CN111363038A - 抗VEGF单域抗体及其人源化、单域抗体和IgG1-Fc构建的融合蛋白和应用 - Google Patents

抗VEGF单域抗体及其人源化、单域抗体和IgG1-Fc构建的融合蛋白和应用 Download PDF

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CN111363038A
CN111363038A CN202010225314.7A CN202010225314A CN111363038A CN 111363038 A CN111363038 A CN 111363038A CN 202010225314 A CN202010225314 A CN 202010225314A CN 111363038 A CN111363038 A CN 111363038A
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叶青
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Beijing Newanbo Biotechnology Co ltd
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Priority to CN202211298088.0A priority patent/CN116715762A/zh
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Abstract

本发明公开了抗VEGF单域抗体及其人源化、单域抗体和IgG1‑Fc构建的融合蛋白和应用。本发明筛选得到一组VEGF的单域抗体，其活性高、具有较强的中和或结合能力。该组单域抗体能特异性结合人VEGF165和VEGF121抗原、结合细胞表面表达VEGF的肿瘤细胞株，有效阻断VEGF抗原与VEGFR结合以及产生相应的信号级联效应。本发明进一步将单域抗体进行人源化改造得到提高亲和力提高的人源化抗体。本发明还将单域抗体或人源化的单域抗体与IgG‑Fc构建得到的融合蛋白。本发明的人源化单域抗体和/或融合蛋白可用于检测或诊断VEGF、阻断VEGF与VEGFR之间相互作用以及用于治疗VEGF表达异常相关疾病。

Description

抗VEGF单域抗体及其人源化、单域抗体和IgG1-Fc构建的融合 蛋白和应用
技术领域
本发明涉及单域抗体，尤其涉及抗VEGF的单域抗体以及用该单域抗体或人源化的单域抗体与IgG1-Fc融合构建的融合蛋白，本发明进一步涉及它们在检测VEGF以及治疗VEGF表达异常相关疾病中的应用，属于抗VEGF的单域抗体、人源化的单域抗体及其应用领域。
背景技术
血管内皮生长因子(Vascular Endothelial Growth Factor,VEGF)家族是一类多功能的细胞因子,在血管生成和***生成中具有直接和间接的调控作用,可促进内皮细胞增殖、促进血管生成以及增加血管的通透性。VEGF家族包括VEGF-A、VEGF-B、VEGF-C、VEGF-D等，其中VEGF-A编码区域，经转录、剪接等步骤后形成5种单体,因单体中所含氨基酸数目不同，分别被命名为VEGF121、VEGF145、VEGF165、VEGF189和VEGF206，相应的含有121个、145个、165个、189个和206个氨基酸。在8个外显子中，1～5被认为具有重要意义，因其能够编码VEGF受体识别的结构域,VEGF121、VEGF145、VEGF165三种单体是分泌性蛋白质,可以从细胞浆中分泌到细胞外。VEGF145和VEGF165可以和所有内皮细胞的KDR/flk-1受体结合。VEGF-B编码VEGF-B mRNA的剪接方式不尽相同，因而产生了2种不同的转录子，根据其含有的氨基酸残基数目分别被命名为VEGF-B167和VEGF-B186。VEGF-B的相关受体为VEGFR-1/FLT-1,在血管生成的过程中可起到促进作用。VEGF-C又称相关蛋白(VRP)，在人类的胚胎及成熟的组织中，均有VEGF-C mRNA表达,成人的VEGF-C除少量表达于脑、肝、胸腺及外周血白细胞外，其余大部分在胎盘、卵巢、心脏及小腺体中表达。VEGF-C与***的关系较为密切，包括对***增生的诱导及对淋巴细胞内皮进行有效的调节，而肿瘤往往最先通过***进行转移，因此，VEGF-C与肿瘤转移密切相关。VEGF-C在发育的胚胎中即能促进血管进行分化、生长，因此与其他促进血管生成的因子相比，其发挥作用的时间的更早。VEGF-C可作用于VEGFR-2和VEGFR-3,诱导内皮细胞增生,尤其是微血管细胞增生。VEGF-D，其mRNA在人体内的表达部位与VEGF-C类似，尤其在骨骼肌与结肠中表达较为丰富，但在胎盘中少见其表达。VEGF-D具有促进内皮细胞迁移的作用，可作为VEGF受体(VGEFRs)、VEGFR-2(Flk-1)及VEGFR-3(Flt-4)的配体，这些配体与VEGF-D结合后发挥促内皮迁移作用，从而促进血管生成。
VEGF能增强微血管的渗透性，血管渗透性增高后可导致血液成分的外漏，包括纤维蛋白原及其他凝固蛋白。当纤维蛋白在血管外聚积后,可使细胞间水肿液体的吸收与清除速度明显减慢,不仅改变了正常组织周围的环境，降低了其抗血管生成的功能，且具备了促血管生成的作用。VEGF还可增加皮肤、胸膜、腹膜、肠系膜等的血管床渗透性,是导致恶性胸腹水的重要原因。VEGF可促使细胞骨架发生变化，从而改变细胞形态。释放氧化亚氮和***素是VEGF激活内皮细胞的重要方式。内皮细胞被激活后即可进行生长与迁移。VEGF也是一种上皮细胞***素，可促进细胞进行有丝***，其原理可能涉及到蛋白激酶c途径及氧化亚氮调节途径。在血管生成的早期，最关键的变化是降解基底膜，降解过程中需要的酶和蛋白,大部分可由VEGF诱导生成。包括基质降解金属蛋白酶,金属蛋白酶间质胶原酶及丝氨酸蛋白酶,激活这些物质，可改变局部的微环境，创造出了适宜内皮细胞的迁移条件.研究证明VEGF可促进内皮细胞uPA受体的表达。此种受体表达增加，可加强蛋白水解作用和组织重塑,这些发现从侧面也证明了VEGF具有促血管生成的作用。此外,uPA本身也可诱导VEGF表达的增加，这就形成了一个表达环路。肿瘤的生长需要源源不断地供血，而VEGF因其能促进血管生成，故在肿瘤生长中必不可少。VEGF也是介导新生血管生成的重要因素，可强烈促使血管内皮细胞进行有丝***，进而形成新的血管，已被认为是促进肿瘤血管生成最强的细胞因子，对于新生血管是必要的。VEGF-C诱导的血管和***数量几乎相同,内皮穿透仅存在于毛细血管，表明VEGF-C不仅在促血管生成方面起作用，在促***方面也具有同样的作用。VEGF-C在肿瘤细胞和***上皮细胞之间起到了一定的作用，使两者进行相互调节，可促使新的***生成，***的增加是肿瘤更容易进行转移在许多实体瘤中，肿瘤的恶性程度与瘤体内血管化程度呈正相关。过量表达VEGF与多种肿瘤病情及预后相关,包括结肠癌，乳腺癌，***癌，肺癌和黑素瘤等。通过阻断VEGF信号途径，可从血管、***两方面抑制肿瘤的生长和转移。VEGF已成为一种重要的抗血管生成的治疗靶点。
专利申请WO9410202A中首次公开了罗氏公司筛选获得VEGF的鼠源A4.6.1抗体，但鼠源单抗对人体有较强的免疫原性，可能会诱发人抗鼠抗体(HAMA)反应，并且其在人体循环***中往往很快地会被清除掉。在WO9845331A和WO9845332A专利申请中通过将A4.6.1产生的6个鼠源CDR与人源VLK1Cl轻链恒定区和VHⅢ-CH1重链恒定区相结合，构建获得了人源化的VEGF抗体。所获得的人源化抗VEGF单克隆抗体与VEGF的亲和力与原抗体相似。罗氏公司贝伐珠单抗就是一种重组人源化单克隆抗体，分子量约为149kD，含有人源抗体的框架区和可结合VEGF的鼠源抗体的互补决定区。它能与VEGF结合并阻止其与内皮细胞表面上的受体结合，从而阻断VEGF导致的内皮细胞扩增和新血管的形成，可减少微血管生成并抑制转移病灶进展，2004年获得美国食品和药物管理局(FDA)的批准，成为美国第一个获得批准上市的抑制肿瘤血管生成的抗体药物。目前FDA已批准用于6种适应症，包括转移性结直肠癌，非小细胞肺癌，胶质母细胞瘤，转移性肾细胞癌，***、复发性或转移性癌，复发性上皮性卵巢癌、输卵管癌或原发性腹膜癌。2010年经国家食品药品监督管理总局(CFDA)批准进入中国，商品名为安维汀。WO9845331A和WO9845332A专利申请中，对抗VEGF抗体进行了片段化处理，并通过突变提高其亲和性，以便药物直接递送到眼内发挥作用，从而制备获得了雷珠单抗。雷珠单抗是重组的VEGF人源化单抗IgG1的抗体片段，不具有抗体Fc区，分子量约为48kD，具有治疗过度血管增生导致的湿性老年性黄斑，是用于眼内治疗的抗体药物。2006年被FDA批准用于治疗老年性黄斑，2012年又被FDA批准用于治疗糖尿病黄斑水肿。
然而，上述单克隆抗体结构复杂，生产周期长，其成本高，价格昂贵。1993年Hamers-Casterman等发现，骆驼抗体有天然缺失轻链，只含有重链，且该重链缺失恒定区的CH1区，因此其可变区直接与铰链区相连接，故又称重链抗体。克隆重链抗体的可变区得到只由一个重链可变区组成的单域抗体，称为VHH抗体(variable domain of heavy chainof heavy-chain antibody，VHH)。VHH晶体直径2.5nm，长4nm，因此,又称为纳米抗体(nanobody)，是自然存在的可与抗原结合的最小片段。纳米抗体一般结构呈椭圆形，体积很小，分子质量为单克隆抗体的1/10(15kD左右)，与普通抗体相比，其稳定性好、亲和力较高，克服了小分子功能抗体的缺点，同时又具有分子质量小免疫原性弱、组织穿透力强，可在酵母菌、大肠杆菌等微生物中大量表达，可进行大规模生产，易于普及和应用，相对价格低廉等单克隆抗体、多克隆抗体不具备的优点，适应用于检测、疾病治疗等方面。因此应用单域抗体技术研发VEGF抗体具有广阔的应用前景。
发明内容
本发明的目的之一是提供一组抗VEGF的单域抗体及其编码基因；
本发明的目的之二是将抗VEGF的单域抗体进行人源化改造得到人源化的单域抗体；
本发明的目的之三是将所述的单域抗体或人源化的单域抗体与人IgG1-Fc融合得到融合蛋白；
本发明的目的之四是将所述的单域抗体或人源化的单域抗体与酶相、放射性同位素、荧光化合物或化学发光化合物中的一种或多种相偶联得到缀合物；
本发明的目的之四将所述的抗VEGF的单域抗体、抗VEGF的人源化的单域抗体、融合蛋白以及缀合物应用于制备检测VEGF的试剂或治疗VEGF表达异常相关疾病；
本发明的上述目的是通过以下技术方案来实现的：
本发明首先提供了抗VEGF的单域抗体，所述单域抗体均由框架区和3个互补决定区组成；所述单域抗体选自NBV1、NBV2、NBV3、NBV4、NBV5、NBV6、NBV7、NBV8、NBV9、NBV10、NBV11、NBV12、NBV13、NBV14、NBV15、NBV16、NBV17、NBV18、NBV19、NBV20、NBV21、NBV22、NBV23、NBV24、NBV25、NBV26、NBV27、NBV28、NBV29、NBV30、NBV31、NBV32、NBV33、NBV34、NBV35、NBV36、NBV37、NBV38、NBV39、NBV40、NBV41或NBV42中的任何一种；
其中，单域抗体NBV1的3个互补决定区的氨基酸序列分别为SEQ ID No.1、SEQ IDNo.2和SEQ ID No.3所示；单域抗体NBV2的3个互补决定区的氨基酸序列分别为SEQ IDNo.4、SEQ ID No.5和SEQ ID No.6所示；单域抗体NBV3的3个互补决定区的氨基酸序列分别为SEQ ID No.7、SEQ ID No.8和SEQ ID No.9所示；单域抗体NBV4的3个互补决定区的氨基酸序列分别为SEQ ID No.10、SEQ ID No.11和SEQ ID No.12所示；单域抗体NBV5的3个互补决定区的氨基酸序列分别为SEQ ID No.13、SEQ ID No.14和SEQ ID No.15所示；单域抗体NBV6的3个互补决定区的氨基酸序列分别为SEQ ID No.16、SEQ ID No.17和SEQ ID No.18所示；单域抗体NBV7的3个互补决定区的氨基酸序列分别为SEQ ID No.19、SEQ ID No.20和SEQ ID No.21所示；单域抗体NBV8的3个互补决定区的氨基酸序列分别为SEQ ID No.22、SEQ ID No.23和SEQ ID No.24所示；单域抗体NBV9的3个互补决定区的氨基酸序列分别为SEQ ID No.25、SEQ ID No.26和SEQ ID No.27所示；单域抗体NBV10的3个互补决定区的氨基酸序列分别为SEQ ID No.28、SEQ ID No.29和SEQ ID No.30所示；单域抗体NBV11的3个互补决定区的氨基酸序列分别为SEQ ID No.31、SEQ ID No.32和SEQ ID No.33所示；单域抗体NBV12的3个互补决定区的氨基酸序列分别为SEQ ID No.34、SEQ ID No.35和SEQ IDNo.36所示；单域抗体NBV13的3个互补决定区的氨基酸序列分别为SEQ ID No.37、SEQ IDNo.38和SEQ ID No.39所示；单域抗体NBV14的3个互补决定区的氨基酸序列分别为SEQ IDNo.40、SEQ ID No.41和SEQ ID No.42所示；单域抗体NBV15的3个互补决定区的氨基酸序列分别为SEQ ID No.43、SEQ ID No.44和SEQ ID No.45所示；单域抗体NBV16的3个互补决定区的氨基酸序列分别为SEQ ID No.46、SEQ ID No.47和SEQ ID No.48所示；单域抗体NBV17的3个互补决定区的氨基酸序列分别为SEQ ID No.49、SEQ ID No.50和SEQ ID No.51所示；单域抗体NBV18的3个互补决定区的氨基酸序列分别为SEQ ID No.52、SEQ ID No.53和SEQID No.54所示；单域抗体NBV19的3个互补决定区的氨基酸序列分别为SEQ ID No.55、SEQID No.56和SEQ ID No.57所示；单域抗体NBV20的3个互补决定区的氨基酸序列分别为SEQID No.58、SEQ ID No.59和SEQ ID No.60所示；单域抗体NBV21的3个互补决定区的氨基酸序列分别为SEQ ID No.61、SEQ ID No.62和SEQ ID No.63所示；单域抗体NBV22的3个互补决定区的氨基酸序列分别为SEQ ID No.64、SEQ ID No.65和SEQ ID No.66所示；单域抗体NBV23的3个互补决定区的氨基酸序列分别为SEQ ID No.67、SEQ ID No.68和SEQ ID No.69所示；单域抗体NBV24的3个互补决定区的氨基酸序列分别为SEQ ID No.70、SEQ ID No.71和SEQ ID No.72所示；单域抗体NBV25的3个互补决定区的氨基酸序列分别为SEQ IDNo.73、SEQ ID No.74和SEQ ID No.75所示；单域抗体NBV26的3个互补决定区的氨基酸序列分别为SEQ ID No.76、SEQ ID No.77和SEQ ID No.78所示；单域抗体NBV27的3个互补决定区的氨基酸序列分别为SEQ ID No.79、SEQ ID No.80和SEQ ID No.81所示；单域抗体NBV28的3个互补决定区的氨基酸序列分别为SEQ ID No.82、SEQ ID No.83和SEQ ID No.84所示；单域抗体NBV29的3个互补决定区的氨基酸序列分别为SEQ ID No.85、SEQ ID No.86和SEQID No.87所示；单域抗体NBV30的3个互补决定区的氨基酸序列分别为SEQ ID No.88、SEQID No.89和SEQ ID No.90所示；单域抗体NBV31的3个互补决定区的氨基酸序列分别为SEQID No.91、SEQ ID No.92和SEQ ID No.93所示；单域抗体NBV32的3个互补决定区的氨基酸序列分别为SEQ ID No.94、SEQ ID No.95和SEQ ID No.96所示；单域抗体NBV33的3个互补决定区的氨基酸序列分别为SEQ ID No.97、SEQ ID No.98和SEQ ID No.99所示；单域抗体NBV34的3个互补决定区的氨基酸序列分别为SEQ ID No.100、SEQ ID No.101和SEQ IDNo.102所示；单域抗体NBV35的3个互补决定区的氨基酸序列分别为SEQ ID No.103、SEQ IDNo.104和SEQ ID No.105所示；单域抗体NBV36的3个互补决定区的氨基酸序列分别为SEQID No.106、SEQ ID No.107和SEQ ID No.108所示；单域抗体NBV37的3个互补决定区的氨基酸序列分别为SEQ ID No.109、SEQ ID No.110和SEQ ID No.111所示；单域抗体NBV38的3个互补决定区的氨基酸序列分别为SEQ ID No.112、SEQ ID No.113和SEQ ID No.114所示；单域抗体NBV39的3个互补决定区的氨基酸序列分别为SEQ ID No.115、SEQ ID No.116和SEQID No.117所示；单域抗体NBV40的3个互补决定区的氨基酸序列分别为SEQ ID No.118、SEQID No.119和SEQ ID No.120所示；单域抗体NBV41的3个互补决定区的氨基酸序列分别为SEQ ID No.121、SEQ ID No.122和SEQ ID No.123所示；单域抗体NBV42的3个互补决定区的氨基酸序列分别为SEQ ID No.124、SEQ ID No.125和SEQ ID No.126所示。
将上述所示的任何一种氨基酸序列中的一个或多个氨基酸进行缺失、取代、***和/或添加所得到的蛋白突变体，该蛋白突变体与突变前的蛋白具有相同的功能，这些蛋白突变体均属本发明的保护范畴之内；此外，与上述所示的任何一种氨基酸序列至少有90％以上同一性的氨基酸序列也属于本发明保护范畴之内。
本发明进一步提供了所述单域抗体的氨基酸序列，其中，单域NBV1的氨基酸序列为SEQ ID No.127所示，单域NBV2的氨基酸序列为SEQ ID No.128所示，单域NBV3的氨基酸序列为SEQ ID No.129所示，单域NBV4的氨基酸序列为SEQ ID No.130所示，单域NBV5的氨基酸序列为SEQ ID No.131所示，单域NBV6的氨基酸序列为SEQ ID No.132所示，单域NBV7的氨基酸序列为SEQ ID No.133所示，单域NBV8的氨基酸序列为SEQ ID No.134所示，单域NBV9的氨基酸序列为SEQ ID No.135所示，单域NBV10的氨基酸序列为SEQ ID No.136所示，单域NBV11的氨基酸序列为SEQ ID No.137所示，单域NBV12的氨基酸序列为SEQ ID No.138所示，单域NBV13的氨基酸序列为SEQ ID No.139所示，单域NBV14的氨基酸序列为SEQ IDNo.140所示，单域NBV15的氨基酸序列为SEQ ID No.141所示，单域抗体NBV16的氨基酸序列为SEQ ID No.142所示，单域抗体NBV17的氨基酸序列为SEQ ID No.143所示，单域抗体NBV18的氨基酸序列为SEQ ID No.144所示，单域抗体NBV19的氨基酸序列为SEQ ID No.145所示，单域抗体NBV20的氨基酸序列为SEQ ID No.146所示，单域抗体NBV21的氨基酸序列为SEQ ID No.147所示，单域抗体NBV22的氨基酸序列为SEQ ID No.148所示，单域抗体NBV23的氨基酸序列为SEQ ID No.149所示，单域抗体NBV24的氨基酸序列为SEQ ID No.150所示，单域抗体NBV25的氨基酸序列为SEQ ID No.151所示，单域抗体NBV26的氨基酸序列为SEQID No.152所示，单域抗体NBV27的氨基酸序列为SEQ ID No.153所示，单域抗体NBV28的氨基酸序列为SEQ ID No.154所示，单域抗体NBV29的氨基酸序列为SEQ ID No.155所示，单域抗体NBV30的氨基酸序列为SEQ ID No.156所示，单域抗体NBV31的氨基酸序列为SEQ IDNo.157所示，单域抗体NBV32的氨基酸序列为SEQ ID No.158所示，单域抗体NBV33的氨基酸序列为SEQ ID No.159所示，单域抗体NBV34的氨基酸序列为SEQ ID No.160所示，单域抗体NBV35的氨基酸序列为SEQ ID No.161所示，单域抗体NBV36的氨基酸序列为SEQ ID No.162所示，单域抗体NBV37的氨基酸序列为SEQ ID No.163所示，单域抗体NBV38的氨基酸序列为SEQ ID No.164所示，单域抗体NBV39的氨基酸序列为SEQ ID No.165所示，单域抗体NBV40的氨基酸序列为SEQ ID No.166所示，单域抗体NBV41的氨基酸序列为SEQ ID No.167所示，单域抗体NBV42的氨基酸序列为SEQ ID No.168所示。
将上述所示的任何一种氨基酸序列中的一个或多个氨基酸进行缺失、取代、***和/或添加所得到的蛋白突变体，该蛋白突变体与突变前的蛋白具有相同的功能，这些蛋白突变体均属本发明保护范畴之内；此外，与上述所示的任何一种氨基酸序列至少有90％以上同一性的氨基酸序列也属于本发明保护范畴之内。
根据单域抗体亲和力测定实验结果可见，本发明的所有VEGF单域抗体的亲和力在50nm、20nm、10nm、1nm、0.1nm、0.01nm，部分VEGF单域抗体亲和力的范围在0.001-37.5nM。根据与VEGF-165抗原和其它相关抗原的结合试验可见，本发明的VEGF单域抗体与人VEGF-165抗原特异性结合,与其它相关蛋白基本不结合，说明本发明的VEGF单域抗体特异性良好。
根据本发明VEGF单域抗体对VEGF与VEGFR的结合竞争抑制作用试验结果可见，不同VEGF单域抗体能够竞争抑制VEGF蛋白与VEGFR蛋白的结合，其中有4株单域抗体(NBV5、NBV6、NBV14以及NBV16)与阳性对照贝划单抗的竞争抑制VEGF蛋白与VEGFR蛋白结合的抑制效率相同。
本发明进一步对单域抗体NBV11、NBV20、NBV32以及NBV35分别进行了人源化改造，得到人源化的抗体；所有人源化后的VEGF单域抗体，其亲和力均没有产生明显的变化；其中，将单域抗体NBV11进行了人源化改造得到了4个人源化的抗体，其氨基酸序列分别为SEQID No.169、SEQ ID No.170、SEQ ID No.171和SEQ ID No.172所示；将单域抗体NBV20进行了人源化改造得到了4个人源化的抗体，其氨基酸序列分别为SEQ ID No.173、SEQ IDNo.174、SEQ ID No.175和SEQ ID No.176所示；将单域抗体NBV32进行了人源化改造得到了4个人源化的抗体，其氨基酸序列分别为SEQ ID No.177、SEQ ID No.178、SEQ ID No.179和SEQ ID No.180所示；将单域抗体NBV35进行了人源化改造得到了3个人源化的抗体，其氨基酸序列分别为SEQ ID No.181、SEQ ID No.182、和SEQ ID No.183所示。
本发明还进一步提供了所述单域抗体的编码基因序列，其中，单域抗体NBV1的编码基因的核苷酸序列为SEQ ID No.184所示，单域抗体NBV2的编码基因的核苷酸序列为SEQID No.185所示，单域抗体NBV3的编码基因的核苷酸序列为SEQ ID No.186所示，单域抗体NBV4的编码基因的核苷酸序列为SEQ ID No.187所示，单域抗体NBV5的编码基因的核苷酸序列为SEQ ID No.188所示，单域抗体NBV6的编码基因的核苷酸序列为SEQ ID No.189所示，单域抗体NBV7的编码基因的核苷酸序列为SEQ ID No.190所示，单域抗体NBV8的编码基因的核苷酸序列为SEQ ID No.191所示，单域抗体NBV9的编码基因的核苷酸序列为SEQ IDNo.192所示，单域抗体NBV10的编码基因的核苷酸序列为SEQ ID No.193所示，单域抗体NBV11的编码基因的核苷酸序列为SEQ ID No.194所示，单域抗体NBV12的编码基因的核苷酸序列为SEQ ID No.195所示，单域抗体NBV13的编码基因的核苷酸序列为SEQ ID No.196所示，单域抗体NBV14的编码基因的核苷酸序列为SEQ ID No.197所示，单域抗体NBV15的编码基因的核苷酸序列为SEQ ID No.198所示，单域抗体NBV16的编码基因的核苷酸序列为SEQ ID No.199所示，单域抗体NBV17的编码基因的核苷酸序列为SEQ ID No.200所示，单域抗体NBV18的编码基因的核苷酸序列为SEQ ID No.201所示，单域抗体NBV19的编码基因的核苷酸序列为SEQ ID No.202所示，单域抗体NBV20的编码基因的核苷酸序列为SEQ IDNo.203所示，单域抗体NBV21的编码基因的核苷酸序列为SEQ ID No.204所示，单域抗体NBV22的编码基因的核苷酸序列为SEQ ID No.205所示，单域抗体NBV23的编码基因的核苷酸序列为SEQ ID No.206所示，单域抗体NBV24的编码基因的核苷酸序列为SEQ ID No.207所示，单域抗体NBV25的编码基因的核苷酸序列为SEQ ID No.208所示，单抗体域NBV26的编码基因的核苷酸序列为SEQ ID No.209所示，单域抗体NBV27的编码基因的核苷酸序列为SEQ ID No.210所示，单域抗体NBV28的编码基因的核苷酸序列为SEQ ID No.211所示，单域抗体NBV29的编码基因的核苷酸序列为SEQ ID No.212所示，单域抗体NBV30的编码基因的核苷酸序列为SEQ ID No.213所示，单域抗体NBV31的编码基因的核苷酸序列为SEQ IDNo.214所示，单域抗体NBV32的编码基因的核苷酸序列为SEQ ID No.215所示，单域NBV33的编码基因的核苷酸序列为SEQ ID No.216所示，单域抗体NBV34的编码基因的核苷酸序列为SEQ ID No.217所示，单域抗体NBV35的编码基因的核苷酸序列为SEQ ID No.218所示，单域抗体NBV36的编码基因的核苷酸序列为SEQ ID No.219所示，单域抗体NBV37的编码基因的核苷酸序列为SEQ ID No.220所示，单域抗体NBV38的编码基因的核苷酸序列为SEQ IDNo.221所示，单域抗体NBV39的编码基因的核苷酸序列为SEQ ID No.222所示，单域抗体NBV40的编码基因的核苷酸序列为SEQ ID No.223所示，单域抗体NBV41的编码基因的核苷酸序列为SEQ ID No.224所示，单域抗体NBV42的编码基因的核苷酸序列为SEQ ID No.225所示。
其中，与上述所示的多核苷酸序列的互补序列在严谨杂交条件能够进行杂交的多核苷酸序列也属于本发明的保护范畴之列；另外，与上述任何一种所示的多核苷酸序列至少有90％以上同一性的多核苷酸序列也属于本发明的保护范畴之列。
本发明进一步提供了一种重组表达载体，所述重组表达载体包含所述单域抗体的编码基因中的一个或多个；优选的，所述重组表达载体可以是重组原核细胞表达载体、重组酵母表达载体、重组真核细胞表达载体或其它重组细胞表达载体。
本发明还提供了重组宿主细胞，其包含上面所述的重组表达载体。
优选的，所述的重组宿主细胞为重组原核表达细胞、重组真核表达细胞，重组真菌细胞或酵重组母细胞，所述重组原核表达细胞优选大肠杆菌。
