CN110964110B - 抗EGFR人源化的单域抗体、Fc融合蛋白、重链Fab蛋白及其应用 - Google Patents

抗EGFR人源化的单域抗体、Fc融合蛋白、重链Fab蛋白及其应用 Download PDF

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CN110964110B
CN110964110B CN201911349020.9A CN201911349020A CN110964110B CN 110964110 B CN110964110 B CN 110964110B CN 201911349020 A CN201911349020 A CN 201911349020A CN 110964110 B CN110964110 B CN 110964110B
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叶青
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Beijing Newanbo Biotechnology Co ltd
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Abstract

本发明公开了抗EGFR人源化的单域抗体、Fc融合蛋白、重链Fab蛋白及其应用。本发明通过设计、构建得到一组抗EGFR人源化的单域抗体，与现有的抗体相比，本发明抗EGFR的单域抗体活性高、具有强的中和或结合能力。由该抗EGFR人源化的单域抗体与人IgG1‑Fc融合得到的Fc融合蛋白或与人IgG‑CH1和人CLκ构建得到的重链Fab蛋白能特异性结合人EGFR抗原、结合细胞表面表达EGFR的肿瘤细胞株，有效阻断EGF抗原与EGFR结合以及产生相应的信号级联效应。本发明所提供的抗EGFR人源化的单域抗体、Fc融合蛋白、重链Fab蛋白能用于检测EGFR或治疗多种与EGFR表达异常相关的肿瘤疾病。

Description

抗EGFR人源化的单域抗体、Fc融合蛋白、重链Fab蛋白及其 应用
技术领域
本发明涉及单域抗体，尤其涉及抗EGFR人源化的单域抗体以及用该单域抗体构建的Fc融合蛋白、重链Fab蛋白及其在检测EGFR以及治疗EGFR表达异常相关疾病中的应用，属于抗EGFR人源化的单域抗体及其应用领域。
背景技术
由于生长因子(Growth factors)和生长因子受体(Growth factor receptors)与肿瘤生长有关，因而作为肿瘤生物治疗的靶点(Mendelsohn J.Targeting the epidermalgrowth factor receptor for cancer therapy.J Clin Oncol 2002；20:1S–3S.PirkerR,Minar W,Filipits M.Integrating epidermal growth factor receptor-targetedtherapies into platinum-based chemotherapy regimens for newly diagnosed non-small-cell lung cancer.Clin Lung Cancer 2008；9:S109–15.)。表皮生长因子受体家族(EGFRs)在许多类型的肿瘤，包括头颈癌、乳腺癌、非小细胞肺癌和胰腺癌等都有过表达或功能失调。EGFR家族成员包括：EGFR(ErbB1),HER2(ErbB2),HER3(ErbB3)and HER4(ErbB4)。表皮生长因子受体(epidermal growth factor receptor，EGFR)是跨膜酪氨酸激酶(transmembrane tyrosine kinase receptors)ErbB家族成员之一，EGFR的配体是EGF和转化生长因子-α(transforming growth factor-alpha，TGF-α)。配体与EGFR结合，诱导EGFR的构象变化和形成二聚体，导致细胞内酪氨酸激酶(TKs)的活化，后续酶促级联反应的结果导致肿瘤细胞的增值、侵袭、转移、新血管生成和程序化死亡降低(Burgess AW,Cho HS,Eigenbrot C,et al.An open-and-shut case Recent insights into the activationof EGF/ErbB receptors.Mol Cell2003；12:541–52.)。EGFR的功能可以通过其受体胞外区特异性单克隆抗体和酪氨酸激酶抑制剂(TKIs)来阻断。单克隆抗体和TKIs的药物代谢动力学和药效学的作用机理是不同的。单克隆抗体结合EGFR的表面，竞争性阻断了EGF与其受体的结合，细胞表面的抗体与受体结合物被细胞内吞并在胞内降解，其结果导致肿瘤细胞表面的EGFR量下调；同时抗体也通过其抗体依赖细胞毒作用等免疫学机制来抗肿瘤。而TKIs是化学小分子物质，通过阻断EGFR胞内三磷酸腺苷结合位点，抑制肿瘤细胞生长。靶向癌细胞上EGFR的抗体药物如：西妥昔单抗(Cetuximab(ErbituxR))，帕尼抗体(Panitumumab)，尼妥珠单抗(Nimotuzumab，泰欣生)，托珠单抗(Necitumumacb)已上市用于转移性结肠癌、非小细胞肺癌和头颈癌并取得了一定的疗效。但由于传统单抗分子量大、对肿瘤组织的渗透性差、生产成本高、药价高昂等，仍然存在诸多满足不了临床应用的需求。
单域抗体(single domain antibody(sdAb)或称纳米抗体(nanobody)是由羊驼血液中发现的缺失轻链的重链抗体可变区片段(VHH)，其具有结构简单，穿透力强，易于表达和纯化，亲和力及稳定性高，毒副反应小等一系列优点。虽然来源于驼类的单域抗体(VHH)结构简单与人类抗体的同源性高，但用于疾病治疗药物，多数需要长期、反复应用，对单域抗体候选药物的基因序列进行人源化优化，以尽可能地降低其可能的免疫原性，这对于提高单域抗体候选药物体的成药性是非常必要的。
发明内容
本发明的目的之一是提供一组抗EGFR人源化的单域抗体及其编码基因；
本发明的目的之二是提供抗EGFR人源化的单域抗体与人IgG1-Fc融合得到的Fc融合蛋白及其编码基因；
本发明的目的之三是提供抗EGFR人源化的单域抗体与人IgG-CH1和人CLκ构建得到的重链Fab蛋白及其编码基因。
本发明的目的之四将所述的抗EGFR人源化的单域抗体、Fc融合蛋白以及重链Fab蛋白应用于制备检测EGFR的试剂或治疗EGFR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EGFR人源化的单域抗体，所述单域抗体的氨基酸序列选自以下(1)-(3)中的任何一种氨基酸序列：
(1)SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ IDNO.8、SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.11或SEQ ID NO.12所示的任何一种氨基酸序列；
(2)将SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7、SEQID NO.8、SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.11或SEQ ID NO.12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、插入和/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；
