CN114395045A - B7h6抗体及其应用 - Google Patents

B7h6抗体及其应用 Download PDF

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CN114395045A
CN114395045A CN202111485380.9A CN202111485380A CN114395045A CN 114395045 A CN114395045 A CN 114395045A CN 202111485380 A CN202111485380 A CN 202111485380A CN 114395045 A CN114395045 A CN 114395045A
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val
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amino acid
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CN114395045B (zh
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田志刚
曹国帅
肖卫华
孙汭
孙昊昱
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Hefei Tiangang Immune Drugs Co ltd
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Hefei Tiangang Immune Drugs Co ltd
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Priority to TW111146966A priority patent/TWI848461B/zh
Priority to CA3227094A priority patent/CA3227094A1/en
Priority to IL310514A priority patent/IL310514A/en
Priority to EP22903484.8A priority patent/EP4378956A1/en
Priority to MX2024003129A priority patent/MX2024003129A/es
Priority to PCT/CN2022/137095 priority patent/WO2023104062A1/zh
Priority to TW113110120A priority patent/TW202426507A/zh
Priority to AU2022404479A priority patent/AU2022404479A1/en
Priority to KR1020247004588A priority patent/KR20240032996A/ko
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Abstract

本发明涉及抗体领域，公开了B7H6的抗体及其用途。所述抗体包括重链CDR1、重链CDR2和重链CDR3，以及轻链CDR1、轻链CDR2和轻链CDR3。本发明的抗体能够与B7H6结合，促进B7H6与NK细胞活化性受体NKp30的结合，以及具有抗癌功能。

Description

B7H6抗体及其应用
技术领域
本发明涉及抗体领域，具体地，涉及一种能够结合B7H6的抗体或其抗原结合片段及用途。
背景技术
NK细胞是机体中非常重要的一种固有样淋巴细胞，在抗病毒、抗肿瘤中发挥重要作用。NK细胞表面表达多种活化性受体，包括NKp30、NKG2D等，识别并结合肿瘤细胞表面表达的配体，这些配体都是由自身基因编码的，在细胞恶性转化(肿瘤)等条件下表达在细胞表面。
NKp30是种重要的NK细胞活化性受体，与其配体结合后，传递活化信号，促进NK细胞杀伤肿瘤。B7H6是已知的唯一一种膜蛋白形式表达的NKp30配体，B7-H6在多种肿瘤组织如黑色素瘤、肝癌等高表达，在正常组织中不表达，具有肿瘤特异性。
肿瘤微环境中TGF-β等细胞因子下调NK细胞表面NKp30表达，造成NK细胞无法利用NKp30-B7H6相互作用精准识别、灭杀肿瘤，进而造成肿瘤免疫逃逸。因而提供一种靶向B7H6的治疗性抗体药物十分必要。
发明内容
本发明的目的是克服现有技术的不足，提供一种能够与人及猴B7H6结合、并促进B7H6与NKp30结合的B7H6抗体及其用途。
本发明提供的抗体通过促进B7H6与NKp30的结合，促进NK细胞的抗癌功能。
在一方面，本发明提供了一种能够结合B7H6的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段包括重链CDR1、重链CDR2和重链CDR3，以及轻链CDR1、轻链CDR2和轻链CDR3，其中，
所述重链CDR1包含选自SEQ ID NO:1、SEQ ID NO:7所示的氨基酸序列及其保守修饰形式的氨基酸序列至少之一；
所述重链CDR2包含选自SEQ ID NO:2、SEQ ID NO:8所示的氨基酸序列及其保守修饰形式的氨基酸序列至少之一；
所述重链CDR3包含选自SEQ ID NO:3、SEQ ID NO:9所示的氨基酸序列及其保守修饰形式的氨基酸序列至少之一；
所述轻链CDR1包含选自SEQ ID NO:4、SEQ ID NO:10所示的氨基酸序列及其保守修饰形式的氨基酸序列至少之一；
所述轻链CDR2包含自SEQ ID NO:5、SEQ ID NO:11所示的氨基酸序列及其保守修饰形式的氨基酸序列至少之一；以及
所述轻链CDR3含选自SEQ ID NO:6、SEQ ID NO:12所示的氨基酸序列及其保守修饰形式的氨基酸序列至少之一。
在某些实施例中，所述的抗体或其抗原结合片段包含：
1)具有SEQ ID NO:1所示的氨基酸序列的重链CDR1，具有SEQ ID NO:2所示的氨基酸序列的重链CDR2，具有SEQ ID NO:3所示的氨基酸序列的重链CDR3，具有SEQ ID NO:4所示的氨基酸序列的轻链CDR1，具有SEQ ID NO:5所示的氨基酸序列的轻链CDR2，具有SEQ IDNO:6所示的氨基酸序列的轻链CDR3：或
2)具有SEQ ID NO:7所示的氨基酸序列的重链CDR1，具有SEQ ID NO:8所示的氨基酸序列的重链CDR2，具有SEQ ID NO:9所示的氨基酸序列的重链CDR3，具有SEQ ID NO:10所示的氨基酸序列的轻链CDR1，具有SEQ ID NO:11所示的氨基酸序列的轻链CDR2，具有SEQID NO:12所示的氨基酸序列的轻链CDR3。
在某些实施例中，所述抗体或其抗原结合片段包括重链可变区和轻链可变区，(i)所述重链可变区包含SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19所示的氨基酸序列及其保守修饰形式的氨基酸序列至少之一至少80％同源性的氨基酸序列；和/或，(ii)所述轻链可变区包含SEQ ID NO:14、SEQ ID NO:16、SEQ ID NO:18、SEQ ID NO:20所示的氨基酸序列及其保修修饰形式的氨基酸序列至少之一至少80％同源性的氨基酸序列。
在某些实施例中，所述重链可变区包含与选自(i)中的重链可变区至少90％，至少95％，至少96％，至少97％，至少98％，至少99％或者100％同源性的氨基酸序列；所述轻链可变区包含与选自(ii)中的轻链可变区至少90％，至少95％，至少96％，至少97％，至少98％，至少99％或者100％同源性的氨基酸序列。
在某些实施例中，所述抗体或其抗原结合片段包括：
1)所述重链可变区如SEQ ID NO:13所示的氨基酸序列，以及所述轻链可变区如SEQ ID NO:14所示的氨基酸序列；
2)所述重链可变区如SEQ ID NO:15所示的氨基酸序列，以及所述轻链可变区如SEQ ID NO:16所示的氨基酸序列；
3)所述重链可变区如SEQ ID NO:17所示的氨基酸序列，以及所述轻链可变区如SEQ ID NO:18所示的氨基酸序列；或
4)所述重链可变区如SEQ ID NO:19所示的氨基酸序列，以及所述轻链可变区如SEQ ID NO:20所示的氨基酸序列。
在某些实施例中，所述抗体或其抗原结合片段包括：
1)所述重链如SEQ ID NO:26所示的氨基酸序列，以及所述轻链如SEQ ID NO:27所示的氨基酸序列；
2)所述重链如SEQ ID NO:28所示的氨基酸序列，以及所述轻链如SEQ ID NO:29所示的氨基酸序列；
3)所述重链如SEQ ID NO:30所示的氨基酸序列，以及所述轻链如SEQ ID NO:31所示的氨基酸序列；或
4)所述重链如SEQ ID NO:32所示的氨基酸序列，以及所述轻链如SEQ ID NO:33所示的氨基酸序列。
在某些实施例中，所述的分离的抗体是IgG1、IgG2或IgG4。
在某些实施例中，所述抗体为单克隆抗体、鼠源抗体、嵌合抗体、人源化抗体人改造抗体、人抗体、Fv、单链抗体(scFv)、Fab、Fab’，Fab’-SH或者F(ab’)2。
在某些实施例中，所述抗体或其抗原结合片段能够结合SEQ ID NO:21所示的氨基酸序列。
在另一方面，本发明提供了一种免疫缀合物，该免疫缀合物含有治疗剂以及与治疗剂偶联的上述的抗体或其抗原结合片段。
在又一方面，本发明提供了一种组合物，其中，所述组合物含有上述的抗体或其抗原结合片段、和/或上述的免疫缀合物、以及药学上可接受的载体。
在再一方面，本发明提供了一种用于检测样品中B7H6的试剂盒，该试剂盒含有上述抗体或其抗原结合片段。
在又一方面，本发明提供了上述的抗体或其抗原结合片段在制备用于检测样品中B7H6的试剂中的用途。
在再一方面，本发明提供了上述的抗体或其抗原结合片段在制备用于预防和/或治疗B7H6介导的疾病的药物中的用途，所述B7H6介导的疾病为癌症或感染性疾病，
任选地，所述癌症为肺癌，肝癌，卵巢癌，宫颈癌，皮肤癌，膀胱癌，结肠癌，乳腺癌，神经胶质瘤，肾癌，胃癌，食道癌，口腔鳞状细胞癌和头颈癌中的至少一种。
在又一方面，本发明提供了一种核酸，其包含编码上述的抗体或其抗原结合片段的核酸。
在再一方面，本发明提供了一种重组载体或转化子，其含有上述的核酸。
通过上述技术方案，本发明提供了能够结合人及猴B7H6的抗B7H6抗体或抗体片段。本发明的抗体具有以下特性的至少一种：能够结合人及猴B7H6；能够促进B7H6与NKp30的相互作用；能够促进NK细胞抗癌功能。
本发明的其它特征和优点将在随后的具体实施方式部分予以详细说明。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
附图是用来提供对本发明的进一步理解，并且构成说明书的一部分，与下面的具体实施方式一起用于解释本发明，但并不构成对本发明的限制。在附图中：
图1为根据本发明具体实施方式的鼠源B7H6抗体结合人B7H6的ELISA结果图；
图2为根据本发明具体实施方式的鼠源B7H6抗体结合HL60-B7H6细胞的结果图；
图3为根据本发明具体实施方式的人源化B7H6抗体结合人B7H6的ELISA结果图；
图4为根据本发明具体实施方式的人源化B7H6抗体结合HL60-B7H6细胞的结果图；
图5为根据本发明具体实施方式的人源化B7H6抗体结合CHO-K1-cyno B7H6细胞的结果图；
图6为根据本发明具体实施方式的人源化B7H6抗体结合猴B7H6的ELISA结果图；
图7为根据本发明具体实施方式的B7H6抗体促进受体NKp30结合的结果图；
图8为根据本发明具体实施方式的B7H6抗体介导补体依赖的细胞毒效应的结果图；
图9为根据本发明具体实施方式的B7H6抗体促进裸鼠对抗人急性髓系白血病U-937肿瘤的结果图。
具体实施方式
下面详细描述本发明的实施例。下面描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。
需要说明的是，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括一个或者更多个该特征。进一步地，在本发明的描述中，除非另有说明，“多个”的含义是两个或两个以上。
以下对本发明的具体实施方式进行详细说明。应当理解的是，此处所描述的具体实施方式仅用于说明和解释本发明，并不用于限制本发明。
在本文中所披露的范围的端点和任何值都不限于该精确的范围或值，这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说，各个范围的端点值之间、各个范围的端点值和单独的点值之间，以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围，这些数值范围应被视为在本文中具体公开。
为了更容易理解本发明，以下具体定义了某些技术和科学术语。除显而易见在本文件中的它处另有明确定义，否则本文中使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常理解的含义。氨基酸残基的缩写是本领域中所用的指代20个常用L-氨基酸之一的标准3字母和/或1字母代码。
在本发明中，在未作相反说明的情况下，使用的术语抗原结合片段也即“抗体片段”，抗体片段通常是指抗原结合性抗体片段，可以包括完整抗体的一部分，一般是抗原结合区或可变区，抗体片段的实例包括Fab、Fab’、F(ab’)2、Fv或scFv，双抗体，线性抗体，单链抗体分子等。
术语“互补决定区”或“CDR”或“CDR序列”是指抗体中负责抗原结合的氨基酸序列，例如，通常包括：轻链可变区中23-34(L1)、50-56(L2)和89-97(L3)附近，和重链可变区中31-35B(H1)、50-65(H2)和95-102(H3)附近的氨基酸残基(Kabat等人，Sequences ofProteins of Immunological Interest，5th Ed.Public Health Service，NationalInstitutes of Health，Bethesda，MD.(1991))；和/或来自“高变环”(例如，轻链可变区中26-32(LI)、50-52(L2)和91-96(L3)，和重链可变区中26-32(H1)、53-55(H2)和96-101(H3)附近的氨基酸残基(Chothia和Lesk J.Mol.Biol.196：901-917(1987))。
术语“保守修饰形式的氨基酸序列”指这样的氨基酸修饰，它不显著影响或改变包含该氨基酸序列的抗体的结合特性，该修饰包括氨基酸置换、增加和缺失。修饰可通过例如定点诱变和PCR介导的诱变等标准技术引入本发明的抗体中。保守氨基酸置换系其中的氨基酸残基被具有相似侧链的氨基酸残基替换的置换。在本领域中已经确定具有相似侧链的氨基酸残基家族。这些家族包括具有碱性侧链的氨基酸(如赖氨酸、精氨酸、组氨酸)，具有酸性侧链的氨基酸(如天冬氨酸、谷氨酸)，具有不带电的极性侧链的氨基酸(如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)，具有非极性侧链的氨基酸(如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)，具有β-支化侧链的氨基酸(如苏氨酸、缬氨酸、异亮氨酸)、和具有芳香侧链的氨基酸(如酪氨酸、苯丙氨酸、色氨酸、组氨酸)。因此，本发明的抗体的CDR区中的一个或多个氨基酸残基可以被来自相同侧链家族的其他氨基酸残基替代，并且使可用此处说明的功能测定方法对经改变的抗体的保留功能进行测试。优选的是，保守修饰在数目上不超过1个或2个。
对于多肽而言，术语“实质同源性”表示，两个多肽或其指定序列在最佳比对及比较(其中适当插入或缺失核苷酸)时有至少约80％的氨基酸、通常至少约90％至95％、且更优选至少约98％至99.5％的氨基酸相同。
两条序列之间的同一性％在最佳比对序列时随这些序列所共享的相同位置数而变化(即同源性％＝相同位置数/总位置数×100)，其中最佳比对系考虑到为达成两条序列的最佳比对而需要引入的空位数及每一空位的长度来确定。两条序列之间的序列比较及同一性百分数测定可使用数学算法来完成，如下文非限制性实施例中所述。
