CN114929740A - 昆虫防控纳米抗体及其用途 - Google Patents

昆虫防控纳米抗体及其用途 Download PDF

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CN114929740A
CN114929740A CN202080091545.1A CN202080091545A CN114929740A CN 114929740 A CN114929740 A CN 114929740A CN 202080091545 A CN202080091545 A CN 202080091545A CN 114929740 A CN114929740 A CN 114929740A
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罗尼·欧伦-本纳罗亚
阿米尔·阿亚莉
杰西卡·蒙塞拉特
杰罗恩·范瑞
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Abstract

本公开提供了昆虫防控纳米抗体。因此，提供了特异性结合昆虫多肽的纳米抗体，所述昆虫多肽选自：包含几丁质结合域(CBD)的多肽、V‑ATP酶亚基c、海藻糖酶、细胞色素p450单加氧酶、几丁质脱乙酰酶、几丁质合成酶和NPC1甾醇转运体，其中所述纳米抗体与所述昆虫多肽的结合赋予所述纳米抗体昆虫防控活性。还提供了编码纳米抗体的多核苷酸、表达纳米抗体的宿主细胞及其使用方法。

Description

昆虫防控纳米抗体及其用途
相关应用
本公开要求2019年11月11日提交的序列号为62/933,533的美国专利申请的优先权，其全部公开内容通过引用并入本文。
序列表声明
与本公开同时提交的、创建于2020年11月9日名为83672_ST25.txt的ASCll文件包括387,592字节，该文件通过引用并入本文。
技术领域
本公开在其一些实施方式中涉及昆虫防控纳米抗体及其用途。
背景技术
在过去的70年里，害虫的治理是通过使用合成农药来实现的。自Paul Mawller于1939年发现DDT的杀虫特性以来，已经开发了数百种杀虫化合物，随之而来的是杀虫剂使用的相对稳定的增加。目前使用的大多数化学杀虫剂很有可能破坏生态系统，它们直接或通过食物链对人类有毒性，并且它们的使用也受到遗传昆虫抗性发展的阻碍。目前使用的最突出的生物解决方案包括减少由昆虫造成的伤害的有益生物体或天敌、充当诱饵或危害繁殖的信息素、释放不育雄性、喷洒苏云金芽孢杆菌毒素或含有苏云金芽孢杆菌毒素基因的转基因作物，该毒素对昆虫是致命的，但对对人类无害。然而，这些生物解决方案也具有缺点，包括低效率，以及违反自然生态平衡和发展遗传昆虫抗性的危险(例如在使用芽孢杆菌毒素的情况下)。
纳米抗体，也称为VHH抗体，是单结构域抗体，实际上包含抗体(HCAb)的重链而完全没有轻链。它们是由Raymond Hamers在骆驼的血液中发现的，在1989年布鲁塞尔自由大学(Vrije University Brussel)记载了该发现。纳米抗体是可用的最小完整抗原结合片段(Cortez–Retamozo等人,2004年；Revets等人,2005年)，具有大约15kDa的尺寸。纳米抗体具有显著的优势，包括在多种表达系统中的高产量，它们的最小尺寸允许对它们的表位的高可达性、高物理化学稳定性、可逆重折叠和在水溶液中的高溶解性、高度均一性、显示没有自发二聚化的迹象，以及能够特异性识别具有亚纳摩尔亲和力的独特表位。以前已经提出了纳米抗体作为杀虫剂的用途(参见例如公开号为EP3415010和EP2609116的欧洲专利申请；公开号为US9516879的美国专利；公开号为US9803003B2的美国专利申请；公开号为WO2014191146的国际专利申请)。
发明内容
根据本公开的一些实施方式的一个方面，提供了一种特异性结合昆虫多肽的纳米抗体，所述昆虫多肽选自：
(i)包含几丁质结合域(CBD)的多肽，其中，所述纳米抗体结合所述CBD；
(ii)V-ATP酶亚基c(V-ATPase subunit c)，其中，所述纳米抗体包含如下序列号中所示的互补性决定区(CDR)：167-169；171和168-169；174和168-169；167、178和169；或者180-182，这些序列在所述纳米抗体上按从N到C的顺序排列；
(iii)海藻糖酶；
(iv)细胞色素p450单加氧酶；
(v)几丁质脱乙酰酶；
(vi)几丁质合成酶；和
(vii)NPC1甾醇转运体，
其中，所述纳米抗体与所述昆虫多肽的结合赋予所述纳米抗体昆虫防控活性。
根据本公开的一些实施例的一个方面，提供了一种包含纳米抗体的组合物，所述纳米抗体特异性结合选自以下的昆虫多肽：
(i)包含几丁质结合域(CBD)的多肽，其中，所述纳米抗体结合所述CBD；
(ii)V-ATP酶亚基c(V-ATPase subunit c)，其中，所述纳米抗体包含如下序列号中所示的互补性决定区(CDR)：167-169；171和168-169；174和168-169；167、178和169；180-182；187-189；或者191-193，这些序列在所述纳米抗体上按从N到C的顺序排列；
(iii)海藻糖酶；
(iv)细胞色素p450单加氧酶；
(v)几丁质脱乙酰酶；
(vi)几丁质合成酶；和
(vii)NPC1甾醇转运体，
以及具有昆虫防控活性的毒素部分。
根据本公开的一些实施方式，所述纳米抗体与所述昆虫多肽的结合赋予所述纳米抗体昆虫防控活性。
根据本公开的一些实施方式，所述纳米抗体下调所述昆虫多肽的活性。
根据本公开的一些实施方式，所述的纳米抗体或组合物被配制用于通过喷雾、灌溉和/或熏蒸递送。
根据本公开的一些实施方式，所述CBD是2型几丁质结合域(ChtBD2)。
根据本公开的一些实施方式，所述CBD包含选自序列号为3-7的氨基酸序列。
根据本公开的一些实施方式，所述纳米抗体特异性结合所述CBD并包含如下序列号中所示的互补性决定区(CDR)：15-17；19-21；23-25；或者15和28-29，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据本公开的一些实施方式，所述纳米抗体特异性结合所述CBD并包含如下序列号中所示的互补性决定区(CDR)：15-17；19-21；23-25；15和28-29；31-33；35-37；39-41；43-45；47-49；51-53；55-57；59-61；63-65；67-69；71-73；75-77；79-81；83-85；87-89；91、80和93；95-97；99-101；103-105；107-109；111-113；115-117；119-121；123-125；127-129；131-133；135-137；139-141；143-145；147-149；151-153；15和156-157；159-161；或者163-165，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据本公开的一些实施方式，所述纳米抗体特异性结合所述V-ATP酶亚基c并包含如下序列号中所示的互补性决定区(CDR)：167-169；171和168-169；174和168-169；167、178和169；或者180-182，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据本公开的一些实施方式，所述纳米抗体特异性结合所述海藻糖酶并包含如下序列号中所示的互补性决定区(CDR)：195-197；199和196-197；201-203；201和205-206；208-210；212和209-210；215-217；219-221；223-225；227-229；231、228和232；234-236；238-240；242-244；246-248；250-252；254-256；258-260；588-590；588-589和592；或者595、589和592，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据本公开的一些实施方式，所述纳米抗体特异性结合所述海藻糖酶并包含如下序列号中所示的互补性决定区(CDR)：195-197；199和196-197；201-203；201和205-206；208-210；212和209-210；215-217；219-221；223-225；227-229；231、228和232；234-236；238-240；242-244；246-248；250-252；254-256；或者258-260，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据本公开的一些实施方式，所述纳米抗体特异性结合所述海藻糖酶并包含如下序列号中所示的互补性决定区(CDR)：201和205-206；208-210；212和209-210；223-225；238-240；242-244；246-248；588-590；或者595、589和592，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据本公开的一些实施方式，所述纳米抗体特异性结合所述细胞色素p450单加氧酶并包含如下序列号中所示的互补性决定区(CDR)：262-264；262-263和266；262、270和264；274-276；280和275-276；282和275-276；282、275和285；287、220和289；291、220和289；239-294和289；296-298；300-302；304-306；308-310；312-314；或者316-318，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据本公开的一些实施方式，所述纳米抗体特异性结合所述细胞色素p450单加氧酶并包含如下序列号中所示的互补性决定区(CDR)：262-264；287、220和289；296-298；300-302；304-306；或者312-314，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据本公开的一些实施方式，所述纳米抗体特异性结合所述几丁质脱乙酰酶并包含如下序列号中所示的互补性决定区(CDR)：320-322；320和324-325；327、321和328；320-321和332；208和336-337；320、324和339；343-345；347-349；351-353；351、347和356；358-360；358-359和362；365、359和366；368-370；372和369-370；374、369和375；377-379；381-383；385和382-383；387-389；387、391和389；394-396；398-400；402-404；或者406-408，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据本公开的一些实施方式，所述纳米抗体特异性结合所述几丁质脱乙酰酶并包含如下序列号中所示的互补性决定区(CDR)：320-322；320和324-325；372和369-370；374、369和375；377-379；387-389；或者398-400，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据本公开的一些实施方式，所述纳米抗体特异性结合所述几丁质合成酶并包含如下序列号中所示的互补性决定区(CDR)：412-414；418-420；422-424；426-428；426-427和430；432-434；436、140和437；439-441；443-445；447-449；356和451-452；454-456；458-460；462-464；466-468；470-472；474-476；478-480；482、479和483；485-487；489-491；493-495；426和497-498；500-502；504-506；或者508-510，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据本公开的一些实施方式，所述纳米抗体特异性结合所述几丁质合成酶并包含如下序列号中所示的互补性决定区(CDR)：412-414；418-420；426-428；432-434；443-445；447-449；466-468；482、479和483；426和497-498；或者504-506，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据本公开的一些实施方式，所述纳米抗体特异性结合所述NPC1甾醇转运体并包含如下序列号中所示的互补性决定区(CDR)：512-514；517和513-514；521-523；521-522和526；531-533；539-541；485和540-541；545和540-451；547-549；552-554；556-558；561-563；565-567；569-571；573-575；485和577-578；580-582；或者584-586，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据本公开的一些实施方式，所述纳米抗体特异性结合所述NPC1甾醇转运体并包含如下序列号中所示的互补性决定区(CDR)：517和513-514；485和540-541；545和540-541；556-558；565-567；573-575；485和577-578；或者580-582，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据本公开的一些实施方式的一个方面，提供了一种编码所述纳米抗体或所述组合物的多核苷酸。
根据本公开的一些实施方式的一个方面，提供了一种核酸构建体，包含所述多核苷酸和用于指导所述多核苷酸表达的顺式作用调节元件。
根据本公开的一些实施方式的一个方面，提供了一种宿主细胞，包含所述纳米抗体或所述组合物或编码其的多核苷酸或核酸构建体。
根据本公开的一些实施方式的一个方面，提供了一种生产昆虫防控纳米抗体的方法，所述方法包括在宿主细胞中表达所述多核苷酸或所述核酸构建体。
根据本公开的一些实施方式，所述方法包括分离所述纳米抗体。
根据本公开的一些实施方式的一个方面，提供了一种昆虫防控方法，所述方法包括使所述昆虫与所述纳米抗体或所述组合物、或编码其的多核苷酸或核酸构建体或表达其的宿主细胞接触。
根据本公开的一些实施方式，所述接触包括将所述纳米抗体或所述组合物直接施用于所述昆虫。
根据本公开的一些实施方式，所述接触包括将所述纳米抗体或所述组合物施用于可能与所述昆虫接触的生物体或表面。
根据本公开的一些实施方式，所述纳米抗体或所述组合物被配制成液体制剂。
根据本公开的一些实施方式，所述纳米抗体或所述组合物配制成干燥制剂。
根据本公开的一些实施方式的一个方面，提供了一种植物，包含所述纳米抗体或所述组合物或编码其的多核苷酸或核酸构建体。
根据本公开的一些实施方式，所述植物是转基因植物。
根据本公开的一些实施方式的一个方面，提供了一种商品产品，包含所述纳米抗体或所述组合物。
根据本公开的一些实施方式，所述商品产品由所述植物生产。
根据本公开的一些实施方式的一个方面，提供了一种生产纳米抗体的方法，所述方法包括用重组或纯化的昆虫多肽免疫骆驼科动物，所述重组或纯化的昆虫多肽选自：
(i)包含几丁质结合域(CBD)的多肽；
(ii)V-ATP酶亚基c；
(iii)海藻糖酶；
(iv)细胞色素p450单加氧酶；
(v)几丁质脱乙酰酶；
(vi)几丁质合成酶；和
(vii)NPC1甾醇转运体；
其中，所述昆虫多肽在昆虫多肽制剂中的纯度为至少80％。
根据本公开的一些实施方式，所述方法包括在所述免疫后分离所述抗体。
据本公开的一些实施方式，所述骆驼科动物是羊驼(llama)。
据本公开的一些实施方式，所述昆虫选自蛾、臭虫、漏斗虫(hopper)、甲虫、蚜虫和蜜蜂。
据本公开的一些实施方式，所述昆虫是蛾。
据本公开的一些实施方式，所述蛾选自棉铃虫(Helicoverpa armigera)和草地夜蛾(Spodoptera frugiperda)。
据本公开的一些实施方式，所述蛾是棉铃虫。
除非另有限定，否则本文中使用的所有技术和/或科学术语具有与本公开所属领域的普通技术人员通常理解的含义相同的含义。尽管可以在本公开的实施方式的实践或测试中使用与本文描述的那些类似或等同的方法和材料，但是下面描述了示例性方法和/或材料。如有冲突，以专利说明书(包括限定)为准。此外，这些材料、方法和实施例仅是说明性的，并不意味着必须是限制性的。
附图说明
本文通过示例的方式参考附图描述了本发明的一些实施例。现在详细地具体参考附图，需要强调的是，所示的细节是示例性的，并且是为了说明性地讨论本公开的实施方式。在这点上，结合附图所作的描述使得本领域技术人员清楚如何实施本公开的实施方式。
在附图中：
图1示出了所产生的抗CBD纳米抗体(本文中称为CB20501、CB20801、CB20901和CB201101)与目标CBD抗原的特异性结合(通过ELISA测定)的图。每个实验进行五次；数据表示为平均值±SE。
图2示出了与PBS对照组相比，用从棉铃虫(H.armigera)幼虫肠道得到的幼虫肠汁培育1小时后产生的抗CBD纳米抗体CB20501、CB20801、CB20901和CB201101的稳定性(通过ELISA测定)的图。每个实验进行三次；数据表示为平均值±SE。
图3示出了与室温(RT)对照组相比，在类似田间条件下培育1-3小时后产生的抗CBD纳米抗体CB20501、CB20801、CB20901和CB201101的稳定性(通过ELISA测定)的图。每个处理包括5次重复；数据表示为平均值±SE。
图4A-C示出了在以单次剂量40μg所示纳米抗体饲喂后，抗CBD纳米抗体CB20501、CB20801、CB20901和CB20110对棉铃虫体长(图4A)、重量(图4B)和死亡率(图4C)的影响。在第7天和第7天或第14天测量死亡率。每个实验包括12个幼虫，重复3-5次，其中误差线表示标准误差偏差。示出了饲喂所示纳米抗体的幼虫的长度相对于饲喂PBS的对照幼虫的长度的百分比(图4A)、饲喂所示纳米抗体的幼虫的重量相对于饲喂PBS的对照幼虫的重量的百分比(图4B)，以及7天和14天后死亡幼虫的百分比(图4C)。统计学意义的差异用a(P<0.05)和b(P<0.01)表示。
图5A-C示出了在用含有荧光Cy3标记的CB20901的人工食物(artificial food)培育之后，昆虫肠道中存在所产生的抗CBD纳米抗体CB20901。图5A示出了与幼虫一起培育48小时和6天后，在幼虫食物中追踪到的荧光标记的纳米抗体。上面板显示亮视野图像，下面板显示反映饲喂给幼虫的食物的Cy3信号的图像。此外，黄色虚线表示两个不同的区域，虚线的右侧表示完整食物，而虚线的左侧表示幼虫粪便。食物和粪便之间荧光强度的差异代表幼虫肠道的吸收。图5B示出了，在用含有Cy3标记的纳米抗体的食物培育48小时之后，或用含有Cy3标记的纳米抗体的食物培育36小时之后，接着用不含纳米抗体的食物培育12小时之后，在活的幼虫的整个肠道中存在Cy3标记的CB20901纳米抗体。图5C示出了，与用不含纳米抗体的食物饲喂48小时的幼虫相比，在用含有Cy3标记的纳米抗体的食物培育36小时之后，接着用不含纳米抗体的食物培育12小时后，Cy3标记的CB20901纳米抗体存在于幼虫肠道中，具体地，存在于围食膜中。上面板显示亮视野图像，下面板显示反映Cy3信号的图像。
图6示出了所产生的抗ATP酶V亚基c(anti-ATPase V subunit c)(本文中称为“VAT”)纳米抗体(本文中称为VAT0101A、VAT0101B、VAT0101C、VAT0101D、VAT0101E、VAT0101F和VAT0101G)与靶VAT抗原的特异性结合(通过ELISA测定)的图。每个实验进行五次；数据表示为平均值±SE。
图7示出了与PBS对照组相比，用从棉铃虫幼虫肠得到的幼虫肠汁培育1小时后产生的抗VAT纳米抗体VAT0101A和VAT0101G的稳定性(通过ELISA测定)的图。每个实验进行三次；数据表示为平均值±SE。
图8示出了与室温(RT)对照组相比，在类似田间条件下培育1-3小时后生成的抗VAT纳米抗体VAT0101A、VAT0101B、VAT0101C、VAT0101D、VAT0101E、VAT0101F和VAT0101G的稳定性(通过ELISA测定)的图。每个处理包括3次重复；数据表示为平均值±SE。
图9A-C示出了在以单次剂量40μg所示纳米抗体饲喂后：抗VAT纳米抗体VAT0101A、VAT0101B、VAT0101C、VAT0101D、VAT0101E、VAT0101F、VAT0101G和VAT0201，所示纳米抗体对棉铃虫体体长(图9A)、重量(图9B)和死亡率(图9C)的影响。如图所示，在第7天或第14天进行测量。每个实验包括12个幼虫，重复3-5次，其中误差线表示标准误差偏差。示出了饲喂所示纳米抗体的幼虫的长度相对于饲喂PBS的对照幼虫的长度的百分比(图9A)、饲喂所述纳米抗体的幼虫的重量相对于饲喂PBS的对照幼虫的重量的百分比(图9B)，以及7天和14天后死亡幼虫的百分比(图9C)。统计学意义的差异用a(P<0.05)和b(P<0.01)表示。
图10示出了所产生的抗海藻糖酶(本文中称为“TRH”)纳米抗体(本文中称为TRH1A、TRH12、TRH13和TRH14)与靶TRH抗原的特异性结合(通过ELISA测定)的图。每个实验进行五次；数据表示为平均值±SE。
图11A-B示出了与室温(RT)对照组相比，在类似田间条件下培育6小时或过夜后(图11A)，以及在50℃(图11B)下培育6小时或过夜后，所产生的抗TRH纳米抗体(本文中称为TRH1A、TRH1D、TRH3B、TRH5A和TRH5C)的稳定性(通过ELISA测定)的图。每个处理包括三次重复；数据表示为平均值±SE。
图12A示出了在第0天和第7天，用含有终浓度为0.4mg/ml的所示纳米抗体的人工食物饲喂之后，抗TRH纳米抗体TRH1A、TRH1D、TRH3B、TRH5A、TRH5B、TRH5C和TRH8对棉铃虫的死亡率的影响。如图所示，在第7天和第14天进行测量。每个实验包括12个幼虫，重复3次，其中误差线表示标准误差偏差。统计学意义的差异用a(P<0.05)表示。
图12B示出了在第0天，用施用了浓度为0.4或1mg/ml的纳米抗体的新鲜棉叶圆片饲喂之后，抗TRH3B纳米抗体对棉铃虫死亡率的影响。在第4天和第7天，将幼虫移至用相似浓度的所述纳米抗体处理的新鲜圆棉片中。如图所示，在第4天、第7天和第10天进行死亡率测量。每个实验包括12个幼虫，重复2次，其中误差线表示标准误差偏差。
图13示出了所产生的抗细胞色素p450单加氧酶(本文中称为“P450”)纳米抗体(本文中称为P45001A、P45003A、P45004、P45005和P45006)与靶P450抗原的特异性结合(通过ELISA测定)的图。每个实验进行五次；数据表示为平均值±SE。
图14示出了在第0天和第7天，用含有终浓度为0.4mg/ml的所述纳米抗体的人工饲料(rtificial diet)饲喂之后，所产生的抗P450纳米抗体(本文中称为P45008)对棉铃虫死亡率的影响。如所示，在第7天和第14天进行测量。每个实验包括12个幼虫，重复3次，其中误差线表示标准误差偏差。
图15示出了所产生的抗几丁质脱乙酰酶(anti-chitin deacetylase)(本文中称为“CDA”)纳米抗体(本文中称为CDA01A、CDA05、CDA07A和CDA10)与靶CDA抗原的特异性结合(通过ELISA测定)的图。每个实验进行5次；数据表示为平均值±SE。
图16A-B示出了与室温(RT)对照组相比，在类似田间条件下培育6小时或过夜后(图16A)，以及在50℃(图16B)下培育6小时或过夜后，所产生的CDA7A纳米抗体的稳定性(通过ELISA测定)的图。每个处理包括3次重复；数据表示为平均值±SE。
图17A示出了在第0天和第7天，用含有终浓度为0.4mg/ml的所述纳米抗体的人工饲料饲喂之后，抗CDA纳米抗体(本文中称为CDA1B、CDA4B、CDA4C和CDA7A)对棉铃虫死亡率的影响。如图所示，在第7天和第14天进行测量。每个实验包括12个幼虫，重复3次，其中误差线表示标准误差偏差。统计学意义的差异用a(P<0.05)表示。
图17B示出了在第0天，用施用了增加的浓度(0.2、0.4、1，2或4mg/ml，如图所示)的纳米抗体的新鲜棉叶圆片饲喂之后，CDA7A纳米抗体对棉铃虫死亡率的影响。在第4天和第7天，将幼虫移至用相似浓度的所述纳米抗体处理的新鲜棉叶圆片中。如图所示，在第4天、第7天和第10天进行死亡率测量。每个实验包括12个幼虫，重复2次，其中误差线表示标准误差偏差。
图18示出了所产生的抗几丁质合成酶(anti-chitin synthase)(本文称为“CHS”)纳米抗体(本文中称为CHS01A、CHS02A、CHS08、CHS13、CHS18、CHS22和CHS24)与靶几丁质抗原的特异性结合(通过ELISA测定)的图。每个实验进行5次；数据表示为平均值±SE。
图19A-B示出了与室温(RT)对照组相比，在类似田间条件下培育6小时或过夜后(图19A)，以及在50℃(图19B)下培育6小时或过夜后，所产生的抗CHS纳米抗体的稳定性(通过ELISA测定)的图。每个处理包括3次重复；数据表示为平均值±SE
图20A示出了在第0天和第7天，用含有终浓度为0.4mg/ml的所述纳米抗体的人工饲料饲喂之后，抗CHS纳米抗体(本文中称为CHS3A、CHS4和CHS7)对棉铃虫死亡率的影响。如图所示，在第7天和第14天进行测量。每个实验包括12个幼虫，重复3次，其中误差线表示标准误差偏差。统计学意义的差异用a(P<0.05)表示。
图20B示出了在第0天，用施用了增加的浓度(0.2、0.4、1，2或4mg/ml，如图所示)的纳米抗体的新鲜棉叶圆片饲喂之后，CHS3A纳米抗体对棉铃虫死亡率的影响。在第4天和第7天，将幼虫移至用相似浓度的所述纳米抗体处理的新鲜棉叶圆片中。如图所示，在第4天、第7天和第10天进行死亡率测量。每个实验包括12个幼虫，重复2次，其中误差线表示标准误差偏差。统计学意义的差异用a(P<0.05)表示。
图21示出了所产生的抗NPC1甾醇转运体(本文中称为“NPC”)纳米抗体(本文中称为NPC01C01、NPC04C01、NPC04D01、NPC07A01、NPC0901、NPC01101和NPC01201)与靶NPC抗原的特异性结合(通过ELISA测定)的图。每个实验进行5次；数据表示为平均值±SE。
图22示出了在第0天和第7天，用含有终浓度为0.4mg/ml的所示纳米抗体的人工饲料饲喂之后，抗NPC纳米抗体(本文中称为TRH1A、TRH1D、TRH3B、TRH5A、TRH5B、TRH5C和TRH8)对棉铃虫的死亡率的影响。如图所示，在第7天和第14天进行测量。每个实验包括12个幼虫，重复3次，其中误差线表示标准误差偏差。
具体实施方式
本公开在其一些实施方式中涉及昆虫防控纳米抗体及其用途。
在详细解释本公开的至少一个实施方式之前，应当理解的是，本公开在其应用中不一定局限于在下面的描述中阐述的或者由实施例示出的细节。本公开能够有其他实施方式，或者能够以各种方式实践或执行。
在过去的70年里，害虫的治理是通过使用合成农药来实现的，其中大多数很有可能破坏生态系统，它们直接或通过食物链对人类有毒性，并且它们的使用也受到遗传昆虫抗性发展的阻碍。目前使用的最突出的生物解决方案也具有许多缺点，包括低效率，以及违反自然生态平衡和发展遗传昆虫抗性的危险。
在将本公开付诸实践的同时，本发明人现在已经开发出几种针对具有昆虫防控活性的昆虫多肽的纳米抗体。因此，本教导的具体实施方式建议将其用作杀虫剂。
因此，根据本公开的第一方面，提供了一种特异性结合昆虫多肽的纳米抗体，所述昆虫多肽选自：
(i)包含几丁质结合域(CBD)的多肽，其中，所述纳米抗体结合所述CBD；
(ii)V-ATP酶亚基c(V-ATPase subunit c)，其中，所述纳米抗体包含如下序列号中所示的互补性决定区(CDR)：167-169；171和168-169；174和168-169；167、178和169；或者180-182，这些序列在所述纳米抗体上按从N到C的顺序排列；
(iii)海藻糖酶；
(iv)细胞色素p450单加氧酶；
(v)几丁质脱乙酰酶；
(vi)几丁质合成酶；以及
(vii)NPC1甾醇转运体，
其中，所述纳米抗体与所述昆虫多肽的结合赋予所述纳米抗体昆虫防控活性。
根据本公开的另外的或替代的方面，提供了一种包含纳米抗体的组合物，所述纳米抗体特异性结合选自由以下昆虫多肽组成的组：
(i)包含几丁质结合域(CBD)的多肽，其中，所述纳米抗体结合所述CBD；
(ii)V-ATP酶亚基c，其中，所述纳米抗体包含如下序列号中所示的互补性决定区(CDR)：167-169；171和168-169；174和168-169；167、178和169；180-182；187-189；或191-193，这些序列在所述纳米抗体上按从N到C的顺序排列；
(iii)海藻糖酶；
(iv)细胞色素p450单加氧酶；
(v)几丁质脱乙酰酶；
(vi)几丁质合成酶；以及
(vii)NPC1甾醇转运体，
以及具有昆虫防控活性的毒素部分。
如本文所使用的，术语“纳米抗体”是指单域抗原结合片段。
根据具体实施方式，纳米抗体是来源于天然存在的抗体重链的单个可变域。纳米抗体通常来源于骆驼科动物中见到的只有重链(不含轻链)的抗体(Hamers-Casterman等人，1993,自然杂志(Nature)363:446-448；Desmyter等人,1996,自然结构生物学杂志(Nat.Struct.Biol.)803-811)，因此，通常称为VHH抗体或VHH序列。骆驼科动物包括旧世界的骆驼科动物(双峰驼(Camelus bactrianus)和单峰驼(Camelus dromedarius))和新世界的骆驼科动物(例如，羊驼(Lama paccos)、大羊驼(Lama glama)、原驼(Lama guanicoe)和瘦驼(Lama vicugna))。骆驼科动物的非限制性实施例包括单峰骆驼(dromedary camels)、双峰骆驼(Bactrian camels)、野生双峰骆驼(wild Bactrian camels)、美洲驼(llamas)、羊驼(alpacas)、小羊驼
Figure BDA0003723677810000141
和红褐色美洲驼(guanacos)。根据具体实施方式，骆驼科动物是美洲驼(llama)。
Figure BDA0003723677810000142
和
Figure BDA0003723677810000143
是Ablynx NV(比利时)的注册商标。
关于VHH或纳米抗体的进一步描述，可参考“单域抗体(Single domainantibodies)”，分子生物学方法杂志(Methods in Molecular Biology)，编辑Saerens和Muyldermans，2012，Vol.911，特别是Vincke和Muyldermans(2012年)的章节，以及作为一般背景技术提及的专利申请的非限制性列表，包括：布鲁塞尔自由大学的WO94/04678、WO95/04079、WO96/34103；联合利华公司的WO94/25591、WO99/37681、WO00/40968、WO00/43507、WO00/65057、WO01/40310、WO01/44301、EP 1134231和WO02/48193；比利时佛朗德生物技术研究院(Vlaams Instituut voor Biotechnologie(VIB))的WO97/49805、WO01/21817、WO03/035694、WO03/054016和WO03/055527；Ablynx N.V的WO04/041867、WO04/041862、WO04/041865、WO04/041863、WO04/062551、WO05/044858、WO06/40153、WO06/079372、WO06/122786、WO06/122787和WO06/122825，以及Ablynx N.V.的进一步公开的专利申请。如本领域技术人员所知，纳米抗体的特征尤其在于在一个或多个框架序列(根据Kabat编号(Kabatnumbering))中存在一个或多个“标志残基(hallmark residues)”，例如在WO 08/020079，在第75页，表A-3中所述，通过引用并入本文。
根据具体实施方式，纳米抗体指能够结合完整分子结合的抗原的表位的完整分子(即包含4个骨架区域和3个互补性决定区)或其功能片段。
如本文所使用的，术语“表位(epitope)”是指抗原上与纳米抗体的互补位结合的任何抗原决定簇。表位决定簇(Epitopic determinants)通常由分子(例如氨基酸或碳水化合物侧链)的化学活性表面基团组成，并且通常具有特定的三维结构特征以及特定的电荷特性。
根据具体实施方式，纳米抗体是完整的(whole或intact)纳米抗体。
根据具体实施方式，纳米抗体是纳米抗体片段。
根据具体实施方式，纳米抗体的尺寸为5-30kDa、10-30KDa或10-20KDa。
根据具体实施方式，纳米抗体的尺寸为约15kDa。
术语“纳米抗体”还包括纳米抗体的天然或合成类似物、同源物、突变体和变体。
通常，完整的纳米抗体包括三个互补性决定区(CDR)(CDR1；CDR2；和CDR3)。
如本文所使用的，术语“互补性决定区”或“CDR”可互换使用，指重链多肽的可变区内发现的抗原结合区。组成CDR的特定纳米抗体中的氨基酸残基的身份可以使用本领域众所周知的方法来确定，包括如下方法：例如Kabat等人定义的序列可变性等方法(参见，例如，Kabat等人，1992，具有免疫学意义的蛋白质序列(Sequences of Proteins ofImmunological Interest)，第5版，公共卫生服务，NIH，华盛顿特区)，如在Riechmann和Muyldermans(1999)的文章中应用于来自骆驼科动物的VHH结构域的方法，Chothia等人定义的结构环区域的位置(例如参见Chothia等人，自然杂志(Nature)342:877-883,1989)，这是使用Oxford Molecular的AbM抗体建模软件实现的Kabat HVR和Chothia的折中(现在参见
Figure BDA0003723677810000151
Martin等人，1989，美国国家科学研究院学报(Proc.Natl Acad Sci USA)86:9268；和万维网网址www.biinf-org.uk/abs)，由接触定义限定的可用的复杂的晶体结构(参见MacCallum等人，分子生物学杂志(J.Mol.Biol.)262:732-745,1996)和“构象定义(conformational definition)”(例如参见Makabe等人，生物化学杂志(Journal ofBiological Chemistry)，283:1156-1166,2008)。
如本文所使用的，“CDR”可指由本领域已知的任何方法(包括方法的组合)定义的CDR。
根据具体实施方式，CDR如Kabat等人所定义，在Riechmann和Muyldermans(1999)的文章中应用于骆驼科动物的VHH域。
纳米抗体可以是单特异性的(能够识别一个表位或蛋白质)、双特异性的(能够结合两个表位或蛋白质)或多特异性的(能够识别多个表位或蛋白质)。
根据具体实施方式，纳米抗体是单特异性纳米抗体。
根据具体实施方式，纳米抗体是多特异性的，例如双特异性的、三特异性的、四特异性的。
根据具体实施方式，纳米抗体是双特异性纳米抗体。产生双特异性纳米抗体的方法是本领域已知的，例如如下文献公开的：Deffar K、Shi H、Li L、Wang X、ZhuX(2009)非洲生物技术(Afr J Biotechnol)8(12):2645–2652；和Zhu，Y等人(2017)科学报告(Scientific reports)，7(1),2602；其内容通过引用全部并入本文。
本文公开的纳米抗体特异性结合本文所述的昆虫多肽。
优选地，纳米抗体特异性结合本文所述的昆虫多肽的至少一个表位。
用于测试结合的测定是本领域公知的，包括但不限于酶联免疫吸附测定(ELISA)、放射免疫分析法(RIA)、流式细胞术(flow cytometry)、分子互作分析(BiaCore)、生物膜干涉技术
Figure BDA0003723677810000162
测定(bio-layer interferometry
Figure BDA0003723677810000161
assay)、高效液相色谱法(HPLC)。
根据具体的实施方式，纳米抗体结合Kd≤10-6M、≤10-7M、≤10-8M、≤10-9M的昆虫多肽，每种可能性代表本公开的一个单独的实施方式。
根据具体实施方式，纳米抗体能够在开放田间或受控条件(例如温室)下与昆虫多肽结合。
术语“昆虫”在本文中以广义的通俗意义使用，包括泛节肢动物超门(superphylumPanarthropoda)的所有种类(分类系统自然(Systema Naturae)，Brands，S.J.(comp.)1989-2005)系统自然2000，阿姆斯特丹，荷兰，[www.sn2000.taxonomy.nl/])，包括节肢动物门(Arthropoda)、缓步动物门(Tardigrada)和有爪动物门(Onychophora)；包括生命周期的所有不同阶段，例如但不限于卵、幼虫、若虫、蛹和成虫。根据具体实施方式，昆虫属于节肢动物门(包括但不限于石蛃目(Archaeognatha)、缨尾目(Thysanura)、古翅类(Paleoptera)和新翅亚部(Neoptera)，也包括蜱、螨和蜘蛛目)，甚至更优选属于六足总纲，最优选为昆虫纲。根据具体实施方式，昆虫属于鳞翅目。昆虫的非限制性实施例包括臭虫、家蝇、蛾子、甲虫、蚱蜢、毛虫、蚜虫、蚊子、跳蚤、马蝇、大黄蜂、蟑螂和蚂蚁，例如但不限于：
·鳞翅目，例如：长翅卷蛾属(Acleris spp.)、茶姬捲葉蛾属(Adoxophyes spp.)、切根夜蛾属(Agrotis spp.)、木棉虫(Alabama argillacea)、脐橙螟属(Amyelois spp.)、大豆夜蛾(Anticarsia gemmatalis)、黄卷蛾属(Archips spp.)、条卷蛾属(Argyrotaeniaspp.)、丫纹夜蛾属(Autographa spp.)、玉米茎蛀褐夜蛾(Busseola fusca)、干果斑螟(Cadra cautella)、桃小食心虫(Carposina nipponensis)、二化螟(Chilosuppressalis)、禾草螟属(Chilo spp.)、大杨卷叶蛾(Choristoneura conflictana)、云杉色卷蛾(Choristoneura fumiferana)、云杉芽卷蛾(Choristoneura occidentalis)、玫瑰色卷蛾(Choristoneura rosaceana)、色卷蛾属(Choristoneura spp.)、葡萄果蠹蛾(Clysia ambiguella)、野螟属(Cnaphalocrocis spp.)、云卷蛾属(Cnephasia spp.)、细卷蛾属(Cochylis spp.)、鞘蛾属(Coleophora spp.)、泛非绒毛螟(Crocidolomiabinotalis)、苹果异形小卷蛾(Cryptophlebia leucotreta)、黄杨绢野螟成虫(Cydalimaperspectalis)、苹果小食心虫(Cydia inopinata)、小卷蛾属(Cydia spp.)、杆草螟属(Diatraea spp.)、苏丹棉铃虫(Diparopsis castanea)、金刚钻属(Earias spp.)、粉斑螟属(Ephestia spp.)、花小卷蛾属(Eucosma spp.)、女贞细卷蛾(Eupoecilia ambiguella)、黄毒蛾属(Euproctis spp.)、切根虫属(Euxoa spp.)、杏小卷蛾(Grapholita prunivora)、小食心虫属(Grapholita spp.)、芽广翅小卷蛾(Hedya nubiferanal)、棉铃虫(Helicoverpa armigera)、美洲棉铃虫(Helicoverpa zea)、夜蛾属(Helicoverpa spp.)、实夜蛾属(Heliothis spp.)、菜心野螟(Hellula undalis)、美国白蛾(Hyphantriacunea)、番茄茎麦蛾(Keiferia lycopersicella)、旋纹潜叶蛾(Leucoptera scitella)、潜叶细蛾属(Lithocolletis spp.)、葡萄花翅小卷蛾(Lobesia botrana)、毒蛾属(Lymantriaspp.)、潜蛾属(Lyonetia spp.)、天幕毛虫属(Malacosoma spp.)、甘蓝夜蛾(Mamestrabrassicae)、烟草天蛾(Manduca sexta)、梨大食心虫(Numonia pyrivorella)、秋尺蛾属(Operophtera spp.)、蔗扁蛾(Opogona sacchari)、玉米螟(Ostrinia nubilalis)、超小卷蛾属(Pammene spp.)、褐卷蛾属(Pandemis spp.)、小眼夜蛾(Panolis flammea)、棕榈蝶蛾(Paysandisia archon)、棉红铃虫(Pectinophora gossypiella)、马铃薯麦蛾(Phthorimaea operculella)、小潜细蛾属(Phyllonorycter spp.)、菜粉蝶(Pierisrapae)、马醉木属(Pieris spp.)、Platynota rostrana、小菜蛾(Plutella xylostella)、小白巢蛾属(Prays spp.)、白禾螟属(Scirpophaga spp.)、蛀茎叶蛾属(Sesamia spp.)、Sesia spp.、长须卷蛾属(Sparganothis spp.)、扁豆甜菜夜蛾(Spodoptera dolichos)、亚热带黏虫(Spodoptera eridania)、甜菜夜蛾(Spodoptera exigua)、草地贪夜蛾(Spodoptera frugiperda)、灰翅夜蛾(Spodoptera littoralis)、斜纹夜蛾(Spodopteralitura)、灰翅夜蛾属(Spodoptera spp.)、透翅蛾属(Synanthedon spp.)、蛾(Teciasolanivora)、苹果异胫小卷蛾(Thaumatotibia leucotreta)、栎列队蛾(Thaumetopoeaprocessionea)、异舟蛾属(Thaumetopoea spp.)、卷蛾属(Tortrix spp.)、粉纹夜蛾(Trichoplusia ni)和巢蛾属(Yponomeuta spp.)；
·鞘翅目，例如：Agrilus anxius、白蜡窄吉丁(Agrilus planipennis)、叩甲属(Agriotes spp.)、Anomala orientalis、星天牛(Anoplophora chinensis)、光肩星天牛(Anoplophora glabripennis)、星天牛属(Anoplophora spp.)、草莓象(Anthonomusbisignifer)、胡椒花象(Anthonomus eugenii)、棉铃象甲(Anthonomus grandis)、苹象(Anthonomus quadrigibbus)、草莓花象(Anthonomus signatus)、花象属(Anthonomusspp.)、粒肩天牛属(Apriona spp.)、栎三锥象(Arrhenodes minutus)、甜菜隐食甲(Atomaria linearis)、甜菜胫跳甲(Chaetocnema tibialis)、梅锥象甲(Conotrachelusnenuphar)、根颈象甲(Cosmopolites spp.)、象鼻虫(Curculio spp.)、云杉大小蠹(Dendroctonus micans)、西伯利亚松毛虫(Dendrolimus sibiricus)、皮蠹属(Dermestesspp.)、玉米根叶甲(Diabrotica virgifera)、Diabrotica virgifera zeae、玉米根叶甲(Diabrotica virgifera)、带斑黄瓜叶甲(Diabrotica balteata)、巴氏根萤叶甲(Diabrotica barberi)、黄瓜十一星叶甲(Diabrotica undecimpunctata howardi)、Diabrotica undecimpunctata tenella、黄瓜点叶甲(Diabrotica undecimpunctata)、黄瓜点叶甲(Diabrotica undecimpunctata)、叶甲属(Diabrotica spp.)、墨西哥豆瓢虫(Epilachna varivestis)、食植瓢虫属(Epilachna spp.)、黄瓜跳甲(Epitrixcucumeris)、Eremnus cerealis、Eremnus spp.、桉象(Gonipterus scutellatus)、Ipsamitinus、落叶松齿小蠹(Ips cembrae)、重齿小蠹(Ips duplicatus)、十二齿小蠹(Ipssexdentatus)、云杉八齿小蠹(Ips typographus)、齿小蠹属(Ips spp.)、马铃薯甲虫(Leptinotarsa decemlineata)、伪马铃薯叶甲(Leptinotarsa juncta)、泰夏那甲虫(Leptinotarsa texana)、稻象属(Lissorhoptrus spp.)、阿根廷茎象甲(Listronotusbonariensis)、鳃角金龟属(Melolontha spp.)、墨天牛属(Monochamus spp.)、白缘象甲(Naupactus leucoloma)、锯谷稻属(Oryzaephilus spp.)、耳象属(Otiorhynchus spp.)、斑象属(Phlyctinus spp.)、木蠹象(Pissodes nemorensis)、白松木蠹象(Pissodesstrobi)、榛梢木蠹象(Pissodes terminalis)、松梢木蠹象(Pissodes spp.)、日本弧丽金龟(Popilia japonica)、云粉蝶属(Popilia spp.)、安第斯马铃薯象甲(Premnotrypesspp.)、栎鬃额小蠹(Pseudopityophthorus minutissimus)、Pseudopityophthoruspruinosus、十节跳甲属(Psylliodes spp.)、谷蠹属(Rhizopertha spp.)、红棕象甲(Rhynchophorus ferrugineus)、棕榈象甲(Rhynchophorus palmarum)、金龟子科家族属(Scarabaeidae family spp.)、小蠹科家族属(Scolytidae family spp.)、米象属(Sitophilus spp.)、麦蛾属(Sitotroga spp.)、芒果果核象(Sternochetus mangiferae)、粉虫属(Tenebrio spp.)、赤拟谷盗(Tribolium castaneum)、拟谷盗属(Tribolium spp.)和皮蠹属(Trogoderma spp.)