(3)与SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7、SEQID NO.8、SEQ ID NO.9、SEQ ID NO.10、SEQ ID NO.11或SEQ ID NO.12中所示的任何一种氨基酸序列至少有75％以上同一性的氨基酸序列。
本发明进一步提供了所述单域抗体的编码基因；所述编码基因的核苷酸序列选自(1)-(3)中的任何一种：
(1)SEQ ID NO.27、SEQ ID NO.28、SEQ ID NO.29、SEQ ID NO.30、SEQ ID NO.31、SEQ ID NO.32、SEQ ID NO.33、SEQ ID NO.34、SEQ ID NO.35或SEQ ID NO.36所示的任何一种多核苷酸序列；
或(2)与SEQ ID NO.27、SEQ ID NO.28、SEQ ID NO.29、SEQ ID NO.30、SEQ IDNO.31、SEQ ID NO.32、SEQ ID NO.33、SEQ ID NO.34、SEQ ID NO.35或SEQ ID NO.36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；
或(3)与SEQ ID NO.27、SEQ ID NO.28、SEQ ID NO.29、SEQ ID NO.30、SEQ IDNO.31、SEQ ID NO.32、SEQ ID NO.33、SEQ ID NO.34、SEQ ID NO.35或SEQ ID NO.36所示的多核苷酸序列至少有75％以上同一性的多核苷酸序列；优选的，与SEQ ID NO.27、SEQ IDNO.28、SEQ ID NO.29、SEQ ID NO.30、SEQ ID NO.31、SEQ ID NO.32、SEQ ID NO.33、SEQ IDNO.34、SEQ ID NO.35或SEQ ID NO.36所示的多核苷酸序列至少有80％以上同一性的多核苷酸序列；进一步优选的，与SEQ ID NO.27、SEQ ID NO.28、SEQ ID NO.29、SEQ ID NO.30、SEQ ID NO.31、SEQ ID NO.32、SEQ ID NO.33、SEQ ID NO.34、SEQ ID NO.35或SEQ IDNO.36所示的多核苷酸序列至少有85％以上同一性的多核苷酸序列；更优选的，与SEQ IDNO.27、SEQ ID NO.28、SEQ ID NO.29、SEQ ID NO.30、SEQ ID NO.31、SEQ ID NO.32、SEQ IDNO.33、SEQ ID NO.34、SEQ ID NO.35或SEQ ID NO.36所示的多核苷酸序列至少有95％以上同一性的多核苷酸序列；最优选的，与SEQ ID NO.27、SEQ ID NO.28、SEQ ID NO.29、SEQ IDNO.30、SEQ ID NO.31、SEQ ID NO.32、SEQ ID NO.33、SEQ ID NO.34、SEQ ID NO.35或SEQID NO.36所示的多核苷酸序列至少有99％以上同一性的多核苷酸序列。
本发明进一步将所述的抗EGFR人源化的单域抗体与人IgG1-Fc融合得到的Fc融合蛋白；其中，所述Fc基因序列可以是来源于IgG，IgA，IgM的Fc基因序列或来源于人IgG1，IgG2，IgG3或IgG4。
所述的Fc融合蛋白的氨基酸序列选自以下(1)-(3)中的任何一种氨基酸序列：
(1)SEQ ID NO.13、SEQ ID NO.14、SEQ ID NO.15、SEQ ID NO.16、SEQ ID NO.17、SEQ ID NO.18、SEQ ID NO.19或SEQ ID NO.20中所示的任何一种氨基酸序列；
(2)将SEQ ID NO.13、SEQ ID NO.14、SEQ ID NO.15、SEQ ID NO.16、SEQ IDNO.17、SEQ ID NO.18、SEQ ID NO.19或SEQ ID NO.20中所示的任何一种氨基酸序列采用一个或多个氨基酸进行缺失、取代、插入和/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；
(3)与SEQ ID NO.13、SEQ ID NO.14、SEQ ID NO.15、SEQ ID NO.16、SEQ IDNO.17、SEQ ID NO.18、SEQ ID NO.19或SEQ ID NO.20中所示的任何一种氨基酸序列至少有75％以上同一性的氨基酸序列。
本发明提供了所述的Fc融合蛋白的编码基因；优选的，所述编码基因的核苷酸序列选自(1)-(3)中的任何一种：
(1)SEQ ID NO.37、SEQ ID NO.38、SEQ ID NO.39、SEQ ID NO.40、SEQ ID NO.41、SEQ ID NO.42、SEQ ID NO.43或SEQ ID NO.44所示的任何一种多核苷酸序列；
或(2)与SEQ ID NO.37、SEQ ID NO.38、SEQ ID NO.39、SEQ ID NO.40、SEQ IDNO.41、SEQ ID NO.42、SEQ ID NO.43或SEQ ID NO.44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；
或(3)与SEQ ID NO.37、SEQ ID NO.38、SEQ ID NO.39、SEQ ID NO.40、SEQ IDNO.41、SEQ ID NO.42、SEQ ID NO.43或SEQ ID NO.44所示的多核苷酸序列至少有75％以上同一性的多核苷酸序列；优选的，与SEQ ID NO.27、SEQ ID NO.28、SEQ ID NO.29、SEQ IDNO.30、SEQ ID NO.31、SEQ ID NO.32、SEQ ID NO.33、SEQ ID NO.34、SEQ ID NO.35或SEQID NO.36所示的多核苷酸序列至少有80％以上同一性的多核苷酸序列；进一步优选的，与SEQ ID NO.27、SEQ ID NO.28、SEQ ID NO.29、SEQ ID NO.30、SEQ ID NO.31、SEQ IDNO.32、SEQ ID NO.33、SEQ ID NO.34、SEQ ID NO.35或SEQ ID NO.36所示的多核苷酸序列至少有85％以上同一性的多核苷酸序列；更优选的，与SEQ ID NO.27、SEQ ID NO.28、SEQID NO.29、SEQ ID NO.30、SEQ ID NO.31、SEQ ID NO.32、SEQ ID NO.33、SEQ ID NO.34、SEQID NO.35或SEQ ID NO.36所示的多核苷酸序列至少有95％以上同一性的多核苷酸序列；最优选的，与SEQ ID NO.27、SEQ ID NO.28、SEQ ID NO.29、SEQ ID NO.30、SEQ ID NO.31、SEQID NO.32、SEQ ID NO.33、SEQ ID NO.34、SEQ ID NO.35或SEQ ID NO.36所示的多核苷酸序列至少有99％以上同一性的多核苷酸序列。
本发明将抗EGFR人源化的单域抗体与人IgG-CH1和人CLκ构建得到的重链Fab蛋白；优选的，所述重链Fab蛋白的氨基酸序列选自以下(1)-(3)中的任何一种氨基酸序列：
(1)SEQ ID NO.21、SEQ ID NO.22、SEQ ID NO.23或SEQ ID NO.24中所示的任何一种氨基酸序列；
(2)将SEQ ID NO.21、SEQ ID NO.22、SEQ ID NO.23或SEQ ID NO.24中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、插入和/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；