在不实质性影响抗体活性(保留至少95％的活性)的前提下，本领域技术人员可以对本发明的序列替换、添加和/或缺失一个或更多个(例如1、2、3、4、5、6、7、8、9或10个或更多个)氨基酸，以获得所述抗体或其功能性片段之序列的变体。它们都被视为包括在本发明保护的范围内。如在可变区将具有类似性质的氨基酸进行替换。本发明所述变体的序列可以与参比序列具有至少80％、85％、90％、95％、96％、97％、98％或99％的一致性(或同源性)。本发明所述的序列一致性可以使用序列分析软件测量。例如使用缺省参数的计算机程序BLAST，尤其是BLASTP或TBLASTN。本发明述及的氨基酸序列均按照N端至C端的方式示出。
如前所述，本发明的抗体可以是全长的(例如，IgG1或IgG4抗体)或可仅包含抗原结合部分(例如，Fab、F(ab’)2或scFv片段)，或可以被修饰以影响功能。本发明包括具有修饰的糖基化模式的抗B7H6抗体。在一些应用中，进行修饰以除去不期望的糖基化位点可以是有用的，或在寡糖链上不存在岩藻糖部分以例如增强抗体依赖性细胞毒性(ADCC)功能的抗体。在另一些应用中，可进行半乳糖基化修饰以改变补体依赖性细胞毒性(CDC)。
本文所使用的术语“功能性片段”尤其是指抗体片段如Fv、scFv(sc指单链)、Fab、F(ab’)2、Fab’、scFv-Fc片段或者双抗体(diabody)、或者通过化学修饰或通过掺入脂质体中应能够增加半寿期的任何片段，所述化学修饰例如添加聚(亚烷基)二醇，如聚乙二醇(“聚乙二醇化，PEG化”)(被称为Fv-PEG、scFv-PEG、Fab-PEG、F(ab’)2-PEG或Fab’-PEG的聚乙二醇化片段)(“PEG”为聚乙二醇)，所述片段具有B7H6结合活性。优选地，所述功能性片段将由其来源抗体的重链可变区或轻链可变区的部分序列构成或者包含它们，所述部分序列足以保留与其来源抗体相同的结合特异性和充分的亲和力，对于B7H6，优选至少等于其来源抗体亲和力的1/100，在更优选方式中至少等于1/10。这种功能片段将包含最少5个氨基酸，优选其来源的抗体序列的10、15、25、50和100个连续氨基酸。
如前所述，本发明提供了一种能够结合B7H6的抗体或其抗原结合片段，其特征在于，该抗体包括重链CDR1、重链CDR2和重链CDR3，以及轻链CDR1、轻链CDR2和轻链CDR3。
根据本发明，所述抗体能够结合B7H6，特别是SEQ ID NO:21所示的氨基酸序列。
本发明的优选实施方式中，为了进一步提高抗体的生物可接受性，还可以对抗体进行人源化，即，所述抗体为嵌合抗体或人源化抗体。术语“嵌合抗体”是指利用重组DNA技术，将来自一个物种(如小鼠)的单克隆抗体的恒定区氨基酸序列替换为来自另一个物种(如人)的抗体的恒定区而获得的重组抗体。术语“人源化抗体”是指利用重组DNA技术，将来自一个物种(如小鼠)的单克隆抗体的恒定区和可变区的非CDR(Fv骨架区(FR))氨基酸序列全部替换为来自另一个物种(如人)的抗体的恒定区和可变区的非CDR氨基酸序列而获得的重组抗体。也即，一个抗体的恒定区被人源化时称为嵌合抗体，而恒定区和可变区的非CDR氨基酸序列全部人源化后称为人源化抗体。人源化的方法可以参照常规的抗体工程技术进行，在此不再赘述。
本发明提供的人源化抗体的重链可变区的序列如SEQ ID NO:17所示，轻链可变区的序列如SEQ ID NO:18所示；或者，重链可变区的序列如SEQ ID NO:19所示，轻链可变区的序列如SEQ ID NO:20所示。
本发明提供的免疫缀合物含有治疗剂以及与治疗剂偶联的如前所述的抗体或其抗原结合片段。所述抗体或其抗原结合片段与治疗剂偶联的方式可以为常规的方式。
本发明提供的组合物含有如前所述的抗体或其抗原结合片段、和/或上述免疫缀合物、以及药学上可接受的载体。在某些实施方式中，所述组合物包括在时间和/或空间上分开的组合，只要其能够共同作用以实现本发明的目的。例如，所述组合物中所含的成分可以以整体施用于受试者，或者分开施用于受试者。当所述组合物中所含的成分分开地施用于受试者时，各个成分可以同时或依次施用于受试者。
术语“药学上可接受的”表明组合物能够给药至受试者而不产生妨碍组合物给药的不良生理反应。例如，“药学上可接受的载体”是指在制备一般安全、无毒、可取的药物组合物中有用的载体，优选地，这些载体或稀释剂的实例包括但不限于：水、盐水、林格氏溶液、葡萄糖、甘露醇、右旋葡萄糖、乳糖、淀粉、硬脂酸镁、纤维素、碳酸镁、0.3％甘油、透明质酸、乙醇、聚亚烷基二醇如聚丙二醇、甘油三酯、5％人血清白蛋白，也可以使用脂质体和非水媒介物，例如不挥发油。
本发明的组合物也可以相互组合、或与一种或多种其它的治疗化合物组合地给药，例如，与化疗剂组合给药。因此，所述组合物还可以含有化疗剂。本发明的抗体或其抗原结合片段、或免疫缀合物还可以与第二治疗剂组合，所述第二治疗剂的示例性试剂包括但不限于抑制B7H6活性的其他试剂(包括其他抗体或其抗原结合片段、肽抑制剂、小分子拮抗剂等)和/或干扰B7H6上游或下游信号转导的试剂。
通常，所述抗体或其抗原结合片段以有效量给药，即足以实现期望的治疗和/或预防效果的量，例如，引起与被治疗的疾病相关的症状的预防或缓解的量，所述疾病例如与B7H6相关的疾病。给药至受试者的组合物的有效量将取决于疾病的类型和严重度，以及取决于个体的特征，例如一般健康状态、年龄、性别、体重和对药物的耐受性；还将取决于疾病的严重程度和类型，本领域技术人员将能够根据这些因素等确定合适的剂量。
本发明提供的用于检测样品中B7H6的试剂盒含有如上所述的抗体或其抗原结合片段。所述样品可以为患有B7H6介导的疾病的患者(特别是移植排斥、自身免疫病、感染性疾病或癌症患者，更优选是患有肺癌，肝癌，卵巢癌，宫颈癌，皮肤癌，膀胱癌，结肠癌，乳腺癌，神经胶质瘤，肾癌，胃癌，食道癌，口腔鳞状细胞癌和头颈癌中的至少一种的患者)的组织。所述试剂盒还可以包括常规用于检测B7H6的试剂，如包被液等。
本发明还提供了如上所述的抗体或其抗原结合片段在制备用于检测样品中B7H6的试剂中的用途。如前所述，所述样品可以为患有B7H6介导的疾病的患者的组织，在此不再赘述。本发明的抗体或其抗原结合片段与B7H6具有良好的亲和性，能够有效地检测样品中的B7H6。
本发明还提供了所述的抗体或其抗原结合片段在制备用于预防和/或治疗B7H6介导的疾病的药物中的用途。优选地，所述B7H6介导的疾病为移植排斥、自身免疫病、感染性疾病或癌症。更优选地，所述癌症为表达B7H6的癌症。进一步优选地，所述癌症为肺癌，肝癌，卵巢癌，宫颈癌，皮肤癌，膀胱癌，结肠癌，乳腺癌，神经胶质瘤，肾癌，胃癌，食道癌，口腔鳞状细胞癌和头颈癌中的至少一种。进一步优选地，所述感染性疾病包括但不限于HIV病毒感染和/或乙型肝炎病毒感染。
本发明还涉及一种预防和/或治疗B7H6介导的疾病(如前所述)的方法，该方法包括：将有效量的本发明的抗体或其抗原结合片段、免疫缀合物和组合物中的至少一种给药至患者。其中，给药的方式可以为口服给药、经鼻给药、皮内给药、皮下给药、肌内给药或静脉给药或腹腔内给药。
本发明中涉及的“患者”或“受试者”一般指哺乳动物，如灵长类动物和/或啮齿类动物，特别是人或鼠。
本领域技术人员可以将编码本发明所述抗体或其抗原结合片段的DNA分子克隆到载体(特别是表达载体)中，进而转化宿主细胞，通过诱导表达可获得所述抗体或其抗原结合片段。因此，本发明还提供了编码上述抗体或其抗原结合片段的(分离的)核酸以及含有该核酸的重组载体和转化子。所述核酸优选为基因工程手段获得的表达盒。
重组载体可以指克隆载体，也可以指表达载体，可以通过将所述核酸与商购的载体(如质粒或病毒载体)可操作地连接而获得，常用的质粒包括pSeTag2、PEE14、pMH3等。
本发明的表达载体可以含有编码的所述抗体的重链可变区、轻链可变区和/或恒定区的DNA序列。然而，也可以分别构建两种表达载体，一种含有重链可变区和恒定区，另一种含有轻链可变区和恒定区，一同转染哺乳动物细胞。在一个优选的实施方式中，所述表达载体进一步含有启动子和编码分泌信号肽的DNA序列，以及至少一种用于筛选的抗药基因。
本发明所述宿主细胞可以为原核宿主细胞、真核宿主细胞或噬菌体。所述原核宿主细胞可以为大肠杆菌、枯草杆菌、链霉菌或奇异变形菌等。所述真核宿主细胞可以为包括巴斯德毕赤酵母、酿酒酵母、裂殖酵母、木霉等真菌，草地粘虫等昆虫细胞，烟草等植物细胞，BHK细胞、CHO细胞、COS细胞、骨髓瘤细胞等哺乳动物细胞。在一些实施方式中，本发明所述宿主细胞优选为哺乳动物细胞，更优选BHK细胞、CHO细胞、NSO细胞或COS细胞。
本发明所涉及的序列说明详见表1。
表1氨基酸序列说明表
Figure BDA0003397356660000091
Figure BDA0003397356660000101
Figure BDA0003397356660000111
Figure BDA0003397356660000121
Figure BDA0003397356660000131
编码本发明的抗体的重链和/或轻链的核酸在本发明的范围内，根据重链和/或轻链的氨基酸序列，本领域技术人员能够很容易得到相应的核酸序列，如表2所示。
表2核苷酸序列说明表
Figure BDA0003397356660000132
Figure BDA0003397356660000141
Figure BDA0003397356660000151
Figure BDA0003397356660000161
Figure BDA0003397356660000171
Figure BDA0003397356660000181
Figure BDA0003397356660000191
Figure BDA0003397356660000201
Figure BDA0003397356660000211
Figure BDA0003397356660000221
Figure BDA0003397356660000231
Figure BDA0003397356660000241
Figure BDA0003397356660000251
以下将通过实施例对本发明进行详细描述。实施例或测试例中，未注明具体条件的实验方法的，均按照常规条件进行。
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1：抗体的制备
生成针对人B7H6的鼠源单克隆抗体，用纯化的重组B7H6胞外区Fc融合蛋白(B7H6-Fc)(重组B7H6胞外区Fc融合蛋白，氨基酸序列如SEQ ID NO:22所示)作为抗原，免疫Balb/c小鼠(9周龄，购自上海莱斯克，体重20g左右)。
免疫小鼠使用纯化抗原和完全弗氏佐剂进行3次免疫，通过尾静脉放血后检测免疫应答。通过ELISA、流式细胞术筛选血清，获取有抗人B7H6免疫球蛋白的小鼠。并对有最高的抗B7H6免疫球蛋白的小鼠取出脾细胞与鼠骨髓瘤细胞SP2/0细胞(ATCC编号CRL-1581)进行融合。融合后的杂交瘤细胞进行抗体筛选，得到鼠单抗。
将候选杂交瘤细胞总数量培养到106，800rpm离心10分钟收集细胞，并以Trizol试剂盒(Invitrogen)提取总RNA；以总RNA为模板，逆转录合成cDNA文库(Invitrogen)，又以cDNA为模板PCR扩增杂交瘤细胞的所对应的可变区核酸序列。PCR扩增反应中所使用的引物序列与抗体可变区第一框架区或信号肽区和恒定区互补(Larrick，J.W.，et al.，(1990)Scand.J.Immunol.，32，121-128和Coloma，J.J.et al.，(1991)BioTechniques，11，152-156)。在50μl反应体系中，分别加入cDNA 2μL，10×PCR缓冲液5μL，上游及下游引物2μL(5μmol)，dNTP 2μL，Taq酶1μLL(Takara，Ex Taq)，H2O 38μL；95℃预变性5min，进入温度循环，进行PCR扩增。反应条件为：94℃变性30S，58℃退火45S，72℃延伸50S，共32个循环，然后72℃延长7min。将扩增产物测序后，得到鼠单抗的重链和轻链可变区序列(包括氨基酸序列和核酸序列)。
实施例2：鼠源B7H6抗体ELISA结合实验
ELISA实验被用于检测B7H6抗体的结合特性。B7H6胞外区Fc融合蛋白(B7H6-Fc)包被到96孔板中，抗体加入后信号的强弱用于判断抗体和B7H6的结合特性。
用PBS缓冲液将B7H6-Fc融合蛋白(氨基酸序列如SEQ ID NO:22所示)稀释为1μg/mL，以100μL/孔的体积加于96孔板中，于4℃放置过夜。将96孔板中PBS缓冲液吸掉，用PBST(pH7.2 PBS含0.1％Tween 20)缓冲液洗板6次后，加入200μL/孔PBS/10％BSA，37℃孵育2h进行封闭。移去封闭液，用PBST洗板6次后，加入100μL/孔用PBST/0.05％BSA稀释至合适浓度的待测B7H6抗体，37℃孵育1h。移去反应体系，用PBST洗板6次后，以100μL/孔用PBST/0.05％BSA稀释HRP(辣根过氧化物酶)标记的抗鼠抗体二抗，37℃孵育1h。用PBST洗板6次后，加入80μL/孔TMB(四甲基联苯胺)，于室温孵育3min，加入80μL/孔4M硫酸终止反应。用酶标仪在450mm处读取吸光值。结果(图1)表明本发明的鼠源抗体12F5(SEQ ID NO:26和SEQID NO:27)、12G4(SEQ ID NO:28和SEQ ID NO:29)能够结合B7H6。
实施例3：鼠源B7H6抗体流式细胞术结合实验
流式细胞术实验被用于检测B7H6抗体的结合特性，于HL60细胞(ATCC编号CCL-240)中过表达B7H6蛋白(HL60-B7H6、SEQ ID NO:21；SEQ ID NO:54)，表达载体为pLVX-EF1a-B7H6-IRES-ZsGreen，抗体加入后信号的强弱用于判断抗体和B7H6的结合特性。
HEK293T细胞按照5×105细胞/孔铺六孔板，用不含双抗的DMEM培养基培养过夜。转染前弃去培养基，加入1mL新鲜的不含双抗的DMEM培养基。将pLVX-EF1a-B7H6-IRES-ZsGreen(pLVX-EF1a-IRES-ZsGreen1载体的酶切位点EcoRI与BamHI之间插入有B7H6蛋白(SEQ ID NO:21)的编码序列(SEQ ID NO:54)、pMD2G、psPAX2载体(共3μg)按照2:1:1的比例加入200μl无血清DMEM培养基中，加入12μg聚醚酰亚胺(PEI，Polysciences有限公司)。混匀后静置16min，然后将全部液体加入铺有HEK293T细胞的六孔板中。培养6h后，弃去培养基，加入新鲜的完全DMEM培养基培养。转染48h后，收细胞培养上清，过0.45μm滤器(Millipore)，即为病毒上清。将病毒上清全部加入含有1×104HL60细胞的6孔板中，加入终浓度4μg/ml的聚凝胺(Sigma)，培养12h。随后弃尽上清，加入新鲜的完全RPMI1640培养基。所得细胞即为HL60-B7H6细胞。
用PBS将HL60-B7H6细胞稀释为2×106/mL，以100μL/管的体积加于1.5ml EP管中，向其中加入10μL/管山羊血清，于4℃封闭30min。加入1μg/管的B7H6抗体，于4℃孵育30min。向EP管中加入1mL PBS，4℃3500rpm×5min离心，弃尽上清，再用PBS洗一遍。离心后弃尽上清，用100μl/管PBS重悬细胞，向其中加入0.1μL/管Alexa-647标记的山羊抗小鼠抗体二抗(Invitrogen)，4℃避光孵育30min。用PBS洗两遍，离心后弃尽上清。用200μL/管PBS重悬细胞，用流式细胞仪进行检测，结果如图2所示，进一步显示本发明的鼠源抗体12F5(SEQ IDNO:26和SEQ ID NO:27)、12G4(SEQ ID NO:28和SEQ ID NO:29)能够结合B7H6。
实施例4：小鼠抗体人源化实验
参照B7H6抗体轻链可变区序列和重链可变区序列，选取与其非CDR区匹配最好的人源化模板。将鼠源抗体CDR区移植到选择的人源化模板上，替换人源模板的CDR区，得到人源化的抗体。然后，以鼠源抗体的三维结构为基础，对包埋残基、与CDR区有直接相互作用的残基，以及对VL和VH的构象有重要影响的残基进行回复突变，得到人源化之后的抗体，人源化B7H6抗体12F5重链可变区的序列如SEQ ID NO:17所示，轻链可变区序列如SEQ ID NO:18所示，或者，人源化B7H6抗体12G4重链可变区的序列如SEQ ID NO:19所示，轻链可变区序列如SEQ ID NO:20所示。
实施例5：人源化B7H6抗体ELISA结合实验
ELISA实验被用于检测人源化B7H6抗体的结合特性。B7H6胞外区Fc融合蛋白(B7H6-Fc)包被到96孔板中，抗体加入后信号的强弱用于判断抗体和B7H6的结合特性。
