thr Lys Ile Glu Ser Leu Lys
465 470 475 480
Glu His Gly Pro Ile Lys Asn Lys Met Ser Glu Ser Pro Asn Lys Thr
485 490 495
Val Ser Glu Glu Lys Ala Lys Gln Tyr Leu Glu Glu Phe His Gln Thr
500 505 510
Ala Leu Glu His Pro Glu Leu Ser Glu Leu Lys Thr Val Thr Gly Thr
515 520 525
Asn Pro Val Phe Ala Gly Ala Asn Tyr Ala Ala Trp Ala Val Asn Val
530 535 540
Ala Gln Val Ile Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys Thr Thr
545 550 555 560
Ala Ala Leu Ser Ile Leu Pro Gly Ile Gly Ser Val Met Gly Ile Ala
565 570 575
Asp Gly Ala Val His His Asn Thr Glu Glu Ile Val Ala Gln Ser Ile
580 585 590
Ala Leu Ser Ser Leu Met Val Ala Gln Ala Ile Pro Leu Val Gly Glu
595 600 605
Leu Val Asp Ile Gly Phe Ala Ala Tyr Asn Phe Val Glu Ser Ile Ile
610 615 620
Asn Leu Phe Gln Val Val His Asn Ser Tyr Asn Arg Pro Ala Tyr Ser
625 630 635 640
Pro Gly His Lys Thr Gln Pro Phe Leu
645
<210> 39
<211> 623
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 39
Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly
1 5 10 15
Val His Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Glu Lys
20 25 30
Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe
35 40 45
Thr Gly Tyr Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu
50 55 60
Glu Trp Ile Gly Leu Ile Thr Pro Tyr Asn Gly Ala Ser Ser Tyr Asn
65 70 75 80
Gln Lys Phe Arg Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser
85 90 95
Thr Ala Tyr Met Asp Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val
100 105 110
Tyr Phe Cys Ala Arg Gly Gly Tyr Asp Gly Arg Gly Phe Asp Tyr Trp
115 120 125
Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Val Gly Gly Ser Gly
130 135 140
Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Glu Leu Thr Gln Ser
145 150 155 160
Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys
165 170 175
Ser Ala Ser Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Ser
180 185 190
Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser
195 200 205
Gly Val Pro Gly Arg Phe Ser Gly Ser Gly Ser Gly Asn Ser Tyr Ser
210 215 220
Leu Thr Ile Ser Ser Val Glu Ala Glu Asp Asp Ala Thr Tyr Tyr Cys
225 230 235 240
Gln Gln Trp Ser Gly Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu
245 250 255
Glu Ile Lys Ala Leu Ala Ala His Arg Val Cys Gly Val Pro Leu Glu
260 265 270
Thr Leu Ala Arg Ser Arg Lys Pro Arg Asp Leu Pro Asp Asp Leu Ser
275 280 285
Cys Ala Tyr Gln Ala Gln Asn Ile Val Ser Leu Phe Val Ala Thr Arg
290 295 300
Ile Leu Phe Ser His Leu Asp Ser Val Phe Thr Leu Asn Leu Asp Glu
305 310 315 320
Gln Glu Pro Glu Val Ala Glu Arg Leu Ser Ala Leu Arg Gln Ile Asn
325 330 335
Glu Asn Asn Pro Gly Met Val Thr Gln Val Leu Thr Val Ala Arg Gln
340 345 350
Ile Tyr Asn Asp Tyr Val Thr His His Pro Gly Leu Ile Pro Glu Gln