本发明进一步将所述的抗VEGF的单域抗体或人源化的单域抗体与IgG-Fc构建融合蛋白；其中，所述Fc基因序列可以是来源于IgG，IgA，IgM的Fc基因序列或来源于IgG1，IgG2，IgG3或IgG4。所述的IgG优选是人IgG及其IgG1、2、3、4的亚类，还可以是人IgM、人IgA或其它动物(如鼠，兔，猴等)免疫球蛋白的Fc片段基因及氨基酸序列。
本发明进一步将所述单域抗体或人源化的单域抗体与酶相(如辣根过氧化物酶、碱性磷酸酶等)、放射性同位素、荧光化合物或化学发光化合物中的一种或多种相偶联得到缀合物，这些缀合物可用于检测VEGF或治疗多种与VEGF表达异常相关疾病。
本发明所提供的抗VEGF的单域抗体、人源化的抗VEGF的单域抗体、VEGF的单域抗体或人源化的单域抗体与IgG-Fc构建的融合蛋白、单域抗体或人源化的单域抗体与酶相、放射性同位素、荧光化合物或化学发光化合物相偶联得到缀合物主要具有如下几方面的用途：
(1)制备检测VEGF中有关的药物或试剂；
(2)制备阻断VEGF与VEGFR之间相互作用的药物或试剂。
(3)制备用于治疗VEGF表达异常相关疾病的药物的应用。优选地，所述的VEGF表达异常相关不同疾病，如肺癌、直结肠癌、乳腺癌、头颈癌等肿瘤疾病，老年眼底黄斑病变等，包括但不仅仅限于这些疾病的检测和治疗的应用。
本发明筛选得到一组VEGF的单域抗体及其人源化单域抗体和融合蛋白。与现有的抗体相比，本发明筛选得到的抗VEGF的单域抗体活性高、具有较强的中和或结合能力。该组单域抗体能特异性结合人VEGF165和VEGF121抗原、结合细胞表面表达VEGF的肿瘤细胞株，有效阻断VEGF抗原与VEGFR结合以及产生相应的信号级联效应，可用于检测和/或治疗多种与VEGF表达异常相关疾病。本发明的VEGF人源化单域抗体和/或融合蛋白可用于治疗老年性眼底湿性黄斑病变疾病，还可用于治疗多种相关肿瘤疾病。
本发明所涉及的术语定义
本文所用的术语“VEGF”是血管内皮生长因子(Vascular Endothelial GrowthFactor,VEGF)家族，是一类多功能的细胞因子,在血管生成和***生成中具有直接和间接的调控作用,可促进内皮细胞增殖、促进血管生成以及增加血管的通透性。
本文所用的术语“单域抗体(sdAb)”是指包含了抗体中单个可变域的片段，也称为纳体(Nanobody)。和完整的抗体一样，它可以选择性的和特定抗原结合。与完整抗体的150－160kDa的质量相比，单域抗体则显得小得多，大约只有12－15kDa。第一个单域抗体是从骆驼的重链抗体中人造工程制作出来的，称为“VHH区段”。
本文所用的术语序列的“同一性(identity)”可以与“相同性”互换使用，指的是序列之间通过序列比对软件例如BLAST确定的相似程度。序列比对的方法和软件对于本领域技术人员是公知的。可以通过对已知序列进行一个或几个氨基酸或碱基的取代、缺失和/或添加而获得经改造的核苷酸序列。例如，通过常规手段(例如保守取代等)，对本发明的序列SEQ ID NO：1-198中一个或多个所示的氨基酸或核苷酸序列进行改造，可以获得与这些具有大于80％、大于85％、大于90％、大于95％或大于99％的序列同一性，并且具有基本相同的性能，这都在本发明的保护范围之内。优选地，本发明通过保守取代获得序列同一性，但并不限于保守取代。
术语“互补的”在此指的是两种包括反向平行核苷酸序列的核苷酸序列，反向平行核苷酸序列能在反向平行核苷酸序列的互补碱基残基之间形成氢键后彼此相互配对。本领域已知的是，当都从5’到3’的方向看序列时，两种互补链的核苷酸序列是彼此反向互补的。本领域也已知的是，两种在给定的条件组下能彼此杂交的序列不必必须是100％完全互补的。
术语“氨基酸序列”是指氨基酸相互连接形成肽链(或多肽)的顺序，氨基酸序列只能按照一个方向读取。氨基酸有100多种不同类型，其中20种常用，本发明不排除氨基酸链上有其他物质，例如糖类、脂类等修饰，本发明也不限于20中常用的氨基酸。
术语“核苷酸序列”是指DNA或RNA中碱基的排列顺序，即在DNA中为A、T、G、C的排列顺序，或者在mRNA中A、U、G、C的排列顺序，也包括rRNA、tRNA、mRNA中碱基的排列顺序。应该理解，本发明请求保护的抗体基因除了DNA序列外，也涵盖RNA(rRNA、tRNA、mRNA)以及它们的互补序列。
本发明中所述的取代可以是保守取代，即将特定的氨基酸残基替换为具有相似物理化学特征的残基。保守取代的非限定性例子包括含脂肪族基团氨基酸残基之间的取代(例如Ile、Val、Leu或Ala间的相互取代)、极性残基之间的取代(例如Lys和Arg、Glu和Asp、Gln和Asn之间的相互取代)等。因氨基酸的缺失、取代、***和/或添加而成的突变体可以通过对编码野生型蛋白质的DNA实施例如作为公知技术的定点诱变(参见例如Nucleic AcidResearch，Vol.10，No.20，p.6487-6500，1982，通过引用其全文引入本说明书)来制作。
在本说明书中，“一个或多个氨基酸”指通过定点诱变方法能够缺失、取代、***和/或添加的程度的氨基酸，并非限定，但优选为20个以下、15个以下、10个以下、或7个以下，更优选为5个以下。就定点诱变方法而言，例如，除希望的变异即特定的不一致之外，还可以使用与要突变的单链噬菌体DNA互补的合成寡核苷酸引物按如下方式进行。
术语“表达载体(Expression vectors)”是指在克隆载体基本骨架的基础上增加表达元件(如启动子、RBS、终止子等)，使目的基因能够表达的载体。表达载体四部分：目的基因、启动子、终止子、标记基因。本发明包括但不限于原核细胞表达载体、真核细胞表达载体或其它细胞表达载体。
术语“框架区(Framework region)”，即骨架区，在免疫球蛋白的H和L链的近N端约有110个氨基酸序列的变化很大，其他部分的氨基酸序列相对恒定，据此可将轻链和重链区分为可变区(V)和恒定区(C)。可变区内包含超变区HVR(hypervariable region)或称互补决定区CDR(Complementarity-determining region)与FR骨架区。
术语“人源化”抗体是指抗体的可变区(VH或VHH)的Fr区部分，恒定区部分(即CH和CL区)或抗体所有全部由人类抗体基因所编码。人源化抗体可以大大减少异源抗体对人类机体造成的免疫副反应。人源化抗体包括嵌合抗体、改型抗体和全人源化抗体等几类。应该理解，本领域技术人员根据实际需要能够制备出本发明单域抗体的合适的人源化形式，这在本发明涵盖的范围之内。
术语“严谨杂交条件”意指在所属领域中已知的低离子强度和高温的条件。通常，在严谨条件下，探针与其靶序列杂交的可检测程度比与其它序列杂交的可检测程度更高(例如超过本底至少2倍)。严谨杂交条件是序列依赖性的，在不同的环境条件下将会不同，较长的序列在较高温度下特异性杂交。通过控制杂交的严谨性或洗涤条件可鉴定与探针100％互补的靶序列。对于核酸杂交的详尽指导可参考有关文献(Tijssen,Techniques inBiochemistry and Molecular Biology-Hybridization with Nucleic Probes,"Overview of principles of hybridization and the strategy of nucleic acidassays.1993)。更具体的，所述严谨条件通常被选择为低于特异序列在规定离子强度pH下的热熔点(Tm)约5-10℃。Tm为在平衡状态下50％与目标互补的探针杂交到目标序列时所处的温度(在指定离子强度、pH和核酸浓度下)(因为目标序列过量存在，所以在Tm下在平衡状态下50％的探针被占据)。严谨条件可为以下条件：其中在pH 7.0到8.3下盐浓度低于约1.0M钠离子浓度，通常为约0.01到1.0M钠离子浓度(或其它盐)，并且温度对于短探针(包括(但不限于)10到50个核苷酸)而言为至少约30℃，而对于长探针(包括(但不限于)大于50个核苷酸)而言为至少约60℃。严谨条件也可通过加入诸如甲酰胺的去稳定剂来实现。对于选择性或特异性杂交而言，正信号可为至少两倍的背景杂交，视情况为10倍背景杂交。例示性严谨杂交条件可如下：50％甲酰胺，5×SSC和1％SDS，在42℃下培养；或5×SSC，1％SDS，在65℃下培养，在0.2×SSC中洗涤和在65℃下于0.1％SDS中洗涤。所述洗涤可进行5、15、30、60、120分钟或更长时间。
术语“突变”和“突变体”在此具有它们的常用含义，指的是在核酸或多肽序列中的遗传的、天然存在的或引入的变化，它们的意义与本领域人员通常所知的意义相同。
术语“宿主细胞”或“重组宿主细胞”意指包含本发明多核苷酸的细胞，而不管使用何种方法进行***以产生重组宿主细胞，例如直接摄取、转导、f配对或所属领域中已知的其它方法。外源性多核苷酸可保持为例如质粒的非整合载体或者可整合入宿主基因组中。
附图说明
图1抗VEGF单域抗体在大肠杆菌中表达试验SDS-PAGE电泳结果，结果显示，8个克隆有6个可以高效表达，另外2个克隆需要优化表达条件后，在进行表达。
图2抗VEGF单域抗体在大肠杆菌中表达后，经镍柱纯化后SDS-PAGE电泳结果，其纯化后蛋白纯度达90％左右。
图3纯化的VEGF单域抗体与人VEGF-165抗原和其它相关抗原的结合试验(ELISA)；结果显示该组抗VEGF单域抗体具有较高的特异性，只与人VEGF165蛋白结果，不与其它相关因子蛋白结合。
图4结果显示VEGF单域抗体能够竞争抑制VEGF蛋白与VEGFR蛋白的结合。
图5VEGF人源化单域抗体-HIgGfc融合蛋白表达纯化SDS-PAGE。
图6NBV11Hm4蛋白的荧光素标记物+辅料多肽X滴眼液，滴眼后12小时眼组织冰冻切片荧光检测结果。
具体实施方式
以下结合具体实施例来进一步描述本发明，本发明的优点和特点将会随着描述而更为清楚。但这些实施例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明的细节和形式进行修改或替换，但这些修改和替换均落入本发明的保护范围内。
实施例1抗VEGF抗原特异性的单域抗体库构建
1)VEGF抗原免疫羊驼
按照常规免疫方法进行，以购买的VEGF抗原(Human VEGF Protein,Human,Recombinant,厂家北京义翘神州，货号HPLC-11066-HNAH)，选择成年健康的羊驼，在颈背部皮下多点注射抗原，加入抗原和等体积福氏佐剂，分4-8次免疫，跟踪观察注射部位包块吸收情况，以确认免疫正确。免疫间隔时间为7-15天，第4次免疫后，采血清，测定抗原免疫效价，当效价达到1-5万倍以上时(ELISA方法)，采全血100ml左右，分离淋巴细胞，-80℃保存备用。
2)羊驼外周血淋巴细胞的分离和RNA的提取
分离羊驼外周血白细胞，应用QIAGEN试剂盒(QIAamp RNA Blood Mini Kit(50)，货号，52304)，按照说明书进行，简述之：1.5ml全血，加入红细胞裂解液5～10ml，混匀，放冰浴中30分钟，待红细胞裂解后，2000rpm，离心10分钟，去上清，再加入红细胞裂解液1～2ml，混匀，放冰浴中10分钟，以裂解残余红细胞，2000rpm，离心10分钟，去净上清，加入0.3ml的裂解液，把白细胞混匀，-80℃保存，备用。
RNA纯化，应用QIAGEN试剂盒(QIAamp RNA Blood Mini Kit(50)，货号，52304)，按照说明书进行，测定得到的RNA浓度。
3)重链抗体可变区-VHH
合成cDNA第一链：用cDNA合成试剂盒(MiniBESTAgarose Gel DNA ExtractionKit Ver.4.0，TAKARA公司)，按照说明书进行。以此模板，分别用两套引物进行PCR扩增重链抗体VHH基因片段。采用巢式PCR方法，第一次PCR扩增中大于800bp的为普通重链基因片段，在800～500bp之间的为缺失轻链的重链抗体基因片段，切胶回收缺失轻链重链抗体基因片段，以此为模板用VHH特异性引物经PCR扩增得到VHH目的基因(～500bp)。
所用引物：
第一轮PCRFd5’引物:YF:CGC CAT CAA GGT ACC AGT TGA
第一轮PCR Bd3’引物:YBN:CAG CCG GCC ATG GCC SMK GTR CAG CTG GTG GAKTCT GGG GGA G
第二轮PCR引物：
YV-BACK：CAT GTG CATGGCCTA GAC TCG CGG CCCAGC CGG CCA TGG CC；YV-FOR：CAT GTG TAG ATT CCT GGC CGG CCT GGC CTG AGG AGA CGG TGA CCT GG
4)VHH片段和噬菌体展示载体的连接及电转化TG1感受态
SfI单酶切VHH片段和pHEN6载体质粒后，将VHH片段及pHEN6载体(Conrath,KEMother.Antimicrob Agents Chemother(Antimicrobial Chemotherapy)2001,45:(10)2807-12.，中国专利ZL20111028003.1)经连接酶连接，电转化至TG1感受态细胞中，涂布平板，经菌落PCR验证抗体***率。重组基因克隆效率检测：取电转化菌液涂布LB/Amp平板上，32℃，过夜培养，次日用菌落PCR的方法验证抗体的连接效率，噬菌体抗体库的连接效率在90％以上。将电转化菌液涂布LB/Amp平板上，32℃，过夜培养物，用2YT培养基洗下，加入15％甘油，-80℃保存。噬菌体库大约0.5-1.0x108；随机挑选30-50个克隆，进行基因测序，其VHH的序列中三个CDR序列重复率小于10％。
5)VHH噬菌体抗体库的制备
抗体库加入辅助噬菌体M13K07(Invitrogen)进行拯救：按照常规方法进行噬菌体抗体库的制备，并保存于-80℃备用。
实施例2抗VEGF的单域抗体的获得
1.针对VEGF特异性单域抗体的筛选
第一轮VEGF蛋白浓度150μg/ml，150μl/孔，1个微孔，4℃过夜孵育。
第二轮VEGF蛋白浓度10～100μg/ml，150μl/孔，5个微孔，4℃过夜孵育。
第三轮VEGF蛋白浓度10～50μg/ml，150μl/孔，5个微孔，4℃过夜孵育。
封闭：1％CPBS，300μl/孔，37℃，孵育2小时。
表1针对VEGF特异性单域抗体的筛选结果
筛选次数 加入噬菌体抗体库总量 洗脱液+Tris-HCl 单菌落个数 洗脱滴度
第一轮 5.6×10<sup>11</sup> 300μl+200μl 10 50/μl
第二轮 5.25×10<sup>11</sup> 150μl/孔+350μl 约2500 2.5×10<sup>4</sup>/μl
第三轮 5.32×10<sup>11</sup> 150μl/孔+350μl 约3100 3.1×10<sup>4</sup>/μl
2.噬菌体ELISA方法挑选阳性克隆
从第3轮筛选生长菌落的琼脂平板上随机挑取单个菌落，接种在含有Amp的2YT液体培养基的96孔培养板中培养，用辅助噬菌体超感染诱导表达噬菌体抗体。收获表达上清，以VEGF为抗原进行ELISA测定，挑选出VEGF阳性孔，经DNA测序以鉴定抗VEGF单域抗体克隆的基因序列。得到包括序列表所示基因序列在内的一系列单域抗体基因序列，用于进一步表达和筛选特异性、高活性的单域抗体。
实施例3特异性VEGF单域抗体表达质粒的构建
PCR扩增实施例3所获得的特异性的VEGF单域抗体基因，而获得带有限制性内切酶BbsI和BamHI位点PCR产物，用限制性内切酶BbsI和BamHI分别处理PCR产物和载体(pSJF2载体)(kim Is.Biosic Biochem.2002,66(5):1148-51，中国专利ZL 201110280031)，经T4连接酶连接重组，而获得能在大肠杆菌中高效表达的质粒sdAb-pSJF2，并进行基因序列测定以确定其序列的正确性。
1)获得VHH目的基因的PCR扩增条件，50μl PCR体系的组成：
Figure BDA0002427446460000081
Figure BDA0002427446460000091
5’引物—GAA GAAGAA GAC AA CAG GCC SVK GTG MAG CTG GWG GAK TCT
3’引物—gaagatctccggatccTGAGGAGACGGTGACCTGGGT
2)目的基因和载体酶切，连接目的基因与载体，转化TG1，PCR鉴定含有目的片段的克隆，基因测序，获得基因序列正确的VEGF单域抗体表达质粒。
实施例4抗VEGF单域抗体的表达和纯化
将实施例3所述的含有质粒VEGFsdAb-pSJF2的菌种接种在含氨基苄青霉素的LB培养板上，37℃过夜。挑选单个菌落接种于15ml的含氨基苄青霉素的LB培养液中，37℃，摇床培养过夜。转种10ml过夜培养物于1L含氨基苄青霉素的2YT培养液中，37℃摇床培养，240转/分，培养到OD值达0.4～0.6时，加入0.5～1.0mM IPTG，继续培养过夜。离心，收菌。加溶菌酶裂解细菌，离心，收上清中可溶性单域抗体蛋白。经Ni+离子亲和层析获得纯度达90％以上的蛋白。图1为表达的抗VEGF单域抗体蛋白SDS-PAGE电泳结果，图2为表达的VEGF-sdAbs经镍柱纯化后SDS-PAGE电泳结果。
实验例1抗VEGF单域抗体亲和力测定实验
1)样品制备
抗原：Bio-VEGF用1×动态缓冲液(1×PBS，含0.05％Tween 20、0.1％BSA，pH 7.2)稀释至10μg/ml；
单域抗体：用1×kinetic缓冲液依次稀释为400nM、200nM、100nM、50nM、25nM、12.5nM、6.25nM；
2)样品测试
待测抗原通过SA sensor进行加载，抗原稀释5个稀释度，所有VEGF单域抗体的亲和力在50nm、20nm、10nm、1nm、0.1nm、0.01nm。部分VEGF单域抗体亲和力见表2,其亲和力范围在0.001-37.5nM。
表2部分VEGF单域抗体亲和力检测结果
Figure BDA0002427446460000092
Figure BDA0002427446460000101
实验例2纯化的VEGF单域抗体与VEGF-165抗原和其它相关抗原的结合实验(ELISA)
1实验过程
分别包被Human VEGF-B Protein(Fc)、Human VEGF-C Protein(His)、HumanVEGF-D Protein(His)、Human PIGF Protein(Fc)、Mouse VEGF164 Protein、Mouse PIGFProtein、Rat VEGF164Protein、对照VEGF 165(His)，浓度2ug/ml，100ul/孔，34个微孔，4℃孵育过夜；加入1％CPBS封闭，300ul/孔。37℃，孵育2h；稀释不同编码的VEGF单域抗体至终浓度为1ug/ml，100ul/孔；稀释Anti-Myc tag antibody(HRP)(1:5000)，100ul/孔，37℃孵育1h；加入TMB显色液，100ul/孔，避光反应10min；加入50ul/孔2M H2SO4终止反应；在450nm波长测OD值。
2实验结果
实验结果见图3，根据实验结果可见纯化的VEGF单域抗体与人VEGF-165抗原特异性结合,与其它相关蛋白基本不结合。
实验例3抗VEGF单域抗体对VEGF与VEGFR的结合竞争抑制作用实验
由于VEGF能够结合VEGFR，产生信号传导和血管内皮细胞后续一系列生物功能和细胞增殖，有功能的VEGF单域抗体应该能够竞争抑制VEGF与VEGFR的结合。按照1μg/ml，100μl/孔包被VEGFR蛋白在可拆酶标板，4℃孵育过夜。加入2％BSA封闭，300μl/孔，37℃，孵育2小时。稀释VEGF单域抗体至终浓度为10μg/ml。加入100μl VEGF(10μg/ml)单域抗体，每孔加入VEGF(5μg/ml)蛋白2μl，混匀。稀释鼠抗myc-IgG-HRP(1:5000)，100μl/孔，37℃孵育1小时。加入TMB显色液，100μl/孔，避光反应10分钟。加入50μl/孔2M H2SO4终止反应。在450nm波长测OD值。
图4的结果显示VEGF单域抗体能够竞争抑制VEGF蛋白与VEGFR蛋白的结合。不同VEGF单域抗体能够竞争抑制VEGF蛋白与VEGFR蛋白的结合，有4株单域抗体(NBV5、NBV6、NBV14以及NBV16)与阳性对照贝划单抗的竞争抑制VEGF蛋白与VEGFR蛋白结合的抑制效率相同，还有3株单域抗体只有较弱的竞争抑制作用。
实验例4纯化的部分VEGF单域抗体与人VEGF-165抗原和人VEGF-121抗原的结合实验(ELISA)
实验过程同实验例2，实验结果见表3。实验结果显示，所检测的部分VEGF单域抗体与人VEGF-165和人VEGF-121抗原结合能力基本相同，从侧面证明这部分测试的VEGF单域抗体结合的抗原表面是人VEGF-165和人VEGF-121抗原共有的。
表3抗VEGF单域抗体特异性结合人VEGF-165和人VEGF-121抗原的实验结果
Figure BDA0002427446460000102
Figure BDA0002427446460000111
实验例5抗VEGF单域抗体的人源化改造实验
人源化方法采用蛋白表面氨基酸人源化(Resurfacing)的方法及VHH人源化通用的抗原结合互补区移植法(CDR grafting to a universal framework)完成。
人源化步骤如下：对抗VEGF单域抗体NBV11、20、32和35同源建模，建模软件为Modeller9。参考可溶性好的人抗体DP-47和同源序列NBBcII10抗体的氨基酸序列，把抗VEGF单域抗体NBV11、20、32和35进行人源化，改造后的序列见表4。
表4抗VEGF单域抗体11、20、32和35的人源化改造
Figure BDA0002427446460000112
Figure BDA0002427446460000121
X*：表示该氨基酸可能继续人源化变化。
将上述的这些VEGF单域抗体人源化后与抗原结合的特性测试，测试结果见表5。
表5 VEGF单域抗体人源化改造后与抗原结合的特性测试结果
Figure BDA0002427446460000122
从表5中的结果可见，所有人源化后的VEGF单域抗体，其亲和力基本上都无明显产生变化，且框架区人源化率符合设计要求。
实验例6人源化的VEGF单域抗体与人IgG1-Fc连接构建融合蛋白实验
1.构建步骤(1)NBV11Hm1(或NBV11Hm4)+人IgG1-Fc(简称NBV11Hmx-HIgG1fc)基因全合成；(2)NBV20Hm1(或NBV20Hm3)+人IgG1-Fc(简称NBV20Hmx+HIgGfc)基因全合成；(3)NBV32Hm1(或NBV32Hm2)+人IgG1-Fc(简称NBV32Hmx+HIgGfc)基因全合成；(4)NBV35Hm1(或NBV35Hm2)+人IgG1-Fc(简称NBV35Hmx+HIgGfc)基因全合成并加入XhoI-EcoRI双酶切，将(1)NBV11Hmx+HIgGfc、(2)NBV20Hmx+HIgGfc、(3)NBV32Hmx+HIgGfc、(4)NBV35Hmx+HIgGfc基因连接至p327.7表达载体(专利公布号CN 104195173 A)，并加入相应酶切位点和终止密码子，用XbaI-SalI双酶切，将另一相同的(1)(2)(3)(4)基因连接至已含有(1)NBV11Hmx+HIgGfc、(2)NBV20Hmx+HIgGfc、(3)NBV32Hmx+HIgGfc、(4)NBV35Hmx+HIgGfc基因(已XhoI-EcoRI双酶切连接)的p327.7表达载体中，最终使一个载体有2条(1)NBV11Hmx+HIgGfc、(2)NBV20Hmx+HIgGfc、(3)NBV32Hmx+HIgGfc、(4)NBV35Hmx+HIgGfc基因序列。
2.VEGF人源化单域抗体Fc融合蛋白的表达与纯化
将上述构建的各个表达载体，分别转染CHO/K1细胞，用MSX筛选稳定的蛋白高表达细胞株，共筛选到3株稳定表达细胞株，用稳定表达细胞株，在500ml摇瓶中培养进行蛋白表达。
蛋白质纯化：将上述细胞表达上清用蛋白A株亲和层析纯化，纯化蛋白置换为柠檬酸(0.05％Tween80，pH6.2)缓冲液。VEGF人源化单域抗体Fc融合蛋白所表达纯化的蛋白见图5(SDS-PAGE还原胶和非还原胶的电泳图)。
从图5的结果可见，VEGF人源化单域抗体融合蛋白表达纯化后鉴定结果与理论推算值接近一致，表达纯化后非还原胶的蛋白带与分子量马克相比，约76KD左右。
实验例7 VEGF单域抗体或VEGF人源化的单域抗体与能够用于眼科滴眼药物辅料多肽混合制备成滴眼液实验
制备过程：大肠杆菌表达纯化的NBV11Hm4蛋白的荧光素标记物+辅料多肽X(见中国专利申请公布号CN107638405A)的混合比例分别为1:1/1:2/1:5/1:10/1:20等不同比例进行配比制备。
用激光将Balb/c小鼠眼底灼伤，将1.中制备的滴眼液，滴于灼伤的小鼠眼内，4小时再滴眼1次，于12小时后，取眼球冰冻切片，用荧光强度测试仪测试不同小鼠眼组织的含有标记荧光的VEGF单域抗体+辅料多肽X的荧光强度，试验同时设有单独标记荧光的VEGF单域抗体，和非药物对照组。
实验结果见图6。根据NBV11Hm4蛋白的荧光素标记物+辅料多肽X实验动物组眼组织切片荧光强度检测结果可见，眼角膜和视网膜的荧光强度比单独NBV11Hm4蛋白的荧光素标记物组和非药物对照组分别高5倍和10-30倍，有显著统计学差别。
实验例8 VEGF人源化单域抗体Fc融合蛋白抑制肿瘤细胞在小鼠体的生长实验
1.动物试验模型的建立：将培养的人结肠癌细胞(LS174T)吹打成单细胞悬液后800转/分钟离心5分钟，弃掉上清，用PBS重悬细胞，调整细胞浓度为2*10^7cell/mL备用。接种Balb/c-nu裸鼠颈背部皮肤，用1mL注射器抽取细胞悬液，在肿瘤预期生长部位回抽无血后注入细胞100μL/只。接种完成后，当肿瘤体积在100-200mm3范围内的动物达到40％时，采用excel软件的RAND函数对瘤体积在该范围内的小鼠进行随机分组，每组10只，分为3组，分别标记为分Z1(NBV20Hm1组)、Z2(贝划珠单抗-阳性对照组)、Z3(PBS对照组)。分组当天为第0天，并开始给药，给药途径为腹腔注射(I.P.)，每周给药2次，连续给药6次，末次给药后3天处死小鼠，取肿瘤组织进行称重。给药方案见表6。
表6试验分组及给药方式
组别 动物数 注射样品 给药途径 剂量 频率/周期
Z1 10 NBV20Hm1 I.P. 5mg/Kg 2次/周×3周
Z2 10 贝伐株单抗注射液 I.P. 5mg/Kg 2次/周×3周
Z3 10 PBS I.P. --------- 2次/周×3周
2.试验动物观察和试验结果统计
肿瘤成瘤后每周2次测量动物体重、肿瘤大小(肿瘤块的最长径和最短径)。动物处死后剥离肿瘤，称量瘤重并拍照。(1)瘤体积计算：肿瘤成瘤后测量肿瘤的最长径(a)和最短径(b)，每周测量2次，通过公式V＝1/2×a×b2计算瘤体积。以时间为横坐标，瘤体积为纵坐标绘制肿瘤生长曲线。
(2)相对肿瘤增殖率T/C(％)：通过计算T/C(％)来评价药物的抗肿瘤作用，T/C(％)＞40％为无效，T/C(％)≤40％，并经统计学处理p＜0.05为有效。T/C(％)的计算方法如下：T/C(％)＝TRTV/CRTV×100％(TRTV治疗组RTV；CRTV阴性对照组RTV)，相对肿瘤体积RTV＝VT/V0，V0为分笼给药时(D 0)测量所得的肿瘤体积，VT为每一次测量所得的肿瘤体积。(3)瘤重抑制率：给药结束后处死小鼠，称瘤重，计算瘤重抑制率。瘤重抑制率％＝(1-T/C)×100％(T为治疗组平均瘤重；C为阴性对照组平均瘤重)(4)统计分析：采用excel软件的TTEST函数对瘤体积、瘤重进行统计学分析，p＜0.05有显著性差异，p≥0.05无显著性差异。从表9.结果可见：VEGF人源化单域抗体Fc融合蛋白-NBV20Hm1试验动物组与阴性对照动物组对于小鼠体内肿瘤生长抑制率有显著差别，与贝伐株单抗注射液阳性对照组动物试验结果相比，没有显著性差别。
