CN109576292B - 一种抗体文库的构建方法及其应用 - Google Patents

一种抗体文库的构建方法及其应用 Download PDF

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CN109576292B
CN109576292B CN201811571758.5A CN201811571758A CN109576292B CN 109576292 B CN109576292 B CN 109576292B CN 201811571758 A CN201811571758 A CN 201811571758A CN 109576292 B CN109576292 B CN 109576292B
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姚永超
李友佳
殷莎
刘广杰
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Shenzhen Aisidi Biological Technology Co ltd
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Abstract

本发明提供了一种抗体文库的构建方法及其应用，所述方法基于synNotch系统控制细胞内基因表达的原理设计，通过大量实验摸索，优化整体方案流程，反复设计验证，将synNotch系统的胞外识别结构域改变为抗体文库，受调控的目标基因改变为筛选标记基因，从而得到了简洁高效，优势诸多的抗体筛选方法，能够筛选针对复杂抗原的多克隆抗体技术，解决肿瘤抗原复杂、多样、易变以及可用靶点有限的问题，具有广阔的应用前景和巨大的市场价值。

Description

一种抗体文库的构建方法及其应用
技术领域
本发明属于生物技术领域，涉及一种抗体文库的构建方法及其应用。
背景技术
抗体是由B细胞表达、结合特定抗原的免疫球蛋白。大多数动物中，抗体由成对的重链和轻链构成。各链由可变区(Fv)和恒定区(Fc)两种不同的区域组成。其中，重链和轻链的Fv区负责与靶抗原的结合，称为抗原结合决定簇。由抗体重链可变区和轻链可变区通过15-20个氨基酸的短肽(linker)连接而成的抗体称为单链抗体(scFv)。抗体药物在肿瘤，自身免疫病两大领域占主导地位。特别是在肿瘤治疗领域，单克隆抗体以及基于单克隆抗体的抗体药物偶联物、双特异性抗体、嵌合抗原受体T(CAR-T)细胞等免疫治疗成为目前最热门的肿瘤治疗手段。当前，抗体或抗体片段的各种筛选技术主要是基于B细胞单克隆技术、蛋白展示技术发展而来。
杂交瘤技术是最早用于建立小鼠B细胞单克隆化的技术。将预定抗原免疫后的动物脾细胞与体外培养、可无限生长的骨髓瘤细胞融合，得到B杂交瘤细胞。这种杂交瘤细胞既能像骨髓瘤细胞一样在体外培养无限增殖，又能像B淋巴细胞那样合成和分泌特异性抗体。通过单克隆化得到单个杂交瘤细胞的细胞系，它所产生的抗体只针对同一抗原表位，这种抗体即所谓单克隆抗体。但由于鼠源抗体对于人来说属于“异种”蛋白，所以当这些单克隆抗体进入人体后，会诱使人体产生针对这些抗体的抗体(即人抗鼠抗体)。人抗鼠抗体会中和鼠源抗体，使得鼠源抗体药物失效。将鼠源抗体的Fc段替换为人源抗体的Fc段得到人鼠嵌合抗体，进一步将嵌合抗体Fv区的FR片段人源化得到人源化抗体，从而降低鼠源单克隆抗的免疫原性。而全人源的单抗更是完全用人类的遗传信息来编码抗体，避免了抗异种蛋白反应。将人B细胞与表达mIL-6和hTERT的鼠骨髓瘤细胞融合而成的人鼠杂交瘤细胞以及用EBV转化人B细胞使其永生化，均是开发全人源单抗的有效手段。
B细胞的单克隆化技术耗时耗力，效率低，无法进行抗体的高通量筛选。蛋白展示技术克服了杂交瘤技术无法进行高通量筛选的缺点，可以从一个巨大的文库中(超过1010个独立克隆)筛选出目的克隆。常用的蛋白展示技术包括噬菌体展示技术、细菌展示技术、核糖体展示技术、酵母展示技术以及哺乳动物细胞展示技术，己经被广泛的应用于新抗体的筛选和抗体亲和力的改进。
噬菌体展示技术由于其相对简单、稳健、方便构建大的人类抗体库等优点，成为迄今为止应用最广泛的展示技术。噬菌体展示技术是将多肽或蛋白质的编码基因插入噬菌体外壳蛋白结构基因的适当位置，在不影响其他外壳蛋白正常功能的情况下，使外源多肽或蛋白与外壳蛋白融合表达，从而使外源多肽或蛋白与外壳蛋白展示在噬菌体表面。展示到噬菌体表面的多肽或蛋白保持相对独立的空间结构和生物活性，可以识别和结合靶分子。噬菌体展示的肽库或蛋白库与固定后的靶分子结合，洗去未结合的噬菌体，然后用酸碱或者竞争的分子洗脱下结合的噬菌体，中和后的噬菌体感染大肠杆菌扩增，经过3-5轮的富集，逐步提高可以特异性识别靶分子的噬菌体比例，最终获得识别靶分子的多肽或者蛋白。将抗体可变区的基因插入噬菌体基因组中，表达的抗体展示到噬菌体的表面，构建噬菌体展示抗体库，可以筛选针对各种抗原的抗体。相对于杂交瘤技术，通过噬菌体展示抗体库技术筛选抗体，可以不经过免疫，缩短抗体生产的周期。也可以筛选在体内免疫原性弱，或者有毒性的抗原的抗体，适用范围广。噬菌体展示抗体库技术不受种属的限制，可以构建各种物种的抗体库。从人天然库中筛选到的抗体，可以不经过人源化过程，直接用于抗体药物研究。噬菌体展示技术以及细菌展示技术受限于展示系统的小容量，更适合展示小肽。因而将其用于抗体文库的展示方面，只能展示抗体的片段，无法进行完整抗体的展示。另外，抗体为真核细胞蛋白，噬菌体和细菌不能保证完全有效地表达真核蛋白。
酵母展示技术已成为筛选人抗体文库以及抗体亲和力成熟的最有力工具之一。最广泛用于酵母展示的系统是酿酒酵母Aga1p/2pα-凝集素系统，它依靠二硫键将GPI锚定的Aga1p蛋白与展示的抗体连接。与噬菌体展示技术相比，酵母展示技术有许多优点。包括使用多色流式细胞仪来定量酵母表面的抗体表达强度以及与荧光标记的抗原结合强度。酵母可表达分泌型的抗体，有助于筛选更高表达，更好的折叠，和适当分泌的克隆。酵母展示可以容纳所有形式的抗体和抗体片段，包括域抗体(dAbs)、scFv、Fabs、甚至IgG。但是，由于转化效率和流式分析技术的限制，酵母展示的人抗体库容大小是有限的(107-109个独立克隆)。然而，最近改进的酵母电穿孔转化技术已经将转化效率提高到1.5×108个转化子/μgDNA，这足以构建多达1010个独立克隆的抗体库。
哺乳动物细胞展示技术成为开发人源抗体最重要的技术。哺乳动物细胞中蛋白质的折叠、分泌和翻译后修饰与人体最为接近。哺乳动物细胞展示系统是表达和分泌人类抗体最自然的系统，有利于抗体的自然折叠、稳定、减少聚集。哺乳动物细胞展示技术与酵母展示技术有相似的优点，可以通过流式细胞仪技术分析哺乳动物细胞表面的抗体情况，并分选出最佳抗体表达信号的细胞。哺乳动物细胞展示技术可以展示全长人源抗体，用于构建包含Fc段完整抗体的抗体库。然而，哺乳动物细胞展示技术一直存在转染效率低、文库容量难以提高的问题。由于这些限制，哺乳动物细胞展示技术最初用途是用于相对较小文库的抗体优化。而最近几年中，随着技术的进步，已经建立了大量基于哺乳动物细胞的文库，用于抗体的筛选。
除了这些主流的抗体筛选技术外，还有一些有鲜明特点的抗体筛选技术，比如：基于生长信号体的抗体筛选技术(Growth Signalobody)、基于嵌合抗原受体的抗体筛选技术(CARbodies)、深度测序抗体发现技术，都展示着巨大的潜力。
得益于多种抗体筛选技术的进步以及人们对疾病的进一步认识，众多抗原靶点的发现，使得抗体药物的开发飞速发展。抗体以及基于抗体的药物对肿瘤的治疗效果与所选择的抗原靶点息息相关，目前FDA批准的抗体药物靶点数量有限，针对这些靶点的抗体均为单克隆抗体或多个单克隆抗体混合而成多样性有限的多克隆抗体。
抗体药物在肿瘤治疗上取得了很好的疗效，但目前还没有任何单克隆抗体能完全解决肿瘤治疗的问题，主要原因在于：(1)目前探明的肿瘤靶点中，没有哪一个靶点可完全覆盖某种肿瘤。肿瘤细胞中可能高表达某个肿瘤抗原，但不等于此肿瘤抗原能在所有肿瘤细胞中都高表达，部分不表达或低表达此肿瘤抗原的肿瘤细胞会对抗体药物产生抗药性，从而导致在肿瘤的治疗过程中，肿瘤的迅速反弹。(2)很少有靶点只表达在肿瘤细胞中，而在正常细胞中不表达。几乎所有的肿瘤抗原，在人的某些正常组织有也有表达，导致抗体药物对正常组织的损伤，具有很大的药副作用。(3)肿瘤细胞的抗原具有极高的突变负荷，即抗原具有多样性且突变率高。肿瘤细胞具有大量各不相同的肿瘤新抗原，这些新抗原即使在同一种肿瘤里，也是不相同的，即便有确定的肿瘤抗原，也可能发生突变，导致抗体药物无效。(4)已探明的靶点有限，针对许多肿瘤，无有效的靶点或靶点组合可用，大量未知的肿瘤靶点并未探明。现有的抗体筛选技术，基本都用于筛选单克隆抗体。单克隆抗体只识别单一肿瘤抗原靶点的一个抗原表位，即便是由多个单克隆抗体混合而成的多克隆抗体，其识别的抗原表位也有限，远远不能覆盖肿瘤细胞复杂的抗原表位。很多抗体筛选技术也可用于筛选多克隆抗体，但其筛选操作便利性、筛选出抗体的成功率、得到的抗体特异性等方面都面临不小的挑战。
因此，开发一种抗体文库的构建方法，用于筛选针对复杂抗原的多克隆抗体技术，从而解决肿瘤抗原复杂、多样、易变以及可用靶点有限的问题，具有广阔的应用前景和巨大的市场价值。
发明内容
针对现有技术的不足及实际的需求，本发明提供一种抗体文库的构建方法及应用，所述方法基于synNotch系统控制细胞内基因表达的原理设计，将synNotch系统的胞外识别结构域改变为胞外抗体文库编码域，受调控的目标基因改变为筛选标记基因，得到受抗原激活的抗体筛选系统，得到了筛选针对复杂抗原的多克隆抗体技术，从而解决肿瘤抗原复杂、多样、易变以及可用靶点有限的问题，具有广阔的应用前景和巨大的市场价值。
为达此目的，本发明采用以下技术方案：
第一方面，本发明提供一种抗体文库的构建方法，所述方法以synNotch系统控制细胞内基因表达为原理，将元件一和元件二插入同一载体或不同载体，转染至细胞内，得到抗体表达细胞文库，即所述抗体文库；
其中，所述元件一包括顺式激活子和筛选标记基因，所述元件二包括胞外抗体文库编码域、Notch核心结构域和胞内转录结构域。
具体地，包括如下步骤：
(1)向细胞中转染带有顺式激活子和筛选标记基因的载体；
(2)向步骤(1)的细胞内转染携带从N端到C端依次为胞外抗体文库编码域、Notch核心结构域、胞内转录结构域的载体，得到抗体表达细胞文库，即所述抗体文库。
步骤(1)和(2)的两个载体可以合在一个载体上，用这个载体来构建抗体文库也是可行的，而分成两个载体进行两步骤转染能够提高转染效率，更容易构建大容量的抗体文库，所述载体包括表达载体。
本发明是基于synNotch系统控制细胞内基因表达的原理设计，synNotch系统包含了天然的细胞间信号传导受体Notch的核心调节结构域，同时含有合成性的胞外识别结构域和合成性的胞内转录结构域；合成性的胞外识别结构域为一单链抗体，当单链抗体识别并结合抗原时，synNotch系统发生诱导性跨膜区域剪切，从而释放胞内转录结构域进入细胞核，结合上游顺式激活子来激活受调控目标基因的表达；因此，synNotch系统改造的细胞，可以通过特定的抗原识别结合来驱动某一特定的基因表达，用来改造T细胞，使得T细胞可以受抗原调控表达细胞因子而杀伤靶细胞，或者T细胞可以受抗原调控表达CAR，从而提高CAR-T细胞对靶细胞的识别精度。
本发明中，发明人为解决现有技术筛选抗体的缺点，提供一种简洁高效的抗体文库构建方法，以synNotch系统控制细胞内基因表达为基本原理，通过大量实验摸索，优化整体方案流程，反复设计验证，将synNotch系统的胞外识别结构域改变为胞外抗体文库编码域，受调控的目标基因改变为筛选标记基因，从而得到受抗原激活的抗体筛选系统；抗体文库的构建方法为先向细胞中转染带有顺式激活子加筛选标记基因的载体，再向这个细胞中转染携带从N端到C端分别为胞外抗体文库编码域、Notch核心结构域、胞内转录结构域的载体，从而得到抗体表达细胞文库。
优选地，所述筛选标记基因包括药物抗性基因、自杀基因、荧光蛋白基因或分子标签中的任意一种或至少两种的组合。
若要进行抗体的正筛选，将筛选标记基因设计为药物抗性基因，则激活的细胞可以在有筛选药物的培养基中存活，其余的细胞死亡，从而筛选出针对目标抗原的抗体表达细胞；若要进行抗体的负筛选，将筛选标记基因设计为自杀基因，则激活的细胞在有筛选药物的培养基中凋亡，其余的细胞存活，从而除去针对目标抗原的抗体表达细胞。
本发明可通过药物进行正筛选的筛选标记基因可以是但不限于嘌呤霉素抗性基因、新霉素抗性基因、杀稻瘟素抗性基因、潮霉素B抗性基因等，分别用药物：用药物嘌呤霉素、G418、杀稻瘟素、潮霉素B进行筛选；可通过药物进行负筛选的筛选标记基因可以是但不限于单纯疱疹病毒胸苷激酶(HSV-TK)基因、胞嘧啶脱氨酶(CD)基因、iCasp9自杀系统基因，分别用药物：更昔洛韦或FIAU、5-氟胞嘧啶、AP1903或AP20187进行筛选。
优选地，所述药物抗性基因包括嘌呤霉素抗性基因、新霉素抗性基因、杀稻瘟素抗性基因或潮霉素B抗性基因中的任意一种或至少两种的组合。
优选地，所述自杀基因包括单纯疱疹病毒胸苷激酶(HSV-TK)基因、胞嘧啶脱氨酶(CD)基因或iCasp9自杀系统基因中的任意一种或至少两种的组合。
优选地，所述荧光蛋白基因包括EGFP、YFP、mCherry、DsRed或BFP中的任意一种或至少两种的组合。
优选地，所述分子标签包括His-tag、Flag-tag、HA-tag、Myc-tag或Strep-tag中的任意一种或至少两种的组合。
本发明的筛选标记基因可以但不限于荧光蛋白基因：EGFP、YFP、mCherry、DsRed、BFP，分子标签，即蛋白/多肽标签包括但不限于：His-tag、Flag-tag、HA-tag、Myc-tag、Strep-tag，通过流式细胞仪检测荧光或抗标签抗体，即可进行正筛选，也可进行负筛选。
所述筛选标记基因的筛选方法包括药物筛选、流式细胞仪检测并分选或磁珠分选中的任意一种或至少两种的组合。
优选地，所述药物抗性基因的筛选药物包括嘌呤霉素、G418、杀稻瘟素或潮霉素B中的任意一种。
优选地，所述自杀基因的筛选药物包括更昔洛韦或FIAU、5-氟胞嘧啶、AP1903或AP20187中的任意一种或至少两种的组合。
优选地，所述荧光蛋白基因的检测和分选方法包括流式细胞仪检测和分选。
优选地，所述分子标签的检测和分选方法包括流式细胞仪检测和分选。
优选地，所述标记基因的分选包括磁珠分选。
优选地，所述胞外抗体文库编码域包括(完整抗体、组成抗体的链或抗体片段)抗体序列、抗体重链序列、抗体轻链序列、抗体可变区序列、单链抗体序列、单域抗体序列或Fab片段序列中的任意一种或至少两种的组合。
本发明中胞外抗体文库编码域部分包括但不限于完整的抗体、组成抗体的链(重链或轻链)、抗体的片段(抗体可变区、单链抗体、单域抗体、Fab段)所构成的文库。胞外抗体文库编码域的来源，可以但不限于为免疫动物制备、来自疾病人群、来自健康人群、接种过疫苗的人群、人工合成。
优选地，所述胞外抗体文库编码域的来源包括免疫动物、疾病人群、健康人群、接种过疫苗的人群或人工合成中的任意一种或至少两种的组合。
优选地，所述Notch核心结构域包括人的Notch、小鼠的Notch、以及与人的Notch或小鼠的Notch相似性不低于85％的序列。
优选地，所述Notch核心结构域包括人的P1391-R1763片段、小鼠的P1390-R1752片段，这些片段的前或后、加或减200个氨基酸以内的片段，以及与这些片段相似性不低于85％的序列。
优选地，所述人的Notch的氨基酸序列如SEQ ID NO.1所示。
优选地，所述小鼠的Notch的氨基酸序列如SEQ ID NO.2所示。
本发明的Notch核心结构域可以来自不限于人的Notch、小鼠的Notch，以及与它们相似性不低于85％的序列；Notch核心结构域可为P1391-R1763片段(人)、P1390-R1752片段(小鼠)，这些片段的前或后、加或减200个氨基酸以内的片段，以及与这些片段相似性不低于85％的序列。
优选地，所述转录结构域包括但不局限于tTA和/或Gal4-VP64。
优选地，所述顺式激活子包括pTet和/或UAS-pSV40。
优选地，所述转录结构域包括tTA和/或Gal4-VP64。
优选地，所述转染的方法包括病毒转染、化学转染试剂转染或电击转染中的任意一种或至少两种的组合。
优选地，所述编码域、结构域或基因包含编码蛋白质的氨基酸序列、编码蛋白的DNA序列或编码蛋白的RNA序列中的任意一种或至少两种的组合。
第二方面，本发明提供一种抗体文库，所述文库由第一方面所述方法构建得到。
第三方面，本发明提供一种筛选抗体的方法，采用第二方面所述抗体文库进行筛选，包括如下步骤：
(1)将抗体文库与抗原接触；
(2)根据筛选标记基因的表达情况，筛选表达目标抗体的细胞；
其中，所述抗体包括单克隆抗体或多克隆抗体；
所述抗原包括野生型细胞、转染特定抗原基因的细胞、结合特定抗原的细胞、溶解在培养基中的抗原、包被在培养器皿上的抗原、包被在微珠上的抗原或包被在培养支架上的抗原中的任意一种或至少两种的组合。
本发明中，筛选抗体的方法是将抗体表达细胞文库与抗原接触，只有能识别抗原的抗体表达细胞，才会激活细胞中筛选标记基因的表达，根据筛选标记基因的表达情况，筛选出表达目标抗体的细胞。
第四方面，本发明提供一种抗体，所述抗体由第三方面所述方法筛选得到。
与现有技术相比，本发明具有如下有益效果：
1、本发明提供的方法既可筛选单克隆抗体，又可筛选多克隆抗体：以往的抗体筛选技术适合于筛选单克隆抗体，即使用于筛选多克隆抗体，所筛选出抗体的多样性、特异性及稳定性也难以保证；本发明的抗体筛选技术，针对抗原筛选出的多克隆抗体具有更大多样性，并且能通过改变筛选条件获得特异性更好、亲和力更高的抗体，通过测序即可获得大量单克隆抗体的编码序列，比以往的单克隆筛选技术更简单；
2，本发明提供的方法不需要表达纯化抗原：以往的抗体筛选技术在进行抗体筛选时，需要表达纯化出抗原蛋白或者抗原表位的肽段，用于免疫动物、铺板进行ELISA筛选抗体、结合抗体进行流式筛选抗体，而这些抗原蛋白或肽段有的不容易纯化、或者和原来抗原蛋白修饰不同、或者不是原来的空间结构，这些都会影响所筛选出抗体的特异性，亲和力；本发明的抗体筛选技术，只需要将目标抗原展示在细胞的表面，即可进行抗体的筛选，要得到展示目标抗原的细胞，只需要将目标抗原的基因转染到细胞中，或者直接用确定表达目标抗原的野生型细胞，十分方便；
3，本发明提供的方法不需要预设抗原：以往的抗体筛选技术需要预先探明抗原，再针对抗原筛选抗体，但是，肿瘤细胞极其复杂，有大量未探明的抗原，而明确的肿瘤抗原靶点十分有限，针对有限抗原靶点筛选出的抗体，无法满足肿瘤治疗的需要；本发明不需要预设抗原，可以以整个肿瘤细胞为抗原，从抗体库中筛选出的多克隆抗体，可以识别肿瘤细胞表面的各种抗原靶点，从而避免了已知抗原靶点有限的问题；