用PBS缓冲液将B7H6-Fc融合蛋白(氨基酸序列如SEQ ID NO:22所示)稀释为1μg/mL，以100μL/孔的体积加于96孔板中，于4℃放置过夜。将96孔板中PBS缓冲液吸掉，用PBST(pH7.2 PBS含0.1％Tween 20)缓冲液洗板6次后，加入200μL/孔PBS/10％BSA，37℃孵育2h进行封闭。移去封闭液，用PBST洗板6次后，加入100μL/孔用PBST/0.05％BSA稀释至合适浓度的待测B7H6抗体，37℃孵育1h。移去反应体系，用PBST洗板6次后，以100μL/孔用PBST/0.05％BSA稀释HRP(辣根过氧化物酶)标记的小鼠抗人IgG(Fab特异性)二抗(Sigma)，37℃孵育1h。用PBST洗板6次后，加入80μL/孔TMB(四甲基联苯胺)，于室温孵育3min，加入80μL孔4M硫酸终止反应。用酶标仪在450mm处读取吸光值。结果(图3)表明本发明的人源化抗体h12F5(SEQ ID NO:30和SEQ ID NO:31)、h12G4(SEQ ID NO:32和SEQ ID NO:33)能够结合B7H6。
实施例6：人源化B7H6抗体流式细胞术结合实验
流式细胞术实验被用于检测B7H6抗体的结合特性，于HL60细胞(ATCC编号CCL-240)中过表达B7H6蛋白(HL60-B7H6)(如实施例3中所示)，抗体加入后信号的强弱用于判断抗体和B7H6的结合特性。
用PBS将HL60-B7H6细胞稀释为2×106/mL，以100μL/管的体积加于1.5mL EP管中，向其中加入10μL/管小鼠血清，于4℃封闭30min。加入1μg/管的B7H6抗体，于4℃孵育30min。向EP管中加入1mL PBS，4℃3500rpm×5min离心，弃尽上清，再用PBS洗一遍。离心后弃尽上清，用100μL/管PBS重悬细胞，向其中加入1μL/管Alexa-647标记的小鼠抗人IgG-Fc二抗(Biolegend)，4℃避光孵育30min。用PBS洗两遍，离心后弃尽上清。用200μL/管PBS重悬细胞，用流式细胞仪进行检测，结果如图4所示，进一步显示本发明的人源化抗体h12F5(SEQID NO:30和SEQ ID NO:31)、h12G4(SEQ ID NO:32和SEQ ID NO:33)能够结合B7H6。
实施例7：人源化B7H6抗体流式细胞术结合实验
流式细胞术实验被用于检测人源化B7H6抗体的结合特性，于CHO-K1细胞(ATCC编号CCL-240)中过表达猴B7H6蛋白(CHO-K1-cyno-B7H6)，抗体加入后信号的强弱用于判断抗体和猴B7H6的结合特性。
HEK293T细胞按照5×105细胞/孔铺六孔板，用不含双抗的DMEM培养基培养过夜。转染前弃去培养基，加入1mL新鲜的不含双抗的DMEM培养基。将pLVX-EF1a-cynoB7H6-IRES-ZsGreen(pLVX-EF1a-IRES-ZsGreen1载体的酶切位点EcoRI与BamHI之间插入有猴B7H6蛋白(SEQ ID NO:24)的编码序列(SEQ ID NO:57)、pMD2G、psPAX2载体(共3μg)按照2:1:1的比例加入200μL无血清DMEM培养基中，加入12μg聚醚酰亚胺(PEI，Polysciences有限公司)。混匀后静置16min，然后将全部液体加入铺有HEK293T细胞的六孔板中。培养6h后，弃去培养基，加入新鲜的完全DMEM培养基培养。转染48h后，收细胞培养上清，过0.45μm滤器(Millipore)，即为病毒上清。将病毒上清全部加入含有1×104CHO-K1细胞的6孔板中，加入终浓度4μg/ml的聚凝胺(Sigma)，培养12h。随后弃尽上清，加入新鲜的完全RPMI1640培养基。所得细胞即为CHO-K1-cyno B7H6细胞。
用PBS将CHO-K1-cynoB7H6细胞稀释为2×106/mL，以100μL/管的体积加于1.5mLEP管中，向其中加入10μL/管小鼠血清，于4℃封闭30min。加入1μg/管的人源化B7H6抗体，于4℃孵育30min。向EP管中加入1mL PBS，4℃3500rpm×5min离心，弃尽上清，再用PBS洗一遍。离心后弃尽上清，用100μL/管PBS重悬细胞，向其中加入1μL/管Alexa-647标记的小鼠抗人IgG-Fc二抗(Biolegend)，4℃避光孵育30min。用PBS洗两遍，离心后弃尽上清。用200μL/管PBS重悬细胞，用流式细胞仪进行检测，结果如图5所示，进一步显示本发明的抗体h12F5、h12G4能够结合猴B7H6。
实施例8：人源化B7H6抗体ELISA结合实验
ELISA实验被用于检测人源化B7H6抗体的结合特性。猴B7H6胞外区Fc融合蛋白(cynoB7H6-Fc)包被到96孔板中，抗体加入后信号的强弱用于判断抗体和猴B7H6的结合特性。
用PBS缓冲液将cynoB7H6-Fc融合蛋白(氨基酸序列如SEQ ID NO:23所示)稀释为1μg/mL，以100μL/孔的体积加于96孔板中，于4℃放置过夜。将96孔板中PBS缓冲液吸掉，用PBST(pH7.2 PBS含0.1％Tween 20)缓冲液洗板6次后，加入200μL/孔PBS/10％BSA，37℃孵育2h进行封闭。移去封闭液，用PBST洗板6次后，加入100μL/孔用PBST/0.05％BSA稀释至合适浓度的待测B7H6抗体，37℃孵育1h。移去反应体系，用PBST洗板6次后，以100μL/孔用PBST/0.05％BSA稀释HRP(辣根过氧化物酶)标记的小鼠抗人IgG(Fab特异性)二抗(Sigma)，37℃孵育1h。用PBST洗板6次后，加入80μL/孔TMB(四甲基联苯胺)，于室温孵育3min，加入80μL/孔4M硫酸终止反应。用酶标仪在450mm处读取吸光值。结果(图6)表明本发明的人源化抗体h12G4能够结合猴B7H6。
实施例9：体外结合亲和力和动力学实验
Biacore方法是公认的客观检测蛋白相互间亲和力和动力学的检测方法，通过Biacore T200分析本发明B7H6抗体表征亲和力及结合动力学。
采用标准氨基偶联法将B7H6胞外段Fc融合蛋白(B7H6-Fc)共价连接至CM5(GE)芯片上。然后将稀释于PBS中的一系列浓度梯度的B7H6抗体与各个循环进样，进样后以10mMNaOH溶液再生。追踪抗原-抗体结合动力学3分钟并追踪解离动力学10分钟，使用GE的BIAevaluation软件以1:1(Langmuir)结合模型分析所得数据。以此法测定的鼠源12F5的KD值为2.2nM，鼠源12G4的KD值为0.15nM。
实施例10：抗体促进B7H6结合NKp30能力的筛选
B7H6抗体是通过和B7H6胞外区的结合，从而促进B7H6和其受体NKp30的信号通路，流式细胞术实验被用于检测B7H6抗体对受体NKp30结合的增强。
用PBS将HL60-B7H6细胞(同上)稀释为2×106/ml，以100μL/管的体积加于1.5mLEP管中，向其中加入10μL/管小鼠血清，于4℃封闭30min。加入4μg/管NKp30胞外区Fc融合蛋白(NKp30-Fc，SEQ ID NO:25，本实验室表达)，4℃孵育30min。加入B7H6抗体，于4℃孵育30min。向EP管中加入1mL PBS，4℃3500rpm×5min离心，弃尽上清，再用PBS洗一遍。离心后弃尽上清，用100μl/管PBS重悬细胞，向其中加入1μL/管647标记的小鼠抗人抗体二抗(Biolegend)，4℃避光孵育30min。用PBS洗两遍，离心后弃尽上清。用200μL/管PBS重悬细胞，用流式细胞仪进行检测，结果如图7所示，可以看出，本发明的鼠源12F5抗体够促进B7H6与受体NKp30的结合。
实施例11：补体依赖的细胞毒检测
补体依赖的细胞毒实验，用于检测B7H6抗体杀伤肿瘤细胞的作用。其中，使用的肿瘤细胞为HL60-B7H6细胞。
(1)肿瘤细胞用无血清RPMI-1640培养基重悬，计数。用完全RPMI-1640培养基稀释为4×105/mL。以25μL/孔的体积将稀释后的细胞加入96孔圆底板中
(2)用无血清RPMI-1640培养基稀释B7H6抗体或对照鼠IgG，以25μL/孔的体积将稀释后的B7H6抗体或鼠IgG加入96孔圆底板中。
(3)用1mL超纯水溶解兔补体(Cedarlane)，然后加入1mL无血清RPMI-1640培养基，混匀后作为工作液。以40μL/孔的体积将补体工作液加入96孔圆底板中。37℃孵育30min。
(4)以110μL/孔的体积将完全RPMI-1640培养基加入96孔圆底板中，以1μL/孔的体积向96孔圆底板中加入7-AAD(BD)，混匀。将孔中所有液体转入流式管，利用流式细胞仪进行检测。结果如图8所示，可以看出，本发明B7H6鼠源12F5抗体有补体依赖细胞毒作用。
实施例12：B7H6抗体促进小鼠抗癌能力
体内药效实验，用于检测B7H6抗体促进裸鼠抗癌功能。其中，使用的肿瘤细胞分别为急性髓系白血病U-937细胞(ATCC编号CRL-1953.2)。
(1)裸鼠右腹侧皮下荷瘤，每只鼠1.5×106U-937细胞，然后将小鼠随机分组。
(2)第5、7、9、11、13天，为小鼠腹腔注射抗体，每只鼠1mg。
(3)每2天测量一次肿瘤体积。
结果如图9所示，可以看出，本发明的鼠源抗体12F5具有抗癌功能。
从以上实验结果可以看出，本发明得到的抗体的能够结合人与猴B7H6；能够促进B7H6和NKp30的相互作用；能够促进小鼠抗癌。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于此。在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，包括各个技术特征以任何其它的合适方式进行组合，这些简单变型和组合同样应当视为本发明所公开的内容，均属于本发明的保护范围。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
SEQUENCE LISTING
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<213> Macaca fascicularis
<400> 24
Met Ala Trp Arg Ala Ala Ser Ser Ala Cys Ala Ala Pro Leu Leu Leu
1 5 10 15
Trp Cys Ala Leu Met Thr Ala Gly Asp Leu Lys Val Glu Met Met Ala
20 25 30
Arg Gly Ile Gln Ala Thr Arg Leu Asn Asp Ser Val Thr Ile Ser Cys
35 40 45
Lys Val Ile Tyr Ser Gln Pro Leu Asn Ile Thr Ser Met Gly Ile Thr
50 55 60
Trp Phe Arg Lys Ser Leu Thr Leu Asp Lys Glu Val Lys Val Phe Glu
65 70 75 80
Phe Phe Gly Asp His Gln Lys Ala Phe Arg Pro Gly Ala Asn Val Ser
85 90 95
Leu Trp Arg Leu Lys Ser Gly Asp Ala Ser Leu Lys Leu Pro Gly Val
100 105 110
Gln Leu Glu Glu Ala Gly Glu Tyr Arg Cys Glu Val Val Val Thr Pro
115 120 125
Leu Lys Ala Gln Gly Thr Val Gln Leu Lys Val Val Ala Ser Pro Thr
130 135 140
Ser Arg Leu Phe Gln Asp Gln Ala Val Val Lys Glu Asn Glu Gly Lys
145 150 155 160
Tyr Met Cys Glu Ser Ser Arg Phe Tyr Pro Lys Asp Ile Asn Ile Thr
165 170 175
Trp Glu Lys Trp Thr Gln Lys Ser Pro His His Val Val Ile Ser Glu
180 185 190
Asn Val Ile Thr Gly Pro Thr Ile Lys Asn Met Asp Gly Thr Phe Asn
195 200 205
Ile Thr Ser Tyr Leu Lys Leu Asn Ser Ser Gln Glu Asp Pro Gly Thr
210 215 220
Val Tyr Arg Cys Val Ile Arg His Glu Ser Leu His Thr Pro Val Ser
225 230 235 240
Ile Asp Phe Ile Leu Thr Ala Pro Gln Gln Ser Leu Ser Glu Pro Glu
245 250 255
Lys Thr Asp Ile Phe Ser Ile Tyr Gly Trp Leu Phe Ser Phe Ile Ala
260 265 270
Ala Gly Leu Ile Leu Leu Ile Val Leu Ile His Trp Lys Lys Val Pro
275 280 285
Leu Arg Glu Glu Glu Gly Arg Asn
290 295
<210> 25
<211> 356
<212> PRT
<213> Homo sapiens
<400> 25
Trp Val Ser Gln Pro Pro Glu Ile Arg Thr Leu Glu Gly Ser Ser Ala
1 5 10 15
Phe Leu Pro Cys Ser Phe Asn Ala Ser Gln Gly Arg Leu Ala Ile Gly
20 25 30
Ser Val Thr Trp Phe Arg Asp Glu Val Val Pro Gly Lys Glu Val Arg
35 40 45
Asn Gly Thr Pro Glu Phe Arg Gly Arg Leu Ala Pro Leu Ala Ser Ser
50 55 60
Arg Phe Leu His Asp His Gln Ala Glu Leu His Ile Arg Asp Val Arg
65 70 75 80
Gly His Asp Ala Ser Ile Tyr Val Cys Arg Val Glu Val Leu Gly Leu
85 90 95
Gly Val Gly Thr Gly Asn Gly Thr Arg Leu Val Val Glu Lys Glu His
100 105 110
Pro Gln Leu Gly Glu Asn Leu Tyr Phe Gln Ser Leu Glu Pro Lys Ser
115 120 125
Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
130 135 140
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
145 150 155 160
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
165 170 175
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
180 185 190
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
195 200 205
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
210 215 220
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro
225 230 235 240
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
245 250 255
Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
260 265 270
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
275 280 285
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
290 295 300