表7对Balb/c-nu裸鼠移植LS174T细胞的抗肿瘤作用结果(平均肿瘤体积统计结果
Figure BDA0002427446460000141
)
Figure BDA0002427446460000142
备注：1.与Z3(PBS对照组)进行比较时，a代表p＜0.05，b代表p≥0.05；与Z2(阳性对照组)比较时c代表p＜0.05，d代表p≥0.05；days：给药天数；T/C(％)：相对肿瘤增殖率。
SEQUENCE LISTING
<110> 北京纽安博生物技术有限公司
<120> 抗VEGF单域抗体及其人源化、单域抗体和IgG1-Fc构建的融合蛋白和应用
<130> BJ-3038-191217A
<160> 225
<170> PatentIn version 3.5
<210> 1
<211> 8
<212> PRT
<213> Artifical sequence
<400> 1
Ser Phe Ser Thr Tyr Ala Val Gly
1 5
<210> 2
<211> 17
<212> PRT
<213> Artifical sequence
<400> 2
Ala Gly Ile Asn Trp Ser Gly Glu Glu Thr Thr Tyr His Asn Ser Val
1 5 10 15
Asn
<210> 3
<211> 16
<212> PRT
<213> Artifical sequence
<400> 3
Ala Ala Asn Arg Arg Asn Tyr Ser Ser Thr Tyr Lys Gly Asn Tyr Asp
1 5 10 15
<210> 4
<211> 8
<212> PRT
<213> Artifical sequence
<400> 4
Thr Phe Ser Ser Tyr Ala Met Gly
1 5
<210> 5
<211> 15
<212> PRT
<213> Artifical sequence
<400> 5
Ala Ala Ile Arg Arg Asp Ala Val Ser Thr Tyr Tyr Ala Asp Ser
1 5 10 15
<210> 6
<211> 11
<212> PRT
<213> Artifical sequence
<400> 6
Ala Met Gly Asp Asp Tyr Val Asp Glu Tyr Asp
1 5 10
<210> 7
<211> 8
<212> PRT
<213> Artifical sequence
<400> 7
Thr Gly Ser His Tyr Asp Leu Ala
1 5
<210> 8
<211> 16
<212> PRT
<213> Artifical sequence
<400> 8
Val Ala Glu Phe Thr Trp Arg Ser Gly Pro Thr Ser Tyr Ala Glu Ser
1 5 10 15
<210> 9
<211> 15
<212> PRT
<213> Artifical sequence
<400> 9
Ala Ser Arg Tyr Phe Tyr Thr Tyr Gly Asp Pro Leu Lys Tyr Asp
1 5 10 15
<210> 10
<211> 6
<212> PRT
<213> Artifical sequence
<400> 10
Ser Ile Asn His Met Ala
1 5
<210> 11
<211> 14
<212> PRT
<213> Artifical sequence
<400> 11
Ala Arg Ala Phe Ser Ser Gly Ser Thr Thr Tyr Ala Asp Ser
1 5 10
<210> 12
<211> 15
<212> PRT
<213> Artifical sequence
<400> 12
Asn Gly Asp Gly Phe Leu Leu Tyr Asp Asp Thr Tyr Tyr Ser Asn
1 5 10 15
<210> 13
<211> 6
<212> PRT
<213> Artifical sequence
<400> 13
Ser Val Phe Asp Met Ala
1 5
<210> 14
<211> 14
<212> PRT
<213> Artifical sequence
<400> 14
Ala Glu Thr Thr Thr Ala Gly Ile Asn Thr Tyr Ala Asp Ser
1 5 10
<210> 15
<211> 9
<212> PRT
<213> Artifical sequence
<400> 15
Asn Ala Lys Asn Gly Trp Arg Thr Leu
1 5
<210> 16
<211> 6
<212> PRT
<213> Artifical sequence
<400> 16
Arg Asp Met Ser Phe Gly
1 5
<210> 17
<211> 14
<212> PRT
<213> Artifical sequence
<400> 17
Ala Tyr Ile Ser Ser Ser Gly Tyr Thr Asn Tyr Val Asp Ala
1 5 10
<210> 18
<211> 4
<212> PRT
<213> Artifical sequence
<400> 18
Asn Thr Leu Val
1
<210> 19
<211> 6
<212> PRT
<213> Artifical sequence
<400> 19
Ser Val Phe Asp Met Ala
1 5
<210> 20
<211> 14
<212> PRT
<213> Artifical sequence
<400> 20
Ala Arg Ile Thr Phe Asp Gly Ile Pro His Tyr Ala Asp Ser
1 5 10
<210> 21
<211> 9
<212> PRT
<213> Artifical sequence
<400> 21
Asn Ala Lys Asn Gly Trp Arg Thr Leu
1 5
<210> 22
<211> 7
<212> PRT
<213> Artifical sequence
<400> 22
Phe Ser Lys Tyr Ala Met Gly
1 5
<210> 23
<211> 15
<212> PRT
<213> Artifical sequence
<400> 23
Gly Asn Ile Tyr Trp Ser Asp Gly Ser Thr His Tyr Gln Asp Ser
1 5 10 15
<210> 24
<211> 18
<212> PRT
<213> Artifical sequence
<400> 24
Ala Ser Arg Gly Ser Asn Asn Gly Gly Ser Tyr Tyr Ser Glu Thr Gly
1 5 10 15
Tyr Asp
<210> 25
<211> 8
<212> PRT
<213> Artifical sequence
<400> 25
Thr Ile Gly Ser Tyr Asp Met Gly
1 5
<210> 26
<211> 14
<212> PRT
<213> Artifical sequence
<400> 26
Ala Ala Ile Thr Trp Ile Ser Asn Thr Asn Tyr Ala Asp Ser
1 5 10
<210> 27
<211> 14
<212> PRT
<213> Artifical sequence
<400> 27
Ala Ala Arg Arg Trp His Arg Ser Ala Pro His Asp Tyr Glu
1 5 10
<210> 28
<211> 6
<212> PRT
<213> Artifical sequence
<400> 28
Ala Arg Tyr Ser Met Gly
1 5
<210> 29
<211> 15
<212> PRT
<213> Artifical sequence
<400> 29
Ala Gly Ile Ser Arg Ser Ser Gly Thr Ile Ile Tyr Gly Gly Ser
1 5 10 15
<210> 30
<211> 16
<212> PRT
<213> Artifical sequence
<400> 30
Ala Ala Arg Glu Ser Leu Leu Ala Val Thr Thr Thr Arg Asp Tyr Pro
1 5 10 15
<210> 31
<211> 7
<212> PRT
<213> Artifical sequence
<400> 31
Ile Pro Tyr Val Pro Asp Met
1 5
<210> 32
<211> 14
<212> PRT
<213> Artifical sequence
<400> 32
Ala Thr Ile Thr Arg Gly Gly Asn Thr Met Tyr Ala Asp Ser
1 5 10
<210> 33
<211> 13
<212> PRT
<213> Artifical sequence
<400> 33
Ala Asp Val Trp Ser Ser Val Ala Leu Lys Leu Val Glu
1 5 10
<210> 34
<211> 6
<212> PRT
<213> Artifical sequence
<400> 34
Ser Asp Tyr Ala Met Ala
1 5
<210> 35
<211> 14
<212> PRT
<213> Artifical sequence
<400> 35
Ala Gly Ile Ser Trp Thr Gly Gly Arg Tyr Tyr Ala Glu Ser
1 5 10
<210> 36
<211> 18
<212> PRT
<213> Artifical sequence
<400> 36
Ala Thr Pro Asn Gln Ala Gly Leu Val Leu Leu Asp Asp Ala Glu Gly
1 5 10 15
Tyr Ala
<210> 37
<211> 7
<212> PRT
<213> Artifical sequence
<400> 37
Val Ser Thr Arg Asn Met Gly
1 5
<210> 38
<211> 15
<212> PRT
<213> Artifical sequence
<400> 38
Ala Arg Ile Gly Ser Asp Gly Ser Thr Tyr Asn Val Asp Ser Val
1 5 10 15
<210> 39
<211> 10
<212> PRT
<213> Artifical sequence
<400> 39
Asn Thr Phe Pro Val Thr Lys Phe Tyr Asp
1 5 10
<210> 40
<211> 8
<212> PRT
<213> Artifical sequence
<400> 40
Arg Phe Ser Ala Tyr Asp Met Gly
1 5
<210> 41
<211> 15
<212> PRT
<213> Artifical sequence
<400> 41
Ala Thr Ile Asn Trp Ser Ala Leu Ser Arg Tyr Tyr Ala Asp Ser
1 5 10 15
<210> 42
<211> 18
<212> PRT
<213> Artifical sequence
<400> 42
Ala Gly Gly Arg Ile Ser Ala Ala Leu Arg Val Pro Asp Arg Asp Ala
1 5 10 15
Tyr Ser
<210> 43
<211> 7
<212> PRT
<213> Artifical sequence
<400> 43
Val Arg Asp Asp Ala Met Gly
1 5
<210> 44
<211> 16
<212> PRT
<213> Artifical sequence
<400> 44
Ala Thr Ile Ser Trp Asn Gly Gly Ser Thr Tyr Tyr Ala Ala Ser Ala
1 5 10 15
<210> 45
<211> 18
<212> PRT
<213> Artifical sequence
<400> 45
Ala Val Ala Ser Arg Tyr Arg Gln Ile Val Leu Asn Thr Glu Glu Lys
1 5 10 15
Tyr Asp
<210> 46
<211> 7
<212> PRT
<213> Artifical sequence
<400> 46
Ser Phe Ser Val Pro His Met
1 5
<210> 47
<211> 15
<212> PRT
<213> Artifical sequence
<400> 47
Ala Thr Ile Ser Arg Gly Gly Val Ser Thr Tyr Ala Ala Asp Ser
1 5 10 15
<210> 48
<211> 14
<212> PRT
<213> Artifical sequence
<400> 48
Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu
1 5 10
<210> 49
<211> 6
<212> PRT
<213> Artifical sequence
<400> 49
Ser His Arg Thr Phe Ala
1 5
<210> 50
<211> 14
<212> PRT
<213> Artifical sequence
<400> 50
Ala Thr Ile Gly Ser Tyr Gly Ser Thr Tyr Tyr Asp Glu Ser
1 5 10
<210> 51
<211> 4
<212> PRT
<213> Artifical sequence
<400> 51
His Thr Gln Val
1
<210> 52
<211> 7
<212> PRT
<213> Artifical sequence
<400> 52
Ser Thr Ser Tyr Val Tyr Asp
1 5
<210> 53
<211> 11
<212> PRT
<213> Artifical sequence
<400> 53
Ala Ile Thr Asp Gly Gly Ser Thr Asp Asn Tyr
1 5 10
<210> 54
<211> 16
<212> PRT
<213> Artifical sequence
<400> 54
Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Tyr Asp
1 5 10 15
<210> 55
<211> 7
<212> PRT
<213> Artifical sequence
<400> 55
Ser Thr Ser Tyr Val Tyr Asp
1 5
<210> 56
<211> 11
<212> PRT
<213> Artifical sequence
<400> 56
Ala Ile Thr Asp Gly Gly Ser Thr Asp Asn Tyr
1 5 10
<210> 57
<211> 17
<212> PRT
<213> Artifical sequence
<400> 57
Ala Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Tyr
1 5 10 15
Asp
<210> 58
<211> 7
<212> PRT
<213> Artifical sequence
<400> 58
Ile Pro Tyr Val Pro Asp Met
1 5
<210> 59
<211> 14
<212> PRT
<213> Artifical sequence
<400> 59
Ala Thr Ile Thr Arg Gly Gly Val Thr Met Tyr Ala Asp Ser
1 5 10
<210> 60
<211> 14
<212> PRT
<213> Artifical sequence
<400> 60
Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu
1 5 10
<210> 61
<211> 7
<212> PRT
<213> Artifical sequence
<400> 61
Ser Thr Ser Tyr Val Tyr Asp
1 5
<210> 62
<211> 12
<212> PRT
<213> Artifical sequence
<400> 62
Ala Ala Ile Ser Arg Gly Gly Val Thr Asp Asn Tyr
1 5 10
<210> 63
<211> 17
<212> PRT
<213> Artifical sequence
<400> 63
Ala Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Tyr
1 5 10 15
Asp
<210> 64
<211> 7
<212> PRT
<213> Artifical sequence
<400> 64
Ile Ser Tyr Val Pro Asp Met
1 5
<210> 65
<211> 14
<212> PRT
<213> Artifical sequence
<400> 65
Ala Thr Ile Thr Arg Gly Gly Asn Thr Met Tyr Ala Asp Ser
1 5 10
<210> 66
<211> 14
<212> PRT
<213> Artifical sequence
<400> 66
Asn Ala Asp Val Trp Ser Ser Val Leu Phe Lys Leu Val Glu
1 5 10
<210> 67
<211> 7
<212> PRT
<213> Artifical sequence
<400> 67
Ser Phe Ser Tyr Ile Tyr Asp
1 5
<210> 68
<211> 12
<212> PRT
<213> Artifical sequence
<400> 68
Ala Ala Ile Ser Arg Gly Ser Ser Thr Asp Asn Tyr
1 5 10
<210> 69
<211> 17
<212> PRT
<213> Artifical sequence
<400> 69
Ala Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Tyr
1 5 10 15
Asp
<210> 70
<211> 7
<212> PRT
<213> Artifical sequence
<400> 70
Ser Phe Ser Tyr Ile Pro Asp
1 5
<210> 71
<211> 12
<212> PRT
<213> Artifical sequence
<400> 71
Ala Ala Ile Ser Arg Gly Ser Ser Thr Asp Asn Tyr
1 5 10
<210> 72
<211> 15
<212> PRT
<213> Artifical sequence
<400> 72
Ala Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu
1 5 10 15
<210> 73
<211> 8
<212> PRT
<213> Artifical sequence
<400> 73
Arg Phe Ser Ala Tyr Asp Met Gly
1 5
<210> 74
<211> 15
<212> PRT
<213> Artifical sequence
<400> 74
Ala Thr Ile Asn Trp Ser Thr Leu Ser Arg Tyr Tyr Ala Asp Ser
1 5 10 15
<210> 75
<211> 18
<212> PRT
<213> Artifical sequence
<400> 75
Ala Gly Gly Arg Ile Ser Ser Glu Leu Arg Val Thr Ala Arg Asp Ala
1 5 10 15
Tyr Thr
<210> 76
<211> 8
<212> PRT
<213> Artifical sequence
<400> 76
Ile Ser Tyr Val Pro Asp Met Gly
1 5
<210> 77
<211> 12
<212> PRT
<213> Artifical sequence
<400> 77
Ala Ala Ile Ser Arg Gly Gly Ser Thr Asp Asn Tyr
1 5 10
<210> 78
<211> 19
<212> PRT
<213> Artifical sequence
<400> 78
Ala Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Ser
1 5 10 15
Ala Tyr Asp
<210> 79
<211> 8
<212> PRT
<213> Artifical sequence
<400> 79
Ile Ser Tyr Val Pro Asp Met Gly
1 5
<210> 80
<211> 12
<212> PRT
<213> Artifical sequence
<400> 80
Ala Thr Ile Ser Arg Gly Gly Val Thr Asp Asn Tyr
1 5 10
<210> 81
<211> 18
<212> PRT
<213> Artifical sequence
<400> 81
Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Ser Ala
1 5 10 15
Tyr Asp
<210> 82
<211> 8
<212> PRT
<213> Artifical sequence
<400> 82
Thr Ser Tyr Val Pro Asp Met Gly
1 5
<210> 83
<211> 12
<212> PRT
<213> Artifical sequence
<400> 83
Ala Thr Ile Ser Arg Gly Gly Val Thr Asp Tyr Tyr
1 5 10
<210> 84
<211> 18
<212> PRT
<213> Artifical sequence
<400> 84
Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Ser Ala
1 5 10 15
Tyr Asp
<210> 85
<211> 6
<212> PRT
<213> Artifical sequence
<400> 85
Ser Ser Tyr His Met Asp
1 5
<210> 86
<211> 15
<212> PRT
<213> Artifical sequence
<400> 86
Ala Ala Ile Ser Trp Thr Gly His Ser Thr Tyr Tyr Ala Asp Ser
1 5 10 15
<210> 87
<211> 14
<212> PRT
<213> Artifical sequence
<400> 87
Ala Ala Thr Arg Arg Ala Thr Met Ile Ala Val Pro Ser Asp
1 5 10
<210> 88
<211> 7
<212> PRT
<213> Artifical sequence
<400> 88
Phe Ile Ser Tyr Val Pro Asp
1 5
<210> 89
<211> 13
<212> PRT
<213> Artifical sequence
<400> 89
Ala Ala Thr Ile Ser Arg Gly Gly Val Thr Asp Tyr Tyr
1 5 10
<210> 90
<211> 16
<212> PRT
<213> Artifical sequence
<400> 90
Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Ser Ala
1 5 10 15
<210> 91
<211> 7
<212> PRT
<213> Artifical sequence
<400> 91
Phe Ile Ser Tyr Val Pro Asp
1 5
<210> 92
<211> 12
<212> PRT
<213> Artifical sequence
<400> 92
Ala Ala Thr Ile Thr Arg Gly Gly Val Thr Asp Tyr
1 5 10
<210> 93
<211> 17
<212> PRT
<213> Artifical sequence
<400> 93
Ala Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Ser
1 5 10 15
Ala
<210> 94
<211> 7
<212> PRT
<213> Artifical sequence
<400> 94
Tyr Thr Ser Tyr Leu Pro Asp
1 5
<210> 95
<211> 12
<212> PRT
<213> Artifical sequence
<400> 95
Ala Ala Thr Ile Thr Arg Gly Gly Val Thr Glu Tyr
1 5 10
<210> 96
<211> 18
<212> PRT
<213> Artifical sequence
<400> 96
Ala Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Ala Val Pro
1 5 10 15
Ser Asp
<210> 97
<211> 8
<212> PRT
<213> Artifical sequence
<400> 97
Thr Phe Gly Ser Tyr Asp Met Ala
1 5
<210> 98
<211> 15
<212> PRT
<213> Artifical sequence
<400> 98
Ala Ala Ile Asn Trp Tyr Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser
1 5 10 15
<210> 99
<211> 22
<212> PRT
<213> Artifical sequence
<400> 99
Ala Ala Glu Lys Gly Phe Ala Ser Leu Arg Leu Trp Ala Leu Ser His
1 5 10 15
Lys Pro His Glu Ser Arg
20
<210> 100
<211> 7
<212> PRT
<213> Artifical sequence
<400> 100
Tyr Ile Pro Tyr Leu Pro Asp
1 5
<210> 101
<211> 11
<212> PRT
<213> Artifical sequence
<400> 101
Ala Thr Ile Thr Arg Gly Gly Val Thr Glu Tyr
1 5 10
<210> 102