·直翅目，例如：蝼蛄属(Gryllotalpa spp.)、飞蝗属(Locusta spp.)和沙漠蝗属(Schistocerca spp.)；
·蜚蠊目，例如：蜚蠊属(Blatta spp.)、小蠊属(Blattella spp.)、大蠊属(Periplaneta spp.)和马德拉蜚蠊(Leucophaea maderae)；
·等翅目，例如：乳白蚁属(Coptotermes spp.)和散白蚁属(Reticulitermesspp.)；
·啮虫目，例如，粉啮虫属(Liposcelis spp.)；
·虱毛目(Phthiraptera)、虱亚目(suborder Anoplura)，例如：血虱属(Haematopinus spp.)、毛虱属(Linognathus spp.)和虱属(Pediculus spp.)，以及嚼虱属(Trichodectes spp.)；
·虱毛目(Phthiraptera)、细角亚目(suborder Ischnocera)，例如：牛虱属(Damalinia spp.)；
·缨翅目(Thysanoptera)，例如：西花蓟马(Frankliniella occidentalis)、Frankliniella platensis、花蓟马属(Frankliniella spp.)、篱蓟马属(Hercinothripsspp.)、带蓟马属(Taeniothrips spp.)、棕榈蓟马(Thrips palmi)、烟蓟马(Thripstabaci)、非洲橘硬蓟马(Scirtothrips aurantii)、柑橘蓟马(Scirtothrips citri)、茶黄蓟马(Scirtothrips dorsalis)和硬蓟马属(Scirtothrips spp.)；
·从半翅目(Hemiptera)、异翅亚目(suborder Heteroptera)，例如：臭虫(Cimexspp.)、可可狄盲蝽(Distantiella theobroma)、棉红蝽属(Dysdercus spp.)、蝽属(Euschistus spp.)、扁盾蝽属(Eurygaster spp.)、稻缘畴属(Leptocorisa spp.)、绿蝽属(Nezara spp.)、皮畴(Piesma spp.)、红猎蝽属(Rhodnius spp.)、可可褐盲蝽(Sahlbergella singularis)、黑蝽属(Scotinophara spp.)、锥蝽属(Triatoma spp.)、盲蝽家族属(Miridae family spp.)例如豆荚草盲蝽(Lygus hesperus)和美国牧草盲蝽(Lygus lineoloris)、长蝽家族属(Lygaeidae family spp.)例如麦长蝽(Blissusleucopterus)，和椿家族属(Pentatomidae family spp.)；
·半翅目(Hemiptera)、胸喙亚目(suborder Sternorrhyncha)，例如：茶园黑刺粉虱(Aleurocanthus spiniferus)、柑黑刺粉虱(Aleurocanthus woglumi)、粉虱属(Aleurocanthus spp.)、橘绵粉虱(Aleurothrixus floccosus)、Aleyrodes brassicae、Aonidella citrina、蚜家族属(Aphididae family spp.)、无网蚜属(Acyrthosiphonspp.)、蚕豆蚜(Aphis fabae)、大豆蚜(Aphis glycines)、棉蚜(Aphis gossypii)、豆蚜(Aphis spp.)、圆盾娇属(Aspidiotus spp.)、烟粉虱(Bemisia tabaci)、蜡蚧属(Ceroplastes spp.)、黑褐圆盾蚧(Chrysomphalus aonidium)、橙褐圆盾蚧(Chrysomphalus dictyospermi)、褐软蜡蚧(Coccus hesperidum)、葡萄根瘤蚜(Daktulosphaira vitifoliae)、柑桔木虱(Diaphorina citri)、苹果绵蚜(Eriosomalarigerum)、Gascardia spp.、茱萸介壳虫(Lacaniumcorni)、厦门秋茄蛎盾蚧(Lepidosaphes spp.)、日本长白盾蚧(Lopholeucaspis japonica)、长管虫牙属(Macrosiphus spp.)、Margarodes prieskaensis、蛛蚧属(Margarodes vitis)、Margarodes vredendalensis、桃蚜(Myzus persicae)、瘤蚜属(Myzus spp.)、橡副珠蜡蚧(Parasaissetia nigra)、瘿绵蚜属(Pemphigus spp.)、根瘤蚜属(Phylloxera spp.)、动性球菌属(Planococcus spp.)、白盾蚧属(Pseudaulacaspis spp.)、粉蚧(Pseudococcusspp.)、木虱属(Psylla spp.)、棉蚧(Pulvinaria aethiopica)、齿盾蚧属(Quadraspidiotus spp.)、缢管蚜属(Rhopalosiphum spp.)、Ripersiella hibisci、黑盔蚧属(Saissetia spp.)、蚜虫(Schizaphis spp.)、麦蚜属(Sitobion spp.)、大桔蚜(Toxoptera citricida)、温室白粉虱(Trialeurodes vaporariorum)、非洲木虱(Triozaerytreae)和柑桔尖盾蚧(Unaspis citri)；
·半翅目(Hemiptera)、颈喙亚目(suborder Auchenorrhyncha)，例如：Circulifer haematoceps、甜菜叶蝉(Circulifer tenellus)、绿尖头叶蝉(Draeculacephala minerva)、茶小绿叶蝉(Empoasca spp.)、桃一点叶蝉(Erythroneuraspp.)、蓝绿尖头叶蝉(Graphocephala atropunctata)、Hishimonus phycitis、蜡蝉(Myndus crudus)、灰飞虱属(Laodelphax spp.)、黑尾叶蝉属(Nephotettix spp.)、褐飞虱属(Nilaparvata spp.)、榆白带叶蝉(Scaphoideus luteolus)、橫帶葉蟬(Scaphoideusspp.)和Xyphon fulgida；
·膜翅目，例如：顶切叶蚁属(Acromyrmex,Atta spp.)、布切叶白蚁属(Attaspp.)、茎叶蜂属(Cephus spp.)、锯角叶蜂家族属(Diprionidae family spp)例如松叶蜂属(Diprion spp.)和松叶蜂(Gilpinia polytoma)、梨实蜂属(Hoplocampa spp.)、毛蚁属(Lasius spp.)、小黄家蚁(Monomorium pharaonis)、新松叶蜂属(Neodiprion spp.)、蚁科家族属(Formicidae family spp.)例如水蚁属(Solenopsis spp.)，和胡蜂属(Vespaspp.)；
·双翅目，例如：白纹伊蚊(Aedes albopictus)、灰色伊蚊(Aedes cinereus)、波里尼西亚斑蚊(Aedes polynesiensis)、伊蚊属(Aedes spp.)、菊花潜蝇(Amauromyzamaculosa)、按实蝇(Anastrepha fraterculus)、墨西哥按实蝇(Anastrepha ludens)、西印度按实蝇(Anastrepha obliqua)、加勒比按实蝇(Anastrepha suspensa)、按实蝇属(Anastrepha spp.)、冈比亚按蚊(Anopheles gambiae)、疟蚊属(Anopheles spp.)、Aschistonyx eppoi、高粱芒蝇(Atherigona soccata)、果实蝇属(Bactrocera spp.)、花园毛蚊(Bibio hortulanus)、红头丽蝇(Calliphora erythrocephala)、腮扁叶蜂(Cephalcialariciphila)、纳塔尔实蝇(Ceratitis rosa)、小条实蝇属(Ceratitis spp.)、金蝇属(Chrysomyia spp.)、库蚊属(Culex spp.)、黄蝇属(Cuterebra spp.)、寡鬃实蝇属(Dacusspp.)、黑腹果蝇(Drosophila melanogaster)、栗瘿蜂(Dryocosmus kuriphilus)、Euphranta canadensis、日本樱桃实蝇(Euphranta japonica)、厕蝇属(Fannia spp.)、胃蝇属(Gastrophilus spp.)、欧洲云杉叶蜂(Gilpinia hercyniae)、舌蝇属(Glossinaspp.)、皮蝇属(Hypoderma spp.)、虱蝇属种(Hippobosca spp.)、番茄斑潛蠅(Liriomyzabryoniae)、拉美豌豆斑潜蝇(Liriomyza huidobrensis)、美洲斑潜蝇(Liriomyzasativae)、三叶草斑潜蝇(Liriomyza trifolii)、斑潜蝇属(Liriomyza spp.)、绿蝇属(Lucilia spp.)、潜蝇属(Melanagromyza spp.)、家蝇属(Musca spp.)、狂蝇属(Oestrusspp.)、瘿蚊属(Orseolia spp.)、瑞典麦秆蝇(Oscinella frit)、Pardalaspiscyanescens、Pardalaspis quinaria、藜泉蝇(Pegomyia hyoscyami)、草种绳属(Phorbiaspp.)、苹绕实蝇(Rhagoletis pomonella)、绕实绳属(Rhagoletis spp.)、尖眼蕈蚊属(Sciara spp.)、刺蝇属(Stomoxys spp.)、虻属(Tabanus spp.)和大蚊属(Tipula spp.)；
·蚤目(Siphonaptera)，例如：角叶蚤属(Ceratophyllus spp.)和印鼠客蚤(Xenopsylla cheopis)；
·缨尾目次纲(infraclass Thysanura)、衣鱼目，例如：衣鱼(Lepismasaccharina)；
根据具体实施方式，昆虫被认为是害虫。如本文所使用的，术语“害虫”是指农业害虫生物体，包括但不限于：蚜虫、蚱蜢、毛虫、甲虫、蛾、臭虫、蓟马、白蝇、家庭害虫生物体，例如蟑螂、蚂蚁、黄蜂、苍蝇、家蟋蟀、臭虫，木虫、粉虱甲虫、蠼螋、蠹鱼、白蚁、吸血害虫，例如如蚊子、跳蚤和虱子等。根据具体实施方式，昆虫是农业害虫生物体。
根据具体实施方式，昆虫选自蛾、臭虫、漏斗虫、甲虫、蚜虫和蜜蜂。
根据具体实施方式，昆虫是蛾。
根据具体实施方式，昆虫是夜蛾。
蛾的非限制性实施例包括棉铃虫(Helicoverpa armigera)、苹果蠹蛾(Cydiapomonella)和草地夜蛾(Spodoptera frugiperda)。
根据具体实施方式，蛾是棉铃虫和/或草地夜蛾。
根据具体实施方式，蛾是棉铃虫。
根据具体的实施方式，昆虫选自由棉铃虫、草地夜蛾、稻绿蝽(Nezara viridula)、稻褐飞虱(Nilaraparvata lugens)、棉蚜(Aphis gossypii)、苹果蠹蛾(Cydiapomonella)、食叶甲虫(Leaf Feeding beetles)(例如马铃薯甲虫(Leptinotarsadecemlineata)或辣根猿叶甲(Phaedon cochleariae))和蜜蜂组成的组。
根据具体实施方式，纳米抗体结合以下昆虫多肽：
(i)包含几丁质结合域(CBD)的多肽，其中，所述纳米抗体结合所述CBD；
(ii)V-ATP酶亚基c，其中，所述纳米抗体包含如下序列号中所示的互补性决定区(CDR)：167-169；171和168-169；174和168-169；167、178和169；180-182；187-189；或191-193，这些序列在所述纳米抗体上按从N到C的顺序排列；
(iii)海藻糖酶；
(iv)细胞色素p450单加氧酶；
(v)几丁质脱乙酰酶；
(vi)几丁质合成酶；和
(vii)NPC1甾醇转运体。
如本文所使用的，术语“特异性结合”是指纳米抗体在生理环境中结合目标昆虫多肽的能力，例如，在生理条件下的昆虫中以比在所述环境中的其它多肽更高的亲和力结合目标昆虫多肽的能力。
根据具体实施方式，纳米抗体结合昆虫多肽，而与非昆虫(例如植物、人)多肽无交叉反应。
根据具体实施方式，纳米抗体特异性结合(i)至(vii)中的一种昆虫多肽，而与其它昆虫多肽无交叉反应。
根据具体实施方式，纳米抗体通过多特异性，例如双特异性纳米抗体的方式，结合(i)至(vii)中的至少两种昆虫多肽。
根据具体实施方式，纳米抗体是包含至少两种纳米抗体的纳米抗体组合，每种纳米抗体结合选自(i)至(vii)的不同昆虫多肽。
根据具体实施方式，纳米抗体或纳米抗体组合结合(i)+(ii)、(i)+(iii)、(i)+(iv)、(i)+(v)、(i)+(vi)、(i)+(vii)、(ii)+(iii)、(ii)+(iv)、(ii)+(v)、(ii)+(vi)、(ii)+(vii)、(iii)+(iv)、(iii)+(v)、(iii)+(vi)、(iii)+(vii)、(iv)+(v)、(iv)+(vi)、(iv)+(vii)、(v)+(vi)、(v)+(vii)、(vi)+(vii)。
根据具体实施方式，纳米抗体结合包含几丁质结合域(CBD)的多肽，其中所述纳米抗体结合所述CBD。
如本文所使用的，术语“几丁质结合域(CBD)”是指能够结合昆虫几丁质(由(1->4)-β-连接的D-氨基葡萄糖残基组成的线性多糖，其中大多数是N-乙酰化的)的昆虫氨基酸域，或其抗原片段，该抗原片段能够引起针对所述片段的特异性免疫应答。CBD的非限制性实施例包括2型几丁质结合域(ChtBD2)和Rebers和Riddiform共有序列。根据其它具体实施方式，CBD包括Rebers和Riddiford共有序列。
如本文所使用的，术语“Rebers和Riddiford共有序列”是指如Pfam数据库条目pfam00379所描述的共有序列(参见Rebers和Riddiford，1988；Anderson，2010；Karouzou等人，2007；Willis，2010，其全部内容通过引用并入本文。
根据具体实施方式，CBD是2型几丁质结合域(ChtBD2)。
如本文所使用的，术语“2型几丁质结合域(ChtBD2)”，也称为围食膜A域(peritrophin A domain)，是指具有形成三个二硫键的六个半胱氨酸的氨基酸序列。这种CBD是本领域已知的，例如公开在以下文献中：Tetreau、Guillaume等人，昆虫生物化学和分子生物学(Insect biochemistry and molecular biology)62(2015):127-141)。根据具体实施方式，ChtBD2具有共有序列：CX11-30CX5-6CX9-24CX12-17CX6-12C(序列号：409)。
根据具体实施方式，ChtBD2包含与选自序列号为3-7的氨基酸序列具有如下同一性的氨基酸序列：至少60％、至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％。
根据具体实施方式，ChtBD2包含与GeneBank收录号XP_021181774(序列号：410)中提供的氨基酸序列具有如下同一性的氨基酸序列：至少60％、至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％。
序列同一性或同源性可以使用任何蛋白质或核酸序列比对算法(例如Blast、ClustalW和MUSCLE)来确定。
根据具体实施方式，ChtBD2包含选自序列号为3-7的氨基酸序列。
包含ChtBD2的多肽的非限制性实施例包括围食膜基质蛋白(PMPs)，例如TcPMP5-B、HaIIM86；以及与围食膜因子类似的角质层蛋白(CPAPs)，例如BmCPAP3-A1、BmCPAP3-A2、BmCPAP3-B、BmCPAP3-C、BmCPAP3-D1和BmCPAP3-D2。
根据具体实施方式，包含ChtBD2的多肽是PMP蛋白。
根据具体实施方式，包含ChtBD2的多肽是TcPMP5-B(赤拟谷盗的PMP5-B)。
根据具体实施方式，包含ChtBD2的多肽包含选自序列号为1-2的氨基酸序列。
根据具体实施方式，包含ChtBD2的多肽由选自序列号为1-2的氨基酸序列组成。
特异性结合ChtBD2及其各自CDR的纳米抗体的非限制性实施例如下表2A-B所示。
根据具体实施方式，所述纳米抗体特异性结合ChtBD2并包含CDR，该CDR具有与如下序列号的氨基酸序列具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％同一性的氨基酸序列：15-17；19-21；23-25；15和28-29；31-33；35-37；39-41；43-45；47-49；51-53；55-57；59-61；63-65；67-69；71-73；75-77；79-81；83-85；87-89；91、80和93；95-97；99-101；103-105；107-109；111-113；115-117；119-121；123-125；127-129；131-133；135-137；139-141；143-145；147-149；151-153；15和156-157；159-161；或者163-165，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据具体实施方式，所述纳米抗体特异性结合ChtBD2并包含CDR，该CDR具有与序列号为15-17；19-21；23-25或15和28-29(这些序列在所述纳米抗体上按从N到C的顺序排列)中所述的氨基酸序列具有如下同一性的氨基酸序列：至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％。
根据具体实施方式，所述纳米抗体特异性结合ChtBD2并包含如下序列号中所述的CDR：15-17；19-21；23-25；15和28-29；31-33；35-37；39-41；43-45；47-49；51-53；55-57；59-61；63-65；67-69；71-73；75-77；79-81；83-85；87-89；91、80和93；95-97；99-101；103-105；107-109；111-113；115-117；119-121；123-125；127-129；131-133；135-137；139-141；143-145；147-149；151-153；15和156-157；159-161；或者163-165，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据具体实施方式，所述纳米抗体特异性结合ChtBD2并包含如下序列号中所述的CDR：15-17；19-21；23-25；或者15和28-29，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据具体实施方式，纳米抗体特异性结合ChtBD2并包含序列号为15-17中所述的CDR，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据具体实施方式，纳米抗体特异性结合ChtBD2并包含序列号为19-21中所述的CDR，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据具体实施方式，纳米抗体特异性结合ChtBD2并包序列号为23-25中所述的CDR，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据具体实施方式，纳米抗体特异性结合ChtBD2并包序列号为15和28-29中所述的CDR，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据具体实施方式，所述纳米抗体包含与如下序列号的氨基酸序列具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％同一性的氨基酸序列：14、18、22、26、30、34、38、42、46、50、54、58、62、66、70、74、78、82、86、90、94、98、102、106、110、114、118、122、126、130、134、138、142、146、150、154、158和162，每种可能性代表本公开的一个单独实施方式。
根据具体实施方式，所述纳米抗体包含与选自序列号为14、18、22和26的氨基酸序列具有如下同一性的氨基酸序列：至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％。
根据具体实施方式，纳米抗体包含选自如下序列号的氨基酸序列：14、18、22、26、30、34、38、42、46、50、54、58、62、66、70、74、78、82、86、90、94、98、102、106、110、114、118、122、126、130、134、138、142、146、150、154、158和162。
根据具体实施方式，纳米抗体包含选自序列号为14、18、22和26的氨基酸序列。
根据具体实施方式，纳米抗体包括序列号：14。
根据具体实施方式，纳米抗体包括序列号：18。
根据具体实施方式，纳米抗体包括序列号：22。
根据具体实施方式，纳米抗体包括序列号：26。
根据具体实施方式，纳米抗体由选自如下序列号的氨基酸序列组成：14、18、22、26、30、34、38、42、46、50、54、58、62、66、70、74、78、82、86、90、94、98、102、106、110、114、118、122、126、130、134、138、142、146、150、154、158和162。
根据具体实施方式，纳米抗体由序列号：14、18、22或26组成。
根据具体实施方式，纳米抗体由序列号：14组成。
根据具体实施方式，纳米抗体由序列号：18组成。
根据具体实施方式，纳米抗体由序列号：22组成。
根据具体实施方式，纳米抗体由序列号：26组成。
根据具体实施方式，纳米抗体结合V-ATP酶亚基c(V-ATPase subunit c)。
特异性结合V-ATP酶亚基c及其各自的CDR的纳米抗体的非限制性实施例如下表3A-B所示。
根据具体实施方式，纳米抗体结合V-ATP酶亚基c，其中，所述纳米抗体包括如下序列号中所述的互补性决定区(CDR)：167-169；171和168-169；174和168-169；167、178和169；180-182；187-189；或者191-193，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据具体实施方式，纳米抗体结合V-ATP酶亚基c，其中，所述纳米抗体包括如下序列号中所述的互补性决定区(CDR)：167-169；171和168-169；174和168-169；167、178和169；或者180-182，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据具体实施方式，纳米抗体结合V-ATP酶亚基c，其中，所述纳米抗体包括如下序列号中所述的互补性决定区(CDR)：167-169；或者167、178和169，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据具体实施方式，纳米抗体结合V-ATP酶亚基c，其中所述纳米抗体包括序列号为167-169(这些序列在所述纳米抗体上按从N到C的顺序排列)中所述的互补决定区(CDR)。
根据具体实施方式，纳米抗体结合V-ATP酶亚基c，其中所述纳米抗体包括序列号为171和168-169(这些序列在所述纳米抗体上按从N到C的顺序排列)中所述的互补决定区(CDR)。
根据具体实施方式，纳米抗体结合V-ATP酶亚基c，其中所述纳米抗体包括序列号为174和168-169(这些序列在所述纳米抗体上按从N到C的顺序排列)中所述的互补决定区(CDR)。
根据具体实施方式，纳米抗体结合V-ATP酶亚基c，其中所述纳米抗体包括序列号为167、178和169(这些序列在所述纳米抗体上按从N到C的顺序排列)中所述的互补决定区(CDR)。
根据具体实施方式，纳米抗体结合V-ATP酶亚基c，其中所述纳米抗体包括序列号为180-182(这些序列在所述纳米抗体上按从N到C的顺序排列)中所述的互补决定区(CDR)。
如本文所使用的，术语“V-ATP酶亚基c”是指昆虫酶Pfam PF03223(一种催化质子电化学电位梯度的酶)的氨基酸序列，或是指或其抗原片段，该抗原片段能够引起针对所述片段的特异性免疫应答。
根据具体实施方式，V-ATP酶亚基c是棉铃虫V-ATP酶亚基c，例如在GeneBank收录号XP_021198264中提供的。
根据具体实施方式，V-ATP酶亚基c包含序列号：8。
根据具体实施方式，V-ATP酶亚基c由序列号：8组成。
根据具体实施方式，所述纳米抗体包含与选自序列号为166、170、172、173、175、176、177、179、183、185、186和190的氨基酸序列具有如下同一性的氨基酸序列：至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％，每种可能性代表本公开的一个单独的实施方式。
根据具体实施方式，所述纳米抗体包含与选自序列号为166、170、172、173、175、176、177和179的氨基酸序列具有如下同一性的氨基酸序列：至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％。
根据具体实施方式，纳米抗体包含与选自序列号为176和177的氨基酸序列具有如下同一性的氨基酸序列：至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％。
根据具体实施方式，纳米抗体包含选自如下序列号的氨基酸序列：166、170、172、173、175、176、177、179、183、185、186和190。
根据具体实施方式，纳米抗体包含选自如下序列号的氨基酸序列：166、170、172、173、175、176、177和179。
根据具体实施方式，纳米抗体包含选自序列号为176和177的氨基酸序列。
根据具体实施方式，纳米抗体包含序列号：166。
根据具体实施方式，纳米抗体包含序列号：170。
根据具体实施方式，纳米抗体包含序列号：172。
根据具体实施方式，纳米抗体包含序列号：173。
根据具体实施方式，纳米抗体包含序列号：175。
根据具体实施方式，纳米抗体包含序列号：176。
根据具体实施方式，纳米抗体包含序列号：177。
根据具体实施方式，纳米抗体包含序列号：179。
根据具体实施方式，纳米抗体由选自如下序列号的氨基酸序列组成：166、170、172、173、175、176、177、179、183、185、186和190。
根据具体实施方式，纳米抗体由选自如下序列号的氨基酸序列组成：166、170、172、173、175、176、177和179。
根据具体实施方式，纳米抗体由选自序列号为176和177的氨基酸序列组成。
根据具体实施方式，纳米抗体由序列号：166组成。
根据具体实施方式，纳米抗体由序列号：170组成。
根据具体实施方式，纳米抗体由序列号：172组成。
根据具体实施方式，纳米抗体由序列号：173组成。
根据具体实施方式，纳米抗体由序列号：175组成。
根据具体实施方式，纳米抗体由序列号：176组成。
根据具体实施方式，纳米抗体由序列号：177组成。
根据具体实施方式，纳米抗体由序列号：179组成。
根据具体实施方式，纳米抗体结合海藻糖酶。
如本文所使用的，术语“海藻糖酶”是指E.C.号为3.2.1.2的昆虫酶(一种催化海藻糖转化为葡萄糖的酶)的氨基酸序列，或其抗原片段，该抗原片段能够引起针对所述片段的特异性免疫应答。
根据具体实施方式，海藻糖酶是棉铃虫海藻糖酶，例如在GeneBank收录号AJK29979中提供的。
根据具体实施方式，海藻糖酶包含序列号：10。
根据具体实施方式，海藻糖酶由SEQ ID NO：10组成。
特异性结合海藻糖酶及其各自CDR的纳米抗体的非限制性实施例如下表4所示。
根据具体实施方式，所述纳米抗体特异性结合海藻糖酶并包含CDR，所述CDR具有与如下序列号的氨基酸序列具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％同一性的氨基酸序列：195-197；199和196-197；201-203；201和205-206；208-210；212和209-210；215-217；219-221；223-225；227-229；231、228和232；234-236；238-240；242-244；246-248；250-252；254-256；258-260；588-590；588-589和592；或者595、589和592，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据具体实施方式，所述纳米抗体特异性结合海藻糖酶并包含CDR，所述CDR具有与如下序列号的氨基酸序列具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％同一性的氨基酸序列：195-197；199和196-197；201-203；201和205-206；208-210；212和209-210；215-217；219-221；223-225；227-229；231、228和232；234-236；238-240；242-244；246-248；250-252；254-256；或者258-260，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据具体实施方式，所述纳米抗体特异性结合海藻糖酶并包含CDR，所述CDR具有与如下序列号的氨基酸序列具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％同一性的氨基酸序列：201和205-206；208-210；212和209-210；223-225；238-240；242-244；246-248；588-590；或者595、589和592，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据具体实施方式，所述纳米抗体特异性结合海藻糖酶并包含CDR，所述CDR具有与如下序列号的氨基酸序列具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％同一性的氨基酸序列：201和205-206；208-210；212和209-210；223-225；588-590；或者595、589和592，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据具体实施方式，所述纳米抗体特异性结合海藻糖酶并包含CDR，所述CDR具有与序列号为201和205-206；208-210；223-225；或者588-590(这些序列在所述纳米抗体上按从N到C的顺序排列)的氨基酸序列具有如下同一性的氨基酸序列：至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％。
根据具体实施方式，所述纳米抗体特异性结合海藻糖酶并包含如下序列号中的CDR：195-197；199和196-197；201-203；201和205-206；208-210；212和209-210；215-217；219-221；223-225；227-229；231、228和232；234-236；238-240；242-244；246-248；250-252；254-256；258-260；588-590；588-589和592；或者595、589和592，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据具体实施方式，所述纳米抗体特异性结合海藻糖酶并包含如下序列号中的CDR：195-197；199和196-197；201-203；201和205-206；208-210；212和209-210；215-217；219-221；223-225；227-229；231、228和232；234-236；238-240；242-244；246-248；250-252；254-256；或者258-260，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据具体实施方式，所述纳米抗体特异性结合海藻糖酶并包含如下序列号中的CDR：201和205-206；208-210；212和209-210；223-225；238-240；242-244；246-248；588-590；或者595、589和592，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据具体实施方式，所述纳米抗体特异性结合海藻糖酶并包含如下序列号中的CDR：201和205-206；208-210；212和209-210；223-225；588-590；或者595、589和592，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据具体实施方式，所述纳米抗体特异性结合海藻糖酶并包含如下序列号中的CDR：201和205-206；208-210；223-225；或者588-590，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据具体实施方式，所述纳米抗体包含与选自如下序列号的氨基酸序列具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％同一性的氨基酸序列：194、198、200、204、207、211、213、214、218、222、226、230、233、237、241、245、249、253、257、587、591、593、594和596，每种可能性代表本公开的一个单独的实施方式。
根据具体实施方式，所述纳米抗体包含与选自如下序列号的氨基酸序列具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％同一性的氨基酸序列：194、198、200、204、207、211、213、214、218、222、226、230、233、237、241、245、249、253和257，每种可能性代表本公开的一个单独的实施方式。
根据具体实施方式，所述纳米抗体包含与选自序列号为204、207、211、213、222、237、241、245、587和594的氨基酸序列具有如下同一性的氨基酸序列：至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％。
根据具体实施方式，所述纳米抗体包含与选自序列号为204、207、211、213、222、587和594的氨基酸序列具有如下同一性的氨基酸序列：至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％。
根据具体实施方式，所述纳米抗体包含与选自序列号为204、207、222和587的氨基酸序列具有如下同一性的氨基酸序列：至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％。
根据具体实施方式，纳米抗体包含选自如下序列号的氨基酸序列：194、198、200、204、207、211、213、214、218、222、226、230、233、237、241、245、249、253、257、587、591、593、594和596。根据具体实施方式，纳米抗体包含选自如下序列号的氨基酸序列：194、198、200、204、207、211、213、214、218、222、226、230、233、237、241、245、249、253和257。
根据具体实施方式，纳米抗体包含选自如下序列号的氨基酸序列：204、207、211、213、222、237、241、245、587和594。
根据具体实施方式，纳米抗体包含选自如下序列号的氨基酸序列：204、207、211、213、222、587和594。
根据具体实施方式，纳米抗体包含选自如下序列号的氨基酸序列：204、207、222和587。
根据具体实施方式，纳米抗体包含序列号：204。
根据具体实施方式，纳米抗体包含序列号：207。
根据具体实施方式，纳米抗体包含序列号：211。
根据具体实施方式，纳米抗体包含序列号：213。
根据具体实施方式，纳米抗体包含序列号：222。
根据具体实施方式，纳米抗体包含序列号：587。
根据具体实施方式，纳米抗体包含序列号：594。
根据具体实施方式，纳米抗体由选自如下序列号的氨基酸序列组成：194、198、200、204、207、211、213、214、218、222、226、230、233、237、241、245、249、253、257、587、591、593、594和596。
根据具体实施方式，纳米抗体由选自如下序列号的氨基酸序列组成：194、198、200、204、207、211、213、214、218、222、226、230、233、237、241、245、249、253和257。
根据具体实施方式，纳米抗体由选自如下序列号的氨基酸序列组成：204、207、211、213、222、237、241、245、587和594。
根据具体实施方式，纳米抗体由选自如下序列号的氨基酸序列组成：204、207、211、213、222、587和594。
根据具体实施方式，纳米抗体由选自如下序列号的氨基酸序列组成：204、207、222和587。
根据具体实施方式，纳米抗体由序列号：204组成。
根据具体实施方式，纳米抗体由序列号：207组成。
根据具体实施方式，纳米抗体由序列号：211组成。
根据具体实施方式，纳米抗体由序列号：213组成。
根据具体实施方式，纳米抗体由序列号：222组成。
根据具体实施方式，纳米抗体由序列号：587组成。
根据具体实施方式，纳米抗体由序列号：594组成。
根据具体实施方式，纳米抗体结合细胞色素p450单加氧酶。
如本文所使用的，术语“细胞色素p450单加氧酶”指E.C.号为1.14的昆虫酶的氨基酸序列，该酶包含血红素作为辅因子，将一个羟基结合到底物中，通过NAD(P)H的伴随氧化将二氧的两个原子还原为一个羟基和一个H2O分子，或还原为其抗原片段，该抗原片段能够引起针对所述片段的特异性免疫应答。
根据具体实施方式，细胞色素p450单加氧酶是棉铃虫细胞色素p450单加氧酶，例如在GeneBank收录号AKS48889中提供的。
根据具体实施方式，细胞色素p450单加氧酶包含序列号：11。
根据具体实施方式，细胞色素p450单加氧酶由序列号：11组成。
特异性结合细胞色素p450单加氧酶及其相应CDR的纳米抗体的非限制性实施例如下表5所示。
根据具体实施方式，所述纳米抗体特异性结合p450单加氧酶并包含CDR，该CDR具有与如下序列号的氨基酸序列具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％同一性的氨基酸序列：262-264；262-263和266；262、270和264；274-276；280和275-276；282和275-276；282、275和285；287、220和289；291、220和289；239-294和289；296-298；300-302；304-306；308-310；312-314；或者316-318，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据具体实施方式，所述纳米抗体特异性结合细胞色素p450单加氧酶并包含CDR，该CDR具有与序列号为262-264；287、220和289；296-298；300-302；304-306；或者312-314(这些序列在所述纳米抗体上按从N到C的顺序排列)的氨基酸序列具有如下同一性的氨基酸序列：至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％。
根据具体实施方式，所述纳米抗体特异性结合细胞色素p450单加氧酶并包含CDR，该CDR具有与序列号为300-302；或者312-314(这些序列在所述纳米抗体上按从N到C的顺序排列)的氨基酸序列具有如下同一性的氨基酸序列：至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％。
根据具体的实施方式，所述纳米抗体特异性结合细胞色素p450单加氧酶并包含CDR，该CDR具有与序列号为312-314(这些序列在所述纳米抗体上按从N到C的顺序排列)的氨基酸序列具有如下同一性的氨基酸序列：至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％。
根据具体的实施方式，纳米抗体特异性结合细胞色素p450单加氧酶并包含如下序列号中的CDR：262-264；262-263和266；262、270和264；274-276；280和275-276；282和275-276；282、275和285；287、220和289；291、220和289；239-294和289；296-298；300-302；304-306；308-310；312-314；或者316-318，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据具体实施方式，所述纳米抗体特异性结合细胞色素p450单加氧酶并包含如下序列号中的CDR：262-264；287、220和289；296-298；300-302；304-306；或者312-314，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据具体实施方式，所述纳米抗体特异性结合细胞色素p450单加氧酶并包含序列号为300-302或312-314(这些序列在所述纳米抗体上按从N到C的顺序排列)中的CDR。
根据具体实施方式，纳米抗体特异性结合细胞色素p450单加氧酶并包含序列号为312-314(这些序列在所述纳米抗体上按从N到C的顺序排列)中的CDR。
根据具体实施方式，所述纳米抗体包含具有与如下序列号的氨基酸序列具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％同一性的氨基酸序列：261、265、267、268、269、271、272、273、277、278、279、281、283、284、286、29、292、295、299、303、307、311和315，每种可能性代表本公开的一个单独实施方式。
根据具体实施方式，所述纳米抗体包含与选自序列号为：261、286、295、299、303和311具有如下同一性的氨基酸序列：至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％。
根据具体实施方式，纳米抗体包含与选自序列号为299和311具有如下同一性的氨基酸序列：至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％。
根据具体实施方式，纳米抗体包含选自如下序列号的氨基酸序列：261、265、267、268、269、271、272、273、277、278、279、281、283、284、286、29、292、295、299、、303、307、311和315。
根据具体实施方式，纳米抗体包含选自如下序列号的氨基酸序列：261、286、295、299、303和311。
根据具体实施方式，纳米抗体包含选自序列号为299和311的氨基酸序列。
根据具体实施方式，纳米抗体包括序列号：311。
根据具体实施方式，纳米抗体包括序列号：299。
根据具体实施方式，纳米抗体由选自如下序列号的氨基酸序列组成：261、265、267、268、269、271、272、273、277、278、279、281、283、284、286、29、292、295、299、303、307、311和315。
根据具体实施方式，纳米抗体由选自如下序列号的氨基酸序列组成：261、286、295、299、303和311。
根据具体实施方式，纳米抗体由选自序列号为299和311的氨基酸序列组成。
根据具体实施方式，纳米抗体由序列号：311组成。
根据具体实施方式，纳米抗体由序列号：299组成。
根据具体实施方式，纳米抗体结合几丁质脱乙酰酶。
如本文所使用的，术语“几丁质脱乙酰酶”是指E.C.号为3.5.1.41的昆虫酶(一种催化几丁质和H2O转化为壳聚糖(chitosan)和醋酸盐(acetate))的氨基酸序列，或其抗原片段，该抗原片段能够引起针对所述片段的特异性免疫应答。
根据具体实施方式，几丁质脱乙酰酶是棉铃虫几丁质脱乙酰酶，例如在GeneBank收录号AJA30435中提供的。
根据具体实施方式，几丁质脱乙酰酶包含序列号：9。
根据具体实施方式，几丁质脱乙酰酶由序列号：9组成。
特异性结合几丁质脱乙酰酶及其相应CDR的纳米抗体的非限制性实施例如下表6所示。
根据具体实施方式，所述纳米抗体特异性结合几丁质脱乙酰酶并包含CDR，该CDR具有与如下序列号的氨基酸序列具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或374％同一性的氨基酸序列：320-322；320和324-325；327、321和328；320-321和332；208和336-337；320、324和339；343-345；347-349；351-353；351、347和356；358-360；358-359和362；365、359和366；368-370；372和369-370；374、369和375；377-379；381-383；385和382-383；387-389；387、391和389；394-396；398-400；402-404；或者406-408，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据具体实施方式，所述纳米抗体特异性结合几丁质脱乙酰酶并包含CDR，该CDR具有与如下序列号的氨基酸序列具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％同一性的氨基酸序列：320-322；320和324-325；372和369-370；374、369和375；377-379；387-389；或者398-400，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据具体实施方式，所述纳米抗体特异性结合几丁质脱乙酰酶并包含CDR，该CDR具有与如下序列号的氨基酸序列具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％同一性的氨基酸序列：320和324-325；372和369-370；374、369和375；或者387-389，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据具体实施方式，所述纳米抗体特异性结合几丁质脱乙酰酶并包含CDR，该CDR具有与序列号为：320和324-325；374、369和375；或者387-389(这些序列在所述纳米抗体上按从N到C的顺序排列)中所述的氨基酸序列具有如下同一性的氨基酸序列：至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％。
根据具体实施方式，所述纳米抗体特异性结合几丁质脱乙酰酶，并包含如下序列号中所述的CDR：320-322；320和324-325；327、321和328；320-321和332；208和336-337；320、324和339；343-345；347-349；351-353；351、347和356；358-360；358-359和362；365、359和366；368-370；372和369-370；374、369和375；377-379；381-383；385和382-383；387-389；387、391和389；394-396；398-400；402-404；或者406-408，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据具体实施方式，所述纳米抗体特异性结合几丁质脱乙酰酶，并包含如下序列号中所述的CDR：320-322；320和324-325；372和369-370；374、369和375；377-379；387-389；或者398-400，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据具体实施方式，所述纳米抗体特异性结合几丁质脱乙酰酶，并包含如下序列号中所述的CDR：320和324-325；372和369-370；374、369和375；或者387-389，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据具体实施方式，所述纳米抗体特异性结合几丁质脱乙酰酶，并包含如下序列号中所述的CDR：320和324-325；374、369和375；或者387-389，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据具体实施方式，所述纳米抗体包含与选自如下序列号的氨基酸序列具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或380％同一性的氨基酸序列：319、323、326、329、331、333、334、338、340、341、342、346、350、354、355、357、361、363、364、367、371、373、376、380、384、386、390、392、393、397、401和405，每个可能性代表本公开的一个单独实施方式。
根据具体实施方式，所述纳米抗体包含与选自序列号为：319、323、371、373、376、386和397的氨基酸序列具有如下同一性的氨基酸序列：至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％。
根据具体实施方式，所述纳米抗体包含与选自序列号为323、371、373和386的氨基酸序列具有如下同一性的氨基酸序列：至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％。
根据具体实施方式，所述纳米抗体包含与选自序列号为323、373和386的氨基酸序列具有如下同一性的氨基酸序列：至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％。
根据具体实施方式，纳米抗体包含选自如下序列号的氨基酸列号：319、323、326、329、331、333、334、338、340、341、342、346、350、354、355、357、361、363、364、367、371、373、376、380、384、386、390、392、393、397、401和405。
根据具体实施方式，纳米抗体包含选自如下序列号的氨基酸列号：319、323、371、373、376、386和397。
根据具体实施方式，纳米抗体包含选自如下序列号的氨基酸列号：323、371、373和386。
根据具体实施方式，纳米抗体包含选自序列号为：323、373和386的氨基酸序列。
根据具体实施方式，纳米抗体包括序列号：323。
根据具体实施方式，纳米抗体包括序列号：371。
根据具体实施方式，纳米抗体包括序列号：373。
根据具体实施方式，纳米抗体包括序列号：386。
根据具体实施方式，纳米抗体由选自如下序列号的氨基酸序列组成：319、323、326、329、331、333、334、338、340、341、342、346、350、354、355、357、361、363、364、367、371、373、376、380、384、386、390、392、393、397、401和405。
根据具体实施方式，纳米抗体由选自如下序列号的氨基酸序列组成：319、323、371、373、376、386和397。
根据具体实施方式，纳米抗体由选自如下序列号的氨基酸序列组成：323、371、373和386。
根据具体实施方式，纳米抗体由序列号：323组成。
根据具体实施方式，纳米抗体由序列号：371组成
根据具体实施方式，纳米抗体由序列号：373组成。
根据具体实施方式，纳米抗体由序列号：386组成。
根据具体实施方式，纳米抗体结合几丁质合成酶。
如本文所使用的，术语“几丁质合成酶”是指E.C.号为2.4.1.16的昆虫酶(一种催化UDP-N-乙酰基-D-氨基葡萄糖和[1,4-(N-乙酰基-β-D-氨基葡萄糖基)]n转化为UDP+[1,4-(N-乙酰基-β-D-氨基葡萄糖基)]n+1的酶)的氨基酸序列，或其抗原片段，该抗原片段能够引起针对所述片段的特异性免疫应答。
根据具体实施方式，几丁质合成酶是棉铃虫几丁质合成酶，例如在GeneBank收录号AKZ08594中提供的。
根据具体实施方式，几丁质合成酶包含序列号：12。
根据具体实施方式，几丁质合成酶由选自序列号为12的氨基酸序列组成。
特异性结合几丁质合成酶及其各自CDR的纳米抗体的非限制性实施例如下表7所示。
根据具体实施方式，所述纳米抗体特异性结合几丁质合成酶并包含CDR，该CDR具有与如下序列号的氨基酸序列具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％同一性的氨基酸序列：412-414；418-420；422-424；426-428；426-427和430；432-434；436、140和437；439-441；443-445；447-449；356和451-452；454-456；458-460；462-464；466-468；470-472；474-476；478-480；482、479和483；485-487；489-491；493-495；426和497-498；500-502；504-506；或者508-510，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据具体实施方式，所述纳米抗体特异性结合几丁质合成酶并包含CDR，该CDR具有与如下序列号的氨基酸序列具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％同一性的氨基酸序列：412-414；418-420；426-428；432-434；443-445；447-449；466-468；482、479和483；426和497-498；或者504-506，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据具体实施方式，所述纳米抗体特异性结合几丁质合成酶并包含CDR，该CDR具有与序列号为426-428；432-434；或者443-445(这些序列在所述纳米抗体上按从N到C的顺序排列)中所述的氨基酸序列具有如下同一性的氨基酸序列：至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％。
根据具体实施方式，所述纳米抗体特异性结合几丁质合成酶并包含CDR，该CDR具有与序列号为426-428；或者443-445(这些序列在所述纳米抗体上按从N到C的顺序排列)中所述的氨基酸序列具有如下同一性的氨基酸序列：至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％。
根据具体实施方式，所述纳米抗体特异性结合几丁质合成酶并包含如下序列号中所述的CDR：412-414；418-420；422-424；426-428；426-427和430；432-434；436、140和437；439-441；443-445；447-449；356和451-452；54-456；458-460；462-464；466-468；470-472；474-476；478-480；482、479和483；485-487；489-491；493-495；426和497-498；500-502；504-506；或者508-510。
根据具体实施方式，所述纳米抗体特异性结合几丁质合成酶并包含如下序列号中所述的CDR：412-414；418-420；426-428；432-434；443-445；447-449；466-468；482、479和483；426和497-498；或者504-506，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据具体实施方式，所述纳米抗体特异性结合几丁质合成酶并包含如下序列号中所述的CDR：426-428；432-434；或者443-445，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据具体实施方式，所述纳米抗体特异性结合几丁质合成酶并包含序列号为426-428或443-445中所述的CDR，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据具体实施方式，所述纳米抗体包含与选自如下序列号的氨基酸序列具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％同一性的氨基酸序列：411、415、416、417、421、425、429、431、435、438、442、446、450、453、457、461、465、469、473、477、481、484、488、492、496、499、503和507，每个可能性代表本公开的一个单独实施方式。
根据具体实施方式，所述纳米抗体包含与选自序列号为411、417、425、431、442、446、465、481、496和503的氨基酸序列具有如下同一性的氨基酸序列：至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％。
根据具体实施方式，所述纳米抗体包含与选自序列号为425、431和442的氨基酸序列具有如下同一性的氨基酸序列：至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％。
根据具体实施方式，纳米抗体包含与选自序列号为425和442的氨基酸序列具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％同一性的氨基酸序列。
根据具体实施方式，纳米抗体包含选自如下序列号的氨基酸序列：411、415、416、417、421、425、429、431、435、438、442、446、450、453、457、461、465、469、473、477、481、484、488、492、496、499、503和507。
根据具体实施方式，纳米抗体包含选自如下序列号的氨基酸序列：411、417、425、431、442、446、465、481、496和503。
根据具体实施方式，纳米抗体包含选自序列号为425、431和442的氨基酸序列。
根据具体实施方式，纳米抗体包含选自序列号为425和442的氨基酸序列。
根据具体实施方式，纳米抗体包括序列号：425。
根据具体实施方式，纳米抗体包括序列号：431。
根据具体实施方式，纳米抗体包括序列号：442。
根据具体实施方式，纳米抗体由如下序列号的氨基酸序列组成：411、415、416、417、421、425、429、431、435、438、442、446、450、453、457、461、465、469、473、477、481、484、488、492、496、499、503和507。
根据具体实施方式，纳米抗体由如下序列号的氨基酸序列组成：411、417、425、431、442、446、465、481、496和503。
根据具体实施方式，纳米抗体由选自序列号为425、431和442的氨基酸序列组成。
根据具体实施方式，纳米抗体由选自序列号为425和442的氨基酸序列组成。
根据具体实施方式，纳米抗体由序列号：425组成。
根据具体实施方式，纳米抗体由序列号：431组成。
根据具体实施方式，纳米抗体由序列号：442组成。
根据具体实施方式，纳米抗体结合NPC1甾醇转运体。
如本文所使用的，术语“NPC1甾醇转运体”指由NPC1基因(基因ID：4864)编码的昆虫蛋白的氨基酸序列，或其抗原片段，其能够引起针对所述片段的特异性免疫应答。
根据具体实施方式，NPC1甾醇转运体是棉铃虫NPC1甾醇转运体，例如在基因库登记号(GeneBank Accession No.)XP_021186115中提供的。
根据具体实施方式，NPC1甾醇转运体包含序列号：13。
根据具体实施方式，NPC1甾醇转运体由序列号：13组成
特异性结合NPC1甾醇转运体及其各自CDR的纳米抗体的非限制性实施例如下表8所示。
根据具体实施方式，所述纳米抗体特异性结合NPC1甾醇转运体并包含CDR，该CDR具有与如下序列号的氨基酸序列具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％同一性的氨基酸序列：512-514；517和513-514；521-523；521-522和526；531-533；539-541；485和540-541；545和540-451；547-549；552-554；556-558；561-563；565-567；569-571；573-575；485和577-578；580-582；或者584-586，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据具体实施方式，所述纳米抗体特异性结合NPC1甾醇转运体并包含CDR，该CDR具有与如下序列号的氨基酸序列具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％同一性的氨基酸序列：517和513-514；485和540-541；545和540-541；556-558；565-567；573-575；485和577-578；或者580-582，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据具体实施方式，所述纳米抗体特异性结合NPC1甾醇转运体并包含CDR，该CDR具有与序列号为485和540-541；或545和540-541(这些序列在所述纳米抗体上按从N到C的顺序排列)的氨基酸序列具有如下同一性的氨基酸序列：至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％。
根据具体实施方式，所述纳米抗体特异性结合NPC1甾醇转运体，并包含如下序列号中的CDR：512-514；517和513-514；521-523；521-522和526；531-533；539-541；485和540-541；545和540-451；547-549；552-554；556-558；561-563；565-567；569-571；573-575；485和577-578；580-582；或者584-586，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据具体实施方式，所述纳米抗体特异性结合NPC1甾醇转运体，并包含如下序列号中的CDR：517和513-514；485和540-541；545和540-541；556-558；565-567；573-575；485和577-578；或者580-582，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据具体的实施方式，纳米抗体特异性结合NPC1甾醇转运体，并包含如下序列号中的CDR：485和540-541；或者545和540-541，这些序列在所述纳米抗体上按从N到C的顺序排列。
根据具体实施方式，所述纳米抗体包含与选自如下序列号的氨基酸序列具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或543％同一性的氨基酸序列：511、515、516、518、519、520、524、525、527、528、529、530、534、535、536、537、538、542、543、544、546、550、551、555、559、560、564、568、572、576、579和583，每种可能性代表本公开的一个单独实施方式。
根据具体实施方式，所述纳米抗体包含与选自序列号为516、543、544、555、564、572、576和579的氨基酸序列具有如下同一性的氨基酸序列：至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％。
根据具体实施方式，纳米抗体包含与选自序列号为543和544的氨基酸序列具有如下同一性的氨基酸序列：至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％。
根据具体实施方式，纳米抗体包含选自如下序列号的氨基酸序列：511、515、516、518、519、520、524、525、527、528、529、530、534、535、536、537、538、542、543、544、546、550、551、555、559、560、564、568、572、576、579和583。
根据具体实施方式，纳米抗体包含选自如下序列号的氨基酸序列：516、543、544、555、564、572、576和579。
根据具体实施方式，纳米抗体包含选自序列号为543和544的氨基酸序列。
根据具体实施方式，纳米抗体包含序列号：543。
根据具体实施方式，纳米抗体包含序列号：544。
根据具体实施方式，纳米抗体由选自如下序列号的氨基酸序列组成：511、515、516、518、519、520、524、525、527、528、529、530、534、535、536、537、538、542、543、544、546、550、551、555、559、560、564、568、572、576、579和583。
根据具体实施方式，纳米抗体由选自如下序列号的氨基酸序列组成：516、543、544、555、564、572、576和579。
根据具体实施方式，纳米抗体由选自序列号为543和544的氨基酸序列组成。
根据具体实施方式，纳米抗体由序列号：543组成。
根据具体实施方式，纳米抗体由序列号：544组成。
应当注意，本公开的一些实施方式的纳米抗体在其最广义上不限于特定的生物来源或特定的制备方法。例如，纳米抗体通常可以通过以下方式获得：(1)通过分离天然存在的重链抗体的VHH域；(2)通过表达编码天然存在的VHH域的核苷酸序列；(3)通过“人源化”天然存在的VHH域，或通过表达编码这种人源化VHH域的核酸；(4)通过“骆驼化(camelization)”来自任何动物物种，特别是来自哺乳动物物种，例如来自人类的天然存在的VH域，或通过表达编码这种骆驼化的VH域的核酸；(5)如本领域所述的“域抗体”或“Dab”的“骆驼化”，或通过编码这种骆驼化的VH域的核酸的表达；(6)使用合成或半合成技术制备本身已知的蛋白质、多肽或其它氨基酸序列；(7)使用本身已知的核酸合成技术制备编码纳米抗体的核酸，随后表达由此获得的核酸；和/或(7)通过前述一种或多种方式的任意组合。纳米抗体的进一步描述，包括纳米抗体的人源化和/或骆驼化，例如可以在WO08/101985和WO08/142164以及本文的其他部分中找到。McMahon，Conor等人，在自然结构与分子生物学(Nature structural&molecular biology)25.3(2018):289中，公开了最近报道的基于酵母表面展示的用于纳米抗体发现的完全体外平台。
根据具体实施方式，纳米抗体是“骆驼化”的。例如，“骆驼化”可以通过提供编码天然存在的VH域的核苷酸序列，然后以本身已知的方式改变核苷酸序列中的一个或多个密码子，使得新核苷酸序列分别编码“骆驼化”的纳米抗体来进行。然后可以以本身已知的方式表达该核酸，从而提供所需的纳米抗体。
从天然存在的VH序列或优选地VHH序列开始，用于获得纳米抗体和/或编码纳米抗体的核酸序列的其它合适的方法和技术对本领域技术人员来说是清楚的，例如可以包括组合一个或多个天然存在的VH序列(例如一个或多个FR序列和/或CDR序列)的一个或多个部分、一个或多个天然存在的VHH序列(例如一个或多个FR序列或CDR序列)的一个或多个部分，和/或一个或多个合成或半合成序列，以合适的方式提供纳米抗体或编码纳米抗体的核酸序列。
本文描述了产生纳米抗体的具体方法。
根据本公开的一个方面，提供了一种生产纳米抗体的方法，该方法包括重组或纯化昆虫多肽免疫骆驼，所述昆虫多肽选自：
(i)包含几丁质结合域(CBD)的多肽；
(ii)V-ATP酶亚基c；
(iii)海藻糖酶；
(iv)细胞色素p450单加氧酶；
(v)几丁质脱乙酰酶；
(vi)几丁质合成酶；和
(vii)NPC1甾醇转运体，
其中，所述昆虫多肽在昆虫多肽制剂中的纯度为至少80％。
如本文所使用的，短语“纯化的昆虫多肽”是指从昆虫中纯化的多肽，使得其与蛋白质制剂中存在的其它多肽相比的纯度至少为80％。
根据具体实施方式，纯化的昆虫蛋白制剂中的昆虫多肽的纯度为至少85％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或100％，每种可能性代表本公开的一个单独实施方式。
根据具体实施方式，所述方法还包括分离所述纳米抗体。
分离纳米抗体的方法是众所周知的，公开于：例如Pardon等人，自然实验室指南(Nat Protoc.)，2014年3月；9(3):674–693，其内容通过引用完全并入本文以及在下面的实施方式中。
一旦获得纳米抗体，可以在体内或体外测定纳米抗体的结合和/或生物活性(例如昆虫防控活性)。这些方法是本领域已知的，并且在上文和下文中进一步公开。
因此，根据具体实施方式，所述方法还包括选择在生物测定中表现出昆虫防控活性的纳米抗体。这种测定是本领域已知的，并将在下文中进一步描述。
根据具体实施方式，纳米抗体是裸纳米抗体。
如本文所使用的，术语“裸纳米抗体”是指不包含异源效应部分(例如，毒素部分，可检测部分)的纳米抗体。
根据具体实施方式，纳米抗体包含异源效应部分，例如毒素部分、可检测部分。效应部分可以是蛋白质或非蛋白质(例如小分子化合物)；后者通常使用纳米主体和缀合体(conjugate partner)上的官能团产生。
因此，例如，各种类型的可检测或报告部分可与本公开的纳米抗体缀合(conjugate)。这些包括但不限于放射性同位素(例如[125]碘)、磷光化学品、化学发光化学品、荧光化学品或多肽(例如藻红蛋白(phycoerythrin)(PE)、异硫氰酸荧光素(fluorescein isothiocyanate，FITC)、Cy-chrome、罗丹明(rhodamine)、绿色荧光蛋白(green fluorescent protein，GFP)、蓝荧光蛋白(blue fluorescent protein，BFP)、德克萨斯红(Texas red)、Cy5、PE-BFP等)、酶(例如，辣根过氧化物酶(horseradishperoxidase，HPR)、β-半乳糖苷酶(beta-galactosidase)和碱性磷酸酶(alkalinephosphatase，AP))、亲和标记物(例如，可由相应抗体(例如，地高辛(digoxigenin，DIG)，其由抗DIG抗体识别)识别的抗原)或对标记物(例如链霉亲和素和生物素)具有高亲和力的分子)，以及可通过正电子发射断层扫描(Positron Emission Tomography，PET)或磁共振成像(Magnetic Resonance Imaging，MRI)检测到的分子(造影剂)。
根据具体实施方式，纳米抗体包括毒素。
如本文所使用的，术语“毒素”或“毒素部分”是指具有昆虫防控活性(如下文所限定)的化合物，包括但不限于多肽、多核苷酸、小分子等。
毒素部分的非限制性实施例包括δ-内毒素(例如：Cry1A、Cry1B、Cry1C、Cry1D、Cry1E、Cry1F、Cry1G、Cry1H、Cry1I、Cry1J、Cry1K、Cry2A、Cry7B、Cry8D、Cry9A、Cry9B、Cry9C、Cry9D、Cry9E、Cry15A、Cry22A、Cry32A、Cry51A、Cyt1A(Crickmore等人，1998；VanFrankhuuyzen，2009))，大肠菌素(如大肠菌素E1、大肠菌素la、大肠菌素A、大肠菌素N)，放线菌素(如海葵溶细胞素(equinatoxinII)、sticholysin II、fragaceatoxin C)，ClyA家族毒素(例如：溶细胞素A、非溶血毒素基因(non-haemolytic tripartite enterotoxin)、溶血素BL(haemolysin BL))、溶血素(haemolysins)(例如：α-溶血素、γ-溶血素、杀白细胞素、坏死性肠炎毒素B(nectrotic enteritis toxin B)、δ-毒素、霍乱弧菌溶细胞素(Vibrio cholerae cytolysin)、创伤弧菌溶血素(Vibrio vulnificus haemolysin))、气溶素家族毒素(aerolysin family toxins)(例如：气溶素、α-毒素、水解素(hydralysin)、ε-毒素、肠毒素、溶血凝集素(haemolytic lectin)、kysenin)、胆甾醇依赖性细胞溶素(cholesterol-dependent cytolysins)(例如：裂解素(perfringolysin)、溶血素(suilysin)、中间链球菌溶素(intermedilysin)、李斯特菌溶素O(listeriolysin O)、凝集素溶素(lectinolysin)、人溶素(anthrolysin)、不耐氧链球菌溶血素(streptolysin))、膜攻击复合物组分/穿孔素(membrane attack complex components/perforins)(例如：Plu-MACPF、Bth-MACPF)、重复子毒素(repeats-in-toxins)(例如：HlyA、双功能溶血素-腺苷酸环化酶毒素(bifunctional haemolysin-adenylyl cyclase toxin)、MARTX)(Dal Peraro和van der Goot，2016)、蜘蛛毒素(spider toxins)、蝎子毒素(scorpion toxins)、马铃薯糖蛋白(patatin)、苏云金芽孢杆菌杀虫蛋白(Bacillus thuringiensis insecticidalprotein)、致病杆菌杀虫蛋白(Xenorhabdus insecticidal protein)、光杆菌杀虫蛋白(Photorhabdus insecticidal protein)、侧孢性杆菌杀虫蛋白(Bacillus laterosporousinsecticidal protein)、蚕腐病芽孢杆菌杀虫蛋白(Bacillus bombysepticusinsecticidal protein)、球形芽孢杆菌杀虫蛋白(Bacillus sphaericus insecticidalprotein)和昆虫防控双链RNA(insect-controlling double-stranded RNAs)。
根据上下文、应用和目的，效应部分，例如毒素部分、可检测部分，可以各种方式连接或缀合到本公开的纳米抗体。
效应部分可以直接或间接(例如当包含在载体中时)偶联到纳米抗体。偶联(coupling)可以是共价或非共价结合。
当效应器部分是多肽时，免疫缀合物(immunoconjugate)可以通过重组方式生产。例如，可以将编码毒素或荧光蛋白的核酸序列与编码纳米抗体的核酸序列框内连接，并在宿主细胞中表达以产生重组结合抗体。可替代地，效应部分可以化学合成(例如，以确定的顺序逐步加入一个或多个氨基酸残基，例如固相肽合成技术)。
还可以使用在本领域中广泛实践的标准化学合成技术(参见例如WorldWideWeb.chemistry.org/portal/chemistry)将效应部分连接到纳米抗体上，例如使用任何合适的直接或间接的化学键，通过肽键(当效应部分是多肽时)，或通过共价键连接到中间连接元件(intervening linker element)上，例如连接肽或其它化学部分，例如有机聚合物。嵌合肽(Chimeric peptides)可以通过在肽的羧基(C)或氨基(N)末端的键连接，或者通过键连接到内部化学基团，例如直链、支链或环状侧链、内部碳原子或氮原子等来连接。在美国专利US3,940,475、US4,289,747和US4,376,110中详细提供了荧光标记的描述。
用于将肽部分缀合至纳米抗体的示例性方法包括但不限于SPDP缀合(SPDPconjugation)、戊二醛缀合(Glutaraldehyde conjugation)和碳二亚胺缀合(Carbodiimide conjugation)。
纳米抗体还可以附着于包含效应部分的颗粒或载体上。共价结合纳米抗体与包封颗粒的方法是本领域已知的，公开于例如专利US5,171,578、US5,204,096和US5,258,499中。
本文所述的任何多肽(例如纳米抗体和蛋白质组合物)可由多核苷酸编码。这些多核苷酸本身可用于或在本文公开的多肽的重组生产中使用。
因此，根据本公开的一个方面，提供了编码纳米抗体的多核苷酸或包含纳米抗体和毒素的组合物。
如本文所使用的，术语“多核苷酸”是指以RNA序列、互补多核苷酸序列(cDNA)、基因组多核苷酸序列和/或复合多核苷酸序列(例如，上述的组合)的形式分离并提供的单链或双链核酸序列。
根据具体实施方式，本文公开的多核苷酸和核酸序列中的任一个可包含保守的核酸取代。保守修饰的多核苷酸是指编码相同或基本相同氨基酸序列的那些核酸，或者在核酸不编码氨基酸序列的情况下，指基本相同或相关(例如，天然连续的)序列。由于遗传密码的简并性，大量功能相同的核酸编码大多数蛋白质。例如，密码子GCA、GCC、GCG和GCU都编码氨基酸丙氨酸。因此，在由密码子指定丙氨酸的每个位置处，密码子可以被改变为所描述的对应密码子中的另一个，而不改变编码的多肽。这种核酸变异是“沉默变异”，是保守修饰的多核苷酸中的一种。根据具体实施方式，本文所述的编码多肽的任何多核苷酸和核酸序列也描述了核酸的沉默变异。本领域技术人员将认识到，在某些情况下，核酸中的每个密码子(除了通常是蛋氨酸(methionine)的唯一密码子的AUG和通常是色氨酸(tryptophan)的唯一密码子的TGG之外)可被修饰以产生功能相同的分子。因此，编码多肽的多核苷酸的沉默变异隐含在相对于表达产物的所述序列中。
根据具体实施方式，本文公开的核酸序列是针对例如哺乳动物或计划表达而优化的密码子。
密码子优化的方法是本领域已知的，公开于例如，日本NIAS(国家农业生物科学研究所(National Institute of Agrobiological Sciences))DNA库(www.Kazusa.or.jp/codon/)的密码子使用数据库中；国际专利申请93/07278；以及Grote等人，核酸研究(Nucleic Acid Res.)，2005年7月1日；W526-W531)。
因此，本公开的一些实施方式包括上文所述的核酸序列；其片段、可与其杂交的序列、与其同源的序列、与其直系同源的序列、编码具有不同密码子用途的类似多肽的序列、以突变为特征的改变的序列，例如一个或多个核苷酸的缺失、插入或取代，无论是天然存在的还是人工诱导的，也可以是随机的或定向的。
为了在细胞中表达外源性多肽，优选将编码该多肽的多核苷酸序列连接到适于在细胞中表达的核酸构建体中。这种核酸构建体包括启动子序列，用于在细胞中以组成型或诱导型方式指导多核苷酸序列的转录。
因此，根据本公开的一个方面，提供了包含多核苷酸和用于指导所述多核苷酸表达的顺式调节元件的核酸构建体。
根据具体实施方式，调节元件是异源调节元件。
本公开的一些实施方式的核酸构建体(本文中也称为“表达载体”)包括其他的序列，这些序列使得该载体适于在原核生物、真核生物或优选两者中复制和整合(例如，穿梭载体)。此外，典型的克隆载体还可含有转录和翻译起始序列、转录和翻译终止子和多聚腺苷酸化信号。举例来说，这样的构建体通常包括5'LTR、tRNA结合位点、包装信号、第二链DNA合成的起点和3'LTR或其一部分。
本公开的一些实施方式的核酸构建体通常包括用于从其所在的细胞中分泌多肽的信号序列。根据具体实施方式，信号序列是本公开的一些实施方式的多肽(例如纳米抗体)的天然信号序列。
真核启动子通常包含两种类型的识别序列，TATA盒和上游启动子元件。TATA盒位于转录起始位点上游25-30个碱基对，被认为参与指导RNA聚合酶开始RNA合成。其它上游启动子元件决定转录起始的速率。
优选地，由本公开的一些实施方式的核酸构建体利用的启动子在转化的特定细胞群中是有活性的。细胞类型特异性和/或组织特异性启动子的实施例包括启动子，例如：肝特异性白蛋白(Pinkert等人，(1987)基因开发(Genes Dev)268-277)、淋巴特异性启动子(Calame等人，免疫学进展(Adv.Immunol.)43:235-275)；特别是T细胞受体(Winoto等人(1989)欧洲分子生物学学会(EMBO J.)8:729-733)和免疫球蛋白(Banerji等人(1983)细胞(Cell)33729-740)的启动子；胰腺特异性启动子(Edlunch等人(1985)科学(Science)230:912-916)或乳腺特异性启动子，如乳清启动子(美国专利US4,873,316和欧洲申请EP264,166)。
在使用植物表达载体的情况下，编码序列的表达可以由多种启动子驱动。例如，可以使用病毒启动子如CaMV的35S-RNA和19S-RNA启动子(Brisson等人(1984)自然(Nature)310:511-514)，或TMV的外壳蛋白启动子(Takamatsu等人(1987)欧洲分子生物学学会(EMBOJ.)6:307-311)。可替代地，可以使用植物启动子，例如RUBISCO(Coruzzi等人(1984)欧洲分子生物学学会(EMBO J.)3:1671-1680，和Brogli等人(1984)科学(Science)224:838-843)，包括AlcR/AlcA(乙醇诱导型)；GR融合、GVG和pOp/LhGR(地塞米松诱导型)；XVE/OlexA(β-雌二醇诱导型)；或热休克促进剂(heat shock promoters)，例如大豆hsp17 5-E或hsp17 3-B(Gurley等人(1986)分子细胞生物学(Mol.Cell.Biol.)6:559-565)。
增强子元件元件可以刺激来自连接的同源或异源启动子的高达1000倍。当放置于转录起始位点的下游或上游时，增强子是有活性的。许多来源于病毒的增强子元件具有广泛的宿主范围，并且在各种组织中是有活性的。例如，SV40早期基因增强子适用于许多细胞类型。适用于本公开的一些实施方式的其它增强子/启动子组合包括：来自多瘤病毒、人或鼠巨细胞病毒(CMV)，来自各种逆转录病毒如鼠白血病病毒、鼠或劳斯肉瘤病毒和HIV的长末端重复序列的那些增强子/启动子。参见，增强子和真核表达(Enhancers andEukaryotic Expression)，冷泉港实验室出版社(Cold Spring Harbor Press)，冷泉港(Cold Spring Harbor)，N.Y.1983，其通过引用并入本文。可包含在AAV表达载体中以获得特异性而无需Cre驱动线的不同神经元细胞类型的特异性增强子已在本领域中有所描述，例如在Hrvtin等人(doi:www.doi.org/10.1101/570895)中描述，其通过引用并入本文。
在表达载体的构建中，当启动子在其天然设置中来自转录起始位点时，启动子优选位于与异源转录起始位点大致相同的距离处。然而，如本领域已知的，在不损失促进剂功能的情况下，可以适应该距离的一些变化。
多聚腺苷酸化序列也可以添加到表达载体中，以提高mRNA翻译的效率。准确和有效的聚腺苷酸化需要两种不同的序列元件：位于聚腺苷酸化位点下游的富含Gu或U的序列，和位于上游11-30个核苷酸的高度保守的六核苷酸序列AAUAAA。适用于本公开的一些实施方式的终止和聚腺苷酸化信号包括源自SV40的信号。