(3)与SEQ ID NO.21、SEQ ID NO.22、SEQ ID NO.23或SEQ ID NO.24中所示的任何一种氨基酸序列至少有75％以上同一性的氨基酸序列。
本发明提供了所述重链Fab蛋白的编码基因；优选的，所述编码基因的核苷酸序列选自(1)-(3)中的任何一种：
(1)SEQ ID NO.45、SEQ ID NO.46、SEQ ID NO.47或SEQ ID NO.48所示的任何一种多核苷酸序列；
或(2)与SEQ ID NO.45、SEQ ID NO.46、SEQ ID NO.47或SEQ ID NO.48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；
或(3)与SEQ ID NO.45、SEQ ID NO.46、SEQ ID NO.47或SEQ ID NO.48所示的多核苷酸序列至少有75％以上同一性的多核苷酸序列；优选的，与SEQ ID NO.45、SEQ IDNO.46、SEQ ID NO.47或SEQ ID NO.48所示的多核苷酸序列至少有80％以上同一性的多核苷酸序列；进一步优选的，与SEQ ID NO.45、SEQ ID NO.46、SEQ ID NO.47或SEQ ID NO.48所示的多核苷酸序列至少有85％以上同一性的多核苷酸序列；更优选的，与SEQ ID NO.45、SEQ ID NO.46、SEQ ID NO.47或SEQ ID NO.48所示的多核苷酸序列至少有95％以上同一性的多核苷酸序列；最优选的，与SEQ ID NO.45、SEQ ID NO.46、SEQ ID NO.47或SEQ IDNO.48所示的多核苷酸序列至少有99％以上同一性的多核苷酸序列。
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、插入和/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、插入和/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。即，以上述合成寡核苷酸为引物合成与噬菌体互补的链，用得到的双链DNA转化宿主细胞。将转化细菌的培养物铺在琼脂上，由含噬菌体的单个细胞形成噬菌斑。然后，收集与该探针杂交的噬菌斑，进行培养，回收DNA。而且，在保持其活性的同时对酶等生物活性肽的氨基酸序列施加一个或多个氨基酸的缺失、取代、插入和/或添加的方法，除了上述的定点突变外，还有用诱变源处理基因的方法，以及选择性地断开基因，然后删除、取代、插入或添加选定核苷酸，再进行连接的方法。
本发明还提供了含有单域抗体的编码基因的表达载体、含有Fc融合蛋白的编码基因的表达载体或含有重链Fab蛋白的编码基因的表达载体；所述的表达载体可以是原核表达载体、真核表达载体或其它的表达载体；
本发明还公开了含有所述表达载体的重组宿主细胞。其中，所述的宿主细胞为原核表达细胞、真核表达细胞，真菌细胞或酵母细胞，所述真核表达细胞优选CHO细胞。
本发明所提供的人源化单域抗体能够有效阻断EGF与EGFR之间相互作用，用本发明的抗EGFR的人源化单域抗体与Fc蛋白融合得到的Fc融合蛋白以及与人IgG-CH1和人CLκ构建得到的重链Fab蛋白活性高、稳定性高、特异性高、结合能力强能够应用于制备检测EGFR的试剂或药物或制备治疗EGFR表达异常相关疾病药物。
作为一种具体的实施方式，可以将抗EGFR人源化的单域抗体、由抗EGFR人源化的单域抗体与人IgG1-Fc融合得到的融合蛋白或抗EGFR人源化的单域抗体与人IgG-CH1和人CLκ构建得到的重链Fab蛋白与细胞毒性剂、酶相、放射性同位素或化学发光化合物中的一种或多种相连得到检测EGFR的试剂或用于治疗EGFR表达异常相关疾病药物。
譬如，将抗EGFR人源化的单域抗体、Fc融合蛋白或重链Fab蛋白用68Ga,89Zr,64Cu,18F,86Y,90Y,111In,99NVTc,125I,124I等放射性同位素进行标记得到标记蛋白用于PET(positron emission tomography)或SPECT的影像学检测。或者将抗EGFR人源化的单域抗体、Fc融合蛋白或重链Fab蛋白用90Y，177Lu,125I,131I,211At,111In,152Sm,186Re,188Re,67Cu,212Pb,225Ac,213Bi,212Bi或67Ga等放射性同位素进行标记得到标记蛋白用于EGFR表达异常相关疾病的治疗。
优选的，所述的EGFR表达异常相关疾病包括结直肠癌、头颈癌、非小细胞肺癌等肿瘤疾病。
所述的化学发光化合物可以是荧光化合物。
本发明通过设计、构建得到的一组抗EGFR人源化的单域抗体及其融合蛋白，与现有的抗体相比，本发明提供的抗EGFR的单域抗体活性高、具有强的中和或结合能力；由该抗EGFR人源化的单域抗体与人IgG1-Fc融合得到的Fc融合蛋白或由抗EGFR人源化的单域抗体与人IgG-CH1和人CLκ构建得到的重链Fab蛋白能特异性结合人EGFR抗原、结合细胞表面表达EGFR的肿瘤细胞株，有效阻断EGF抗原与EGFR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EGFR表达异常相关的疾病。
本发明所涉及的术语定义
本文所用的术语“EGFR”，是表皮生长因子受体家族(EGFRs)的成员，EGFR家族成员包括：EGFR(ErbB1),HER2(ErbB2),HER3(ErbB3)and HER4(ErbB4)。表皮生长因子受体(epidermal growth factor receptor，EGFR)是跨膜酪氨酸激酶(transmembranetyrosine kinase receptors)ErbB家族成员之一，EGFR的配体是EGF和转化生长因子-α(transforming growth factor-alpha，TGF-α)。配体与EGFR结合，诱导EGFR的构象变化和形成二聚体，导致细胞内酪氨酸激酶(TKs)的活化，后续酶促级联反应的结果导致肿瘤细胞的增值、侵袭、转移、新血管生成和程序化死亡降低。
针对EGFR的新抗体及其Fc融合蛋白是本文的研发对象，最终也是本文的保护对象，本文的范围涉及所得到的抗EGFR人源化单域抗体及其Fc融合蛋白、重链Fab、以该抗体为成分的物质(如，药物组合物、试剂盒、载体等等)、应用(如，诊断应用、治疗应用、制备应用等等)，然而，本领域技术人员应该理解，本文的保护对象并不限于举例的这些内容。
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(例如1、2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18或更多个)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。
在一些实施方式中，本发明的多肽、药物组合物，应该理解，本发明并不限于此，本发明的上述物质可以包含2个单域抗体、3个单域抗体，或多个单域抗体，这些多个单域抗体之间相同或不同，此外，除了包含本发明的单域抗体之外，还可以包含本发明以外的其他抗体或单域抗体，并没有超出本发明的范围。
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插入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为表达的抗EGFR-人源化sdAbs经镍柱纯化后SDS-PAGE结果。