4、本发明提供的方法可方便进行负筛选：抗体负筛选的目的是去除不需要的抗体，比如去除可能识别正常细胞的抗体，因为肿瘤细胞和正常细胞有大量相同的抗原，因此针对肿瘤细胞筛选出的抗体除了可以识别肿瘤细胞外，还可能识别正常的细胞，从而使得开发的抗体药物具有伤害正常组织的副作用，以往的抗体筛选技术筛选单克隆抗体，可以将筛出的抗体一个一个去验证对正常细胞的作用，去除不需要的抗体，这种方法效率低，不适合多克隆抗体的筛选；本发明的抗体筛选技术，可以通过用负筛选标记基因，用负筛选药物或流式细胞仪阴性分选等方式，很方便进行负筛选；
5、本发明提供的方法筛选方式多样：本发明提供的抗体筛选技术，可以用药物进行正负筛选，也可用流式细胞仪来进行筛选，还可以药物筛选和流式细胞仪筛选配合进行，根据自己的研究条件和研究经验自由选择和搭配，更有利于获得目标抗体。
附图说明
图1为本发明的synNotch系统控制细胞内基因表达的原理图；
图2为本发明的基于synNotch的抗体筛选系统；
图3为本发明的顺式激活子加筛选标记基因的载体示意图；
图4为本发明的pTet顺式激活子调控荧光蛋白融合嘌呤霉素抗性基因的载体示意图；
图5为本发明的tTA表达载体示意图；
图6为本发明的荧光显微镜拍摄tTA激活pTet顺式激活子表达绿色荧光蛋白图；
图7为本发明的荧光显微镜拍摄tTA激活pTet顺式激活子表达红色荧光蛋白图；
图8为本发明的抗体文库的通用表达载体示意图；
图9为本发明的抗CD19单链抗体的表达载体示意图；
图10为本发明的抗GPC3单链抗体的表达载体示意图；
图11为本发明的CD19和GFPC3抗原表达载体示意图；
图12为本发明的流式细胞仪检测CD19和GFPC3抗原在K562细胞中的表达图；
图13为本发明的用抗体筛选系统从混合的抗体表达细胞文库中筛选CD19抗体表达细胞结果图；
图14为本发明的用抗体筛选系统从混合的抗体表达细胞文库中筛选GPC3抗体表达细胞结果图；
图15为本发明的pTet顺式激活子调控绿色荧光蛋白的载体示意图；
图16为本发明的抗Raji单链抗体基因文库表达载体示意图；
图17为本发明的用于负筛选的抗原表达载体示意图；
图18为本发明的流式细胞仪检测CD19抗原表达载体和负筛选抗原表达载体在K562细胞中的表达结果图；
图19为本发明的流式细胞仪检测筛选前抗体表达细胞文库诱导激活情况图；
图20为本发明的流式细胞仪检测筛选后抗体表达细胞文库诱导激活情况图；
图21为本发明的pTet顺式激活子调控荧光蛋白融合正负筛选基因的载体示意图；
图22为本发明的可进行药物初筛选的单链抗体基因文库表达载体示意图；
图23为本发明的用抗体筛选系统进行药物正筛选原理图；
图24为本发明的流式细胞仪检测进行正筛选的抗体表达细胞结果图；
图25为本发明的用抗体筛选系统进行药物负筛选原理图；
图26为本发明的流式细胞仪检测进行负筛选的抗体表达细胞结果图；
图27为本发明的单链抗体与抗体恒定区融合表达载体示意图；
图28为本发明的流式细胞仪通过本发明抗体筛选方法筛选出的抗体与抗原结合效率结果图。
具体实施方式
为更进一步阐述本发明所采取的技术手段及其效果，以下结合附图并通过具体实施方式来进一步说明本发明的技术方案，但本发明并非局限在实施例范围内。
实施例1筛选已知的CD19和GPC3抗体
synNotch系统控制细胞内基因表达的原理图见图1，synNotch系统细胞外的单链抗体识别并结合抗原时，synNotch系统发生诱导性跨膜区域剪切，从而释放胞内转录结构域进入细胞核，结合上游顺式激活子来激活受调控目标基因的表达；
基于synNotch的抗体筛选系统的原理图见图2，在synNotch系统的的基础上，将胞外识别结构域改变为胞外抗体文库编码域，受调控的目标基因改变为筛选标记基因，从而得到受抗原激活的抗体筛选系统；
从已知的CD19和GPC3混合抗体库中分别筛选特异性针对CD19和GPC3的抗体；
(1)构建稳定转染顺式激活子和筛选标记基因的单克隆细胞系
本实施例中，顺式激活子采用pTet(SEQ ID NO.3)，pTet接收到tTA信号后，即可启动筛选基因的表达，筛选标记用荧光蛋白融合嘌呤霉素抗性基因(SEQ ID NO.4)，二者之间用可自动断裂的2A(SEQ ID NO.5)序列连接，顺式激活子加筛选标记基因的载体示意图见图3；
本实施例分别用两种荧光蛋白EGFP(SEQ ID NO.6)和mCherry(SEQ ID NO.7)与嘌呤霉素抗性基因融合构建两种筛选标记，终止子采用SV40多聚A(SEQ ID NO.8)，通过人工合成直接合成整个序列(图4)的编码基因，构建到慢病毒载体中，包装慢病毒，分别转染到293T细胞中，3天后将转染的细胞单克隆化；
要对单克隆的细胞系进行筛选，获得正确的单克隆细胞，需要构建tTA表达载体(图5)，tTA表达载体由ef1-α启动子(SEQ ID NO.9)，tTA基因(SEQ ID NO.10)，终止子构成，将tTA表达载体构建到慢病毒载体中，包装成慢病毒；
将待筛选的单克隆细胞分别取出一部分细胞转染tTA表达慢病毒，2-3天后，荧光显微镜观察，有EGFP(图6，筛选稳定转染pTet顺式激活子调控绿色荧光蛋白融合嘌呤霉素抗性基因的单克隆细胞时，显微镜观察绿色荧光表达情况。)或mCherry(图7，筛选稳定转染pTet顺式激活子调控红色荧光蛋白融合嘌呤霉素抗性基因的单克隆细胞时，显微镜观察红色荧光表达情况。)表达而转染之前无荧光表达的细胞系，均为稳定转染pTet顺式激活子和筛选标记基因的单克隆细胞系；
(2)构建抗体表达细胞文库：首先构建抗体基因文库表达载体中除胞外抗体文库编码域基因外的中间载体，方便后续插入胞外抗体文库编码域基因(图8，抗体文库的通用表达载体示意图)，人工合成中间载体各组成部分的序列，从N端到C端依次串联的部分分别为mPGK1启动子(SEQ ID NO.11)，CD8α信号肽(SEQ ID NO.12)编码序列，抗体基因文库编码域(带有β-半乳糖苷酶(lacZ)N端α片段的编码区序列，前面Asc I限制性内切酶位点，后面为Not I限制性内切酶位点，方便抗体库序列的插入，并能通过蓝白斑显示片段插入情况)，NOTCH1片段(SEQ ID NO.2中含跨膜区的P1390-R1752片段)编码序列，tTA编码序列，终止子，将合成的序列构建到慢病毒载体中，以备进一步插入抗体基因序列；
合成已知的抗人CD19单链抗体(SEQ ID NO.13)和抗人GPC3单链抗体(SEQ IDNO.14)的编码序列，两端带有Asc I和Not I限制性内切酶位点，分别插入到中间载体的AscI和NotI限制性内切酶位点之间；将插入有抗人CD19单链抗体基因的载体(图9)包装成慢病毒，转染到带EGFP筛选标记的单克隆细胞系中，得到CD19单链抗体表达细胞；
将插入有抗人GPC3单链抗体基因的载体(图10)包装成慢病毒，转染到带mCherry筛选标记的单克隆细胞系中，得到GPC3单链抗体表达细胞。
(3)构建CD19和GFPC3抗原表达细胞：合成CD19(SEQ ID NO.15)和GPC3(SEQ IDNO.16)抗原的编码序列，分别构建到表达载体上，表达载体中通过两个2A序列将CD19或GPC3、蓝色荧光蛋白(SEQ ID NO.17)、嘌呤霉素抗性基因三者融合(图11)；
将构建好的表达载体包装成慢病毒，通过病毒转染到K562细胞中，转染2天后，培养基中加入1μg/mL的嘌呤霉素筛选得到稳定表达CD19或GPC3的K562细胞。流式细胞仪检测蓝色荧光蛋白的表达几乎达到100％(图12)，表明抗原在K562细胞中已稳定表达。
(4)从混合的抗体表达细胞文库中筛选CD19抗体表达细胞：将CD19单链抗体表达细胞和GPC3单链抗体表达细胞1:1混合，再将混合细胞与CD19抗原表达细胞1:1混合，混合细胞培养2天后，在培养基中加入1μg/mL的嘌呤霉素进行筛选，经过8-10天的筛选，抗体表达细胞已几乎全为表达CD19抗体带有绿色荧光的细胞(图13)，将CD19单链抗体和绿色荧光共同表达的细胞与GPC3单链抗体和红色荧光共同表达的细胞1:1混合，经过与CD19抗原表达细胞混合并加嘌呤霉素进行筛选，得到几乎全为表达CD19抗体带有绿色荧光的细胞；
(5)从混合的细胞中筛选表达抗GPC3抗体的细胞：将CD19抗体表达细胞和GPC3抗体表达细胞1:1混合，再将混合细胞与GPC3抗原表达细胞1:1混合，混合细胞培养2天后，在培养基中加入1μg/mL的嘌呤霉素进行筛选，经过8-10天的筛选，抗体表达细胞已几乎全为表达GPC3抗体带有红色荧光的细胞(图14)，将CD19单链抗体和绿色荧光共同表达的细胞与GPC3单链抗体和红色荧光共同表达的细胞1:1混合，经过与GPC3抗原表达细胞混合并加嘌呤霉素进行筛选，得到几乎全为表达GPC3抗体带有红色荧光的细胞；
上述实验结果显示，可以通过本抗体筛选方法从已知的抗体库中，筛选出针对目标抗原的单链抗体。
实施例2.制备抗体文库并用流式细胞仪筛选CD19抗体表达细胞
(1)构建稳定转染顺式激活子和筛选标记基因的单克隆细胞系
本实施例采用顺式激活子pTet，pTet接收到tTA信号后，启动筛选标记绿色荧光蛋白基因的表达，通过人工合成整个序列(图15)，构建到慢病毒载体中。包装慢病毒，转染到293T细胞中，3天后将转染的细胞单克隆化，将待筛选的单克隆细胞分别取出一部分细胞转染表达tTA的慢病毒，2-3天后，荧光显微镜观察，挑选有绿色荧光表达而转染之前无荧光表达的细胞系，为稳定转染转染顺式激活子和筛选标记基因的单克隆细胞系。
(2)构建抗体表达细胞文库
用CD19阳性的Raji细胞免疫C57BL/6J小鼠，两周时加强免疫一次，第四周再免疫一次，3天后处死小鼠，分离出小鼠的脾脏淋巴细胞；用RNA提取纯化试剂盒提取脾脏淋巴细胞的RNA；
将提取的RNA用逆转录试剂盒分别通过轻链的反转录引物(SEQ ID NO.18)和重链的反转录引物(SEQ ID NO.19)进行反转录，用简并引物分别扩增重链和轻链，再通过重叠PCR的方法将轻链和重链连接起来，形成轻链-Linker-重链的scFv文库，将scFv文库的DNA片段通过Asc I和Not I双酶切，插入到抗体基因文库表达载体中间载体的Asc I和Not I限制性内切酶位点之间，得到抗体基因文库表达载体(图16)，Raji细胞免疫小鼠制备的单链抗体克隆到抗体基因文库表达载体上；
将抗体基因文库表达载体包装成慢病毒，转染到步骤(1)已稳定转染顺式激活子和筛选标记基因的单克隆细胞系中，得到抗体表达细胞文库。
(3)构建抗原表达细胞；
表达CD19的抗原表达细胞制备见实施例1，此抗原表达细胞用于抗CD19抗体的正筛选，合成一个穿膜结构域(SEQ ID NO.20)的编码基因，替换正筛选载体中的CD19抗原基因，得到用于负筛选的抗原表达载体(图17)；
将构建好的抗原表达载体分别包装成慢病毒，通过病毒转染到K562细胞中；转染2天后，培养基中加入1μg/mL的嘌呤霉素筛选分别得到正筛选抗原表达细胞系和负筛选抗原表达细胞系。流式细胞仪检测蓝色荧光蛋白的表达几乎达到100％(图18)，表明抗原在K562细胞中已稳定表达。
(4)流式细胞仪筛选表达CD19抗体的细胞；
将抗体表达细胞文库和表达CD19抗原的正筛选细胞1:1混合，混合细胞培养2天后，抗体表达细胞文库有少量表达绿色荧光蛋白的细胞出现(图19)，将抗体表达细胞文库和表达CD19抗原的正筛选细胞混合培养2天后，抗体表达细胞文库有少量表达绿色荧光蛋白的细胞出现，用分选型的流式细胞仪，分选出表达绿色荧光蛋白的细胞，继续培养，直到大部分绿色荧光消失；
再将分选出的抗体表达细胞文库和负筛选抗原表达细胞1:1混合，3天后，用分选型流式细胞仪分选出绿色荧光蛋白和蓝色荧光蛋白双阴性的细胞，即为表达CD19抗体的细胞，此时的抗体表达细胞与负筛选抗原表达细胞共培养时无绿色荧光蛋白表达，与CD19抗原表达细胞共培养时有绿色荧光蛋白表达(图20)，经过正负筛选后的抗体表达细胞与负筛选抗原表达细胞共培养时无绿色荧光蛋白表达，与CD19抗原表达细胞共培养时有绿色荧光蛋白表达。
实施例3.制备抗体文库并用药物筛选CD19抗体表达细胞
(1)构建稳定转染顺式激活子和筛选标记基因的单克隆细胞系；
本实施例中，顺式激活子pTet，筛选标记用iCasp9负筛选系统(SEQ ID NO.21)、绿色荧光蛋白、嘌呤霉素抗性基因，三者之间用可自动断裂的2A序列连接；
通过人工合成整个编码序列(图21)，构建到慢病毒载体中，包装慢病毒，转染到293T细胞中，3天后将转染的细胞单克隆化；将待筛选的单克隆细胞分别取出一部分细胞转染实施例1中表达tTA的慢病毒，2-3天后，荧光显微镜观察，挑选有绿色荧光表达而转染之前无荧光表达的细胞系，为稳定转染有顺式激活子和筛选标记基因的单克隆细胞系。
(2)构建抗体表达细胞文库；
首先构建抗体基因文库表达载体中除胞外抗体文库编码域基因外的中间载体，方便后续插入胞外抗体文库基因(图22)，人工合成中间载体各组成部分的序列，从N端到C端依次串联的部分分别为mPGK1启动子，CD8α信号肽编码序列，抗体基因文库编码域(带有β-半乳糖苷酶(lacZ)N端α片段的编码区序列，前面Asc I限制性内切酶位点，后面为Not I限制性内切酶位点，方便抗体库序列的插入，并能通过蓝白斑显示片段插入情况)，NOTCH1片段编码序列，tTA编码序列，终止子，EFS启动子(SEQ ID NO.22)，杀稻瘟素抗性(SEQ IDNO.23)基因，终止子；
将合成的序列构建到慢病毒载体中，以备进一步插入抗体基因文库序列。将Raji细胞免疫小鼠的scFv文库DNA片段通过Asc I和Not I双酶切，插入到抗体基因文库表达载体中间载体的Asc I和Not I限制性内切酶位点之间，得到抗体基因文库表达载体,将抗体基因文库表达载体包装成慢病毒，转染到步骤(1)已稳定转染顺式激活子和筛选标记基因的单克隆细胞系中，2天后在培养基中加入10μg/mL的杀稻瘟菌素进行初筛，得到抗体表达细胞文库；
(3)药物筛选表达CD19抗体的细胞；
将抗体表达细胞文库和表达CD19抗原的正筛选细胞1:1混合，混合细胞培养2天后，在培养基中加入1μg/mL的嘌呤霉素进行筛选，经过6-8天的正筛选，(正筛选原理图见图23)，抗体文库表达细胞已几乎全为表达绿色荧光蛋白的细胞(图24)，将抗体文库表达细胞和表达CD19抗原的正筛选细胞混合细胞经过嘌呤霉素进行正筛选，抗体表达细胞已几乎全为表达绿色荧光蛋白的细胞；
再在混合细胞的培养基中加入10μg/mL的杀稻瘟菌素，直到蓝色荧光的正筛选抗原细胞完全消失，继续培养经过正筛选后的抗体文库表达细胞直到绿色荧光蛋白几乎不表达时，将抗体文库表达细胞和负筛选抗原表达细胞1:1混合，并在培养基中加入10nM的AP1903进行负筛选(负筛选原理图见图25)，直到培养的抗体表达细胞中不再表达绿色荧光蛋白(图26)，将抗体文库表达细胞和负筛选细胞混合细胞经过AP1903负筛选，抗体表达细胞已几乎全都为不表达绿色荧光蛋白的细胞；
最后在混合细胞的培养基中加入10μg/mL的杀稻瘟菌素，直到蓝色荧光的负筛选抗原细胞完全消失，此时，抗体文库表达细胞即为表达CD19抗体的细胞。
实施例4.抗体结合性质的鉴定
(1)克隆筛选出的CD19抗体表达细胞的抗体编码序列；
将经过筛选的CD19抗体表达细胞用DNeasy Blood&Tissue Kit基组提取试剂盒(Qiagen)按照试剂盒的操作指南提出细胞的基因组，以提取的基因组为模板，用引物对(SEQ ID NO.24，SEQ ID NO.25)扩增scFv的编码基因，用Asc I和Not I双酶切后，插入到经AscI和Not I双酶切的表达载体上，与人的IgG1恒定区(SEQ ID NO.26)融合(图27)；
将连接好的载体转染到感受态细胞中，涂在含100mg/L氨苄青霉素的LB固体培养基的培养皿上，第二天，从培养皿上挑出20个左右的单克隆分别接种于含100mg/L氨苄青霉素的LB液体培养基中摇瓶培养，培养皿上的剩余菌落用LB培养基洗脱下来，用质粒提取试剂盒按照试剂盒说明书提取多克隆的scFv表达质粒，摇瓶培养的单克隆菌过夜培养后也分别提取单克隆的scFv表达质粒。
(2)表达纯化抗体；
分别将多克隆的scFv表达质粒和单克隆的scFv表达质粒用PEI转染到293T细胞中，转染72小时后，收集培养上清；将培养上清与结合缓冲液1:1混合、过滤后待用，先用5-10倍体积的结合缓冲液平衡Pteoein A柱，再将准备好的培养上清样品上样，然后用结合缓冲液冲洗柱子，直到结合液中不含蛋白，最后用洗脱液过柱，收集洗出液，直至洗出液中不含蛋白，透析并浓缩所收集的抗体。
(3)流式细胞仪检测抗体的结合；
100μL表达CD19和不表达CD19的K562细胞悬液，铺到V型细胞板，500g，离心3min，沉积细胞，弃去上清加入100μL纯化的抗体溶液(定量IgG浓度为0.5μg/mL)，4℃孵育30min，500g，离心3min，用200μL PBS洗三遍；
按照实验设计孔，每孔加入80μl FITC标记的羊抗人二抗(1:150稀释)，4℃孵育30min，500g，离心3min，弃去上清，200μL PBS洗涤3次，最后一次洗完加入200μL PBS悬浮细胞，悬浮细胞用过滤网过滤后上流式细胞仪检测，结果显示，无论多克隆的scFv或单克隆的scFv都能不同程度特异性结合表达CD19抗原的K562细胞(图28)，筛选得到多克隆的scFv或单克隆的scFv都能不同程度特异性结合表达CD19抗原的K562细胞而不结合无CD19抗原表达的K562细胞。
综上所述，本发明提供了一种抗体文库的构建方法及其应用，基于synNotch系统控制细胞内基因表达的原理设计，通过大量实验摸索，优化整体方案流程，反复设计验证，将synNotch系统的胞外识别结构域改变为抗体文库，受调控的目标基因改变为筛选标记基因，从而得到了筛选针对复杂抗原的多克隆抗体技术，解决肿瘤抗原复杂、多样、易变以及可用靶点有限的问题，具有广阔的应用前景和巨大的市场价值。
申请人声明，本发明通过上述实施例来说明本发明的详细方法，但本发明并不局限于上述详细方法，即不意味着本发明必须依赖上述详细方法才能实施。所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明产品各原料的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。
SEQUENCE LISTING
<110> 深圳市爱思迪生物科技有限公司
<120> 一种抗体文库的构建方法及其应用
<130> 2018年
<160> 26
<170> PatentIn version 3.3
<210> 1
<211> 2556
<212> PRT
<213> 人工合成
<220>
<221> misc_feature
<222> (891)..(891)
<223> Xaa can be any naturally occurring amino acid
<400> 1