除了已经描述的元件之外，本公开的一些实施方式的表达载体通常可包含旨在增加克隆核酸的表达水平或有助于鉴定携带重组DNA的细胞的其它专用元素。例如，许多动物病毒含有在允许的细胞类型中促进病毒基因组的染色体外复制的DNA序列。只要质粒或宿主细胞基因组携带的基因提供了合适的因子，携带这些病毒复制子的质粒就可以附加地复制(replicated episomally)。
载体可以包含或可以不包含真核复制子。如果存在真核复制子，那么使用适当的可选择标记物，载体在真核细胞中可扩增。如果载体不包含真核复制子，则不进行附加体扩增(episomal amplification)是可能的。相反，重组DNA整合到工程化细胞的基因组中，其中启动子指导所需核酸的表达。
本公开的一些实施方式的表达载体可进一步包括另外的多核苷酸序列，其允许例如来自单个mRNA的若干蛋白质的翻译，例如内部核糖体进入位点(IRES)和用于启动子-嵌合多肽的基因组整合的序列。
应当理解，包含在表达载体中的各个元件可以以多种构型排列。例如，增强子元件、启动子等，甚至编码多肽的多核苷酸序列可以以“头对尾”构型排列，可以以反向补体，或者以互补构型，作为反平行链存在。虽然表达载体的非编码元件更可能出现这样的多种构型，但是也可以设想表达载体内的编码序列的替代构型。
哺乳动物表达载体的实施例包括但不限于pcDNA3、pcDNA3.1(+/-)、pGL3、pZeoSV2(+/-)、pSecTag2、pDisplay、pEF/myc/cyto、pCMV/myc/cyto、pCR3 1、pSinRep5、DH26S、DHBB、pNMT1、pNMT41、pNMT81，它们可从Invitrogen获得，pCI可从Promega获得，pMbac、pPbac、RS-RSV和RS-CMV可从Strategene获得，pTRES及其衍生物可从Clontech获得。
也可使用含有来自真核病毒如逆转录病毒的调节元件的表达载体。SV40载体包括pSVT7和pMT2。来自于牛乳头状瘤病毒的载体包括pBV-1MTHA，来自于EB病毒的载体包括pHEBO和p2O5。其它示例性载体包括pMSG、pAV009/A+、pMTO10/A+、pMAMneo-5、杆状病毒pDSVE，以及其他允许在SV-40早期启动子、SV-40晚期启动子、金属硫蛋白启动子、鼠乳腺肿瘤病毒启动子、劳斯肉瘤病毒启动子(Rous sarcoma virus promoter)、多角体蛋白启动子(polyhedrin promoter)、或其他显示对真核细胞中的表达有效的启动子。
如上所述，病毒是特殊的感染因子(specialized infectious agents)，在许多情况下，它们已经进化以逃避宿主防御机制。通常，病毒在特定的细胞类型中感染和繁殖。病毒载体的靶向特异性利用其天然特异性以特异性靶向预定的细胞类型，从而将重组基因引入被感染的细胞中。因此，本公开的一些实施方式使用的载体的类型将取决于转换的细胞类型。根据转化的细胞类型选择合适载体的能力完全在普通技术人员的能力范围内，因此，本文没有提供对选择考虑的一般描述。
可以用本公开一些实施方式的核酸构建体稳定或瞬时地转化细胞。在稳定转化中，本公开一些实施方式的核酸分子被整合到细胞基因组中，因此其代表稳定的遗传性状。在瞬时转化中，核酸分子由转化的细胞表达，但它不整合到基因组中，因此，它代表瞬时性状。
可使用各种方法将本公开的一些实施方式的表达载体引入细胞中。这些方法一般描述于：Sambrook等人，分子克隆：实验室手册(Molecular Cloning:A LaboratoryManual)，冷泉港实验室出版社(Cold Spring Harbor Press)，纽约(1989，1992)；Ausubel等人，分子生物学实验(Current Protocols in Molecular Biology)，约翰威立国际出版公司(John Wiley and Sons,Baltimore,Md.)(1989)；Chang等人，体细胞基因治疗(Somatic Gene Therapy)，CRC出版公司，密歇根州安阿伯(1995)；Vega等人，基因靶向(Gene Targeting)，CRC出版公司，密歇根州安阿伯(1995)，载体：分子克隆载体及其用途概述，巴特沃思出版公司(Butterworths)，波士顿麻省(Boston Mass.)(1988)；以及Gilboa等人(生物技术期刊(Biotechniques)4(6):504-512,1986)，这些方法包括例如，稳定或瞬时转染、脂转染、电穿孔和用重组病毒载体感染。另外，参见美国专利US5,464,764和US5,487,992，用于正-负选择方法(positive-negative selection method)。
在使用植物表达载体的情况下，可以使用Ti质粒、Ri质粒、植物病毒载体、直接DNA转化、微量注射、电穿孔和本领域技术人员熟知的其它技术，将构建体引入植物细胞中。例如，参见Weissbach&Weissbach，1988，植物分子生物学方法(Methods for PlantMolecular Biology)，美国学术出版社(Academic Press)，纽约，第八节，421-463。
通过病毒感染引入核酸与比其它方法(例如脂肪转移和电穿孔)相比具有几个优势，因为病毒的传染性可以获得更高的转染效率。
目前优选的体内核酸转移技术包括用病毒或非病毒构建体转染，例如腺病毒、慢病毒、单纯疱疹病毒I型病毒或腺相关病毒(AAV)和基于脂质的系统。用于脂质介导的基因转移的有用脂质例如是DOTMA、DOPE和DC-Chol(Tonkinson等人，癌症研究，14(1):54-65(1996))。用于基因治疗的最优选构建体是病毒，最优选腺病毒、AAV、慢病毒或逆转录病毒。病毒构建体(例如逆转录病毒构建体)包括至少一个转录启动子/增强子或基因座限定元件(locus-defining element)，或通过其它方式(例如交替剪接、核RNA输出或信使的翻译后修饰(post-translational modification of messenger))控制基因表达的其它元件。此类载体构建体还包括包装信号、长末端重复序列(LTR)或其部分、以及适用于所用病毒的正链和负链引物结合位点，除非其已经存在于病毒构建体中。此外，此类构建体通常包括用于从其所在的宿主细胞分泌肽的信号序列。优选地，用于此目的的信号序列是哺乳动物信号序列或本公开一些实施方式的多肽变体的信号序列。可选地，构建体还可包括引导聚腺苷酸化的信号，以及一个或多个限制性位点和翻译终止序列。例如，这样的构建体通常包括5'LTR、tRNA结合位点、包装信号、第二链DNA合成起点和3'LTR或其一部分。可使用其它非病毒载体，例如阳离子脂质、聚赖氨酸和树突状聚合物。
除了包含用于插入的编码序列的转录和翻译的必要元件之外，本公开一些实施方式的表达构建体还可包括经工程改造以增强所表达的肽的稳定性、生产、纯化、产率或毒性的序列。例如，可以对包含本公开一些实施方式的多肽和异源蛋白的融合蛋白或可切割融合蛋白的表达进行工程改造。可以设计这样的融合蛋白，使得融合蛋白可以通过亲和层析容易地分离；例如，通过固定在对异源蛋白特异性的柱上。在多肽和异源蛋白之间工程化切割位点的情况下，通过用干扰切割位点的适当酶或试剂处理而将多肽从色谱柱中释放(例如，参见Booth等人，(1988)免疫学通讯免疫(Immunol.Lett.)19:65-70；和Gardella等人，(1990)生物与生化(J.Biol.Chem.)265:15854-15859)。
本公开还考虑了包含本文所述的多肽、多核苷酸和核酸构建体的细胞。
因此，根据本公开的一个方面，提供了一种宿主细胞，其包含纳米抗体或包含纳米抗体和毒素的组合物或编码其的多核苷酸或核酸构建体。
如上所述，多种原核或真核细胞可用作宿主表达系统以表达本公开一些实施方式的多肽。这些包括但不限于微生物，例如用含有编码序列的重组噬菌体DNA、质粒DNA或粘粒DNA表达载体转化的细菌；用含有所述编码序列的重组酵母表达载体转化的酵母；用含有编码序列的重组病毒表达载体(例如，花椰菜花叶病毒，CaMV；烟草花叶病毒(TMV))感染或用重组质粒表达载体(例如Ti质粒)转化的植物细胞系统。哺乳动物表达系统也可用于表达本公开一些实施方式的多肽。
根据具体实施方式，所述细胞是哺乳动物细胞。
根据具体实施方式，所述细胞是骆驼科动物细胞。
合适的哺乳动物细胞包括原代细胞和永生化细胞系。
根据其它具体实施方式，哺乳动物细胞是永生化细胞系。
合适的哺乳动物细胞系包括但不限于HeLa细胞(例如，美国典型培养物保藏中心(American Type Culture Collection)(ATCC)编号CCL-2)、CHO细胞(例如，ATCC编号CRL9618、CCL61、CRL9096)、HEK293细胞(例如，ATCC编号1573)、Vero细胞、NIH 3T3细胞(例如，ATCC编号CRL-1658)、Huh-7细胞、BHK细胞(例如，ATCC编号CCL10)，PC12细胞(ATCC编号CRL1721)、COS细胞、COS-7细胞(ATCC编号CRL1651)、RAT1细胞、小鼠L细胞(ATCC编号CCLI.3)，人胚肾(HEK)细胞(ATCC编号CRL1573)、HLHepG2细胞、NS0、Sp2/0、BHK、Namalwa等。
根据具体实施方式，所述细胞是大肠杆菌，例如SHuffle T7 Express&BL21。
根据具体实施方式，细胞是植物细胞。
根据本公开的一个方面，提供了一种生产昆虫防控纳米抗体的方法，该方法包括在宿主细胞中表达本文公开的多核苷酸或核酸构建体。
根据具体实施方式，所述方法还包括分离所述纳米抗体。
任何重组多肽(例如纳米抗体)的分离或回收可通过本领域已知的任何方法来实现。根据具体实施方式，重组多肽的回收或分离在培养适当时间后进行。短语“回收重组多肽”或“分离重组多肽”是指收集包含该多肽的整个发酵培养基，不需要另外的分离或纯化步骤。尽管如此，本公开一些实施方式的多肽可以使用各种标准蛋白纯化技术纯化，例如但不限于亲和层析、离子交换层析、过滤、电泳、疏水相互作用层析、凝胶过滤层析、反相层析、伴刀豆球蛋白A层析、混合模式层析、金属亲和层析、凝集素亲和层析聚焦和差异溶解(differential solubilization)。
根据具体实施方式，在合成和纯化之后，可以在体内或体外测定纳米抗体的结合和/或昆虫防控活性。这些方法在本领域是已知的，并在上文和下文中进一步公开。
本文公开的组合物(例如纳米抗体、包含纳米抗体和毒素的组合物、编码其的多核苷酸和核酸构建体，以及表达其的宿主细胞)可以以适当的方式配制，例如冻干、冷冻干燥、干燥，或者在水性载体、介质或合适的稀释剂(例如盐水或其它缓冲液)中配制。根据具体的实施方式，配制的组合物可以是粉尘或颗粒材料、粉末、凝胶、乳剂、糊剂、丸剂、片剂或油(植物或矿物)中的悬浮液，或水或油/水乳剂、胶囊悬浮液、可乳化浓缩物的形式，或为可湿性粉剂、可湿性颗粒、水分散颗粒、气溶胶、泡沫、淤浆或可流动浓缩物。
根据具体实施方式，组合物被配制成液体浓缩物、干粉、片剂、胶囊悬浮液、浆液或“湿滤饼”，其可在最终使用之前由最终用户适当稀释、分散、悬浮、乳化或以其它方式适当重构。
根据具体实施方式，组合物被配制成液体制剂。
根据具体实施方式，组合物被配制成干制剂。
根据具体实施方式，组合物被配制用于通过喷雾、灌溉和/或熏蒸递送。
根据具体实施方式，本文公开的组合物(例如纳米抗体、包含纳米抗体和毒素的组合物、编码其的多核苷酸和核酸构建体，以及表达其的宿主细胞)在储存和使用期间都是稳定的，这意味着组合物的完整性在组合物的储存和/或使用条件下得以保持，所述条件可包括升高的温度、冻融循环、pH或离子强度的变化、紫外线照射、有害化学物质的存在等。
根据具体的实施方式，组合物的完整性和活性在组合物的储存和/或使用条件下得以保持，所述条件可包括升高的温度、冻融循环、pH或离子强度的变化、紫外线照射、有害化学物质的存在等。
根据具体的实施方式，组合物的完整性和活性在开阔田地或受控条件下(例如温室)得以保持。
应当注意的是，本文公开的组合物可以用各种载体配制，所述载体用于增加例如递送、稳定性、渗透性等。
如本文所使用的“载体”是指任何固体、半固体或液体载体，化合物(例如纳米抗体和/或毒素，包含其的组合物)可以适当地掺入、包含、固定、吸附、吸收、结合、包封、嵌入、附着或包含在其中。这种载体的非限制性实施例包括纳米胶囊、微胶囊、纳米球、微球、纳米颗粒、微粒、脂质体、囊泡、珠、凝胶、弱离子树脂颗粒、脂质体、耳蜗递送载体、小颗粒、颗粒、纳米管、巴克球(bucky-balls)、作为油包水乳液一部分的水滴，作为水包油乳液一部分的油滴、有机材料如软木、木材或其它植物衍生材料(例如，以种子壳、木屑、纸浆、球体、珠、薄片或任何其它合适的形式)、纸或纸板、无机材料如滑石、粘土、微晶纤维素、二氧化硅、氧化铝、硅酸盐和沸石，或甚至微生物细胞(如酵母细胞)或其合适的部分或片段。
根据具体实施方式，载体是这样的，即它们具有立即或逐渐或缓慢释放的特性，例如在几分钟、几小时、几天或几周内释放。此外，载体可由这样的材料(例如聚合物)制成，所述材料随时间(例如，数分钟、数小时、数天或数周)破裂或缓慢降解(例如，由于长时间暴露于高温或低温、日光(sunlight)、高湿度或低湿度或其他环境因素或条件)，从而从载体释放化合物(例如纳米抗体和/或毒素，包含它们的组合物)。根据具体实施方式，载体与化合物偶联、结合、连接或以其他方式附着或结合。根据具体实施方式，载体共价偶联至化合物上。
本文公开的组合物(例如纳米抗体、包含纳米抗体和毒素的组合物，和编码其的多核苷酸或核酸构建体，以及表达其的宿主细胞)可以配制在组合物中，例如与合适的生理学上可接受的载体或赋形剂混合的农用化学品或杀虫组合物。
在本文中，术语“活性成分”是指纳米抗体、包含纳米抗体和毒素的组合物、编码其的多核苷酸或核酸构建体，以及表达其的宿主细胞，对生物效应负责。
本文所用的“农用化学制剂”指用于农业用途的组合物，其包含一种或多种所述的活性成分以及其它化学成分，例如农业上可接受的载体和赋形剂。
在下文中，短语“生理上可接受的载体”是指不会对生物体造成显著刺激并且不会消除所施用的化合物的生物活性和性质的载体或稀释剂。助剂包含在这些短语中。
根据具体实施方式，用农业上可接受的载体配制组合物。合适的农用载体可以是固体或液体，并且是本领域公知的。术语“农业上可接受的载体”涵盖所有助剂，例如惰性组分、分散剂、表面活性剂、增粘剂、粘合剂等，这些助剂通常用于杀虫剂制剂技术中，并且是本领域技术人员熟知的。
在本文中，术语“赋形剂”是指添加到组合物中以进一步促进活性成分的施用的惰性物质。赋形剂的非限制性实施例包括碳酸钙、磷酸钙、各种糖和各种类型的淀粉、纤维素衍生物、表面活性剂、明胶、植物油、聚乙二醇、润湿剂、分散剂、缓冲剂、酸化剂等。
根据具体实施方式，本文公开的组合物(例如纳米抗体、包含纳米抗体和毒素的组合物、和编码其的多核苷酸或核酸构建体，以及表达其的宿主细胞)可以是组合物中唯一的活性物质。
根据其它具体实施方式，除了本文公开的组合物(例如，纳米抗体、包含纳米抗体和毒素的组合物、和编码它的多核苷酸或核酸构建体，以及表达它的宿主细胞)之外，所述组合物还包含一种或多种其它活性剂。这种试剂的非限制性实施例包括除草剂、杀虫剂、植物生长调节剂、安全剂等。
根据具体实施方式，本文公开的组合物和另外的活性剂在共同制剂中。
根据具体实施方式，本文公开的组合物和另外的活性剂在分开的容器中。
根据其它具体实施方式，组合物可包含昆虫引诱剂。引诱剂可以是信息素，例如雄性或雌性信息素。作为一个实施例，在本公开中可以使用“昆虫信息素及其在害虫管理中的应用”一书(Howse等人，查普曼和霍尔出版公司(Chapman and Hall)，1998)中提到的信息素。
引诱剂可以存在于制剂中，或者可以与制剂分开施用，以确保昆虫被吸引到施用制剂的部位。
本公开一些实施方式的纳米抗体和包含纳米抗体和毒素的组合物被赋予昆虫防控活性。
如本文所使用的，术语“昆虫防控”是指防止、抑制或降低昆虫进食、生长、移动、传播、发育、存活和/或繁殖的能力，和/或限制与昆虫相关的损害，其可能表现为，例如杀灭昆虫、降低昆虫存活率或寿命、降低昆虫的生育力力和/或繁殖力，减少或阻止昆虫的进食、减少或阻止昆虫的生长、减少或阻止昆虫的发育、减少或阻止昆虫的活动性和/或防止昆虫的侵扰。根据具体实施方式，昆虫防控活动通过杀灭昆虫来表现。
测定昆虫防控活性的方法是本领域技术人员所熟知的，也公开在下面的实施例部分中，这些实施例包括但不限于，在存在纳米抗体或组合物存在下体外培养幼虫，并测定死亡率、重量、长度、化蛹和成虫出现时间，与不存在纳米抗体或组合物的情况进行比较。
本文公开的纳米抗体本身可以具有昆虫防控活性，或者它可以通过向昆虫递送毒素来发挥其昆虫防控活性。
因此，根据具体实施方式，纳米抗体与昆虫多肽的结合赋予纳米抗体昆虫防控活性。
根据具体实施方式，纳米抗体下调其结合的昆虫多肽的活性。
如本文所使用的，“下调活性”是指与不存在纳米抗体的情况相比，存在纳米抗体的情况下昆虫多肽的生物功能降低了至少5％，这是通过适于确定昆虫多肽活性的方法来确定的。因此，例如测定包含CBD的多肽的活性可通过例如ELISA、蛋白质印迹分析、免疫沉淀或流式细胞术实现；测定V-ATP酶亚基c的活性可以通过例如ATP酶活性测定和/或ATP依赖性质子转运体测定来实现；测定海藻糖酶的活性可以通过例如铁-亚铁氰化反应来实现，用于确定还原糖或在与海藻糖酶培育之后使用葡萄糖氧化酶-过氧化物酶定量释放的葡萄糖；测定细胞色素p450单加氧酶的活性可以通过，例如药物或替代化合物的氧化的测量、血红素测定或P450-Glo发光测定来实现；测定几丁质脱乙酰化酶的活性可通过，例如部分O-羟乙基化的几丁质(乙二醇几丁质)作为底物，在N-乙酰基中进行放射性标记的放射性测定，或测定由几丁质脱乙酰酶在各种几丁质底物上的作用释放的乙酸盐的Bergmeyer酶测定法来实现；测定几丁质合成酶的活性可以通过，例如使用[14C]UDP-N-乙酰基-D-氨基葡萄糖(GlcNAc)作为底物的放射性测定，随后在酸沉淀后量化不溶性14C标记的几丁质来实现；测定NPC1甾醇转运体的活性可以通过例如胆甾醇吸收测定、甾醇定量测定来实现。
根据其它具体实施方式，与不存在纳米抗体的情况相比，减少了至少10％、至少20％、至少30％、至少40％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少99％或100％，例如可以通过上文所述的任何方法测定。
根据另外的或可替代的实施方式，纳米抗体是靶向剂，其用于向昆虫提供例如具有昆虫防控活性的毒素的特定递送。
以上进一步描述了毒素的非限制性实施例和将它们偶联到纳米抗体的方法。
由于本公开的一些实施方式的纳米抗体和包含纳米抗体和毒素的组合物被赋予昆虫防控活性，本公开还包括昆虫防控方法。
因此，根据本公开的一个方面，提供了一种昆虫防控方法，该方法包括使所述昆虫与所述纳米抗体或包含所述纳米抗体和所述毒素的组合物、编码其的多核苷酸或核酸构建体或表达其的宿主细胞的组合物接触。
根据具体实施方式，接触包括将纳米抗体或包含纳米抗体和毒素的组合物直接施用于昆虫。
根据具体实施方式，接触包括将纳米抗体或包含纳米抗体和毒素的组合物施用于可能与所述昆虫接触的生物体或表面。
根据本公开的另一方面，提供了一种防止商品产品的昆虫侵染的方法，该方法包括向产品中加入纳米抗体或包含纳米抗体和毒素的组合物。
根据本公开的另一方面，提供了一种防止商品产品的昆虫侵染的方法，该方法包括将产品包装在包含纳米抗体或包含纳米抗体和毒素的组合物的包装材料中。
接触或添加可以使用本领域已知的任何合适的方法来实现，包括但不限于喷涂(包括高体积(HV)、低体积(LV)和超低体积(ULV)喷涂)、雾化、刷涂、敷涂(dressing)、滴涂(dripping)、涂敷(coating)、浸染(dipping)、沉浸(immersing)、浸渍(submerging)、包覆(encrusting)、铺展(spreading)、发泡、雾化、注入、添加到培养物中，灌溉、以小液滴、雾或气溶胶的形式施用，在生物体的细胞中重组表达核酸构建体(如上文进一步公开的)。
根据具体实施方式，接触或添加通过喷涂、灌溉和/或熏蒸来实现。
根据具体实施方式，接触或添加通过将多核苷酸或核酸构建体引入生物体的细胞中来实现。
根据具体实施方式，所述生物体是植物。
如本文所使用的，术语“表面”是指可与昆虫接触的任何物体。非限制性表面包括网(例如蚊帐)、光源、彩色物体、从对比背景温室、户外露营设施、土壤等中突出的形状或轮廓。
根据具体实施方式，商品产品由植物生产。
本文中使用的术语“植物”包括整个植物、嫁接植物、植物和植物部分的祖先和后代，包括种子、芽、茎、根(包括块茎)、根茎、接穗、果实、蔬菜、花和植物细胞、组织和器官。植物可以是任何形式，包括悬浮培养物、胚胎、分生组织、愈伤组织、叶、配子体、孢子体、花粉和小孢子。
根据具体实施方式，植物是作物。本文中使用的“作物”是指作为食物、牲畜饲料、燃料原料或用于任何其它经济目的而种植收获的植物物种或品种。作为非限制性实施例，作物可以是玉米、谷物(例如小麦、黑麦、大麦和燕麦)、高粱、水稻、甜菜和饲料甜菜、水果(例如，梨果类水果(例如苹果和梨)、柑橘类水果(例如橙子、柠檬、柠檬、葡萄柚、或柑橘)、核果类水果(例如桃子、油桃或李子))、坚果(例如杏仁或核桃)、软质水果(例如樱桃、草莓、黑莓或覆盆子)、大蕉科或葡萄藤、豆科作物(例如豆荚、小扁豆、豌豆和大豆)、油料作物(如向日葵、红花、油菜籽、芥花籽油、蓖麻或橄榄)、葫芦(例如黄瓜、甜瓜或南瓜)、纤维植物(例如棉花、亚麻或大麻)、燃料作物(例如甘蔗、芒属或柳枝稷(switchgrass))、蔬菜(例如马铃薯、蕃茄、胡椒、莴苣、菠菜、洋葱、胡萝卜、茄子、芦笋或卷心菜)、观赏植物(例如花卉(例如，矮牵牛、天竺葵、玫瑰、郁金香、百合或菊花)、灌木、阔叶树(例如，杨树或柳树)和常绿植物(例如针叶树)、草(例如草坪、草坪或牧草))例如花(例如，矮牵牛花、雪茄花、玫瑰、郁金香、百合或菊花)、灌木、阔叶树(例如，杨树或柳树)和常绿植物(例如针叶树)、草(例如草坪、草皮或饲草)或其它有用的植物(例如咖啡、茶、烟草、啤酒花、胡椒、橡胶或乳胶植物)。
根据具体实施方式，植物可选自玉米、大豆、紫苜蓿、棉花、向日葵、甘蓝型油籽，例如芥属植物油籽(例如油菜(Brassica napus)(例如芥花籽油、油菜籽)、芜菁(Brassicarapa)、芥菜(Brassica juncea)(例如(田)芥菜)和埃塞俄比亚芥(Brassica carinata))、棕榈科(Arecaceae sp.)(例如油椰、椰子)、大米、小麦、甜菜、甘蔗、燕麦、黑麦、大麦、谷子和高粱、黑小麦、亚麻、坚果、葡萄和藤蔓，以及来自各种植物分类群的各种水果和蔬菜，例如：蔷薇科植物(Rosaceae sp.)(例如梨果类水果(例如苹果树和梨)、但也有核果类水果(例如杏、樱桃、杏仁、李子和桃)，以及浆果(例如草莓、覆盆子、红醋栗和黑醋栗)以及醋栗)、茶藨子科(Ribesioidae sp.)、胡桃科(Juglandaceae sp.)、桦木科(Betulaceaesp.)、漆树科(Anacardiaceae sp.)、壳斗科(Fagaceae sp.)、桑科(Moraceae sp.)、木樨科(Oleaceae sp.)(例如橄榄树)、猕猴桃科(Actinidaceae sp.)、樟科(Lauraceae sp.)(例如鳄梨、肉桂、樟脑)、芭蕉科(Musaceae sp.)(例如香蕉树和种植园)、茜草科(Rubiaceaesp.)(例如咖啡)、山茶科(Theaceae sp.)(例如茶)、梧桐科(Sterculiceae sp.)、芸香科(Rutaceae sp.)(例如柠檬、橙子、芦柑和葡萄柚)、茄科(例如蕃茄、马铃薯、胡椒、辣椒、茄子、烟草)、百合科(Liliaceae sp.)、菊科(例如莴苣、朝鲜蓟和菊苣，包括菊苣根、欧洲菊苣或普通菊苣)、伞形科(例如胡萝卜、欧芹、芹菜和块根芹)、葫芦科(例如黄瓜，包括小黄瓜、南瓜、西瓜、葫芦和甜瓜)、葱科(例如青蒜和洋葱)、十字花科(Cruciferae sp.)(例如白甘蓝、红甘蓝、西兰花、花椰菜、抱子甘蓝、小白菜、球茎甘蓝、萝卜、辣根、水芥和大白菜)、豆科(Leguminosae sp.)(例如花生、豌豆、小扁豆和蚕豆(beans)，例如扁豆和蚕豆(broadbeans))、藜科(Chenopodiaceae sp.)(例如牛皮菜、饲料甜菜、菠菜、甜菜根)、亚麻科(Linaceae sp.)(例如大麻)、大麻科(Cannabeacea sp.)(例如大麻制品)、锦葵科(Malvaceae sp.)(例如秋葵、可可)、罂粟科(Papaveraceae)(例如罂粟)、天门冬科(Asparagaceae)(例如芦笋)、花园和树林中的有用植物和观赏植物，包括草皮、草坪、草和甜叶菊，以及这些植物的转基因类型。
根据具体实施方式，植物可以是植物的可收获部分，选自水果、花、坚果、蔬菜、具有不可食用果皮的水果或蔬菜，例如鳄梨、香蕉、大蕉、柠檬、葡萄柚、甜瓜、橙子、菠萝、猕猴桃，番石榴、芦柑、芒果和南瓜。
根据具体实施方式，植物是森林植物，例如在www.fao.org/3/3/i0640e/i0640e13.pdf中描述的。
根据具体实施方式，植物是观赏植物。
根据具体的实施方式，植物可以是观赏植物的切花，优选地选自水仙百合(Alstroemeria)、康乃馨、菊花、小苍兰、非洲菊(Gerbera)、唐菖蒲(Gladiolus)、满天星(baby's breath)(Gypsophila spp.)、向日葵属植物(Helianthus)、绣球花(Hydrangea)、百合(Lilium)、洋桔梗(Lisianthus)、玫瑰和夏季花卉。
根据具体实施方式，植物是切割草或木材。
根据具体实施方式，植物是棉花。
根据具体实施方式，植物是转基因植物。
根据具体实施方式，植物是重组表达纳米抗体或包含纳米抗体和毒素的组合物的转基因植物。
产生转基因植物的方法是本领域公知的，也在上文中有所描述。
接下来，本公开还包括包含纳米抗体或包含纳米抗体和毒素的组合物、或编码其的多核苷酸或核酸构建体或表达其的宿主细胞的产品。
根据具体实施方式，与不包含纳米抗体或不包含纳米抗体和毒素、或编码其的多核苷酸或核酸构建体或表达其的宿主细胞的组合物的产品相比，这种产品更能抵抗昆虫侵扰或损害。
因此，根据本公开的一个方面，提供了包含纳米抗体或包含纳米抗体和毒素的组合物，或编码其的多核苷酸或核酸构建体的植物。
包含纳米抗体或包含纳米抗体和毒素的组合物、或编码其的多核苷酸或核酸构建体的植物。
根据本公开的另一方面，提供了包含纳米抗体或包含纳米抗体和毒素的组合物的商品产品。
根据本公开的另一方面，提供了覆盖有纳米抗体或包含纳米抗体和毒素的组合物的表面
根据本公开的另一个方面，提供了一种包装产品，其包括包含在包装材料内的商品产品，该包装材料包含纳米抗体或包含纳米抗体和毒素的组合物。
下面的表1-8列出了可用于本公开的具体实施方式的多肽和纳米抗体。
表1：目标蛋白的氨基酸序列
Figure BDA0003723677810000641
Figure BDA0003723677810000651
Figure BDA0003723677810000661
表2A：抗CBD纳米抗体的氨基酸序列
Figure BDA0003723677810000662
Figure BDA0003723677810000671
表2B：抗CBD纳米抗体的氨基酸序列
Figure BDA0003723677810000672
Figure BDA0003723677810000681
Figure BDA0003723677810000691
Figure BDA0003723677810000701
表3A：抗V-ATP酶亚基纳米抗体的氨基酸序列
Figure BDA0003723677810000702
Figure BDA0003723677810000711
表3B：抗V-ATP酶亚基纳米抗体的氨基酸序列
Figure BDA0003723677810000712
Figure BDA0003723677810000721
表4：抗海藻糖酶纳米抗体的氨基酸序列
Figure BDA0003723677810000722
Figure BDA0003723677810000731
Figure BDA0003723677810000741
表5：抗细胞色素p450纳米抗体的氨基酸序列
Figure BDA0003723677810000742
Figure BDA0003723677810000751
Figure BDA0003723677810000761
Figure BDA0003723677810000771
表6：抗几丁质脱乙酰酶纳米抗体的氨基酸序列
Figure BDA0003723677810000772
Figure BDA0003723677810000781
Figure BDA0003723677810000791
Figure BDA0003723677810000801
表7：抗几丁质合成酶纳米抗体的氨基酸序列
Figure BDA0003723677810000811
Figure BDA0003723677810000821
Figure BDA0003723677810000831
表8：抗NPC1甾醇转运体纳米抗体的氨基酸序列
Figure BDA0003723677810000832
Figure BDA0003723677810000841
Figure BDA0003723677810000851
Figure BDA0003723677810000861
Figure BDA0003723677810000871
如本文所使用的，术语“约(about)”指±10％
术语“包括(comprises、comprising、includes、including)”、“具有(having)”及其同源词(conjugates)意为“包括但不限于”。
术语“由……组成(consisting of)”是为“包括并限于”。
术语“基本上由……组成(consisting essentially of)”意为组合物、方法或结构可包括另外的成分、步骤和/或部分，但前提是另外的成分、步骤和/或部分不会实质性地改变所要求保护的组合物、方法或结构的基本和新颖特性。
如本文所使用的，单数形式“一个(a、an)”和“所述(the)”包括复数，除非上下文另有明确的指示。例如，术语“化合物(a compound)”或“至少一种化合物(at least onecompound)”可以包括多种化合物，包括其混合物。
在整个申请中，本公开的各个实施方式可以以范围的形式呈现。应当理解，范围形式的描述仅仅是为了方便和简洁，不应当被解释为对本公开的范围的不可改变的限制。因此，范围的描述应当被认为已经具体公开了所有可能的子范围以及该范围内的单个数值。例如，对诸如1至6的范围的描述应当被认为已经具体公开了子范围，诸如1至3、1至4、1至5、2至4、2至6、3至6等，以及该范围内的单个数字，例如1、2、3、4、5和6。不管范围的宽度如何，都适用。
每当本文指出数值范围时，意为包括在所指出的范围内的任何引用的数字(分数或整数)。表述“第一指示数和第二指示数之间的范围(ranging/ranges between a firstindicate number and a second indicate number)”和“从第一指示数到第二指示数的范围(ranging/ranges from afirst,indicate number“to”a second indicate number)”在本文中可互换使用，意为包括第一指示数字和第二指示数字以及它们之间的所有数和整数。
如本文所使用的，术语“方法”是指用于完成给定任务的方式、手段、技术和程序，包括但不限于化学、药理学、生物学、生物化学和医学领域的从业人员已知的或者容易从已知的方式、手段、技术和程序开发的那些方式、手段、技术和程序。
当提及特定的序列表时，这种提及应理解为还包括基本上与其互补序列相对应的序列，包括由例如测序错误、克隆错误或导致碱基替换、碱基缺失或碱基添加的其它改变引起的微小序列变异，只要这种变异的频率为50个核苷酸中少于1个；可替代地，100个核苷酸中少于1个；可替代地，200个核苷酸中少于1个；可替代地，500个核苷酸中少于1个；可替代地，1000个核苷酸中少于1个；可替代地，5000个核苷酸中少于一个；可替代地，10,000个核苷酸中少于一个。
应当理解，为了清楚起见，在单独实施方式的上下文中描述的本公开的某些特征也可以在单个实施方式中组合地提供。相反，为了简洁起见，在单个实施方式的上下文中描述的本公开的各个特征也可以单独提供，或者以任何合适的子组合提供，或者以本公开的任何其它描述的实施方式中的合适的方式提供。在各个实施方式的上下文中描述的某些特征不应被认为是这些实施方式的必要特征，除非该实施方式在没有这些元件的情况下不起作用。
如上文所述以及如以下权利要求部分所要求的本公开的各个实施方式和方面在以下实施例中得到实验支持。
实施例
现在参考下面的实施例，这些实施例与上面的描述一起以非限制性的方式说明了本公开的一些实施方式。
通常，本文使用的命名和本公开中使用的实验室方法包括分子、生物化学、微生物和重组DNA技术。这些技术在文献中有详尽的解释。参见，例如：“分子克隆：实验室手册(Molecular Cloning:A laboratory Manual)”Sambrook等人，(1989)；“分子生物学实验(Current Protocols in Molecular Biology)”，第I-III卷，Ausubel,R.M.编(1994)；Ausubel等人，“分子生物学实验(Current Protocols in Molecular Biology)”，约翰威立国际出版公司(John Wiley and Sons,Baltimore,Md.)，巴尔的摩，马里兰州(1989)；Perbal，“分子克隆实用指南(A Practical Guide to Molecular Cloning)”，约翰威立国际出版公司，纽约(1988)；Watson等人，“重组DNA(Recombinant DNA)”，科学美国人书籍(Scientific American Books)，纽约；Birren等人(编辑)，“基因组分析：实验室手册系列(Genome Analysis:A Laboratory Manual Series)”，1-4卷，冷泉港实验室出版社(ColdSpring Harbor Press)，纽约(1998)；如以下美国专利中所述的方法：US4,666,828、US 4,683,202、US 4,801,531、US 5,192,659和US 5,272,057；“细胞生物学：实验室手册(CellBiology:A Laboratory Handbook)”，第I-III卷，Cellis,J.E.编辑(1994)；Freshney的“动物细胞培养-基础技术手册(Culture of Animal Cells-A Manual of BasicTechnique)”，Freshney,Wiley-Liss,N.Y(1994)，第三版；“当前免疫学方案”卷I-III，Coligan J.E.编辑(1994)；Stites等人编辑，“基础和临床免疫学(Basic and ClinicalImmunology)”(第八版)，Appleton&Lange出版社，诺沃克，CT(1994)；Mishell和Shiigi编辑，“细胞免疫学中的选定方法(Selected Methods in Cellular Immunology)”，曼出版公司，纽约(1980)；可用的免疫测定在专利和科学文献中被广泛描述，参见，例如：美国专利US3,791,932、US3,839,153、US3,850,752、US3,850,578、US3,853,987、US3,867,517、US3,879,262、US3,901,654、US3,935,074、US3,984,533、US3,996,345、US4,034,074、US4,098,876、US4,879,219、US5,011,771和US5,281,521；“寡核苷酸合成(OligonucleotideSynthesis)”，Gait,M.J.编辑(1984)；“核酸杂交(Nucleic Acid Hybridization)”，Hames,B.D.和Higgins S.J.编辑(1985)；“转录与翻译(Transcription and Translation)”，Hames,B.D.,and Higgins S.J.编辑(1984)；“动物细胞培养(Animal Cell Culture)”，Freshney,R.I.编辑(1986)；“固定化细胞和酶(Immobilized Cells and Enzymes)”，IRL出版社(1986)；“分子克隆实用指南(A Practical Guide to Molecular Cloning)”，Perbal,B.(1984)和“酶学方法”，317卷，美国学术出版社；“PCR方案：方法和应用指南”，美国学术出版社，圣地亚哥，CA(1990)；Marshak等人，CSHL出版社(1996)；所有这些都通过引用的方式并入本文，如同在本文中完全阐述一样。在本文中还提供了其它的一般参考资料。其中的过程被认为是本领域公知的，是为了方便读者而提供的。其中包含的所有信息通过引用并入本文。
材料和方法
用于免疫的蛋白质靶标制备-来自棉铃虫的靶蛋白质的DNA序列(参见上文表1)进行了优化和合成。将合成的序列用组氨酸标签(His tag)克隆到pET-28a或His-30a载体中，并在大肠杆菌菌株BL21 Star(DE3)中表达。将单个菌落接种到含有卡那霉素的LB培养基中；将培养物在37℃下，以200转/分(rpm)培育，然后用异丙基β-D-1-硫代半乳糖苷(IPTG)诱导。SDS-PAGE分析用于监测蛋白质表达。随后，将储存在甘油中的BL21 Star(DE3)接种到含有卡那霉素的TB培养基中，并在37℃下培养。当OD600达到1.2时，用IPTG在15℃下诱导细胞培养16小时。通过离心收获细胞，用裂解缓冲液(20mM NaPi pH7.4，500mM NaCl，20mM咪唑，蛋白酶抑制剂鸡尾酒-cOmpleteTM，不含EDTA的蛋白酶抑制剂鸡尾酒-罗氏公司，目录号(Cat No.)：4693132001)重悬浮颗粒，然后进行超声处理和离心。使用尿素溶解沉淀物；保留变性的上清液用于将来的纯化。随后，使用Ni-NTA柱(GE-Healthcare，目录号：17-5318-02)从上清液中纯化蛋白质：用10床体积的洗涤缓冲液(20mL NaPi pH7.4，500mM NaCl，50mL咪唑)洗涤柱，并用洗脱缓冲液(20mL pH7.4，500mM NaCl，500mM咪唑)洗脱蛋白质。洗脱后，将馏分置于膜截留值为3.5kDa(Tivan Biotech MEGA3-50)的MEGA管中，并在1L PBSpH7.4中于4℃透析过夜，随后在相同条件下进行第二轮透析。一些靶蛋白(例如，V-ATP酶亚基C靶蛋白)在以等分试样储存之前，也通过0.22μm过滤器重新折叠和灭菌。使用微量蛋白核酸仪(Nano-drop)测定浓度。通过标准SDS-PAGE测定蛋白质纯度和分子量。
免疫-在第0天、第7天、第14天、第21天、第28天和第35天，对羊驼(llama)进行皮下注射，每次注射约125μg或150μg的靶蛋白与Gerbu佐剂P(Gerbu Biotech公司，#1-0025)。在第40天，从羊驼收集约100毫升抗凝血以制备淋巴细胞。
构建VHH抗体库(VHH library)-从羊驼淋巴细胞构建VHH抗体库以筛选抗原特异性纳米抗体的存在。为此，将来自外周血淋巴细胞的总RNA用作模板，用寡聚(dT)引物合成第一链cDNA。使用该cDNA，通过聚合酶链式反应(PCR)扩增VHH编码序列，用PstI(来自Thermofcher的#ER0611)和NotI(来自Sigma的#11037668001)消化，并克隆到噬菌体载体pMECS的PstI和NotI位点。VHH抗体库包含约108个独立的转化体，其中约80-92％的转化体含有具有正确插入大小的载体。
特异性靶纳米抗体的分离和测序-将抗体库在用靶抗原(100mM NaHCO3 pH 8.2中，100g/ml)包被的固相上淘选(panned)3轮。在每一轮淘选后，通过比较从抗原包被孔中洗脱的噬菌体颗粒的数量与从阴性对照(未包被的封闭)孔中洗脱的噬菌体颗粒的数量，来评估抗原特异性噬菌体的富集。这些实验表明，到第三轮时，噬菌体群体富集了抗原特异性噬菌体。用于淘选和ELISA筛选的抗原与用于免疫的抗原相同，使用未包被的封闭孔作为阴性对照。生成的纳米抗体的氨基酸序列如上表2A-6所示。
纳米抗体表达和纯化-通过GenScript优化和合成纳米抗体DNA序列。将合成的序列用组氨酸标签克隆到pET-28b+载体中，用于在大肠杆菌中表达蛋白质。随后，用重组质粒转化大肠杆菌SHuffle T7。将单个菌落接种到含有卡那霉素的LB培养基中；将培养物在37℃下，以220转/分(rpm)培育，然后用1MIPTG诱导。SDS-PAGE分析用于监测表达。随后，将储存在甘油中的SHuffle T7接种到含有卡那霉素的TB培养基中，并在37℃下培养。当OD600达到约1.2时，用1MIPTG在30℃下诱导细胞培养20-22小时。离心收集细胞，用裂解缓冲液(20mM NaPi pH7.5，500mM NaCl，20mM咪唑)重悬浮细胞颗粒，然后进行超声处理和离心。使用尿素溶解沉淀物，保留变性的上清液用于将来的纯化。用Ni-NTA柱纯化纳米抗体，用10床体积的洗涤缓冲液(20mM NaPi pH7.5，500mM NaCl，50mM咪唑)洗涤柱，并用洗脱缓冲液(20mM NaPi pH7.5，500mM NaCl，500mM咪唑)洗脱蛋白质。随后，用透析的缓冲液(PBS＝7.4)在4℃透析洗脱馏分过夜。浓度通过微量蛋白核酸仪(Nano-drop)测定。蛋白质纯度和分子量通过标准SDS-PAGE测定。
将所产生的纳米抗体与靶蛋白质结合-通过在4℃过夜培育，用10g/ml的靶蛋白包被Maxisorb 96孔板。用PBS洗涤包被的板，并在37℃下，在含有PBS+0.5％吐温+1％BSA的封闭缓冲液中封闭1小时，然后在室温下与2g/ml的被测纳米抗体一起培育1小时。用PBS洗涤后，将板与MonoRabTMRabbit Anti-Camelid VHH Antibody-HRP(Genscript公司，目录号：A01860)在封闭缓冲液中培育。最后，用PBS洗涤板，用TMB溶液培育30分钟，然后加入H2SO4，在450nm处读取吸光度。
稳定性测定-通过在自然日光、自然UV辐射和高夏季室外/田间温度中培育，在“类似田间条件(field-like conditions)”下测试生成的纳米抗体的稳定性。此外，通过将纳米抗体与从棉铃虫4号收集的管腔汁在起始幼虫肠道中培育来评估对酶促蛋白水解活性和高pH的稳定性(参见，例如：Purcell,John P.,John T.Greenplate和R.Douglas Sammons.，昆虫生物化学与分子生物学(Insect Biochemistry and Molecular Biology)22.1(1992):41-47；Pang,A.S.,&Gringorten,J.L.(1998)欧洲微生物学通讯(FEMSmicrobiology letters)，167(2),281-285；以及Ma,Gang等人，昆虫生物化学与分子生物学(Insect Biochemistry and Molecular Biology)35.7(2005):729-739)。具体地，通过在4℃过夜培育，用2g/ml的测试纳米抗体包被Maxisorb 96孔板。用PBS洗涤包被的板，并在37℃下，在含有PBS+0.5％吐温+1％BSA的封闭缓冲液中封闭1小时。将包被的板与PBS在“类似田间条件”下培育1-3小时，或者在存在腔肠液的情况下在室温下培育1小时。培育后，用PBS洗涤板，与MonoRabTMRabbit Anti-Camelid VHH Antibody-HRP在封闭缓冲液中培育。最后，用PBS洗涤板，用TMB溶液培育30分钟，然后加入H2SO4，在450nm处读取吸光度。
昆虫培养-从以色列的棉花采集棉铃虫，在24℃、70％相对湿度的实验室中，以10小时白天/14小时夜晚周期饲养。将所收集的幼虫放在具有沃德饲料粉末(Ward’s dietpowder)Stonefly Heliothis Diet(产品编号：38-0600，沃德自然科学(Ward’s NaturalScience)，Rochester,NY)的人工饲料中饲养，辅以维生素混合物(烟酸40μM、泛酸钙5μM、核黄素5μM、盐酸硫胺素4μM、盐酸吡哆醇5μM、叶酸2.5μM、D-生物素4μM和氰钴胺素5μM)、山梨酸0.2％、对羟基苯甲酸甲酯0.4％、抗坏血酸0.25％、啤酒酵母1％、白醋4％和亚麻籽油0.5％。
喂养生物测定-将0.5克Heliothis人工饲料加入24孔板的每个孔中，并用浓度为0.4mg/ml的100μl受试纳米抗体悬浮液或PBS PBS覆盖，作为阴性对照。在每孔中，以12次重复的方式加入一只24-48小时龄的幼虫。用聚氧乙烯醚密封箔(HJ-BIOANALITIC，目录号：900371)涂覆板，并在实验室中于24℃和70％相对湿度下在黑暗中培育。每个纳米抗体的每个研究重复3-5次。培育7天后，将幼虫移动到直径为5cm的较大的培养皿中，培养皿中有相同的人工食物，但没有添加纳米抗体。在培育7天和14天后测量每只幼虫的体长和体重，然后观察化蛹和成虫出现的时间。
棉花生长-棉花品种Akalpi(陆地棉(G.hirsutum)和G.hirsutum(G.barbadense)之间的种间杂交(Inter-specific hybrid，ISH))；1432Intercott Hazera)在昼夜25/18℃的控制温度下生长。由SAS自动化系统监测和维持温度。此外，灌溉施肥提供Sheffer7-7-7加钙；保持30％的浸出率。害虫防控完全是通过使用粘性陷阱(sticky-colored traps)以机械方式进行的。种植周期持续时间为6-8周。此后，将植物转移实验室，作为生物测定实验的植物模型。
叶片生物测定-1ml灭菌琼脂添加到12孔板的每个孔中，并在每个孔的琼脂上放置2cm
Figure BDA0003723677810000931
棉叶圆片(cotton leave disc)。每个圆叶片上覆盖50μl浓度为0.4mg/ml、1mg/ml、2mg/ml或4mg/ml的测试纳米抗体悬浮液，或PBS作为阴性对照。在每孔中，以12次重复的方式加入一只24-48小时龄的幼虫。用聚氧乙烯醚密封箔(HJ-BIOANALITIC，目录号：900371)密封板，并在实验室中于24℃和70％相对湿度下培育。培育4天后，将幼虫移至具有新鲜棉叶圆片的新板上，用相同浓度的纳米抗体或PBS处理，在第7天，将幼虫再次移至更大的孔(6孔板)中，加入琼脂和用100μl相同浓度的相同纳米抗体处理的新鲜的2.8cm褐色棉叶圆片。在第4天、第7天和第10天评估幼虫死亡率。每个纳米抗体的每个研究重复2-3次。
免疫荧光-按照制造商的说明，将浓度为1mg/ml的纯化的抗CBD纳米抗体(本文称为“CB20901”)缀合到荧光标记的Cy3(ab188287-Cy3快速缀合试剂盒，英国剑桥)上。如上所述进行饲养生物测定。具体地，用100μl Cy3荧光标记的纳米抗体饲养12只幼体，用100μlPBS饲养12只幼体作为对照。培育36小时后，将处理过的幼虫转移到干净的新鲜制备的Heliothis人工饲料中再饲养12小时，以消除假阳性荧光信号，随后进行第一次成像。在下一步骤中，将幼虫放回到纳米抗体/PBS-食物中，直到它们6天龄，然后以没有纳米抗体的食物饲养12小时，最后对活幼体和提取的肠道进行第二次成像。为了有效的成像比较，设置为休眠：尼康Eclipse80i显微镜，NIS-Element软件，Camara DS-Ri2，借用1X_zoom_2。0x_exposure 10ms_gain_7.6x(用于食物成像)，lent 1X_Zoom 1.4x_exposure 200ms_gain_2.0x(用于48小时龄幼虫)，借出1x_zoom1.4X_exposure 400ms_gain_6.2x(用于六天龄活幼虫和提取的肠道)。
实施例1
抗几丁质结合域纳米抗体
产生了几种针对棉铃虫几丁质结合域(CBD)抗原的纳米抗体，本文称为Ha-PMP5B1和Ha-PMP5B2(参见上文表2A-B)。Ha-PMP5B1蛋白含有来自棉铃虫的具有五种不同CBD的506个氨基酸，Ha-CBD蛋白是Ha-CBD的部分蛋白，含有具有两种CBD的192个氨基酸。使用包被有靶抗原的板进行的ELISA测定证实了产生的纳米抗体结合了靶CBD抗原(图1)。具体实施方式公开了通过口服给药向棉铃虫幼虫施用纳米抗体，从而纳米抗体被吸收到幼虫肠腔(其具有高pH并含有高含量的蛋白水解酶)中，直到到达幼虫肠道的围食膜中的靶位点；研究了抗CBD纳米抗体在管腔中的稳定性。使用包被有所产生的抗CBD纳米抗体并在从棉铃虫得到的管腔汁液存在下培育1小时的板进行的ELISA测定表明，40-70％的纳米抗体在培育1小时后保持完整(图2)，因此表明了所产生的抗CBD纳米抗体在内腔条件下的存活能力。此外，还在模拟野外条件的条件下测试了所生成的抗CBD纳米抗体的稳定性，方法是在室外与自然太阳辐射和30℃的外部高温下培育1-3小时。结果表明，在30℃、60-70％湿度和UV指数为4-5的类似田间条件下培育3小时后，与对照相比，仅观察到纳米抗体含量略有降低(图3)。
为了评估所产生的纳米抗体的杀虫活性，使年龄不超过48小时的幼棉铃虫幼虫在补充有单剂量的抗CBD产生的纳米抗体的人工食物上生长。在昆虫的整个生命周期中进行观察，包括在第7天和第14天测量幼虫的重量和长度，以及随后观察从蛹转化为成虫的个体数量。结果表明，用几种抗CBD纳米抗体饲养7天的幼虫表现出长度减少(图4A)和/或重量减轻(图4B)和/或死亡率增加(图4C)，这取决于所测试的纳米抗体。
为了跟踪幼虫肠道中的纳米抗体轨迹，使棉铃虫幼虫在补充有荧光标记的Cy3抗CBD纳米抗体CB12901的人工食品上生长。如图5A所示，标记的纳米抗体在食品中至少可以稳定一周。此外，在饲养后，在食物和幼虫粪便之间观察到荧光信号的降低，表明被标记的纳米抗体被幼虫肠道摄取(图5A)。此外，在将幼虫转移到不含纳米抗体的食物中12小时后，在幼虫中检测到标记的纳米抗体，这表明标记的纳米抗体保留在幼虫肠道中(图5B)。此外，在饲养6天后，证实了标记的纳米抗体到达围食膜基质的能力：次定时，从所有肠道，特别是从围养基质和腔肠分离后的围食膜基质中观察到阳性信号(图5C)。
实施例2
抗V-ATP酶亚单元C纳米抗体
产生了几种针对棉铃虫V-AT酶亚单元C(本文中称为“VAT”)抗原的纳米抗体(参见上文表3A-B)。使用包被有靶抗原的板进行的ELISA测定证实了产生的纳米抗体结合了靶VAT抗原(图6)。接着，研究了抗VAT纳米抗体在管腔条件和类似田间条件下的稳定性(图7-8)。结果表明，40-70％的纳米抗体在管腔汁液存在下培育1小时后保持完整，因此表明了所产生的抗VAT纳米抗体在管腔条件下的存活能力。此外，结果表明，在30℃、60-70％湿度和UV指数为4-5的类似田间条件下培育3小时后，与对照相比，仅观察到纳米抗体含量略有降低。
为了评估所产生的纳米抗体的杀虫活性，使年龄不超过48小时的幼棉铃虫幼虫在补充有单剂量的抗VAT产生的纳米抗体的人工食品上生长。在昆虫的整个生命周期中进行观察，包括在第7天和第14天测量幼虫的重量和长度，以及随后观察从蛹转化为成虫的个体数量。结果显示，用所产生的抗VAT纳米抗体饲养7天的幼虫表现出长度减少(图9A)和/或重量减轻和/或死亡率增加(图9C)，这取决于所测试的纳米抗体。
此外，测定了存活并完成从幼虫到蛹阶段然后变为成虫的完整生命周期的幼虫的百分比。如下文表9所示，用抗VAT纳米抗体处理7天的幼虫具有较低的幼虫百分比，这些幼虫完成了成虫的完整生命周期。重要的是，尽管幼虫仅在实验的25-30天中的前7天暴露于抗VAT纳米抗体，但结果显示它们对棉铃虫具有长期稳定的作用，并且影响它们的蛹和成虫的后期发育阶段。
表9：完成从幼虫到成虫的完整生命周期的幼虫的百分比
％成虫
VAT0101A 58
VAT0101B 42
VAT0101C 33
VAT0101D 16
VAT0101E 33
VAT0101F 42
VAT0101G 58
VAT0202 58
PBS 67
实施例3
抗海藻糖酶纳米抗体
产生了几种针对棉铃虫海藻糖酶(本文称为“TRH”)抗原的纳米抗体(参见上文表4)。使用包被有靶抗原的板进行的ELISA测定证实了产生的纳米抗体结合了靶海藻糖酶抗原(图10)。接着，研究了抗TRH纳米抗体在类似田间条件下和50℃高温下的稳定性(图11A-B)。结果表明，与对照组相比，在30℃、60-70％湿度和UV指数为4-5的田间条件下培育过夜不影响纳米抗体含量或仅导致纳米抗体含量略有下降。在50℃下培育过夜后，显示了类似的结果。
为了评估所产生的纳米抗体的杀虫活性，使年龄不超过48小时的幼棉铃虫幼虫在补充有抗TRH产生的纳米抗体的人工食品上生长；在第7天和第14天测量死亡率。结果显示，用产生的抗TRH纳米抗体处理后幼虫死亡率显著增加(图12A)。随后，使年龄不超过48小时的幼棉铃虫幼虫棉叶圆片上生长，并在第0天、第4天和第7天施用补充有所产生的抗TRH纳米抗体，在第4天、7天和10天测量死亡率。结果显示，在用抗TRH纳米抗体处理后，幼虫死亡率显著增加，呈剂量依赖性(图12B)。
此外，对昆虫的整个生命周期进行了观察，包括幼虫的重量、长度测量，以及随后观察从蛹转化为成虫的个体数量。此外，测定了存活并完成从幼虫到蛹阶段然后变为成虫的完整生命周期的幼虫的百分比。
实施例4
抗细胞色素P450单加氧酶纳米抗体
产生了几种针对棉铃虫细胞色素p450单加氧酶(本文称为“P450”)抗原的纳米抗体(参见上文表5)。使用包被有靶抗原的板进行的ELISA测定证实了产生的纳米抗体结合了靶P450抗原。接着，研究了抗P450纳米抗体在内腔条件和类似田间条件下的稳定性。
为了评估所产生的纳米抗体的杀虫活性，使年龄不超过48小时的幼棉铃虫幼虫在补充有抗P450产生的纳米抗体的人工食品上生长；并在第7天和第14天测量死亡率。结果显示，用抗P450纳米抗体处理后幼虫死亡率显著增加。
此外，对昆虫的整个生命周期进行了观察，包括在第7天和第14天测量幼虫的重量和长度，以及随后观察从蛹转化为成虫的个体数量。此外，测定了存活并完成从幼虫到蛹阶段然后变为成虫的完整生命周期的幼虫的百分比。
实施例5
抗几丁质脱乙酰酶纳米抗体
产生了几种针对棉铃虫几丁质脱乙酰酶(本文称为“CDA”)抗原的纳米抗体(参见上文表6)。使用包被有靶抗原的板进行的ELISA测定证实了产生的纳米抗体结合了靶CDA抗原(图15)。接着，研究了抗CDA纳米抗体在类似田间条件下和50℃高温下的稳定性(图16A-B)。结果表明，与对照组相比，在30℃、60-70％湿度和UV指数为4-5的田间条件下培育过夜，纳米抗体的含量没有变化。在50℃下培育过夜后，显示了类似的结果。
为了评估所产生的纳米抗体的杀虫活性，使年龄不超过48小时的幼棉铃虫幼虫在补充有所产生的抗CDA纳米抗体的人工食品上生长；在第7天和第14天测量死亡率。结果显示，用抗CDA纳米抗体处理后幼虫死亡率显著增加(图17A)。随后，使年龄不超过48小时的幼棉铃虫幼虫棉叶圆片上生长，并在第0天、第4天和第7天施用补充有所产生的抗CDA纳米抗体，在第4天、第7天和第10天测量死亡率。结果显示，在用抗CDA纳米抗体处理后，幼虫死亡率显著增加，呈剂量依赖性(图17B)。
此外，对昆虫的整个生命周期进行了观察，包括在第7天和第14天测量幼虫的重量和长度，以及随后观察从蛹转化为成虫的个体数量。此外，测定了存活并完成从幼虫到蛹阶段然后变为成虫的完整生命周期的幼虫的百分比。
实施例6
抗几丁质合成酶纳米抗体
产生了几种针对棉铃虫几丁质合成酶(本文称为“CHS”)抗原的纳米抗体(参见上文表7)。使用包被有靶抗原的板进行的ELISA测定证实了产生的纳米抗体结合了靶CHS抗原(图18)。接着，研究了抗CHS纳米抗体在类似田间条件下和50℃高温下的稳定性(图19A-B)。结果表明，与对照相比，在30℃、60-70％湿度和UV指数为4-5的田间条件下培育过夜，纳米抗体的含量仅略有下降。在50℃下培育过夜后，显示了类似的结果。
为了评估所产生的纳米抗体的杀虫活性，使年龄不超过48小时的幼棉铃虫幼虫在补充有所产生的抗-CHS纳米抗体的人工食品上生长；在第7天和第14天测量死亡率。结果显示，用抗CHS纳米抗体处理后幼虫死亡率显著增加(图20A)。随后，使年龄不超过48小时的幼棉铃虫幼虫在棉叶圆片上生长，并在第0天、第4天和第7天施用补充有抗-CHS纳米抗体，在第4天、第7天和第10天测量死亡率。结果显示，在用抗CDA纳米抗体处理后，幼虫死亡率显著增加，呈剂量依赖性(图20B)。
此外，对昆虫的整个生命周期进行了观察，包括在第7天和第14天测量幼虫的重量和长度，以及随后观察从蛹转化为成虫的个体数量。此外，测定了存活并完成从幼虫到蛹阶段然后变为成虫的完整生命周期的幼虫的百分比。
实施例7
抗NPC1甾醇转运体纳米抗体
产生了几种针对棉铃虫NPC1甾醇转运体(本文中称为“NPC1”)抗原的纳米抗体(参见上文表8)。使用包被有靶抗原的板进行的ELISA测定证实了产生的纳米抗体结合了靶NPC1抗原(图21)。接着，研究了抗NPC1甾醇转运体纳米抗体在内腔条件和类似田间条件下的稳定性。
为了评估所产生的纳米抗体的杀虫活性，使年龄不超过48小时的幼棉铃虫幼虫在补充有所产生的抗NPC1纳米抗体的人工食品上生长；在第7天和第14天测量死亡率。结果显示，用抗NPC1纳米抗体处理后幼虫死亡率显著增加。
此外，对昆虫的整个生命周期进行了观察，包括在第7天和第14天测量幼虫的重量和长度，以及随后观察从蛹转化为成虫的个体数量。此外，测定了存活并完成从幼虫到蛹阶段然后变为成虫的完整生命周期的幼虫的百分比。
尽管已经结合本公开的具体实施方式描述了本公开，但是显然，对于本领域技术人员来说，许多替代、修改和变化将是显而易见的。因此，旨在包含落入所附权利要求的精神和广泛范围内的所有这些替换、修改和变化。
本说明书中提及的所有出版物、专利和专利申请通过引用全部并入本说明书中，结合程度如同每个单独的出版物、专利或专利申请都通过引用具体和单独地结合到本说明书中。此外，本申请中任何参考的引用或标识不应解释为承认此类参考可作为本公开的现有技术。就所使用的章节标题而言，它们不应被理解为必要的限制。此外，本公开的任何一个或多个优先权文件的全部内容过引用的方式整体并入本文。
序列表
<110> IBI-AG创新生物杀虫剂有限公司（IBI-Ag Innovative Bio InsecticidesLtd.）
罗尼·欧伦-本纳罗亚（OREN-BENAROYA, Rony）
阿米尔·阿亚莉（AYALI, Amir）
杰西卡·蒙塞拉特（MONSERRATE, Jessica）
杰罗恩·范瑞（VAN RIE, Jeroen）
<120> 昆虫防控纳米抗体及其用途
<130> 83672
<150> US 62/933,533
<151> 2019-11-11
<160> 596
<170> PatentIn version 3.5
<210> 1
<211> 526
<212> PRT
<213> Artificial sequence
<220>
<223> A polypeptide comprising Chitin binding domain CBD - HaPMP5B1
<400> 1
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Gly Asp Arg Gly Ile Ser Glu Pro Gly Asn Asp
20 25 30
Gln Gly Asn Asp Asn Asp Ser Asn Asp Asn Asn Ser Ser Asn Glu Gln
35 40 45
Gly Gly Val Cys Asn Cys Asn Pro Glu Glu Ala Pro Ala Ile Cys Ala
50 55 60
Ser Pro Gly Ser Glu Gly Val Leu Val Ala His Glu Asn Cys Glu Lys
65 70 75 80
Tyr Tyr Ile Cys Asn His Gly Arg Pro Val Val Ala Ser Cys Ser Gly
85 90 95
Asn Leu Leu Phe Asn Pro Tyr Thr Asn Glu Cys Gly Trp Pro Arg Asp
100 105 110
Val Asp Cys Gly Asp Arg Ile Glu Pro Gly Cys Thr Gly Cys Asn Asp
115 120 125
Asn Asn Asn Asn Asp Asp Asp Asp Ser Asp Cys Asp Gly Asp Asp Pro
130 135 140
Val Pro Pro Pro Ala Asp Asn Asp Asp Ser Glu Ser Ala Asp Ile Asp
145 150 155 160
Asp Leu Pro Pro Pro Gly Asp Asp Ala Ser Val Arg Pro Pro Val Asp
165 170 175
Glu Gly Thr Cys Asn Cys Asn Pro Glu Gln Ala Pro Ser Ile Cys Ala
180 185 190
Glu Asp Asp Ser Asp Gly Val Leu Val Ala His Glu Asp Cys Asn Lys
195 200 205
Phe Tyr Lys Cys His Asn Gly Lys Pro Val Ala Leu Tyr Cys Pro Gly
210 215 220
Asn Leu Leu Tyr Asn Pro Asn Thr Glu Gln Cys Asp Trp Pro Glu Lys
225 230 235 240
Val Asp Cys Gly Asp Arg Val Ile Pro Asp Pro Glu Asp Asn Thr Val
245 250 255
Gly Gly Asn Asn Asp Gly Glu Asp Asp Ser Glu Gly Val Leu Val Ala
260 265 270
His Glu Asn Cys Asn Gln Phe Tyr Lys Cys Ser Gly Gly Lys Pro Val
275 280 285
Ala Leu Leu Cys Pro Gly Asn Leu Leu Phe Asn Pro Asn Thr Asp Gln
290 295 300
Cys Asp Trp Pro Trp Glu Val Asp Cys Gly Asp Arg Ile Ile Pro Asp
305 310 315 320
Pro Asp Arg Thr His Cys Gly Ser His Cys Ser Thr His Cys Ser Thr
325 330 335
His Cys Gly Ser Leu Leu Arg Leu Pro Leu Arg Leu His Cys Gly Ser
340 345 350
His Cys Gly Ser His Cys Cys Thr Asn Thr Ala Thr Asn Arg Arg Arg
355 360 365
Asn Met Gln Leu Gln Ser Trp Ser Thr Phe His Leu Cys Ser Arg Arg
370 375 380
Leu Leu Ile Ala His Glu Asp Cys Asn Lys Phe Tyr Ile Cys Asp His
385 390 395 400
Gly Lys Pro Val Ala Leu Ser Cys Pro Gly Asn Leu Leu Tyr Asn Pro
405 410 415
Tyr Thr Glu Lys Cys Asp Trp Pro Glu Asn Val Glu Cys Gly Asp Arg
420 425 430
Ala Pro Asp Pro Asp Ala Ser Gln Ala Pro Ala Ile Cys Ala Asp Ser
435 440 445
Gly Ser Glu Gly Val Leu Val Ala His Glu Asn Cys Asp Gln Tyr Tyr
450 455 460
Ile Cys Asp Gly Gly Arg Pro Val Ala Arg Pro Cys Gln Gly Gly Leu
465 470 475 480
Leu Tyr Asn Pro Leu Thr Gln Tyr Cys Asp Gly Gln Glu Met Ser Thr
485 490 495
Ala Val Thr Gly Leu Ser Leu Met Thr Ala Pro Val Ile Pro Glu Met
500 505 510
Arg Pro Asp Cys Ala Val Ser Gln Thr Pro Lys Glu Ala Trp
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<210> 2
<211> 220
<212> PRT
<213> Artificial sequence
<220>
<223> A polypeptide comprising Chitin binding domain CBD - HaPMP5B1
<400> 2
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Asp Arg Val Ile Pro Asp Pro Glu Asp Asn Thr
20 25 30
Val Gly Gly Asn Asn Asp Gly Glu Asp Asp Ser Glu Gly Val Leu Val
35 40 45
Ala His Glu Asn Cys Asn Gln Phe Tyr Lys Cys Ser Gly Gly Lys Pro
50 55 60
Val Ala Leu Leu Cys Pro Gly Asn Leu Leu Phe Asn Pro Asn Thr Asp
65 70 75 80
Gln Cys Asp Trp Pro Trp Glu Val Asp Cys Gly Asp Arg Ile Ile Pro
85 90 95
Asp Pro Asp Arg Thr His Cys Gly Ser His Cys Ser Thr His Cys Ser
100 105 110
Thr His Cys Gly Ser Leu Leu Arg Leu Pro Leu Arg Leu His Cys Gly
115 120 125
Ser His Cys Gly Ser His Cys Cys Thr Asn Thr Ala Thr Asn Arg Arg
130 135 140
Arg Asn Met Gln Leu Gln Ser Trp Ser Thr Phe His Leu Cys Ser Arg
145 150 155 160
Arg Leu Leu Ile Ala His Glu Asp Cys Asn Lys Phe Tyr Ile Cys Asp
165 170 175
His Gly Lys Pro Val Ala Leu Ser Cys Pro Gly Asn Leu Leu Tyr Asn
180 185 190
Pro Tyr Thr Glu Lys Cys Asp Trp Pro Glu Asn Val Glu Cys Gly Asp
195 200 205
Arg Ala Pro Asp Pro Asp Ala Ser Gln Ala Pro Ala
210 215 220
<210> 3
<211> 45
<212> PRT
<213> Artificial sequence
<220>
<223> Chitin binding domain (CBD)
<400> 3
Ser Glu Gly Val Leu Val Ala His Glu Asn Cys Glu Lys Tyr Tyr Ile
1 5 10 15
Cys Asn His Gly Arg Pro Val Val Ala Ser Cys Ser Gly Asn Leu Leu
20 25 30
Phe Asn Pro Tyr Thr Asn Glu Cys Gly Trp Pro Arg Asp
35 40 45
<210> 4
<211> 33
<212> PRT
<213> Artificial sequence
<220>
<223> Chitin binding domain (CBD)
<400> 4
Cys Asn Lys Phe Tyr Lys Cys His Asn Gly Lys Pro Val Ala Leu Tyr
1 5 10 15
Cys Pro Gly Asn Leu Leu Tyr Asn Pro Asn Thr Glu Gln Cys Asp Trp
20 25 30
Pro
<210> 5
<211> 45
<212> PRT
<213> Artificial sequence
<220>
<223> Chitin binding domain (CBD)
<400> 5
Ser Glu Gly Val Leu Val Ala His Glu Asn Cys Asn Gln Phe Tyr Lys
1 5 10 15
Cys Ser Gly Gly Lys Pro Val Ala Leu Leu Cys Pro Gly Asn Leu Leu
20 25 30
Phe Asn Pro Asn Thr Asp Gln Cys Asp Trp Pro Trp Glu
35 40 45
<210> 6
<211> 37
<212> PRT
<213> Artificial sequence
<220>
<223> Chitin binding domain (CBD)
<400> 6
Glu Asp Cys Asn Lys Phe Tyr Ile Cys Asp His Gly Lys Pro Val Ala
1 5 10 15
Leu Ser Cys Pro Gly Asn Leu Leu Tyr Asn Pro Tyr Thr Glu Lys Cys
20 25 30
Asp Trp Pro Glu Asn
35
<210> 7
<211> 41
<212> PRT
<213> Artificial sequence
<220>
<223> Chitin binding domain (CBD)
<400> 7
Ser Glu Gly Val Leu Val Ala His Glu Asn Cys Asp Gln Tyr Tyr Ile
1 5 10 15
Cys Asp Gly Gly Arg Pro Val Ala Arg Pro Cys Gln Gly Gly Leu Leu
20 25 30
Tyr Asn Pro Leu Thr Gln Tyr Cys Asp
35 40
<210> 8
<211> 400
<212> PRT
<213> Artificial sequence
<220>
<223> V-ATPase sub unit c
<400> 8
Met His His His His His His Met Ser Glu Tyr Trp Val Ile Ser Ala
1 5 10 15
Pro Gly Asp Lys Thr Cys Gln Gln Thr Trp Asp Thr Leu Asn Asn Ala
20 25 30
Thr Lys Ser Gly Asn Leu Ser Ala Asn Tyr Lys Phe Pro Ile Pro Asp
35 40 45
Leu Lys Val Gly Thr Leu Asp Gln Leu Val Gly Leu Ser Asp Asp Leu
50 55 60
Gly Lys Leu Asp Thr Phe Val Glu Ser Val Thr Arg Lys Val Ala Gln
65 70 75 80
Tyr Leu Gly Glu Val Leu Glu Asp Gln Arg Asp Lys Leu His Glu Asn
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Leu Met Ala Asn Asn Ser Asp Met Pro Ser Tyr Leu Thr Arg Phe Gln
100 105 110
Trp Asp Met Ala Lys Tyr Pro Ile Lys Gln Ser Leu Arg Asn Ile Ala
115 120 125
Asp Ile Ile Ser Lys Gln Val Gly Gln Ile Asp Ser Asp Leu Lys Gln
130 135 140
Lys Ser Ala Ala Tyr Asn Ala Leu Lys Gly Asn Leu Gln Asn Leu Glu
145 150 155 160
Lys Lys Gln Thr Gly Ser Leu Leu Thr Arg Asn Leu Ala Asp Leu Val
165 170 175
Lys Arg Glu His Phe Ile Leu Asp Ser Glu Tyr Leu Thr Thr Leu Leu
180 185 190
Val Ile Val Pro Lys Ser Met Phe Asn Asp Trp Thr Ala Asn Tyr Glu
195 200 205
Lys Ile Thr Asp Met Ile Val Pro Arg Ser Ser Gln Leu Ile His Gln
210 215 220
Asp Asn Asp Tyr Gly Leu Phe Asn Val Thr Leu Phe Lys Lys Val Val
225 230 235 240
Glu Glu Phe Lys His His Ala Arg Glu Arg Lys Phe Val Val Arg Glu
245 250 255
Phe Ser Tyr Asn Glu Ala Asp Met Ala Ala Ala Arg Thr Arg Ser Pro
260 265 270
Ser Ser Ser Pro Thr Arg Arg Ser Ser Ser Ile Leu Arg Lys Phe Ile
275 280 285
Asn Phe Leu Gly Pro Leu Val Arg Trp Leu Lys Val Asn Phe Ser Glu
290 295 300
Cys Phe Cys Ala Trp Ile His Val Lys Ala Leu Arg Val Phe Val Glu
305 310 315 320
Ser Val Leu Arg Tyr Gly Leu Pro Val Asn Phe Gln Ala Val Val Met
325 330 335
Val Pro Ser Arg Lys Asn Thr Lys Lys Leu Arg Glu Val Leu Gln Thr
340 345 350
Leu Tyr Ala His Leu Asp His Ser Ala His Gln His Thr Ser Ser Ala
355 360 365
Gln Asp Asn Ala Glu Leu Ala Gly Leu Gly Phe Gly Ser Ser Glu Tyr
370 375 380
Phe Pro Tyr Val Phe Tyr Lys Ile Asn Val Asp Met Leu Asp Lys Asn
385 390 395 400
<210> 9
<211> 516
<212> PRT
<213> Artificial sequence
<220>
<223> Chitin deacetylase
<400> 9
Met Glu Thr Arg Val Lys Arg Gln Glu Glu Asp Gly Gly Asp Glu Val
1 5 10 15
Asn Ala Glu Gln Leu Cys Asp Gly Arg Pro Ala Asp Glu Tyr Phe Arg
20 25 30
Leu Thr Thr Glu Gly Asp Cys Arg Asp Val Val Arg Cys Thr Arg Ser
35 40 45
Gly Leu Lys Gln Ile Thr Cys Pro Ser Gly Leu Ala Phe Asp Leu Asp
50 55 60
Lys Gln Thr Cys Asp Trp Lys Gly Lys Val Thr Asn Cys Asp Lys Leu
65 70 75 80
Glu Lys Pro Arg Lys Val Leu Pro Ile Leu Lys Thr Asp Glu Pro Ile
85 90 95
Cys Pro Glu Gly Lys Leu Ala Cys Gly Ser Gly Asp Cys Ile Glu Lys
100 105 110
Glu Leu Phe Cys Asn Gly Lys Pro Asp Cys Lys Asp Glu Ser Asp Glu
115 120 125
Asn Ala Cys Thr Val Asp Val Asp Pro Asn Arg Ala Pro Asp Cys Asp
130 135 140
Pro Asn Gln Cys Ala Leu Pro Asp Cys Phe Cys Ser Ala Asp Gly Thr
145 150 155 160
Arg Ile Pro Gly Gly Ile Glu Val Asn Gln Val Pro Gln Met Ile Thr
165 170 175
Ile Thr Phe Asn Gly Ala Val Asn Val Asp Asn Ile Asp Leu Tyr Glu
180 185 190
Gln Ile Phe Asn Gly Asn Arg His Asn Pro Asn Gly Cys Gln Ile Arg
195 200 205
Gly Thr Phe Phe Val Ser His Lys Tyr Thr Asn Tyr Ala Ala Val Gln
210 215 220
Glu Leu His Arg Lys Gly His Glu Ile Ser Val Phe Ser Ile Thr His
225 230 235 240
Lys Asp Asp Pro Gln Tyr Trp Ser Ser Gly Ser Tyr Asp Asp Trp Leu
245 250 255
Ala Glu Met Ala Gly Ala Arg Leu Ile Val Glu Arg Phe Ala Asn Ile
260 265 270
Thr Asp Ser Ser Ile Ile Gly Val Arg Ala Pro Tyr Leu Arg Val Gly
275 280 285
Gly Asn Lys Gln Phe Glu Met Met Ala Asp Gln Tyr Phe Val Tyr Asp
290 295 300
Ala Ser Ile Thr Ala Pro Leu Gly Arg Val Pro Ile Trp Pro Tyr Thr
305 310 315 320
Leu Tyr Phe Arg Met Pro His Lys Cys Asn Gly Asn Ala His Asn Cys
325 330 335
Pro Ser Arg Ser His Pro Val Trp Glu Met Val Met Asn Glu Leu Asp
340 345 350
Arg Arg Asp Asp Pro Thr Phe Asp Glu Ser Leu Pro Gly Cys His Val
355 360 365
Val Asp Ser Cys Ser Asn Ile Gln Thr Gly Glu Gln Phe Ala Arg Leu
370 375 380
Leu Arg His Asn Ser Asn Arg His Tyr Ser Thr Asn Arg Ala Pro Leu
385 390 395 400
Gly Phe His Phe His Ala Ser Trp Leu Lys Ser Lys Lys Glu Phe Arg
405 410 415
Asp Glu Leu Ile Lys Phe Ile Glu Glu Met Leu Glu Lys Asn Asp Val
420 425 430
Tyr Phe Thr Ser Leu Ile Gln Val Ile Gln Trp Met Gln Asn Pro Thr
435 440 445
Glu Leu Thr Ser Leu Arg Asp Phe Gln Glu Trp Lys Gln Asp Lys Cys
450 455 460
Asp Val Lys Gly Gln Pro Phe Cys Ser Leu Pro Asn Ala Cys Pro Leu
465 470 475 480
Thr Thr Arg Glu Leu Pro Gly Glu Thr Leu Arg Leu Phe Thr Cys Met
485 490 495
Glu Cys Pro Asn Asn Tyr Pro Trp Ile Leu Asp Pro Thr Gly Glu Gly
500 505 510
Phe Asn Val Lys
515
<210> 10
<211> 548
<212> PRT
<213> Artificial sequence
<220>
<223> Trehalase
<400> 10
Met Asp Leu Pro Leu Thr Cys Thr Lys Pro Val Tyr Cys Asn Ser Asn
1 5 10 15
Leu Leu His Gln Ile Gln Met Ala Arg Leu Tyr Asn Asp Ser Lys Thr
20 25 30
Phe Val Asp Leu Gln Met Asn Phe Asp Glu Asn Lys Thr Leu Thr Asp
35 40 45
Phe Glu Thr Phe Phe Asn Leu His Asn Lys Asn Pro Thr Lys Glu Gln
50 55 60
Leu Met Glu Phe Val Asn Glu Tyr Phe Ser Asn Asp Asn Glu Leu Glu
65 70 75 80
Pro Trp Gln Pro Lys Asp Phe Ser Asp Asn Pro Ala Phe Leu Ala Lys
85 90 95
Ile Lys Asp Asp Ala Leu Arg Glu Phe Gly Lys Gly Ile Asn Asn Ile
100 105 110
Trp Pro Leu Leu Ala Arg Lys Val Lys Ala Glu Val Phe Gln Lys Pro
115 120 125
Asp Gln Phe Ser Leu Val Pro Leu Thr His Gly Phe Ile Ile Pro Gly
130 135 140
Gly Arg Phe Lys Glu Ile Tyr Tyr Trp Asp Thr Phe Trp Ile Ile Glu
145 150 155 160
Gly Leu Leu Ile Ser Gly Met Gln Glu Thr Ala Lys Gly Met Ile Glu
165 170 175
Asn Leu Ile Glu Leu Leu Asn Leu Phe Gly His Ile Pro Asn Gly Ser
180 185 190
Arg Gly Tyr Tyr Gln Gln Arg Ser Gln Pro Pro Met Leu Asn Ala Met
195 200 205
Val Ala Thr Tyr Tyr Met Tyr Thr Lys Asp Leu Glu Phe Leu Arg Asn
210 215 220
Asn Ile Ala Tyr Leu Glu Lys Glu Leu Asp Phe Trp Met Asp Asn Arg
225 230 235 240
Val Val Ser Val Asn Arg Gly Gly Lys Asn Tyr Thr Leu Leu Arg Tyr