图2为表达的抗EGFR人源化单域抗体-Fc融合蛋白经蛋白A株亲和层析纯化后SDS-PAGE结果；1.EG2M1-EG10M1-Fc-p327.7表达纯化后还原性蛋白带；2.EG2M1-Fc-EG10M1-p327.7表达纯化后还原性蛋白带；3.EG2M1-EG10M1-Fc-p327.7表达纯化后非还原性蛋白带；4.EG2M1-Fc-EG10M1-p327.7表达纯化后非还原性蛋白带；5.蛋白质分子量标准(Marker)。箭头所示分子量为50KD。
图3为表达的抗EGFR人源化单域抗体Fab，经镍柱亲和层析纯化后SDS-PAGE结果；1.蛋白分子量标准(Marker)；2.sdAb1-CH1—sdAb2-CL-p327.7表达蛋白非还原(20mM咪唑)；3.sdAb1-CH1—sdAb2-CL-p327.7表达蛋白非还原(50mM咪唑)；4.sdAb1-CH1—sdAb2-CL-p327.7表达蛋白非还原(100mM咪唑)；5.sdAb1-CH1—sdAb2-CL-p327.7表达蛋白非还原型(250mM咪唑)；6.sdAb1-CH1—sdAb2-CL-p327.7表达蛋白非还原型(500mM咪唑)；7.sdAb1-CH1—sdAb2-CL-p327.7表达蛋白还原型(500mM咪唑)；8.sdAb1-CH1—sdAb2-CL-p327.7表达蛋白还原型(250mM咪唑)；9.sdAb1-CH1—sdAb2-CL-p327.7表达蛋白还原型(100mM咪唑)；10.sdAb1-CH1—sdAb2-CL-p327.7表达蛋白还原型(50mM咪唑)；11.sdAb1-CH1—sdAb2-CL-p327.7表达蛋白还原型(20mM咪唑。
图4抗体修饰及89Zr标记路线图。
图5给予89Zr-EGFR37.0抗体后各时间点各组织放射性物质摄取％ID/g值柱状图(小动物PET/CT扫描)。
图6给予89Zr-EGFR37.2抗体后各时间点各组织放射性物质摄取％ID/g值柱状图(小动物PET/CT扫描)。
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EGFR抗原特异性的人源化单域抗体设计构建
(1)原抗EGFR单域抗体
2个来源于美洲驼(Llama)抗EGFR的单域抗体氨基酸序列分别为SEQ ID NO:1-2所示。
(2)抗EGFR单域抗体的人源化
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EGFR单域抗体EG2、EG10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EGFR单域抗体EG2和EG10进行人源化。人源化结果见下表1。
表1.EG2和EG10人源化结果
Figure BDA0002334199540000091
*注释：根据文献研究报道，超过80％，其免疫原性就接近人源抗体。
实施例2抗EGFR人源化单域抗体表达质粒的构建
基因合成实施例1所列出的抗EGFR单域抗体EG2，EG10，EG2M1，EG2M2，EG10，EG10M1和EG10M2基因，然后PCR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，中国专利公布号CN102321175A(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)合成VHH目的基因的PCR扩增条件，50μl PCR体系的组成：
Figure BDA0002334199540000092
Figure BDA0002334199540000101
PCR反应条件：
Figure BDA0002334199540000102
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单域抗体表达质粒。
实施例3抗EGFR人源化单域抗体的表达和纯化
将实施例2所述的含有质粒sdAb-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95％以上的蛋白。图1为表达纯化的抗EGFR单域抗体蛋白经镍柱纯化后SDS-PAGE电泳结果。
实施例4抗EGFR人源化单域抗体Fc融合蛋白的载体构建
(1)第一结构：sdAb1-linker-sdAb2-Hinger-CH2-CH3(IgG1-Fc)。
sdAb1＝EG2M1或EG2M2或EG2M3或EG2M4或EG2M5或EG10M1或EG10M2或EG10M3或EG10M4或EG10M5。
linker＝GGGGSGGGGS或GGSGGSGGGGS或GGGGSGGGGSGGGGS sdAb2＝EG10M1或EG10M2或EG10M3或EG10M4或EG10M5或EG2M1或EG2M2或EG2M3或EG2M4或EG2M5。
构建步骤：
EG2M1(sdAb1)+(GGGGS)*2(linker)+EG10M1(sdAb2)+人IgG1-Fc基因全合成，加入XhoI-EcoRI双酶切，将sdAb1-linker-sdAb2-Fc基因连接至p327.7表达载体(专利公布号CN104195173A)，并加入相应酶切位点和终止密码子,用XbaI-SalI双酶切，将另一sdAb1-linker-sdAb2-Fc基因连接至已含有EG2M1-EG10M1-Fc(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EG2M1-EG10M1-Fc序列。
(2)第二结构：sdAb1-Hing-CH2-CH3(IgG1-Fc)-linker-sdAb2。
sdAb1＝EG2M1或EG2M2或EG2M3或EG2M4或EG2M5或EG10M1或EG10M2或EG10M3或EG10M4或EG10M5。
linker＝GGGGSGGGGS或GGSGGSGGGGS或GGGGSGGGGSGGGGS。
sdAb2＝EG10M1或EG10M2或EG10M3或EG10M4或EG10M5或EG2M1或EG2M2或EG2M3或EG2M4或EG2M5。
构建步骤：
EG2M1(sdAb1)+人IgG1-Fc+(GGGGS)*2(linker)+EG10M1(sdAb2)基因全合成，加入XhoI-EcoRI双酶切，将sdAb1-Fc-linker-sdAb2连接至p327.7表达载体，并加入相应酶切位点和终止密码子，用XbaI-SaII双酶切将另一sdAb1-Fc-linker-sdAb2基因连接至已含有EG2M1-Fc-EG10M1(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EG2M1-Fc-EG10M1序列。
(3)第三结构：通过构建重链Fab，其中一个单域抗体与人IgG-CH1相连接(sdAb1-CH1)，并且在C末端连接6个His，另一个单域抗体与人CLκ相连接(sdAb2-CL)，即sdAb1-CH1//sdAb2-CL，这种Fab在体内半衰期短，有利于制备核素标记的体内检测药物应用。
sdAb1＝EG2M1或EG2M2或EG2M3或EG2M4或EG2M5或EG10M1或EG10M2或EG10M3或EG10M4或EG10M5；
sdAb2＝EG10M1或EG10M2或EG10M3或EG10M4或EG10M5或EG2M1或EG2M2或EG2M3或EG2M4或EG2M5。
构建步骤：
1)EG2M1(sdAb1)+CH1+6His基因全合成，加入XhoI-EcoRI双酶切，将sdAb1-CH1-6His基因连接至p327.7表达载体，并加入相应酶切位点和终止密码子；
2)EG10M1(sdAb2)+CL基因全合成，用XbaI-SalI双酶切后，将sdAb2-CL连接至已含有sdAb1-Ch1-6His基因的p327.7表达载体中。
本发明所提供的抗EGFR人源化单域抗体、Fc融合蛋白以及重链Fab蛋白的氨基酸和基因的序列表见图2.