Met Pro Pro Leu Leu Ala Pro Leu Leu Cys Leu Ala Leu Leu Pro Ala
1 5 10 15
Leu Ala Ala Arg Gly Pro Arg Cys Ser Gln Pro Gly Glu Thr Cys Leu
20 25 30
Asn Gly Gly Lys Cys Glu Ala Ala Asn Gly Thr Glu Ala Cys Val Cys
35 40 45
Gly Gly Ala Phe Val Gly Pro Arg Cys Gln Asp Pro Asn Pro Cys Leu
50 55 60
Ser Thr Pro Cys Lys Asn Ala Gly Thr Cys His Val Val Asp Arg Arg
65 70 75 80
Gly Val Ala Asp Tyr Ala Cys Ser Cys Ala Leu Gly Phe Ser Gly Pro
85 90 95
Leu Cys Leu Thr Pro Leu Asp Asn Ala Cys Leu Thr Asn Pro Cys Arg
100 105 110
Asn Gly Gly Thr Cys Asp Leu Leu Thr Leu Thr Glu Tyr Lys Cys Arg
115 120 125
Cys Pro Pro Gly Trp Ser Gly Lys Ser Cys Gln Gln Ala Asp Pro Cys
130 135 140
Ala Ser Asn Pro Cys Ala Asn Gly Gly Gln Cys Leu Pro Phe Glu Ala
145 150 155 160
Ser Tyr Ile Cys His Cys Pro Pro Ser Phe His Gly Pro Thr Cys Arg
165 170 175
Gln Asp Val Asn Glu Cys Gly Gln Lys Pro Arg Leu Cys Arg His Gly
180 185 190
Gly Thr Cys His Asn Glu Val Gly Ser Tyr Arg Cys Val Cys Arg Ala
195 200 205
Thr His Thr Gly Pro Asn Cys Glu Arg Pro Tyr Val Pro Cys Ser Pro
210 215 220
Ser Pro Cys Gln Asn Gly Gly Thr Cys Arg Pro Thr Gly Asp Val Thr
225 230 235 240
His Glu Cys Ala Cys Leu Pro Gly Phe Thr Gly Gln Asn Cys Glu Glu
245 250 255
Asn Ile Asp Asp Cys Pro Gly Asn Asn Cys Lys Asn Gly Gly Ala Cys
260 265 270
Val Asp Gly Val Asn Thr Tyr Asn Cys Pro Cys Pro Pro Glu Trp Thr
275 280 285
Gly Gln Tyr Cys Thr Glu Asp Val Asp Glu Cys Gln Leu Met Pro Asn
290 295 300
Ala Cys Gln Asn Gly Gly Thr Cys His Asn Thr His Gly Gly Tyr Asn
305 310 315 320
Cys Val Cys Val Asn Gly Trp Thr Gly Glu Asp Cys Ser Glu Asn Ile
325 330 335
Asp Asp Cys Ala Ser Ala Ala Cys Phe His Gly Ala Thr Cys His Asp
340 345 350
Arg Val Ala Ser Phe Tyr Cys Glu Cys Pro His Gly Arg Thr Gly Leu
355 360 365
Leu Cys His Leu Asn Asp Ala Cys Ile Ser Asn Pro Cys Asn Glu Gly
370 375 380
Ser Asn Cys Asp Thr Asn Pro Val Asn Gly Lys Ala Ile Cys Thr Cys
385 390 395 400
Pro Ser Gly Tyr Thr Gly Pro Ala Cys Ser Gln Asp Val Asp Glu Cys
405 410 415
Ser Leu Gly Ala Asn Pro Cys Glu His Ala Gly Lys Cys Ile Asn Thr
420 425 430
Leu Gly Ser Phe Glu Cys Gln Cys Leu Gln Gly Tyr Thr Gly Pro Arg
435 440 445
Cys Glu Ile Asp Val Asn Glu Cys Val Ser Asn Pro Cys Gln Asn Asp
450 455 460
Ala Thr Cys Leu Asp Gln Ile Gly Glu Phe Gln Cys Met Cys Met Pro
465 470 475 480
Gly Tyr Glu Gly Val His Cys Glu Val Asn Thr Asp Glu Cys Ala Ser
485 490 495
Ser Pro Cys Leu His Asn Gly Arg Cys Leu Asp Lys Ile Asn Glu Phe
500 505 510
Gln Cys Glu Cys Pro Thr Gly Phe Thr Gly His Leu Cys Gln Tyr Asp
515 520 525
Val Asp Glu Cys Ala Ser Thr Pro Cys Lys Asn Gly Ala Lys Cys Leu
530 535 540
Asp Gly Pro Asn Thr Tyr Thr Cys Val Cys Thr Glu Gly Tyr Thr Gly
545 550 555 560
Thr His Cys Glu Val Asp Ile Asp Glu Cys Asp Pro Asp Pro Cys His
565 570 575
Tyr Gly Ser Cys Lys Asp Gly Val Ala Thr Phe Thr Cys Leu Cys Arg
580 585 590
Pro Gly Tyr Thr Gly His His Cys Glu Thr Asn Ile Asn Glu Cys Ser
595 600 605
Ser Gln Pro Cys Arg Leu Arg Gly Thr Cys Gln Asp Pro Asp Asn Ala
610 615 620
Tyr Leu Cys Phe Cys Leu Lys Gly Thr Thr Gly Pro Asn Cys Glu Ile
625 630 635 640
Asn Leu Asp Asp Cys Ala Ser Ser Pro Cys Asp Ser Gly Thr Cys Leu
645 650 655
Asp Lys Ile Asp Gly Tyr Glu Cys Ala Cys Glu Pro Gly Tyr Thr Gly
660 665 670
Ser Met Cys Asn Ser Asn Ile Asp Glu Cys Ala Gly Asn Pro Cys His
675 680 685
Asn Gly Gly Thr Cys Glu Asp Gly Ile Asn Gly Phe Thr Cys Arg Cys
690 695 700
Pro Glu Gly Tyr His Asp Pro Thr Cys Leu Ser Glu Val Asn Glu Cys
705 710 715 720
Asn Ser Asn Pro Cys Val His Gly Ala Cys Arg Asp Ser Leu Asn Gly
725 730 735
Tyr Lys Cys Asp Cys Asp Pro Gly Trp Ser Gly Thr Asn Cys Asp Ile
740 745 750
Asn Asn Asn Glu Cys Glu Ser Asn Pro Cys Val Asn Gly Gly Thr Cys
755 760 765
Lys Asp Met Thr Ser Gly Ile Val Cys Thr Cys Arg Glu Gly Phe Ser
770 775 780
Gly Pro Asn Cys Gln Thr Asn Ile Asn Glu Cys Ala Ser Asn Pro Cys
785 790 795 800
Leu Asn Lys Gly Thr Cys Ile Asp Asp Val Ala Gly Tyr Lys Cys Asn
805 810 815
Cys Leu Leu Pro Tyr Thr Gly Ala Thr Cys Glu Val Val Leu Ala Pro
820 825 830
Cys Ala Pro Ser Pro Cys Arg Asn Gly Gly Glu Cys Arg Gln Ser Glu
835 840 845
Asp Tyr Glu Ser Phe Ser Cys Val Cys Pro Thr Ala Gly Ala Lys Gly
850 855 860
Gln Thr Cys Glu Val Asp Ile Asn Glu Cys Val Leu Ser Pro Cys Arg
865 870 875 880
His Gly Ala Ser Cys Gln Asn Thr His Gly Xaa Tyr Arg Cys His Cys
885 890 895
Gln Ala Gly Tyr Ser Gly Arg Asn Cys Glu Thr Asp Ile Asp Asp Cys
900 905 910
Arg Pro Asn Pro Cys His Asn Gly Gly Ser Cys Thr Asp Gly Ile Asn
915 920 925
Thr Ala Phe Cys Asp Cys Leu Pro Gly Phe Arg Gly Thr Phe Cys Glu
930 935 940
Glu Asp Ile Asn Glu Cys Ala Ser Asp Pro Cys Arg Asn Gly Ala Asn
945 950 955 960
Cys Thr Asp Cys Val Asp Ser Tyr Thr Cys Thr Cys Pro Ala Gly Phe
965 970 975
Ser Gly Ile His Cys Glu Asn Asn Thr Pro Asp Cys Thr Glu Ser Ser
980 985 990
Cys Phe Asn Gly Gly Thr Cys Val Asp Gly Ile Asn Ser Phe Thr Cys
995 1000 1005
Leu Cys Pro Pro Gly Phe Thr Gly Ser Tyr Cys Gln His Val Val
1010 1015 1020
Asn Glu Cys Asp Ser Arg Pro Cys Leu Leu Gly Gly Thr Cys Gln
1025 1030 1035
Asp Gly Arg Gly Leu His Arg Cys Thr Cys Pro Gln Gly Tyr Thr
1040 1045 1050
Gly Pro Asn Cys Gln Asn Leu Val His Trp Cys Asp Ser Ser Pro
1055 1060 1065
Cys Lys Asn Gly Gly Lys Cys Trp Gln Thr His Thr Gln Tyr Arg
1070 1075 1080
Cys Glu Cys Pro Ser Gly Trp Thr Gly Leu Tyr Cys Asp Val Pro
1085 1090 1095
Ser Val Ser Cys Glu Val Ala Ala Gln Arg Gln Gly Val Asp Val
1100 1105 1110
Ala Arg Leu Cys Gln His Gly Gly Leu Cys Val Asp Ala Gly Asn
1115 1120 1125
Thr His His Cys Arg Cys Gln Ala Gly Tyr Thr Gly Ser Tyr Cys
1130 1135 1140
Glu Asp Leu Val Asp Glu Cys Ser Pro Ser Pro Cys Gln Asn Gly
1145 1150 1155
Ala Thr Cys Thr Asp Tyr Leu Gly Gly Tyr Ser Cys Lys Cys Val
1160 1165 1170
Ala Gly Tyr His Gly Val Asn Cys Ser Glu Glu Ile Asp Glu Cys
1175 1180 1185
Leu Ser His Pro Cys Gln Asn Gly Gly Thr Cys Leu Asp Leu Pro
1190 1195 1200
Asn Thr Tyr Lys Cys Ser Cys Pro Arg Gly Thr Gln Gly Val His
1205 1210 1215
Cys Glu Ile Asn Val Asp Asp Cys Asn Pro Pro Val Asp Pro Val
1220 1225 1230
Ser Arg Ser Pro Lys Cys Phe Asn Asn Gly Thr Cys Val Asp Gln
1235 1240 1245
Val Gly Gly Tyr Ser Cys Thr Cys Pro Pro Gly Phe Val Gly Glu
1250 1255 1260
Arg Cys Glu Gly Asp Val Asn Glu Cys Leu Ser Asn Pro Cys Asp
1265 1270 1275
Ala Arg Gly Thr Gln Asn Cys Val Gln Arg Val Asn Asp Phe His
1280 1285 1290
Cys Glu Cys Arg Ala Gly His Thr Gly Arg Arg Cys Glu Ser Val
1295 1300 1305
Ile Asn Gly Cys Lys Gly Lys Pro Cys Lys Asn Gly Gly Thr Cys
1310 1315 1320
Ala Val Ala Ser Asn Thr Ala Arg Gly Phe Ile Cys Lys Cys Pro
1325 1330 1335
Ala Gly Phe Glu Gly Ala Thr Cys Glu Asn Asp Ala Arg Thr Cys
1340 1345 1350
Gly Ser Leu Arg Cys Leu Asn Gly Gly Thr Cys Ile Ser Gly Pro
1355 1360 1365
Arg Ser Pro Thr Cys Leu Cys Leu Gly Pro Phe Thr Gly Pro Glu
1370 1375 1380
Cys Gln Phe Pro Ala Ser Ser Pro Cys Leu Gly Gly Asn Pro Cys
1385 1390 1395
Tyr Asn Gln Gly Thr Cys Glu Pro Thr Ser Glu Ser Pro Phe Tyr
1400 1405 1410
Arg Cys Leu Cys Pro Ala Lys Phe Asn Gly Leu Leu Cys His Ile
1415 1420 1425
Leu Asp Tyr Ser Phe Gly Gly Gly Ala Gly Arg Asp Ile Pro Pro
1430 1435 1440
Pro Leu Ile Glu Glu Ala Cys Glu Leu Pro Glu Cys Gln Glu Asp
1445 1450 1455
Ala Gly Asn Lys Val Cys Ser Leu Gln Cys Asn Asn His Ala Cys
1460 1465 1470
Gly Trp Asp Gly Gly Asp Cys Ser Leu Asn Phe Asn Asp Pro Trp
1475 1480 1485
Lys Asn Cys Thr Gln Ser Leu Gln Cys Trp Lys Tyr Phe Ser Asp
1490 1495 1500
Gly His Cys Asp Ser Gln Cys Asn Ser Ala Gly Cys Leu Phe Asp
1505 1510 1515
Gly Phe Asp Cys Gln Arg Ala Glu Gly Gln Cys Asn Pro Leu Tyr
1520 1525 1530
Asp Gln Tyr Cys Lys Asp His Phe Ser Asp Gly His Cys Asp Gln
1535 1540 1545
Gly Cys Asn Ser Ala Glu Cys Glu Trp Asp Gly Leu Asp Cys Ala
1550 1555 1560
Glu His Val Pro Glu Arg Leu Ala Ala Gly Thr Leu Val Val Val
1565 1570 1575
Val Leu Met Pro Pro Glu Gln Leu Arg Asn Ser Ser Phe His Phe
1580 1585 1590
Leu Arg Glu Leu Ser Arg Val Leu His Thr Asn Val Val Phe Lys
1595 1600 1605
Arg Asp Ala His Gly Gln Gln Met Ile Phe Pro Tyr Tyr Gly Arg
1610 1615 1620
Glu Glu Glu Leu Arg Lys His Pro Ile Lys Arg Ala Ala Glu Gly
1625 1630 1635
Trp Ala Ala Pro Asp Ala Leu Leu Gly Gln Val Lys Ala Ser Leu
1640 1645 1650
Leu Pro Gly Gly Ser Glu Gly Gly Arg Arg Arg Arg Glu Leu Asp