245 250 255
Tyr Ala Pro Ser Lys Gly Pro Arg Pro Glu Ser Tyr Tyr Glu Asp Tyr
260 265 270
Ser Asn Thr Glu Gly Phe Ser Glu Glu Asp Ser Thr Asn Phe Cys Ile
275 280 285
Asp Ile Lys Ser Ala Ala Glu Ser Gly Trp Asp Phe Ser Thr Arg Trp
290 295 300
Phe Leu Met Pro Asp Gly Ser Asn Asn Gly Thr Leu Thr Asp Leu His
305 310 315 320
Thr Arg Tyr Ile Ile Pro Val Asp Leu Asn Ala Ile Phe Ala Gly Ala
325 330 335
Ala Gln Tyr Val Ser Asn Phe His Ala Leu Leu Lys Asn Pro Gln Lys
340 345 350
Ala Ala Arg Tyr Gly Gln Leu Ala Gln Thr Trp Arg Asp Asn Ile Gln
355 360 365
Ala Val Leu Trp Asn Asp Gln Asp Ala Met Trp Tyr Asp Phe Asn Ile
370 375 380
Arg Asp Asn Leu His Arg Arg Tyr Tyr Tyr Ser Ser Asn Ala Ala Pro
385 390 395 400
Leu Trp Gln Asn Ala Val Asn Pro Asp Phe Leu Lys Leu Asn Ala Asp
405 410 415
Arg Ile Leu Lys Ala Ile Thr Glu Ser Gly Gly Val Asp Phe Pro Gly
420 425 430
Gly Val Pro Thr Ser Leu Ile Arg Ser Gly Glu Gln Trp Asp Phe Pro
435 440 445
Asn Val Trp Pro Pro Glu Val Ser Ile Glu Val Ala Ala Ile Glu Asn
450 455 460
Ile Gly Thr Pro Glu Ala Ile Thr Leu Ala Gln Glu Val Ala Gln Thr
465 470 475 480
Phe Val Arg Ser Cys His Trp Gly Phe Gln Lys Tyr Lys Gln Met Phe
485 490 495
Glu Lys Tyr Asp Ala Glu Thr Pro Gly Arg Phe Gly Gly Gly Gly Glu
500 505 510
Tyr Asn Val Gln Phe Gly Phe Gly Trp Ser Asn Gly Val Val Leu Glu
515 520 525
Phe Leu Asn Lys Tyr Gly Ser Gln Leu Thr Ala Asp Asp Ser Asn Asn
530 535 540
Thr Asn Asn Ser
545
<210> 11
<211> 483
<212> PRT
<213> Artificial sequence
<220>
<223> cytochrome p450 monooxygenase
<400> 11
Met Arg Thr Phe Asn Tyr Trp Lys Lys Arg Asn Val Arg Gly Pro Glu
1 5 10 15
Pro Val Val Phe Phe Gly Asn Leu Lys Asp Ser Ala Leu Arg Lys Lys
20 25 30
Asn Met Gly Val Val Met Glu Glu Leu Tyr Asn Met Phe Pro Glu Glu
35 40 45
Lys Val Ile Gly Ile Tyr Arg Met Thr Ser Pro Cys Leu Leu Val Arg
50 55 60
Asp Leu Asp Val Ile Lys His Ile Met Ile Lys Asp Phe Glu Val Phe
65 70 75 80
Ser Asp Arg Gly Val Glu Phe Ser Lys Glu Gly Leu Gly Ser Asn Leu
85 90 95
Phe His Ala Asp Gly Glu Thr Trp Arg Ala Leu Gly Asn Arg Phe Thr
100 105 110
Pro Ile Phe Thr Ser Gly Lys Leu Lys Asn Met Phe Tyr Leu Met His
115 120 125
Glu Gly Ala Asp Asn Phe Ile Asp His Val Ser Ala Glu Cys Glu Lys
130 135 140
Asn Gln Glu Phe Glu Val His Ser Leu Leu Gln Thr Tyr Thr Met Ser
145 150 155 160
Thr Ile Ala Ala Cys Ala Phe Gly Ile Ser Tyr Asp Ser Ile Gly Asp
165 170 175
Lys Val Lys Ala Leu Asp Ile Val Asp Lys Ile Ile Ser Glu Pro Ser
180 185 190
Tyr Ala Ile Glu Leu Asp Met Met Tyr Pro Gly Leu Leu Ser Lys Leu
195 200 205
Asn Leu Ser Ile Phe Pro Thr Val Val Lys Asn Phe Phe Lys Ser Leu
210 215 220
Val Asp Asn Ile Val Ala Gln Arg Asn Gly Lys Pro Ser Gly Arg Asn
225 230 235 240
Asp Phe Met Asp Leu Ile Leu Glu Leu Arg Gln Leu Gly Glu Val Thr
245 250 255
Ser Asn Lys Tyr Gly Ser Ser Ala Ser Ser Leu Glu Ile Thr Asp Glu
260 265 270
Val Ile Cys Ala Gln Ala Phe Val Phe Tyr Ile Ala Gly Tyr Glu Thr
275 280 285
Ser Ala Thr Thr Met Ala Tyr Met Ile Tyr Gln Leu Ala Leu Asn Pro
290 295 300
Asp Ile Gln Asn Lys Leu Ile Ala Glu Val Asp Glu Val Leu Lys Ala
305 310 315 320
Asn Asp Gly Lys Val Thr Tyr Asp Thr Val Lys Glu Met Lys Tyr Leu
325 330 335
Asn Lys Ala Phe Asp Glu Thr Leu Arg Met Tyr Ser Ile Val Glu Pro
340 345 350
Leu Gln Arg Lys Ala Thr Arg Asp Tyr Lys Ile Pro Gly Thr Asp Val
355 360 365
Val Ile Glu Lys Asp Thr Ile Val Leu Ile Ser Pro Arg Gly Ile His
370 375 380
Tyr Asp Pro Lys Tyr Tyr Asp Asn Pro Lys Gln Phe Asn Pro Asp Arg
385 390 395 400
Phe Asp Ala Glu Glu Val Gly Lys Arg His Pro Cys Ala Tyr Leu Pro
405 410 415
Phe Gly Leu Gly Gln Arg Asn Cys Ile Gly Met Arg Phe Gly Arg Leu
420 425 430
Gln Ser Leu Leu Cys Ile Thr Lys Ile Leu Ser Lys Phe Arg Ile Glu
435 440 445
Pro Ser Lys Asn Thr Asp Arg Asn Leu Gln Val Glu Pro His Arg Gly
450 455 460
Leu Ile Gly Pro Lys Gly Gly Ile Arg Val Asn Ala Ile Pro Arg Lys
465 470 475 480
Leu Val Ser
<210> 12
<211> 415
<212> PRT
<213> Artificial sequence
<220>
<223> Chitin synthase
<400> 12
Asn Cys Tyr Phe His Gly Thr Val Pro Asp Tyr Leu Tyr Phe Glu Ser
1 5 10 15
Pro Pro Val Phe Thr Leu Ser Asp Phe Ile Ser Arg Gln Met Ala Trp
20 25 30
Ile Cys Arg Thr Phe Gly Leu Asn Glu Lys Leu Phe Val Met Pro Met
35 40 45
Tyr Asn Gly Leu Leu Ile Asp Gln Ser Met Ala Leu Asn Arg Lys Arg
50 55 60
Asn Asp Gln Arg Asp Val Lys Thr Glu Asp Leu Ala Glu Ile Glu Lys
65 70 75 80
Glu Lys Gly Asp Glu Tyr Tyr Glu Thr Ile Ser Val His Thr Asp Asn
85 90 95
Thr Gly Ser Ser Pro Lys Ala Ile Lys Ser Ser Asp Gln Ile Thr Arg
100 105 110
Ile Tyr Ala Cys Ala Thr Met Trp His Glu Thr Lys Asp Glu Met Met
115 120 125
Glu Phe Leu Lys Ser Ile Leu Arg Leu Asp Glu Asp Gln Cys Ala Arg
130 135 140
Arg Val Ala Gln Lys Tyr Leu Arg Val Val Asp Pro Asp Tyr Tyr Glu
145 150 155 160
Phe Glu Thr His Ile Phe Leu Asp Asp Ala Phe Glu Ile Ser Asp His
165 170 175
Ser Asp Asp Asp Ser Gln Val Asn Arg Phe Val Lys Leu Leu Val Asp
180 185 190
Thr Ile Asp Glu Ala Ala Ser Glu Val His Gln Thr Asn Ile Arg Asp
195 200 205
Val His Val Leu Pro Ser Arg Ser Ser Phe Asn Gly Thr Ala Asp Ile
210 215 220
Leu Asp Arg Lys Glu Val Met Ala Glu Asn Thr Tyr Leu Leu Thr Leu
225 230 235 240
Asp Gly Asp Ile Asp Phe Gln Pro His Ala Val Arg Leu Leu Ile Asp
245 250 255
Leu Met Lys Lys Asn Lys Asn Leu Gly Ala Ala Cys Gly Arg Ile His
260 265 270
Pro Val Gly Ser Gly Pro Met Val Trp Tyr Gln Met Phe Glu Tyr Ala
275 280 285
Ile Gly His Trp Leu Gln Lys Ala Thr Glu His Met Ile Gly Cys Val
290 295 300
Leu Cys Ser Pro Gly Cys Phe Ser Leu Phe Arg Gly Lys Ala Leu Met
305 310 315 320
Asp Asp Asn Val Met Lys Lys Tyr Thr Leu Arg Ser Asp Glu Ala Arg
325 330 335
His Tyr Val His Thr Ile Arg Gly Arg Ser Met Val Met Tyr Ala Ile
340 345 350
Thr Ala Thr Trp Leu Ser Cys Arg Ile Leu Ser Cys Leu Arg Cys Tyr
355 360 365
Thr His Cys Pro Glu Gly Phe Asn Glu Phe Tyr Asn Arg Arg Arg Trp
370 375 380
Val Pro Ser Thr Ile Ala Asn Ile Met Asp Leu Leu Ala Asp Cys Lys
385 390 395 400
His Thr Ile Lys Ile Asn Asp Asn Ile Ser Ser Pro Tyr Ile Ala
405 410 415
<210> 13
<211> 240
<212> PRT
<213> Artificial sequence
<220>
<223> 13 NPC1-sterol transporter
<400> 13
Asp Ser Phe Gln Thr Lys Tyr Phe Gln Tyr Leu Asn Arg Tyr Leu Asn
1 5 10 15
Ile Gly Pro Pro Val Tyr Phe Val Val Thr Glu Gly Leu Asn Tyr Ser
20 25 30
Asp Met Asp Thr Gln Asn Met Ile Cys Gly Thr Arg Phe Cys Arg Pro
35 40 45
Asp Ser Leu Ser Met Gln Leu Tyr Ala Ala Tyr Arg Asn Pro Asn Glu
50 55 60
Thr Tyr Ile Ala Gln Pro Pro Asn Ser Trp Leu Asp Asp Tyr Phe Asp
65 70 75 80
Trp Ser Ala Leu Pro Asn Cys Cys Lys Tyr Phe Pro Ser Asn Ser Ser
85 90 95
Phe Cys Pro Asn Asp Arg Gly Ala Pro Cys Lys Ala Cys Gly Ile Ala
100 105 110
Leu Glu Gly Asp Glu Gln Arg Pro Asn Ser Thr Glu Phe Glu Arg Tyr
115 120 125
Val Pro Phe Phe Leu Gln Asp Ile Pro Asp Thr Ser Gly Ser Gly Cys
130 135 140
Val Lys Gly Gly His Ala Ala Tyr Gly Gln Ala Val Asn Tyr Lys Met
145 150 155 160
Phe Asn Lys Thr Gln Ala His Val Gly Ala Thr Tyr Tyr Gln Gly Tyr
165 170 175
His Thr Val Leu Lys Thr Ser Leu Asp Tyr Tyr Ser Ala Leu Lys Gly
180 185 190
Ala Arg Glu Val Ala Ala Asn Leu Thr Glu Thr Leu Asn Arg Asn Leu
195 200 205
Lys His Gln Leu Asn Gly Thr Thr Ile Asn Val Phe Pro Tyr Ser Val
210 215 220
Phe Tyr Val Phe Tyr Glu Gln Tyr Leu Thr Met Trp Pro Asp Thr Leu
225 230 235 240
<210> 14
<211> 144
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 14
Met Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly
1 5 10 15
Asp Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Ser Phe Ser Ile
20 25 30
Tyr Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Asn Glu Arg Glu Phe
35 40 45
Val Thr Ala Ile Ser Pro Ser Gly Gly Ser Thr His Tyr Ala Asp Phe
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr Met
65 70 75 80
Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Phe
85 90 95
Cys Ala Ser Thr Ser Ser Gln His Tyr Glu Asp Thr Glu Glu Ser Tyr
100 105 110
Lys Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala
115 120 125
Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 15
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 15
Gly Arg Ser Phe Ser Ile Tyr Thr
1 5
<210> 16
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 16
Ile Ser Pro Ser Gly Gly Ser Thr
1 5
<210> 17
<211> 17
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 17
Ala Ser Thr Ser Ser Gln His Tyr Glu Asp Thr Glu Glu Ser Tyr Lys
1 5 10 15
Tyr
<210> 18
<211> 140
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 18
Met Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Glu Ala Ala
1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Ala Ala Leu Gly Ser Asn Leu Arg Ile
20 25 30
Asn Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu
35 40 45
Val Ala Thr Ile Thr Asn Gly Gly Arg Lys Asn Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Phe Arg Gly Asn Ala Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn
85 90 95
Ala Gly Leu Leu Asp Pro Pro Tyr Ser Ala Pro Gly Asp Tyr Trp Gly
100 105 110
Glu Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp
115 120 125
Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 19
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 19
Leu Gly Ser Asn Leu Arg Ile Asn Thr
1 5
<210> 20
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 20
Thr Ile Thr Asn Gly Gly Arg Lys Asn Tyr
1 5 10
<210> 21
<211> 15
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 21
Asn Ala Gly Leu Leu Asp Pro Pro Tyr Ser Ala Pro Gly Asp Tyr
1 5 10 15
<210> 22
<211> 144
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 22
Met Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly
1 5 10 15
Gly Ser Leu Thr Leu Ser Cys Ala Ala Ser Gly Arg Ser Phe Ser Thr
20 25 30
Tyr Ala Met Gly Trp Ser Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe
35 40 45
Leu Ala Gly Ile Ser Arg Gly Gly Gly Thr Thr Val Tyr Ala Asp Ser
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Thr Val
65 70 75 80
Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Ala Ala Val Tyr Tyr
85 90 95
Cys Ala Ala Ala Leu Arg Pro Phe Asp Gly Ser Gly Glu Arg Arg Tyr
100 105 110
Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala
115 120 125
Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 23
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 23
Gly Arg Ser Phe Ser Thr Tyr Ala
1 5
<210> 24
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 24
Gly Ile Ser Arg Gly Gly Gly Thr Thr
1 5
<210> 25
<211> 16
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 25
Ala Ala Leu Arg Pro Phe Asp Gly Ser Gly Glu Arg Arg Tyr Asp Tyr
1 5 10 15
<210> 26
<211> 143
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 26
Met Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly
1 5 10 15
Asp Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Ser Phe Ser Ile
20 25 30
Tyr Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe
35 40 45
Val Ala Ala Ile Ser Pro Ser Gly Val Ser Thr Asp Tyr Ala Asp Ser
50 55 60
Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Met
65 70 75 80
Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr
85 90 95
Cys Ala Ala Gly Gly Arg His Tyr Thr Arg His Pro Tyr Asp Tyr Asp
100 105 110
Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr
115 120 125
Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 27
<400> 27
000
<210> 28
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 28
Ala Ile Ser Pro Ser Gly Val Ser Thr Asp
1 5 10
<210> 29
<211> 16
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 29
Ala Ala Gly Gly Arg His Tyr Thr Arg His Pro Tyr Asp Tyr Asp Tyr
1 5 10 15
<210> 30
<211> 143
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 30
Met Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Thr Gly
1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Ser Phe Ser Ser
20 25 30
Tyr Ser Met Gly Trp Phe Arg Gln Gly Pro Gly Lys Gly Arg Glu Trp
35 40 45
Val Ala Asp Ile Asn Glu Ser Gly Ser Ser Thr Ser Tyr Tyr Asp Pro
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val
65 70 75 80
Tyr Leu Gln Met Asn Asp Leu Lys Pro Glu Asp Thr Ala Asp Tyr Tyr
85 90 95
Cys Ala Ala Leu Val Thr Gly Gly Asp Thr Asp Leu Gly Glu Trp Asp
100 105 110
Phe Trp Gly Pro Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr
115 120 125
Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 31
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 31
Gly Arg Ser Phe Ser Ser Tyr Ser
1 5
<210> 32
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 32
Ile Asn Glu Ser Gly Ser Ser Thr
1 5
<210> 33
<211> 16
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 33
Ala Ala Leu Val Thr Gly Gly Asp Thr Asp Leu Gly Glu Trp Asp Phe
1 5 10 15
<210> 34
<211> 144
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 34
Met Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly
1 5 10 15
Asp Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Gly Thr Phe Ser Ser
20 25 30
Tyr Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe
35 40 45
Val Ala Arg Ile Gly Val Ser Glu Gly Tyr Leu Tyr Tyr Ala Asp Ser
50 55 60
Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Ser Ala Lys Asn Thr Gly
65 70 75 80
Tyr Leu Gln Met Asn Ala Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr
85 90 95
Cys Ala Ala Gly Pro Arg Arg Tyr Trp Thr Arg Glu Pro Asp Ala Tyr
100 105 110
Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala
115 120 125
Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 35
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 35
Gly Gly Thr Phe Ser Ser Tyr Val
1 5
<210> 36
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 36
Ile Gly Val Ser Glu Gly Tyr Leu
1 5
<210> 37
<211> 17
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 37
Ala Ala Gly Pro Arg Arg Tyr Trp Thr Arg Glu Pro Asp Ala Tyr Asp
1 5 10 15
Tyr
<210> 38
<211> 145
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 38
Met Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly
1 5 10 15
Gly Ser Leu Lys Leu Ser Cys Gly Ala Ser Gly Arg Thr Phe Ser Thr
20 25 30
Asn Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe
35 40 45
Val Ala Thr Ile Ser Ala Gly Gly Ser Leu Thr Tyr Tyr Ala Asp Ser
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val
65 70 75 80
Tyr Leu Arg Met Asn Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr
85 90 95
Cys Ala Ala Asp Gln Asp Ser Gly Arg Leu Pro Leu Ile Asn Ser Gly
100 105 110
Tyr Glu Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala
115 120 125
Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His
130 135 140
His
145
<210> 39
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 39
Gly Arg Thr Phe Ser Thr Asn Ala
1 5
<210> 40
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 40
Ile Ser Ala Gly Gly Ser Leu Thr
1 5
<210> 41
<211> 18
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 41
Ala Ala Asp Gln Asp Ser Gly Arg Leu Pro Leu Ile Asn Ser Gly Tyr
1 5 10 15
Glu Tyr
<210> 42
<211> 145
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 42
Met Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly
1 5 10 15
Ala Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser
20 25 30
Tyr Gly Thr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg
35 40 45
Glu Phe Val Ala Ala Ile Leu Trp Thr Gly Ser Ser Ser Tyr Asp Ser
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val
65 70 75 80
Tyr Leu Gln Met Ile Ser Leu Asn Pro Glu Asp Thr Ala Val Tyr Tyr
85 90 95
Cys Ala Ala Arg Ser Arg Tyr Thr Gly Ser Tyr Tyr Glu Glu Ser Thr
100 105 110
Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala
115 120 125
Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His
130 135 140
His
145
<210> 43
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 43
Gly Arg Thr Phe Ser Ser Tyr Gly Thr Ala
1 5 10
<210> 44
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 44
Ile Leu Trp Thr Gly Ser Ser Ser
1 5
<210> 45
<211> 18
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 45
Ala Ala Arg Ser Arg Tyr Thr Gly Ser Tyr Tyr Glu Glu Ser Thr Tyr
1 5 10 15
Asn Tyr
<210> 46
<211> 147
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 46
Met Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly
1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Asp Phe Ser Asn
20 25 30
Tyr Asn Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Asp Val
35 40 45
Val Ala Thr Ile Arg Arg Ser Gly Asp Ile Thr Ser Tyr Thr Asp Ser
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val
65 70 75 80
Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ala Tyr Tyr
85 90 95
Cys Ala Ala Arg Thr Gly Ser Phe Leu Thr Val Leu Ile Thr Thr Pro
100 105 110
Gly Asn Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His
130 135 140
His His His
145
<210> 47
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 47
Gly Arg Asp Phe Ser Asn Tyr Asn
1 5
<210> 48
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 48
Ile Arg Arg Ser Gly Asp Ile Thr
1 5
<210> 49
<211> 20
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 49
Ala Ala Arg Thr Gly Ser Phe Leu Thr Val Leu Ile Thr Thr Pro Gly
1 5 10 15
Asn Tyr Asn Tyr
20
<210> 50
<211> 140
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 50
Met Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly
1 5 10 15
Asp Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ala
20 25 30
Phe Arg Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Ala Leu
35 40 45
Val Ala Asp Ile Ser Arg Leu Ser Thr Arg Thr Tyr Tyr Ala Asp Ser
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val
65 70 75 80
Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr
85 90 95
Cys Ala Ala Asp Leu Glu Gly Val Gly Pro Met Trp Glu Tyr Trp Val
100 105 110
Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp
115 120 125
Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 51
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 51
Gly Arg Thr Phe Ser Ala Phe Arg
1 5
<210> 52
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 52
Ile Ser Arg Leu Ser Thr Arg Thr
1 5
<210> 53
<211> 13
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 53
Ala Ala Asp Leu Glu Gly Val Gly Pro Met Trp Glu Tyr
1 5 10
<210> 54
<211> 144
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 54
Met Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly
1 5 10 15
Asn Ser Leu Arg Leu Ser Cys Thr Tyr Ser Gly Arg Thr Phe Ser Thr
20 25 30
Arg Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu
35 40 45
Val Ala Gly Ile Gly Trp Asn Gly Ala Thr Gln Tyr Tyr Ala Asp Ser
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Ser Ala Ser Asn Thr Val
65 70 75 80
Ala Leu Gln Met Asn Ser Leu Glu Pro Glu Asp Thr Ala Val Tyr Tyr
85 90 95
Cys Ala Ala His Gly Arg Glu Tyr Val Thr Pro Ser Tyr Asn Asn Tyr
100 105 110
Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala
115 120 125
Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 55
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 55
Gly Arg Thr Phe Ser Thr Arg Ala
1 5
<210> 56
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 56
Ile Gly Trp Asn Gly Ala Thr Gln
1 5
<210> 57
<211> 17
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 57
Ala Ala His Gly Arg Glu Tyr Val Thr Pro Ser Tyr Asn Asn Tyr Asp
1 5 10 15
Tyr
<210> 58
<211> 143
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 58
Met Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Thr Gln Ala Gly
1 5 10 15
Asp Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Ser Phe Val Arg
20 25 30
Tyr Thr Thr Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe
35 40 45
Val Ala Ser Ile Ser Trp Ser Arg Gly Ser Thr Tyr Tyr Ala Asp Ser
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Lys Asp Asn Ala Glu Asn Thr Val
65 70 75 80
Tyr Leu Gln Met Asn Ser Pro Glu Pro Glu Asp Thr Ala Val Tyr Tyr
85 90 95
Cys Ala Gly Asn Ser Arg Gly Ala Thr Thr Phe Ala Gln Tyr Tyr Asp
100 105 110
Asp Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr
115 120 125
Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 59
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 59
Gly Arg Ser Phe Val Arg Tyr Thr
1 5
<210> 60
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 60
Ile Ser Trp Ser Arg Gly Ser Thr
1 5
<210> 61
<211> 16
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 61
Ala Gly Asn Ser Arg Gly Ala Thr Thr Phe Ala Gln Tyr Tyr Asp Asp
1 5 10 15
<210> 62
<211> 137
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 62
Met Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly
1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Leu
20 25 30
Thr Arg Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Ile
35 40 45
Val Ala His Ile Met Arg Ser Ser Asp Ser Thr Phe Tyr Gly Asp Ser
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val
65 70 75 80
Tyr Leu Gln Met Asn Arg Leu Asn Pro Glu Asp Thr Ala Val Tyr Tyr
85 90 95
Cys Ala Ala Ala Gln Trp Ala Gly Tyr Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp
115 120 125
Tyr Gly Ser His His His His His His
130 135
<210> 63
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 63
Gly Arg Thr Phe Ser Leu Thr Arg
1 5
<210> 64
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 64
Ile Met Arg Ser Ser Asp Ser Thr
1 5
<210> 65
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 65
Ala Ala Ala Gln Trp Ala Gly Tyr Asp Tyr
1 5 10
<210> 66
<211> 143
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 66
Met Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Pro Ala Gly
1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Thr Ser
20 25 30
Ser Thr Met Ala Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Phe
35 40 45
Val Ala Ala Ile Ser Pro Arg Gly Leu Ser Gln Asp Tyr Gly His Ser
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Glu Asn Thr Val
65 70 75 80
Tyr Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Leu Tyr Tyr
85 90 95
Cys Ala Ala Thr Ser Gly Ser Tyr Ser Ser Ser Arg Asn Asp Tyr Tyr
100 105 110
Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr
115 120 125
Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 67
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 67
Gly Arg Thr Phe Thr Ser Ser Thr
1 5
<210> 68
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 68
Ile Ser Pro Arg Gly Leu Ser Gln
1 5
<210> 69
<211> 16
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 69
Ala Ala Thr Ser Gly Ser Tyr Ser Ser Ser Arg Asn Asp Tyr Tyr Tyr
1 5 10 15
<210> 70
<211> 137
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 70
Met Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Ser Asn
20 25 30
Tyr Arg Met Ala Trp Phe Arg Gln Gly Leu Gly Lys Glu Arg Glu Phe
35 40 45
Val Ala His Ile Met Arg Asn Ser Asp Thr Thr Trp Tyr Thr Glu Ser
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val
65 70 75 80
Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr
85 90 95
Cys Ala Ala Ser Asn Ala Gly Thr Phe Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp
115 120 125
Tyr Gly Ser His His His His His His
130 135
<210> 71
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 71
Gly Phe Thr Leu Ser Asn Tyr Arg
1 5
<210> 72
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 72
Ile Met Arg Asn Ser Asp Thr Thr
1 5
<210> 73
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 73
Ala Ala Ser Asn Ala Gly Thr Phe Asp Tyr
1 5 10
<210> 74
<211> 136
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 74
Met Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Ala Ser Ser Gly Ala Asn Ile Arg Leu
20 25 30
Tyr Gly Met Ala Trp Tyr Arg Gln Pro Pro Gly Glu Glu Arg Glu Leu
35 40 45
Val Ala Ser Ile Thr Val Gly Gly Ser Ile Thr Tyr Ala Glu Ser Val
50 55 60
Lys Ala Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asp Met Val Phe
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Asn Ala Met Asn Pro Trp Tyr Tyr Trp Ala Trp Gly Gln Gly Thr Gln
100 105 110
Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr
115 120 125
Gly Ser His His His His His His
130 135
<210> 75
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 75
Gly Ala Asn Ile Arg Leu Tyr Gly
1 5
<210> 76
<211> 7
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 76
Ile Thr Val Gly Gly Ser Ile
1 5
<210> 77
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 77
Asn Ala Met Asn Pro Trp Tyr Tyr Trp Ala
1 5 10
<210> 78
<211> 141
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 78
Met Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly
1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Leu Ser Ser
20 25 30
Tyr Asp Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Asp Phe
35 40 45
Val Ala Gly Ile Asp Trp Ser Gly Gly Ser Thr Asn Tyr Glu Arg Ser
50 55 60
Val Lys Gly Arg Phe Thr Ile Thr Arg Asp Asn Ala Lys Asn Thr Val
65 70 75 80
Leu Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr
85 90 95
Cys Ala Ala Ala Arg Ala Asn Ser Asp Leu Gly Ile Tyr Asp Tyr Trp
100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr
115 120 125
Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 79
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 79
Gly Gly Thr Leu Ser Ser Tyr Asp
1 5
<210> 80
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 80
Ile Asp Trp Ser Gly Gly Ser Thr
1 5
<210> 81
<211> 14
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 81
Ala Ala Ala Arg Ala Asn Ser Asp Leu Gly Ile Tyr Asp Tyr
1 5 10
<210> 82
<211> 138
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 82
Met Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Glu Ala Gly
1 5 10 15
Gly Ser Leu Gly Leu Ala Cys Thr Thr Ser Gly Ile Ile Phe Ser Arg
20 25 30
Asn Asp Met Gly Trp Phe Arg Gln Ala Pro Gly Glu Gln Arg Thr Ala
35 40 45
Val Ala Thr Ile Thr Arg Ser Ser Ser Thr Asn Tyr Ala Gly Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Glu Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Ser Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Ile Pro Thr Ala Thr Gln Pro Tyr Val Tyr Trp Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro
115 120 125
Asp Tyr Gly Ser His His His His His His
130 135
<210> 83
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 83
Gly Ile Ile Phe Ser Arg Asn Asp
1 5
<210> 84
<211> 7
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 84
Ile Thr Arg Ser Ser Ser Thr
1 5
<210> 85
<211> 12
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 85
Ala Thr Ile Pro Thr Ala Thr Gln Pro Tyr Val Tyr
1 5 10
<210> 86
<211> 144
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 86
Met Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Thr Gly
1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Ser Phe Ser Ser
20 25 30
Tyr Ser Met Gly Trp Phe Arg Gln Gly Pro Gly Lys Gly Arg Glu Trp
35 40 45
Val Ala Asp Ile Asn Glu Ser Gly Thr Thr Thr Asn Tyr Trp Asp Pro
50 55 60
Val Lys Gly Arg Phe Ile Ile Ser Arg Asp Asn Ala Gln Asn Met Val
65 70 75 80
Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr
85 90 95
Cys Ala Ala Gly Pro Arg Thr Arg Trp Thr Arg Glu Pro Asp Ala Tyr
100 105 110
Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala
115 120 125
Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 87
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 87
Gly Arg Ser Phe Ser Ser Tyr Ser
1 5
<210> 88
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 88
Ile Asn Glu Ser Gly Thr Thr Thr
1 5
<210> 89
<211> 17
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 89
Ala Ala Gly Pro Arg Thr Arg Trp Thr Arg Glu Pro Asp Ala Tyr Asp
1 5 10 15
Tyr
<210> 90
<211> 141
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 90
Met Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly
1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Thr Phe Ser Thr
20 25 30
Tyr Asp Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu Phe
35 40 45
Val Ala Gly Ile Asp Trp Ser Gly Gly Ser Thr Asn Tyr Val Asn Phe
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Met
65 70 75 80
Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr
85 90 95
Cys Ala Ala Ala Val Gly Asp Ser Glu Met Ala Thr Tyr Asp Tyr Trp
100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr
115 120 125
Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 91
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 91
Gly Arg Thr Phe Ser Thr Tyr Asp
1 5
<210> 92
<400> 92
000
<210> 93
<211> 14
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 93
Ala Ala Ala Val Gly Asp Ser Glu Met Ala Thr Tyr Asp Tyr
1 5 10
<210> 94
<211> 137
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 94
Met Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Val Gly
1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Val Ala Ser Glu Pro Thr Phe Arg Pro
20 25 30
Asn Arg Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu
35 40 45
Val Ala His Ile Met Trp Ser Ser Gly Ser Thr Trp Tyr Gly Asp Ser
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val
65 70 75 80
Ile Leu Gln Met Asn Ser Leu Asn Pro Glu Asp Thr Ala Val Tyr Tyr
85 90 95
Cys Ala Ala Ala Gln Arg Ala Gly Tyr Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp
115 120 125
Tyr Gly Ser His His His His His His
130 135
<210> 95
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 95
Glu Pro Thr Phe Arg Pro Asn Arg
1 5
<210> 96
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 96
Ile Met Trp Ser Ser Gly Ser Thr Trp
1 5
<210> 97
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 97
Ala Ala Gln Arg Ala Gly Tyr Asp Tyr
1 5
<210> 98
<211> 143
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 98
Met Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly
1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Ser Leu Ser Ser
20 25 30
Tyr Ser Met Gly Trp Ile Arg Gln Ala Pro Gly Lys Glu Arg Asp Phe
35 40 45
Val Thr Ala Ile Arg Trp Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Ile Ala Lys Asn Ala Val
65 70 75 80
Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr
85 90 95
Cys Ala Ala Arg Leu Gly Gly Arg Ser Trp Asp Ala Gly Asp Tyr Gln
100 105 110
Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr
115 120 125
Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 99
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 99
Gly Arg Ser Leu Ser Ser Tyr Ser
1 5
<210> 100
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 100
Ile Arg Trp Ser Gly Gly Ser Thr
1 5
<210> 101
<211> 16
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 101
Ala Ala Arg Leu Gly Gly Arg Ser Trp Asp Ala Gly Asp Tyr Gln Tyr
1 5 10 15
<210> 102
<211> 135
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 102
Met Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ser Gly
1 5 10 15
Gly Ser Leu Phe Leu Ser Cys Ala Ala Ser Gly Phe Val Phe Glu Thr
20 25 30
Ser Pro Met Thr Trp Val Arg Gln Ala Pro Gly Gln Gly Val Arg Trp
35 40 45
Val Gly Ser Ile Thr Thr Asp Gly Arg Arg Ala Asp Tyr Glu Asp Ala
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Met Leu
65 70 75 80
Tyr Leu Glu Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Met Tyr Phe
85 90 95
Cys Arg Glu Ser Arg Asp Leu Asn Ala Val Thr Arg Gly Thr Gln Val
100 105 110
Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly
115 120 125
Ser His His His His His His
130 135
<210> 103
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 103
Gly Phe Val Phe Glu Thr Ser Pro
1 5
<210> 104
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 104
Ile Thr Thr Asp Gly Arg Arg Ala
1 5
<210> 105
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 105
Arg Glu Ser Arg Asp Leu Asn Ala
1 5
<210> 106
<211> 139
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 106
Met Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Thr Gly
1 5 10 15
Asp Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Arg Thr Gly Ser Ile
20 25 30
Asn Arg Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Ile
35 40 45
Val Ser His Ile Phe Trp Ser Asn Val Gly Thr Trp Ser Ala Glu Ser
50 55 60
Val Lys Gly Arg Phe Ile Ile Ser Arg Asp Asn Ala Lys Asn Thr Val
65 70 75 80
Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Ser
85 90 95
Cys Ala Ala Ala Thr Gly Ser Ala Tyr Asn Tyr Trp Val Pro Ser Arg
100 105 110
Gly Asp Pro Gly His Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val
115 120 125
Pro Asp Tyr Gly Ser His His His His His His
130 135
<210> 107
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 107
Gly Arg Thr Gly Ser Ile Asn Arg
1 5
<210> 108
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 108
Ile Phe Trp Ser Asn Val Gly Thr
1 5
<210> 109
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 109
Ala Ala Ala Thr Gly Ser Ala Tyr Asn Tyr
1 5 10
<210> 110
<211> 133
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 110
Met Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Gln Val Gln Ala Gly
1 5 10 15
Asp Ser Leu Arg Leu Ser Cys Val Thr Ser Gly Met Ser Phe Ser Thr
20 25 30
Ser Ala Met Gly Trp Tyr Arg Gln Ala Ser Gly Lys Gln Arg Glu Trp
35 40 45
Val Ala Ile Ile Arg Glu Asp Ser Thr Thr Asn Tyr Ser Ser Phe Ala
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Asn Lys Thr Val Tyr
65 70 75 80
Leu Leu Met Asn Ser Leu Glu Pro Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Arg Thr Tyr Thr Gly Gly Tyr Trp Gly Gln Gly Thr Gln Val Thr Val
100 105 110
Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser His
115 120 125
His His His His His
130
<210> 111
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 111
Gly Met Ser Phe Ser Thr Ser Ala
1 5
<210> 112
<211> 7
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 112
Ile Arg Glu Asp Ser Thr Thr
1 5
<210> 113
<211> 7
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 113
Arg Thr Tyr Thr Gly Gly Tyr
1 5
<210> 114
<211> 145
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 114
Met Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly
1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser
20 25 30
Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe
35 40 45
Val Ala Gly Ile Ser Trp Ser Gly Arg Ser Thr Tyr Tyr Ala Asp Ser
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ala Lys Asn Thr Val
65 70 75 80
Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr
85 90 95
Cys Ala Ala Pro Asp Thr Ala Ala Gln Phe Thr Thr Pro Leu Tyr Glu
100 105 110
Tyr Ala Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala
115 120 125
Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His
130 135 140
His
145
<210> 115
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 115
Gly Arg Thr Phe Ser Ser Tyr Ala
1 5
<210> 116
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 116
Ile Ser Trp Ser Gly Arg Ser Thr
1 5
<210> 117
<211> 16
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 117
Ala Ala Pro Asp Thr Ala Ala Gln Phe Thr Thr Pro Leu Tyr Glu Tyr
1 5 10 15
<210> 118
<211> 143
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 118
Met Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly
1 5 10 15
Asp Ser Leu Arg Leu Ala Cys Ala Ser Ser Ser Arg Thr Phe Ser Thr
20 25 30
Tyr Thr Met Gly Trp Phe Arg Gln Thr Pro Gly Arg Glu Arg Asp Phe
35 40 45
Val Ala Ala Ile Ser Pro Ser Gly Ala Thr Ala Asp Tyr Ala Asp Ser
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu
65 70 75 80
Tyr Leu Gln Met Asp Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr
85 90 95
Cys Ala Ala Arg Tyr Leu Ser Trp Ser Arg Met Asn His Glu Tyr Pro
100 105 110
Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr
115 120 125
Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 119
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 119
Ser Arg Thr Phe Ser Thr Tyr Thr
1 5
<210> 120
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 120
Ile Ser Pro Ser Gly Ala Thr Ala
1 5
<210> 121
<211> 16
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 121
Ala Ala Arg Tyr Leu Ser Trp Ser Arg Met Asn His Glu Tyr Pro Tyr
1 5 10 15
<210> 122
<211> 146
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 122
Met Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly
1 5 10 15
Asp Ser Leu Arg Leu Ser Cys Ala Ala Ser Glu Arg Thr Phe Ser Ser
20 25 30
Tyr Val Met Gly Trp Phe Arg Gln Ala Pro Arg Lys Glu Arg Glu Phe
35 40 45
Val Ala Ala Met Thr Trp Ser Gly Ser Ser Arg Ile Tyr Tyr Ala Asp
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr
65 70 75 80
Ala Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Ala Lys Asp Ala Tyr Gly Gly Ile Ser Phe Arg Pro Asn
100 105 110
Thr Tyr His Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala
115 120 125
Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His
130 135 140
His His
145
<210> 123
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 123
Glu Arg Thr Phe Ser Ser Tyr Val
1 5
<210> 124
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 124
Met Thr Trp Ser Gly Ser Ser Arg Ile
1 5
<210> 125
<211> 18
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 125
Ala Ala Lys Asp Ala Tyr Gly Gly Ile Ser Phe Arg Pro Asn Thr Tyr
1 5 10 15
His Ser
<210> 126
<211> 130
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 126
Met Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Ala Ala Leu Gly Asn Ile Val Asn Ile
20 25 30
Asn Asn Met Gly Trp Tyr Arg Gln Ala Pro Gly Gly Gln Arg Glu Leu
35 40 45
Val Ala Thr Ile Thr Arg Gly Ala Ile Lys Asn Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Phe Arg Gly Asn Ala Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Val
85 90 95
Ala Asp Ser Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala
100 105 110
Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His