355 360 365
Thr Ser Ala Gly Ala Gln Ala Ala Asp Ile Leu Ser Leu Phe Cys Pro
370 375 380
Asp Ala Asp Lys Pro Cys Val Ala Ser Asn Asn Asp Gln Ala Asn Ile
385 390 395 400
Asn Ile Glu Ser Arg Ser Gly Arg Ser Tyr Leu Pro Glu Asn Arg Ala
405 410 415
Val Ile Thr Pro Gln Gly Val Thr Asn Trp Thr Tyr Gln Glu Leu Glu
420 425 430
Ala Thr His Gln Ala Leu Thr Arg Glu Gly Tyr Val Phe Val Gly Tyr
435 440 445
His Gly Thr Asn His Val Ala Ala Gln Thr Ile Val Asn Arg Ile Ala
450 455 460
Pro Val Pro Arg Gly Asn Asn Thr Glu Asn Glu Glu Lys Trp Gly Gly
465 470 475 480
Leu Tyr Val Ala Thr His Ala Glu Val Ala His Gly Tyr Ala Arg Ile
485 490 495
Lys Glu Gly Thr Gly Glu Tyr Gly Leu Pro Thr Arg Ala Glu Arg Glu
500 505 510
Ala Arg Gly Val Met Leu Arg Val Tyr Ile Pro Arg Ala Ser Leu Glu
515 520 525
Arg Phe Tyr Arg Thr Asn Thr Pro Leu Glu Asn Ala Glu Arg His Ile
530 535 540
Thr Gln Val Ile Gly His Ser Leu Pro Leu Arg Asn Glu Ala Phe Thr
545 550 555 560
Gly Pro Glu Ser Ala Gly Gly Glu Asp Glu Thr Val Ile Gly Trp Asp
565 570 575
Met Ala Ile His Ala Val Ala Ile Pro Ser Thr Ile Pro Gly Asn Ala
580 585 590
Tyr Glu Glu Leu Ala Ile Asp Glu Glu Ala Val Ala Lys Glu Gln Ser
595 600 605
Ile Ser Ala Lys Pro Pro Tyr Lys Glu Gln Lys Asp Glu Leu Lys
610 615 620
<210> 40
<211> 526
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 40
Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly
1 5 10 15
Val His Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Glu Lys
20 25 30
Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe
35 40 45
Thr Gly Tyr Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu
50 55 60
Glu Trp Ile Gly Leu Ile Thr Pro Tyr Asn Gly Ala Ser Ser Tyr Asn
65 70 75 80
Gln Lys Phe Arg Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser
85 90 95
Thr Ala Tyr Met Asp Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val
100 105 110
Tyr Phe Cys Ala Arg Gly Gly Tyr Asp Gly Arg Gly Phe Asp Tyr Trp
115 120 125
Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Val Gly Gly Ser Gly
130 135 140
Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Glu Leu Thr Gln Ser
145 150 155 160
Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys
165 170 175
Ser Ala Ser Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Ser
180 185 190
Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser
195 200 205
Gly Val Pro Gly Arg Phe Ser Gly Ser Gly Ser Gly Asn Ser Tyr Ser
210 215 220
Leu Thr Ile Ser Ser Val Glu Ala Glu Asp Asp Ala Thr Tyr Tyr Cys
225 230 235 240
Gln Gln Trp Ser Gly Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu
245 250 255
Glu Ile Lys Ile Phe Pro Lys Gln Tyr Pro Ile Ile Asn Phe Thr Thr
260 265 270