表2抗EGFR人源化单域抗体、Fc融合蛋白、重链Fab蛋白的序列表
序号 名称 氨基酸序列 编码基因序列
1 EG2 SEQ ID NO.1 SEQ ID NO.25
2 EG10 SEQ ID NO.2 SEQ ID NO.26
3 EG2M1 SEQ ID NO.3 SEQ ID NO.27
4 EG2M2 SEQ ID NO.4 SEQ ID NO.28
5 EG2M3 SEQ ID NO.5 SEQ ID NO.29
6 EG2M4 SEQ ID NO.6 SEQ ID NO.30
7 EG2M5 SEQ ID NO.7 SEQ ID NO.31
8 EG10M1 SEQ ID NO.8 SEQ ID NO.32
9 EG10M2 SEQ ID NO.9 SEQ ID NO.33
10 EG10M3 SEQ ID NO.10 SEQ ID NO.34
11 EG10M4 SEQ ID NO.11 SEQ ID NO.35
12 EG10M5 SEQ ID NO.12 SEQ ID NO.36
13 EG2M1-EG10M1-Fc SEQ ID NO.13 SEQ ID NO.37
14 EG2M2-EG10M2-Fc SEQ ID NO.14 SEQ ID NO.38
15 EG10M1-EG2M1-Fc SEQ ID NO.15 SEQ ID NO.39
16 EG10M2-EG2M2-Fc SEQ ID NO.16 SEQ ID NO.40
17 EG2M1-Fc-EG10M1 SEQ ID NO.17 SEQ ID NO.41
18 EG2M2-Fc-EG10M2 SEQ ID NO.18 SEQ ID NO42
19 EG10M1-Fc-EG2M1 SEQ ID NO.19 SEQ ID NO.43
20 EG10M2-Fc-EG2M2 SEQ ID NO.20 SEQ ID NO.44
21 EG2M1-CH1//EG10M1-CL SEQ ID NO.21 SEQ ID NO.45
22 EG2M2-CH1//EG10M2-CL SEQ ID NO.22 SEQ ID NO.46
23 EG10M1-CH1//EG2M1-CL SEQ ID NO.23 SEQ ID NO.47
24 EG10M2-CH1//EG2M2-CL SEQ ID NO.24 SEQ ID NO.48
实施例5抗EGFR人源化单域抗体Fc融合蛋白的表达与纯化
将表达载体转染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。抗EGFR人源化单域抗体Fc融合蛋白构建的第一结构表达载体和第二结构表达载体所表达纯化的蛋白见图2。
将表达载体sdAb1-CH1-sdAb2-CL-p327.7转染CHO/K1细胞，MSx筛选稳定的高表达细胞株，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。用镍柱亲和层析纯化蛋白后，置换为柠檬酸(0.05％Tween80，pH6.2)缓冲液，其表达纯化的蛋白见图3。抗EGFR人源化单域抗体三种融合蛋白表达纯化后鉴定结果与理论推算值接近一致。
第一结构EG2M1-EG10M1-Fc-p327.7表达载体表达的蛋白理论推算值为：含有968个氨基酸；分子量(Mw)，经Hinger二硫键连接分子量为10.5702 KD，等电点(pI)为8.08，纯化后蛋白电泳SDS-PAGE还原后分子量为50KD左右与理论推算值一致。
第二结构EG2M1-Fc-EG10M1-p327.7表达载体表达的蛋白理论推算值为：含有976个氨基酸；分子量(Mw)，经Hinger二硫键连接分子量为10.6219 KD，等电点(pI)为8.08，纯化后蛋白电泳SDS-PAGE还原后分子量为50KD左右与理论推算值一致。
第三结构sdAb1-CH1-sdAb2-CL-p327.7表达载体表达的蛋白理论推算值为：含有468个氨基酸；分子量(Mw)为5.0499 KD，等电点(pI)为8.66，纯化后蛋白电泳SDS-PAGE非还原的分子量为50KD左右，还原后分子量为25KD与理论推算值一致。
试验例1纯化的抗EGFR人源化单域抗体Fc融合蛋白与EGFR蛋白结合试验(ELISA)
0.05 M NaHCO3(pH 9.5)稀释EGFR-His抗原(EGFR Protein，Human，Recombinant，Cat：10001-H08B，北京义翘神州生物技术有限公司)至1μg/ml，1000μl抗原包被96孔板，4℃过夜。300μl0.5％BSA-PBS封闭96孔板，37℃，2小时。
加入不同稀释浓度的纯化的抗EGFR人源化单域抗体Fc融合蛋白，按100μl/孔加入，37℃，1小时。用0.05％PBST洗板三次。加入1∶5000倍稀释的鼠抗人IgG-HRP，按100μl/孔加入，37℃，1小时。用0.05％PBST洗板三次。加TMB100μl，避光室温静置20分钟。加入1mol/LHCl 100μl终止反应。用酶标仪测定450nm波长下的样品OD值。表3为抗EGFR人源化单域抗体Fc融合蛋白与BCMA蛋白结合的浓度梯度(ELISA)及试验结果。
表3各种抗EGFR单域抗体及融合蛋白结合EGFR蛋白活性试验结果
蛋白组别 EG2 EG2M1 EG2M2 EG2M1-EG10M1-Fc EG2M2-EM10M2-Fc EG10M1-EG2M1-Fc EG2M1-Fc-EG10M1 EG2M2-Fc-EG10M2 EG2M1-CH1-EG10M1-CL
(ug/ml)
5.000 3.690 3.799 3.689 4.200 4.310 3.987 4.018 4.245 4.105
2.500 3.109 3.089 3.101 3.996 4.123 3.698 3.899 3.957 3.878
1.000 2.520 2.569 2.467 3.109 3.098 3.011 3.056 3.124 3.044
0.500 1.459 1.534 1.519 2.998 2.688 2.236 2.345 2.153 2.236
0.100 0.532 0.499 0.520 1.569 1.601 1.298 1.386 1.373 1.420
0.050 0.089 0.091 0.101 1.093 1.011 0.987 0.896 0.921 0.861
0.010 0.068 0.078 0.088 0.156 0.210 0.196 0.201 0.189 0.108
0.000 0.009 0.013 0.029 0.089 0.076 0.089 0.078 0.092 0.068
注：EG2M1-EG10M1-Fc(SEQ ID NO.13)，EG2M2-EG10M2-Fc(SEQ ID NO.14)，EG10M1-EG2M1-Fc(SEQ ID NO.15)，EG2M1-Fc-EG10M1(SEQ ID NO.17)，EG2M2-Fc-EG10M2(SEQ ID NO.18)，EG2M1-CH1-EG10M1-CL(SEQ ID NO.21)。
试验例2抗EGFR人源化单域抗体及其融合蛋白的亲和力测定试验
1)样品制备
抗原：Bio-EGFR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；
人源化单域抗体：用1×kinetic缓冲液依次稀释为400nM、200nM、100nM、50nM、25nM、12.5nM、0nM；
人源化单域抗体融合蛋白：用1×kinetic缓冲液依次稀释为235.3nM、117.6nM、58.8nM、29.4nM、0nM浓度。
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，所有抗EGFR人源化单域抗体及其融合蛋白的亲和力在50nm、20nm、10nm、1nm、0.1nm、0.01nm。检测结果见表4。
表4抗EGFR人源化单域抗体及其融合蛋白与人EGFR的亲和力分析结果
Figure BDA0002334199540000141
Figure BDA0002334199540000151
试验例3放射性同位素标记EGFR人源化单域抗体融合蛋白试验
(1)抗体DFO修饰：反应瓶中取1mL抗体溶液(上述三种结构蛋白中的一种2m/ml)+1mL 0.5M NaHCO3/Na2CO3溶液，测pH值至碱性；37℃搅拌反应40min。PD10柱纯化。(2)抗体标记：取少许89Zr，加入2M Na2CO3溶液，调节pH中性；(3)抗体质控：玻璃纤维纸，展开剂；柠檬酸钠体系。抗体标记物在原点，游离89Zr在前沿。抗体修饰及89Zr标记路线图见图4。(4)三种抗体结构的蛋白标记同位素89Zr，在小鼠肿瘤动物模型体内重要器官和肿瘤部位组织的分布结果见表5-表7和图5-图6。
表5给予89Zr-EGFR37.0抗体后各时间点各组织放射性物质摄取％ID/g值(mean±SD,n＝6)
Figure BDA0002334199540000152
表6给予89Zr-EGFR37.2抗体后各时间点各组织放射性物质摄取％ID/g值(mean±SD,n＝6)
Figure BDA0002334199540000153
表7给予89Zr-EGFR37.3后各组织放射性物质摄取％ID值
Figure BDA0002334199540000154
SEQUENCE LISTING
<110> 北京纽安博生物技术有限公司
<120> 抗EGFR人源化的单域抗体、Fc融合蛋白、重链Fab蛋白及其应用
<130> BJ-4013-190916A
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195 200 205
Ser Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
210 215 220
Asp Thr Ala Val Tyr Tyr Cys Ala Ala Asp Thr Phe Arg Ser Phe Val
225 230 235 240
Val Gly Asn Val Lys Glu Trp Gly Gln Gly Thr Gln Val Thr Val Ser
245 250 255
Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu
260 265 270
Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Gln Leu
275 280 285
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser
290 295 300
His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu
305 310 315 320
Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
325 330 335
Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
340 345 350
Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Ala Leu Pro Ala Pro Ile
355 360 365
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
370 375 380
Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
385 390 395 400
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
405 410 415
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
420 425 430
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
435 440 445
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu
450 455 460
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
465 470 475 480
Pro Gly
<210> 14
<211> 483
<212> PRT
<213> Artifical sequence
<400> 14
Gln Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asp
1 5 10 15
Ser Leu Arg Val Ser Cys Ala Ala Ser Gly Arg Asp Phe Ser Asp Tyr
20 25 30
Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val
35 40 45
Ala Ala Ile Ser Arg Asn Gly Leu Thr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Met Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Val Asn Ser Ala Gly Thr Tyr Val Ser Pro Arg Ser Arg Glu Tyr
100 105 110
Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Glu Ser Gly Gly
130 135 140
Gly Leu Val Gln Pro Gly Gly Ser Leu Thr Leu Ser Cys Ala Ala Ser
145 150 155 160
Gly Gly Thr Phe Ser Ser Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro
165 170 175
Gly Lys Gly Leu Glu Phe Val Ala Ala Ile Ser Gly Arg Ser Ser Ile
180 185 190
Arg Asn Tyr Asp Asp Ser Val Lys Gly Arg Phe Ala Ile Ser Arg Asp
195 200 205
Asn Ser Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
210 215 220
Asp Thr Ala Val Tyr Tyr Cys Ala Ala Asp Thr Val Phe Arg Ser Phe
225 230 235 240
Val Val Gly Asn Val Lys Glu Trp Gly Gln Gly Thr Leu Val Thr Val
245 250 255
Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
260 265 270
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Gln
275 280 285
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
290 295 300
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
305 310 315 320
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
325 330 335