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
305 310 315 320
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
325 330 335
Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
340 345 350
Ser Pro Gly Lys
355
<210> 26
<211> 451
<212> PRT
<213> Artificial Sequence
<220>
<223> synthetic polypeptide
<400> 26
Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Tyr Met Tyr Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe
50 55 60
Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Thr Leu Tyr Tyr Gly Asn Asp Trp Tyr Phe Asp Val Trp Gly
100 105 110
Ala Gly Thr Thr Val Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser
115 120 125
Val Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val
130 135 140
Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu
145 150 155 160
Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala
165 170 175
Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr
180 185 190
Ser Ser Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro
195 200 205
Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr
210 215 220
Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly
225 230 235 240
Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met
245 250 255
Ile Ser Leu Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu
260 265 270
Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val
275 280 285
His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu
290 295 300
Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly
305 310 315 320
Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile
325 330 335
Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val
340 345 350
Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr
355 360 365
Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu
370 375 380
Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro
385 390 395 400
Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val
405 410 415
Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val
420 425 430
His Glu Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr
435 440 445
Pro Gly Lys
450
<210> 27
<211> 213
<212> PRT
<213> Artificial Sequence
<220>
<223> synthetic polypeptide
<400> 27
Gln Ile Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Asn Tyr Met
20 25 30
His Trp Tyr Gln Gln Lys Ser Gly Thr Ser Pro Lys Arg Trp Ile Tyr
35 40 45
Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Val Thr
85 90 95
Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Ala Asp Ala Ala Pro
100 105 110
Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly
115 120 125
Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn
130 135 140
Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn
145 150 155 160
Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser
165 170 175
Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr
180 185 190
Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser Phe
195 200 205
Asn Arg Asn Glu Cys
210
<210> 28
<211> 450
<212> PRT
<213> Artificial Sequence
<220>
<223> synthetic polypeptide
<400> 28
Glu Val Leu Leu Gln Gln Ser Gly Pro Glu Val Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Thr Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Asn Met Asp Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile
35 40 45
Gly Asp Ile Asn Pro Asn Asn Gly Gly Thr Leu Tyr Asn Gln Lys Phe
50 55 60
Arg Gly Arg Val Thr Leu Ile Val Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Thr Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Glu Val Phe Tyr Gly Asn Tyr Ala Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Leu Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val
115 120 125
Tyr Pro Leu Ala Pro Val Cys Gly Asp Thr Thr Gly Ser Ser Val Thr
130 135 140
Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Leu Thr
145 150 155 160
Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Thr Ser
180 185 190
Ser Thr Trp Pro Ser Gln Ser Ile Thr Cys Asn Val Ala His Pro Ala
195 200 205
Ser Ser Thr Lys Val Asp Lys Lys Ile Glu Pro Arg Gly Pro Thr Ile
210 215 220
Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile
245 250 255
Ser Leu Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp
260 265 270
Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His
275 280 285
Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg
290 295 300
Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys
305 310 315 320
Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu
325 330 335
Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr
340 345 350
Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu
355 360 365
Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp
370 375 380
Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu
405 410 415
Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His
420 425 430
Glu Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro
435 440 445
Gly Lys
450
<210> 29
<211> 218
<212> PRT
<213> Artificial Sequence
<220>
<223> synthetic polypeptide
<400> 29
Asp Ile Val Leu Thr Gln Ser Pro Val Ser Leu Ala Val Pro Leu Gly
1 5 10 15
Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp
20 25 30
Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Ala Ala Ser Thr Leu His Ser Gly Ile Pro Val
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His
65 70 75 80
Pro Val Glu Glu Glu Asp Ala Ala Ser Tyr Tyr Cys Gln Gln Ser Lys
85 90 95
Glu Asp Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105 110
Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln
115 120 125
Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr
130 135 140
Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln
145 150 155 160
Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr
165 170 175
Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg
180 185 190
His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro
195 200 205
Ile Val Lys Ser Phe Asn Arg Asn Glu Cys
210 215
<210> 30
<211> 451
<212> PRT
<213> Artificial Sequence
<220>
<223> synthetic polypeptide
<400> 30
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Glu Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe
50 55 60
Lys Ser Arg Val Thr Leu Thr Val Asp Lys Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Arg Thr Leu Tyr Tyr Gly Asn Asp Trp Tyr Phe Asp Val Trp Gly
100 105 110
Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
130 135 140
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
145 150 155 160
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
195 200 205
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
210 215 220
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
225 230 235 240
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
260 265 270
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
275 280 285
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
290 295 300
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
305 310 315 320
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
325 330 335
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
340 345 350
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
355 360 365
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
385 390 395 400
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