Ala Gly Ala Thr Val Gln Ser Tyr Thr Asn Phe Ile Arg Ala Val Arg
275 280 285
Gly Arg Leu Thr Thr Gly Ala Asp Val Arg His Glu Ile Pro Val Leu
290 295 300
Pro Asn Arg Val Gly Leu Pro Ile Asn Gln Arg Phe Ile Leu Val Glu
305 310 315 320
Leu Ser Asn His Ala Glu Leu Ser Val Thr Leu Ala Leu Asp Val Thr
325 330 335
Asn Ala Tyr Val Val Gly Tyr Arg Ala Gly Asn Ser Ala Tyr Phe Phe
340 345 350
His Pro Asp Asn Gln Glu Asp Ala Glu Ala Ile Thr His Leu Phe Thr
355 360 365
Asp Val Gln Asn Arg Tyr Thr Phe Ala Phe Gly Gly Asn Tyr Asp Arg
370 375 380
Leu Glu Gln Leu Ala Gly Asn Leu Arg Glu Asn Ile Glu Leu Gly Asn
385 390 395 400
Gly Pro Leu Glu Glu Ala Ile Ser Ala Leu Tyr Tyr Tyr Ser Thr Gly
405 410 415
Gly Thr Gln Leu Pro Thr Leu Ala Arg Ser Phe Ile Ile Cys Ile Gln
420 425 430
Met Ile Ser Glu Ala Ala Arg Phe Gln Tyr Ile Glu Gly Glu Met Arg
435 440 445
Thr Arg Ile Arg Tyr Asn Arg Arg Ser Ala Pro Asp Pro Ser Val Ile
450 455 460
Thr Leu Glu Asn Ser Trp Gly Arg Leu Ser Thr Ala Ile Gln Glu Ser
465 470 475 480
Asn Gln Gly Ala Phe Ala Ser Pro Ile Gln Leu Gln Arg Arg Asn Gly
485 490 495
Ser Lys Phe Ser Val Tyr Asp Val Ser Ile Leu Ile Pro Ile Ile Ala
500 505 510
Leu Met Val Tyr Arg Cys Ala Pro Pro Pro Ser Ser Gln Phe
515 520 525
<210> 41
<211> 383
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 41
Met Glu Trp Ser Trp Val Phe Leu Phe Phe Leu Ser Val Thr Thr Gly
1 5 10 15
Val His Ser Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Glu Lys
20 25 30
Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe
35 40 45
Thr Gly Tyr Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Ser Leu
50 55 60
Glu Trp Ile Gly Leu Ile Thr Pro Tyr Asn Gly Ala Ser Ser Tyr Asn
65 70 75 80
Gln Lys Phe Arg Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser
85 90 95
Thr Ala Tyr Met Asp Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val
100 105 110
Tyr Phe Cys Ala Arg Gly Gly Tyr Asp Gly Arg Gly Phe Asp Tyr Trp
115 120 125
Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Val Gly Gly Ser Gly
130 135 140
Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Glu Leu Thr Gln Ser
145 150 155 160
Pro Ala Ile Met Ser Ala Ser Pro Gly Glu Lys Val Thr Met Thr Cys
165 170 175
Ser Ala Ser Ser Ser Val Ser Tyr Met His Trp Tyr Gln Gln Lys Ser
180 185 190
Gly Thr Ser Pro Lys Arg Trp Ile Tyr Asp Thr Ser Lys Leu Ala Ser
195 200 205
Gly Val Pro Gly Arg Phe Ser Gly Ser Gly Ser Gly Asn Ser Tyr Ser
210 215 220
Leu Thr Ile Ser Ser Val Glu Ala Glu Asp Asp Ala Thr Tyr Tyr Cys
225 230 235 240
Gln Gln Trp Ser Gly Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu
245 250 255
Glu Ile Lys Lys Glu Ser Arg Ala Lys Lys Phe Gln Arg Gln His Met
260 265 270
Asp Ser Asp Ser Ser Pro Ser Ser Ser Ser Thr Tyr Cys Asn Gln Met