1655 1660 1665
Pro Met Asp Val Arg Gly Ser Ile Val Tyr Leu Glu Ile Asp Asn
1670 1675 1680
Arg Gln Cys Val Gln Ala Ser Ser Gln Cys Phe Gln Ser Ala Thr
1685 1690 1695
Asp Val Ala Ala Phe Leu Gly Ala Leu Ala Ser Leu Gly Ser Leu
1700 1705 1710
Asn Ile Pro Tyr Lys Ile Glu Ala Val Gln Ser Glu Thr Val Glu
1715 1720 1725
Pro Pro Pro Pro Ala Gln Leu His Phe Met Tyr Val Ala Ala Ala
1730 1735 1740
Ala Phe Val Leu Leu Phe Phe Val Gly Cys Gly Val Leu Leu Ser
1745 1750 1755
Arg Lys Arg Arg Arg Gln His Gly Gln Leu Trp Phe Pro Glu Gly
1760 1765 1770
Phe Lys Val Ser Glu Ala Ser Lys Lys Lys Arg Arg Glu Pro Leu
1775 1780 1785
Gly Glu Asp Ser Val Gly Leu Lys Pro Leu Lys Asn Ala Ser Asp
1790 1795 1800
Gly Ala Leu Met Asp Asp Asn Gln Asn Glu Trp Gly Asp Glu Asp
1805 1810 1815
Leu Glu Thr Lys Lys Phe Arg Phe Glu Glu Pro Val Val Leu Pro
1820 1825 1830
Asp Leu Asp Asp Gln Thr Asp His Arg Gln Trp Thr Gln Gln His
1835 1840 1845
Leu Asp Ala Ala Asp Leu Arg Met Ser Ala Met Ala Pro Thr Pro
1850 1855 1860
Pro Gln Gly Glu Val Asp Ala Asp Cys Met Asp Val Asn Val Arg
1865 1870 1875
Gly Pro Asp Gly Phe Thr Pro Leu Met Ile Ala Ser Cys Ser Gly
1880 1885 1890
Gly Gly Leu Glu Thr Gly Asn Ser Glu Glu Glu Glu Asp Ala Pro
1895 1900 1905
Ala Val Ile Ser Asp Phe Ile Tyr Gln Gly Ala Ser Leu His Asn
1910 1915 1920
Gln Thr Asp Arg Thr Gly Glu Thr Ala Leu His Leu Ala Ala Arg
1925 1930 1935
Tyr Ser Arg Ser Asp Ala Ala Lys Arg Leu Leu Glu Ala Ser Ala
1940 1945 1950
Asp Ala Asn Ile Gln Asp Asn Met Gly Arg Thr Pro Leu His Ala
1955 1960 1965
Ala Val Ser Ala Asp Ala Gln Gly Val Phe Gln Ile Leu Ile Arg
1970 1975 1980
Asn Arg Ala Thr Asp Leu Asp Ala Arg Met His Asp Gly Thr Thr
1985 1990 1995
Pro Leu Ile Leu Ala Ala Arg Leu Ala Val Glu Gly Met Leu Glu
2000 2005 2010
Asp Leu Ile Asn Ser His Ala Asp Val Asn Ala Val Asp Asp Leu
2015 2020 2025
Gly Lys Ser Ala Leu His Trp Ala Ala Ala Val Asn Asn Val Asp
2030 2035 2040
Ala Ala Val Val Leu Leu Lys Asn Gly Ala Asn Lys Asp Met Gln
2045 2050 2055
Asn Asn Arg Glu Glu Thr Pro Leu Phe Leu Ala Ala Arg Glu Gly
2060 2065 2070
Ser Tyr Glu Thr Ala Lys Val Leu Leu Asp His Phe Ala Asn Arg
2075 2080 2085
Asp Ile Thr Asp His Met Asp Arg Leu Pro Arg Asp Ile Ala Gln
2090 2095 2100
Glu Arg Met His His Asp Ile Val Arg Leu Leu Asp Glu Tyr Asn
2105 2110 2115
Leu Val Arg Ser Pro Gln Leu His Gly Ala Pro Leu Gly Gly Thr
2120 2125 2130
Pro Thr Leu Ser Pro Pro Leu Cys Ser Pro Asn Gly Tyr Leu Gly
2135 2140 2145
Ser Leu Lys Pro Gly Val Gln Gly Lys Lys Val Arg Lys Pro Ser
2150 2155 2160
Ser Lys Gly Leu Ala Cys Gly Ser Lys Glu Ala Lys Asp Leu Lys
2165 2170 2175
Ala Arg Arg Lys Lys Ser Gln Asp Gly Lys Gly Cys Leu Leu Asp
2180 2185 2190
Ser Ser Gly Met Leu Ser Pro Val Asp Ser Leu Glu Ser Pro His
2195 2200 2205
Gly Tyr Leu Ser Asp Val Ala Ser Pro Pro Leu Leu Pro Ser Pro
2210 2215 2220
Phe Gln Gln Ser Pro Ser Val Pro Leu Asn His Leu Pro Gly Met
2225 2230 2235
Pro Asp Thr His Leu Gly Ile Gly His Leu Asn Val Ala Ala Lys
2240 2245 2250
Pro Glu Met Ala Ala Leu Gly Gly Gly Gly Arg Leu Ala Phe Glu
2255 2260 2265
Thr Gly Pro Pro Arg Leu Ser His Leu Pro Val Ala Ser Gly Thr
2270 2275 2280
Ser Thr Val Leu Gly Ser Ser Ser Gly Gly Ala Leu Asn Phe Thr
2285 2290 2295
Val Gly Gly Ser Thr Ser Leu Asn Gly Gln Cys Glu Trp Leu Ser
2300 2305 2310
Arg Leu Gln Ser Gly Met Val Pro Asn Gln Tyr Asn Pro Leu Arg
2315 2320 2325
Gly Ser Val Ala Pro Gly Pro Leu Ser Thr Gln Ala Pro Ser Leu
2330 2335 2340
Gln His Gly Met Val Gly Pro Leu His Ser Ser Leu Ala Ala Ser
2345 2350 2355
Ala Leu Ser Gln Met Met Ser Tyr Gln Gly Leu Pro Ser Thr Arg
2360 2365 2370
Leu Ala Thr Gln Pro His Leu Val Gln Thr Gln Gln Val Gln Pro
2375 2380 2385
Gln Asn Leu Gln Met Gln Gln Gln Asn Leu Gln Pro Ala Asn Ile
2390 2395 2400
Gln Gln Gln Gln Ser Leu Gln Pro Pro Pro Pro Pro Pro Gln Pro
2405 2410 2415
His Leu Gly Val Ser Ser Ala Ala Ser Gly His Leu Gly Arg Ser
2420 2425 2430
Phe Leu Ser Gly Glu Pro Ser Gln Ala Asp Val Gln Pro Leu Gly
2435 2440 2445
Pro Ser Ser Leu Ala Val His Thr Ile Leu Pro Gln Glu Ser Pro
2450 2455 2460
Ala Leu Pro Thr Ser Leu Pro Ser Ser Leu Val Pro Pro Val Thr
2465 2470 2475
Ala Ala Gln Phe Leu Thr Pro Pro Ser Gln His Ser Tyr Ser Ser
2480 2485 2490
Pro Val Asp Asn Thr Pro Ser His Gln Leu Gln Val Pro Glu His
2495 2500 2505
Pro Phe Leu Thr Pro Ser Pro Glu Ser Pro Asp Gln Trp Ser Ser
2510 2515 2520
Ser Ser Pro His Ser Asn Val Ser Asp Trp Ser Glu Gly Val Ser
2525 2530 2535
Ser Pro Pro Thr Ser Met Gln Ser Gln Ile Ala Arg Ile Pro Glu
2540 2545 2550
Ala Phe Lys
2555
<210> 2
<211> 2531
<212> PRT
<213> 人工合成
<400> 2
Met Pro Arg Leu Leu Thr Pro Leu Leu Cys Leu Thr Leu Leu Pro Ala
1 5 10 15
Leu Ala Ala Arg Gly Leu Arg Cys Ser Gln Pro Ser Gly Thr Cys Leu
20 25 30
Asn Gly Gly Arg Cys Glu Val Ala Asn Gly Thr Glu Ala Cys Val Cys
35 40 45
Ser Gly Ala Phe Val Gly Gln Arg Cys Gln Asp Ser Asn Pro Cys Leu
50 55 60
Ser Thr Pro Cys Lys Asn Ala Gly Thr Cys His Val Val Asp His Gly
65 70 75 80
Gly Thr Val Asp Tyr Ala Cys Ser Cys Pro Leu Gly Phe Ser Gly Pro
85 90 95
Leu Cys Leu Thr Pro Leu Asp Asn Ala Cys Leu Ala Asn Pro Cys Arg
100 105 110
Asn Gly Gly Thr Cys Asp Leu Leu Thr Leu Thr Glu Tyr Lys Cys Arg
115 120 125
Cys Pro Pro Gly Trp Ser Gly Lys Ser Cys Gln Gln Ala Asp Pro Cys
130 135 140
Ala Ser Asn Pro Cys Ala Asn Gly Gly Gln Cys Leu Pro Phe Glu Ser
145 150 155 160
Ser Tyr Ile Cys Arg Cys Pro Pro Gly Phe His Gly Pro Thr Cys Arg
165 170 175
Gln Asp Val Asn Glu Cys Ser Gln Asn Pro Gly Leu Cys Arg His Gly
180 185 190
Gly Thr Cys His Asn Glu Ile Gly Ser Tyr Arg Cys Ala Cys Arg Ala
195 200 205
Thr His Thr Gly Pro His Cys Glu Leu Pro Tyr Val Pro Cys Ser Pro
210 215 220
Ser Pro Cys Gln Asn Gly Gly Thr Cys Arg Pro Thr Gly Asp Thr Thr
225 230 235 240
His Glu Cys Ala Cys Leu Pro Gly Phe Ala Gly Gln Asn Cys Glu Glu
245 250 255
Asn Val Asp Asp Cys Pro Gly Asn Asn Cys Lys Asn Gly Gly Ala Cys
260 265 270
Val Asp Gly Val Asn Thr Tyr Asn Cys Arg Cys Pro Pro Glu Trp Thr
275 280 285
Gly Gln Tyr Cys Thr Glu Asp Val Asp Glu Cys Gln Leu Met Pro Asn
290 295 300
Ala Cys Gln Asn Gly Gly Thr Cys His Asn Thr His Gly Gly Tyr Asn
305 310 315 320
Cys Val Cys Val Asn Gly Trp Thr Gly Glu Asp Cys Ser Glu Asn Ile
325 330 335
Asp Asp Cys Ala Ser Ala Ala Cys Phe Gln Gly Ala Thr Cys His Asp
340 345 350
Arg Val Ala Ser Phe Tyr Cys Glu Cys Pro His Gly Arg Thr Gly Leu
355 360 365
Leu Cys His Leu Asn Asp Ala Cys Ile Ser Asn Pro Cys Asn Glu Gly
370 375 380
Ser Asn Cys Asp Thr Asn Pro Val Asn Gly Lys Ala Ile Cys Thr Cys
385 390 395 400
Pro Ser Gly Tyr Thr Gly Pro Ala Cys Ser Gln Asp Val Asp Glu Cys
405 410 415
Ala Leu Gly Ala Asn Pro Cys Glu His Ala Gly Lys Cys Leu Asn Thr
420 425 430
Leu Gly Ser Phe Glu Cys Gln Cys Leu Gln Gly Tyr Thr Gly Pro Arg
435 440 445
Cys Glu Ile Asp Val Asn Glu Cys Ile Ser Asn Pro Cys Gln Asn Asp
450 455 460
Ala Thr Cys Leu Asp Gln Ile Gly Glu Phe Gln Cys Ile Cys Met Pro
465 470 475 480
Gly Tyr Glu Gly Val Tyr Cys Glu Ile Asn Thr Asp Glu Cys Ala Ser
485 490 495
Ser Pro Cys Leu His Asn Gly His Cys Met Asp Lys Ile Asn Glu Phe
500 505 510
Gln Cys Gln Cys Pro Lys Gly Phe Asn Gly His Leu Cys Gln Tyr Asp
515 520 525
Val Asp Glu Cys Ala Ser Thr Pro Cys Lys Asn Gly Ala Lys Cys Leu
530 535 540
Asp Gly Pro Asn Thr Tyr Thr Cys Val Cys Thr Glu Gly Tyr Thr Gly
545 550 555 560
Thr His Cys Glu Val Asp Ile Asp Glu Cys Asp Pro Asp Pro Cys His
565 570 575
Tyr Gly Ser Cys Lys Asp Gly Val Ala Thr Phe Thr Cys Leu Cys Gln
580 585 590
Pro Gly Tyr Thr Gly His His Cys Glu Thr Asn Ile Asn Glu Cys His
595 600 605
Ser Gln Pro Cys Arg His Gly Gly Thr Cys Gln Asp Arg Asp Asn Ser
610 615 620
Tyr Leu Cys Leu Cys Leu Lys Gly Thr Thr Gly Pro Asn Cys Glu Ile
625 630 635 640
Asn Leu Asp Asp Cys Ala Ser Asn Pro Cys Asp Ser Gly Thr Cys Leu
645 650 655
Asp Lys Ile Asp Gly Tyr Glu Cys Ala Cys Glu Pro Gly Tyr Thr Gly
660 665 670
Ser Met Cys Asn Val Asn Ile Asp Glu Cys Ala Gly Ser Pro Cys His
675 680 685
Asn Gly Gly Thr Cys Glu Asp Gly Ile Ala Gly Phe Thr Cys Arg Cys
690 695 700
Pro Glu Gly Tyr His Asp Pro Thr Cys Leu Ser Glu Val Asn Glu Cys
705 710 715 720
Asn Ser Asn Pro Cys Ile His Gly Ala Cys Arg Asp Gly Leu Asn Gly
725 730 735
Tyr Lys Cys Asp Cys Ala Pro Gly Trp Ser Gly Thr Asn Cys Asp Ile
740 745 750