405 410 415
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
420 425 430
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
435 440 445
Pro Gly Lys
450
<210> 31
<211> 213
<212> PRT
<213> Artificial Sequence
<220>
<223> synthetic polypeptide
<400> 31
Glu Ile Val Leu Thr Gln Ser Pro Asp Phe Gln Ser Val Thr Pro Lys
1 5 10 15
Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Asn Tyr Met
20 25 30
His Trp Tyr Gln Gln Lys Pro Asp Gln Ser Pro Lys Arg Leu Ile Tyr
35 40 45
Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Asn Ser Leu Glu Ala Glu
65 70 75 80
Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Val Thr
85 90 95
Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro
100 105 110
Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr
115 120 125
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys
130 135 140
Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu
145 150 155 160
Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser
165 170 175
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala
180 185 190
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe
195 200 205
Asn Arg Gly Glu Cys
210
<210> 32
<211> 450
<212> PRT
<213> Artificial Sequence
<220>
<223> synthetic polypeptide
<400> 32
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Asn Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Asp Ile Asn Pro Asn Asn Gly Gly Thr Leu Tyr Asn Gln Lys Phe
50 55 60
Arg Gly Arg Val Thr Leu Thr Val Asp Lys Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Arg Leu Arg Ser Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Glu Val Phe Tyr Gly Asn Tyr Ala Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly Lys
450
<210> 33
<211> 218
<212> PRT
<213> Artificial Sequence
<220>
<223> synthetic polypeptide
<400> 33
Asp Ile Val Leu Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly
1 5 10 15
Gln Pro Ala Ser Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp
20 25 30
Gly Asp Ser Tyr Met Asn Trp Tyr Leu Gln Lys Pro Gly Gln Pro Pro
35 40 45
Gln Leu Leu Ile Tyr Ala Ala Ser Thr Leu His Ser Gly Val Pro Asp
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser
65 70 75 80
Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln Gln Ser Lys
85 90 95
Glu Asp Pro Arg Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg
100 105 110
Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
115 120 125
Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr
130 135 140
Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
145 150 155 160
Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr
165 170 175
Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys
180 185 190
His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
195 200 205
Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 34
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic nucleotide
<400> 34
gggtacacat ttaccagcta ctat 24
<210> 35
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic nucleotide
<400> 35
attaatccat ctaacggggg aacc 24
<210> 36
<211> 42
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic nucleotide
<400> 36
actaggacc ctgtattacg gtaacgactg gtatttcgat gtg 42
<210> 37
<211> 15
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic nucleotide
<400> 37
tcttctgtta actat 15
<210> 38
<211> 9
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic nucleotide
<400> 38
gacacctcc 9
<210> 39
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic nucleotide
<400> 39
cagcaatgga gcagtaatcc agtcaca 27
<210> 40
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic nucleotide
<400> 40
gggtacacat ttacagacta caat 24
<210> 41
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic nucleotide
<400> 41
atcaatccaa acaatggcgg tacc 24
<210> 42
<211> 39
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic nucleotide
<400> 42
gctcgctctg aagtctttta tgggaattat gctgactac 39
<210> 43
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic nucleotide
<400> 43
cagagcgtgg actatgatgg cgacagctat 30
<210> 44
<211> 9
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic nucleotide
<400> 44
gccgcctcc 9
<210> 45
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic nucleotide
<400> 45
cagcagagc aaagaggatc ctagaacc 27
<210> 46
<211> 363
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic nucleotide
<400> 46
caggtgcagt tgcaacaatc tggtgcagag ctggtgaaac ctggtgcatc agtgaagctt 60
tcatgtaaag cttccgggta cacatttacc agctactata tgtactgggt taagcagcgt 120
cctggacagg gtctggagtg gatcggcgag attaatccat ctaacggggg aaccaacttt 180
aacgaaaagt tcaagtctaa ggctacactg acagtcgata aatcctcatc caccgcctac 240
atgcagctct cttctctgac ttcagaggac agcgccgtgt actactgcac taggaccctg 300
tattacggta acgactggta tttcgatgtg tggggtgccg gtacaacagt gaccgtgtcc 360
tca 363
<210> 47
<211> 318
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic nucleotide
<400> 47
caaatcgtgc tgacccagtc tccagcaata atgtccgcat ctcctggtga gaaagtgacc 60
atgacttgtt cagcttcctc ttctgttaac tatatgcact ggtaccaaca gaagtctggc 120
acctccccaa agcggtggat ttatgacacc tccaagctgg ctagcggggt acccgcaagg 180
ttctccggtt caggcagcgg cacttcttac tccctgacta ttagctctat ggaggcagag 240
gatgccgcta cctactattg ccagcaatgg agcagtaatc cagtcacatt tggggccgga 300
accaagctgg agctgaag 318
<210> 48
<211> 360
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic nucleotide
<400> 48
gaagttctgc ttcagcagtc tggcccagaa gttgttaagc caggagcctc cgtgaaaatt 60
acttgcaagg cctccgggta cacatttaca gactacaata tggactgggt gaagcagtcc 120
catggaaaga gtctggaatg gatcggcgat atcaatccaa acaatggcgg taccctctat 180
aaccagaaat ttcgcggacg tgtgaccttg atcgtcgaca aaagtagttc tactgcctac 240
atggagctgc gcagcctgac tagtgacgac accgccgtct actattgcgc tcgctctgaa 300
gtcttttatg ggaattatgc tgactactgg ggccagggta ccactttgac cgtctccagt 360
<210> 49
<211> 333
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic nucleotide
<400> 49
gacattgtgc tgactcagag tcctgtaagt ctggcagttc ccctcggcca aagagctacc 60
attagttgta aggcttccca gagcgtggac tatgatggcg acagctatat gaattggtat 120
caacagaagc caggacagcc acccaagctg ctgatctacg ccgcctccac cctgcattca 180
ggtatccctg tgcgattttc tggtagtggc tccggcacag atttcaccct gaatatccat 240
ccagttgagg aggaggatgc tgcctcttac tactgtcagc agagcaaaga ggatcctaga 300
accttcggcg gtggcactaa gctggaaatt aag 333
<210> 50
<211> 363
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic nucleotide
<400> 50
gaagttcaac tggttcagag cggagctgag gtaaagaagc caggtgccag cgtgaaagtt 60
agttgcaagg catccggata cactttcact agctattata tgtattgggt gcgacaggcc 120
ccagggcaag gtttggagtg gatcggagag atcaatcctt caaatggcgg cacaaatttc 180
aacgaaaagt tcaaatctcg cgtgactctg acagtggaca agtccatctc cacagcatat 240
atggagctga gccggcttag atccgatgac actgctgtct actattgtac aagaacactg 300
tactatggca acgactggta cttcgatgtc tggggacagg ggaccactgt gaccgtctcc 360
agt 363
<210> 51
<211> 318
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic nucleotide
<400> 51
gaaatcgtgc tgacccagag cccagatttc cagagtgtca caccaaagga gaaggtaaca 60
attacatgca gtgccagcag tagcgtgaac tatatgcatt ggtatcagca gaaacccgat 120
cagtccccta agcgactgat ctacgatacc tccaagctgg ccagcggtgt tccaagtagg 180
ttctctggca gcgggtctgg gacagactac actctcacta tcaactctct ggaagctgag 240
gatgctgcta cttattattg ccagcagtgg agctcaaatc ccgttacctt tggccagggt 300