<211> 17
<212> PRT
<213> Artifical sequence
<400> 102
Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Ala Val Pro Ser
1 5 10 15
Asp
<210> 103
<211> 8
<212> PRT
<213> Artifical sequence
<400> 103
Pro Ser Ser Tyr Tyr Gly Val Ser
1 5
<210> 104
<211> 15
<212> PRT
<213> Artifical sequence
<400> 104
Ala Gly Ile Ser Arg Ser Gly Gly Thr Asp Asn Tyr Ala Asn Phe
1 5 10 15
<210> 105
<211> 16
<212> PRT
<213> Artifical sequence
<400> 105
Ala Ala Ala Thr Asn Val Tyr Ala Ser Ala Thr Leu Ser Ala Tyr Asp
1 5 10 15
<210> 106
<211> 7
<212> PRT
<213> Artifical sequence
<400> 106
Tyr Ile Pro Tyr Leu Pro Asp
1 5
<210> 107
<211> 11
<212> PRT
<213> Artifical sequence
<400> 107
Ala Thr Ile Thr Arg Gly Gly Val Thr Glu Tyr
1 5 10
<210> 108
<211> 18
<212> PRT
<213> Artifical sequence
<400> 108
Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Ala Val Glu Tyr
1 5 10 15
Ser Asp
<210> 109
<211> 7
<212> PRT
<213> Artifical sequence
<400> 109
Tyr Ser Thr Tyr Leu Pro Asp
1 5
<210> 110
<211> 12
<212> PRT
<213> Artifical sequence
<400> 110
Ala Ala Ile Thr Arg Gly Gly His Ser Thr Tyr Tyr
1 5 10
<210> 111
<211> 18
<212> PRT
<213> Artifical sequence
<400> 111
Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Ala Val Glu Tyr
1 5 10 15
Ser Asp
<210> 112
<211> 7
<212> PRT
<213> Artifical sequence
<400> 112
Val Ser Ser Tyr Asp Met Gly
1 5
<210> 113
<211> 14
<212> PRT
<213> Artifical sequence
<400> 113
Ala Ala Ile Thr Trp Ile Ser Asn Thr Asn Tyr Ala Asp Ser
1 5 10
<210> 114
<211> 14
<212> PRT
<213> Artifical sequence
<400> 114
Ala Ala Arg Arg Tyr Tyr Arg Ser Gly Ala Gln Asp Tyr Glu
1 5 10
<210> 115
<211> 7
<212> PRT
<213> Artifical sequence
<400> 115
Ser Ser Thr Tyr Leu Pro Asp
1 5
<210> 116
<211> 11
<212> PRT
<213> Artifical sequence
<400> 116
Ala Ile Thr Arg Gly Gly His Ser Thr Tyr Tyr
1 5 10
<210> 117
<211> 17
<212> PRT
<213> Artifical sequence
<400> 117
Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Tyr Ser
1 5 10 15
Asp
<210> 118
<211> 7
<212> PRT
<213> Artifical sequence
<400> 118
Ile Gly Ser Tyr Asp Met Gly
1 5
<210> 119
<211> 14
<212> PRT
<213> Artifical sequence
<400> 119
Ala Ala Ile Thr Trp Ile Ser Asn Thr Asn Tyr Ala Asp Ser
1 5 10
<210> 120
<211> 14
<212> PRT
<213> Artifical sequence
<400> 120
Ala Ala Arg Arg Trp Tyr Arg Ser Ala Pro Gln Asp Tyr Glu
1 5 10
<210> 121
<211> 7
<212> PRT
<213> Artifical sequence
<400> 121
Ser Ser Thr Tyr Leu Pro Asp
1 5
<210> 122
<211> 11
<212> PRT
<213> Artifical sequence
<400> 122
Ala Ile Thr Arg Gly Gly Val Ser Thr Tyr Tyr
1 5 10
<210> 123
<211> 18
<212> PRT
<213> Artifical sequence
<400> 123
Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Tyr Pro
1 5 10 15
Ser Asp
<210> 124
<211> 7
<212> PRT
<213> Artifical sequence
<400> 124
Ile Gly Ser Tyr Asp Met Gly
1 5
<210> 125
<211> 14
<212> PRT
<213> Artifical sequence
<400> 125
Ala Ala Ile Thr Trp Ile Ser Asn Thr Asn Tyr Ala Asp Ser
1 5 10
<210> 126
<211> 14
<212> PRT
<213> Artifical sequence
<400> 126
Ala Ala Arg Arg Tyr Tyr Arg Ser Ala Pro Gln Asp Tyr Asp
1 5 10
<210> 127
<211> 124
<212> PRT
<213> Vicugna pacos
<400> 127
Gln Val Gln Leu Val 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 Pro Ser Phe Ser Thr Tyr
20 25 30
Ala Val Gly Trp Phe Arg Gln Val Pro Arg Arg Glu Arg Ala Phe Val
35 40 45
Ala Gly Ile Asn Trp Ser Gly Glu Glu Thr Thr Tyr His Asn Ser Val
50 55 60
Asn 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 Gln Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Asn Arg Arg Asn Tyr Ser Ser Thr Tyr Lys Gly Asn Tyr Asp
100 105 110
Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 128
<211> 119
<212> PRT
<213> Vicugna pacos
<400> 128
Gln Val Gln Leu Val 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 Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Ile Arg Arg Asp Ala Val Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asp Met Val 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 Met Gly Asp Asp Tyr Val Asp Glu Tyr Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Gln Val Thr Val Ser Ser
115
<210> 129
<211> 123
<212> PRT
<213> Vicugna pacos
<400> 129
Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ala Cys Val Ala Ser Arg Val Thr Gly Ser His Tyr
20 25 30
Asp Leu Ala Trp Phe Arg Gln Gly Pro Gly Lys Glu Arg Glu Val Val
35 40 45
Ala Glu Phe Thr Trp Arg Ser Gly Pro Thr Ser Tyr Ala Glu Ser Val
50 55 60
Lys Gly Arg Phe Ala Val Ser Lys Asp Asn Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asp Asn Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ser Arg Tyr Phe Tyr Thr Tyr Gly Asp Pro Leu Lys Tyr Asp Tyr
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 130
<211> 122
<212> PRT
<213> Vicugna pacos
<400> 130
Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Arg Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Ile Asn
20 25 30
His Met Ala Trp Tyr Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Arg Ala Phe Ser Ser Gly Ser Thr Thr Tyr Ala Asp Ser Val Arg
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asp 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
Gly Asp Gly Phe Leu Leu Tyr Asp Asp Thr Tyr Tyr Ser Asn Ala Trp
100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 131
<211> 115
<212> PRT
<213> Vicugna pacos
<400> 131
Gln Val Lys Leu Glu 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 Ile Ala Phe Ser Val Phe
20 25 30
Asp Met Ala Trp Tyr Arg Gln Ala Pro Gly Asn Gln Arg Glu Leu Val
35 40 45
Ala Glu Thr Thr Thr Ala Gly Ile Asn Thr 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 Leu Arg Pro Glu Asp Thr Ala Val Tyr Ser Cys Asn
85 90 95
Ala Lys Asn Gly Trp Arg Thr Leu Trp Gly Gln Gly Thr Gln Val Thr
100 105 110
Val Ser Ser
115
<210> 132
<211> 111
<212> PRT
<213> Vicugna pacos
<400> 132
Gly Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Val Ser Gly Ser Thr Phe Arg Asp Met
20 25 30
Ser Phe Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Arg Leu
35 40 45
Ala Tyr Ile Ser Ser Ser Gly Tyr Thr Asn Tyr Val Asp Ala Val Lys
50 55 60
Asp 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 Ile Tyr Tyr Cys Asn
85 90 95
Thr Leu Val Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
100 105 110
<210> 133
<211> 115
<212> PRT
<213> Vicugna pacos
<400> 133
Gln Val Lys Leu Glu 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 Ile Ala Phe Ser Val Phe
20 25 30
Asp Met Ala Trp Tyr Arg Gln Ala Pro Gly Asn Gln Arg Glu Leu Val
35 40 45
Ala Arg Ile Thr Phe Asp Gly Ile Pro His 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 Leu Arg Pro Glu Asp Thr Ala Val Tyr Ser Cys Asn
85 90 95
Ala Lys Asn Gly Trp Arg Thr Leu Trp Gly Gln Gly Thr Gln Val Thr
100 105 110
Val Ser Ser
115
<210> 134
<211> 126
<212> PRT
<213> Vicugna pacos
<400> 134
Gln Val Gln Leu Val Asp Ser Gly Gly Gly Tyr Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Gly Ser Phe Ser Lys Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Gln Phe Val
35 40 45
Gly Asn Ile Tyr Trp Ser Asp Gly Ser Thr His Tyr Gln Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Leu Ser Lys Asp Tyr Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Glu Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ser Arg Gly Ser Asn Asn Gly Gly Ser Tyr Tyr Ser Glu Thr Gly
100 105 110
Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 135
<211> 121
<212> PRT
<213> Vicugna pacos
<400> 135
Gln Val Gln Leu Glu 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 Ile Gly Ser Tyr
20 25 30
Asp Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Ile Thr Trp Ile Ser Asn Thr Asn Tyr Ala Asp Ser Val Glu
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Lys Asp Lys Thr Thr Met 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 Arg Arg Trp His Arg Ser Ala Pro His Asp Tyr Glu Phe Trp Gly
100 105 110
Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 136
<211> 124
<212> PRT
<213> Vicugna pacos
<400> 136
Gln Val Gln Leu Val Asp Ser Gly Gly Gly Leu Val Leu Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asp Thr Phe Ala Arg Tyr
20 25 30
Ser Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Ser Arg Ser Ser Gly Thr Ile Ile Tyr Gly Gly Ser Val
50 55 60
Lys Gly Arg Phe Ile Ile Phe Arg Asp Asn Val Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Gly Leu Lys Pro Asp Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Ala Arg Glu Ser Leu Leu Ala Val Thr Thr Thr Arg Asp Tyr Pro
100 105 110
Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 137
<211> 121
<212> PRT
<213> Vicugna pacos
<400> 137
Gln Val Lys Leu Glu 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 Pro Tyr Val Pro
20 25 30
Asp Met His Trp Tyr Arg Gln Ala Pro Gly Gln Gln Arg Gln Leu Val
35 40 45
Ala Thr Ile Thr Arg Gly Gly Asn Thr Met 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 Thr Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn
85 90 95
Ala Asp Val Trp Ser Ser Val Ala Leu Lys Leu Val Glu Tyr Trp Gly
100 105 110
Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 138
<211> 125
<212> PRT
<213> Vicugna pacos
<400> 138
Gln Val Gln Leu Val Asp Ser Gly Gly Gly Phe Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Asp Tyr
20 25 30
Ala Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Ser Trp Thr Gly Gly Arg Tyr Tyr Ala Glu Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Ala Phe Leu
65 70 75 80
Gln Met Asn Thr Leu Lys Pro Glu Asp Thr Gly Ile Tyr His Cys Ala
85 90 95
Thr Pro Asn Gln Ala Gly Leu Val Leu Leu Asp Asp Ala Glu Gly Tyr
100 105 110
Ala Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 139
<211> 117
<212> PRT
<213> Vicugna pacos
<400> 139
Gln Val Gln Leu Val Asp Ser Gly Gly Gly Leu Val Gln Ala Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Phe Thr Val Ser Thr Arg
20 25 30
Asn Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Val Leu Val
35 40 45
Ala Arg Ile Gly Ser Asp Gly Ser Thr Tyr Asn Val Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Ala Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Trp Cys Asn
85 90 95
Thr Phe Pro Val Thr Lys Phe Tyr Asp Tyr Trp Gly Gln Gly Thr Gln
100 105 110
Val Thr Val Ser Ser
115
<210> 140
<211> 126
<212> PRT
<213> Vicugna pacos
<400> 140
Glu Val Lys Leu Val Asp 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 Arg Phe Ser Ala Tyr
20 25 30
Asp Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Thr Ile Asn Trp Ser Ala Leu Ser 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 Ser
65 70 75 80
Leu His Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Gly Gly Arg Ile Ser Ala Ala Leu Arg Val Pro Asp Arg Asp Ala
100 105 110
Tyr Ser Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 141
<211> 126
<212> PRT
<213> Vicugna pacos
<400> 141
Gln Val Gln Leu Val Asp 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 Val Arg Asp Asp
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 Gly Gly Ser Thr Tyr Tyr Ala Ala Ser Ala
50 55 60
Lys Gly Arg Phe Ile Val 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 Val Ala Ser Arg Tyr Arg Gln Ile Val Leu Asn Thr Glu Glu Lys
100 105 110
Tyr Asp Ala Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 142
<211> 122
<212> PRT
<213> Vicugna pacos
<400> 142
Gln Val Lys Leu Val 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 Ser Ser Phe Ser Val Pro
20 25 30
His Met Asp Trp Phe Arg Gln Ala Pro Gly Gln Glu Arg Glu Phe Val
35 40 45
Ala Thr Ile Ser Arg Gly Gly Val Ser Thr Tyr Ala Ala Asp Ser Val
50 55 60
Lys Gly Arg Ser Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Met Tyr
65 70 75 80
Leu Gln Met Asn Asn Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Tyr Trp
100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 143
<211> 111
<212> PRT
<213> Vicugna pacos
<400> 143
Gln Val Lys Leu Glu 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 Thr Thr Phe Ser His Arg
20 25 30
Thr Phe Ala Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val
35 40 45
Ala Thr Ile Gly Ser Tyr Gly Ser Thr Tyr Tyr Asp Glu Ser Val Lys
50 55 60
Asp Arg Phe Thr Ile Ala Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Gln Asp Thr Ala Val Tyr Tyr Cys His
85 90 95
Thr Gln Val Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
100 105 110
<210> 144
<211> 124
<212> PRT
<213> Vicugna pacos
<400> 144
Glu Val Lys Leu Glu 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 Thr Ser Tyr Val Tyr