Asn Asn Asn Glu Cys Glu Ser Asn Pro Cys Val Asn Gly Gly Thr Cys
755 760 765
Lys Asp Met Thr Ser Gly Tyr Val Cys Thr Cys Arg Glu Gly Phe Ser
770 775 780
Gly Pro Asn Cys Gln Thr Asn Ile Asn Glu Cys Ala Ser Asn Pro Cys
785 790 795 800
Leu Asn Gln Gly Thr Cys Ile Asp Asp Val Ala Gly Tyr Lys Cys Asn
805 810 815
Cys Pro Leu Pro Tyr Thr Gly Ala Thr Cys Glu Val Val Leu Ala Pro
820 825 830
Cys Ala Thr Ser Pro Cys Lys Asn Ser Gly Val Cys Lys Glu Ser Glu
835 840 845
Asp Tyr Glu Ser Phe Ser Cys Val Cys Pro Thr Gly Trp Gln Gly Gln
850 855 860
Thr Cys Glu Val Asp Ile Asn Glu Cys Val Lys Ser Pro Cys Arg His
865 870 875 880
Gly Ala Ser Cys Gln Asn Thr Asn Gly Ser Tyr Arg Cys Leu Cys Gln
885 890 895
Ala Gly Tyr Thr Gly Arg Asn Cys Glu Ser Asp Ile Asp Asp Cys Arg
900 905 910
Pro Asn Pro Cys His Asn Gly Gly Ser Cys Thr Asp Gly Ile Asn Thr
915 920 925
Ala Phe Cys Asp Cys Leu Pro Gly Phe Gln Gly Ala Phe Cys Glu Glu
930 935 940
Asp Ile Asn Glu Cys Ala Ser Asn Pro Cys Gln Asn Gly Ala Asn Cys
945 950 955 960
Thr Asp Cys Val Asp Ser Tyr Thr Cys Thr Cys Pro Val Gly Phe Asn
965 970 975
Gly Ile His Cys Glu Asn Asn Thr Pro Asp Cys Thr Glu Ser Ser Cys
980 985 990
Phe Asn Gly Gly Thr Cys Val Asp Gly Ile Asn Ser Phe Thr Cys Leu
995 1000 1005
Cys Pro Pro Gly Phe Thr Gly Ser Tyr Cys Gln Tyr Asp Val Asn
1010 1015 1020
Glu Cys Asp Ser Arg Pro Cys Leu His Gly Gly Thr Cys Gln Asp
1025 1030 1035
Ser Tyr Gly Thr Tyr Lys Cys Thr Cys Pro Gln Gly Tyr Thr Gly
1040 1045 1050
Leu Asn Cys Gln Asn Leu Val Arg Trp Cys Asp Ser Ala Pro Cys
1055 1060 1065
Lys Asn Gly Gly Arg Cys Trp Gln Thr Asn Thr Gln Tyr His Cys
1070 1075 1080
Glu Cys Arg Ser Gly Trp Thr Gly Val Asn Cys Asp Val Leu Ser
1085 1090 1095
Val Ser Cys Glu Val Ala Ala Gln Lys Arg Gly Ile Asp Val Thr
1100 1105 1110
Leu Leu Cys Gln His Gly Gly Leu Cys Val Asp Glu Gly Asp Lys
1115 1120 1125
His Tyr Cys His Cys Gln Ala Gly Tyr Thr Gly Ser Tyr Cys Glu
1130 1135 1140
Asp Glu Val Asp Glu Cys Ser Pro Asn Pro Cys Gln Asn Gly Ala
1145 1150 1155
Thr Cys Thr Asp Tyr Leu Gly Gly Phe Ser Cys Lys Cys Val Ala
1160 1165 1170
Gly Tyr His Gly Ser Asn Cys Ser Glu Glu Ile Asn Glu Cys Leu
1175 1180 1185
Ser Gln Pro Cys Gln Asn Gly Gly Thr Cys Ile Asp Leu Thr Asn
1190 1195 1200
Ser Tyr Lys Cys Ser Cys Pro Arg Gly Thr Gln Gly Val His Cys
1205 1210 1215
Glu Ile Asn Val Asp Asp Cys His Pro Pro Leu Asp Pro Ala Ser
1220 1225 1230
Arg Ser Pro Lys Cys Phe Asn Asn Gly Thr Cys Val Asp Gln Val
1235 1240 1245
Gly Gly Tyr Thr Cys Thr Cys Pro Pro Gly Phe Val Gly Glu Arg
1250 1255 1260
Cys Glu Gly Asp Val Asn Glu Cys Leu Ser Asn Pro Cys Asp Pro
1265 1270 1275
Arg Gly Thr Gln Asn Cys Val Gln Arg Val Asn Asp Phe His Cys
1280 1285 1290
Glu Cys Arg Ala Gly His Thr Gly Arg Arg Cys Glu Ser Val Ile
1295 1300 1305
Asn Gly Cys Arg Gly Lys Pro Cys Lys Asn Gly Gly Val Cys Ala
1310 1315 1320
Val Ala Ser Asn Thr Ala Arg Gly Phe Ile Cys Arg Cys Pro Ala
1325 1330 1335
Gly Phe Glu Gly Ala Thr Cys Glu Asn Asp Ala Arg Thr Cys Gly
1340 1345 1350
Ser Leu Arg Cys Leu Asn Gly Gly Thr Cys Ile Ser Gly Pro Arg
1355 1360 1365
Ser Pro Thr Cys Leu Cys Leu Gly Ser Phe Thr Gly Pro Glu Cys
1370 1375 1380
Gln Phe Pro Ala Ser Ser Pro Cys Val Gly Ser Asn Pro Cys Tyr
1385 1390 1395
Asn Gln Gly Thr Cys Glu Pro Thr Ser Glu Asn Pro Phe Tyr Arg
1400 1405 1410
Cys Leu Cys Pro Ala Lys Phe Asn Gly Leu Leu Cys His Ile Leu
1415 1420 1425
Asp Tyr Ser Phe Thr Gly Gly Ala Gly Arg Asp Ile Pro Pro Pro
1430 1435 1440
Gln Ile Glu Glu Ala Cys Glu Leu Pro Glu Cys Gln Val Asp Ala
1445 1450 1455
Gly Asn Lys Val Cys Asn Leu Gln Cys Asn Asn His Ala Cys Gly
1460 1465 1470
Trp Asp Gly Gly Asp Cys Ser Leu Asn Phe Asn Asp Pro Trp Lys
1475 1480 1485
Asn Cys Thr Gln Ser Leu Gln Cys Trp Lys Tyr Phe Ser Asp Gly
1490 1495 1500
His Cys Asp Ser Gln Cys Asn Ser Ala Gly Cys Leu Phe Asp Gly
1505 1510 1515
Phe Asp Cys Gln Leu Thr Glu Gly Gln Cys Asn Pro Leu Tyr Asp
1520 1525 1530
Gln Tyr Cys Lys Asp His Phe Ser Asp Gly His Cys Asp Gln Gly
1535 1540 1545
Cys Asn Ser Ala Glu Cys Glu Trp Asp Gly Leu Asp Cys Ala Glu
1550 1555 1560
His Val Pro Glu Arg Leu Ala Ala Gly Thr Leu Val Leu Val Val
1565 1570 1575
Leu Leu Pro Pro Asp Gln Leu Arg Asn Asn Ser Phe His Phe Leu
1580 1585 1590
Arg Glu Leu Ser His Val Leu His Thr Asn Val Val Phe Lys Arg
1595 1600 1605
Asp Ala Gln Gly Gln Gln Met Ile Phe Pro Tyr Tyr Gly His Glu
1610 1615 1620
Glu Glu Leu Arg Lys His Pro Ile Lys Arg Ser Thr Val Gly Trp
1625 1630 1635
Ala Thr Ser Ser Leu Leu Pro Gly Thr Ser Gly Gly Arg Gln Arg
1640 1645 1650
Arg Glu Leu Asp Pro Met Asp Ile Arg Gly Ser Ile Val Tyr Leu
1655 1660 1665
Glu Ile Asp Asn Arg Gln Cys Val Gln Ser Ser Ser Gln Cys Phe
1670 1675 1680
Gln Ser Ala Thr Asp Val Ala Ala Phe Leu Gly Ala Leu Ala Ser
1685 1690 1695
Leu Gly Ser Leu Asn Ile Pro Tyr Lys Ile Glu Ala Val Lys Ser
1700 1705 1710
Glu Pro Val Glu Pro Pro Leu Pro Ser Gln Leu His Leu Met Tyr
1715 1720 1725
Val Ala Ala Ala Ala Phe Val Leu Leu Phe Phe Val Gly Cys Gly
1730 1735 1740
Val Leu Leu Ser Arg Lys Arg Arg Arg Gln His Gly Gln Leu Trp
1745 1750 1755
Phe Pro Glu Gly Phe Lys Val Ser Glu Ala Ser Lys Lys Lys Arg
1760 1765 1770
Arg Glu Pro Leu Gly Glu Asp Ser Val Gly Leu Lys Pro Leu Lys
1775 1780 1785
Asn Ala Ser Asp Gly Ala Leu Met Asp Asp Asn Gln Asn Glu Trp
1790 1795 1800
Gly Asp Glu Asp Leu Glu Thr Lys Lys Phe Arg Phe Glu Glu Pro
1805 1810 1815
Val Val Leu Pro Asp Leu Ser Asp Gln Thr Asp His Arg Gln Trp
1820 1825 1830
Thr Gln Gln His Leu Asp Ala Ala Asp Leu Arg Met Ser Ala Met
1835 1840 1845
Ala Pro Thr Pro Pro Gln Gly Glu Val Asp Ala Asp Cys Met Asp
1850 1855 1860
Val Asn Val Arg Gly Pro Asp Gly Phe Thr Pro Leu Met Ile Ala
1865 1870 1875
Ser Cys Ser Gly Gly Gly Leu Glu Thr Gly Asn Ser Glu Glu Glu
1880 1885 1890
Glu Asp Ala Pro Ala Val Ile Ser Asp Phe Ile Tyr Gln Gly Ala
1895 1900 1905
Ser Leu His Asn Gln Thr Asp Arg Thr Gly Glu Thr Ala Leu His
1910 1915 1920
Leu Ala Ala Arg Tyr Ser Arg Ser Asp Ala Ala Lys Arg Leu Leu
1925 1930 1935
Glu Ala Ser Ala Asp Ala Asn Ile Gln Asp Asn Met Gly Arg Thr
1940 1945 1950
Pro Leu His Ala Ala Val Ser Ala Asp Ala Gln Gly Val Phe Gln
1955 1960 1965
Ile Leu Leu Arg Asn Arg Ala Thr Asp Leu Asp Ala Arg Met His
1970 1975 1980
Asp Gly Thr Thr Pro Leu Ile Leu Ala Ala Arg Leu Ala Val Glu
1985 1990 1995
Gly Met Leu Glu Asp Leu Ile Asn Ser His Ala Asp Val Asn Ala
2000 2005 2010
Val Asp Asp Leu Gly Lys Ser Ala Leu His Trp Ala Ala Ala Val
2015 2020 2025
Asn Asn Val Asp Ala Ala Val Val Leu Leu Lys Asn Gly Ala Asn
2030 2035 2040
Lys Asp Met Gln Asn Asn Lys Glu Glu Thr Pro Leu Phe Leu Ala
2045 2050 2055
Ala Arg Glu Gly Ser Tyr Glu Thr Ala Lys Val Leu Leu Asp His
2060 2065 2070
Phe Ala Asn Arg Asp Ile Thr Asp His Met Asp Arg Leu Pro Arg
2075 2080 2085
Asp Ile Ala Gln Glu Arg Met His His Asp Ile Val Arg Leu Leu
2090 2095 2100
Asp Glu Tyr Asn Leu Val Arg Ser Pro Gln Leu His Gly Thr Ala
2105 2110 2115
Leu Gly Gly Thr Pro Thr Leu Ser Pro Thr Leu Cys Ser Pro Asn
2120 2125 2130
Gly Tyr Leu Gly Asn Leu Lys Ser Ala Thr Gln Gly Lys Lys Ala
2135 2140 2145
Arg Lys Pro Ser Thr Lys Gly Leu Ala Cys Gly Ser Lys Glu Ala
2150 2155 2160
Lys Asp Leu Lys Ala Arg Arg Lys Lys Ser Gln Asp Gly Lys Gly
2165 2170 2175
Cys Leu Leu Asp Ser Ser Ser Met Leu Ser Pro Val Asp Ser Leu
2180 2185 2190
Glu Ser Pro His Gly Tyr Leu Ser Asp Val Ala Ser Pro Pro Leu
2195 2200 2205
Leu Pro Ser Pro Phe Gln Gln Ser Pro Ser Met Pro Leu Ser His
2210 2215 2220
Leu Pro Gly Met Pro Asp Thr His Leu Gly Ile Ser His Leu Asn
2225 2230 2235
Val Ala Ala Lys Pro Glu Met Ala Ala Leu Ala Gly Gly Ser Arg
2240 2245 2250
Leu Ala Phe Glu Pro Pro Pro Pro Arg Leu Ser His Leu Pro Val
2255 2260 2265
Ala Ser Ser Ala Ser Thr Val Leu Ser Thr Asn Gly Thr Gly Ala
2270 2275 2280
Met Asn Phe Thr Val Gly Ala Pro Ala Ser Leu Asn Gly Gln Cys
2285 2290 2295
Glu Trp Leu Pro Arg Leu Gln Asn Gly Met Val Pro Ser Gln Tyr
2300 2305 2310
Asn Pro Leu Arg Pro Gly Val Thr Pro Gly Thr Leu Ser Thr Gln
2315 2320 2325
Ala Ala Gly Leu Gln His Ser Met Met Gly Pro Leu His Ser Ser
2330 2335 2340
Leu Ser Thr Asn Thr Leu Ser Pro Ile Ile Tyr Gln Gly Leu Pro
2345 2350 2355
Asn Thr Arg Leu Ala Thr Gln Pro His Leu Val Gln Thr Gln Gln
2360 2365 2370
Val Gln Pro Gln Asn Leu Gln Leu Gln Pro Gln Asn Leu Gln Pro