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
340 345 350
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Ala Leu Pro Ala Pro
355 360 365
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
370 375 380
Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
385 390 395 400
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
405 410 415
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
420 425 430
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
435 440 445
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
450 455 460
Leu His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
465 470 475 480
Ser Pro Gly
<210> 15
<211> 483
<212> PRT
<213> Artifical sequence
<400> 15
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 Ala Ala Ser Gly Gly Thr Phe Ser Ser Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val
35 40 45
Ala Ala Ile Ser Gly Arg Ser Ser Ile Arg Asn Tyr Asp Asp Ser Val
50 55 60
Lys Gly Arg Phe Ala Ile Ser Arg Asp Ser Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Asp Thr Val Phe Arg Ser Phe Val Val Gly Asn Val Lys Glu
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Gly Gly Gly Gly Ser
115 120 125
Gly Gly Gly Gly Ser Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu
130 135 140
Val Gln Pro Gly Asp Ser Leu Arg Val Ser Cys Ala Ala Ser Gly Arg
145 150 155 160
Asp Phe Ser Asp Tyr Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys
165 170 175
Gly Leu Glu Phe Val Ala Ala Ile Ser Arg Asn Gly Leu Thr Thr Arg
180 185 190
Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp
195 200 205
Lys Asn Met Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
210 215 220
Ala Val Tyr Tyr Cys Ala Val Asn Ser Ala Gly Thr Tyr Val Ser Pro
225 230 235 240
Arg Ser Arg Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val
245 250 255
Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
260 265 270
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Gln
275 280 285
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
290 295 300
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
305 310 315 320
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
325 330 335
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
340 345 350
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Ala Leu Pro Ala Pro
355 360 365
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
370 375 380
Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
385 390 395 400
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
405 410 415
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
420 425 430
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
435 440 445
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
450 455 460
Leu His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
465 470 475 480
Ser Pro Gly
<210> 16
<211> 483
<212> PRT
<213> Artifical sequence
<400> 16
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 Ala Ala Ser Gly Gly Thr Phe Ser Ser Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val
35 40 45
Ala Ala Ile Ser Gly Arg Ser Ser Ile Arg Asn Tyr Asp Asp Ser Val
50 55 60
Lys Gly Arg Phe Ala Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Asp Thr Val Phe Arg Ser Phe Val Val Gly Asn Val Lys Glu
100 105 110
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser
115 120 125
Gly Gly Gly Gly Ser Gln Val Lys Leu Val Glu Ser Gly Gly Gly Leu
130 135 140
Val Gln Pro Gly Asp Ser Leu Arg Val Ser Cys Ala Ala Ser Gly Arg
145 150 155 160
Asp Phe Ser Asp Tyr Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys
165 170 175
Gly Leu Glu Phe Val Ala Ala Ile Ser Arg Asn Gly Leu Thr Thr Arg
180 185 190
Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
195 200 205
Lys Asn Met Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
210 215 220
Ala Val Tyr Tyr Cys Ala Val Asn Ser Ala Gly Thr Tyr Val Ser Pro
225 230 235 240
Arg Ser Arg Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val
245 250 255
Ser Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
260 265 270
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Gln
275 280 285
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
290 295 300
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
305 310 315 320
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
325 330 335
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
340 345 350
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Ala Leu Pro Ala Pro
355 360 365
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
370 375 380
Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val
385 390 395 400
Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val
405 410 415
Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
420 425 430
Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr
435 440 445
Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
450 455 460
Leu His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
465 470 475 480
Ser Pro Gly
<210> 17
<211> 486
<212> PRT
<213> Artifical sequence
<400> 17
Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asp
1 5 10 15
Ser Leu Arg Val Ser Cys Ala Ala Ser Gly Arg Asp Phe Ser Asp Tyr
20 25 30
Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val
35 40 45
Ala Ala Ile Ser Arg Asn Gly Leu Thr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Met Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Val Asn Ser Ala Gly Thr Tyr Val Ser Pro Arg Ser Arg Glu Tyr
100 105 110
Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Asp Lys Thr
115 120 125
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
130 135 140
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Gln Leu Met Ile Ser Arg
145 150 155 160
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
165 170 175
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
180 185 190
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
195 200 205
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
210 215 220
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Ile Glu Lys Thr Ile
225 230 235 240
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
245 250 255
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
260 265 270
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
275 280 285
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
290 295 300
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
305 310 315 320
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala Leu
325 330 335
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly
340 345 350
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gln Val Gln Leu Val
355 360 365
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Thr Leu Ser
370 375 380
Cys Ala Ala Ser Gly Gly Thr Phe Ser Ser Tyr Ala Met Gly Trp Phe
385 390 395 400
Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val Ala Ala Ile Ser Gly
405 410 415
Arg Ser Ser Ile Arg Asn Tyr Asp Asp Ser Val Lys Gly Arg Phe Ala
420 425 430