accaagctcg aaattaaa 318
<210> 52
<211> 360
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic nucleotide
<400> 52
gaagtgcagc ttgtacagag tggagcagag gtcaagaaac caggagcttc cgtgaaggtg 60
agctgcaagg cctccggcta tacctttact gattacaaca tggactgggt ccgtcaggct 120
cctggaaaag gactggagtg gattggtgac attaatccta ataacggcgg cacactttac 180
aatcagaagt ttcgtggtag agttacactg actgtggata aatcaatcag caccgcctat 240
atggagctgt cccgactgcg gtccgacgat actgcagtgt attactgcgc caggtcagag 300
gtgttttacg gtaactacgc agactattgg ggccagggga caacagtgac agtcagcagc 360
<210> 53
<211> 333
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic nucleotide
<400> 53
gacatagtat tgacccagac accattgtct ttgagcgtta ccccaggcca gccagcttca 60
atatcttgca aggcctctca gtccgtcgac tatgatggcg attcctacat gaattggtac 120
ctccaaaagc caggccaacc accacagctg ttgatttatg ccgcttctac actccacagt 180
ggcgtgcccg atcgattttc aggatccggc tcagggaccg atttcaccct taagatttct 240
cgagtggaag ccgaggatgt gggcgtgtat tactgccagc agagcaaaga ggatcctaga 300
acttttggcc agggaaccaa gctggagatt aaa 333
<210> 54
<211> 1365
<212> DNA
<213> Homo sapiens
<400> 54
atgacgtgga gggctgccgc ctccacgtgc gcggcgctcc tgattctgct gtgggcgctg 60
acgaccgaag gtgatctgaa agtagagatg atggcagggg ggactcagat cacacccctg 120
aatgacaatg tcaccatatt ctgcaatatc ttttattccc aacccctcaa catcacgtct 180
atgggtatca cctggttttg gaagagtctg acgtttgaca aagaagtcaa agtctttgaa 240
ttttttggag atcaccaaga ggcattccga cctggagcca ttgtgtctcc atggaggctg 300
aagagtgggg acgcctcact gcggctgcct ggaatccagc tggaggaagc aggagagtac 360
cgatgtgagg tggtggtcac ccctctgaag gcacagggaa cagtccagct tgaagttgtg 420
gcttccccag ccagcagatt gttgctggat caagtgggca tgaaagagaa tgaagacaaa 480
tatatgtgtg agtcaagtgg gttctaccca gaggctatta atataacatg ggagaagcag 540
acccagaagt ttccccatcc catagagatt tctgaggatg tcatcactgg tcccaccatc 600
aagaatatgg atggcacatt taatgtcact agctgcttga agctgaactc ctctcaggaa 660
gaccctggga ctgtctacca gtgtgtggta cggcatgcgt ccttgcatac ccccttgagg 720
agcaacttta ccctgactgc tgctcggcac agtctttctg aaactgagaa gacagataat 780
ttttccattc attggtggcc tatttcattc attggtgttg gactggtttt attaattgtt 840
ttgattcctt ggaaaaagat atgtaacaaa tcatcttcag cctatactcc tctcaagtgc 900
attctgaaac actggaactc ctttgacact cagactctga agaaagagca cctcatattc 960
ttttgcactc gggcatggcc gtcttaccag ctgcaggatg gggaggcttg gcctcctgag 1020
ggaagtgtta atattaatac tattcaacaa ctagatgttt tctgcagaca ggagggcaaa 1080
tggtccgagg ttccttatgt gcaagccttc tttgccttgc gagacaaccc agatctttgt 1140
cagtgttgta gaattgaccc tgctctccta acagttacat caggcaagtc catagatgat 1200
aattccacaa agtctgagaa acaaacccct agggaacact cggatgcagt tccggatgcc 1260
ccaatccttc ctgtctcccc tatctgggaa cctcctccag ccacaacatc aacaactcca 1320
gttctatcct cccaaccccc aactttactg ttacccctac agtaa 1365
<210> 55
<211> 1413
<212> DNA
<213> Homo sapiens
<400> 55
gacctgaagg tggaaatgat ggcagggggt actcagataa cacctctcaa tgataatgtc 60
acaatttttt gtaacatctt ttactctcag ccacttaata tcacctccat gggaatcact 120
tggttttgga agtcattgac cttcgataag gaggtgaagg tatttgagtt tttcggggac 180
caccaggaag ctttccgacc aggagcaatt gtgtcccctt ggcgcttgaa gtctggcgac 240
gccagcttga gacttcctgg aatccaattg gaggaagctg gggagtacag atgtgaggtt 300
gtcgtgactc ctctgaaagc tcagggtacc gtgcagttgg aggttgtcgc ctcacccgct 360
tcccggcttc tgctggacca ggttggaatg aaggagaacg aagataagta tatgtgcgag 420
agttccggat tttatcccga ggccatcaac attacatggg agaaacagac ccaaaagttt 480
cctcacccca ttgaaatcag cgaggatgtg attaccggac ccaccattaa aaatatggat 540
ggcactttca acgttacctc atgtttgaaa ttgaacagta gtcaggaaga ccctggaaca 600
gtctatcagt gcgtcgtgag acacgcatcc ctccatacac ctctgcgttc aaacttcact 660
ctcactgccg ccaggcatag cctgagcgag accgagaaga ccgataactt ctctctggag 720
cctaagagct gcgacaagac ccacacctgt ccaccctgtc cagcccctga gctgctgggg 780
gggccctccg tgttcctttt ccctccaaaa cccaaggaca ccctgatgat ctctagaacc 840
cccgaagtca catgcgtggt ggtagatgtc tctcatgagg accccgaggt gaagttcaat 900
tggtacgtcg atggagtgga ggtgcataac gccaagacta agcctaggga agagcagtat 960
aattcaacct accgcgtcgt gtctgtgctc accgtgctgc atcaagactg gctcaacggc 1020
aaagaataca agtgcaaggt ctctaacaag gctctgcccg ctcccatcga aaagaccatc 1080
tccaaggcta agggccaacc cagggagccc caagtctaca cactgcctcc atcacgtgaa 1140
gagatgacta aaaatcaagt tagtttgacc tgcttggtta agggcttcta tccatctgat 1200
atagcagtcg aatgggagag caacggccag cctgaaaaca actacaagac aactccaccc 1260
gtcttggact ccgacggtag ttttttcctg tactccaaac tgaccgtgga taagagcaga 1320
tggcagcagg gcaacgtgtt ctcatgttcc gtcatgcatg aagctctcca caaccactac 1380
acccagaagt ctctgagtct ctctcccggt aaa 1413
<210> 56
<211> 1398
<212> DNA
<213> Macaca fascicularis
<400> 56
gatttgaagg tggagatgat ggctcgagga attcaggcca ccagactcaa cgacagcgtc 60
acaatctctt gcaaagtgat ctatagtcag cccctcaaca taaccagtat gggaattacc 120
tggtttcgca aatcactgac tttggataaa gaggttaaag tctttgaatt ttttggcgat 180
caccagaagg ccttcagacc cggagcaaat gtgtccctgt ggaggctgaa gtctggcgac 240
gctagcctga aacttccagg agttcagctc gaagaggccg gcgagtacag gtgcgaagtt 300
gtagtaaccc ctctcaaagc acagggcacc gttcagctga aggtggtggc ttcacctact 360
tctagacttt tccaggatca ggccgtggtt aaggagaacg agggcaaata tatgtgcgag 420
tcctctcgtt tttatcccaa agacatcaat atcacctggg agaaatggac acagaaatca 480
cctcaccacg ttgtaattag cgagaacgtg atcacaggcc ccacaataaa gaacatggac 540
ggaactttca atataacctc ctacttgaag ctgaactcct ctcaagaaga tcctggcact 600
gtgtatagat gtgtgattag gcacgagagc ctccacacac ccgtctctat tgacttcata 660
ttgactgcac cacagcagtc tctgtctgaa ccagaaaaga ccgatccaaa atcctgtgac 720
aagactcata catgtcctcc ttgtccagca ccagagctgc ttggtggccc tagcgtgttc 780
ctgttccctc ccaagcctaa ggacaccctg atgatttcaa gaacccccga ggtgacttgt 840
gtggtcgtgg atgtgagcca cgaggatcct gaagtcaaat tcaattggta cgttgatggc 900
gtggaggtgc acaatgccaa aaccaagcca cgagaggagc agtacaacag cacctacaga 960
gtggtgtctg tgctgaccgt gttgcatcag gactggctta atggaaaaga atacaagtgt 1020
aaggtctcca ataaagctct ccctgcccca atcgagaaga ctatctctaa ggctaaagga 1080
cagccaaggg aaccccaggt ttacacactg cccccaagcc gagatgaatt gacaaaaaat 1140
caggtttcct tgacctgcct ggtgaagggt ttctacccat ctgatatcgc cgttgagtgg 1200
gagagcaatg gacagcctga gaacaattac aaaacaaccc ctcctgtgtt ggatagtgat 1260
ggcagcttct ttctgtattc taagcttacc gtggataagt ctagatggca gcaaggaaat 1320
gtgttttcct gcagcgtaat gcacgaagca cttcataatc attatacaca gaagtctctc 1380
tctcttagcc cagggaag 1398
<210> 57
<211> 888
<212> DNA
<213> Macaca fascicularis
<400> 57
atggcatgga gagctgcatc ttccgcttgt gctgctcctt tgcttctgtg gtgtgctttg 60
atgactgccg gagacctgaa agtcgaaatg atggctagag gaatccaagc tactcgcctg 120
aatgatagtg tgacaatctc atgtaaagta atttattccc agcctttgaa catcacctct 180
atgggcatta cctggtttcg caagagtctc accctggata aagaggtaaa agtgttcgag 240
ttctttggag accatcaaaa agctttccgc cctggagcta atgtgagcct ttggcgcctg 300
aagagtggag acgccagtct gaaattgcct ggtgtgcagc tcgaagaggc tggtgaatat 360
aggtgcgaag tggtggtgac tcctctgaag gctcagggga ccgtccagct taaagtggtg 420
gccagtccta ccagtcgcct gtttcaggac caggcagtgg tcaaggagaa cgaaggtaaa 480
tatatgtgtg aatcttctcg tttctaccca aaggatatta atatcacctg ggagaagtgg 540
acccaaaaga gcccacacca tgtggtcatc tcagagaatg tgatcaccgg ccccaccatc 600
aagaatatgg atggaacttt caatatcaca tcttacctga agctgaattc aagccaagag 660
gatcctggaa ccgtatatag gtgcgtcatc agacatgaga gcctccacac acccgtgagc 720
atcgacttca ttctgacagc tccacagcag agtctcagtg aacctgagaa aacagacatt 780
ttttctatct atgggtggct tttttctttt atcgccgctg ggctgatctt gctgatcgtg 840