2375 2380 2385
Pro Ser Gln Pro His Leu Ser Val Ser Ser Ala Ala Asn Gly His
2390 2395 2400
Leu Gly Arg Ser Phe Leu Ser Gly Glu Pro Ser Gln Ala Asp Val
2405 2410 2415
Gln Pro Leu Gly Pro Ser Ser Leu Pro Val His Thr Ile Leu Pro
2420 2425 2430
Gln Glu Ser Gln Ala Leu Pro Thr Ser Leu Pro Ser Ser Met Val
2435 2440 2445
Pro Pro Met Thr Thr Thr Gln Phe Leu Thr Pro Pro Ser Gln His
2450 2455 2460
Ser Tyr Ser Ser Ser Pro Val Asp Asn Thr Pro Ser His Gln Leu
2465 2470 2475
Gln Val Pro Glu His Pro Phe Leu Thr Pro Ser Pro Glu Ser Pro
2480 2485 2490
Asp Gln Trp Ser Ser Ser Ser Pro His Ser Asn Ile Ser Asp Trp
2495 2500 2505
Ser Glu Gly Ile Ser Ser Pro Pro Thr Thr Met Pro Ser Gln Ile
2510 2515 2520
Thr His Ile Pro Glu Ala Phe Lys
2525 2530
<210> 3
<211> 316
<212> DNA
<213> 人工合成
<400> 3
ggcgatctga cggttcacta aacgagctct gcttatatag gcctcccacc gtacacgcct 60
acctcgacat acgttctcta tcactgatag ggagtaaact cgacatacgt tctctatcac 120
tgatagggat aaactcgaca tacgttctct atcactgata gggagtaaac tcgacatacg 180
ttctctatca ctgataggga gtaaactcga catacgttct ctatcactga tagggagtaa 240
actcgacatc gttctctatc actgataggg agtaaactcg acatacgttc tctatcactg 300
atagggagta aactcg 316
<210> 4
<211> 199
<212> PRT
<213> 人工合成
<400> 4
Met Thr Glu Tyr Lys Pro Thr Val Arg Leu Ala Thr Arg Asp Asp Val
1 5 10 15
Pro Arg Ala Val Arg Thr Leu Ala Ala Ala Phe Ala Asp Tyr Pro Ala
20 25 30
Thr Arg His Thr Val Asp Pro Asp Arg His Ile Glu Arg Val Thr Glu
35 40 45
Leu Gln Glu Leu Phe Leu Thr Arg Val Gly Leu Asp Ile Gly Lys Val
50 55 60
Trp Val Ala Asp Asp Gly Ala Ala Val Ala Val Trp Thr Thr Pro Glu
65 70 75 80
Ser Val Glu Ala Gly Ala Val Phe Ala Glu Ile Gly Pro Arg Met Ala
85 90 95
Glu Leu Ser Gly Ser Arg Leu Ala Ala Gln Gln Gln Met Glu Gly Leu
100 105 110
Leu Ala Pro His Arg Pro Lys Glu Pro Ala Trp Phe Leu Ala Thr Val
115 120 125
Gly Val Ser Pro Asp His Gln Gly Lys Gly Leu Gly Ser Ala Val Val
130 135 140
Leu Pro Gly Val Glu Ala Ala Glu Arg Ala Gly Val Pro Ala Phe Leu
145 150 155 160
Glu Thr Ser Ala Pro Arg Asn Leu Pro Phe Tyr Glu Arg Leu Gly Phe
165 170 175
Thr Val Thr Ala Asp Val Glu Val Pro Glu Gly Pro Arg Thr Trp Cys
180 185 190
Met Thr Arg Lys Pro Gly Ala
195
<210> 5
<211> 18
<212> PRT
<213> 人工合成
<400> 5
Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro
1 5 10 15
Gly Pro
<210> 6
<211> 239
<212> PRT
<213> 人工合成
<400> 6
Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu
1 5 10 15
Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly
20 25 30
Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile
35 40 45
Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr
50 55 60
Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys
65 70 75 80
Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu
85 90 95
Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu
100 105 110
Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly
115 120 125
Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr
130 135 140
Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn
145 150 155 160
Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser
165 170 175
Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly
180 185 190
Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu
195 200 205
Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe
210 215 220
Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys
225 230 235
<210> 7
<211> 236
<212> PRT
<213> 人工合成
<400> 7
Met Val Ser Lys Gly Glu Glu Asp Asn Met Ala Ile Ile Lys Glu Phe
1 5 10 15
Met Arg Phe Lys Val His Met Glu Gly Ser Val Asn Gly His Glu Phe
20 25 30
Glu Ile Glu Gly Glu Gly Glu Gly Arg Pro Tyr Glu Gly Thr Gln Thr
35 40 45
Ala Lys Leu Lys Val Thr Lys Gly Gly Pro Leu Pro Phe Ala Trp Asp
50 55 60
Ile Leu Ser Pro Gln Phe Met Tyr Gly Ser Lys Ala Tyr Val Lys His
65 70 75 80
Pro Ala Asp Ile Pro Asp Tyr Leu Lys Leu Ser Phe Pro Glu Gly Phe
85 90 95
Lys Trp Glu Arg Val Met Asn Phe Glu Asp Gly Gly Val Val Thr Val
100 105 110
Thr Gln Asp Ser Ser Leu Gln Asp Gly Glu Phe Ile Tyr Lys Val Lys
115 120 125
Leu Arg Gly Thr Asn Phe Pro Ser Asp Gly Pro Val Met Gln Lys Lys
130 135 140
Thr Met Gly Trp Glu Ala Ser Ser Glu Arg Met Tyr Pro Glu Asp Gly
145 150 155 160
Ala Leu Lys Gly Glu Ile Lys Gln Arg Leu Lys Leu Lys Asp Gly Gly
165 170 175
His Tyr Asp Ala Glu Val Lys Thr Thr Tyr Lys Ala Lys Lys Pro Val
180 185 190
Gln Leu Pro Gly Ala Tyr Asn Val Asn Ile Lys Leu Asp Ile Thr Ser
195 200 205
His Asn Glu Asp Tyr Thr Ile Val Glu Gln Tyr Glu Arg Ala Glu Gly
210 215 220
Arg His Ser Thr Gly Gly Met Asp Glu Leu Tyr Lys
225 230 235
<210> 8
<211> 189
<212> DNA
<213> 人工合成
<400> 8
ggatcataat cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac 60
acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg 120
cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt 180
tttcactgc 189
<210> 9
<211> 1265
<212> DNA
<213> 人工合成
<400> 9
aagctttgca aagatggata aagttttaaa cagagaggaa tctttgcagc taatggacct 60
tctaggtctt gaaaggagtg ggaattggct ccggtgcccg tcagtgggca gagcgcacat 120
cgcccacagt ccccgagaag ttggggggag gggtcggcaa ttgaaccggt gcctagagaa 180
ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg 240
gtgggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt 300
ttgccgccag aacacaggta agtgccgtgt gtggttcccg cgggcctggc ctctttacgg 360
gttatggccc ttgcgtgcct tgaattactt ccacctggct gcagtacgtg attcttgatc 420
ccgagcttcg ggttggaagt gggtgggaga gttcgaggcc ttgcgcttaa ggagcccctt 480
cgcctcgtgc ttgagttgag gcctggcctg ggcgctgggg ccgccgcgtg cgaatctggt 540
ggcaccttcg cgcctgtctc gctgctttcg ataagtctct agccatttaa aatttttgat 600
gacctgctgc gacgcttttt ttctggcaag atagtcttgt aaatgcgggc caagatctgc 660
acactggtat ttcggttttt ggggccgcgg gcggcgacgg ggcccgtgcg tcccagcgca 720
catgttcggc gaggcggggc ctgcgagcgc ggccaccgag aatcggacgg gggtagtctc 780
aagctggccg gcctgctctg gtgcctggcc tcgcgccgcc gtgtatcgcc ccgccctggg 840
cggcaaggct ggcccggtcg gcaccagttg cgtgagcgga aagatggccg cttcccggcc 900
ctgctgcagg gagctcaaaa tggaggacgc ggcgctcggg agagcgggcg ggtgagtcac 960
ccacacaaag gaaaagggcc tttccgtcct cagccgtcgc ttcatgtgac tccacggagt 1020
accgggcgcc gtccaggcac ctcgattagt tctcgagctt ttggagtacg tcgtctttag 1080
gttgggggga ggggttttat gcgatggagt ttccccacac tgagtgggtg gagactgaag 1140
ttaggccagc ttggcacttg atgtaattct ccttggaatt tgcccttttt gagtttggat 1200
cttggttcat tctcaagcct cagacagtgg ttcaaagttt ttttcttcca tttcaggtgt 1260
cgtga 1265
<210> 10
<211> 248
<212> PRT
<213> 人工合成
<400> 10
Met Ser Arg Leu Asp Lys Ser Lys Val Ile Asn Ser Ala Leu Glu Leu
1 5 10 15
Leu Asn Glu Val Gly Ile Glu Gly Leu Thr Thr Arg Lys Leu Ala Gln
20 25 30
Lys Leu Gly Val Glu Gln Pro Thr Leu Tyr Trp His Val Lys Asn Lys
35 40 45
Arg Ala Leu Leu Asp Ala Leu Ala Ile Glu Met Leu Asp Arg His His
50 55 60
Thr His Phe Cys Pro Leu Glu Gly Glu Ser Trp Gln Asp Phe Leu Arg
65 70 75 80
Asn Asn Ala Lys Ser Phe Arg Cys Ala Leu Leu Ser His Arg Asp Gly
85 90 95
Ala Lys Val His Leu Gly Thr Arg Pro Thr Glu Lys Gln Tyr Glu Thr
100 105 110
Leu Glu Asn Gln Leu Ala Phe Leu Cys Gln Gln Gly Phe Ser Leu Glu
115 120 125
Asn Ala Leu Tyr Ala Leu Ser Ala Val Gly His Phe Thr Leu Gly Cys
130 135 140
Val Leu Glu Asp Gln Glu His Gln Val Ala Lys Glu Glu Arg Glu Thr
145 150 155 160
Pro Thr Thr Asp Ser Met Pro Pro Leu Leu Arg Gln Ala Ile Glu Leu
165 170 175
Phe Asp His Gln Gly Ala Glu Pro Ala Phe Leu Phe Gly Leu Glu Leu
180 185 190
Ile Ile Cys Gly Leu Glu Lys Gln Leu Lys Cys Glu Ser Gly Gly Pro
195 200 205
Ala Asp Ala Leu Asp Asp Phe Asp Leu Asp Met Leu Pro Ala Asp Ala
210 215 220
Leu Asp Asp Phe Asp Leu Asp Met Leu Pro Ala Asp Ala Leu Asp Asp
225 230 235 240
Phe Asp Leu Asp Met Leu Pro Gly
245
<210> 11
<211> 501
<212> DNA
<213> 人工合成
<400> 11
gggtagggga ggcgcttttc ccaaggcagt ctggagcatg cgctttagca gccccgctgg 60
gcacttggcg ctacacaagt ggcctctggc ctcgcacaca ttccacatcc accggtaggc 120
gccaaccggc tccgttcttt ggtggcccct tcgcgccacc ttctactcct cccctagtca 180
ggaagttccc ccccgccccg cagctcgcgt cgtgcaggac gtgacaaatg gaagtagcac 240
gtctcactag tctcgtgcag atggacagca ccgctgagca atggaagcgg gtaggccttt 300
ggggcagcgg ccaatagcag ctttgctcct tcgctttctg ggctcagagg ctgggaaggg 360
gtgggtccgg gggcgggctc aggggcgggc tcaggggcgg ggcgggcgcc cgaaggtcct 420
ccggaggccc ggcattctgc acgcttcaaa agcgcacgtc tgccgcgctg ttctcctctt 480