115 120 125
His His
130
<210> 127
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 127
Gly Asn Ile Val Asn Ile Asn Asn
1 5
<210> 128
<211> 7
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 128
Ile Thr Arg Gly Ala Ile Lys
1 5
<210> 129
<211> 5
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 129
Val Ala Asp Ser Ser
1 5
<210> 130
<211> 143
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 130
Met Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Ala Gly
1 5 10 15
Gly Phe Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Ile Phe Asp Asp
20 25 30
Thr Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe
35 40 45
Val Ala Ala Val Ser Pro Ser Gly Val Ser Thr Asp Tyr Thr Asp Ser
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Thr Val
65 70 75 80
Phe Leu Gln Met Ser Arg Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr
85 90 95
Cys Ala Ala Arg Leu Arg His Tyr Ser Asn Asp Gln His Glu Tyr Asp
100 105 110
Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr
115 120 125
Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 131
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 131
Gly Leu Ile Phe Asp Asp Thr Ala
1 5
<210> 132
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 132
Val Ser Pro Ser Gly Val Ser Thr
1 5
<210> 133
<211> 16
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 133
Ala Ala Arg Leu Arg His Tyr Ser Asn Asp Gln His Glu Tyr Asp Ser
1 5 10 15
<210> 134
<211> 136
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 134
Met Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly
1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ile Val Phe Ser Ile
20 25 30
Thr Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu
35 40 45
Val Ala Thr Ile Ala Ser Gly Val Arg Ala Asp Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Asn Ala Asn Arg Phe Ser Leu Gly Asn Tyr Trp Gly Gln Gly Thr Gln
100 105 110
Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr
115 120 125
Gly Ser His His His His His His
130 135
<210> 135
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 135
Gly Ile Val Phe Ser Ile Thr Thr
1 5
<210> 136
<211> 7
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 136
Ile Ala Ser Gly Val Arg Ala
1 5
<210> 137
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 137
Asn Ala Asn Arg Phe Ser Leu Gly Asn Tyr
1 5 10
<210> 138
<211> 144
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 138
Met Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Thr Gly
1 5 10 15
Asp Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Phe Ser Ala
20 25 30
Tyr Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe
35 40 45
Val Ala Ala Ile Ser Arg Ser Gly Ser Ser Thr His Tyr Ala Asn Ser
50 55 60
Val Lys Gly His Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Thr Val
65 70 75 80
Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Arg Tyr Tyr
85 90 95
Cys Ala Gly Glu Arg Thr Gly His Phe Thr Asp Leu Tyr Tyr Glu Tyr
100 105 110
Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala
115 120 125
Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 139
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 139
Gly Gly Thr Phe Ser Ala Tyr Thr
1 5
<210> 140
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 140
Ile Ser Arg Ser Gly Ser Ser Thr
1 5
<210> 141
<211> 17
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 141
Ala Gly Glu Arg Thr Gly His Phe Thr Asp Leu Tyr Tyr Glu Tyr Asp
1 5 10 15
Tyr
<210> 142
<211> 140
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 142
Met Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly
1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Ala Ala Pro Gly Ser Asn Phe Arg Ile
20 25 30
Asn Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu
35 40 45
Val Ala Thr Ile Ala Asn Ser Gly Arg Ile Asn Ser Ala Asn Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Phe Arg Gly Asn Ala Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn
85 90 95
Ala Asp Val Val Glu Ser Ser Asn Tyr Gln Thr Leu Asn Tyr Trp Gly
100 105 110
Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp
115 120 125
Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 143
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 143
Gly Ser Asn Phe Arg Ile Asn Thr
1 5
<210> 144
<211> 7
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 144
Ile Ala Asn Ser Gly Arg Ile
1 5
<210> 145
<211> 15
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 145
Asn Ala Asp Val Val Glu Ser Ser Asn Tyr Gln Thr Leu Asn Tyr
1 5 10 15
<210> 146
<211> 143
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 146
Met Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly
1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Val Ala Ser Arg Ala Ser Gly Ser Ile
20 25 30
Phe Gly Ala Gln Thr Met Ala Trp Tyr Arg Gln Ala Ser Gly Glu Arg
35 40 45
Arg Glu Leu Val Ala Thr Ile Thr Ser Ser Gly Ser Thr Asn Tyr Ala
50 55 60
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser
65 70 75 80
Thr Met Phe Leu Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val
85 90 95
Tyr Tyr Cys Asn Val Gly Phe Arg Ser Arg Tyr Ser Tyr Asp Ser Ser
100 105 110
Val Trp Gly Glu Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr
115 120 125
Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 147
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 147
Arg Ala Ser Gly Ser Ile Phe Gly Ala Gln Thr
1 5 10
<210> 148
<211> 7
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 148
Ile Thr Ser Ser Gly Ser Thr
1 5
<210> 149
<211> 14
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 149
Asn Val Gly Phe Arg Ser Arg Tyr Ser Tyr Asp Ser Ser Val
1 5 10
<210> 150
<211> 144
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 150
Met Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly
1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Thr
20 25 30
Ser Asn Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe
35 40 45
Val Gly Ala Ile Ser Pro Ser Gly Arg Ser Thr Asp Tyr Ala Ser Ser
50 55 60
Val Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Pro Lys Asn Thr Met
65 70 75 80
Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr
85 90 95
Cys Ala Ala Arg Arg Ser Pro Ser Tyr Thr Arg Val Gly Asp Glu Tyr
100 105 110
Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala
115 120 125
Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 151
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 151
Gly Arg Thr Phe Ser Thr Ser Asn
1 5
<210> 152
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 152
Ile Ser Pro Ser Gly Arg Ser Thr
1 5
<210> 153
<211> 17
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 153
Ala Ala Arg Arg Ser Pro Ser Tyr Thr Arg Val Gly Asp Glu Tyr Asp
1 5 10 15
Tyr
<210> 154
<211> 148
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 154
Met Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly
1 5 10 15
Asp Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Ser Phe Ser Ile
20 25 30
Tyr Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe
35 40 45
Val Ser Leu Ile Met Arg Ser Gly Gly Ile Ile Tyr Ala Asp Phe Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Leu Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Gly Gly Ser Thr Asn Ser Arg Ala Tyr Asn Tyr Tyr Lys Leu
100 105 110
Ser Leu Ala Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser
115 120 125
Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His
130 135 140
His His His His
145
<210> 155
<400> 155
000
<210> 156
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 156
Ile Met Arg Ser Gly Gly Ile Ile
1 5
<210> 157
<211> 22
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 157
Ala Ala Gly Gly Ser Thr Asn Ser Arg Ala Tyr Asn Tyr Tyr Lys Leu
1 5 10 15
Ser Leu Ala Tyr Asp Tyr
20
<210> 158
<211> 144
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 158
Met Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly
1 5 10 15
Asp Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Phe Ser Thr
20 25 30
Tyr Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe
35 40 45
Val Ala Ala Ile Ser Arg Thr Gly Arg Gly Thr Asp Tyr Ala Asp Ser
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Thr Val
65 70 75 80
Ser Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr
85 90 95
Cys Ala Glu Arg Ser Ser Val His Tyr Ser Gly Ile Ala Ala Asp Tyr
100 105 110
Asp Tyr Trp Ser Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala
115 120 125
Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 159
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 159
Gly Gly Thr Phe Ser Thr Tyr Thr
1 5
<210> 160
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 160
Ile Ser Arg Thr Gly Arg Gly Thr
1 5
<210> 161
<211> 17
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 161
Ala Glu Arg Ser Ser Val His Tyr Ser Gly Ile Ala Ala Asp Tyr Asp
1 5 10 15
Tyr
<210> 162
<211> 144
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 162
Met Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
1 5 10 15
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Asn Leu Ser Arg
20 25 30
Ser Ala Met Gly Trp Phe Arg Gln Asn Pro Gly Glu Glu Arg Glu Phe
35 40 45
Val Ala Ala Ile Asp Trp Trp Gly Asp Ser Thr Tyr Tyr Gly Asp Ser
50 55 60
Val Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu
65 70 75 80
Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr
85 90 95
Cys Ala Ala Gly Leu Arg Pro Phe Asp Gly Ser Trp Glu Arg Arg Tyr
100 105 110
Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala
115 120 125
Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 163
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 163
Gly Arg Asn Leu Ser Arg Ser Ala
1 5
<210> 164
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 164
Ile Asp Trp Trp Gly Asp Ser Thr
1 5
<210> 165
<211> 17
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 165
Ala Ala Gly Leu Arg Pro Phe Asp Gly Ser Trp Glu Arg Arg Tyr Asp
1 5 10 15
Tyr
<210> 166
<211> 135
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 166
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Ser Gly Tyr
20 25 30
Ile Met Ser Trp Phe Arg Gln Pro Pro Gly Lys Glu Arg Glu Leu Val
35 40 45
Ala Ala Ile Thr Tyr Val Gly Ser Thr Tyr Val Glu Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Ser Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Arg
85 90 95
Ala Arg Glu Gly Glu Gln Leu Asp Phe Trp Gly Gln Gly Thr Gln Val
100 105 110
Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly
115 120 125
Ser His His His His His His
130 135
<210> 167
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 167
Gly Ser Thr Phe Ser Gly Tyr Ile
1 5
<210> 168
<211> 7
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 168
Ile Thr Tyr Val Gly Ser Thr
1 5
<210> 169
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 169
Arg Ala Arg Glu Gly Glu Gln Leu Asp Phe
1 5 10
<210> 170
<211> 135
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 170
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Leu Ser Gly Tyr
20 25 30
Ile Met Ser Trp Phe Arg Gln Pro Pro Gly Lys Glu Arg Glu Leu Val
35 40 45
Ala Ala Ile Thr Tyr Val Gly Ser Thr Tyr Val Glu Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Ser Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Arg
85 90 95
Ala Arg Glu Gly Glu Gln Leu Asp Phe Trp Gly Gln Gly Thr Gln Val
100 105 110
Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly
115 120 125
Ser His His His His His His
130 135
<210> 171
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 171
Ser Gly Ser Thr Phe Ser Gly Tyr
1 5
<210> 172
<211> 135
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 172
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ser Gly Asp
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Ser Gly Tyr
20 25 30
Ile Met Ser Trp Phe Arg Gln Pro Pro Gly Lys Glu Arg Glu Leu Val
35 40 45
Ala Ala Ile Thr Tyr Val Gly Ser Thr Tyr Val Glu Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Ser Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Arg
85 90 95
Ala Arg Glu Gly Glu Gln Leu Asp Phe Trp Gly Gln Gly Thr Gln Val
100 105 110
Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly
115 120 125
Ser His His His His His His
130 135
<210> 173
<211> 135
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 173
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Ser Thr Phe Ser Gly Tyr
20 25 30
Ile Met Ser Trp Phe Arg Gln Pro Pro Gly Lys Glu Arg Glu Leu Val
35 40 45
Ala Ala Ile Thr Tyr Val Gly Ser Thr Tyr Val Glu Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Ser Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Arg
85 90 95
Ala Arg Glu Gly Glu Gln Leu Asp Phe Trp Gly Gln Gly Thr Gln Val
100 105 110
Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly
115 120 125
Ser His His His His His His
130 135
<210> 174
<211> 12
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 174
Ala Ser Gly Ser Thr Phe Ser Gly Tyr Ile Met Ser
1 5 10
<210> 175
<211> 135
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 175
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Ser Gly Tyr
20 25 30
Ile Met Ser Trp Phe Arg Gln Pro Pro Gly Lys Glu Arg Glu Leu Val
35 40 45
Ala Ala Ile Thr Tyr Val Gly Ser Thr Tyr Val Glu Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Ser Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Arg
85 90 95
Ala Arg Glu Gly Glu Gln Leu Asp Phe Trp Gly Gln Gly Thr Gln Val
100 105 110
Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly
115 120 125
Ser His His His His His His
130 135
<210> 176
<211> 135
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 176
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Thr Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Ser Gly Tyr
20 25 30
Ile Met Ser Trp Phe Arg Gln Pro Pro Gly Lys Glu Arg Glu Leu Val
35 40 45
Ala Ala Ile Thr Tyr Val Gly Ser Thr Tyr Val Glu Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Ser Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Arg
85 90 95
Ala Arg Glu Gly Glu Gln Leu Asp Phe Trp Gly Gln Gly Thr Gln Val
100 105 110
Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly
115 120 125
Ser His His His His His His
130 135
<210> 177
<211> 135
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 177
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Ser Gly Tyr
20 25 30
Ile Met Ser Trp Phe Arg Gln Pro Pro Gly Lys Glu Arg Glu Leu Val
35 40 45
Ala Ala Ile Thr Tyr Val Gly Ser Thr Trp Tyr Gln Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Ser Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Arg
85 90 95
Ala Arg Glu Gly Glu Gln Leu Asp Phe Trp Gly Gln Gly Thr Gln Val
100 105 110
Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly
115 120 125
Ser His His His His His His
130 135
<210> 178
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 178
Ala Ile Thr Tyr Val Gly Ser Thr Trp Tyr Gln
1 5 10
<210> 179
<211> 142
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 179
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Thr Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Asn Leu Asn Tyr Ile Asn
20 25 30
Val Trp Ser Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Arg Val
35 40 45
Ala Gly Ile Ala Thr Gly Gly Gly Arg Ile Ser Tyr Ser Glu Ser Val
50 55 60
Lys Gly Arg Phe Ile Ile Ser Arg Asp Asp Ala Thr Asn Thr Val Ser
65 70 75 80
Leu Gln Met Ser Gly Leu Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Asn Ala Phe Gly Ser Asp Pro Asp Phe Ser Asp Tyr Lys His Asp Tyr
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro
115 120 125
Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 180
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 180
Gly Asn Leu Asn Tyr Ile Asn Val
1 5
<210> 181
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 181
Ile Ala Thr Gly Gly Gly Arg Ile
1 5
<210> 182
<211> 16
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 182
Asn Ala Phe Gly Ser Asp Pro Asp Phe Ser Asp Tyr Lys His Asp Tyr
1 5 10 15
<210> 183
<211> 135
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 183
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Ser Gly Tyr
20 25 30
Ile Met Ser Trp Phe Arg Gln Pro Pro Gly Lys Glu Arg Glu Leu Val
35 40 45
Ala Ala Ile Thr Tyr Val Gly Ser Thr Tyr Val Glu Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Ser Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Arg
85 90 95
Ala Arg Glu Gly Glu Gln Leu Asp Phe Trp Gly Gln Gly Thr Gln Val
100 105 110
Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly
115 120 125
Ser His His His His His His
130 135
<210> 184
<400> 184
000
<210> 185
<211> 135
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 185
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Thr Phe Ser Gly Tyr
20 25 30
Ile Met Ser Trp Phe Arg Gln Pro Pro Gly Lys Glu Arg Glu Leu Val
35 40 45
Ala Ala Ile Thr Tyr Val Gly Ser Thr Tyr Val Glu Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Thr Val Ser Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Arg
85 90 95
Ala Arg Glu Gly Glu Gln Leu Asp Phe Trp Gly Gln Gly Thr Gln Val
100 105 110
Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly
115 120 125
Ser His His His His His His
130 135
<210> 186
<211> 136
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 186
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Thr Ile Phe Ser Ala Lys
20 25 30
Ala Leu Gly Trp His Arg Gln Ala Pro Gly Lys Gln Arg Glu Val Val
35 40 45
Ala Gly Ile Thr Ser Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Val Lys Phe Glu Asp Thr Ala Val Tyr Tyr Cys Val
85 90 95
Leu Tyr Asp Leu Ile Lys Asp Arg Thr Tyr Trp Gly Gln Gly Thr Gln
100 105 110
Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr
115 120 125
Gly Ser His His His His His His
130 135
<210> 187
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 187
Gly Thr Ile Phe Ser Ala Lys Ala
1 5
<210> 188
<211> 7
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 188
Ile Thr Ser Gly Gly Ser Thr
1 5
<210> 189
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 189
Val Leu Tyr Asp Leu Ile Lys Asp Arg Thr Tyr
1 5 10
<210> 190
<211> 137
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 190
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Thr Ser Thr Ser Ile Phe Ser Ile Asn
20 25 30
Val Met Asp Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val
35 40 45
Ala Gly Ile Thr Ser Gly Asp Asn Thr Asn Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Asn Asn Thr Val Trp Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Arg
85 90 95
Gly Arg Val Tyr Asn Gly Gly Trp Tyr Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp
115 120 125
Tyr Gly Ser His His His His His His
130 135
<210> 191
<211> 7
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 191
Thr Ser Ile Phe Ser Gly Thr
1 5
<210> 192
<211> 7
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 192
Ile Thr Ser Gly Asp Asn Thr
1 5
<210> 193
<211> 12
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 193
Arg Gly Arg Val Tyr Asn Gly Gly Trp Tyr Asp Tyr
1 5 10
<210> 194
<211> 141
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 194
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ala Cys Ala Ala Ser Glu Arg Thr Phe Ser Ser Tyr
20 25 30
Val Met Gly Trp Phe Arg Gln Gly Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Ile Ser Trp Ser Gly Gly Ala Arg Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Ser Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Val Cys
85 90 95
Ala Ala Arg Arg Thr Tyr Ser Pro Arg Thr Leu Glu Tyr Asp Phe Trp
100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr
115 120 125
Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 195
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 195
Glu Arg Thr Phe Ser Ser Tyr Val
1 5
<210> 196
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 196
Ile Ser Trp Ser Gly Gly Ala Arg
1 5
<210> 197
<211> 15
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 197
Ala Ala Arg Arg Thr Tyr Ser Pro Arg Thr Leu Glu Tyr Asp Phe
1 5 10 15
<210> 198
<211> 141
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 198
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Thr Phe Ser Thr Tyr
20 25 30
Val Met Gly Trp Phe Arg Gln Gly Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Ile Ser Trp Ser Gly Gly Ala Arg Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Ser Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Val Cys
85 90 95
Ala Ala Arg Arg Thr Tyr Ser Pro Arg Thr Leu Glu Tyr Asp Phe Trp
100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr
115 120 125
Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 199
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 199
Gly Asp Thr Phe Ser Thr Tyr Val
1 5
<210> 200
<211> 140
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 200
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr
20 25 30
Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Phe Glu Trp Val
35 40 45
Ser Ile Ile Asn Thr Asp Gly Ile Gly Ser Arg Tyr Ala Asp Ser Val
50 55 60
Met Gly Arg Phe Thr Ile Ser Arg Asp Asn Glu Lys Lys Met Met Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Arg Gly Asn Ala Ala Leu Thr Val Ile Arg Gly Arg Pro Trp Gly
100 105 110
Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp
115 120 125
Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 201
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 201
Gly Phe Thr Phe Ser Asp Tyr Val
1 5
<210> 202
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 202
Ile Asn Thr Asp Gly Ile Gly Ser
1 5
<210> 203
<211> 14
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 203
Ala Arg Gly Asn Ala Ala Leu Thr Val Ile Arg Gly Arg Pro
1 5 10
<210> 204
<211> 140
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 204
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr
20 25 30
Val Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Phe Glu Trp Val
35 40 45
Ser Ile Ile Asn Thr Asp Gly Asn Gly Ser Arg Tyr Ala Asp Ser Val
50 55 60
Met Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Lys Met Met Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Met Tyr Tyr Cys
85 90 95
Ala Lys Gly Asn Ala Ala Leu Ser Leu Ile Arg Gly Arg Pro Trp Gly
100 105 110
Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp
115 120 125
Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 205
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 205
Ile Asn Thr Asp Gly Asn Gly Ser
1 5
<210> 206
<211> 14
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 206
Ala Lys Gly Asn Ala Ala Leu Ser Leu Ile Arg Gly Arg Pro
1 5 10
<210> 207
<211> 147
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 207
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Thr Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala His Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Ile Ser Trp Gly Arg Gly Asn Thr Tyr Tyr Gly Asp Ser Leu
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asp Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Asp Pro Gly Arg Ala Tyr Val Ser Gly Asn Tyr Tyr Ser Ala
100 105 110
Ala Thr Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His
130 135 140
His His His
145
<210> 208
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 208
Gly Arg Thr Phe Ser Thr Tyr Ala
1 5
<210> 209
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 209
Ile Ser Trp Gly Arg Gly Asn Thr
1 5
<210> 210
<211> 21
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 210
Ala Ala Asp Pro Gly Arg Ala Tyr Val Ser Gly Asn Tyr Tyr Ser Ala
1 5 10 15
Ala Thr Tyr Asp Tyr
20
<210> 211
<211> 147
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 211
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr
20 25 30
Ala Met Gly Trp Phe Arg Arg Phe Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Ile Ser Trp Gly Arg Gly Asn Thr Tyr Tyr Gly Asp Ser Leu
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asp Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Asp Pro Gly Arg Ala Tyr Val Ser Gly Asn Tyr Tyr Ser Ala
100 105 110
Ala Thr Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His
130 135 140
His His His
145
<210> 212
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 212
Gly Arg Thr Phe Ser Ser Tyr Ala
1 5
<210> 213
<211> 147
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 213
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Thr Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala His Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Ile Ser Trp Gly Arg Gly Asn Thr Tyr Tyr Gly Asp Ser Leu
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asp Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Asp Pro Gly Arg Ala Tyr Val Ser Gly Asn Tyr Tyr Ser Ala
100 105 110
Ala Thr Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His
130 135 140
His His His
145
<210> 214
<211> 145
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 214
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Ser Phe Ser Thr Ile
20 25 30
Pro Met Gly Trp Phe Arg Gln Ala Pro Glu Met Glu Arg Gly Phe Val
35 40 45
Ala Ala Ile Ser Tyr Arg Gly Thr Tyr Thr Tyr Tyr Thr Asn Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Gly Ser Pro Gly Ile Ser Ala Tyr Trp Gly Asp Leu Ser Asn
100 105 110
Trp Lys Asn Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala
115 120 125
Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His
130 135 140
His
145
<210> 215
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 215
Gly Gly Ser Phe Ser Thr Ile Pro
1 5
<210> 216
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 216
Ile Ser Tyr Arg Gly Thr Tyr Thr
1 5
<210> 217
<211> 19
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 217
Ala Ala Gly Ser Pro Gly Ile Ser Ala Tyr Trp Gly Asp Leu Ser Asn
1 5 10 15
Trp Lys Asn
<210> 218
<211> 143
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 218
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Ser Phe Ser Ser His
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Thr Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Thr Ile Ser Trp Asn Ser Gly Ser Thr Phe Tyr Ala Asp Ser Leu
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Thr Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Ser Gly Arg Gly Ile Thr Ala Ser Ala Phe Arg Tyr Asp
100 105 110
Val Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr
115 120 125
Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 219
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 219
Gly Arg Ser Phe Ser Ser His Ala
1 5
<210> 220
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 220
Ile Ser Trp Asn Ser Gly Ser Thr
1 5
<210> 221
<211> 17
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 221
Ala Ala Ala Ser Gly Arg Gly Ile Thr Ala Ser Ala Phe Arg Tyr Asp
1 5 10 15
Val
<210> 222
<211> 148
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 222
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr
20 25 30
Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Ile Arg Trp Ser Gly Ser Ile Thr Tyr Tyr Ser Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Asp Pro Gly Arg Ile Phe Gly Ser Gly Ser Tyr Tyr Gly Asn
100 105 110
Arg Asn Thr Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser
115 120 125
Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His
130 135 140
His His His His
145
<210> 223
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 223
Gly Arg Thr Phe Ser Ser Tyr Thr
1 5
<210> 224
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 224
Ile Arg Trp Ser Gly Ser Ile Thr
1 5
<210> 225
<211> 22
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 225
Ala Ala Asp Pro Gly Arg Ile Phe Gly Ser Gly Ser Tyr Tyr Gly Asn
1 5 10 15
Arg Asn Thr Tyr Asp Tyr
20
<210> 226
<211> 146
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 226
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Glu Arg Thr Phe Arg Thr Tyr
20 25 30
Thr Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Ile Arg Trp Asn Gly Asp Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Met Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Asn Cys
85 90 95
Ala Ala Arg Ala Pro Ser Gly Gly Tyr Tyr Tyr Pro Asn Ala Leu Ser
100 105 110
Glu Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala
115 120 125
Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His
130 135 140
His His
145
<210> 227
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 227
Glu Arg Thr Phe Arg Thr Tyr Thr
1 5
<210> 228
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 228
Ile Arg Trp Asn Gly Asp Ser Thr
1 5
<210> 229
<211> 20
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 229
Ala Ala Arg Ala Pro Ser Gly Gly Tyr Tyr Tyr Pro Asn Ala Leu Ser
1 5 10 15
Glu Tyr Asn Tyr
20
<210> 230
<211> 146
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 230
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Glu Arg Thr Phe Arg Thr Tyr
20 25 30
Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Ile Arg Trp Asn Gly Asp Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Met Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Ser Cys
85 90 95
Ala Ala Arg Ala Tyr Ser Ile Gly Tyr Tyr Tyr Pro Asn Ala Leu Ser
100 105 110
Glu Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala
115 120 125
Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His
130 135 140
His His
145
<210> 231
<211> 13
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 231
Ala Ala Ser Glu Arg Thr Phe Arg Thr Tyr Thr Met Gly
1 5 10
<210> 232
<211> 20
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 232
Ala Ala Arg Ala Tyr Ser Ile Gly Tyr Tyr Tyr Pro Asn Ala Leu Ser
1 5 10 15
Glu Tyr Asn Tyr
20
<210> 233
<211> 145
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 233
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Val Thr Ile Ser Arg Tyr
20 25 30
Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Gly Leu Ile Arg Trp Ser Asn Gly Asn Thr Tyr Tyr Thr Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Leu Arg Ser Gly Tyr Val Gly Ser Ala Tyr Ser Gln Gln Ala
100 105 110
Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala
115 120 125
Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His
130 135 140
His
145
<210> 234
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 234
Gly Val Thr Ile Ser Arg Tyr Thr
1 5
<210> 235
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 235
Ile Arg Trp Ser Asn Gly Asn Thr
1 5
<210> 236
<211> 19
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 236
Ala Thr Leu Arg Ser Gly Tyr Val Gly Ser Ala Tyr Ser Gln Gln Ala
1 5 10 15
Tyr Asp Tyr
<210> 237
<211> 138
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 237
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Thr Ser Thr Val Ser Asp Tyr
20 25 30
His Met Gly Trp Phe Arg Gln Gly Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Ile Ser Trp Ser Gly Asp Ser Thr Asp Tyr Ala Asp Ser Val
50 55 60
Lys Ala Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Thr Met Ser
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ala Tyr Tyr Cys
85 90 95
Ala Ala Arg Arg Val Phe Lys Ala Thr Trp Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro
115 120 125
Asp Tyr Gly Ser His His His His His His
130 135
<210> 238
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 238
Thr Ser Thr Val Ser Asp Tyr His
1 5
<210> 239
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 239
Ile Ser Trp Ser Gly Asp Ser Thr
1 5
<210> 240
<211> 12
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 240
Ala Ala Arg Arg Val Phe Lys Ala Thr Trp Asp Tyr
1 5 10
<210> 241
<211> 147
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 241
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Thr Cys Ala Ala Thr Val Arg Ile Phe Ser Ser Tyr
20 25 30
Thr Met Gly Trp Phe Arg Gln Ser Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Gly Ala Ile Thr Trp Ser Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Asp Pro Gly Arg Ser Tyr Val Leu Ser Arg Tyr Tyr Asp Gln
100 105 110
Ala Ser Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His
130 135 140
His His His
145
<210> 242
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 242
Val Arg Ile Phe Ser Ser Tyr Thr
1 5
<210> 243
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 243
Ile Thr Trp Ser Gly Gly Asn Thr
1 5
<210> 244
<211> 21
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 244
Ala Ala Asp Pro Gly Arg Ser Tyr Val Leu Ser Arg Tyr Tyr Asp Gln
1 5 10 15
Ala Ser Tyr Asp Tyr
20
<210> 245
<211> 148
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 245
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Pro Phe Ser Ser Tyr
20 25 30
Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Ile Ser Trp Ser Gly Gly Thr Thr Tyr Tyr Thr Asp Ser Val
50 55 60
Gln Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Arg Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Thr Asp Val Gly Arg Ile Tyr Gly Gly Gly Ser Leu Tyr Ser Ser
100 105 110
Ala Phe Ser Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser
115 120 125
Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His
130 135 140
His His His His
145
<210> 246
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 246
Gly Arg Pro Phe Ser Ser Tyr Thr
1 5
<210> 247
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 247
Ile Ser Trp Ser Gly Gly Thr Thr
1 5
<210> 248
<211> 22
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 248
Ala Thr Asp Val Gly Arg Ile Tyr Gly Gly Gly Ser Leu Tyr Ser Ser
1 5 10 15
Ala Phe Ser Tyr Asp Tyr
20
<210> 249
<211> 140
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 249
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Ala
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Phe Ser Asp Tyr
20 25 30
Val Met Gly Trp Phe Arg Gln Pro Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ser Ile Asn Tyr Gly Gly Asp Lys Ile Ser Tyr Ala Asp Ser Leu
50 55 60
Glu Gly Arg Phe Thr Ile Leu Arg Asp Asn Thr Lys Asp Thr Thr Ser
65 70 75 80
Leu Gln Met Asn Thr Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Lys Trp Gly Tyr Lys Thr Gly Pro Thr Tyr Ser Tyr Trp Gly
100 105 110
Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp
115 120 125
Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 250
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 250
Gly Gly Thr Phe Ser Asp Tyr Val
1 5
<210> 251
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 251
Ile Asn Tyr Gly Gly Asp Lys Ile
1 5
<210> 252
<211> 14
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 252
Ala Ala Lys Trp Gly Tyr Lys Thr Gly Pro Thr Tyr Ser Tyr
1 5 10
<210> 253
<211> 132
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 253
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Asn Val
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Ala
35 40 45
Ala Ala Ile Ser Gly Ser Gly Gly Thr Thr Tyr Tyr Ala Ala Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Lys Met Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Thr Tyr Asp Gly Arg Arg Gly Gln Gly Thr Gln Val Thr Val Ser
100 105 110
Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His
115 120 125
His His His His
130
<210> 254
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 254
Gly Arg Thr Phe Ser Asn Val Ala
1 5
<210> 255
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 255
Ile Ser Gly Ser Gly Gly Thr Thr
1 5
<210> 256
<211> 6
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 256
Ser Thr Tyr Asp Gly Arg
1 5
<210> 257
<211> 145
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 257
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Ser Arg Asn
20 25 30
Ala Ile Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Ile Ser Trp Asn Ala Ile Thr Thr Ala Tyr Gly Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Phe Arg Gly Asn Thr Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Arg Tyr Ser Ser Gly Ser Tyr Tyr Tyr Ala Arg Thr Tyr Glu
100 105 110
Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala
115 120 125
Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His
130 135 140
His
145
<210> 258
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 258
Gly Leu Thr Phe Ser Arg Asn Ala
1 5
<210> 259
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 259
Ile Ser Trp Asn Ala Ile Thr Thr Ala
1 5
<210> 260
<211> 19
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 260
Ala Ala Arg Tyr Ser Ser Gly Ser Tyr Tyr Tyr Ala Arg Thr Tyr Glu
1 5 10 15
Tyr Asp Tyr
<210> 261
<211> 138
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 261
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Ser Arg Ser Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val
35 40 45
Ala Asp Ile Ala Trp Ser Asp Gly Arg Ile Tyr Tyr Ser Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Glu Ile Asp Ser Leu Lys Pro Gly Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ser Arg Ala Arg Gly Thr Val Leu Tyr Asn Tyr Trp Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro
115 120 125
Asp Tyr Gly Ser His His His His His His
130 135
<210> 262
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 262
Gly Arg Thr Ser Arg Ser Tyr Ala
1 5
<210> 263
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 263
Ile Ala Trp Ser Asp Gly Arg Ile
1 5
<210> 264
<211> 12
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 264
Ala Ser Arg Ala Arg Gly Thr Val Leu Tyr Asn Tyr
1 5 10
<210> 265
<211> 138