20 25 30
Asp Met His Trp Tyr Arg Gln Ala Pro Gly Gln Gln Arg Gln Leu Val
35 40 45
Ala Ala Ile Thr Asp Gly Gly Ser Thr Asp Asn 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 Thr Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Tyr Asp
100 105 110
Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 145
<211> 125
<212> PRT
<213> Vicugna pacos
<400> 145
Glu Val Lys Leu Glu 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 Thr Ser Tyr Val Tyr
20 25 30
Asp Met His Trp Tyr Arg Gln Ala Pro Gly Gln Gln Arg Gln Leu Val
35 40 45
Ala Ala Ile Thr Asp Gly Gly Ser Thr Asp Asn 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 Thr Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Tyr
100 105 110
Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 146
<211> 121
<212> PRT
<213> Vicugna pacos
<400> 146
Gln Val Lys Leu Glu 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 Pro Tyr Val Pro
20 25 30
Asp Met His Trp Tyr Arg Gln Ala Pro Gly Gln Gln Arg Gln Leu Val
35 40 45
Ala Thr Ile Thr Arg Gly Gly Val Thr Met 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 Thr Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn
85 90 95
Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Tyr Trp Gly
100 105 110
Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 147
<211> 125
<212> PRT
<213> Vicugna pacos
<400> 147
Glu Val Lys Leu Glu 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 Thr Ser Tyr Val Tyr
20 25 30
Asp Met His Trp Tyr Arg Gln Ala Pro Gly Gln Gln Arg Gln Leu Val
35 40 45
Ala Ala Ile Ser Arg Gly Gly Val Thr Asp Asn 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 Thr Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Tyr
100 105 110
Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 148
<211> 121
<212> PRT
<213> Vicugna pacos
<400> 148
Glu Val Gln Leu Glu Asp 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 Ser Tyr Val Pro
20 25 30
Asp Met His Trp Tyr Arg Gln Ala Pro Gly Gln Gln Arg Gln Leu Val
35 40 45
Ala Thr Ile Thr Arg Gly Gly Asn Thr Met 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 Thr Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn
85 90 95
Ala Asp Val Trp Ser Ser Val Leu Phe Lys Leu Val Glu Tyr Trp Gly
100 105 110
Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 149
<211> 125
<212> PRT
<213> Vicugna pacos
<400> 149
Glu Val Lys Leu Glu 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 Phe Ser Tyr Ile Tyr
20 25 30
Asp Met His Trp Tyr Arg Gln Ala Pro Gly Gln Gln Arg Gln Leu Val
35 40 45
Ala Ala Ile Ser Arg Gly Ser Ser Thr Asp Asn 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 Thr Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Tyr
100 105 110
Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 150
<211> 123
<212> PRT
<213> Vicugna pacos
<400> 150
Glu Val Lys Leu Glu 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 Phe Ser Tyr Ile Pro
20 25 30
Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Gln Gln Arg Gln Leu Val
35 40 45
Ala Ala Ile Ser Arg Gly Ser Ser Thr Asp Asn 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 Thr Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Tyr
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 151
<211> 126
<212> PRT
<213> Vicugna pacos
<400> 151
Gln Val Gln Leu Val 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 Arg Phe Ser Ala Tyr
20 25 30
Asp Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Thr Ile Asn Trp Ser Thr Leu Ser 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 His Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Gly Gly Arg Ile Ser Ser Glu Leu Arg Val Thr Ala Arg Asp Ala
100 105 110
Tyr Thr Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 152
<211> 127
<212> PRT
<213> Vicugna pacos
<400> 152
Gln Val Gln Leu Glu 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 Ser Tyr Val Pro
20 25 30
Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Gln Gln Arg Gln Leu Val
35 40 45
Ala Ala Ile Ser Arg Gly Gly Ser Thr Asp Asn 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 Thr Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Ser
100 105 110
Ala Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 153
<211> 126
<212> PRT
<213> Vicugna pacos
<400> 153
Gln Val Gln Leu Glu 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 Ser Tyr Val Pro
20 25 30
Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Gln Gln Arg Gln Leu Val
35 40 45
Ala Thr Ile Ser Arg Gly Gly Val Thr Asp Asn 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 Thr Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Ser Ala
100 105 110
Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 154
<211> 126
<212> PRT
<213> Vicugna pacos
<400> 154
Gln Val Gln Leu Val 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 Thr Ser Tyr Val Pro
20 25 30
Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Gln Gln Arg Gln Leu Val
35 40 45
Ala Thr Ile Ser Arg Gly Gly Val Thr Asp 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 Thr Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Ser Ala
100 105 110
Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 155
<211> 122
<212> PRT
<213> Vicugna pacos
<400> 155
Gln Val Gln Leu Val 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 Ser Ser Phe Ser Ser Tyr
20 25 30
His Met Asp Trp Phe Arg Gln Ala Pro Gly Gln Glu Arg Glu Phe Val
35 40 45
Ala Ala Ile Ser Trp Thr Gly His Ser Thr Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Ser Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Met 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 Ala Thr Arg Arg Ala Thr Met Ile Ala Val Pro Ser Asp Tyr Trp
100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 156
<211> 125
<212> PRT
<213> Vicugna pacos
<400> 156
Gln Val Gln Leu Val 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 Ile Ser Tyr Val Pro
20 25 30
Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Gln Gln Arg Gln Leu Val
35 40 45
Ala Ala Thr Ile Ser Arg Gly Gly Val Thr Asp 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 Thr Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr
85 90 95
Cys Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Ser
100 105 110
Ala Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 157
<211> 125
<212> PRT
<213> Vicugna pacos
<400> 157
Gln Val Gln Leu Val 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 Ile Ser Tyr Val Pro
20 25 30
Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Gln Gln Arg Gln Leu Val
35 40 45
Ala Ala Thr Ile Thr Arg Gly Gly Val Thr Asp 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 Thr Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Ser
100 105 110
Ala Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 158
<211> 126
<212> PRT
<213> Vicugna pacos
<400> 158
Gln Val Lys Leu Glu 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 Tyr Thr Ser Tyr Leu Pro
20 25 30
Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Gln Gln Arg Gln Leu Val
35 40 45
Ala Ala Thr Ile Thr Arg Gly Gly Val Thr Glu 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 Thr Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Ala Val Pro
100 105 110
Ser Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 159
<211> 128
<212> PRT
<213> Vicugna pacos
<400> 159
Gln Val Lys Leu Glu 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 Gly Ser Tyr
20 25 30
Asp Met Ala Trp Phe Arg Gln Val Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Ile Asn Trp Tyr Gly Gly Tyr Thr Tyr Tyr Ala Asp Ser 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 Asn Leu Lys Pro Ser Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Glu Lys Gly Phe Ala Ser Leu Arg Leu Trp Ala Leu Ser His
100 105 110
Lys Pro His Glu Ser Arg Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 160
<211> 124
<212> PRT
<213> Vicugna pacos
<400> 160
Gln Val Lys Leu Glu 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 Tyr Ile Pro Tyr Leu Pro
20 25 30
Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Gln Gln Arg Gln Leu Val
35 40 45
Ala Thr Ile Thr Arg Gly Gly Val Thr Glu 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 Thr Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn
85 90 95
Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Ala Val Pro Ser Asp
100 105 110
Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 161
<211> 124
<212> PRT
<213> Vicugna pacos
<400> 161
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Thr Leu Ser Cys Val Phe Ser Gly Arg Pro Ser Ser Tyr Tyr
20 25 30
Gly Val Ser Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Gly Ile Ser Arg Ser Gly Gly Thr Asp Asn Tyr Ala Asn Phe Val
50 55 60
Lys Gly Arg Phe Thr Val Ser Lys Asp Asn Gly Lys Asn Thr Val 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 Ala Ala Thr Asn Val Tyr Ala Ser Ala Thr Leu Ser Ala Tyr Asp
100 105 110
Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 162
<211> 125
<212> PRT
<213> Vicugna pacos
<400> 162
Gln Val Lys Leu Glu 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 Tyr Ile Pro Tyr Leu Pro
20 25 30
Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Gln Gln Arg Gln Leu Val
35 40 45
Ala Thr Ile Thr Arg Gly Gly Val Thr Glu 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 Thr Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn
85 90 95
Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Ala Val Glu Tyr Ser
100 105 110
Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 163
<211> 126
<212> PRT
<213> Vicugna pacos
<400> 163
Gln Val Lys Leu Glu 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 Tyr Ser Thr Tyr Leu Pro
20 25 30
Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Gln Gln Arg Gln Leu Val
35 40 45
Ala Ala Ile Thr Arg Gly Gly His 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 Tyr
65 70 75 80
Leu Gln Met Thr Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Ala Val Glu Tyr
100 105 110
Ser Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 164
<211> 121
<212> PRT
<213> Vicugna pacos
<400> 164
Gln Val Lys Leu Glu 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 Val Ser Ser Tyr
20 25 30
Asp Met Gly Trp Phe Arg Gln Ser Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Ile Thr Trp Ile Ser Asn Thr Asn Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Asp Arg Asp Lys 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 Arg Arg Tyr Tyr Arg Ser Gly Ala Gln Asp Tyr Glu Tyr Trp Gly
100 105 110
Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 165
<211> 124
<212> PRT
<213> Vicugna pacos
<400> 165
Gln Val Lys Leu Glu 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 Ser Thr Tyr Leu Pro
20 25 30
Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Gln Gln Arg Gln Leu Val
35 40 45
Ala Ile Thr Arg Gly Gly His Ser Thr Tyr 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 Thr Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn
85 90 95
Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Tyr Ser Asp
100 105 110
Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 166
<211> 121
<212> PRT
<213> Vicugna pacos
<400> 166
Asp Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Asp
1 5 10 15
Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Gly Thr Ile Gly Ser Tyr
20 25 30
Asp Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Ile Thr Trp Ile Ser Asn Thr Asn Tyr Ala Asp Ser Val Glu
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Lys Ala Lys Ser Thr Met Tyr Leu
65 70 75 80
Gln Met His Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Arg Arg Trp Tyr Arg Ser Ala Pro Gln Asp Tyr Glu Tyr Trp Gly
100 105 110
Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 167
<211> 125
<212> PRT
<213> Vicugna pacos
<400> 167
Gln Val Lys Leu Glu 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 Ser Thr Tyr Leu Pro
20 25 30
Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Gln Gln Arg Gln Leu Val
35 40 45
Ala Ile Thr Arg Gly Gly Val Ser Thr Tyr 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 Thr Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn
85 90 95
Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Tyr Pro Ser
100 105 110
Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 168