cctcatctcc gggcctttcg a 501
<210> 12
<211> 19
<212> PRT
<213> 人工合成
<400> 12
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala
<210> 13
<211> 242
<212> PRT
<213> 人工合成
<400> 13
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
35 40 45
Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu
115 120 125
Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys
130 135 140
Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg
145 150 155 160
Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser
165 170 175
Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile
180 185 190
Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln
195 200 205
Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly
210 215 220
Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val
225 230 235 240
Ser Ser
<210> 14
<211> 243
<212> PRT
<213> 人工合成
<400> 14
Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly
1 5 10 15
Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser
20 25 30
Asn Ala Asn Thr Tyr Leu His Trp Tyr Leu Gln Lys Pro Gly Gln Ser
35 40 45
Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro
50 55 60
Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile
65 70 75 80
Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Asn
85 90 95
Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105 110
Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
115 120 125
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
130 135 140
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
145 150 155 160
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
165 170 175
Gly Ala Leu Asp Pro Lys Thr Gly Asp Thr Ala Tyr Ser Gln Lys Phe
180 185 190
Lys Gly Arg Val Thr Leu Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr
195 200 205
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
210 215 220
Thr Arg Phe Tyr Ser Tyr Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr
225 230 235 240
Val Ser Ser
<210> 15
<211> 557
<212> PRT
<213> 人工合成
<400> 15
Met Pro Pro Pro Arg Leu Leu Phe Phe Leu Leu Phe Leu Thr Pro Met
1 5 10 15
Glu Val Arg Pro Glu Glu Pro Leu Val Val Lys Val Glu Glu Gly Asp
20 25 30
Asn Ala Val Leu Gln Cys Leu Lys Gly Thr Ser Asp Gly Pro Thr Gln
35 40 45
Gln Leu Thr Trp Ser Arg Glu Ser Pro Leu Lys Pro Phe Leu Lys Leu
50 55 60
Ser Leu Gly Leu Pro Gly Leu Gly Ile His Met Arg Pro Leu Ala Ile
65 70 75 80
Trp Leu Phe Ile Phe Asn Val Ser Gln Gln Met Gly Gly Phe Tyr Leu
85 90 95
Cys Gln Pro Gly Pro Pro Ser Glu Lys Ala Trp Gln Pro Gly Trp Thr
100 105 110
Val Asn Val Glu Gly Ser Gly Glu Leu Phe Arg Trp Asn Val Ser Asp
115 120 125
Leu Gly Gly Leu Gly Cys Gly Leu Lys Asn Arg Ser Ser Glu Gly Pro
130 135 140
Ser Ser Pro Ser Gly Lys Leu Met Ser Pro Lys Leu Tyr Val Trp Ala
145 150 155 160
Lys Asp Arg Pro Glu Ile Trp Glu Gly Glu Pro Pro Cys Leu Pro Pro
165 170 175
Arg Asp Ser Leu Asn Gln Ser Leu Ser Gln Asp Leu Thr Met Ala Pro
180 185 190
Gly Ser Thr Leu Trp Leu Ser Cys Gly Val Pro Pro Asp Ser Val Ser
195 200 205
Arg Gly Pro Leu Ser Trp Thr His Val His Pro Lys Gly Pro Lys Ser
210 215 220
Leu Leu Ser Leu Glu Leu Lys Asp Asp Arg Pro Ala Arg Asp Met Trp
225 230 235 240
Val Met Glu Thr Gly Leu Leu Leu Pro Arg Ala Thr Ala Gln Asp Ala
245 250 255
Gly Lys Tyr Tyr Cys His Arg Gly Asn Leu Thr Met Ser Phe His Leu
260 265 270
Glu Ile Thr Ala Arg Pro Val Leu Trp His Trp Leu Leu Arg Thr Gly
275 280 285
Gly Trp Lys Val Ser Ala Val Thr Leu Ala Tyr Leu Ile Phe Cys Leu
290 295 300
Cys Ser Leu Val Gly Ile Leu His Leu Gln Arg Ala Leu Val Leu Arg
305 310 315 320
Arg Lys Arg Lys Arg Met Thr Asp Pro Thr Arg Arg Phe Phe Lys Val
325 330 335
Thr Pro Pro Pro Gly Ser Gly Pro Gln Asn Gln Tyr Gly Asn Val Leu
340 345 350
Ser Leu Pro Thr Pro Thr Ser Gly Leu Gly Arg Ala Gln Arg Trp Ala
355 360 365
Ala Gly Leu Gly Gly Thr Ala Pro Ser Tyr Gly Asn Pro Ser Ser Asp
370 375 380
Val Gln Ala Asp Gly Ala Leu Gly Ser Arg Ser Pro Pro Gly Val Gly
385 390 395 400
Pro Glu Glu Glu Glu Gly Glu Gly Tyr Glu Glu Pro Asp Ser Glu Glu
405 410 415
Asp Ser Glu Phe Tyr Glu Asn Asp Ser Asn Leu Gly Gln Asp Gln Leu
420 425 430
Ser Gln Asp Gly Ser Gly Tyr Glu Asn Pro Glu Asp Glu Pro Leu Gly
435 440 445
Pro Glu Asp Glu Asp Ser Phe Ser Asn Ala Glu Ser Tyr Glu Asn Glu
450 455 460
Asp Glu Glu Leu Thr Gln Pro Val Ala Arg Thr Met Asp Phe Leu Ser
465 470 475 480
Pro His Gly Ser Ala Trp Asp Pro Ser Arg Glu Ala Thr Ser Leu Ala
485 490 495
Gly Ser Gln Ser Tyr Glu Asp Met Arg Gly Ile Leu Tyr Ala Ala Pro
500 505 510
Gln Leu Arg Ser Ile Arg Gly Gln Pro Gly Pro Asn His Glu Glu Asp
515 520 525
Ala Asp Ser Tyr Glu Asn Met Asp Asn Pro Asp Gly Pro Asp Pro Ala
530 535 540
Trp Gly Gly Gly Gly Arg Met Gly Thr Trp Ser Thr Arg
545 550 555
<210> 16
<211> 603
<212> PRT
<213> 人工合成
<400> 16
Met Ala Gly Thr Val Arg Thr Ala Cys Leu Val Val Ala Met Leu Leu
1 5 10 15
Ser Leu Asp Phe Pro Gly Gln Ala Gln Pro Pro Pro Pro Pro Pro Asp
20 25 30
Ala Thr Cys His Gln Val Arg Ser Phe Phe Gln Arg Leu Gln Pro Gly
35 40 45
Leu Lys Trp Val Pro Glu Thr Pro Val Pro Gly Ser Asp Leu Gln Val
50 55 60
Cys Leu Pro Lys Gly Pro Thr Cys Cys Ser Arg Lys Met Glu Glu Lys
65 70 75 80
Tyr Gln Leu Thr Ala Arg Leu Asn Met Glu Gln Leu Leu Gln Ser Ala
85 90 95
Ser Met Glu Leu Lys Phe Leu Ile Ile Gln Asn Ala Ala Val Phe Gln
100 105 110
Glu Ala Phe Glu Ile Val Val Arg His Ala Lys Asn Tyr Thr Asn Ala
115 120 125
Met Phe Lys Asn Asn Tyr Pro Ser Leu Thr Pro Gln Ala Phe Glu Phe
130 135 140
Val Gly Glu Phe Phe Thr Asp Val Ser Leu Tyr Ile Leu Gly Ser Asp
145 150 155 160
Ile Asn Val Asp Asp Met Val Asn Glu Leu Phe Asp Ser Leu Phe Pro
165 170 175
Val Ile Tyr Thr Gln Leu Met Asn Pro Gly Leu Pro Asp Ser Ala Leu
180 185 190
Asp Ile Asn Glu Cys Leu Arg Gly Ala Arg Arg Asp Leu Lys Val Phe
195 200 205
Gly Asn Phe Pro Lys Leu Ile Met Thr Gln Val Ser Lys Ser Leu Gln
210 215 220
Val Thr Arg Ile Phe Leu Gln Ala Leu Asn Leu Gly Ile Glu Val Ile
225 230 235 240
Asn Thr Thr Asp His Leu Lys Phe Ser Lys Asp Cys Gly Arg Met Leu
245 250 255
Thr Arg Met Trp Tyr Cys Ser Tyr Cys Gln Gly Leu Met Met Val Lys
260 265 270
Pro Cys Gly Gly Tyr Cys Asn Val Val Met Gln Gly Cys Met Ala Gly
275 280 285
Val Val Glu Ile Asp Lys Tyr Trp Arg Glu Tyr Ile Leu Ser Leu Glu
290 295 300
Glu Leu Val Asn Gly Met Tyr Arg Ile Tyr Asp Met Glu Asn Val Leu
305 310 315 320
Leu Gly Leu Phe Ser Thr Ile His Asp Ser Ile Gln Tyr Val Gln Lys
325 330 335
Asn Ala Gly Lys Leu Thr Thr Thr Glu Thr Glu Lys Lys Ile Trp His
340 345 350
Phe Lys Tyr Pro Ile Phe Phe Leu Cys Ile Gly Leu Asp Leu Gln Ile
355 360 365
Gly Lys Leu Cys Ala His Ser Gln Gln Arg Gln Tyr Arg Ser Ala Tyr
370 375 380
Tyr Pro Glu Asp Leu Phe Ile Asp Lys Lys Val Leu Lys Val Ala His
385 390 395 400
Val Glu His Glu Glu Thr Leu Ser Ser Arg Arg Arg Glu Leu Ile Gln
405 410 415
Lys Leu Lys Ser Phe Ile Ser Phe Tyr Ser Ala Leu Pro Gly Tyr Ile
420 425 430
Cys Ser His Ser Pro Val Ala Glu Asn Asp Thr Leu Cys Trp Asn Gly
435 440 445
Gln Glu Leu Val Glu Arg Tyr Ser Gln Lys Ala Ala Arg Asn Gly Met
450 455 460
Lys Asn Gln Phe Asn Leu His Glu Leu Lys Met Lys Gly Pro Glu Pro
465 470 475 480
Val Val Ser Gln Ile Ile Asp Lys Leu Lys His Ile Asn Gln Leu Leu
485 490 495
Arg Thr Met Ser Met Pro Lys Gly Arg Val Leu Asp Lys Asn Leu Asp
500 505 510
Glu Glu Gly Phe Glu Ser Gly Asp Cys Gly Asp Asp Glu Asp Glu Cys
515 520 525
Ile Gly Gly Ser Gly Asp Gly Met Ile Lys Val Lys Asn Gln Leu Arg
530 535 540
Phe Leu Ala Glu Leu Ala Tyr Asp Leu Asp Val Asp Asp Ala Pro Gly
545 550 555 560
Asn Ser Gln Gln Ala Thr Pro Lys Asp Asn Glu Ile Ser Thr Phe His
565 570 575
Asn Leu Gly Asn Val His Ser Pro Leu Lys Leu Leu Thr Ser Met Ala
580 585 590
Ile Ser Val Val Cys Phe Phe Phe Leu Val His
595 600
<210> 17
<211> 233
<212> PRT
<213> 人工合成
<400> 17
Met Ser Glu Leu Ile Lys Glu Asn Met His Met Lys Leu Tyr Met Glu
1 5 10 15
Gly Thr Val Asp Asn His His Phe Lys Cys Thr Ser Glu Gly Glu Gly
20 25 30
Lys Pro Tyr Glu Gly Thr Gln Thr Met Arg Ile Lys Val Val Glu Gly
35 40 45
Gly Pro Leu Pro Phe Ala Phe Asp Ile Leu Ala Thr Ser Phe Leu Tyr
50 55 60
Gly Ser Lys Thr Phe Ile Asn His Thr Gln Gly Ile Pro Asp Phe Phe
65 70 75 80
Lys Gln Ser Phe Pro Glu Gly Phe Thr Trp Glu Arg Val Thr Thr Tyr
85 90 95
Glu Asp Gly Gly Val Leu Thr Ala Thr Gln Asp Thr Ser Leu Gln Asp