ctcatccact ggaaaaaagt gccactgcga gaagaagaag gcagaaac 888
<210> 58
<211> 1068
<212> DNA
<213> Homo sapiens
<400> 58
tgggtgagtc aaccacctga gattagaact ctggagggca gctctgcatt cttgccatgc 60
tccttcaacg caagtcaggg gaggctggct atcggttccg taacatggtt tagggatgag 120
gtggtgccag gaaaagaagt gaggaacgga accccagagt tcagaggaag actggcccct 180
ctggcaagtt cccgctttct gcatgatcac caagccgaac ttcacattcg cgacgtcagg 240
gggcacgacg catctatata cgtgtgcaga gtggaggtgc ttggtctggg tgtgggcacc 300
ggaaacggca ctcgactcgt cgttgagaaa gagcatccac agctgggaga aaatctgtac 360
tttcagagcc tcgaacctaa gtcttgtgac aagacccata cctgtcctcc ctgtcctgct 420
cctgaactgt tggggggccc ctctgtgttc ctgttccctc caaagcccaa ggacacactg 480
atgattagca ggacaccaga agtcacatgt gtcgtggttg atgtgtccca cgaggatccc 540
gaggttaaat ttaattggta tgtggacggt gttgaggtgc ataacgccaa gaccaagcct 600
cgagaagagc agtacaattc cacttatagg gtagtctccg tattgaccgt gctccatcag 660
gactggctga atggcaaaga gtacaaatgc aaggtctcca ataaggccct tccagcccca 720
attgaaaaga ctatttcaaa ggccaagggg caacccagag agcctcaggt gtatacactt 780
cctccctcac gtgaggagat gacaaagaac caagtgagtc tgacatgtct cgtgaaaggc 840
ttttaccctt cagacattgc tgtggagtgg gaaagcaacg gccaacccga gaacaactac 900
aagaccacac ctcccgtgct ggacagcgat gggtctttct ttctctacag caagctgact 960
gtggacaaaa gtcgctggca gcaaggcaat gtcttcagct gctctgtgat gcacgaagcc 1020
ctccataatc attataccca gaagtctctg agcctgagcc caggaaag 1068
<210> 59
<211> 1353
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic nucleotide
<400> 59
caggtgcagt tgcaacaatc tggtgcagag ctggtgaaac ctggtgcatc agtgaagctt 60
tcatgtaaag cttccgggta cacatttacc agctactata tgtactgggt taagcagcgt 120
cctggacagg gtctggagtg gatcggcgag attaatccat ctaacggggg aaccaacttt 180
aacgaaaagt tcaagtctaa ggctacactg acagtcgata aatcctcatc caccgcctac 240
atgcagctct cttctctgac ttcagaggac agcgccgtgt actactgcac taggaccctg 300
tattacggta acgactggta tttcgatgtg tggggtgccg gtacaacagt gaccgtgtcc 360
tcagccaaaa caacagcccc tagtgtatat ccacttgccc ctgtctgcgg cgacactaca 420
gggtcttctg tgacactcgg gtgtctggtc aaggggtatt tccctgagcc agtgaccctc 480
acttggaact caggctccct ttctagcggc gtccacacat ttccagccgt gcttcagtct 540
gatctgtata ccctgagcag cagcgtgacc gtcacctcct ccacatggcc ttcacaaagc 600
ataacttgta atgtcgccca ccctgcctca tctaccaaag tcgacaagaa gatcgagcct 660
cgaggcccca ctatcaaacc ctgtccaccc tgtaagtgcc ccgctccaaa cttgctgggc 720
ggaccatccg ttttcatctt ccctccaaag atcaaagacg tgctgatgat ttcactcagc 780
cctattgtca cttgtgtagt ggtggacgtg tcagaagatg atcctgacgt gcagatttcc 840
tggtttgtca acaatgttga ggtgcataca gctcagacac agacccaccg cgaagattat 900
aactctactt tgagggtcgt ttccgccctg ccaattcagc accaggactg gatgtctgga 960
aaggaattca aatgtaaagt gaataataag gacctccccg ctcccatcga acggactatc 1020
tctaagccta aagggagcgt acgagctcct caggtgtatg tcctgccccc cccagaggaa 1080
gaaatgacca agaaacaggt gacccttacc tgtatggtta ctgactttat gcctgaggac 1140
atctatgtag agtggaccaa caacgggaag actgagttga actacaaaaa cacagaacct 1200
gtgcttgata gtgatggttc ttatttcatg tatagcaagc tcagagtcga aaaaaaaaac 1260
tgggtggaac gcaattctta ctcctgttcc gtggtgcacg aggggttgca caatcaccac 1320
actacaaaaa gcttcagtag gacccccggc aag 1353
<210> 60
<211> 639
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic nucleotide
<400> 60
caaatcgtgc tgacccagtc tccagcaata atgtccgcat ctcctggtga gaaagtgacc 60
atgacttgtt cagcttcctc ttctgttaac tatatgcact ggtaccaaca gaagtctggc 120
acctccccaa agcggtggat ttatgacacc tccaagctgg ctagcggggt acccgcaagg 180
ttctccggtt caggcagcgg cacttcttac tccctgacta ttagctctat ggaggcagag 240
gatgccgcta cctactattg ccagcaatgg agcagtaatc cagtcacatt tggggccgga 300
accaagctgg agctgaagcg agctgatgca gctcccacag tatctatttt tcctccatct 360
tctgagcagc tcacatctgg tggggcctcc gtggtctgct tcttgaataa cttctatcct 420
aaggacatta acgtgaaatg gaagatcgac ggatccgaac ggcagaacgg agtgcttaac 480
agctggaccg accaggatag taaggattct acatattcca tgtcttccac cctcacactc 540
acaaaggacg agtatgagag acacaacagc tacacatgtg aagccactca caaaacctcc 600
acatctccaa ttgtgaagtc ctttaatagg aatgagtgt 639
<210> 61
<211> 1350
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic nucleotide
<400> 61
gaagttctgc ttcagcagtc tggcccagaa gttgttaagc caggagcctc cgtgaaaatt 60
acttgcaagg cctccgggta cacatttaca gactacaata tggactgggt gaagcagtcc 120
catggaaaga gtctggaatg gatcggcgat atcaatccaa acaatggcgg taccctctat 180
aaccagaaat ttcgcggacg tgtgaccttg atcgtcgaca aaagtagttc tactgcctac 240
atggagctgc gcagcctgac tagtgacgac accgccgtct actattgcgc tcgctctgaa 300
gtcttttatg ggaattatgc tgactactgg ggccagggta ccactttgac cgtctccagt 360
gccaaaacaa cagcccctag tgtatatcca cttgcccctg tctgcggcga cactacaggg 420
tcttctgtga cactcgggtg tctggtcaag gggtatttcc ctgagccagt gaccctcact 480
tggaactcag gctccctttc tagcggcgtc cacacatttc cagccgtgct tcagtctgat 540
ctgtataccc tgagcagcag cgtgaccgtc acctcctcca catggccttc acaaagcata 600
acttgtaatg tcgcccaccc tgcctcatct accaaagtcg acaagaagat cgagcctcga 660
ggccccacta tcaaaccctg tccaccctgt aagtgccccg ctccaaactt gctgggcgga 720
ccatccgttt tcatcttccc tccaaagatc aaagacgtgc tgatgatttc actcagccct 780
attgtcactt gtgtagtggt ggacgtgtca gaagatgatc ctgacgtgca gatttcctgg 840
tttgtcaaca atgttgaggt gcatacagct cagacacaga cccaccgcga agattataac 900
tctactttga gggtcgtttc cgccctgcca attcagcacc aggactggat gtctggaaag 960
gaattcaaat gtaaagtgaa taataaggac ctccccgctc ccatcgaacg gactatctct 1020
aagcctaaag ggagcgtacg agctcctcag gtgtatgtcc tgcccccccc agaggaagaa 1080
atgaccaaga aacaggtgac ccttacctgt atggttactg actttatgcc tgaggacatc 1140
tatgtagagt ggaccaacaa cgggaagact gagttgaact acaaaaacac agaacctgtg 1200
cttgatagtg atggttctta tttcatgtat agcaagctca gagtcgaaaa aaaaaactgg 1260
gtggaacgca attcttactc ctgttccgtg gtgcacgagg ggttgcacaa tcaccacact 1320
acaaaaagct tcagtaggac ccccggcaag 1350
<210> 62
<211> 654
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic nucleotide
<400> 62
gacattgtgc tgactcagag tcctgtaagt ctggcagttc ccctcggcca aagagctacc 60
attagttgta aggcttccca gagcgtggac tatgatggcg acagctatat gaattggtat 120
caacagaagc caggacagcc acccaagctg ctgatctacg ccgcctccac cctgcattca 180
ggtatccctg tgcgattttc tggtagtggc tccggcacag atttcaccct gaatatccat 240
ccagttgagg aggaggatgc tgcctcttac tactgtcagc agagcaaaga ggatcctaga 300
accttcggcg gtggcactaa gctggaaatt aagcgagctg atgcagctcc cacagtatct 360
atttttcctc catcttctga gcagctcaca tctggtgggg cctccgtggt ctgcttcttg 420
aataacttct atcctaagga cattaacgtg aaatggaaga tcgacggatc cgaacggcag 480
aacggagtgc ttaacagctg gaccgaccag gatagtaagg attctacata ttccatgtct 540
tccaccctca cactcacaaa ggacgagtat gagagacaca acagctacac atgtgaagcc 600
actcacaaaa cctccacatc tccaattgtg aagtccttta ataggaatga gtgt 654
<210> 63
<211> 1353
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic nucleotide
<400> 63
gaagttcaac tggttcagag cggagctgag gtaaagaagc caggtgccag cgtgaaagtt 60
agttgcaagg catccggata cactttcact agctattata tgtattgggt gcgacaggcc 120
ccagggcaag gtttggagtg gatcggagag atcaatcctt caaatggcgg cacaaatttc 180
aacgaaaagt tcaaatctcg cgtgactctg acagtggaca agtccatctc cacagcatat 240
atggagctga gccggcttag atccgatgac actgctgtct actattgtac aagaacactg 300
tactatggca acgactggta cttcgatgtc tggggacagg ggaccactgt gaccgtctcc 360
agtgccagta caaagggacc atctgtgttt cctctggcac cttcatctaa gagcacttct 420
ggtggtactg ctgctctggg ctgccttgta aaggactatt tcccagagcc cgtgaccgtc 480