<211> 121
<212> PRT
<213> Vicugna pacos
<400> 168
Gln Val Lys Leu Glu 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 Ile Gly Ser Tyr
20 25 30
Asp Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Phe Val
35 40 45
Ala Ala Ile Thr Trp Ile Ser Asn Thr Asn Tyr Ala Asp Ser Val Glu
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Lys Ala Lys Ser Thr Met Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Val Cys Ala
85 90 95
Ala Arg Arg Tyr Tyr Arg Ser Ala Pro Gln Asp Tyr Asp Phe Trp Gly
100 105 110
Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 169
<211> 121
<212> PRT
<213> artifical sequence
<400> 169
Glu Val Lys Leu Glu 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 Pro Tyr Val Pro
20 25 30
Asp Met His Trp Phe Arg Gln Ala Pro Gly Gln Gly Leu Gln Leu Val
35 40 45
Ala Thr Ile Thr Arg Gly Gly Asn Thr Met Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn
85 90 95
Ala Asp Val Trp Ser Ser Val Ala Leu Lys Leu Val Glu Tyr Trp Gly
100 105 110
Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 170
<211> 121
<212> PRT
<213> artifical sequence
<400> 170
Glu Val Lys Leu Glu 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 Pro Tyr Val Pro
20 25 30
Asp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Gln Leu Val
35 40 45
Ala Thr Ile Thr Arg Gly Gly Asn Thr Met Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn
85 90 95
Ala Asp Val Trp Ser Ser Val Ala Leu Lys Leu Val Glu Tyr Trp Gly
100 105 110
Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 171
<211> 121
<212> PRT
<213> artifical sequence
<400> 171
Gln Val Lys Leu Glu 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 Pro Tyr Val Pro
20 25 30
Asp Met His Trp Phe Arg Gln Ala Pro Gly Gln Gly Leu Gln Leu Val
35 40 45
Ala Thr Ile Thr Arg Gly Gly Asn Thr Met Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn
85 90 95
Ala Asp Val Trp Ser Ser Val Ala Leu Lys Leu Val Glu Tyr Trp Gly
100 105 110
Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 172
<211> 121
<212> PRT
<213> artifical sequence
<400> 172
Glu Val Lys Leu Glu 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 Pro Tyr Val Pro
20 25 30
Asp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Val
35 40 45
Ala Thr Ile Thr Arg Gly Gly Asn Thr Met Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn
85 90 95
Ala Asp Val Trp Ser Ser Val Ala Leu Lys Leu Val Glu Tyr Trp Gly
100 105 110
Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 173
<211> 121
<212> PRT
<213> artifical sequence
<400> 173
Glu Val Lys Leu Glu 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 Pro Tyr Val Pro
20 25 30
Asp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Gln Leu Val
35 40 45
Ala Thr Ile Thr Arg Gly Gly Val Thr Met Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn
85 90 95
Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Tyr Trp Gly
100 105 110
Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 174
<211> 121
<212> PRT
<213> artifical sequence
<400> 174
Gln Val Lys Leu Glu 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 Pro Tyr Val Pro
20 25 30
Asp Met His Trp Tyr Arg Gln Ala Pro Gly Gln Gly Leu Gln Leu Val
35 40 45
Ala Thr Ile Thr Arg Gly Gly Val Thr Met Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn
85 90 95
Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Tyr Trp Gly
100 105 110
Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 175
<211> 121
<212> PRT
<213> artifical sequence
<400> 175
Glu Val Lys Leu Glu 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 Pro Tyr Val Pro
20 25 30
Asp Met His Trp Tyr Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Val
35 40 45
Ala Thr Ile Thr Arg Gly Gly Val Thr Met Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn
85 90 95
Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Tyr Trp Gly
100 105 110
Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 176
<211> 120
<212> PRT
<213> artifical sequence
<400> 176
Gln Val Lys Leu Glu 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 Pro Tyr Val Pro
20 25 30
Asp Met His Trp Tyr Arg Gln Ala Pro Gly Gln Gly Leu Gln Val Ala
35 40 45
Thr Ile Thr Arg Gly Gly Val Thr Met Tyr Ala Asp Ser Val Lys Gly
50 55 60
Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu Gln
65 70 75 80
Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Asn Ala
85 90 95
Asp Val Trp Ser Ser Val Ser Leu Lys Leu Val Glu Tyr Trp Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 177
<211> 126
<212> PRT
<213> artifical sequence
<400> 177
Glu Val Lys Leu Glu 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 Tyr Thr Ser Tyr Leu Pro
20 25 30
Asp Met Gly Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Val
35 40 45
Ala Ala Thr Ile Thr Arg Gly Gly Val Thr Glu Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Ala Val Pro
100 105 110
Ser Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 178
<211> 126
<212> PRT
<213> artifical sequence
<400> 178
Glu Val Lys Leu Glu 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 Tyr Thr Ser Tyr Leu Pro
20 25 30
Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Val
35 40 45
Ala Ala Thr Ile Thr Arg Gly Gly Val Thr Glu Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Ala Val Pro
100 105 110
Ser Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 179
<211> 126
<212> PRT
<213> artifical sequence
<400> 179
Gln Val Lys Leu Glu 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 Tyr Thr Ser Tyr Leu Pro
20 25 30
Asp Met Gly Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Gln Leu Val
35 40 45
Ala Ala Thr Ile Thr Arg Gly Gly Val Thr Glu Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Thr Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Ala Val Pro
100 105 110
Ser Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 180
<211> 126
<212> PRT
<213> artifical sequence
<400> 180
Glu Val Lys Leu Glu 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 Tyr Thr Ser Tyr Leu Pro
20 25 30
Asp Met Gly Trp Tyr Arg Gln Ala Pro Gly Gln Gly Leu Gln Leu Val
35 40 45
Ala Ala Thr Ile Thr Arg Gly Gly Val Thr Glu Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn 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
Ala Asn Ala Asp Val Trp Ser Ser Val Ser Leu Lys Leu Ala Val Pro
100 105 110
Ser Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120 125
<210> 181
<211> 124
<212> PRT
<213> artifical sequence
<400> 181
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Thr Leu Ser Cys Val Phe Ser Gly Arg Pro Ser Ser Tyr Tyr
20 25 30
Gly Val Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val
35 40 45
Ala Gly Ile Ser Arg Ser Gly Gly Thr Asp Asn Tyr Ala Asn Phe Val
50 55 60
Lys Gly Arg Phe Thr Val Ser Lys Asp Asn Ser Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Thr Asn Val Tyr Ala Ser Ala Thr Leu Ser Ala Tyr Asp
100 105 110
Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 182
<211> 124
<212> PRT
<213> artifical sequence
<400> 182
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Thr Leu Ser Cys Val Phe Ser Gly Arg Pro Ser Ser Tyr Tyr
20 25 30
Gly Val Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Gly Ile Ser Arg Ser Gly Gly Thr Asp Asn Tyr Ala Asn Phe Val
50 55 60
Lys Gly Arg Phe Thr Val Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Thr Asn Val Tyr Ala Ser Ala Thr Leu Ser Ala Tyr Asp
100 105 110
Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 183
<211> 124
<212> PRT
<213> artifical sequence
<400> 183
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Thr Leu Ser Cys Val Phe Ser Gly Arg Pro Ser Ser Tyr Tyr
20 25 30
Gly Val Ser Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val
35 40 45
Ala Gly Ile Ser Arg Ser Gly Gly Thr Asp Asn Tyr Ala Asn Phe Val
50 55 60
Lys Gly Arg Phe Thr Val Ser Lys Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Asn Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Ala Thr Asn Val Tyr Ala Ser Ala Thr Leu Ser Ala Tyr Asp
100 105 110
Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 184
<211> 372
<212> DNA
<213> Vicugna pacos
<400> 184
caggtgcagc tggtggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60
tcctgtgcag cctctggacc atccttcagt acctatgccg tgggctggtt ccgccaggtt 120
ccccggagag agcgtgcgtt tgttgcaggt attaattgga gtggcgaaga aacaacatat 180
cataactccg tgaacggccg attcaccatc tccagagaca acgccaagaa tacggtgtat 240
ctacaaatga acagcctgca acctgaggac acggccgttt attactgtgc agccaaccgt 300
cggaattact ctagcaccta taaagggaac tatgactact ggggccaggg gacccaggtc 360
accgtctcct ca 372
<210> 185
<211> 357
<212> DNA
<213> Vicugna pacos
<400> 185
caggtacagc tggtggagtc tgggggagga ttggtgcagg ctgggggctc tctgagactc 60
tcctgtgcag cctctggacg caccttcagt agctatgcca tgggctggtt ccgccaggct 120
ccagggaagg agcgtgagtt tgtagcagct attaggcggg atgctgttag cacatactat 180
gcagactccg tgaagggccg attcaccatc tccagagaca acgccaagga tatggtgtat 240
ctgcaaatga acaacctgaa acctgaggac acggccgttt attactgtgc gatgggggat 300
gactatgtag atgagtatga ctactggggc cagggaaccc aggtcaccgt ctcctca 357
<210> 186
<211> 369
<212> DNA
<213> Vicugna pacos
<400> 186
caggtgaagc tggaggagtc tgggggagga ttggtgcagg ctgggggctc tctgagactc 60
gcctgtgtag cctccagagt taccggtagt cactatgacc tggcctggtt ccgccagggt 120
ccagggaagg agcgtgaagt tgtagcagag tttacctggc gtagtggtcc cacatcgtat 180
gcagagtccg tgaagggccg attcgccgtc tccaaagata acgccaagaa cacactgtat 240
ctgcaaatgg acaacctgag acctgaggac acggccgttt attactgtgc atcccgatac 300
ttttacacct atggcgaccc cctgaagtat gactattggg gccagggaac ccaggtcacc 360
gtctcctca 369
<210> 187
<211> 366
<212> DNA
<213> Vicugna pacos
<400> 187
caggtaaagc tggaggagtc tgggggaggc ttggtgcgcc ctggggggtc tctgagactc 60
tcctgtgcag cctccggaag catcttcagt atcaatcaca tggcctggta ccgccaggct 120
ccagggaagg agcgcgagtt cgtcgcacgt gcttttagta gtggttcgac aacctatgca 180
gactccgtga ggggccgatt caccatctcc agagacgacg ccaagaacac ggtgtatcta 240
caaatgaaca gtctgaaacc tgaggacacg gccgtctatt actgtaatgg agatgggttt 300
ctattatacg acgatactta ctactccaac gcctggggcc aggggaccca ggtcaccgtc 360
tcctca 366
<210> 188
<211> 345
<212> DNA
<213> Vicugna pacos
<400> 188
caggtaaagc tggaggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60
tcctgtgcag cctctggaat tgccttcagt gtctttgaca tggcctggta ccgccaggct 120
ccagggaacc agcgcgagtt ggtcgcagag actactactg ctggtattaa cacatatgca 180
gactccgtga agggccgatt caccatctcc agagacaacg ccaagaacac ggtgtatctg 240
cagatgaaca gcctgagacc cgaggacacg gccgtctata gctgtaatgc gaaaaatggg 300
tggcggaccc tctggggcca ggggacccag gtcaccgtct cctca 345
<210> 189
<211> 333
<212> DNA
<213> Vicugna pacos
<400> 189
ggtgtgcagc tggtggagtc tgggggaggc ttggtgcagg ctgggggctc tctgagactc 60
tcctgtgtag tttctggaag caccttcagg gacatgtcct tcggctggta ccgccaggct 120
ccagggaagc agcgcgagcg gctcgcatac atttcgagca gtggctacac aaactatgta 180
gatgctgtga aggaccgatt caccatctcc agagacaacg ccaagaacac ggtgtacctc 240
caaatgaaca gcctgaaacc tgaggacacg gccatctatt actgcaatac ccttgtgtac 300
tggggccagg ggacccaggt caccgtctcc tca 333
<210> 190
<211> 345
<212> DNA
<213> Vicugna pacos
<400> 190
caggtaaagc tggaagagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60
tcctgtgcag cctctggaat tgccttcagt gtctttgaca tggcctggta ccgccaggct 120
ccagggaacc agcgcgagtt ggtcgcacgc atcacttttg atggtatccc gcactacgca 180
gactccgtga agggccgatt caccatctcc agagacaacg ccaagaacac ggtgtatctg 240
cagatgaaca gcctgagacc cgaggacacg gccgtctata gctgtaatgc gaaaaatggg 300
tggcggaccc tctggggcca ggggacccag gtcaccgtct cctca 345
<210> 191
<211> 378
<212> DNA
<213> Vicugna pacos
<400> 191
caggtacagc tggtggattc tgggggaggc tatgtgcagc cgggggggtc tctgagactc 60
tcctgtgcag cctctggagg cagcttcagt aaatatgcca tgggatggtt ccgccaggct 120
ccagggaagg agcgtcagtt tgtaggaaat atttactgga gtgatggtag cacacactat 180
caagactccg tgaagggccg attcaccctc tccaaagact acgccaagaa cacggtgtat 240
ctagaaatga acaacctgaa acctgaggac acggccgttt attactgcgc gtctcggggc 300
tccaataatg gtggttctta ctacagcgag actgggtatg attactgggg ccaggggact 360
caggtcaccg tctcctca 378
<210> 192
<211> 363
<212> DNA
<213> Vicugna pacos
<400> 192
caggtgcagc tggaggagtc tgggggagga ttggtgcagg ctgggggctc tctgagactc 60
tcctgtgcgg cctctggagg caccatcggt agctatgaca tgggctggtt ccgccaagct 120
ccagggaagg agcgcgagtt tgtagcagcc attacctgga tttctaacac aaactatgca 180