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 265
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Ser Arg Ser Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu His Val
35 40 45
Ala Asp Ile Ala Trp Ser Asp Gly Arg Ile Tyr Tyr Ser Asp Ser Val
50 55 60
Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Glu Ile Asp Ser Leu Lys Pro Gly Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ser Arg Ala Arg Gly Thr Val Ser Tyr Asn Tyr Trp Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro
115 120 125
Asp Tyr Gly Ser His His His His His His
130 135
<210> 266
<211> 12
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 266
Ala Ser Arg Ala Arg Gly Thr Val Ser Tyr Asn Tyr
1 5 10
<210> 267
<211> 138
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 267
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Ser Arg Ser Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val
35 40 45
Ala Asp Ile Ala Trp Ser Asp Gly Arg Ile Tyr Tyr Ser Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Glu Ile Asp Ser Leu Lys Pro Gly Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ser Arg Ala Arg Gly Thr Val Leu Tyr Asn Tyr Trp Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro
115 120 125
Asp Tyr Gly Ser His His His His His His
130 135
<210> 268
<211> 138
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 268
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Thr Leu Ser Cys Ala Ala Ser Gly Arg Thr Ser Arg Ser Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val
35 40 45
Ala Asp Ile Ala Trp Ser Asp Gly Arg Ile Tyr Tyr Ser Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Glu Ile Asp Ser Leu Lys Pro Gly Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ser Arg Ala Arg Gly Thr Val Ser Tyr Asn Tyr Trp Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro
115 120 125
Asp Tyr Gly Ser His His His His His His
130 135
<210> 269
<211> 138
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 269
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Ser Arg Ser Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val
35 40 45
Ala Asp Ile Ser Trp Ser Asp Gly Arg Ile Tyr Tyr Ser Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Glu Ile Asp Ser Leu Lys Pro Gly Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ser Arg Ala Arg Gly Ser Val Leu Tyr Asn Tyr Trp Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro
115 120 125
Asp Tyr Gly Ser His His His His His His
130 135
<210> 270
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 270
Ile Ser Trp Ser Asp Gly Arg Ile
1 5
<210> 271
<211> 138
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 271
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Ser Arg Ser Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val
35 40 45
Ala Asp Ile Ala Trp Ser Asp Gly Arg Ile Tyr Tyr Ser Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Glu Ile Asp Ser Leu Lys Pro Gly Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ser Arg Ala Arg Gly Thr Val Ser Tyr Asn Tyr Trp Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro
115 120 125
Asp Tyr Gly Ser His His His His His His
130 135
<210> 272
<211> 138
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 272
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Ser Arg Ser Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Val
35 40 45
Ala Asp Ile Ala Trp Ser Asp Gly Arg Ile Tyr Tyr Ser Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Glu Ile Asp Ser Leu Lys Pro Gly Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ser Arg Ala Arg Gly Thr Val Ser Tyr Asn Tyr Trp Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro
115 120 125
Asp Tyr Gly Ser His His His His His His
130 135
<210> 273
<211> 135
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 273
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Tyr Ser Gly Asn Val Phe Ser Ile Asn
20 25 30
Leu Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Leu
35 40 45
Ala Gly Ile Asn Arg Gly Gly Arg Thr Asn Tyr Thr Asp Val Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Thr Ile Tyr Leu
65 70 75 80
Gln Met Asn Gly Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys Ala
85 90 95
Ala Ser Arg Asp Pro Tyr Thr Gly Tyr Trp Gly Gln Gly Thr Gln Val
100 105 110
Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly
115 120 125
Ser His His His His His His
130 135
<210> 274
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 274
Gly Asn Val Phe Ser Ile Asn Leu
1 5
<210> 275
<211> 7
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 275
Ile Asn Arg Gly Gly Arg Thr
1 5
<210> 276
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 276
Ala Ala Ser Arg Asp Pro Tyr Thr Gly Tyr
1 5 10
<210> 277
<211> 135
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 277
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Tyr Ser Gly Asn Val Phe Ser Ile Asn
20 25 30
Leu Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Leu
35 40 45
Ala Gly Ile Asn Arg Gly Gly Arg Thr Asn Tyr Thr Asp Val Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Lys Glu Asn Ala Lys Asn Thr Ile Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys Ala
85 90 95
Ala Ser Arg Asp Pro Tyr Thr Gly Tyr Trp Gly Gln Gly Thr Gln Val
100 105 110
Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly
115 120 125
Ser His His His His His His
130 135
<210> 278
<211> 135
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 278
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Tyr Ser Gly Asn Val Phe Ser Ile Asn
20 25 30
Leu Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Leu
35 40 45
Ala Gly Ile Asn Arg Gly Gly Arg Thr Asn Tyr Thr Asp Val Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Thr Ile Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys Ala
85 90 95
Ala Ser Arg Asp Pro Tyr Thr Gly Tyr Trp Gly Gln Gly Thr Gln Val
100 105 110
Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly
115 120 125
Ser His His His His His His
130 135
<210> 279
<211> 135
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 279
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Ser Val Asp Ser Ile Asn
20 25 30
Leu Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Leu
35 40 45
Ala Gly Ile Asn Arg Gly Gly Arg Thr Asn Tyr Thr Asp Val Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Thr Ile Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys Ala
85 90 95
Ala Ser Arg Asp Pro Tyr Thr Gly Tyr Trp Gly Gln Gly Thr Gln Val
100 105 110
Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly
115 120 125
Ser His His His His His His
130 135
<210> 280
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 280
Gly Ser Val Asp Ser Ile Asn Leu
1 5
<210> 281
<211> 135
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 281
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Pro Ser Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Tyr Ser Gly Asn Val Phe Ser Ile Asn
20 25 30
Leu Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Leu
35 40 45
Ala Gly Ile Asn Arg Gly Gly Arg Thr Asn Tyr Thr Asp Val Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Thr Ile Tyr Leu
65 70 75 80
Gln Met Asn Gly Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys Ala
85 90 95
Ala Ser Arg Asp Pro Tyr Thr Gly Tyr Trp Gly Gln Gly Thr Gln Val
100 105 110
Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly
115 120 125
Ser His His His His His His
130 135
<210> 282
<211> 7
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 282
Asn Val Phe Ser Ile Asn Leu
1 5
<210> 283
<211> 135
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 283
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Tyr Ser Gly Asn Val Phe Ser Ile Asn
20 25 30
Leu Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Leu
35 40 45
Ala Gly Ile Thr Arg Gly Gly Arg Thr Asn Tyr Thr Asp Val Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Glu Ser Ala Lys Asn Thr Ile Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys Ala
85 90 95
Ala Ser Arg Asp Pro Tyr Thr Gly Tyr Trp Gly Gln Gly Thr Gln Val
100 105 110
Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly
115 120 125
Ser His His His His His His
130 135
<210> 284
<211> 135
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 284
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Tyr Ser Gly Asn Val Phe Ser Ile Asn
20 25 30
Leu Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Leu
35 40 45
Ala Gly Ile Thr Arg Gly Gly Arg Thr Asn Tyr Thr Asp Val Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Glu Asn Ala Lys Asn Thr Ile Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys Ala
85 90 95
Ala Ser Arg Asp Pro Tyr Thr Gly Tyr Trp Gly Gln Gly Thr Gln Val
100 105 110
Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly
115 120 125
Ser His His His His His His
130 135
<210> 285
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 285
Cys Ala Ala Ser Arg Asp Pro Tyr Thr Gly Tyr
1 5 10
<210> 286
<211> 143
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 286
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Lys Arg Ser Phe Ser Ser His
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Ala Arg Glu Phe Val
35 40 45
Ala Thr Ile Ser Trp Asn Ser Gly Ser Thr Phe Tyr Ser Asp Ser Ser
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Ser Gly Arg Gly Ile Thr Ala Ser Asp Phe Arg Tyr Asp
100 105 110
Ala Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr
115 120 125
Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 287
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 287
Lys Arg Ser Phe Ser Ser His Ala
1 5
<210> 288
<400> 288
000
<210> 289
<211> 17
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 289
Ala Ala Ala Ser Gly Arg Gly Ile Thr Ala Ser Asp Phe Arg Tyr Asp
1 5 10 15
Ala
<210> 290
<211> 143
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 290
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Gly Ser Gly Arg Thr Phe Ser Ser His
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Thr Ile Ser Trp Asn Ser Gly Ser Thr Phe Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Ser Gly Arg Gly Ile Thr Ala Ser Asp Phe Arg Tyr Asp
100 105 110
Ala Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr
115 120 125
Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 291
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 291
Gly Arg Thr Phe Ser Ser His Ala
1 5
<210> 292
<211> 143
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 292
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Ser Phe Ser Arg His
20 25 30
Ala Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Thr Ile Ser Trp Asn Ala Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Thr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Ser Gly Arg Gly Ile Thr Ala Ser Asp Phe Arg Tyr Asp
100 105 110
Ala Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr
115 120 125
Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 293
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 293
Gly Arg Ser Phe Ser Arg His Ala
1 5
<210> 294
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 294
Ile Ser Trp Asn Ala Gly Ser Thr
1 5
<210> 295
<211> 141
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 295
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Arg Ile
20 25 30
Asn Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Val Ile Ser Trp Asn Ser Ile Ala Asp Tyr Ala Asp Ser Val Lys
50 55 60
Ala Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Ser Ala Pro Phe Arg Ser Lys Asn Pro Thr Leu Tyr Leu Tyr Trp
100 105 110
Gly Arg Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr
115 120 125
Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 296
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 296
Gly Arg Thr Phe Ser Arg Ile Asn
1 5
<210> 297
<211> 7
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 297
Ile Ser Trp Asn Ser Ile Ala
1 5
<210> 298
<211> 16
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 298
Ala Ala Ser Ala Pro Phe Arg Ser Lys Asn Pro Thr Leu Tyr Leu Tyr
1 5 10 15
<210> 299
<211> 147
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<220>
<221> misc_feature
<222> (39)..(39)
<223> Xaa can be any naturally occurring amino acid
<400> 299
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ser Gly Asp
1 5 10 15
Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Arg Thr Phe Asn Pro Val
20 25 30
Ala Met Ala Trp Phe Arg Xaa Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Gly Thr Ile Thr Trp Gly Ile Gly Ser Thr His Tyr Ala Val Pro Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Lys Glu Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Arg Leu Gln Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Arg Thr Ser Leu Leu Arg Arg Ala Asp Glu Ile Pro Ser Val
100 105 110
Ala Asn Tyr Asp Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His
130 135 140
His His His
145
<210> 300
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 300
Gly Arg Thr Phe Asn Pro Val Ala
1 5
<210> 301
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 301
Ile Thr Trp Gly Ile Gly Ser Thr
1 5
<210> 302
<211> 21
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 302
Ala Ala Arg Thr Ser Leu Leu Arg Arg Ala Asp Glu Ile Pro Ser Val
1 5 10 15
Ala Asn Tyr Asp Ser
20
<210> 303
<211> 143
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<220>
<221> misc_feature
<222> (3)..(3)
<223> Xaa can be any naturally occurring amino acid
<400> 303
Gln Val Xaa Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Met Tyr
20 25 30
Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Lys Ile Ser Thr Ser Gly Arg Tyr Thr Asp Tyr Val Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Leu Ser Arg Asp Asn Val Lys Asn Thr Ile Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Arg Leu Pro Arg Pro Asp Thr Trp Ser Gln Gly Lys Thr Asp
100 105 110
Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr
115 120 125
Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 304
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 304
Gly Arg Thr Phe Ser Met Tyr Thr
1 5
<210> 305
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 305
Ile Ser Thr Ser Gly Arg Tyr Thr
1 5
<210> 306
<211> 17
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 306
Ala Ala Arg Leu Pro Arg Pro Asp Thr Trp Ser Gln Gly Lys Thr Asp
1 5 10 15
Tyr
<210> 307
<211> 144
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<220>
<221> misc_feature
<222> (39)..(39)
<223> Xaa can be any naturally occurring amino acid
<400> 307
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Asn Asn
20 25 30
Pro Met Gly Trp Phe Arg Xaa Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Thr Lys Thr Gly Gly Asn Ser Gly Ile Thr Val Tyr Ser Asn Ser
50 55 60
Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Leu Ala Lys Asn Thr Val
65 70 75 80
Tyr Leu Gln Met Asp Gly Leu Lys Leu Glu Asp Thr Ala Ile Tyr Tyr
85 90 95
Cys Ala Ala Ser Gly Thr Pro Leu Ala Leu Arg Ser Glu Lys Asn Tyr
100 105 110
Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala
115 120 125
Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 308
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 308
Gly Arg Thr Phe Ser Asn Asn Pro
1 5
<210> 309
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 309
Lys Thr Gly Gly Asn Ser Gly Ile Thr
1 5
<210> 310
<211> 17
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 310
Ala Ala Ser Gly Thr Pro Leu Ala Leu Arg Ser Glu Lys Asn Tyr Asp
1 5 10 15
Tyr
<210> 311
<211> 136
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<220>
<221> misc_feature
<222> (3)..(3)
<223> Xaa can be any naturally occurring amino acid
<400> 311
Gln Val Xaa Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Asn Ile Asn
20 25 30
Thr Met Gly Trp Tyr Arg Gln Ala Pro Gly Asn Gln Arg Glu Leu Val
35 40 45
Ala Ala Ile Ser Ser Asp Gly Arg Pro Asn His Arg Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn
85 90 95
Thr Val Pro Ala Ser Arg Ala Gly Gly Tyr Trp Gly Gln Gly Thr Gln
100 105 110
Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr
115 120 125
Gly Ser His His His His His His
130 135
<210> 312
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 312
Gly Ser Ile Phe Asn Ile Asn Thr
1 5
<210> 313
<211> 7
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 313
Ile Ser Ser Asp Gly Arg Pro
1 5
<210> 314
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 314
Asn Thr Val Pro Ala Ser Arg Ala Gly Gly Tyr
1 5 10
<210> 315
<211> 143
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 315
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Ala Gln Ala Gly Asp
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Thr Ile Asp Asn Gly
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Ser Val
35 40 45
Ala Ala Ile Asn Trp Ser Gly Ser Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Ala Ile Ser Arg Asp Asn Val Lys His Glu Val Tyr
65 70 75 80
Leu Gln Met Asn Arg Leu Arg Arg Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Lys Ser Ile Gly Thr Tyr Ser Ser Ser Ser Ala Tyr Asp
100 105 110
Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr
115 120 125
Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 316
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 316
Gly Arg Thr Ile Asp Asn Gly Ala
1 5
<210> 317
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 317
Ile Asn Trp Ser Gly Ser Ser Thr
1 5
<210> 318
<211> 17
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 318
Ala Ala Ala Lys Ser Ile Gly Thr Tyr Ser Ser Ser Ser Ala Tyr Asp
1 5 10 15
Tyr
<210> 319
<211> 143
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 319
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Asn Thr Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Met Asn Arg Asp Gly Ser Thr Val Leu Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Ala Ile Ser Lys Asp Asn Ala Lys Asn Thr Gly Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys
85 90 95
Ala Ala Asn Ala Arg Tyr Ser Asp Tyr Thr Asp Gly Gln Ser Phe Val
100 105 110
Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr
115 120 125
Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 320
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 320
Gly Arg Thr Phe Asn Thr Tyr Ala
1 5
<210> 321
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 321
Met Asn Arg Asp Gly Ser Thr Val
1 5
<210> 322
<211> 17
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 322
Ala Ala Asn Ala Arg Tyr Ser Asp Tyr Thr Asp Gly Gln Ser Phe Val
1 5 10 15
Ser
<210> 323
<211> 143
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 323
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Gln Ala Ser Gly Arg Thr Phe Asn Thr Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Met Asn Arg Asp Gly Ser Thr Ile Leu Tyr Gly Asp Ser Val
50 55 60
Lys Gly Arg Phe Ala Ile Ser Arg Asp Asn Ala Lys Asn Thr Gly Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys
85 90 95
Ala Ala Asn Ala Arg Tyr Ser Asp Tyr Thr His Gly Gln Ser Phe Val
100 105 110
Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr
115 120 125
Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 324
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 324
Met Asn Arg Asp Gly Ser Thr Ile
1 5
<210> 325
<211> 17
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 325
Ala Ala Asn Ala Arg Tyr Ser Asp Tyr Thr His Gly Gln Ser Phe Val
1 5 10 15
Ser
<210> 326
<211> 143
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 326
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Gln Ala Ser Gly Arg Thr Phe Asp Ala Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Met Asn Arg Asp Gly Ser Thr Val Leu Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Ala Ile Ser Lys Asp Asn Ala Lys Asn Thr Gly Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys
85 90 95
Ala Ala Asn Ala Arg Tyr Ser Asp Tyr Thr Asp Gly Gln Ser Phe Ile
100 105 110
Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr
115 120 125
Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 327
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 327
Gly Arg Thr Phe Asp Ala Tyr Ala
1 5
<210> 328
<211> 17
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 328
Ala Ala Asn Ala Arg Tyr Ser Asp Tyr Thr Asp Gly Gln Ser Phe Ile
1 5 10 15
Ser
<210> 329
<211> 143
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 329
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Gln Ala Ser Gly Arg Thr Phe Asn Thr Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Met Asn Arg Asp Gly Ser Thr Val Leu Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Ala Ile Ser Lys Asp Asn Ala Lys Asn Thr Gly Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys
85 90 95
Ala Ala Asn Ala Arg Tyr Ser Asp Tyr Thr Asp Gly Gln Ser Phe Val
100 105 110
Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr
115 120 125
Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 330
<400> 330
000
<210> 331
<211> 143
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 331
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Thr Phe Asn Thr Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Met Asn Arg Asp Gly Ser Thr Val Leu Tyr Ala Asp Tyr Val
50 55 60
Lys Gly Arg Phe Ala Ile Ser Lys Asp Asn Ala Lys Asn Thr Gly Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys
85 90 95
Ala Ala Asn Ala Arg Tyr Ser Asp Tyr Thr Asp Glu Gln Ser Phe Val
100 105 110
Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr
115 120 125
Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 332
<211> 17
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 332
Ala Ala Asn Ala Arg Tyr Ser Asp Tyr Thr Asp Glu Gln Ser Phe Val
1 5 10 15
Ser
<210> 333
<211> 143
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 333
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Gln Ala Ser Gly Arg Thr Phe Asn Thr Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Met Asn Arg Asp Gly Ser Thr Val Leu Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Ala Ile Ser Lys Asp Asn Ala Lys Ser Thr Gly Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys
85 90 95
Ala Ala Asn Ala Arg Tyr Ser Asp Tyr Thr Asp Gly Gln Ser Phe Val
100 105 110
Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr
115 120 125
Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 334
<211> 143
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 334
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Lys Ala Ser Gly Arg Thr Phe Ser Thr Tyr
20 25 30
Ala Val Gly Trp Phe Arg Gln Ala Pro Gly Lys Pro Arg Glu Phe Val
35 40 45
Ala Ala Met Asn Ser Arg Gly Ser Thr Ile Asn Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Ala Ile Ser Arg Asp Asn Ala Lys Asn Thr Gly Tyr
65 70 75 80
Leu Gln Met Asp Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys
85 90 95
Ala Ala Asp Ala Arg Tyr Ser Asp Tyr Thr Asp Gly Gln Ser Phe Lys
100 105 110
Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr
115 120 125
Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 335
<400> 335
000
<210> 336
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 336
Met Asn Ser Arg Gly Ser Thr Ile
1 5
<210> 337
<211> 17
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 337
Ala Ala Asp Ala Arg Tyr Ser Asp Tyr Thr Asp Gly Gln Ser Phe Lys
1 5 10 15
Ser
<210> 338
<211> 143
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 338
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Gln Ala Ser Gly Arg Thr Phe Asn Thr Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Met Asn Arg Asp Gly Ser Thr Ile Leu Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Ala Ile Ser Lys Asp Asn Ala Lys Asn Thr Gly Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys
85 90 95
Ala Ala Asn Ala Arg Tyr Ser Asp Tyr Thr Asn Gly His Ser Phe Val
100 105 110
Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr
115 120 125
Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 339
<211> 17
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 339
Ala Ala Asn Ala Arg Tyr Ser Asp Tyr Thr Asn Gly His Ser Phe Val
1 5 10 15
Ser
<210> 340
<211> 143
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 340
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Gln Ala Ser Gly Arg Thr Phe Asn Thr Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Met Asn Arg Asp Gly Ser Thr Val Leu Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Ala Ile Ser Lys Asp Asn Ala Arg Asn Thr Gly Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys
85 90 95
Ala Ala Asn Ala Arg Tyr Ser Asp Tyr Thr Asp Gly Gln Ser Phe Val
100 105 110
Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr
115 120 125
Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 341
<211> 143
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 341
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Gln Ala Ser Gly Arg Thr Phe Asn Thr Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Met Asn Arg Asp Gly Ser Thr Val Leu Tyr Arg Asp Ser Val
50 55 60
Lys Gly Arg Phe Ala Ile Ser Lys Asp Asn Ala Arg Asn Thr Gly Tyr
65 70 75 80
Leu Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys
85 90 95
Ala Ala Asn Ala Arg Tyr Ser Asp Tyr Thr Asp Gly Gln Ser Phe Val
100 105 110
Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr
115 120 125
Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 342
<211> 143
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 342
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Gln Ala Ser Glu Arg Ser Phe Ser Thr Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Met Asn Arg Asn Gly Asn Thr Ile Asn Tyr Leu Asp Ser Val
50 55 60
Lys Gly Arg Phe Ala Ile Ser Arg Asp Asn Ala Lys Ser Thr Gly Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Ser Ala Asn Ala Arg Leu Ser Asp Tyr Thr Asn Pro Gln Ser Phe Val
100 105 110
Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr
115 120 125
Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 343
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 343
Glu Arg Ser Phe Ser Thr Tyr Ala
1 5
<210> 344
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 344
Met Asn Arg Asn Gly Asn Thr Ile
1 5
<210> 345
<211> 17
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 345
Ser Ala Asn Ala Arg Leu Ser Asp Tyr Thr Asn Pro Gln Ser Phe Val
1 5 10 15
Ser
<210> 346
<211> 140
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 346
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Ala Phe Ser Ser Tyr
20 25 30
Gly Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Ile Asn Ser Asn Gly Arg Ser Thr Tyr Tyr Ala Asp Thr Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Gly Arg Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Asp Arg Gln Ser Met Lys Gly Tyr Glu Tyr Gly Tyr Trp Gly
100 105 110
Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp
115 120 125
Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 347
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 347
Gly Arg Ala Phe Ser Ser Tyr Gly
1 5
<210> 348
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 348
Ile Asn Ser Asn Gly Arg Ser Thr
1 5
<210> 349
<211> 14
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 349
Ala Ala Asp Arg Gln Ser Met Lys Gly Tyr Glu Tyr Gly Tyr
1 5 10
<210> 350
<211> 140
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 350
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Ala Phe Ser Arg Tyr
20 25 30
Gly Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Ile Thr Ser Asn Gly Arg Ser Thr Tyr Tyr Ala Asp Thr Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Arg Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Thr Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Asp Arg Lys Ser Met Thr Gly Tyr Glu Tyr Gly Tyr Trp Gly
100 105 110
Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp
115 120 125
Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 351
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 351
Gly Arg Ala Phe Ser Arg Tyr Gly
1 5
<210> 352
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 352
Ile Thr Ser Asn Gly Arg Ser Thr
1 5
<210> 353
<211> 14
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 353
Ala Ala Asp Arg Lys Ser Met Thr Gly Tyr Glu Tyr Gly Tyr
1 5 10
<210> 354
<211> 140
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 354
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Ala Phe Ser Arg Tyr
20 25 30
Gly Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Ile Thr Ser Asn Gly Arg Ser Thr Tyr Tyr Ala Asp Thr Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Gly Arg Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Thr Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Asp Arg Lys Ser Met Thr Gly Tyr Glu Tyr Gly Tyr Trp Gly
100 105 110
Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp
115 120 125
Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 355
<211> 140
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 355
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Ala Phe Ser Arg Tyr
20 25 30
Gly Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Gly Ala Ile Asn Ser Asn Gly Arg Ser Thr Tyr Tyr Ala Asp Thr Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Arg Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Asp Arg Gln Ser Met Thr Arg Tyr Glu Tyr Gly Tyr Trp Gly
100 105 110
Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp
115 120 125
Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 356
<211> 14
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 356
Ala Ala Asp Arg Gln Ser Met Thr Arg Tyr Glu Tyr Gly Tyr
1 5 10
<210> 357
<211> 144
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 357
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Asp Phe Val
35 40 45
Ala Ala Val Ser Gly Ile Ala Arg Arg Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Ser Thr Ile Ser Arg Asp Asn Gly Arg Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ala Thr Ser Arg Met Thr Ser Val Thr Thr Leu Asn Asp Tyr
100 105 110
Gly Tyr Trp Gly Gln Gly Thr Pro Val Thr Val Ser Ser Ala Ala Ala
115 120 125
Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 358
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 358
Gly Phe Thr Phe Ser Asn Tyr Ala
1 5
<210> 359
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 359
Val Ser Gly Ile Ala Arg Arg Thr
1 5
<210> 360
<211> 18
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 360
Ala Arg Ala Thr Ser Arg Met Thr Ser Val Thr Thr Leu Asn Asp Tyr
1 5 10 15
Gly Tyr
<210> 361
<211> 144
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 361
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Asp Phe Val
35 40 45
Ala Ala Val Ser Gly Ile Ala Arg Arg Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Ser Thr Ile Ser Arg Asp Asn Gly Arg Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ala Thr Ser Arg Met Thr Ser Val Thr Thr Leu Asp Asp Tyr
100 105 110
Gly Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala
115 120 125
Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 362
<211> 18
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 362
Ala Arg Ala Thr Ser Arg Met Thr Ser Val Thr Thr Leu Asp Asp Tyr
1 5 10 15
Gly Tyr
<210> 363
<211> 144
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 363
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Asp Phe Val
35 40 45
Ala Ala Val Ser Gly Ile Ala Arg Arg Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Ser Thr Ile Ser Arg Asp Asn Gly Arg Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ala Thr Ser Arg Met Thr Ser Val Thr Thr Leu Asn Asp Tyr
100 105 110
Gly Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala
115 120 125
Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 364
<211> 144
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 364
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Val Ser Gly Ile Ala Arg Arg Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Ser Thr Ile Ser Arg Asp Asn Gly Arg Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ala Thr Ser Arg Met Thr Ser Val Thr Thr Leu Asn Asp Tyr
100 105 110
Ala Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala
115 120 125
Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 365
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 365
Gly Phe Thr Phe Ser Ser Tyr Ala
1 5
<210> 366
<211> 18
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 366
Ala Arg Ala Thr Ser Arg Met Thr Ser Val Thr Thr Leu Asn Asp Tyr
1 5 10 15
Ala Tyr
<210> 367
<211> 141
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 367
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ser Gly Gly
1 5 10 15
Ser Leu Thr Leu Ser Cys Ser Ala Ser Gly Ser Leu Phe Ser Ile Asn
20 25 30
Ala Met Gly Trp Tyr Arg Gln Ala Pro Arg Lys Gln His Glu Leu Val
35 40 45
Ala Thr Met Met Asp Gly Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Lys Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys Val
85 90 95
Ala Asp Arg Leu Gly Ser Gly Arg Tyr Ala Tyr Gly Ile Asp Tyr Trp
100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr
115 120 125
Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 368
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 368
Gly Ser Leu Phe Ser Ile Asn Ala
1 5
<210> 369
<211> 7
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 369
Met Met Asp Gly Gly Ser Thr
1 5
<210> 370
<211> 16
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 370
Val Ala Asp Arg Leu Gly Ser Gly Arg Tyr Ala Tyr Gly Ile Asp Tyr
1 5 10 15
<210> 371
<211> 141
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 371
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ala Cys Ala Ala Ser Gly Ser Ile Phe Ser Ile Asn
20 25 30
Ala Met Gly Trp Tyr Arg Gln Ala Pro Arg Lys Gln Arg Glu Leu Val
35 40 45
Ala Thr Met Met Asp Gly Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Lys Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys Val
85 90 95
Ala Asp Arg Leu Gly Ser Gly Arg Tyr Ala Tyr Gly Ile Asp Tyr Trp
100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr
115 120 125
Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 372
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 372
Gly Ser Ile Phe Ser Ile Asn Ala
1 5
<210> 373
<211> 141
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 373
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Val Ser Ile Asn
20 25 30
Gly Met Gly Trp Tyr Arg Gln Ala Pro Arg Lys Gln Arg Glu Leu Val
35 40 45
Ala Thr Met Met Asp Gly Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Lys Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys Ala
85 90 95
Ala Asp Arg Leu Gly Ser Gly Arg Tyr Ala Tyr Ala Ile Asp Tyr Trp
100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr
115 120 125
Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 374
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 374
Gly Ser Ile Val Ser Ile Asn Gly
1 5
<210> 375
<211> 16
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 375
Ala Ala Asp Arg Leu Gly Ser Gly Arg Tyr Ala Tyr Ala Ile Asp Tyr
1 5 10 15
<210> 376
<211> 140
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 376
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Arg Thr Val Ser Asn Tyr
20 25 30
Gly Met Ala Trp Phe Arg Gln Val Ala Gly Lys Glu Arg Ala Phe Val
35 40 45
Ala Ala Ile Asn Asn Arg Gly Asp Ser Lys Tyr Tyr Ala Glu Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ala Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Val Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Asp Arg Arg Ser Leu Val Arg Tyr Glu Tyr Asn Tyr Trp Gly
100 105 110
Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp
115 120 125
Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 377
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 377
Gly Arg Thr Val Ser Asn Tyr Gly
1 5
<210> 378
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 378
Ile Asn Asn Arg Gly Asp Ser Lys
1 5
<210> 379
<211> 14
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 379
Ala Ala Asp Arg Arg Ser Leu Val Arg Tyr Glu Tyr Asn Tyr
1 5 10
<210> 380
<211> 141
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 380
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Arg Tyr
20 25 30
Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Ile Arg Trp Ser Ser Gly Asn Thr Tyr Tyr Thr Asp Ala Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Glu Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Val Ala Asp Asp Gly Leu Ser Tyr Ala Ser Ser Ser Tyr Leu Tyr Trp
100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr
115 120 125
Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 381
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 381
Gly Arg Thr Phe Ser Arg Tyr Thr
1 5
<210> 382
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 382
Ile Arg Trp Ser Ser Gly Asn Thr
1 5
<210> 383
<211> 15
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 383
Val Ala Asp Asp Gly Leu Ser Tyr Ala Ser Ser Ser Tyr Leu Tyr
1 5 10 15
<210> 384
<211> 141
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 384
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr
20 25 30
Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Ile Arg Trp Ser Ser Gly Asn Thr Tyr Tyr Thr Asp Ala Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Thr Glu Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Val Ala Asp Asp Gly Leu Ser Tyr Ala Ser Ser Ser Tyr Leu Tyr Trp
100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr
115 120 125
Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 385
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 385
Gly Phe Thr Phe Ser Arg Tyr Thr
1 5
<210> 386
<211> 140
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 386
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Thr Phe Gly Asn Tyr
20 25 30
Gly Leu Ala Trp Phe Arg Gln Pro Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Ile Asn Asn Arg Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Ala Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Ser Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Asp Arg Thr Ser Leu His Ser Tyr Arg Tyr Thr Tyr Trp Gly
100 105 110
Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp
115 120 125
Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 387
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 387
Gly Arg Thr Phe Gly Asn Tyr Gly
1 5
<210> 388
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 388
Ile Asn Asn Arg Gly Gly Asn Thr
1 5
<210> 389
<211> 14
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 389
Ala Ala Asp Arg Thr Ser Leu His Ser Tyr Arg Tyr Thr Tyr
1 5 10
<210> 390
<211> 140
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 390
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Thr Phe Gly Asn Tyr
20 25 30
Gly Leu Ala Trp Phe Arg Gln Pro Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Ile Ser Asn Arg Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Ala Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Ser Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Asp Arg Thr Ser Leu His Ser Tyr Arg Tyr Thr Tyr Trp Gly
100 105 110
Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp
115 120 125
Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 391
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 391
Ile Ser Asn Arg Gly Gly Asn Thr
1 5
<210> 392
<211> 140
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 392
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp
1 5 10 15
Ser Pro Lys Leu Ser Cys Val Ala Ser Gly Arg Thr Phe Gly Asn Tyr
20 25 30
Gly Leu Ala Trp Phe Arg Gln Pro Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Ile Ser Asn Arg Gly Gly Asn Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Ala Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Ser Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Asp Arg Thr Ser Leu His Ser Tyr Arg Tyr Thr Tyr Trp Gly
100 105 110
Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp
115 120 125
Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 393
<211> 142
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 393
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Glu Arg Thr Phe Ser Thr Tyr
20 25 30
Asn Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Ser Val
35 40 45
Ser Arg Ile Asn Trp Asn Gly Gly Phe Thr Gly Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Ala Met Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Cys Gly Ser Ala Tyr Pro Cys Arg Pro Glu Glu Tyr Thr Tyr
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro
115 120 125
Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 394
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 394
Glu Arg Thr Phe Ser Thr Tyr Asn
1 5
<210> 395
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 395
Ile Asn Trp Asn Gly Gly Phe Thr
1 5
<210> 396
<211> 14
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 396
Ala Ala Cys Gly Ser Ala Tyr Pro Cys Arg Pro Glu Glu Tyr
1 5 10
<210> 397
<211> 139
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 397
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Leu Ser Ser Tyr
20 25 30
Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Ile Val
35 40 45
Ala Ala Ser Ser Trp Ser Gly Gly Arg Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Met Ser Arg Asn Asn Ala Glu Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Asn Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Asp Ser Ser Arg Ala Pro Arg Thr Tyr Asn Tyr Trp Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val
115 120 125
Pro Asp Tyr Gly Ser His His His His His His
130 135
<210> 398
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 398
Gly Arg Thr Leu Ser Ser Tyr Thr
1 5
<210> 399
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 399
Ser Ser Trp Ser Gly Gly Arg Thr
1 5
<210> 400
<211> 13
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 400
Ala Ala Asp Ser Ser Arg Ala Pro Arg Thr Tyr Asn Tyr
1 5 10
<210> 401
<211> 140
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 401
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Thr Tyr
20 25 30
Asn Met Ser Trp Phe Arg Gln Gly Pro Gly Lys Glu Arg Ile Phe Val
35 40 45
Ala Thr Ile Ser Trp Ser Gly Arg Val Thr Asp Tyr Ala Asp Ser Val
50 55 60
Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Met Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Asp Ser Asn Ser Arg Arg Ser Arg Asp Tyr Asp Tyr Trp Gly
100 105 110
Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp
115 120 125
Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 402
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 402
Gly Arg Thr Phe Ser Thr Tyr Asn
1 5
<210> 403
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 403
Ile Ser Trp Ser Gly Arg Val Thr
1 5
<210> 404
<211> 14
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 404
Ala Ala Asp Ser Asn Ser Arg Arg Ser Arg Asp Tyr Asp Tyr
1 5 10
<210> 405
<211> 134
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 405
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Thr Thr Arg Arg Ile Asn
20 25 30
Ser Met Arg Trp Tyr Arg Gln Thr Pro Gly Asn Glu Arg Asp Leu Val
35 40 45
Ala Gly Ile Thr Glu Gly Gly Phe Thr Ala Tyr Val Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Tyr
85 90 95
Ala Ala Tyr Leu Gly Ala Ala Tyr Trp Gly Gln Gly Thr Gln Val Thr
100 105 110
Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser
115 120 125
His His His His His His
130
<210> 406
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 406
Gly Thr Thr Arg Arg Ile Asn Ser
1 5
<210> 407
<211> 7
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 407
Ile Thr Glu Gly Gly Phe Thr
1 5
<210> 408
<211> 6
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 408
Tyr Leu Gly Ala Ala Tyr
1 5
<210> 409
<211> 133
<212> PRT
<213> Artificial sequence
<220>
<223> type 2 chitin-binding domain (ChtBD2)
<220>
<221> misc_feature
<222> (2)..(12)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (43)..(47)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (54)..(62)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (87)..(98)
<223> Xaa can be any naturally occurring amino acid
<220>
<221> misc_feature
<222> (116)..(121)
<223> Xaa can be any naturally occurring amino acid
<400> 409
Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Cys Cys Cys
1 5 10 15
Cys Cys Cys Cys Cys Cys Cys Cys Cys Cys Cys Cys Cys Cys Cys Cys
20 25 30
Cys Cys Cys Cys Cys Cys Cys Cys Cys Cys Xaa Xaa Xaa Xaa Xaa Cys
35 40 45
Cys Cys Cys Cys Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Cys
50 55 60
Cys Cys Cys Cys Cys Cys Cys Cys Cys Cys Cys Cys Cys Cys Cys Cys
65 70 75 80
Cys Cys Cys Cys Cys Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa
85 90 95
Xaa Xaa Cys Cys Cys Cys Cys Cys Cys Cys Cys Cys Cys Cys Cys Cys
100 105 110
Cys Cys Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Cys Cys Cys Cys Cys Cys
115 120 125
Cys Cys Cys Cys Cys
130
<210> 410
<211> 1137
<212> PRT
<213> Artificial sequence
<220>
<223> type 2 chitin-binding domain (ChtBD2)
<400> 410
Met Lys Asp Ala Val Ile Leu Leu Leu Cys Ala Ile Ala Leu Ala Asp
1 5 10 15
Gly Phe Asn His Glu Leu Leu Ser Asn Ser Arg Ser Cys Asn Cys Asn
20 25 30
Pro Ser Glu Ala Gln Gln Ile Cys Glu Ala Asn Tyr Gly Lys Asp Asp
35 40 45
Val Leu Ile Ala His Glu Asn Cys Asp Lys Phe Tyr Lys Cys Ala Asn
50 55 60
Gly Lys Pro Phe Pro Tyr Lys Cys Pro Asn Asn Leu Leu Tyr Asp Pro
65 70 75 80
Tyr Lys Glu Glu Cys Glu Trp Pro Trp Leu Val Asp Cys Gly Asp Arg
85 90 95
Pro Ile Ser Glu Gly Ser Asp Gln Glu Asp Gly Gly Val Asp Asn Asp
100 105 110
Asn Asn Asp Ser Asn Asp Asn Asn Gly Val Asp Asp Gly Trp Thr Cys
115 120 125
Asn Cys Asn Pro Ser Glu Ala Pro Ser Ile Cys Ala Ala Glu Gly Ser
130 135 140
Ser Gly Val Leu Val Ala His Glu Asn Cys Asp Gln Phe Tyr Met Cys
145 150 155 160
Asp Ala Gly Arg Pro Val Thr Phe Asp Cys Pro Pro Thr Leu Met Tyr
165 170 175
Asp Pro His Arg Glu Glu Cys Tyr Trp Pro His Glu Val Asp Cys Gly
180 185 190
Asp Arg Gly Ile Ser Glu Pro Gly Asn Asp Gln Gly Asn Asp Asn Asp
195 200 205
Gly Asn Asp Asn Asn Ser Ser Asn Glu Gln Gly Gly Val Cys Asn Cys
210 215 220
Asn Pro Glu Glu Ala Pro Ala Ile Cys Ala Ser Pro Gly Ser Glu Gly
225 230 235 240
Val Leu Val Ala His Glu Asn Cys Glu Lys Tyr Tyr Ile Cys Asn His
245 250 255
Gly Arg Pro Val Val Ala Ser Cys Ser Gly Asn Leu Leu Phe Asn Pro
260 265 270
Tyr Thr Asn Glu Cys Gly Trp Pro Arg Asp Val Asp Cys Gly Asp Arg
275 280 285
Ile Glu Pro Gly Cys Thr Gly Cys Asn Asp Asn Asn Asn Asn Asp Asp
290 295 300
Asn Asp Ser Asp Cys Asp Gly Asp Asp Pro Val Pro Pro Pro Ala Asp
305 310 315 320
Asn Asp Asp Ser Glu Ser Ala Asp Ile Asp Asp Leu Pro Pro Pro Gly
325 330 335
Asp Asp Ala Ser Val Arg Pro Pro Val Asp Glu Gly Thr Cys Asn Cys
340 345 350
Asn Pro Glu Gln Ala Pro Ser Ile Cys Ala Glu Asp Asp Ser Asp Gly
355 360 365
Val Leu Val Ala His Glu Asp Cys Asn Lys Phe Tyr Lys Cys His Asn
370 375 380
Gly Lys Pro Val Ala Leu Tyr Cys Pro Gly Asn Leu Leu Tyr Asn Pro
385 390 395 400
Asn Thr Glu Gln Cys Asp Trp Pro Glu Lys Val Asp Cys Gly Asp Arg
405 410 415
Val Ile Pro Asp Pro Glu Asp Asn Asn Asn Asn Asp Ser Ser Gly Gly
420 425 430
Asn Asn Asp Gly Gly Asn Asp Gly Gly Asn Gly Gly Gly Gly Asn Cys
435 440 445
Asp Pro Ser Glu Ala Pro Ala Ile Cys Ala Glu Asp Asp Ser Glu Gly
450 455 460
Val Leu Val Ala His Glu Asn Cys Asn Gln Phe Tyr Lys Cys Ser Gly
465 470 475 480
Gly Lys Pro Val Ala Leu Leu Cys Pro Gly Asn Leu Leu Phe Asn Pro
485 490 495
Asn Thr Asp Gln Cys Asp Trp Pro Trp Glu Val Asp Cys Gly Asp Arg
500 505 510
Ile Ile Pro Asp Pro Asp Gln Lys Pro Asp Pro Glu Asp Ser Ser Asp
515 520 525
Asp Ser Ser Ala Asp Ile Asp Asp Leu Pro Pro Pro Gly Asp Asp Val
530 535 540
Thr Thr Arg Pro Pro Gly Thr Cys Asn Cys Asn Pro Glu Glu Ala Pro
545 550 555 560
Ser Ile Cys Ala Gln Asp Gly Ser Asn Gly Thr Leu Ile Ala His Glu
565 570 575
Asp Cys Asn Lys Phe Tyr Ile Cys Asp His Gly Lys Pro Val Ala Leu
580 585 590
Ser Cys Pro Gly Asn Leu Leu Tyr Asn Pro Tyr Thr Glu Lys Cys Asp
595 600 605
Trp Pro Glu Asn Val Glu Cys Gly Asp Ser Asp Gly Ile Leu Ile Ala
610 615 620
His Glu Asp Cys Asn Lys Phe Tyr Ile Cys Asp His Gly Lys Pro Val
625 630 635 640
Val Leu Ser Cys Pro Gly Asn Leu Phe Tyr Asn Pro Tyr Thr Glu Gln
645 650 655
Cys Asp Trp Pro Val Asn Val Glu Cys Gly Asp Arg Val Thr Pro Asp
660 665 670
Pro Asp Ala Thr Pro Ala Pro Thr Ala Ala Pro Thr Ser Thr Thr Thr
675 680 685
Val Ala Pro Thr Ala Ala Pro Ser Thr Thr Thr Thr Val Ala Pro Thr
690 695 700
Ala Ala Pro Thr Thr Thr Thr Thr Val Ala Pro Thr Thr Thr Thr Thr
705 710 715 720
Val Ala Pro Thr Thr Thr Thr Thr Val Ala Pro Thr Ala Ala Pro Thr
725 730 735
Thr Thr Thr Thr Val Ala Pro Thr Ala Ala Pro Thr Thr Thr Thr Thr
740 745 750
Val Ala Pro Thr Ala Ala Pro Thr Thr Thr Thr Thr Val Ala Ser Thr
755 760 765
Ala Ala Pro Thr Ala Ala Pro Thr Ala Ala Pro Thr Ala Ala Ser Thr
770 775 780
Pro Asp Asp Ser Asp Cys Asp Asp Asp Asn Asn Gly Gly Asp Asp Thr
785 790 795 800
Cys Asn Cys Asn Pro Asp Glu Ala Ala Ser Ile Cys Ser Val Gly Asn
805 810 815
Ser Asp Gly Ile His Val Ala His Glu Asn Cys Asn Trp Phe Tyr Lys
820 825 830
Cys Asp Asn Gly Arg Pro Val Pro Phe Arg Cys Pro Ser Gly Leu Met
835 840 845
Tyr Asn Pro Tyr Thr Gln Ile Cys Asp Trp Pro Trp Asp Val Glu Cys
850 855 860
Gly Asp Arg Val Ile Ala Asp Asp Asp Asp Ser Ser Glu Glu Asp Asn
865 870 875 880
Asp Asn Asp Asn Asp Ser Gly Val Val Gly Pro Cys Asn Cys Asn Pro
885 890 895
Glu Glu Ala Pro Ala Ile Cys Ala Ala Glu Gly Ser Asn Gly Val His
900 905 910
Val Ala His Gln Asn Cys Asn Gln Tyr Tyr Met Cys Asp Asn Gly Arg
915 920 925
Pro Val Ala Phe Thr Cys Asn Gly Phe Leu Leu Tyr Asn Pro Tyr Thr
930 935 940
Gln Gln Cys Asp Trp Pro His Leu Val Glu Cys Gly Asp Arg Val Ile
945 950 955 960
Pro Glu Pro Gly Asp Glu Asp Asp Glu Asp Cys Asp Asp Asp Asp Asp
965 970 975
Asn Ser Asn Asn Val Ile Asn Asp Asp Pro Ser Gln Ala Pro Ala Ile
980 985 990
Cys Ala Asp Ser Gly Ser Glu Gly Val Leu Val Ala His Glu Asn Cys
995 1000 1005
Asp Gln Tyr Tyr Ile Cys Asp Gly Gly Arg Pro Val Ala Arg Pro
1010 1015 1020
Cys Gln Gly Gly Leu Leu Tyr Asn Pro Leu Thr Gln Tyr Cys Asp
1025 1030 1035
Trp Pro Gly Asn Val Asn Cys Gly Asp Arg Ile Ile Pro Asp Asp
1040 1045 1050
Cys Ala Cys Asn Pro Arg Asn Ala Pro Arg Leu Cys Ser Lys Pro
1055 1060 1065
Asp Ser Glu Gly Ser Leu Val Ala His Glu Asn Cys Asn Gln Phe
1070 1075 1080
Tyr Ile Cys Ala His Ser Val Pro Val Glu His Phe Cys Pro Val
1085 1090 1095
Gly Leu Tyr Tyr Asn Ile Glu Leu Glu Leu Cys Asp Trp Ala Gln
1100 1105 1110
Asn Val Asn Cys Glu Asn Arg Asn Leu Pro Ser Leu Asn Lys His
1115 1120 1125
Trp Glu Ser Arg Gln Thr Leu Arg Lys
1130 1135
<210> 411
<211> 134
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 411
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ser Ala Ser Thr Ser Ile Ala Ser Ile Asn
20 25 30
Ala Val Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val
35 40 45
Ala Val Ile Val Asn Gly Ser Thr Thr Arg Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Gly Asp Asn Ala Glu Asn Thr Val Pro Leu
65 70 75 80
Leu Met Ser Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Gly
85 90 95
Ala Arg Asp Leu Ser Gly Thr Tyr Trp Gly Gln Gly Thr Gln Val Thr
100 105 110
Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser
115 120 125
His His His His His His
130
<210> 412
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 412
Thr Ser Ile Ala Ser Ile Asn Ala
1 5
<210> 413
<211> 7
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 413
Ile Val Asn Gly Ser Thr Thr
1 5
<210> 414
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 414
Gly Ala Arg Asp Leu Ser Gly Thr Tyr
1 5
<210> 415
<211> 134
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 415
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ser Ala Ser Thr Ser Ile Ala Ser Ile Asn
20 25 30
Ala Val Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val
35 40 45
Ala Val Ile Val Asn Gly Ser Thr Thr Arg Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Gly Asp Asn Ala Lys Asn Thr Val Pro Leu
65 70 75 80
Gln Met Ser Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Gly
85 90 95
Ala Arg Asp Leu Ser Gly Thr Tyr Trp Gly Gln Gly Thr Gln Val Thr
100 105 110
Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser
115 120 125
His His His His His His
130
<210> 416
<211> 134
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 416
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ser Ala Ser Thr Ser Ile Ala Ser Ile Asn
20 25 30
Ala Val Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val
35 40 45
Ala Val Ile Val Asn Gly Ser Thr Thr Arg Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Gly Asp Asn Ala Lys Asn Thr Val Pro Leu
65 70 75 80
Gln Met Ser Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys Gly
85 90 95
Ala Arg Asp Leu Ser Gly Thr Tyr Trp Gly Gln Gly Thr Gln Val Thr
100 105 110
Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser
115 120 125
His His His His His His
130
<210> 417
<211> 136
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 417
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Gly Thr Phe Ser Arg Tyr
20 25 30
Val Met Gly Trp Phe Arg Gln Gly Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Ser Trp Ser Gly Ile Ser Thr Tyr Tyr Ala Asp Phe Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Asp Pro Gly Arg Gly Tyr Asp Tyr Trp Gly Gln Gly Thr Gln
100 105 110
Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr
115 120 125
Gly Ser His His His His His His
130 135
<210> 418
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 418
Arg Gly Thr Phe Ser Arg Tyr Val
1 5
<210> 419
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 419
Ile Ser Trp Ser Gly Ile Ser Thr
1 5
<210> 420
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 420
Ala Ala Asp Pro Gly Arg Gly Tyr Asp Tyr
1 5 10
<210> 421
<211> 136
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 421
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Ser Phe Ser Arg Ser
20 25 30
Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Thr Ser Trp Ser Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Gln Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Asp Val Gly Arg Gly Tyr His Tyr Trp Gly Gln Gly Thr Gln
100 105 110
Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr
115 120 125
Gly Ser His His His His His His
130 135
<210> 422
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 422
Gly Arg Ser Phe Ser Arg Ser Val
1 5
<210> 423
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 423
Thr Ser Trp Ser Gly Gly Gly Thr
1 5
<210> 424
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 424
Ala Ala Asp Val Gly Arg Gly Tyr His Tyr
1 5 10
<210> 425
<211> 136
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 425
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Gly Thr Phe Ser Gly Leu
20 25 30
Thr Met Gly Trp Phe Arg Gln Ala Pro Gln Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Ile Ser Trp Thr Gly Arg Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Met Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Val Tyr Phe Cys
85 90 95
Ala Ala Asp Trp Ala Ser Gly Thr Pro Tyr Trp Gly Gln Gly Thr Gln
100 105 110
Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr
115 120 125
Gly Ser His His His His His His
130 135
<210> 426
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 426
Gly Gly Thr Phe Ser Gly Leu Thr
1 5
<210> 427
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 427
Ile Ser Trp Thr Gly Arg Ser Thr
1 5
<210> 428
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 428
Ala Ala Asp Trp Ala Ser Gly Thr Pro Tyr
1 5 10
<210> 429
<211> 136
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 429
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Thr
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Phe Ser Gly Leu
20 25 30
Thr Met Gly Trp Phe Arg Gln Ala Pro Gln Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Ile Ser Trp Thr Gly Arg Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Val Lys Asn Met Val Tyr
65 70 75 80
Leu Gln Met Val Ala Leu Asn Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Asp Trp Ala Ser Ala Thr Pro Tyr Trp Gly Gln Gly Thr Gln
100 105 110
Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr
115 120 125
Gly Ser His His His His His His
130 135
<210> 430
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 430
Ala Ala Asp Trp Ala Ser Ala Thr Pro Tyr
1 5 10
<210> 431
<211> 149
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 431
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ser Gly Glu
1 5 10 15
Ser Leu Thr Leu Ala Cys Val Ile Ser Gly Ile Thr Leu Glu Arg Tyr
20 25 30
Thr Val Gly Trp Phe His Gln Ala Pro Gly Lys Asn Pro Glu Gly Val
35 40 45
Ser Cys Ile Gly Lys Ser Asn Asp Glu Thr Phe Tyr Thr Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ser Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Thr Pro Ser Asp Ala Gly Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Lys Ala Pro Val Thr Ala Tyr Asp Cys Ser Leu Tyr Leu
100 105 110
Tyr Thr Trp Arg Ser Thr Tyr Arg Gly Gln Gly Thr Gln Val Thr Val
115 120 125
Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser His
130 135 140
His His His His His
145
<210> 432
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 432
Gly Ile Thr Leu Glu Arg Tyr Thr
1 5
<210> 433
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 433
Ile Gly Lys Ser Asn Asp Glu Thr
1 5
<210> 434
<211> 23
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 434
Ala Ala Ala Lys Ala Pro Val Thr Ala Tyr Asp Cys Ser Leu Tyr Leu
1 5 10 15
Tyr Thr Trp Arg Ser Thr Tyr
20
<210> 435
<211> 141
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 435
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Thr Leu Ser Cys Ala Ala Ser Ala Arg Thr Phe Ser Thr Tyr
20 25 30
Ala Leu Ala Trp Phe Arg Gln Thr Pro Gly Lys Gly Arg Glu Phe Val
35 40 45
Gly Ala Ile Ser Arg Ser Gly Ser Ser Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Ala Ile Ser Arg Asp Asn Ala Gln Arg Ala Ile Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Gly Ser Arg Ser Tyr Tyr Asn Ile Pro Tyr Tyr Asp Tyr Trp
100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr
115 120 125
Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 436
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 436
Ala Arg Thr Phe Ser Thr Tyr Ala
1 5
<210> 437
<211> 15
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 437
Ala Ala Gly Ser Arg Ser Tyr Tyr Asn Ile Pro Tyr Tyr Asp Tyr
1 5 10 15
<210> 438
<211> 147
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 438
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Ala
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Val Arg Thr Phe Ser Ser Tyr
20 25 30
Ala Val Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Thr Trp Ser Gly Gly Ser Lys Tyr Tyr Arg Asp Ala Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ala Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Ser Cys
85 90 95
Ala Ala Thr Ser Ala Arg Tyr Thr Ser Gly Ala Leu Tyr Tyr Arg Asp
100 105 110
Arg Gln Tyr Asn Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His
130 135 140
His His His
145
<210> 439
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 439
Val Arg Thr Phe Ser Ser Tyr Ala
1 5
<210> 440
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 440
Ile Thr Trp Ser Gly Gly Ser Lys
1 5
<210> 441
<211> 21
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 441
Ala Ala Thr Ser Ala Arg Tyr Thr Ser Gly Ala Leu Tyr Tyr Arg Asp
1 5 10 15
Arg Gln Tyr Asn Tyr
20
<210> 442
<211> 145
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 442
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Thr Arg Thr Phe Gly Asn Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Ile Asn Arg Arg Gly Thr Thr Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Asp Arg Phe Thr Ile Ser Thr Asp Tyr Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Glu Met Ile Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Val Asp Arg Ser Thr Gly Trp Gln Pro Ser Thr Ser Arg Tyr Asp
100 105 110
Tyr Ala Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala
115 120 125
Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His
130 135 140
His
145
<210> 443
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 443
Thr Arg Thr Phe Gly Asn Tyr Ala
1 5
<210> 444
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 444
Ile Asn Arg Arg Gly Thr Thr Thr
1 5
<210> 445
<211> 19
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 445
Ala Val Asp Arg Ser Thr Gly Trp Gln Pro Ser Thr Ser Arg Tyr Asp
1 5 10 15
Tyr Ala Ser
<210> 446
<211> 143
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 446
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Ser
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Arg Tyr His
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Ser Thr Ser Gly Gly Met Thr Tyr Tyr Pro Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Gly Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Phe His Gly Asp Lys Gly Tyr Gly Ser Ser Trp Tyr Tyr Asp
100 105 110
Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr
115 120 125
Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 447
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 447
Gly Arg Thr Phe Arg Tyr His Ala
1 5
<210> 448
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 448
Ile Ser Thr Ser Gly Gly Met Thr
1 5
<210> 449
<211> 17
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 449
Ala Lys Phe His Gly Asp Lys Gly Tyr Gly Ser Ser Trp Tyr Tyr Asp
1 5 10 15
Tyr
<210> 450
<211> 143
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 450
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Arg Ile
35 40 45
Ser Ile Ile Asn Ser Gly Gly Gly Ser Thr Ser Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Asn Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Gln Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Gly Val Arg Arg Asn Trp Gly Leu Gly Thr His Ser Gly Glu Tyr Val
100 105 110
Tyr Trp Gly Arg Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr
115 120 125
Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 451
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 451
Ile Asn Ser Gly Gly Gly Ser Thr
1 5
<210> 452
<211> 17
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 452
Gly Val Arg Arg Asn Trp Gly Leu Gly Thr His Ser Gly Glu Tyr Val
1 5 10 15
Tyr
<210> 453
<211> 139
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 453
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Val Arg Glu Gly Val
35 40 45
Ser Thr Ile Lys Ser Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Leu Asp Asn Ala Lys Arg Thr Val Ser
65 70 75 80
Leu Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Gly Ala Arg Arg Trp Pro Tyr Asp Tyr Ile Tyr Trp Gly Arg
100 105 110
Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val
115 120 125
Pro Asp Tyr Gly Ser His His His His His His
130 135
<210> 454
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 454
Gly Phe Thr Phe Asp Asp Tyr Ala
1 5
<210> 455
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 455
Ile Lys Ser Ser Asp Gly Ser Thr
1 5
<210> 456
<211> 13
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 456
Ala Ala Gly Ala Arg Arg Trp Pro Tyr Asp Tyr Ile Tyr
1 5 10
<210> 457
<211> 144
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 457
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Ala Gly Ala
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Glu Arg Ile Phe Leu Asn Tyr
20 25 30
Asn Met Ala Trp Val Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Ile Thr Trp Ser Gly Ser Asn Ile Asp Tyr Ala Asp Thr Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asp Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Asp Pro Ser Tyr Trp Lys Ile Arg Thr Thr Leu Asn Gly Leu
100 105 110
Asp Lys Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala
115 120 125
Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 458
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 458
Glu Arg Ile Phe Leu Asn Tyr Asn
1 5
<210> 459
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 459
Ile Thr Trp Ser Gly Ser Asn Ile
1 5
<210> 460
<211> 18
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 460
Ala Ala Asp Pro Ser Tyr Trp Lys Ile Arg Thr Thr Leu Asn Gly Leu
1 5 10 15
Asp Lys
<210> 461
<211> 138
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 461
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Phe Val Gln Ala Gly Asp
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Arg Phe
20 25 30
Pro Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Ile Ser Trp Ser Gly Gly Ser Thr Leu Ile Thr Asp Ser Val
50 55 60
Lys Asp Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Met Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Arg Arg Asp Ser Thr Thr Ser Tyr Val Thr Trp Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro
115 120 125
Asp Tyr Gly Ser His His His His His His
130 135
<210> 462
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 462
Gly Arg Thr Phe Ser Arg Phe Pro
1 5
<210> 463
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 463
Ile Ser Trp Ser Gly Gly Ser Thr
1 5
<210> 464
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 464
Ala Arg Arg Asp Ser Thr Thr Ser Tyr Val Thr
1 5 10
<210> 465
<211> 137
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 465
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Val Ser Gly Gly Thr Phe Thr Thr Tyr
20 25 30
Thr Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gln Ile Ser Trp Ser Gly Gly Ile Thr Ala Tyr Leu Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Gly Arg Lys Tyr Glu Asp Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp
115 120 125
Tyr Gly Ser His His His His His His
130 135
<210> 466
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 466
Gly Gly Thr Phe Thr Thr Tyr Thr
1 5
<210> 467
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 467
Ile Ser Trp Ser Gly Gly Ile Thr
1 5
<210> 468
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 468
Ala Arg Arg Gly Arg Lys Tyr Glu Asp Asp Tyr
1 5 10
<210> 469
<211> 136
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 469
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr
20 25 30
Ile Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Val Arg Glu Phe Val
35 40 45
Ala Ser Ile Ser Trp Ser Gly Gly Phe Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Ser Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Asp Pro Gly Gln Gly Tyr Lys Tyr Trp Gly Gln Gly Thr Gln
100 105 110
Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr
115 120 125
Gly Ser His His His His His His
130 135
<210> 470
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 470
Gly Arg Thr Phe Ser Ser Tyr Ile
1 5
<210> 471
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 471
Ile Ser Trp Ser Gly Gly Phe Thr
1 5
<210> 472
<211> 10
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 472
Ala Ala Asp Pro Gly Gln Gly Tyr Lys Tyr
1 5 10
<210> 473
<211> 138
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 473
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Ser Phe Arg Ser Tyr
20 25 30
Thr Met Gly Trp Leu Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Ile Ser Met Ser Gly Val Val Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Ala Lys Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Arg Pro Asp Arg Thr Gly Lys Ala Asp Tyr Ser Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro
115 120 125
Asp Tyr Gly Ser His His His His His His
130 135
<210> 474
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 474
Gly Arg Ser Phe Arg Ser Tyr Thr
1 5
<210> 475
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 475
Ile Ser Met Ser Gly Val Val Thr
1 5
<210> 476
<211> 12
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 476
Ala Ala Arg Pro Asp Arg Thr Gly Lys Ala Asp Tyr
1 5 10
<210> 477
<211> 137
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 477
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Phe Ser Arg Ser
20 25 30
Ile Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Arg Val
35 40 45
Ala Ala Ile Ser Trp Ser Gly Ser Leu Thr Phe Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Val Tyr
65 70 75 80
Leu Gln Met Asp Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Asp Lys Leu Gly Gly Thr Trp Asp Ser Trp Gly Pro Gly Thr
100 105 110
Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp
115 120 125
Tyr Gly Ser His His His His His His
130 135
<210> 478
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 478
Gly Gly Thr Phe Ser Arg Ser Ile
1 5
<210> 479
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 479
Ile Ser Trp Ser Gly Ser Leu Thr
1 5
<210> 480
<211> 11
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 480
Ala Ala Asp Lys Leu Gly Gly Thr Trp Asp Ser
1 5 10
<210> 481
<211> 138
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 481
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Arg Thr Phe Ser Ser Tyr
20 25 30
Ser Met Ala Trp Phe Arg Gln Ala Ser Gly Lys Glu Arg Glu Ile Val
35 40 45
Ala Ala Val Ser Arg Phe Gly Lys Phe Lys Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Thr Asp Gly Ser Ser Trp Tyr Leu Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro
115 120 125
Asp Tyr Gly Ser His His His His His His
130 135
<210> 482
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 482
Gly Arg Thr Phe Ser Ser Tyr Ser
1 5
<210> 483
<211> 12
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 483
Ala Lys Thr Asp Gly Ser Ser Trp Tyr Leu Asp Tyr
1 5 10
<210> 484
<211> 143
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 484
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Arg Thr Phe Ser Ser Tyr
20 25 30
Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Ile Ser Arg Ser Gly Gly Asn Thr Tyr Phe Gly Asp Ser Ala
50 55 60
Glu Ala Arg Phe Thr Ile Ser Arg Asp Asn Thr Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Ser Ser Leu Arg Pro Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Tyr Arg Leu Val Ala Gly Ser Thr Ser Arg Tyr Thr Tyr Asp
100 105 110
Gln Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr
115 120 125
Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 485
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 485
Gly Arg Thr Phe Ser Ser Tyr Val
1 5
<210> 486
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 486
Ile Ser Arg Ser Gly Gly Asn Thr
1 5
<210> 487
<211> 17
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 487
Ala Arg Tyr Arg Leu Val Ala Gly Ser Thr Ser Arg Tyr Thr Tyr Asp
1 5 10 15
Gln
<210> 488
<211> 141
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 488
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp
1 5 10 15
Ser Leu Arg Leu Thr Cys Ala His Ser Gly Arg Pro Phe Ser Ser Ser
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Ile Ser Arg Gly Gly Leu Ser Lys Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Phe Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Ile Tyr Tyr Cys
85 90 95
Ala Gly Ser Leu Arg Asp Arg Pro Thr Lys Asp Glu Tyr Val Val Trp
100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr
115 120 125
Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 489
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 489
Gly Arg Pro Phe Ser Ser Ser Ala
1 5
<210> 490
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 490
Ile Ser Arg Gly Gly Leu Ser Lys
1 5
<210> 491
<211> 15
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 491
Ala Gly Ser Leu Arg Asp Arg Pro Thr Lys Asp Glu Tyr Val Val
1 5 10 15
<210> 492
<211> 138
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 492
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Asp
1 5 10 15
Ser Leu Lys Leu Ser Cys Val Ala Ser Gly Arg Ser Arg Tyr Ala Leu
20 25 30
Gly Trp Phe Arg Gln Ala Pro Gly Lys Ala Arg Glu Phe Val Gly Ala
35 40 45
Ala Arg Gly Gly Ala Gly Asn Thr Tyr Tyr His Glu Ser Val Lys Gly
50 55 60
Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Leu Thr Val Tyr Leu Gln
65 70 75 80
Met Asn Asp Leu Lys Pro Glu Asp Thr Ala Val Tyr Thr Cys Ala Ala
85 90 95
Gly Lys Asp Phe Gly Thr Ala Val Ser Trp Thr Ser Trp Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro
115 120 125
Asp Tyr Gly Ser His His His His His His
130 135
<210> 493
<211> 6
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 493
Gly Arg Ser Arg Tyr Ala
1 5
<210> 494
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 494
Ala Arg Gly Gly Ala Gly Asn Thr
1 5
<210> 495
<211> 14
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 495
Ala Ala Gly Lys Asp Phe Gly Thr Ala Val Ser Trp Thr Ser
1 5 10
<210> 496
<211> 138
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 496
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Thr Phe Ser Gly Leu
20 25 30
Thr Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Asp Phe Val
35 40 45
Ala Ala Ile Ser Trp Asn Gly Arg Phe Thr Tyr Tyr Lys Asp Ser Val
50 55 60
Lys Gly Arg Phe Ala Ile Ser Arg Asp Asn Ala Lys Ser Thr Val Tyr
65 70 75 80
Leu Gln Met Thr Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Gly Ala Asp Val Leu Ser Gly Arg Gly Tyr Arg Tyr Trp Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro
115 120 125
Asp Tyr Gly Ser His His His His His His
130 135
<210> 497
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 497
Ile Ser Trp Asn Gly Arg Phe Thr
1 5
<210> 498
<211> 12
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 498
Gly Ala Asp Val Leu Ser Gly Arg Gly Tyr Arg Tyr
1 5 10
<210> 499
<211> 135
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 499
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Arg Ala Phe Ser Thr Tyr
20 25 30
Gly Met Gly Trp Phe Arg Gln Ala Pro Arg Lys Glu Arg Glu Phe Ile
35 40 45
Thr Ala Ile Asn Arg Asn Gly Asp Arg Thr Trp Tyr Ala Asp Ser Val
50 55 60
Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Met Val Tyr
65 70 75 80
Leu Gln Met Asp Ser Leu Lys Thr Glu Asp Thr Gly Ile Tyr Tyr Cys
85 90 95
His Thr Arg Arg Phe Gly Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val
100 105 110
Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly
115 120 125
Ser His His His His His His
130 135
<210> 500
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 500
Gly Arg Ala Phe Ser Thr Tyr Gly
1 5
<210> 501
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 501
Ile Asn Arg Asn Gly Asp Arg Thr
1 5
<210> 502
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 502
His Thr Arg Arg Phe Gly Tyr Asp Tyr
1 5
<210> 503
<211> 140
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 503
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Trp Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Ile Ser Gly Leu Ser Arg Tyr Tyr Ala
20 25 30
Met Gly Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Ser Val Ala