agttggaatt caggcgccct taccagcggg gttcacactt tcccagctgt gttgcagtct 540
tcagggcttt acagtctcag cagtgttgtc accgtccctt cctcctcact gggcacccaa 600
acctatattt gtaatgtcaa ccataaacca agtaacacca aggtggacaa gaaggtggag 660
cccaagtcat gtgataaaac tcacacttgt ccaccatgcc ctgctcccga actgttgggc 720
ggacctagcg tgtttctctt tccacccaaa ccaaaggata ccctgatgat cagcaggact 780
cccgaggtga cttgtgtggt ggttgacgtg tcccatgagg atcctgaagt gaagttcaat 840
tggtacgtgg acggcgtgga ggttcataac gcaaagacca agcccaggga agagcagtat 900
aactctactt accgggtggt ttcagtcttg actgtgttgc accaggactg gctgaatgga 960
aaggagtata agtgtaaagt gagtaacaaa gccctgccag ctcccattga gaagacaatc 1020
tcaaaagcta agggtcagcc ccgcgaacca caggtgtata cactcccacc ctcccgcgag 1080
gagatgacca aaaaccaggt gtccctcacc tgcctggtca agggatttta ccccagcgat 1140
attgctgtcg agtgggagtc aaacggtcag cccgagaata attataaaac tacccctccc 1200
gtgcttgaca gcgatggatc tttcttcctg tactcaaaac tgaccgtcga caaatcccga 1260
tggcagcaag gtaacgtgtt ctcttgcagc gtaatgcacg aggctcttca caatcactat 1320
actcagaagt cactcagcct tagtcctgga aag 1353
<210> 64
<211> 639
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic nucleotide
<400> 64
gaaatcgtgc tgacccagag cccagatttc cagagtgtca caccaaagga gaaggtaaca 60
attacatgca gtgccagcag tagcgtgaac tatatgcatt ggtatcagca gaaacccgat 120
cagtccccta agcgactgat ctacgatacc tccaagctgg ccagcggtgt tccaagtagg 180
ttctctggca gcgggtctgg gacagactac actctcacta tcaactctct ggaagctgag 240
gatgctgcta cttattattg ccagcagtgg agctcaaatc ccgttacctt tggccagggt 300
accaagctcg aaattaaacg tacagtcgca gcacctagtg tcttcatctt tcctccctct 360
gacgagcagt tgaagagcgg aactgcaagc gtcgtttgcc tcctgaacaa tttctatccc 420
cgggaggcta aggtccagtg gaaagtggat aatgctctcc agagcggtaa ctcacaagag 480
tctgttaccg agcaggactc caaggattct acctatagtc tgagctctac cctgacactg 540
tccaaggccg attacgagaa acacaaagtg tatgcctgcg aagtaaccca ccagggcctg 600
agttccccag tgaccaagtc tttcaatagg ggggagtgt 639
<210> 65
<211> 1350
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic nucleotide
<400> 65
gaagtgcagc ttgtacagag tggagcagag gtcaagaaac caggagcttc cgtgaaggtg 60
agctgcaagg cctccggcta tacctttact gattacaaca tggactgggt ccgtcaggct 120
cctggaaaag gactggagtg gattggtgac attaatccta ataacggcgg cacactttac 180
aatcagaagt ttcgtggtag agttacactg actgtggata aatcaatcag caccgcctat 240
atggagctgt cccgactgcg gtccgacgat actgcagtgt attactgcgc caggtcagag 300
gtgttttacg gtaactacgc agactattgg ggccagggga caacagtgac agtcagcagc 360
gccagtacaa agggaccatc tgtgtttcct ctggcacctt catctaagag cacttctggt 420
ggtactgctg ctctgggctg ccttgtaaag gactatttcc cagagcccgt gaccgtcagt 480
tggaattcag gcgcccttac cagcggggtt cacactttcc cagctgtgtt gcagtcttca 540
gggctttaca gtctcagcag tgttgtcacc gtcccttcct cctcactggg cacccaaacc 600
tatatttgta atgtcaacca taaaccaagt aacaccaagg tggacaagaa ggtggagccc 660
aagtcatgtg ataaaactca cacttgtcca ccatgccctg ctcccgaact gttgggcgga 720
cctagcgtgt ttctctttcc acccaaacca aaggataccc tgatgatcag caggactccc 780
gaggtgactt gtgtggtggt tgacgtgtcc catgaggatc ctgaagtgaa gttcaattgg 840
tacgtggacg gcgtggaggt tcataacgca aagaccaagc ccagggaaga gcagtataac 900
tctacttacc gggtggtttc agtcttgact gtgttgcacc aggactggct gaatggaaag 960
gagtataagt gtaaagtgag taacaaagcc ctgccagctc ccattgagaa gacaatctca 1020
aaagctaagg gtcagccccg cgaaccacag gtgtatacac tcccaccctc ccgcgaggag 1080
atgaccaaaa accaggtgtc cctcacctgc ctggtcaagg gattttaccc cagcgatatt 1140
gctgtcgagt gggagtcaaa cggtcagccc gagaataatt ataaaactac ccctcccgtg 1200
cttgacagcg atggatcttt cttcctgtac tcaaaactga ccgtcgacaa atcccgatgg 1260
cagcaaggta acgtgttctc ttgcagcgta atgcacgagg ctcttcacaa tcactatact 1320
cagaagtcac tcagccttag tcctggaaag 1350
<210> 66
<211> 654
<212> DNA
<213> Artificial Sequence
<220>
<223> synthetic nucleotide
<400> 66
gacatagtat tgacccagac accattgtct ttgagcgtta ccccaggcca gccagcttca 60
atatcttgca aggcctctca gtccgtcgac tatgatggcg attcctacat gaattggtac 120
ctccaaaagc caggccaacc accacagctg ttgatttatg ccgcttctac actccacagt 180
ggcgtgcccg atcgattttc aggatccggc tcagggaccg atttcaccct taagatttct 240
cgagtggaag ccgaggatgt gggcgtgtat tactgccagc agagcaaaga ggatcctaga 300
acttttggcc agggaaccaa gctggagatt aaacgtacag tcgcagcacc tagtgtcttc 360
atctttcctc cctctgacga gcagttgaag agcggaactg caagcgtcgt ttgcctcctg 420
aacaatttct atccccggga ggctaaggtc cagtggaaag tggataatgc tctccagagc 480
ggtaactcac aagagtctgt taccgagcag gactccaagg attctaccta tagtctgagc 540
tctaccctga cactgtccaa ggccgattac gagaaacaca aagtgtatgc ctgcgaagta 600
acccaccagg gcctgagttc cccagtgacc aagtctttca atagggggga gtgt 654

Claims (16)

1.一种能够结合B7H6的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段包括重链CDR1、重链CDR2和重链CDR3，以及轻链CDR1、轻链CDR2和轻链CDR3，其中，
所述重链CDR1包含选自SEQ ID NO:1、SEQ ID NO:7所示的氨基酸序列及其保守修饰形式的氨基酸序列至少之一；
所述重链CDR2包含选自SEQ ID NO:2、SEQ ID NO:8所示的氨基酸序列及其保守修饰形式的氨基酸序列至少之一；
所述重链CDR3包含选自EQ ID NO:3、SEQ ID NO:9所示的氨基酸序列及其保守修饰形式的氨基酸序列至少之一；
所述轻链CDR1包含选自EQ ID NO:4、SEQ ID NO:10所示的氨基酸序列及其保守修饰形式的氨基酸序列至少之一；
所述轻链CDR2包含自SEQ ID NO:5、SEQ ID NO:11所示的氨基酸序列及其保守修饰形式的氨基酸序列至少之一；以及
所述轻链CDR3含选自SEQ ID NO:6、SEQ ID NO:12所示的氨基酸序列及其保守修饰形式的氨基酸序列至少之一。
2.根据权利要求1所述的抗体或其抗原结合片段，其中，所述的抗体或其抗原结合片段包含：
1)具有SEQ ID NO:1所示的氨基酸序列的重链CDR1，具有SEQ ID NO:2所示的氨基酸序列的重链CDR2，具有SEQ ID NO:3所示的氨基酸序列的重链CDR3，具有SEQ ID NO:4所示的氨基酸序列的轻链CDR1，具有SEQ ID NO:5所示的氨基酸序列的轻链CDR2，具有SEQ ID NO:6所示的氨基酸序列的轻链CDR3：或
2)具有SEQ ID NO:7所示的氨基酸序列的重链CDR1，具有SEQ ID NO:8所示的氨基酸序列的重链CDR2，具有SEQ ID NO:9所示的氨基酸序列的重链CDR3，具有SEQ ID NO:10所示的氨基酸序列的轻链CDR1，具有SEQ ID NO:11所示的氨基酸序列的轻链CDR2，具有SEQ IDNO:12所示的氨基酸序列的轻链CDR3。
3.根据权利要求1或2所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段包括重链可变区和轻链可变区，(i)所述重链可变区包含SEQ ID NO:13、SEQ ID NO:15、SEQ ID NO:17、SEQ ID NO:19所示的氨基酸序列及其保守修饰形式的氨基酸序列至少之一至少80％同源性的氨基酸序列；和/或，(ii)所述轻链可变区包含SEQ ID NO:14、SEQ IDNO:16、SEQ ID NO:18、SEQ ID NO:20所示的氨基酸序列及其保修修饰形式的氨基酸序列至少之一至少80％同源性的氨基酸序列。
4.根据权利要求1-3任一项所述的抗体或其抗原结合片段，其中，所述重链可变区包含与选自(i)中的重链可变区至少90％，至少95％，至少96％，至少97％，至少98％，至少99％或者100％同源性的氨基酸序列；所述轻链可变区包含与选自(ii)中的轻链可变区至少90％，至少95％，至少96％，至少97％，至少98％，至少99％或者100％同源性的氨基酸序列。
5.根据权利要求1-4任一项所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段包括：
1)所述重链可变区如SEQ ID NO:13所示的氨基酸序列，以及所述轻链可变区如SEQ IDNO:14所示的氨基酸序列；
2)所述重链可变区如SEQ ID NO:15所示的氨基酸序列，以及所述轻链可变区如SEQ IDNO:16所示的氨基酸序列；
3)所述重链可变区如SEQ ID NO:17所示的氨基酸序列，以及所述轻链可变区如SEQ IDNO:18所示的氨基酸序列；或
4)所述重链可变区如SEQ ID NO:19所示的氨基酸序列，以及所述轻链可变区如SEQ IDNO:20所示的氨基酸序列。
6.根据权利要求1-5任一项所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段包括：
1)所述重链如SEQ ID NO:26所示的氨基酸序列，以及所述轻链如SEQ ID NO:27所示的氨基酸序列；
2)所述重链如SEQ ID NO:28所示的氨基酸序列，以及所述轻链如SEQ ID NO:29所示的氨基酸序列；
3)所述重链如SEQ ID NO:30所示的氨基酸序列，以及所述轻链如SEQ ID NO:31所示的氨基酸序列；或
4)所述重链如SEQ ID NO:32所示的氨基酸序列，以及所述轻链如SEQ ID NO:33所示的氨基酸序列。
7.根据权利要求1-5任一项所述的抗体或其抗原结合片段，其中，所述的分离的抗体是IgG1、IgG2或IgG4。
8.根据权利要求1-5中任一项所述的抗体或其抗原结合片段，其中，所述抗体为单克隆抗体、鼠源抗体、嵌合抗体、人源化抗体人改造抗体、人抗体、Fv、单链抗体(scFv)、Fab、Fab’，Fab’-SH或者F(ab’)2。
9.根据权利要求1-8任一项所述的抗体或其抗原结合片段，其中，所述抗体或其抗原结合片段能够结合SEQ ID NO:21所示的氨基酸序列。
10.一种免疫缀合物，其中，该免疫缀合物含有治疗剂以及与治疗剂偶联的权利要求1-9中任一项所述的抗体或其抗原结合片段。
11.一种组合物，其中，所述组合物含有权利要求1-9中任一项所述的抗体或其抗原结合片段、和/或权利要求10所述的免疫缀合物、以及药学上可接受的载体。
12.一种用于检测样品中B7H6的试剂盒，该试剂盒含有权利要求1-9中任一项所述抗体或其抗原结合片段。
13.权利要求1-9中任一项所述的抗体或其抗原结合片段在制备用于检测样品中B7H6的试剂中的用途。
14.权利要求1-9中任一项所述的抗体或其抗原结合片段在制备用于预防和/或治疗B7H6介导的疾病的药物中的用途，所述B7H6介导的疾病为癌症或感染性疾病，
任选地，所述癌症为肺癌，肝癌，卵巢癌，宫颈癌，皮肤癌，膀胱癌，结肠癌，乳腺癌，神经胶质瘤，肾癌，胃癌，食道癌，口腔鳞状细胞癌和头颈癌中的至少一种。
15.一种核酸，其包含编码权利要求1-9中任一项所述的抗体或其抗原结合片段的核酸。
16.一种重组载体或转化子，其含有权利要求15中所述的核酸。
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