gactccgtgg agggccgatt caccatctcc agagacaaag acaagacgac gatgtatctg 240
caaatgaata gcctgaaacc tgaggacacg gccgtttatt actgtgcagc acgccgttgg 300
catcgctccg ccccacacga ctatgagttt tggggccagg ggacccaggt caccgtctcc 360
tca 363
<210> 193
<211> 372
<212> DNA
<213> Vicugna pacos
<400> 193
caggtacagc tggtggattc tgggggagga ttggtgctgg ctgggggctc tctgagactc 60
tcctgtgcag cctctggaga caccttcgct aggtattcta tgggctggtt ccgccaggct 120
ccagggaagg agcgtgagtt tgttgcaggt attagcaggt cgagcggtac cataatttat 180
ggaggctccg tgaagggccg attcatcatc ttcagagaca atgtcaagaa cacggtgtat 240
ctacaaatga acggtttgaa acctgacgac acggccgttt atttctgtgc agcccgagag 300
agcttactgg cagtgactac cacacgggat tatccgtact ggggccaggg gacccaggtc 360
accgtctcct ca 372
<210> 194
<211> 363
<212> DNA
<213> Vicugna pacos
<400> 194
caggtgaagc tggaggagtc tgggggaggg ttggtgcagg ctggggggtc tctgagactc 60
tcctgtgcag cctctggaag cataccatat gtccctgaca tgcactggta ccgccaggct 120
ccagggcaac agcgccaatt ggtcgcaact attactcgtg gaggcaacac aatgtatgct 180
gactccgtga agggccgatt caccatctcc agagacaacg ccaagaacac ggtatatcta 240
caaatgacct ccctgaaacc tgaggacacg gccgtctact actgtaatgc agacgtttgg 300
tcgagtgttg cattgaagct tgtggaatac tggggccagg ggacccaggt caccgtctcc 360
tca 363
<210> 195
<211> 375
<212> DNA
<213> Vicugna pacos
<400> 195
caggtacagc tggtggattc tgggggagga tttgtgcagg ctgggggctc tctgagactc 60
tcctgtgcag cctctggacg caccttcagt gactatgcca tggcctggtt ccgccaggct 120
ccagggaagg agcgtgaatt tgtagcaggt attagctgga ctggtggcag atactatgct 180
gagtccgtga agggccgatt caccatctcc agagacaacg ccaagaacac ggcgtttctg 240
caaatgaaca cgctgaaacc tgaggacacg ggcatttatc actgtgcaac tcccaaccaa 300
gccgggcttg tgctgttaga tgacgccgaa gggtacgcct actggggcca ggggacccag 360
gtcaccgtct cctca 375
<210> 196
<211> 351
<212> DNA
<213> Vicugna pacos
<400> 196
caggtacagc tggtggattc tgggggaggg ttggtgcagg ctggggggtc tctgagactc 60
tcctgcgcag tctctggatt caccgtcagc acccggaaca tgggctggta ccgccaggct 120
ccagggaagc agcgtgtttt ggtcgcacgt attggtagtg atggtagtac ctacaatgta 180
gactccgtga agggccgatt caccatctcc agagacaact ctaagaacgc ggtgtatctg 240
caaatgaaca gcctgaagcc tgaggacacg gccgtgtact ggtgtaacac ctttcccgtc 300
acaaagttct atgactactg gggccagggg acccaggtca ccgtctcctc a 351
<210> 197
<211> 378
<212> DNA
<213> Vicugna pacos
<400> 197
gaggtgaagc tggtggattc tgggggagga ttggtgcagg ctgggggctc tctgagactc 60
tcctgtgcag cctctggact ccgcttcagt gcctatgaca tgggctggtt ccgtcaggct 120
ccagggaagg agcgtgagtt tgtagcaact attaactgga gtgctcttag ccgatactat 180
gcagactctg tgaagggccg attcaccatc tccagagaca acgccaagaa cacggtatct 240
ctgcacatga acagcctgaa atctgaggac acggccgttt attactgtgc aggagggaga 300
attagtgcgg cactacgtgt cccggaccgg gatgcgtata gctactgggg ccagggtacc 360
caggtcaccg tctcctca 378
<210> 198
<211> 378
<212> DNA
<213> Vicugna pacos
<400> 198
caggtacagc tggtggattc tgggggaggt ttggtgcagg ctgggggctc tctgagactc 60
tcctgtgtcg cttctggacg caccgtcagg gacgatgcca tgggctggtt ccgccaggct 120
ccagggaagg agcgtgagtt tgtagcaact attagctgga atggtggtag tacatactat 180
gccgcctccg caaaggggcg tttcatagtc tctagagaca atgccaagaa caccatgtat 240
ctgcaaatga acagcctgaa acctgaggac acggccgtgt actactgtgc agtagcgtct 300
cgttatcggc agatagtact taataccgag gagaagtatg acgcctgggg ccaggggacc 360
caggtcaccg tctcctca 378
<210> 199
<211> 426
<212> DNA
<213> Vicugna pacos
<400> 199
caggtgcagc tggtggagtc tgggggagga ttggtgcagg ctgggggctc cctgagactc 60
tcctgtacag cctctggaag ctctttcagt gtccctcaca tggactggtt ccgccaggct 120
ccaggccagg agcgtgagtt tgtagcaact attagccgtg gaggtgtcag tacttacgca 180
gcagactccg tgaagggccg aagcaccatc tccagagaca acgccaaaaa cacgatgtac 240
cttcaaatga acaacctgaa acctgaggac acggccgttt attactgtaa tgcagacgtt 300
tggtcgagtg tttcattgaa attggtggag tactggggcc aggggaccca ggtcaccgtc 360
tcctcaggat ccgaacaaaa actgatcagc gaagaagatc tgaaccatca ccatcaccat 420
tagtga 426
<210> 200
<211> 333
<212> DNA
<213> Vicugna pacos
<400> 200
caggtgaagc tggaggagtc tgggggaggc ttggtgcagg ctggggggtc tctgagactc 60
tcctgtgcag cctctacaac caccttcagt caccgtactt ttgcctggta ccgccaggct 120
ccagggaagc agcgcgagtt ggtcgcaact attggaagtt acggcagtac gtactatgac 180
gagtccgtga aggaccgatt caccatcgcc agagacaacg ccaagaacac ggtgtatctg 240
caaatgaaca gcctgaaacc tcaggacacg gccgtctatt actgtcatac ccaggtgtac 300
tggggccagg ggacccaggt caccgtctcc tca 333
<210> 201
<211> 372
<212> DNA
<213> Vicugna pacos
<400> 201
gaggtgaagc tggaggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60
tcctgtgcag cctctggaag cacttcctat gtctatgaca tgcactggta ccgccaggct 120
ccagggcaac agcgccaatt ggtcgcagca attactgatg gaggctccac ggataactat 180
gcagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacggtatat 240
ctgcaaatga cctccctgaa acctgaggac acggccgtct actactgtaa tgcagacgtt 300
tggtcgagtg tttcattgaa attggtggag tacgactact ggggccaggg gacccaggtc 360
accgtctcct ca 372
<210> 202
<211> 375
<212> DNA
<213> Vicugna pacos
<400> 202
gaggtgaagc tggaggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60
tcctgtgcag cctctggaag cacttcctat gtctatgaca tgcactggta ccgccaggct 120
ccagggcaac agcgccaatt ggtcgcagca attactgatg gaggctccac ggataactat 180
gcagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacggtatat 240
ctgcaaatga cctccctgaa acctgaggac acggccgtct actactgtgc aaatgcagac 300
gtttggtcga gtgtttcatt gaaattggtg gagtacgact actggggcca ggggacccag 360
gtcaccgtct cctca 375
<210> 203
<211> 363
<212> DNA
<213> Vicugna pacos
<400> 203
caggtgaagc tggaggagtc tgggggaggc ttggtgcagg ctggggggtc tctgagactc 60
tcctgtgcag cctctggaag catcccctat gtccctgaca tgcactggta ccgccaggct 120
ccagggcaac agcgccaatt ggtcgcaact attactcgtg gaggcgtcac gatgtatgca 180
gactccgtga agggccgatt caccatctcc agagacaacg ccaagaacac ggtatatctg 240
caaatgacct ccctgaaacc tgaggacacg gccgtctact actgtaatgc agacgtttgg 300
tcgagtgttt cattgaaatt ggtggagtac tggggccagg ggacccaggt caccgtctcc 360
tca 363
<210> 204
<211> 375
<212> DNA
<213> Vicugna pacos
<400> 204
gaggtgaagc tggaggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60
tcctgtgcag cctctggaag cacttcctat gtctatgaca tgcactggta ccgccaggct 120
ccagggcaac agcgccaatt ggtcgcagca atttctcgtg gaggcgtcac ggataactat 180
gcagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacggtatat 240
ctgcaaatga cctccctgaa acctgaggac acggccgtct actactgtgc aaatgcagac 300
gtttggtcga gtgtttcatt gaaattggtg gagtacgact actggggcca ggggacccag 360
gtcaccgtct cctca 375
<210> 205
<211> 363
<212> DNA
<213> Vicugna pacos
<400> 205
gaggtgcagc tggaggattc tgggggaggc ttggtgcagg ctggggggtc tctgagactc 60
tcctgtgcag cctctggaag catctcatat gtccctgaca tgcactggta ccgccaggct 120
ccagggcaac agcgccaatt ggtcgcaact attactcgtg gaggcaacac aatgtatgca 180
gactccgtga agggccgatt caccatctcc agagacaacg ccaagaacac ggtatatctg 240
caaatgacct ccctgaaacc tgaggacacg gccgtgtact actgtaatgc agacgtttgg 300
tcgagtgttt tattcaaact tgtggagtac tggggtcagg ggacccaggt caccgtctcc 360
tca 363
<210> 206
<211> 375
<212> DNA
<213> Vicugna pacos
<400> 206
gaggtgaagc tggaggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60
tcctgtgcag cctctggaag cttctcctat atctatgaca tgcactggta ccgccaggct 120
ccagggcaac agcgccaatt ggtcgcagca atttctcgtg gaagctccac ggataactat 180
gcagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacggtatat 240
ctgcaaatga cctccctgaa acctgaggac acggccgtct actactgtgc aaatgcagac 300
gtttggtcga gtgtttcatt gaaattggtg gagtacgact actggggcca ggggacccag 360
gtcaccgtct cctca 375
<210> 207
<211> 369
<212> DNA
<213> Vicugna pacos
<400> 207
gaggtgaagc tggaggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60
tcctgtgcag cctctggaag cttctcctat atccctgaca tgggctggta ccgccaggct 120
ccagggcaac agcgccaatt ggtcgcagca atttctcgtg gaagctccac ggataactat 180
gcagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacggtatat 240
ctgcaaatga cctccctgaa acctgaggac acggccgtct actactgtgc aaatgcagac 300
gtttggtcga gtgtttcatt gaaattggtg gagtactggg gccaggggac ccaggtcacc 360
gtctcctca 369
<210> 208
<211> 378
<212> DNA
<213> Vicugna pacos
<400> 208
caggtgcagc tggtggagtc tgggggagga ttggtgcagg ctgggggctc tctgagactc 60
tcctgtgcag cctctggact ccgcttcagt gcctatgaca tgggctggtt ccgtcaggct 120
ccagggaagg agcgtgagtt tgtagcaact attaactgga gtactcttag ccgatattat 180
gcagactctg tgaagggccg attcaccatc tccagagaca acgccaagaa cacggtatat 240
ctgcacatga acagcctgaa acctgaggac acggccgttt attactgtgc aggagggaga 300
attagttcgg aactacgtgt cacggcccgg gatgcgtata cctactgggg ccagggtacc 360
caggtcaccg tctcctca 378
<210> 209
<211> 381
<212> DNA
<213> Vicugna pacos
<400> 209
caggtgcagc tggaggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60
tcctgtgcag cctctggaag catctcctat gtccctgaca tgggctggta ccgccaggct 120
ccagggcaac agcgccaatt ggtcgcagca atttctcgtg gaggctccac ggataactat 180
gcagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacggtatat 240
ctgcaaatga cctccctgaa acctgaggac acggccgtct actactgtgc aaatgcagac 300
gtttggtcga gtgtttcatt gaaattggtg gagtccgcct atgattactg gggccagggg 360
acccaggtca ccgtctcctc a 381
<210> 210
<211> 378
<212> DNA
<213> Vicugna pacos
<400> 210
caggtgcagc tggaggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60
tcctgtgcag cctctggaag catctcctat gtccctgaca tgggctggta ccgccaggct 120
ccagggcaac agcgccaatt ggtcgcaact atttctcgtg gaggcgtcac ggataactat 180
gcagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacggtatat 240
ctgcaaatga cctccctgaa acctgaggac acggccgtct actactgtaa tgcagacgtt 300
tggtcgagtg tttcattgaa attggtggag tccgcctatg attactgggg ccaggggacc 360
caggtcaccg tctcctca 378
<210> 211
<211> 378
<212> DNA
<213> Vicugna pacos
<400> 211
caggtgcagc tggtggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60
tcctgtgcag cctctggaag cacctcctat gtccctgaca tgggctggta ccgccaggct 120
ccagggcaac agcgccaatt ggtcgcaact atttctcgtg gaggcgtcac ggattattat 180
gcagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacggtatat 240
ctgcaaatga cctccctgaa acctgaggac acggccgtct actactgtaa tgcagacgtt 300
tggtcgagtg tttcattgaa attggtggag tccgcctatg attactgggg ccaggggacc 360
caggtcaccg tctcctca 378
<210> 212
<211> 366
<212> DNA
<213> Vicugna pacos
<400> 212
caggtgcagc tggtggagtc tgggggagga ttggtgcagg ctgggggctc cctgagactc 60
tcctgtacag cctctggaag ctctttcagt agttatcaca tggactggtt ccgccaggct 120
ccaggccagg agcgtgagtt tgtagcagct attagctgga ctggtcatag tacttactat 180
gcagactccg tgaagggccg aagcaccatc tccagagaca acgccaaaaa cacgatgtac 240
cttcaaatga acaacctgaa acctgaggac acggccgttt attactgtgc agcgaccagg 300
cgcgcgacta tgatcgccgt accgtctgat tactggggcc aggggaccca ggtcaccgtc 360
tcctca 366
<210> 213
<211> 375
<212> DNA
<213> Vicugna pacos
<400> 213
caggtgcagc tggtggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60
tcctgtgcag cctctggatt catctcctat gtccctgaca tgggctggta ccgccaggct 120
ccagggcaac agcgccaatt ggtcgcagca actatttctc gtggaggcgt cacggattac 180
tatgcagact ccgtgaaggg ccgattcacc atctccagag acaacgccaa gaacacggta 240
tatctgcaaa tgacctccct gaaacctgag gacacggccg tctactactg taatgcagac 300
gtttggtcga gtgtttcatt gaaattggtg gagtccgcct attggggcca ggggacccag 360
gtcaccgtct cctca 375
<210> 214
<211> 375
<212> DNA
<213> Vicugna pacos
<400> 214
caggtgcagc tggtggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60
tcctgtgcag cctctggatt catctcctat gtccctgaca tgggctggta ccgccaggct 120
ccagggcaac agcgccaatt ggtcgcagca actattactc gtggaggcgt cacggattac 180
gcagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacggtatat 240
ctgcaaatga cctccctgaa acctgaggac acggccgtct actactgtgc aaatgcagac 300
gtttggtcga gtgtttcatt gaaattggtg gagtccgcct attggggcca ggggacccag 360
gtcaccgtct cctca 375
<210> 215
<211> 378
<212> DNA
<213> Vicugna pacos
<400> 215
caggtgaagc tggaggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60
tcctgtgcag cctctggata cacctcctat ctccctgaca tgggctggta ccgccaggct 120
ccagggcaac agcgccaatt ggtcgcagca actattactc gtggaggcgt cacggaatac 180
gcagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacggtatat 240
ctgcaaatga cctccctgaa acctgaggac acggccgtct actactgtgc aaatgcagac 300
gtttggtcga gtgtttcatt gaaattggcc gtgccgtccg actattgggg ccaggggacc 360
caggtcaccg tctcctca 378
<210> 216
<211> 383
<212> DNA
<213> Vicugna pacos
<400> 216
caggtgaagc tggaggagtc tgggggagga ttggtgcagg ctgggggctc tctgagactc 60
tcctgtgcag cctctggacg caccttcggt agctatgata tggcctggtt ccgccaggtt 120
ccagggaagg aacgagagtt tgtagcggct attaactggt acggtggcta cacatactac 180
gcagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cgcggtgtat 240