275 280 285
Met Arg Arg Arg Asn Met Thr Gln Gly Arg Cys Lys Pro Val Asn Thr
290 295 300
Phe Val His Glu Pro Leu Val Asp Val Gln Asn Val Cys Phe Gln Glu
305 310 315 320
Lys Val Thr Cys Lys Asn Gly Gln Gly Asn Cys Tyr Lys Ser Asn Ser
325 330 335
Ser Met His Ile Thr Asp Cys Arg Leu Thr Asn Gly Ser Arg Tyr Pro
340 345 350
Asn Cys Ala Tyr Arg Thr Ser Pro Lys Glu Arg His Ile Ile Val Ala
355 360 365
Cys Glu Gly Ser Pro Tyr Val Pro Val His Phe Asp Ala Ser Val
370 375 380
<210> 42
<211> 850
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 42
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
20 25 30
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
35 40 45
Thr Phe Asn Lys Asn Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys
50 55 60
Gly Leu Glu Trp Val Gly Arg Ile Arg Asn Lys Thr Asn Asn Tyr Ala
65 70 75 80
Thr Tyr Tyr Ala Asp Ser Val Lys Ala Arg Phe Thr Ile Ser Arg Asp
85 90 95
Asp Ser Lys Asn Ser Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu
100 105 110
Asp Thr Ala Val Tyr Tyr Cys Val Ala Gly Asn Ser Phe Ala Tyr Trp
115 120 125
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly
130 135 140
Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser
145 150 155 160
Pro Asp Ser Leu Ala Val Ser Leu Gly Glu Arg Ala Thr Ile Asn Cys
165 170 175
Lys Ser Ser Gln Ser Leu Leu Tyr Ser Ser Asn Gln Lys Asn Tyr Leu
180 185 190
Ala Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr
195 200 205
Trp Ala Ser Ser Arg Glu Ser Gly Val Pro Asp Arg Phe Ser Gly Ser
210 215 220
Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu
225 230 235 240
Asp Val Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Asn Tyr Pro Leu Thr
245 250 255
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Ser Gly Ile Leu Asp Tyr
260 265 270
Ser Phe Thr Gly Gly Ala Gly Arg Asp Ile Pro Pro Pro Gln Ile Glu
275 280 285
Glu Ala Cys Glu Leu Pro Glu Cys Gln Val Asp Ala Gly Asn Lys Val
290 295 300
Cys Asn Leu Gln Cys Asn Asn His Ala Cys Gly Trp Asp Gly Gly Asp
305 310 315 320
Cys Ser Leu Asn Phe Asn Asp Pro Trp Lys Asn Cys Thr Gln Ser Leu
325 330 335
Gln Cys Trp Lys Tyr Phe Ser Asp Gly His Cys Asp Ser Gln Cys Asn
340 345 350
Ser Ala Gly Cys Leu Phe Asp Gly Phe Asp Cys Gln Leu Thr Glu Gly
355 360 365
Gln Cys Asn Pro Leu Tyr Asp Gln Tyr Cys Lys Asp His Phe Ser Asp
370 375 380
Gly His Cys Asp Gln Gly Cys Asn Ser Ala Glu Cys Glu Trp Asp Gly
385 390 395 400
Leu Asp Cys Ala Glu His Val Pro Glu Arg Leu Ala Ala Gly Thr Leu
405 410 415
Val Leu Val Val Leu Leu Pro Pro Asp Gln Leu Arg Asn Asn Ser Phe
420 425 430
His Phe Leu Arg Glu Leu Ser His Val Leu His Thr Asn Val Val Phe
435 440 445
Lys Arg Asp Ala Gln Gly Gln Gln Met Ile Phe Pro Tyr Tyr Gly His