Ile Ser Arg Asp Ser Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser
435 440 445
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Asp Thr Val
450 455 460
Phe Arg Ser Phe Val Val Gly Asn Val Lys Glu Trp Gly Gln Gly Thr
465 470 475 480
Gln Val Thr Val Ser Ser
485
<210> 18
<211> 486
<212> PRT
<213> Artifical sequence
<400> 18
Gln Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asp
1 5 10 15
Ser Leu Arg Val Ser Cys Ala Ala Ser Gly Arg Asp Phe Ser Asp Tyr
20 25 30
Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val
35 40 45
Ala Ala Ile Ser Arg Asn Gly Leu Thr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Met Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Val Asn Ser Ala Gly Thr Tyr Val Ser Pro Arg Ser Arg Glu Tyr
100 105 110
Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Asp Lys Thr
115 120 125
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser
130 135 140
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Gln Leu Met Ile Ser Arg
145 150 155 160
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
165 170 175
Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
180 185 190
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val
195 200 205
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
210 215 220
Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Ile Glu Lys Thr Ile
225 230 235 240
Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
245 250 255
Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
260 265 270
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
275 280 285
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
290 295 300
Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg
305 310 315 320
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala Leu
325 330 335
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly
340 345 350
Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gln Val Gln Leu Val
355 360 365
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Thr Leu Ser
370 375 380
Cys Ala Ala Ser Gly Gly Thr Phe Ser Ser Tyr Ala Met Gly Trp Phe
385 390 395 400
Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val Ala Ala Ile Ser Gly
405 410 415
Arg Ser Ser Ile Arg Asn Tyr Asp Asp Ser Val Lys Gly Arg Phe Ala
420 425 430
Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu Gln Met Asn Ser
435 440 445
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Asp Thr Val
450 455 460
Phe Arg Ser Phe Val Val Gly Asn Val Lys Glu Trp Gly Gln Gly Thr
465 470 475 480
Leu Val Thr Val Ser Ser
485
<210> 19
<211> 486
<212> PRT
<213> Artifical sequence
<400> 19
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 Ala Ala Ser Gly Gly Thr Phe Ser Ser Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val
35 40 45
Ala Ala Ile Ser Gly Arg Ser Ser Ile Arg Asn Tyr Asp Asp Ser Val
50 55 60
Lys Gly Arg Phe Ala Ile Ser Arg Asp Ser Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Asp Thr Val Phe Arg Ser Phe Val Val Gly Asn Val Lys Glu
100 105 110
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Asp Lys Thr His Thr
115 120 125
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
130 135 140
Leu Phe Pro Pro Lys Pro Lys Asp Gln Leu Met Ile Ser Arg Thr Pro
145 150 155 160
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
165 170 175
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
180 185 190
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
195 200 205
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
210 215 220
Lys Val Ser Asn Lys Ala Leu Pro Ala Ile Glu Lys Thr Ile Ser Lys
225 230 235 240
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
245 250 255
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
260 265 270
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
275 280 285
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
290 295 300
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
305 310 315 320
Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His Asn
325 330 335
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Ser
340 345 350
Gly Gly Gly Gly Ser Gly Gly Gly Ser Gln Val Lys Leu Glu Glu Ser
355 360 365
Gly Gly Gly Leu Val Gln Pro Gly Asp Ser Leu Arg Val Ser Cys Ala
370 375 380
Ala Ser Gly Arg Asp Phe Ser Asp Tyr Val Met Gly Trp Phe Arg Gln
385 390 395 400
Ala Pro Gly Lys Gly Leu Glu Phe Val Ala Ala Ile Ser Arg Asn Gly
405 410 415
Leu Thr Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser
420 425 430
Arg Asp Asn Asp Lys Asn Met Leu Tyr Leu Gln Met Asn Ser Leu Arg
435 440 445
Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Val Asn Ser Ala Gly Thr
450 455 460
Tyr Val Ser Pro Arg Ser Arg Glu Tyr Asp Tyr Trp Gly Gln Gly Thr
465 470 475 480
Gln Val Thr Val Ser Ser
485
<210> 20
<211> 486
<212> PRT
<213> Artifical sequence
<400> 20
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 Ala Ala Ser Gly Gly Thr Phe Ser Ser Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val
35 40 45
Ala Ala Ile Ser Gly Arg Ser Ser Ile Arg Asn Tyr Asp Asp Ser Val
50 55 60
Lys Gly Arg Phe Ala Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Asp Thr Val Phe Arg Ser Phe Val Val Gly Asn Val Lys Glu
100 105 110
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Asp Lys Thr His Thr
115 120 125
Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe
130 135 140
Leu Phe Pro Pro Lys Pro Lys Asp Gln Leu Met Ile Ser Arg Thr Pro
145 150 155 160
Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
165 170 175
Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr
180 185 190
Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val
195 200 205
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys
210 215 220
Lys Val Ser Asn Lys Ala Leu Pro Ala Ile Glu Lys Thr Ile Ser Lys
225 230 235 240
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser
245 250 255
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys
260 265 270
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
275 280 285
Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
290 295 300
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln
305 310 315 320
Gln Gly Asn Val Phe Ser Cys Ser Val Leu His Glu Ala Leu His Asn
325 330 335
His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Gly Gly Gly Ser
340 345 350
Gly Gly Gly Gly Ser Gly Gly Gly Ser Gln Val Lys Leu Val Glu Ser
355 360 365
Gly Gly Gly Leu Val Gln Pro Gly Asp Ser Leu Arg Val Ser Cys Ala
370 375 380
Ala Ser Gly Arg Asp Phe Ser Asp Tyr Val Met Gly Trp Phe Arg Gln
385 390 395 400
Ala Pro Gly Lys Gly Leu Glu Phe Val Ala Ala Ile Ser Arg Asn Gly
405 410 415
Leu Thr Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser
420 425 430
Arg Asp Asn Ser Lys Asn Met Leu Tyr Leu Gln Met Asn Ser Leu Arg
435 440 445
Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Val Asn Ser Ala Gly Thr
450 455 460
Tyr Val Ser Pro Arg Ser Arg Glu Tyr Asp Tyr Trp Gly Gln Gly Thr
465 470 475 480
Leu Val Thr Val Ser Ser
485
<210> 21
<211> 467