ctacaaatga acaacctgaa accgtcggac acggccgttt attactgtgc agcggagaaa 300
ggcttcgcat cgctcaggct gtgggcatta tctcacaaac cccatgagtc aaggggccag 360
gggacccagg tcaccgtctc ctc 383
<210> 217
<211> 372
<212> DNA
<213> Vicugna pacos
<400> 217
caggtgaagc tggaggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60
tcctgtgcag cctctggata catcccctat ctccctgaca tgggctggta ccgccaggct 120
ccagggcaac agcgccaatt ggtcgcaact attactcgtg gaggcgtcac ggaatacgca 180
gactccgtga agggccgatt caccatctcc agagacaacg ccaagaacac ggtatatctg 240
caaatgacct ccctgaaacc tgaggacacg gccgtctact actgtaatgc agacgtttgg 300
tcgagtgttt cattgaaatt ggccgtgccg tccgactatt ggggccaggg gacccaggtc 360
accgtctcct ca 372
<210> 218
<211> 372
<212> DNA
<213> Vicugna pacos
<400> 218
caggtgcagc tggtggagtc tgggggagga ttggtgcagc ctgggggctc tctgaccctc 60
tcctgtgtat tctctggccg tcccagtagc tactatggcg tgagctggtt ccgccaggct 120
ccagggaagg agcgtgagtt tgtagcagga atttcgagga gtggtggtac tgacaactat 180
gcaaatttcg tgaagggccg attcaccgtc tccaaagaca acggcaagaa cacggtgtat 240
ctccaaatga acaatctgaa acctgaggac acggccgttt attactgtgc agccgctaca 300
aatgtctatg cctccgcgac gttgtccgcc tatgattatt ggggccaggg gacccaggtc 360
accgtctcct ca 372
<210> 219
<211> 375
<212> DNA
<213> Vicugna pacos
<400> 219
caggtgaagc tggaggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60
tcctgtgcag cctctggata catcccctat ctccctgaca tgggctggta ccgccaggct 120
ccagggcaac agcgccaatt ggtcgcaact attactcgtg gaggcgtcac ggaatacgca 180
gactccgtga agggccgatt caccatctcc agagacaacg ccaagaacac ggtatatctg 240
caaatgacct ccctgaaacc tgaggacacg gccgtctact actgtaatgc agacgtttgg 300
tcgagtgttt cattgaaatt ggccgtggag tactccgact attggggcca ggggacccag 360
gtcaccgtct cctca 375
<210> 220
<211> 378
<212> DNA
<213> Vicugna pacos
<400> 220
caggtgaagc tggaggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60
tcctgtgcag cctctggata ctccacctat ctccctgaca tgggctggta ccgccaggct 120
ccagggcaac agcgccaatt ggtcgcagct attactcgtg gaggccactc cacgtactac 180
gcagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacggtatat 240
ctgcaaatga cctccctgaa acctgaggac acggccgtct actactgtaa tgcagacgtt 300
tggtcgagtg tttcattgaa attggccgtg gagtactccg actattgggg ccaggggacc 360
caggtcaccg tctcctca 378
<210> 221
<211> 363
<212> DNA
<213> Vicugna pacos
<400> 221
caggtgaagc tggaggagtc tgggggagga ttggtgcagg ctgggggctc tctgagactc 60
tcctgtgcgg cctcgggacg caccgtcagt agctatgaca tgggctggtt ccgccaatct 120
ccagggaagg agcgcgagtt tgtagcagcc attacctgga tttcaaacac aaactatgca 180
gactccgtga agggccgatt caccatcgac agagacaaag ccaagaacac ggtgtatctg 240
caaatgaaca gcctgaaacc tgaggacacg gccgtttatt actgtgcagc acgccgttat 300
tatcgctctg gcgcacaaga ctatgagtac tggggccagg ggacccaggt caccgtctcc 360
tca 363
<210> 222
<211> 372
<212> DNA
<213> Vicugna pacos
<400> 222
caggtgaagc tggaggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60
tcctgtgcag cctctggatc atccacctat ctccctgaca tgggctggta ccgccaggct 120
ccagggcaac agcgccaatt ggtcgctatt actcgtggag gccactccac gtactacgca 180
gactccgtga agggccgatt caccatctcc agagacaacg ccaagaacac ggtatatctg 240
caaatgacct ccctgaaacc tgaggacacg gccgtctact actgtaatgc agacgtttgg 300
tcgagtgttt cattgaaatt ggtggagtac tccgactatt ggggccaggg gacccaggtc 360
accgtctcct ca 372
<210> 223
<211> 363
<212> DNA
<213> Vicugna pacos
<400> 223
gatgtgaagc tggaggagtc tgggggagga ttggtgcagg ctggggactc tctgagactc 60
tcctgtgtgg cctctggagg caccatcggt agctatgaca tgggctggtt ccgccaagct 120
ccagggaagg agcgcgagtt tgtagcagcc attacctgga tttctaacac aaactatgca 180
gactccgtgg agggccgatt caccatctcc agagacaaag ccaagagcac gatgtatctg 240
caaatgcata gcctgaaacc tgaggacacg gccgtttatt actgtgcagc acgccgttgg 300
tatcgctccg ccccacaaga ctatgagtac tggggccagg ggacccaggt caccgtctcc 360
tca 363
<210> 224
<211> 375
<212> DNA
<213> Vicugna pacos
<400> 224
caggtgaagc tggaggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60
tcctgtgcag cctctggatc atccacctat ctccctgaca tgggctggta ccgccaggct 120
ccagggcaac agcgccaatt ggtcgctatt actcgtggag gcgtgtccac gtactacgca 180
gactccgtga agggccgatt caccatctcc agagacaacg ccaagaacac ggtatatctg 240
caaatgacct ccctgaaacc tgaggacacg gccgtctact actgtaatgc agacgtttgg 300
tcgagtgttt cattgaaatt ggtggagtac ccatccgact attggggcca ggggacccag 360
gtcaccgtct cctca 375
<210> 225
<211> 363
<212> DNA
<213> Vicugna pacos
<400> 225
caggtgaagc tggaggagtc tgggggagga ttggtgcagg ctgggggctc tctgagactc 60
tcctgtgcgg cctctggagg caccatcggt agctatgaca tgggctggtt ccgccaagct 120
ccagggaagg agcgcgagtt tgtagcagcc attacctgga tttctaacac aaactatgca 180
gactccgtgg agggccgatt caccatctcc agagacaaag ccaagagcac gatgtatctg 240
caaatgaata gcctgaaacc tgaggacacg gccgtttatg tctgtgcagc acgccgttat 300
tatcgctccg ccccacaaga ctatgacttt tggggccagg ggacccaggt caccgtctcc 360
tca 363

Claims (10)

1.一组抗VEGF的单域抗体，所述单域抗体均由框架区和3个互补决定区组成，其特征在于，所述单域抗体选自NBV1、NBV2、NBV3、NBV4、NBV5、NBV6、NBV7、NBV8、NBV9、NBV10、NBV11、NBV12、NBV13、NBV14、NBV15、NBV16、NBV17、NBV18、NBV19、NBV20、NBV21、NBV22、NBV23、NBV24、NBV25、NBV26、NBV27、NBV28、NBV29、NBV30、NBV31、NBV32、NBV33、NBV34、NBV35、NBV36、NBV37、NBV38、NBV39、NBV40、NBV41或NBV42中的任何一种；
其中，单域抗体NBV1的3个互补决定区的氨基酸序列分别为SEQ ID No.1、SEQ ID No.2和SEQ ID No.3所示；单域抗体NBV2的3个互补决定区的氨基酸序列分别为SEQ ID No.4、SEQ ID No.5和SEQ ID No.6所示；单域抗体NBV3的3个互补决定区的氨基酸序列分别为SEQID No.7、SEQ ID No.8和SEQ ID No.9所示；单域抗体NBV4的3个互补决定区的氨基酸序列分别为SEQ ID No.10、SEQ ID No.11和SEQ ID No.12所示；单域抗体NBV5的3个互补决定区的氨基酸序列分别为SEQ ID No.13、SEQ ID No.14和SEQ ID No.15所示；单域抗体NBV6的3个互补决定区的氨基酸序列分别为SEQ ID No.16、SEQ ID No.17和SEQ ID No.18所示；单域抗体NBV7的3个互补决定区的氨基酸序列分别为SEQ ID No.19、SEQ ID No.20和SEQ IDNo.21所示；单域抗体NBV8的3个互补决定区的氨基酸序列分别为SEQ ID No.22、SEQ IDNo.23和SEQ ID No.24所示；单域抗体NBV9的3个互补决定区的氨基酸序列分别为SEQ IDNo.25、SEQ ID No.26和SEQ ID No.27所示；单域抗体NBV10的3个互补决定区的氨基酸序列分别为SEQ ID No.28、SEQ ID No.29和SEQ ID No.30所示；单域抗体NBV11的3个互补决定区的氨基酸序列分别为SEQ ID No.31、SEQ ID No.32和SEQ ID No.33所示；单域抗体NBV12的3个互补决定区的氨基酸序列分别为SEQ ID No.34、SEQ ID No.35和SEQ ID No.36所示；单域抗体NBV13的3个互补决定区的氨基酸序列分别为SEQ ID No.37、SEQ ID No.38和SEQID No.39所示；单域抗体NBV14的3个互补决定区的氨基酸序列分别为SEQ ID No.40、SEQID No.41和SEQ ID No.42所示；单域抗体NBV15的3个互补决定区的氨基酸序列分别为SEQID No.43、SEQ ID No.44和SEQ ID No.45所示；单域抗体NBV16的3个互补决定区的氨基酸序列分别为SEQ ID No.46、SEQ ID No.47和SEQ ID No.48所示；单域抗体NBV17的3个互补决定区的氨基酸序列分别为SEQ ID No.49、SEQ ID No.50和SEQ ID No.51所示；单域抗体NBV18的3个互补决定区的氨基酸序列分别为SEQ ID No.52、SEQ ID No.53和SEQ ID No.54所示；单域抗体NBV19的3个互补决定区的氨基酸序列分别为SEQ ID No.55、SEQ ID No.56和SEQ ID No.57所示；单域抗体NBV20的3个互补决定区的氨基酸序列分别为SEQ IDNo.58、SEQ ID No.59和SEQ ID No.60所示；单域抗体NBV21的3个互补决定区的氨基酸序列分别为SEQ ID No.61、SEQ ID No.62和SEQ ID No.63所示；单域抗体NBV22的3个互补决定区的氨基酸序列分别为SEQ ID No.64、SEQ ID No.65和SEQ ID No.66所示；单域抗体NBV23的3个互补决定区的氨基酸序列分别为SEQ ID No.67、SEQ ID No.68和SEQ ID No.69所示；单域抗体NBV24的3个互补决定区的氨基酸序列分别为SEQ ID No.70、SEQ ID No.71和SEQID No.72所示；单域抗体NBV25的3个互补决定区的氨基酸序列分别为SEQ ID No.73、SEQID No.74和SEQ ID No.75所示；单域抗体NBV26的3个互补决定区的氨基酸序列分别为SEQID No.76、SEQ ID No.77和SEQ ID No.78所示；单域抗体NBV27的3个互补决定区的氨基酸序列分别为SEQ ID No.79、SEQ ID No.80和SEQ ID No.81所示；单域抗体NBV28的3个互补决定区的氨基酸序列分别为SEQ ID No.82、SEQ ID No.83和SEQ ID No.84所示；单域抗体NBV29的3个互补决定区的氨基酸序列分别为SEQ ID No.85、SEQ ID No.86和SEQ ID No.87所示；单域抗体NBV30的3个互补决定区的氨基酸序列分别为SEQ ID No.88、SEQ ID No.89和SEQ ID No.90所示；单域抗体NBV31的3个互补决定区的氨基酸序列分别为SEQ IDNo.91、SEQ ID No.92和SEQ ID No.93所示；单域抗体NBV32的3个互补决定区的氨基酸序列分别为SEQ ID No.94、SEQ ID No.95和SEQ ID No.96所示；单域抗体NBV33的3个互补决定区的氨基酸序列分别为SEQ ID No.97、SEQ ID No.98和SEQ ID No.99所示；单域抗体NBV34的3个互补决定区的氨基酸序列分别为SEQ ID No.100、SEQ ID No.101和SEQ ID No.102所示；单域抗体NBV35的3个互补决定区的氨基酸序列分别为SEQ ID No.103、SEQ ID No.104和SEQ ID No.105所示；单域抗体NBV36的3个互补决定区的氨基酸序列分别为SEQ IDNo.106、SEQ ID No.107和SEQ ID No.108所示；单域抗体NBV37的3个互补决定区的氨基酸序列分别为SEQ ID No.109、SEQ ID No.110和SEQ ID No.111所示；单域抗体NBV38的3个互补决定区的氨基酸序列分别为SEQ ID No.112、SEQ ID No.113和SEQ ID No.114所示；单域抗体NBV39的3个互补决定区的氨基酸序列分别为SEQ ID No.115、SEQ ID No.116和SEQ IDNo.117所示；单域抗体NBV40的3个互补决定区的氨基酸序列分别为SEQ ID No.118、SEQ IDNo.119和SEQ ID No.120所示；单域抗体NBV41的3个互补决定区的氨基酸序列分别为SEQID No.121、SEQ ID No.122和SEQ ID No.123所示；单域抗体NBV42的3个互补决定区的氨基酸序列分别为SEQ ID No.124、SEQ ID No.125和SEQ ID No.126所示；
或者将上述所示的任何一种氨基酸序列中的一个或多个氨基酸进行缺失、取代、***和/或添加所得到的蛋白突变体，该蛋白突变体与突变前的蛋白具有相同的功能；或者与上述所示的任何一种氨基酸序列至少有90％以上同一性的氨基酸序列。
2.按照权利要求1所述的单域抗体，其特征在于，单域NBV1的氨基酸序列为SEQ IDNo.127所示，单域NBV2的氨基酸序列为SEQ ID No.128所示，单域NBV3的氨基酸序列为SEQID No.129所示，单域NBV4的氨基酸序列为SEQ ID No.130所示，单域NBV5的氨基酸序列为SEQ ID No.131所示，单域NBV6的氨基酸序列为SEQ ID No.132所示，单域NBV7的氨基酸序列为SEQ ID No.133所示，单域NBV8的氨基酸序列为SEQ ID No.134所示，单域NBV9的氨基酸序列为SEQ ID No.135所示，单域NBV10的氨基酸序列为SEQ ID No.136所示，单域NBV11的氨基酸序列为SEQ ID No.137所示，单域NBV12的氨基酸序列为SEQ ID No.138所示，单域NBV13的氨基酸序列为SEQ ID No.139所示，单域NBV14的氨基酸序列为SEQ ID No.140所示，单域NBV15的氨基酸序列为SEQ ID No.141所示，单域抗体NBV16的氨基酸序列为SEQ IDNo.142所示，单域抗体NBV17的氨基酸序列为SEQ ID No.143所示，单域抗体NBV18的氨基酸序列为SEQ ID No.144所示，单域抗体NBV19的氨基酸序列为SEQ ID No.145所示，单域抗体NBV20的氨基酸序列为SEQ ID No.146所示，单域抗体NBV21的氨基酸序列为SEQ ID No.147所示，单域抗体NBV22的氨基酸序列为SEQ ID No.148所示，单域抗体NBV23的氨基酸序列为SEQ ID No.149所示，单域抗体NBV24的氨基酸序列为SEQ ID No.150所示，单域抗体NBV25的氨基酸序列为SEQ ID No.151所示，单域抗体NBV26的氨基酸序列为SEQ ID No.152所示，单域抗体NBV27的氨基酸序列为SEQ ID No.153所示，单域抗体NBV28的氨基酸序列为SEQID No.154所示，单域抗体NBV29的氨基酸序列为SEQ ID No.155所示，单域抗体NBV30的氨基酸序列为SEQ ID No.156所示，单域抗体NBV31的氨基酸序列为SEQ ID No.157所示，单域抗体NBV32的氨基酸序列为SEQ ID No.158所示，单域抗体NBV33的氨基酸序列为SEQ IDNo.159所示，单域抗体NBV34的氨基酸序列为SEQ ID No.160所示，单域抗体NBV35的氨基酸序列为SEQ ID No.161所示，单域抗体NBV36的氨基酸序列为SEQ ID No.162所示，单域抗体NBV37的氨基酸序列为SEQ ID No.163所示，单域抗体NBV38的氨基酸序列为SEQ ID No.164所示，单域抗体NBV39的氨基酸序列为SEQ ID No.165所示，单域抗体NBV40的氨基酸序列为SEQ ID No.166所示，单域抗体NBV41的氨基酸序列为SEQ ID No.167所示，单域抗体NBV42的氨基酸序列为SEQ ID No.168所示；
或者将上述所示的任何一种氨基酸序列中的一个或多个氨基酸进行缺失、取代、***和/或添加所得到的蛋白突变体，该蛋白突变体与突变前的蛋白具有相同的功能；或者与上述所示的任何一种氨基酸序列至少有90％以上同一性的氨基酸序列。
3.人源化后的抗VEGF的单域抗体，其特征在于，(1)将单域抗体NBV11进行了人源化改造得到了4个人源化的抗体，其氨基酸序列分别为SEQ ID No.169、SEQ ID No.170、SEQ IDNo.171和SEQ ID No.172所示；或(2)将单域抗体NBV20进行了人源化改造得到了4个人源化的抗体，其氨基酸序列分别为SEQ ID No.173、SEQ ID No.174、SEQ ID No.175和SEQ IDNo.176所示；或(3)将单域抗体NBV32进行了人源化改造得到了4个人源化的抗体，其氨基酸序列分别为SEQ ID No.177、SEQ ID No.178、SEQ ID No.179和SEQ ID No.180所示；或(4)将单域抗体NBV35进行了人源化改造得到了3个人源化的抗体，其氨基酸序列分别为SEQ IDNo.181、SEQ ID No.182、和SEQ ID No.183所示。
4.权利要求1或2任何一项所述单域抗体的编码基因；优选的，单域抗体NBV1的编码基因的核苷酸序列为SEQ ID No.184所示，单域抗体NBV2的编码基因的核苷酸序列为SEQ IDNo.185所示，单域抗体NBV3的编码基因的核苷酸序列为SEQ ID No.186所示，单域抗体NBV4的编码基因的核苷酸序列为SEQ ID No.187所示，单域抗体NBV5的编码基因的核苷酸序列为SEQ ID No.188所示，单域抗体NBV6的编码基因的核苷酸序列为SEQ ID No.189所示，单域抗体NBV7的编码基因的核苷酸序列为SEQ ID No.190所示，单域抗体NBV8的编码基因的核苷酸序列为SEQ ID No.191所示，单域抗体NBV9的编码基因的核苷酸序列为SEQ IDNo.192所示，单域抗体NBV10的编码基因的核苷酸序列为SEQ ID No.193所示，单域抗体NBV11的编码基因的核苷酸序列为SEQ ID No.194所示，单域抗体NBV12的编码基因的核苷酸序列为SEQ ID No.195所示，单域抗体NBV13的编码基因的核苷酸序列为SEQ ID No.196所示，单域抗体NBV14的编码基因的核苷酸序列为SEQ ID No.197所示，单域抗体NBV15的编码基因的核苷酸序列为SEQ ID No.198所示，单域抗体NBV16的编码基因的核苷酸序列为SEQ ID No.199所示，单域抗体NBV17的编码基因的核苷酸序列为SEQ ID No.200所示，单域抗体NBV18的编码基因的核苷酸序列为SEQ ID No.201所示，单域抗体NBV19的编码基因的核苷酸序列为SEQ ID No.202所示，单域抗体NBV20的编码基因的核苷酸序列为SEQ IDNo.203所示，单域抗体NBV21的编码基因的核苷酸序列为SEQ ID No.204所示，单域抗体NBV22的编码基因的核苷酸序列为SEQ ID No.205所示，单域抗体NBV23的编码基因的核苷酸序列为SEQ ID No.206所示，单域抗体NBV24的编码基因的核苷酸序列为SEQ ID No.207所示，单域抗体NBV25的编码基因的核苷酸序列为SEQ ID No.208所示，单抗体域NBV26的编码基因的核苷酸序列为SEQ ID No.209所示，单域抗体NBV27的编码基因的核苷酸序列为SEQ ID No.210所示，单域抗体NBV28的编码基因的核苷酸序列为SEQ ID No.211所示，单域抗体NBV29的编码基因的核苷酸序列为SEQ ID No.212所示，单域抗体NBV30的编码基因的核苷酸序列为SEQ ID No.213所示，单域抗体NBV31的编码基因的核苷酸序列为SEQ IDNo.214所示，单域抗体NBV32的编码基因的核苷酸序列为SEQ ID No.215所示，单域NBV33的编码基因的核苷酸序列为SEQ ID No.216所示，单域抗体NBV34的编码基因的核苷酸序列为SEQ ID No.217所示，单域抗体NBV35的编码基因的核苷酸序列为SEQ ID No.218所示，单域抗体NBV36的编码基因的核苷酸序列为SEQ ID No.219所示，单域抗体NBV37的编码基因的核苷酸序列为SEQ ID No.220所示，单域抗体NBV38的编码基因的核苷酸序列为SEQ IDNo.221所示，单域抗体NBV39的编码基因的核苷酸序列为SEQ ID No.222所示，单域抗体NBV40的编码基因的核苷酸序列为SEQ ID No.223所示，单域抗体NBV41的编码基因的核苷酸序列为SEQ ID No.224所示，单域抗体NBV42的编码基因的核苷酸序列为SEQ ID No.225所示；
或者与上述所示的任何一种多核苷酸序列的互补序列在严谨杂交条件能够进行杂交的多核苷酸序列；或者与上述所示的任何一种的多核苷酸序列至少有90％以上同一性的多核苷酸序列。
5.一种重组表达载体，其特征在于，所述重组表达载体包含权利要求4或5所述的编码基因中的一个或多个。
6.一种融合蛋白，其特征在于，将权利要求1-3任何一种的单域抗体与IgG-Fc构建得到的融合蛋白；优选的，所述Fc基因序列是来源于IgG，IgA，IgM的Fc基因序列或来源于IgG1，IgG2，IgG3或IgG4；所述的IgG优选是人IgG及其IgG1、2、3、4的亚类、人IgM、人IgA或其它动物免疫球蛋白的Fc片段。
7.一种缀合物，其特征在于，将权利要求1-3任何一种的单域抗体与酶相、放射性同位素、荧光化合物或化学发光化合物中的一种或多种相偶联得到缀合物。
8.权利要求1-3任何一项所述的单域抗体、权利要求4所述的编码基因、权利要求6所述的融合蛋白以及权利要求7所述的缀合物在制备检测或诊断与VEGF有关的的药物或试剂中的用途。
9.权利要求1-3任何一项所述的单域抗体、权利要求4所述的编码基因、权利要求6所述的融合蛋白以及权利要求7所述的缀合物在制备阻断VEGF与VEGFR之间相互作用的药物或试剂中的用途。
10.权利要求1-3任何一项所述的单域抗体、权利要求4所述的编码基因、权利要求6所述的融合蛋白以及权利要求7所述的缀合物在制备用于治疗VEGF表达异常相关疾病的药物的应用；优选的，所述的VEGF表达异常相关疾病包括各种肿瘤疾病或老年眼底黄斑病变。
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