450 455 460
Glu Glu Glu Leu Arg Lys His Pro Ile Lys Arg Ser Thr Val Gly Trp
465 470 475 480
Ala Thr Ser Ser Leu Leu Pro Gly Thr Ser Gly Gly Arg Gln Arg Arg
485 490 495
Glu Leu Asp Pro Met Asp Ile Arg Gly Ser Ile Val Tyr Leu Glu Ile
500 505 510
Asp Asn Arg Gln Cys Val Gln Ser Ser Ser Gln Cys Phe Gln Ser Ala
515 520 525
Thr Asp Val Ala Ala Phe Leu Gly Ala Leu Ala Ser Leu Gly Ser Leu
530 535 540
Asn Ile Pro Tyr Lys Ile Glu Ala Val Lys Ser Glu Pro Val Glu Pro
545 550 555 560
Pro Leu Pro Ser Gln Leu His Leu Met Tyr Val Ala Ala Ala Ala Phe
565 570 575
Val Leu Leu Phe Phe Val Gly Cys Gly Val Leu Leu Ser Arg Lys Arg
580 585 590
Arg Arg Met Ala Pro Lys Lys Lys Arg Lys Val Ser Arg Leu Asp Lys
595 600 605
Ser Lys Val Ile Asn Ser Ala Leu Glu Leu Leu Asn Glu Val Gly Ile
610 615 620
Glu Gly Leu Thr Thr Arg Lys Leu Ala Gln Lys Leu Gly Val Glu Gln
625 630 635 640
Pro Thr Leu Tyr Trp His Val Lys Asn Lys Arg Ala Leu Leu Asp Ala
645 650 655
Leu Ala Ile Glu Met Leu Asp Arg His His Thr His Phe Cys Pro Leu
660 665 670
Glu Gly Glu Ser Trp Gln Asp Phe Leu Arg Asn Asn Ala Lys Ser Phe
675 680 685
Arg Cys Ala Leu Leu Ser His Arg Asp Gly Ala Lys Val His Leu Gly
690 695 700
Thr Arg Pro Thr Glu Lys Gln Tyr Glu Thr Leu Glu Asn Gln Leu Ala
705 710 715 720
Phe Leu Cys Gln Gln Gly Phe Ser Leu Glu Asn Ala Leu Tyr Ala Leu
725 730 735
Ser Ala Val Gly His Phe Thr Leu Gly Cys Val Leu Glu Asp Gln Glu
740 745 750
His Gln Val Ala Lys Glu Glu Arg Glu Thr Pro Thr Thr Asp Ser Met
755 760 765
Pro Pro Leu Leu Arg Gln Ala Ile Glu Leu Phe Asp His Gln Gly Ala
770 775 780
Glu Pro Ala Phe Leu Phe Gly Leu Glu Leu Ile Ile Cys Gly Leu Glu
785 790 795 800
Lys Gln Leu Lys Cys Glu Ser Gly Gly Pro Ala Asp Ala Leu Asp Asp
805 810 815
Phe Asp Leu Asp Met Leu Pro Ala Asp Ala Leu Asp Asp Phe Asp Leu
820 825 830
Asp Met Leu Pro Ala Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu
835 840 845
Pro Gly
850
<210> 43
<211> 852
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 43
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro Gln Ser Leu Glu Glu Ser Gly Gly Asp Leu Val
20 25 30
Lys Pro Gly Ala Ser Leu Thr Leu Thr Cys Thr Ala Ser Gly Phe Ser
35 40 45
Phe Ser Gly Asp Tyr Tyr Met Cys Trp Val Arg Gln Ala Pro Gly Lys
50 55 60
Gly Leu Glu Trp Ile Ala Cys Ile Gly Gly Gly Ser Asn Thr Ala Thr
65 70 75 80
Tyr Tyr Ala Thr Trp Ala Lys Gly Arg Phe Thr Ile Ser Lys Thr Ser
85 90 95
Ser Thr Thr Val Thr Leu Gln Met Thr Ser Leu Thr Ala Ala Asp Thr
100 105 110
Ala Thr Tyr Phe Cys Ala Arg Asp Leu Gly Phe Val Asp Tyr Ala Leu
115 120 125
Glu Leu Trp Gly Pro Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly
130 135 140
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Val Met
145 150 155 160