35 40 45
Thr Ile Ser Leu Arg Gly Gly Arg Thr Tyr Tyr Ala Asp Ser Val Glu
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Met Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Val
85 90 95
Ala Asp Thr Thr Trp Gly Ala Pro Arg Ser Arg Tyr His Tyr Trp Gly
100 105 110
Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp
115 120 125
Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 504
<211> 7
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 504
Gly Leu Ser Arg Tyr Tyr Ala
1 5
<210> 505
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 505
Ile Ser Leu Arg Gly Gly Arg Thr
1 5
<210> 506
<211> 15
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 506
Val Ala Asp Thr Thr Trp Gly Ala Pro Arg Ser Arg Tyr His Tyr
1 5 10 15
<210> 507
<211> 141
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 507
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Tyr
20 25 30
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Val
35 40 45
Ser Ala Ile Asn Ser Gly Gly Gly Ser Thr Thr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Met Gly Ser Ser Ser Arg Gly Asn Arg Tyr Leu Glu Val Trp
100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr
115 120 125
Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 508
<211> 15
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 508
Ala Lys Met Gly Ser Ser Ser Arg Gly Asn Arg Tyr Leu Glu Val
1 5 10 15
<210> 509
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 509
Ile Asn Ser Gly Gly Gly Ser Thr
1 5
<210> 510
<211> 15
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 510
Ala Lys Met Gly Ser Ser Ser Arg Gly Asn Arg Tyr Leu Glu Val
1 5 10 15
<210> 511
<211> 142
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 511
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp
1 5 10 15
Ser Leu Arg Leu Ser Cys Glu Ala Ser Ala Arg Thr Phe Ile Ala Tyr
20 25 30
Ala Val Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Ile Ser Trp Asn Gly Gly Met Thr Asp Tyr Ala Asp Phe Val
50 55 60
Lys Gly Arg Phe Ala Ile Ser Arg Asp Asn Ala Lys Thr Val Ser Leu
65 70 75 80
Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Thr
85 90 95
Ala Gly Pro Ala Arg Arg Ser Tyr Ser Tyr Arg Asp Gly Tyr Asp Tyr
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro
115 120 125
Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 512
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 512
Ala Arg Thr Phe Ile Ala Tyr Ala
1 5
<210> 513
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 513
Ile Ser Trp Asn Gly Gly Met Thr
1 5
<210> 514
<211> 17
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 514
Thr Ala Gly Pro Ala Arg Arg Ser Tyr Ser Tyr Arg Asp Gly Tyr Asp
1 5 10 15
Tyr
<210> 515
<211> 142
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 515
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Glu Ala Ser Ala Arg Thr Phe Ile Ala Tyr
20 25 30
Ala Val Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Ile Ser Trp Asn Gly Gly Met Thr Asp Tyr Ala Asp Phe Val
50 55 60
Lys Gly Arg Phe Ala Ile Ser Arg Asp Ser Ala Lys Thr Val Ser Leu
65 70 75 80
Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Thr
85 90 95
Ala Gly Pro Ala Arg Arg Ser Tyr Ser Tyr Arg Asp Gly Tyr Asp Tyr
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro
115 120 125
Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 516
<211> 142
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 516
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Val Gly Gly
1 5 10 15
Ser Leu Arg Ile Ser Cys Val Ala Ser Gly Arg Thr Gly Ser Tyr Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Ile Ser Trp Asn Gly Gly Met Thr Asp Tyr Ala Asp Phe Val
50 55 60
Lys Gly Arg Phe Ala Ile Ser Arg Asp Asn Ala Lys Thr Val Ser Leu
65 70 75 80
Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Thr
85 90 95
Ala Gly Pro Ala Arg Arg Ser Tyr Ser Tyr Arg Asp Gly Tyr Asp Tyr
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro
115 120 125
Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 517
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 517
Gly Arg Thr Gly Ser Tyr Tyr Ala
1 5
<210> 518
<211> 142
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 518
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Glu Ala Ser Ala Arg Thr Phe Ile Ala Tyr
20 25 30
Ala Val Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Ile Ser Trp Asn Gly Gly Met Thr Asp Tyr Ala Asp Phe Val
50 55 60
Lys Gly Arg Phe Ala Ile Ser Arg Asp Asn Ala Lys Thr Val Ser Leu
65 70 75 80
Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Thr
85 90 95
Ala Gly Pro Ala Arg Arg Ser Tyr Ser Tyr Arg Asp Gly Tyr Asp Tyr
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro
115 120 125
Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 519
<211> 142
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 519
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ser Gly Gly
1 5 10 15
Ser Leu Thr Leu Ser Cys Ala Ala Ser Ala Arg Thr Phe Ile Ala Tyr
20 25 30
Ala Val Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Ile Ser Trp Asn Gly Gly Met Thr Asp Tyr Ala Asp Phe Val
50 55 60
Lys Gly Arg Phe Ala Ile Ser Arg Asp Asn Ala Lys Thr Val Ser Leu
65 70 75 80
Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Leu Tyr Tyr Cys Thr
85 90 95
Ala Gly Pro Ala Arg Arg Ser Tyr Ser Tyr Arg Asp Gly Tyr Asp Tyr
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro
115 120 125
Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 520
<211> 141
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 520
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Thr Asn
20 25 30
Ala Met Gly Trp Tyr Arg Gln Ala Pro Asp Lys Gln Arg Glu Phe Leu
35 40 45
Ala Val Ile Thr Pro Arg Gly Arg Thr Ala Tyr Ala Asp Ser Ala Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Met Asn Ser Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Phe Glu Asp Thr Ala Val Tyr Tyr Cys Tyr
85 90 95
Ala Gly Arg Tyr Arg Ser Tyr Asp Ala Arg Phe Ala Thr Asp Ile Trp
100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr
115 120 125
Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 521
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 521
Gly Ser Ile Phe Ser Thr Asn Ala
1 5
<210> 522
<211> 7
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 522
Ile Thr Pro Arg Gly Arg Thr
1 5
<210> 523
<211> 16
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 523
Tyr Ala Gly Arg Tyr Arg Ser Tyr Asp Ala Arg Phe Ala Thr Asp Ile
1 5 10 15
<210> 524
<211> 141
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 524
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Thr Asn
20 25 30
Ala Met Gly Trp Tyr Arg Gln Ala Pro Asp Lys Gln Arg Glu Phe Leu
35 40 45
Ala Val Ile Thr Pro Arg Gly Arg Thr Ala Tyr Ala Asp Ser Ala Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Met Asn Ser Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Phe Glu Asp Thr Ala Val Tyr Tyr Cys Tyr
85 90 95
Ala Gly Arg Tyr Arg Ser Tyr Asp Ala Arg Phe Ala Thr Asp Ile Trp
100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr
115 120 125
Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 525
<211> 141
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 525
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Thr Asn
20 25 30
Ala Met Gly Trp Tyr Arg Gln Ala Pro Asp Lys Gln Arg Glu Phe Leu
35 40 45
Ala Val Ile Thr Pro Arg Gly Arg Thr Ala Tyr Ala Asp Ser Ala Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Met Asn Ser Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Phe Glu Asp Thr Ala Val Tyr Tyr Cys Tyr
85 90 95
Ala Gly Arg Tyr Arg Ser Phe Asp Ala Arg Phe Ala Thr Asp Ile Trp
100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr
115 120 125
Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 526
<211> 16
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 526
Tyr Ala Gly Arg Tyr Arg Ser Phe Asp Ala Arg Phe Ala Thr Asp Ile
1 5 10 15
<210> 527
<211> 141
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 527
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Arg Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Thr Asn
20 25 30
Ala Met Gly Trp Tyr Arg Gln Ala Pro Asp Lys Gln Arg Glu Phe Leu
35 40 45
Ala Val Ile Thr Pro Arg Gly Arg Thr Ala Tyr Ala Asp Ser Ala Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Met Asn Ser Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Phe Glu Asp Thr Ala Val Tyr Tyr Cys Tyr
85 90 95
Ala Gly Arg Tyr Arg Ser Phe Asp Ala Arg Phe Ala Thr Asp Ile Trp
100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr
115 120 125
Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 528
<211> 141
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 528
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Thr Asn
20 25 30
Ala Met Gly Trp Tyr Arg Gln Ala Pro Asp Lys Gln Arg Glu Phe Leu
35 40 45
Ala Val Ile Thr Pro Arg Gly Arg Thr Ala Tyr Ala Asp Ser Ala Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Met Asn Ser Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Phe Glu Asp Thr Ala Val Tyr Tyr Cys Tyr
85 90 95
Ala Gly Arg Tyr Arg Ser Phe Asp Ala Arg Phe Ala Thr Asp Ile Trp
100 105 110
Ser Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr
115 120 125
Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 529
<211> 141
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 529
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Thr Asn
20 25 30
Ala Met Gly Trp Tyr Arg Gln Ala Pro Asp Lys Gln Arg Glu Phe Leu
35 40 45
Ala Val Ile Thr Pro Arg Gly Arg Thr Ala Tyr Ala Asp Ser Ala Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Val Asn Ser Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Phe Glu Asp Thr Ala Val Tyr Tyr Cys Tyr
85 90 95
Ala Gly Arg Tyr Arg Ser Phe Asp Ala Arg Phe Ala Thr Asp Ile Trp
100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr
115 120 125
Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 530
<211> 139
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 530
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val His Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Ala Phe Leu Thr Gly Ala
20 25 30
Thr Val Gly Trp Tyr Arg Gln Ala Pro Gly Lys Leu Arg Glu Leu Val
35 40 45
Ala Ala Ile Ile Thr Gly Gly Thr Thr Thr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Ser Ile Ser Arg Asp Tyr Thr Lys Arg Ala Leu Ile Leu
65 70 75 80
Gln Met Asp Ser Leu Arg Pro Asp Asp Thr Ala Val Tyr Phe Cys Ser
85 90 95
Ile Arg Gly Phe Tyr Arg Gln Thr Gln Phe Arg Glu Ile Trp Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val
115 120 125
Pro Asp Tyr Gly Ser His His His His His His
130 135
<210> 531
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 531
Gly Ala Phe Leu Thr Gly Ala Thr
1 5
<210> 532
<211> 7
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 532
Ile Ile Thr Gly Gly Thr Thr
1 5
<210> 533
<211> 14
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 533
Ser Ile Arg Gly Phe Tyr Arg Gln Thr Gln Phe Arg Glu Ile
1 5 10
<210> 534
<211> 139
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 534
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Ala Phe Leu Thr Gly Ala
20 25 30
Thr Val Gly Trp Tyr Arg Gln Ala Pro Gly Lys Leu Arg Glu Leu Val
35 40 45
Ala Ala Ile Ile Thr Gly Gly Thr Thr Thr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Ser Ile Ser Arg Asp Tyr Thr Lys Arg Ala Leu Ile Leu
65 70 75 80
Gln Met Asp Ser Leu Arg Pro Asp Asp Thr Ala Val Tyr Phe Cys Ser
85 90 95
Ile Arg Gly Phe Tyr Arg Gln Thr Gln Phe Arg Glu Ile Trp Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val
115 120 125
Pro Asp Tyr Gly Ser His His His His His His
130 135
<210> 535
<211> 139
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 535
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Ala Phe Leu Thr Gly Ala
20 25 30
Thr Val Gly Trp Tyr Arg Gln Ala Pro Gly Lys Leu Arg Glu Leu Val
35 40 45
Ala Ala Ile Ile Thr Gly Gly Thr Thr Thr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Ser Ile Ser Arg Asp Tyr Thr Lys Arg Ala Leu Ile Leu
65 70 75 80
Gln Met Asp Ser Leu Arg Pro Asp Asp Thr Ala Val Tyr Phe Cys Ser
85 90 95
Ile Arg Gly Phe Tyr Arg Gln Thr Gln Phe Arg Glu Ile Trp Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val
115 120 125
Pro Asp Tyr Gly Ser His His His His His His
130 135
<210> 536
<211> 139
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 536
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Glu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Ala Phe Leu Thr Gly Ala
20 25 30
Thr Val Gly Trp Tyr Arg Gln Ala Pro Gly Lys Leu Arg Glu Leu Val
35 40 45
Ala Ala Ile Ile Thr Gly Gly Thr Thr Thr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Ser Ile Ser Arg Asp Tyr Thr Lys Arg Ala Leu Ile Leu
65 70 75 80
Gln Met Asp Ser Leu Arg Pro Asp Asp Thr Ala Val Tyr Phe Cys Ser
85 90 95
Ile Arg Gly Phe Tyr Arg Gln Thr Gln Phe Arg Glu Ile Trp Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val
115 120 125
Pro Asp Tyr Gly Ser His His His His His His
130 135
<210> 537
<211> 139
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 537
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Glu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Ala Phe Leu Thr Gly Ala
20 25 30
Thr Val Gly Trp Tyr Arg Gln Thr Pro Gly Asn Leu Arg Glu Leu Val
35 40 45
Ala Ala Ile Ile Thr Gly Gly Thr Thr Thr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Ser Ile Ser Arg Asp Tyr Thr Lys Arg Ala Leu Ile Leu
65 70 75 80
Gln Met Asp Ser Leu Arg Pro Asp Asp Thr Ala Val Tyr Phe Cys Ser
85 90 95
Ile Arg Gly Phe Tyr Arg Gln Thr Gln Phe Arg Glu Ile Trp Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val
115 120 125
Pro Asp Tyr Gly Ser His His His His His His
130 135
<210> 538
<211> 143
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 538
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Asn Tyr
20 25 30
Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Ile
35 40 45
Gly Ala Ile Asn Arg Ser Ser Thr Arg Leu Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Ser Ile Ser Arg Asp Asn Ala Lys Ser Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Asp Leu Val Ser Ile Leu Gly Lys Gly Tyr Arg Asp Val Asp
100 105 110
Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr
115 120 125
Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 539
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 539
Gly Arg Thr Phe Ser Asn Tyr Val
1 5
<210> 540
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 540
Ile Asn Arg Ser Ser Thr Arg Leu
1 5
<210> 541
<211> 17
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 541
Ala Ala Asp Leu Val Ser Ile Leu Gly Lys Gly Tyr Arg Asp Val Asp
1 5 10 15
Tyr
<210> 542
<211> 143
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 542
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Asn Tyr
20 25 30
Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Ile
35 40 45
Gly Ala Ile Asn Arg Ser Ser Thr Arg Leu Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Ser Ile Ser Arg Asp Asn Ala Lys Ser Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Asp Leu Val Ser Ile Leu Gly Lys Gly Tyr Arg Asp Val Asp
100 105 110
Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr
115 120 125
Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 543
<211> 143
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 543
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr
20 25 30
Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Ile
35 40 45
Gly Ala Ile Asn Arg Ser Ser Thr Arg Leu Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Ser Ile Ser Arg Asp Asn Ala Lys Ser Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Asp Leu Val Ser Ile Leu Gly Lys Gly Tyr Arg Asp Val Asp
100 105 110
Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr
115 120 125
Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 544
<211> 143
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 544
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Val Ser Cys Ala Ala Ser Gly Phe Ser Phe Ser Asn Tyr
20 25 30
Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Leu Ile
35 40 45
Gly Ala Ile Asn Arg Ser Ser Thr Arg Leu Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Ser Ile Ser Arg Asp Asn Ala Lys Ser Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Asp Leu Val Ser Ile Leu Gly Lys Gly Tyr Arg Asp Val Asp
100 105 110
Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr
115 120 125
Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 545
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 545
Gly Phe Ser Phe Ser Asn Tyr Val
1 5
<210> 546
<211> 144
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 546
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Thr Phe Ser Pro Tyr
20 25 30
Thr Met Gly Trp Phe Arg Arg Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Ile Ser Trp Gly Ala Gly Val Lys Ser Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Glu Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Met Leu Lys Pro Asp Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
Ala Ala Lys Arg Pro Ile Ser Gly Ser Tyr Ser Asn Glu Arg Asp Tyr
100 105 110
Ala Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala
115 120 125
Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 547
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 547
Gly Arg Thr Phe Ser Pro Tyr Thr
1 5
<210> 548
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 548
Ile Ser Trp Gly Ala Gly Val Lys
1 5
<210> 549
<211> 18
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 549
Ala Ala Lys Arg Pro Ile Ser Gly Ser Tyr Ser Asn Glu Arg Asp Tyr
1 5 10 15
Ala Tyr
<210> 550
<211> 144
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 550
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Arg Thr Phe Ser Pro Tyr
20 25 30
Thr Met Gly Trp Phe Arg Arg Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Ile Ser Trp Gly Ala Gly Val Lys Ser Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Glu Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Met Leu Lys Pro Asp Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
Ala Ala Lys Arg Pro Ile Ser Gly Ser Tyr Ser Asn Glu Arg Asp Tyr
100 105 110
Ala Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala
115 120 125
Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 551
<211> 134
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 551
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Glu
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Gly Thr Asn Phe Leu Ser Ser Arg Phe
20 25 30
Glu Met Gly Trp Tyr Arg Gln Ile Pro Gly Lys Gln Arg Glu Leu Val
35 40 45
Ala Arg Ile Phe Arg Asp Gly Asn Thr Asp Tyr Val Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Thr Ala Lys Asn Thr Ile Asp Leu
65 70 75 80
Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Gly Tyr Phe Cys His
85 90 95
Val His Ile Leu Gly Arg Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr
100 105 110
Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser
115 120 125
His His His His His His
130
<210> 552
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 552
Asn Phe Leu Ser Ser Arg Phe Glu
1 5
<210> 553
<211> 7
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 553
Ile Phe Arg Asp Gly Asn Thr
1 5
<210> 554
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 554
His Val His Ile Leu Gly Arg Asp Tyr
1 5
<210> 555
<211> 144
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 555
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp
1 5 10 15
Ser Leu Arg Ile Ser Cys Lys Ala Ser Gly Arg Thr Phe Ser Ser Tyr
20 25 30
Pro Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Ile Ser Arg Ser Gly Gly Arg Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Leu Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Leu Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Lys Thr Thr Met Gly Leu Pro Val Gly Gly Thr Tyr Glu Tyr
100 105 110
Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala
115 120 125
Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 556
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 556
Gly Arg Thr Phe Ser Ser Tyr Pro
1 5
<210> 557
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 557
Ile Ser Arg Ser Gly Gly Arg Thr
1 5
<210> 558
<211> 18
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 558
Ala Ala Lys Thr Thr Met Gly Leu Pro Val Gly Gly Thr Tyr Glu Tyr
1 5 10 15
Asp Tyr
<210> 559
<211> 144
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 559
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp
1 5 10 15
Ser Leu Arg Ile Ser Cys Lys Ala Ser Gly Arg Thr Phe Ser Ser Tyr
20 25 30
Pro Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Ile Ser Arg Ser Gly Gly Arg Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Leu Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Lys Thr Thr Met Gly Leu Pro Val Gly Gly Thr Tyr Glu Tyr
100 105 110
Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala
115 120 125
Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 560
<211> 142
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 560
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Asp Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Ser Phe Val
35 40 45
Ala Ala Ile Asp Ser Asn Gly Ser Asn Thr Tyr Tyr Ala Asp Ser Leu
50 55 60
Lys Gly Arg Phe Thr Ile Ala Arg Asp Asn Ala Lys Ser Met Val Phe
65 70 75 80
Leu Arg Met Asn Asn Leu Glu Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Gly Gln Asn Phe Trp Thr Phe Thr Thr Thr Pro Pro Pro Tyr
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro
115 120 125
Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 561
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 561
Gly Phe Thr Leu Asp Asp Tyr Ala
1 5
<210> 562
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 562
Ile Asp Ser Asn Gly Ser Asn Thr
1 5
<210> 563
<211> 16
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 563
Ala Ala Gly Gln Asn Phe Trp Thr Phe Thr Thr Thr Pro Pro Pro Tyr
1 5 10 15
<210> 564
<211> 145
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 564
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp
1 5 10 15
Ser Leu Arg Ile Ser Cys Lys Ala Ser Gly Arg Thr Phe Ser Asn Tyr
20 25 30
Pro Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Ile Ser Arg Ser Gly Gly Gly Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Lys Arg Asp Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Tyr Phe Gly Asn Leu Gly Gly Gly Val Gly Arg Ser Ser Asp
100 105 110
Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala
115 120 125
Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His
130 135 140
His
145
<210> 565
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 565
Gly Arg Thr Phe Ser Asn Tyr Pro
1 5
<210> 566
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 566
Ile Ser Arg Ser Gly Gly Gly Thr
1 5
<210> 567
<211> 19
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 567
Ala Ala Tyr Phe Gly Asn Leu Gly Gly Gly Val Gly Arg Ser Ser Asp
1 5 10 15
Tyr Asp Tyr
<210> 568
<211> 134
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 568
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Glu
1 5 10 15
Ser Leu Thr Leu Ser Cys Ala Ala Ser Asn Val Leu Val Ser Lys Phe
20 25 30
Thr Val Ala Trp Phe Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val
35 40 45
Ala Asp Ile Ala Arg Ala Gly Phe Thr Ser Tyr Ala Asp Phe Val Arg
50 55 60
Gly Arg Phe Ser Ile Ser Arg Asp Asn Ala Gln Asn Thr Val Thr Leu
65 70 75 80
Gln Met Asn Ser Leu Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn
85 90 95
Cys His Val Leu Gly Arg Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr
100 105 110
Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser
115 120 125
His His His His His His
130
<210> 569
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 569
Asn Val Leu Val Ser Lys Phe Thr
1 5
<210> 570
<211> 7
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 570
Ile Ala Arg Ala Gly Phe Thr
1 5
<210> 571
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 571
Asn Cys His Val Leu Gly Arg Asp Tyr
1 5
<210> 572
<211> 134
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 572
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Ile Arg Thr Phe Ser Thr Tyr
20 25 30
Ala Phe Ala Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val
35 40 45
Ala Gly Ile Ser Ser Gly Ser Arg Thr Asn Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Lys Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Asn Leu Gln Pro Glu Asp Thr Ala Val Tyr Tyr Cys Thr
85 90 95
Lys Gly Arg Thr Asn Ile Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr
100 105 110
Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser
115 120 125
His His His His His His
130
<210> 573
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 573
Ile Arg Thr Phe Ser Thr Tyr Ala
1 5
<210> 574
<211> 7
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 574
Ile Ser Ser Gly Ser Arg Thr
1 5
<210> 575
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 575
Thr Lys Gly Arg Thr Asn Ile Asp Tyr
1 5
<210> 576
<211> 144
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 576
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Ser Tyr
20 25 30
Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Thr Ile Ser Thr Gly Gly Gly Thr Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Gly Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Lys Met Ala Thr Gly Thr Ala Ser Ile Arg Thr Tyr Glu Tyr
100 105 110
Ala Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala
115 120 125
Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 577
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 577
Ile Ser Thr Gly Gly Gly Thr Thr
1 5
<210> 578
<211> 18
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 578
Ala Ala Lys Met Ala Thr Gly Thr Ala Ser Ile Arg Thr Tyr Glu Tyr
1 5 10 15
Ala Tyr
<210> 579
<211> 142
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 579
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Thr Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Ile Leu Trp Gly Gly Val Ile Ala Asp Ser Val Lys Gly Arg
50 55 60
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Asp Leu Gln Met
65 70 75 80
Phe Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Arg
85 90 95
Arg Gly Gly Leu Asn Asn His Leu Gly Ser Val Gly Asn Tyr Asp Tyr
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro
115 120 125
Tyr Asp Val Pro Asp Tyr Gly Ser His His His His His His
130 135 140
<210> 580
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 580
Gly Phe Thr Phe Ser Arg Tyr Ala
1 5
<210> 581
<211> 6
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 581
Ile Leu Trp Gly Gly Val
1 5
<210> 582
<211> 19
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 582
Ala Arg Arg Arg Gly Gly Leu Asn Asn His Leu Gly Ser Val Gly Asn
1 5 10 15
Tyr Asp Tyr
<210> 583
<211> 134
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 583
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Ser Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Glu Ile Ile Gly Ala Thr Val Ser Ser Ser
20 25 30
Ser Met Ala Trp Tyr Arg Gln Ala Pro Gly Leu Gln Arg Glu Leu Val
35 40 45
Ala Gly Ile Thr Thr Pro Ser Asn Pro His Tyr Ala Ala Ser Val Arg
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Gly Ala Arg Asn Leu Asn Tyr Leu
65 70 75 80
Gln Ile Asp Ser Ala Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys His
85 90 95
Ala Ala Ile Arg Gly Ser Ile Tyr Arg Gly Gln Gly Thr Gln Val Thr
100 105 110
Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Gly Ser
115 120 125
His His His His His His
130
<210> 584
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 584
Gly Ala Thr Val Ser Ser Ser Ser
1 5
<210> 585
<211> 7
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 585
Ile Thr Thr Pro Ser Asn Pro
1 5
<210> 586
<211> 9
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 586
His Ala Ala Ile Arg Gly Ser Ile Tyr
1 5
<210> 587
<211> 138
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 587
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Arg Ala Phe Thr Asn Tyr
20 25 30
His Met Gly Trp Phe Arg Gln Thr Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Leu Ile Ser Trp Ser Gly Asp Ser Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg Thr Val Ala
65 70 75 80
Leu Gln Met Asp Ser Leu Lys Pro Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Ser Arg Leu Val Gly Gln Ser Gln Tyr Glu Val Trp Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro
115 120 125
Asp Tyr Gly Ser His His His His His His
130 135
<210> 588
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 588
Gly Arg Ala Phe Thr Asn Tyr His
1 5
<210> 589
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 589
Ile Ser Trp Ser Gly Asp Ser Thr
1 5
<210> 590
<211> 12
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 590
Ala Ser Arg Leu Val Gly Gln Ser Gln Tyr Glu Val
1 5 10
<210> 591
<211> 138
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 591
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Ala Phe Thr Asn Tyr
20 25 30
His Met Gly Trp Phe Arg Gln Thr Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Leu Ile Ser Trp Ser Gly Asp Ser Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg Thr Val Ala
65 70 75 80
Leu Gln Met Asp Ser Leu Lys Pro Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Ser Arg Leu Val Gly Gln Ser Gln Tyr Glu Ile Trp Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro
115 120 125
Asp Tyr Gly Ser His His His His His His
130 135
<210> 592
<211> 12
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 592
Ala Ser Arg Leu Val Gly Gln Ser Gln Tyr Glu Ile
1 5 10
<210> 593
<211> 138
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 593
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Ala Phe Thr Asn Tyr
20 25 30
His Met Gly Trp Phe Arg Gln Thr Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Leu Ile Ser Trp Ser Gly Asp Ser Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg Thr Val Ala
65 70 75 80
Leu Gln Met Asp Ser Leu Lys Pro Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Ser Arg Leu Val Gly Gln Ser Gln Tyr Glu Val Trp Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro
115 120 125
Asp Tyr Gly Ser His His His His His His
130 135
<210> 594
<211> 138
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 594
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Thr Arg Ala Phe Thr Asn Tyr
20 25 30
His Met Gly Trp Phe Arg Gln Thr Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Leu Ile Ser Trp Ser Gly Asp Ser Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg Thr Val Ala
65 70 75 80
Leu Gln Met Asp Ser Leu Lys Pro Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Ser Arg Leu Val Gly Gln Ser Gln Tyr Glu Ile Trp Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro
115 120 125
Asp Tyr Gly Ser His His His His His His
130 135
<210> 595
<211> 8
<212> PRT
<213> Artificial sequence
<220>
<223> Complementarity-determining region amino acid sequence
<400> 595
Thr Arg Ala Phe Thr Asn Tyr His
1 5
<210> 596
<211> 138
<212> PRT
<213> Artificial sequence
<220>
<223> nanobody amino acid sequence
<400> 596
Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Thr Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Ala Phe Thr Asn Tyr
20 25 30
His Met Gly Trp Phe Arg Gln Thr Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Leu Ile Ser Trp Ser Gly Asp Ser Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Arg Thr Val Ala
65 70 75 80
Leu Gln Met Asp Ser Leu Lys Pro Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Ser Arg Leu Val Gly Gln Ser Gln Tyr Glu Val Trp Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser Ala Ala Ala Tyr Pro Tyr Asp Val Pro
115 120 125
Asp Tyr Gly Ser His His His His His His
130 135

Claims (42)

1.一种特异性结合昆虫多肽的纳米抗体，所述昆虫多肽选自：
(i)几丁质合成酶；
(ii)几丁质脱乙酰酶；
(iii)海藻糖酶；
(iv)细胞色素p450单加氧酶；
(v)NPC1甾醇转运体；
(vi)包含几丁质结合域(CBD)的多肽，其中所述纳米抗体结合所述CBD；和
(vii)V-ATP酶亚基c，其中，所述纳米抗体包含如下序列号中所示的互补性决定区(CDR)：167-169；171和168-169；174和168-169；167、178和169；或者180-182，这些序列在所述纳米抗体上按从N到C的顺序排列；
其中，所述纳米抗体与所述昆虫多肽的结合赋予所述纳米抗体昆虫防控活性。
2.一种包含纳米抗体的组合物，所述纳米抗体特异性结合选自以下的昆虫多肽：
(i)几丁质合成酶；
(ii)几丁质脱乙酰酶；
(iii)海藻糖酶；
(iv)细胞色素p450单加氧酶；
(v)NPC1甾醇转运体；
(vi)包含几丁质结合域(CBD)的多肽，其中，所述纳米抗体结合所述CBD；和
(vii)V-ATP酶亚基c，其中，所述纳米抗体包含如下序列号中所示的互补性决定区(CDR)：167-169；171和168-169；174和168-169；167、178和169；180-182；187-189；或191-193，这些序列在所述纳米抗体上按从N到C的顺序排列；
以及具有昆虫防控活性的毒素部分。
3.根据权利要求2所述的组合物，其中，所述纳米抗体与所述昆虫多肽的结合赋予所述纳米抗体昆虫防控活性。
4.根据权利要求1所述的纳米抗体或根据权利要求3所述的组合物，其中，所述纳米抗体下调所述昆虫多肽的活性。
5.根据权利要求1至4中任一项所述的纳米抗体或组合物，被配制用于通过喷雾、灌溉和/或熏蒸递送。
6.根据权利要求1至5中任一项所述的纳米抗体，其中所述CBD是2型几丁质结合域。
7.根据权利要求1至6中任一项所述的纳米抗体，其中所述CBD包含选自序列号为3-7的氨基酸序列。
8.根据权利要求1至7中任一项所述的纳米抗体，其中，所述纳米抗体特异性结合所述CBD并包含如下序列号中所示的互补性决定区(CDR)：15-17；19-21；23-25；或者15和28-29，这些序列在所述纳米抗体上按从N到C的顺序排列。
9.根据权利要求2和5至7中任一项所述的纳米抗体，其中，所述纳米抗体特异性结合所述CBD并包含如下序列号中所示的互补性决定区(CDR)：15-17；19-21；23-25；15和28-29；31-33；35-37；39-41；43-45；47-49；51-53；55-57；59-61；63-65；67-69；71-73；75-77；79-81；83-85；87-89；91、80和93；95-97；99-101；103-105；107-109；111-113；115-117；119-121；123-125；127-129；131-133；135-137；139-141；143-145；147-149；151-153；15和156-157；159-161；或者163-165，这些序列在所述纳米抗体上按从N到C的顺序排列。
10.根据权利要求2至4中任一项所述的纳米抗体，其中，所述纳米抗体特异性结合所述V-ATP酶亚基c并包含如下序列号中所示的互补性决定区(CDR)：167-169；171和168-169；174和168-169；167、178和169；或180-182，这些序列在所述纳米抗体上按从N到C的顺序排列。
11.根据权利要求1至3和5中任一项所述的纳米抗体，其中，所述纳米抗体特异性结合所述海藻糖酶并包含如下序列号中所示的互补性决定区(CDR)：195-197；199和196-197；201-203；201和205-206；208-210；212和209-210；215-217；219-221；223-225；227-229；231、228和232；234-236；238-240；242-244；246-248；250-252；254-256；258-260；588-590；588-589和592；或者595、589和592，这些序列在所述纳米抗体上按从N到C的顺序排列。
12.根据权利要求1至3和5中任一项所述的纳米抗体，其中，所述纳米抗体特异性结合所述海藻糖酶并包含如下序列号中所示的互补性决定区(CDR)：195-197；199和196-197；201-203；201和205-206；208-210；212和209-210；215-217；219-221；223-225；227-229；231、228和232；234-236；238-240；242-244；246-248；250-252；254-256；或者258-260，这些序列在所述纳米抗体上按从N到C的顺序排列。
13.根据权利要求1至3和5中任一项所述的纳米抗体，其中，所述纳米抗体特异性结合所述海藻糖酶并包含如下序列号中所示的互补性决定区(CDR)：201和205-206；208-210；212和209-210；223-225；238-240；242-244；246-248；588-590；或者595、589和592，这些序列在所述纳米抗体上按从N到C的顺序排列。
14.根据权利要求1至3和5中任一项所述的纳米抗体，其中，所述纳米抗体特异性结合所述细胞色素p450单加氧酶并包含如下序列号中所示的互补性决定区(CDR)：262-264；262-263和266；262、270和264；274-276；280和275-276；282和275-276；282、275和285；287、220和289；291、220和289；239-294和289；296-298；300-302；304-306；308-310；312-314；或者316-318，这些序列在所述纳米抗体上按从N到C的顺序排列。
15.根据权利要求1至3和5中任一项所述的纳米抗体，其中，所述纳米抗体特异性结合所述细胞色素p450单加氧酶并包含如下序列号中所示的互补性决定区(CDR)：262-264；287、220和289；296-298；300-302；304-306；或者312-314，这些序列在所述纳米抗体上按从N到C的顺序排列。
16.根据权利要求1至3和5中任一项所述的纳米抗体，其中，所述纳米抗体特异性结合所述几丁质脱乙酰酶并包含如下序列号中所示的互补性决定区(CDR)：320-322；320和324-325；327、321和328；320-321和332；208和336-337；320、324和339；343-345；347-349；351-353；351、347和356；358-360；358-359和362；365、359和366；368-370；372和369-370；374、369和375；377-379；381-383；385和382-383；387-389；387、391和389；394-396；398-400；402-404；或者406-408，这些序列在所述纳米抗体上按从N到C的顺序排列。
17.根据权利要求1至3和5中任一项所述的纳米抗体，其中，所述纳米抗体特异性结合所述几丁质脱乙酰酶并包含如下序列号中所示的互补性决定区(CDR)：320-322；320和324-325；372和369-370；374、369和375；377-379；387-389；或者398-400，这些序列在所述纳米抗体上按从N到C的顺序排列。
18.根据权利要求1至3和5中任一项所述的纳米抗体，其中，所述纳米抗体特异性结合所述几丁质合成酶并包含如下序列号中所示的互补性决定区(CDR)：412-414；418-420；422-424；426-428；426-427和430；432-434；436、140和437；439-441；443-445；447-449；356和451-452；454-456；458-460；462-464；466-468；470-472；474-476；478-480；482、479和483；485-487；489-491；493-495；426和497-498；500-502；504-506；或者508-510，这些序列在所述纳米抗体上按从N到C的顺序排列。
19.根据权利要求1至3和5中任一项所述的纳米抗体，其中，所述纳米抗体特异性结合所述几丁质合成酶并包含如下序列号中所示的互补性决定区(CDR)：412-414；418-420；426-428；432-434；443-445；447-449；466-468；482、479和483；426和497-498；或者504-506，这些序列在所述纳米抗体上按从N到C的顺序排列。
20.根据权利要求1至3和5中任一项所述的纳米抗体，其中，所述纳米抗体特异性结合所述NPC1甾醇转运体并包含如下序列号中所示的互补性决定区(CDR)：512-514；517和513-514；521-523；521-522和526；531-533；539-541；485和540-541；545和540-451；547-549；552-554；556-558；561-563；565-567；569-571；573-575；485和577-578；580-582；或者584-586，这些序列在所述纳米抗体上按从N到C的顺序排列。
21.根据权利要求1至3和5中任一项所述的纳米抗体，其中，所述纳米抗体特异性结合所述NPC1甾醇转运体并包含如下序列号中所示的互补性决定区(CDR)：517和513-514；485和540-541；545和540-541；556-558；565-567；573-575；485和577-578；或者580-582，这些序列在所述纳米抗体上按从N到C的顺序排列。
22.一种多核苷酸，编码根据权利要求1至21中任一项所述的纳米抗体或组合物。
23.一种核酸构建体，包含根据权利要求22所述的多核苷酸和用于指导所述多核苷酸表达的顺式作用调节元件。
24.一种宿主细胞，包含根据权利要求1至21中任一项所述的纳米抗体或组合物或编码其的多核苷酸或核酸构建体。
25.一种生产昆虫防控纳米抗体的方法，所述方法包括在宿主细胞中表达根据权利要求22所述的多核苷酸或根据权利要求23所述的核酸构建体。
26.根据权利要求25所述的方法，包括分离所述纳米抗体。
27.一种昆虫防控方法，所述方法包括使所述昆虫与根据权利要求1至21中任一项所述的纳米抗体或组合物、编码其的多核苷酸或核酸构建体或表达其的宿主细胞接触。
28.根据权利要求27所述的方法，其中，所述接触包括将所述纳米抗体或所述组合物直接施用于所述昆虫。
29.根据权利要求27所述的方法，其中，所述接触包括将所述纳米抗体或所述组合物施用于可以与所述昆虫接触的生物体或表面。
30.根据权利要求1至29中任一项所述的纳米抗体、组合物或方法，其中，所述纳米抗体或所述组合物被配制成液体制剂。
31.根据权利要求1至29中任一项所述的纳米抗体、组合物或方法，其中，所述纳米抗体或所述组合物配制成干制剂。
32.一种植物，包含根据权利要求1至21中任一项所述的纳米抗体或组合物或编码其的多核苷酸或核酸构建体。
33.根据权利要求32所述的植物，所述植物是转基因植物。
34.一种商品产品，包含根据权利要求1至21中任一项所述的纳米抗体或组合物。
35.根据权利要求34所述的商品产品，其中所述商品产品由根据权利要求32-33中任一项所述的植物生产。
36.一种生产纳米抗体的方法，所述方法包括用重组或纯化的昆虫多肽免疫骆驼科动物，所述重组或纯化的昆虫多肽选自：
(i)几丁质合成酶；
(ii)几丁质脱乙酰酶；
(iii)海藻糖酶；
(iv)细胞色素p450单加氧酶；
(v)NPC1甾醇转运体；
(vi)包含几丁质结合域(CBD)的多肽；和
(vii)V-ATP酶亚基c；
其中，所述昆虫多肽在昆虫多肽制剂中的纯度为至少80％。
37.根据权利要求36所述的方法，包括在所述免疫后分离所述抗体。
38.根据权利要求36至37中任一项所述的方法，其中所述骆驼科动物是羊驼。
39.根据权利要求1至38中任一项所述的纳米抗体、组合物、多核苷酸、核酸构建体、宿主细胞、植物、产品或方法，其中，所述昆虫选自蛾、臭虫、漏斗虫、甲虫、蚜虫和蜜蜂。
40.根据权利要求1至38中任一项所述的纳米抗体、组合物、多核苷酸、核酸构建体、宿主细胞、植物、产品或方法，其中所述昆虫是蛾。
41.根据权利要求39至40中任一项所述的纳米抗体、组合物、多核苷酸、核酸构建体、宿主细胞、植物、产品或方法，其中，所述蛾选自棉铃虫和草地夜蛾。
42.根据权利要求39至40中任一项所述的纳米抗体、组合物、多核苷酸、核酸构建体、宿主细胞、植物、产品或方法，其中，所述蛾是棉铃虫。
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