100 105 110
Gly Cys Leu Ile Tyr Asn Val Lys Ile Arg Gly Val Asn Phe Thr Ser
115 120 125
Asn Gly Pro Val Met Gln Lys Lys Thr Leu Gly Trp Glu Ala Phe Thr
130 135 140
Glu Thr Leu Tyr Pro Ala Asp Gly Gly Leu Glu Gly Arg Asn Asp Met
145 150 155 160
Ala Leu Lys Leu Val Gly Gly Ser His Leu Ile Ala Asn Ile Lys Thr
165 170 175
Thr Tyr Arg Ser Lys Lys Pro Ala Lys Asn Leu Lys Met Pro Gly Val
180 185 190
Tyr Tyr Val Asp Tyr Arg Leu Glu Arg Ile Lys Glu Ala Asn Asn Glu
195 200 205
Thr Tyr Val Glu Gln His Glu Val Ala Val Ala Arg Tyr Cys Asp Leu
210 215 220
Pro Ser Lys Leu Gly His Lys Leu Asn
225 230
<210> 18
<211> 17
<212> DNA
<213> 人工合成
<400> 18
agtggcctca caggtat 17
<210> 19
<211> 16
<212> DNA
<213> 人工合成
<400> 19
acagtcactg agctgc 16
<210> 20
<211> 50
<212> PRT
<213> 人工合成
<400> 20
Asn Ala Val Gly Gln Asp Thr Gln Glu Val Ile Val Val Pro His Ser
1 5 10 15
Leu Pro Phe Lys Val Val Val Ile Ser Ala Ile Leu Ala Leu Val Val
20 25 30
Leu Thr Ile Ile Ser Leu Ile Ile Leu Ile Met Leu Trp Gln Lys Lys
35 40 45
Pro Arg
50
<210> 21
<211> 395
<212> PRT
<213> 人工合成
<400> 21
Met Gly Val Gln Val Glu Thr Ile Ser Pro Gly Asp Gly Arg Thr Phe
1 5 10 15
Pro Lys Arg Gly Gln Thr Cys Val Val His Tyr Thr Gly Met Leu Glu
20 25 30
Asp Gly Lys Lys Val Asp Ser Ser Arg Asp Arg Asn Lys Pro Phe Lys
35 40 45
Phe Met Leu Gly Lys Gln Glu Val Ile Arg Gly Trp Glu Glu Gly Val
50 55 60
Ala Gln Met Ser Val Gly Gln Arg Ala Lys Leu Thr Ile Ser Pro Asp
65 70 75 80
Tyr Ala Tyr Gly Ala Thr Gly His Pro Gly Ile Ile Pro Pro His Ala
85 90 95
Thr Leu Val Phe Asp Val Glu Leu Leu Lys Leu Glu Ser Gly Gly Gly
100 105 110
Ser Gly Phe Gly Asp Val Gly Ala Leu Glu Ser Leu Arg Gly Asn Ala
115 120 125
Asp Leu Ala Tyr Ile Leu Ser Met Glu Pro Cys Gly His Cys Leu Ile
130 135 140
Ile Asn Asn Val Asn Phe Cys Arg Glu Ser Gly Leu Arg Thr Arg Thr
145 150 155 160
Gly Ser Asn Ile Asp Cys Glu Lys Leu Arg Arg Arg Phe Ser Ser Leu
165 170 175
His Phe Met Val Glu Val Lys Gly Asp Leu Thr Ala Lys Lys Met Val
180 185 190
Leu Ala Leu Leu Glu Leu Ala Arg Gln Asp His Gly Ala Leu Asp Cys
195 200 205
Cys Val Val Val Ile Leu Ser His Gly Cys Gln Ala Ser His Leu Gln
210 215 220
Phe Pro Gly Ala Val Tyr Gly Thr Asp Gly Cys Pro Val Ser Val Glu
225 230 235 240
Lys Ile Val Asn Ile Phe Asn Gly Thr Ser Cys Pro Ser Leu Gly Gly
245 250 255
Lys Pro Lys Leu Phe Phe Ile Gln Ala Cys Gly Gly Glu Gln Lys Asp
260 265 270
His Gly Phe Glu Val Ala Ser Thr Ser Pro Glu Asp Glu Ser Pro Gly
275 280 285
Ser Asn Pro Glu Pro Asp Ala Thr Pro Phe Gln Glu Gly Leu Arg Thr
290 295 300
Phe Asp Gln Leu Asp Ala Ile Ser Ser Leu Pro Thr Pro Ser Asp Ile
305 310 315 320
Phe Val Ser Tyr Ser Thr Phe Pro Gly Phe Val Ser Trp Arg Asp Pro
325 330 335
Lys Ser Gly Ser Trp Tyr Val Glu Thr Leu Asp Asp Ile Phe Glu Gln
340 345 350
Trp Ala His Ser Glu Asp Leu Gln Ser Leu Leu Leu Arg Val Ala Asn
355 360 365
Ala Val Ser Val Lys Gly Ile Tyr Lys Gln Met Pro Gly Cys Phe Asn
370 375 380
Phe Leu Arg Lys Lys Leu Phe Phe Lys Thr Ser
385 390 395
<210> 22
<211> 256
<212> DNA
<213> 人工合成
<400> 22
taggtcttga aaggagtggg aattggctcc ggtgcccgtc agtgggcaga gcgcacatcg 60
cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt gatccggtgc ctagagaagg 120
tggcgcgggg taaactggga aagtgatgtc gtgtactggc tccgcctttt tcccgagggt 180
gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttcg caacgggttt 240
gccgccagaa cacagg 256
<210> 23
<211> 132
<212> PRT
<213> 人工合成
<400> 23
Met Ala Lys Pro Leu Ser Gln Glu Glu Ser Thr Leu Ile Glu Arg Ala
1 5 10 15
Thr Ala Thr Ile Asn Ser Ile Pro Ile Ser Glu Asp Tyr Ser Val Ala
20 25 30
Ser Ala Ala Leu Ser Ser Asp Gly Arg Ile Phe Thr Gly Val Asn Val
35 40 45
Tyr His Phe Thr Gly Gly Pro Cys Ala Glu Leu Val Val Leu Gly Thr
50 55 60
Ala Ala Ala Ala Ala Ala Gly Asn Leu Thr Cys Ile Val Ala Ile Gly
65 70 75 80
Asn Glu Asn Arg Gly Ile Leu Ser Pro Cys Gly Arg Cys Arg Gln Val
85 90 95
Leu Leu Asp Leu His Pro Gly Ile Lys Ala Ile Val Lys Asp Ser Asp
100 105 110
Gly Gln Pro Thr Ala Val Gly Ile Arg Glu Leu Leu Pro Ser Gly Tyr
115 120 125
Val Trp Glu Gly
130
<210> 24
<211> 21
<212> DNA
<213> 人工合成
<400> 24
gcattctgca cgcttcaaaa g 21
<210> 25
<211> 22
<212> DNA
<213> 人工合成
<400> 25
gaagctgtag tccaggatgt gg 22
<210> 26
<211> 227
<212> PRT
<213> 人工合成
<400> 26
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
1 5 10 15
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
20 25 30
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
35 40 45
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
50 55 60
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
65 70 75 80
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
85 90 95
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
100 105 110
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
115 120 125
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
130 135 140
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
145 150 155 160
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
210 215 220
Pro Gly Lys
225

Claims (14)

1.一种抗体文库的构建方法，其特征在于，所述方法以synNotch系统控制细胞内基因表达为原理，将元件一和元件二插入同一载体或不同载体，转染至细胞内，得到抗体表达细胞文库，即所述抗体文库；
其中，所述元件一包括顺式激活子和筛选标记基因，所述元件二包括胞外抗体文库编码域、Notch核心结构域和胞内转录结构域；
所述筛选标记基因包括药物抗性基因、自杀基因、荧光蛋白基因或分子标签中的任意一种或至少两种的组合；
所述Notch核心结构域包括人的Notch和/或小鼠的Notch；
所述人的Notch的氨基酸序列如SEQ ID NO.1所示；
所述小鼠的Notch的氨基酸序列如SEQ ID NO.2所示；
所述顺式激活子包括pTet和/或UAS-pSV40；
所述转录结构域包括tTA和/或Gal4-VP64；
若要进行抗体的正筛选，将筛选标记基因设计为药物抗性基因，则激活的细胞可以在有筛选药物的培养基中存活，其余的细胞死亡，从而筛选出针对目标抗原的抗体表达细胞；若要进行抗体的负筛选，将筛选标记基因设计为自杀基因，则激活的细胞在有筛选药物的培养基中凋亡，其余的细胞存活，从而除去针对目标抗原的抗体表达细胞；通过流式细胞仪检测荧光或抗标签抗体，即可进行正筛选，也可进行负筛选。
2.根据权利要求1所述的方法，其特征在于，所述药物抗性基因包括嘌呤霉素抗性基因、新霉素抗性基因、杀稻瘟素抗性基因或潮霉素B抗性基因中的任意一种或至少两种的组合。
3.根据权利要求1所述的方法，其特征在于，所述自杀基因包括单纯疱疹病毒胸苷激酶基因、胞嘧啶脱氨酶基因或iCasp9自杀系统基因中的任意一种或至少两种的组合。
4.根据权利要求1所述的方法，其特征在于，所述荧光蛋白基因包括EGFP、YFP、mCherry、DsRed或BFP中的任意一种或至少两种的组合。
5.根据权利要求1所述的方法，其特征在于，所述分子标签包括His-tag、Flag-tag、HA-tag、Myc-tag或Strep-tag中的任意一种或至少两种的组合。
6.根据权利要求1所述的方法，其特征在于，所述筛选标记基因的筛选方法包括药物筛选、流式细胞仪检测并分选或磁珠分选中的任意一种或至少两种的组合。
7.根据权利要求1所述的方法，其特征在于，所述药物抗性基因的筛选药物包括嘌呤霉素、G418、杀稻瘟素或潮霉素B中的任意一种或至少两种的组合。
8.根据权利要求1所述的方法，其特征在于，所述自杀基因的筛选药物包括更昔洛韦或FIAU、5-氟胞嘧啶、AP1903或AP20187中的任意一种或至少两种的组合。
9.根据权利要求1所述的方法，其特征在于，所述胞外抗体文库编码域包括抗体序列、抗体重链序列、抗体轻链序列、抗体可变区序列、单链抗体序列、单域抗体序列或Fab片段序列中的任意一种或至少两种的组合。
10.根据权利要求1所述的方法，其特征在于，所述胞外抗体文库编码域的来源包括免疫动物、疾病人群、健康人群、接种过疫苗的人群或人工合成中的任意一种或至少两种的组合。
11.根据权利要求1所述的方法，其特征在于，所述转染的方法包括病毒转染、化学转染试剂转染或电击转染中的任意一种或至少两种的组合。
12.根据权利要求1所述的方法，其特征在于，所述编码域、结构域或基因包含编码蛋白质的氨基酸序列、编码蛋白的DNA序列或编码蛋白的RNA序列中的任意一种或至少两种的组合。
13.一种抗体文库，其特征在于，所述文库由权利要求1-12中任一项所述方法构建得到。
14.一种筛选抗体的方法，其特征在于，采用权利要求13所述抗体文库进行筛选，包括如下步骤：
(1)将抗体文库与抗原接触；
(2)根据筛选标记基因的表达情况，筛选表达目标抗体的细胞；
其中，所述抗体包括单克隆抗体或多克隆抗体；
所述抗原包括野生型细胞、转染特定抗原基因的细胞、结合特定抗原的细胞、溶解在培养基中的抗原、包被在培养器皿上的抗原、包被在微珠上的抗原或包被在培养支架上的抗原中的任意一种或至少两种的组合。
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CN114437232B (zh) * 2020-11-05 2023-11-24 中国科学院分子细胞科学卓越创新中心 一种细胞表面大分子定量展示系统及其制备方法和用途
CN115197968A (zh) * 2021-04-09 2022-10-18 新乡医学院 抗原结合域自动优化的嵌合抗原受体修饰细胞文库的构建、筛选方法及其应用
WO2023025208A1 (zh) * 2021-08-24 2023-03-02 赛斯尔擎生物技术（上海）有限公司 一种修饰细胞的方法
CN114621929B (zh) * 2021-12-21 2023-05-30 河南省肿瘤医院 一种抗肿瘤树突状细胞及其制备方法、表达载体及应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108864290A (zh) * 2017-05-08 2018-11-23 上海津曼特生物科技有限公司 双特异性重组蛋白及其应用

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DK2981607T3 (da) * 2013-04-03 2020-11-16 Memorial Sloan Kettering Cancer Center Effektiv generering af tumormålrettede t-celler afledt af pluripotente stamceller
AU2016222887B2 (en) * 2015-02-24 2022-07-14 The Regents Of The University Of California Binding-triggered transcriptional switches and methods of use thereof
JP6878408B2 (ja) * 2015-09-01 2021-05-26 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニアＴｈｅ Ｒｅｇｅｎｔｓ Ｏｆ Ｔｈｅ Ｕｎｉｖｅｒｓｉｔｙ Ｏｆ Ｃａｌｉｆｏｒｎｉａ モジュールポリペプチドライブラリならびにその作製方法及びその使用
CN113774495A (zh) * 2015-09-25 2021-12-10 阿布维特罗有限责任公司 用于对天然配对t细胞受体序列进行t细胞受体靶向鉴别的高通量方法
CN109576292B (zh) * 2018-12-21 2022-11-29 深圳市爱思迪生物科技有限公司 一种抗体文库的构建方法及其应用

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108864290A (zh) * 2017-05-08 2018-11-23 上海津曼特生物科技有限公司 双特异性重组蛋白及其应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch Receptors;Leonardo Morsut;《Cell》;20161231;第164卷(第4期);第18页图3B以及图3B的注释、第9页第2段最后1句、摘要、第6页最后1段 *

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