Thr Gln Thr Pro Ala Ser Val Glu Val Ala Val Gly Gly Thr Val Thr
165 170 175
Ile Lys Cys Gln Ala Ser Glu Asn Met Tyr Asn Ser Leu Ala Trp Tyr
180 185 190
Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Arg Ala Ser
195 200 205
Thr Leu Glu Ser Gly Val Pro Ser Arg Phe Lys Gly Ser Gly Ser Gly
210 215 220
Thr Glu Tyr Thr Leu Thr Ile Ser Asp Leu Glu Cys Ala Asp Ala Ala
225 230 235 240
Thr Tyr Tyr Cys Gln Cys Thr Phe Tyr Ser His Asn Asn Asn Tyr Gly
245 250 255
Gly Ala Phe Gly Gly Gly Thr Glu Val Val Val Lys Ser Gly Ile Leu
260 265 270
Asp Tyr Ser Phe Thr Gly Gly Ala Gly Arg Asp Ile Pro Pro Pro Gln
275 280 285
Ile Glu Glu Ala Cys Glu Leu Pro Glu Cys Gln Val Asp Ala Gly Asn
290 295 300
Lys Val Cys Asn Leu Gln Cys Asn Asn His Ala Cys Gly Trp Asp Gly
305 310 315 320
Gly Asp Cys Ser Leu Asn Phe Asn Asp Pro Trp Lys Asn Cys Thr Gln
325 330 335
Ser Leu Gln Cys Trp Lys Tyr Phe Ser Asp Gly His Cys Asp Ser Gln
340 345 350
Cys Asn Ser Ala Gly Cys Leu Phe Asp Gly Phe Asp Cys Gln Leu Thr
355 360 365
Glu Gly Gln Cys Asn Pro Leu Tyr Asp Gln Tyr Cys Lys Asp His Phe
370 375 380
Ser Asp Gly His Cys Asp Gln Gly Cys Asn Ser Ala Glu Cys Glu Trp
385 390 395 400
Asp Gly Leu Asp Cys Ala Glu His Val Pro Glu Arg Leu Ala Ala Gly
405 410 415
Thr Leu Val Leu Val Val Leu Leu Pro Pro Asp Gln Leu Arg Asn Asn
420 425 430
Ser Phe His Phe Leu Arg Glu Leu Ser His Val Leu His Thr Asn Val
435 440 445
Val Phe Lys Arg Asp Ala Gln Gly Gln Gln Met Ile Phe Pro Tyr Tyr
450 455 460
Gly His Glu Glu Glu Leu Arg Lys His Pro Ile Lys Arg Ser Thr Val
465 470 475 480
Gly Trp Ala Thr Ser Ser Leu Leu Pro Gly Thr Ser Gly Gly Arg Gln
485 490 495
Arg Arg Glu Leu Asp Pro Met Asp Ile Arg Gly Ser Ile Val Tyr Leu
500 505 510
Glu Ile Asp Asn Arg Gln Cys Val Gln Ser Ser Ser Gln Cys Phe Gln
515 520 525
Ser Ala Thr Asp Val Ala Ala Phe Leu Gly Ala Leu Ala Ser Leu Gly
530 535 540
Ser Leu Asn Ile Pro Tyr Lys Ile Glu Ala Val Lys Ser Glu Pro Val
545 550 555 560
Glu Pro Pro Leu Pro Ser Gln Leu His Leu Met Tyr Val Ala Ala Ala
565 570 575
Ala Phe Val Leu Leu Phe Phe Val Gly Cys Gly Val Leu Leu Ser Arg
580 585 590
Lys Arg Arg Arg Met Ala Pro Lys Lys Lys Arg Lys Val Ser Arg Leu
595 600 605
Asp Lys Ser Lys Val Ile Asn Ser Ala Leu Glu Leu Leu Asn Glu Val
610 615 620
Gly Ile Glu Gly Leu Thr Thr Arg Lys Leu Ala Gln Lys Leu Gly Val
625 630 635 640
Glu Gln Pro Thr Leu Tyr Trp His Val Lys Asn Lys Arg Ala Leu Leu
645 650 655
Asp Ala Leu Ala Ile Glu Met Leu Asp Arg His His Thr His Phe Cys
660 665 670
Pro Leu Glu Gly Glu Ser Trp Gln Asp Phe Leu Arg Asn Asn Ala Lys
675 680 685
Ser Phe Arg Cys Ala Leu Leu Ser His Arg Asp Gly Ala Lys Val His
690 695 700
Leu Gly Thr Arg Pro Thr Glu Lys Gln Tyr Glu Thr Leu Glu Asn Gln
705 710 715 720
Leu Ala Phe Leu Cys Gln Gln Gly Phe Ser Leu Glu Asn Ala Leu Tyr
725 730 735
Ala Leu Ser Ala Val Gly His Phe Thr Leu Gly Cys Val Leu Glu Asp
740 745 750
Gln Glu His Gln Val Ala Lys Glu Glu Arg Glu Thr Pro Thr Thr Asp
755 760 765
Ser Met Pro Pro Leu Leu Arg Gln Ala Ile Glu Leu Phe Asp His Gln
770 775 780
Gly Ala Glu Pro Ala Phe Leu Phe Gly Leu Glu Leu Ile Ile Cys Gly
785 790 795 800
Leu Glu Lys Gln Leu Lys Cys Glu Ser Gly Gly Pro Ala Asp Ala Leu
805 810 815
Asp Asp Phe Asp Leu Asp Met Leu Pro Ala Asp Ala Leu Asp Asp Phe
820 825 830
Asp Leu Asp Met Leu Pro Ala Asp Ala Leu Asp Asp Phe Asp Leu Asp
835 840 845
Met Leu Pro Gly
850

Claims (17)

1.一种分泌性表达免疫毒素的嵌合抗原受体表达系统，其特征在于包含嵌合抗原受体表达元件和免疫毒素表达元件，所述嵌合抗原受体和免疫毒素分别识别肿瘤细胞表面相同或不同的抗原或抗原表位，嵌合抗原受体和免疫毒素的表达方式为组成性的表达方式，或者为受表达调控元件调控的条件诱导性的表达方式，所述表达调控元件选自synNotch 系统、四环素介导的调控系统、雌激素受体介导的调控系统中的一种或几种。
2.如权利要求 1 所述的嵌合抗原受体表达系统，其特征在于所述嵌合抗原受体包括抗原结合区和功能区，所述的免疫毒素包括抗原识别肽段和杀伤肿瘤细胞的活性肽段，嵌合抗原受体的抗原结合区和免疫毒素的抗原识别肽段选自抗体、抗体的抗原结合片段或其任意组合。
3.如权利要求 2 所述的嵌合抗原受体表达系统，其特征在于所述嵌合抗原受体的抗原结合区和免疫毒素的抗原识别片段选自 Fab、Fab'、(Fab')2、Fv、scFv、VH 中的一种或几种。
4.如权利要求 2 所述的嵌合抗原受体表达系统，其特征在于所述免疫毒素的活性肽段选自假单胞菌外毒素、白喉毒素、霍乱毒素、炭疽毒素、蓖麻毒素、白树毒素、皂草素中的一种或几种。
5.如权利要求 1所述的嵌合抗原受体表达系统，其特征在于所述 synNotch 系统的胞外域与嵌合抗原受体和免疫毒素识别肿瘤细胞表面的抗原表位不同。
6.如权利要求 1 所述的嵌合抗原受体表达系统，其特征在于所述肿瘤细胞表面抗原选自Glypican-3、B7-H3、Mesothelin、EGFRvIII 中的一种或几种。
7.如权利要求 6 所述的嵌合抗原受体表达系统，其特征在于所述 synNotch 系统靶向肿瘤细胞表面抗原后，其胞内转录因子激活嵌合抗原受体和免疫毒素的表达，synNotch的抗原识别片段和嵌合抗原受体以及免疫毒素分别识别并靶向肿瘤细胞不同的表面抗原或抗原表位，或者相同肿瘤抗原的不同抗原表位。
8.如权利要求 7所述的嵌合抗原受体表达系统，其特征在于所述 synNotch 系统的synNotch 蛋白包括胞外抗原识别区和核心控制区，所述 synNotch 蛋白核心控制区的氨基酸序列如 SEQ ID No：29 所示或 SEQ ID No：29 的同效突变体；嵌合抗原受体包括抗原结合区和功能区，所述功能区的氨基酸序列如 SEQ ID No：1、3、5 所示或 SEQ ID No：1、3、5 的同效突变体； 免疫毒素由抗原识别肽段和杀伤肿瘤细胞的活性肽段构成，所述杀伤肿瘤细胞的活性肽段的 氨基酸序列如 SEQ ID No：17、19、21、23、25、27 所示或 SEQ ID No：17、19、21、23、 25、27 的同效突变体，所述的同效突变体是指突变体的序列和所述的序列有 90%或以上的相 似性、但是突变体的功能性质和所述序列相同。
9. 如权利要求8所述的嵌合抗原受体表达系统，其特征在于所述 synNotch 蛋白的胞外抗原识别区、嵌合抗原受体的胞外抗原识别区和免疫毒素抗原识别肽段，选自 SEQ IDNo：7、9、 11、13、15 中的一种或几种。
10.如权利要求1所述的嵌合抗原受体表达系统，其特征在于所述四环素介导的调控系统通过施用四环素打开或关闭嵌合抗原受体和免疫毒素的表达。
11.如权利要求1所述的嵌合抗原受体表达系统，其特征在于所述雌激素受体介导的调控系统通过施用雌激素或其类似物打开或关闭嵌合抗原受体和免疫毒素的表达。
12.如权利要求 1-11 任一项所述的嵌合抗原受体表达系统，其特征在于所述系统为能够表达权利要求 1 所述嵌合抗原受体和免疫毒素的重组质粒、表达框、病毒载体或细胞。
13.如权利要求 12 所述的的嵌合抗原受体表达系统，其特征在于所述细胞选自自体的或异体的的 T 细胞、NK 细胞、细胞毒性 T 淋巴细胞或调节 T 细胞、记忆性 T 细胞、双特异性T 细 胞、CIK 细胞、NKT 细胞、γδ-T 细胞。
14.如权利要求 1-13 任一所述的嵌合抗原受体和免疫毒素表达系统在制备抗肿瘤药物中的应用。
15.如权利要求 14 所述的应用，其特征在于以所述的嵌合抗原受体和免疫毒素表达系统作为药物活性成分。
16.如权利要求 14 所述的应用，其特征在于所述肿瘤选自肝癌、肺癌、结直肠癌、胃癌、卵巢癌、胰腺癌、前列腺癌、肾细胞癌、霍奇金淋巴瘤、非霍奇金淋巴瘤、白血病、多发性骨髓瘤、腺癌、乳腺癌、头颈部癌、胶质母细胞瘤、神经母细胞瘤、肉瘤、多形性胶质母细胞瘤。
17.一种对免疫细胞进行遗传修饰和基因编辑的方法，其特征在于利用权利要求 1-13任一所述的嵌合抗原受体和免疫毒素表达系统使免疫细胞共表达嵌合抗原受体和免疫毒素。
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