<212> PRT
<213> Artifical sequence
<400> 21
Gln Val Lys Leu Glu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asp
1 5 10 15
Ser Leu Arg Val Ser Cys Ala Ala Ser Gly Arg Asp Phe Ser Asp Tyr
20 25 30
Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val
35 40 45
Ala Ala Ile Ser Arg Asn Gly Leu Thr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Met Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Val Asn Ser Ala Gly Thr Tyr Val Ser Pro Arg Ser Arg Glu Tyr
100 105 110
Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Thr Lys Gly
115 120 125
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
130 135 140
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
145 150 155 160
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
165 170 175
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
180 185 190
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
195 200 205
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys
210 215 220
Ser Cys Asp Lys Thr His His His His His His Gln Val Gln Leu Val
225 230 235 240
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Thr Leu Ser
245 250 255
Cys Ala Ala Ser Gly Gly Thr Phe Ser Ser Tyr Ala Met Gly Trp Phe
260 265 270
Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val Ala Ala Ile Ser Gly
275 280 285
Arg Ser Ser Ile Arg Asn Tyr Asp Asp Ser Val Lys Gly Arg Phe Ala
290 295 300
Ile Ser Arg Asp Ser Ala Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser
305 310 315 320
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Asp Thr Val
325 330 335
Phe Arg Ser Phe Val Val Asn Val Lys Glu Trp Gly Gln Gly Thr Gln
340 345 350
Val Thr Val Ser Ser Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val
355 360 365
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser
370 375 380
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln
385 390 395 400
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val
405 410 415
Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu
420 425 430
Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu
435 440 445
Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg
450 455 460
Gly Glu Cys
465
<210> 22
<211> 468
<212> PRT
<213> Artifical sequence
<400> 22
Gln Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Asp
1 5 10 15
Ser Leu Arg Val Ser Cys Ala Ala Ser Gly Arg Asp Phe Ser Asp Tyr
20 25 30
Val Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val
35 40 45
Ala Ala Ile Ser Arg Asn Gly Leu Thr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Met Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Val Asn Ser Ala Gly Thr Tyr Val Ser Pro Arg Ser Arg Glu Tyr
100 105 110
Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Lys Gly
115 120 125
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
130 135 140
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
145 150 155 160
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
165 170 175
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
180 185 190
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
195 200 205
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys
210 215 220
Ser Cys Asp Lys Thr His His His His His His Gln Val Gln Leu Val
225 230 235 240
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Thr Leu Ser
245 250 255
Cys Ala Ala Ser Gly Gly Thr Phe Ser Ser Tyr Ala Met Gly Trp Phe
260 265 270
Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val Ala Ala Ile Ser Gly
275 280 285
Arg Ser Ser Ile Arg Asn Tyr Asp Asp Ser Val Lys Gly Arg Phe Ala
290 295 300
Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr Leu Gln Met Asn Ser
305 310 315 320
Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Ala Asp Thr Val
325 330 335
Phe Arg Ser Phe Val Val Gly Asn Val Lys Glu Trp Gly Gln Gly Thr
340 345 350
Leu Val Thr Val Ser Ser Glu Ile Lys Arg Thr Val Ala Ala Pro Ser
355 360 365
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala
370 375 380
Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val
385 390 395 400
Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser
405 410 415
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr
420 425 430
Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys
435 440 445
Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn
450 455 460
Arg Gly Glu Cys
465
<210> 23
<211> 467
<212> PRT
<213> Artifical sequence
<400> 23
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 Ala Ala Ser Gly Gly Thr Phe Ser Ser Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val
35 40 45
Ala Ala Ile Ser Gly Arg Ser Ser Ile Arg Asn Tyr Asp Asp Ser Val
50 55 60
Lys Gly Arg Phe Ala Ile Ser Arg Asp Ser Ala Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Asp Thr Val Phe Arg Ser Phe Val Val Asn Val Lys Glu Trp
100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His His His His His His Gln Val Lys Leu Glu Glu Ser Gly
225 230 235 240
Gly Gly Leu Val Gln Pro Gly Asp Ser Leu Arg Val Ser Cys Ala Ala
245 250 255
Ser Gly Arg Asp Phe Ser Asp Tyr Val Met Gly Trp Phe Arg Gln Ala
260 265 270
Pro Gly Lys Gly Leu Glu Phe Val Ala Ala Ile Ser Arg Asn Gly Leu
275 280 285
Thr Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg
290 295 300
Asp Asn Asp Lys Asn Met Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala
305 310 315 320
Glu Asp Thr Ala Val Tyr Tyr Cys Ala Val Asn Ser Ala Gly Thr Tyr
325 330 335
Val Ser Pro Arg Ser Arg Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Gln
340 345 350
Val Thr Val Ser Ser Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val
355 360 365
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser
370 375 380
Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln
385 390 395 400
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val
405 410 415
Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu
420 425 430
Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu
435 440 445
Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg
450 455 460
Gly Glu Cys
465
<210> 24
<211> 468
<212> PRT
<213> Artifical sequence
<400> 24
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 Ala Ala Ser Gly Gly Thr Phe Ser Ser Tyr
20 25 30
Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Leu Glu Phe Val
35 40 45
Ala Ala Ile Ser Gly Arg Ser Ser Ile Arg Asn Tyr Asp Asp Ser Val
50 55 60
Lys Gly Arg Phe Ala Ile Ser Arg Asp Asn Ser Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Ala Asp Thr Val Phe Arg Ser Phe Val Val Gly Asn Val Lys Glu
100 105 110
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Lys Gly Pro Ser
115 120 125
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
130 135 140
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
145 150 155 160
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
195 200 205
Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys
210 215 220
Asp Lys Thr His His His His His His Gln Val Lys Leu Val Glu Ser
225 230 235 240
Gly Gly Gly Leu Val Gln Pro Gly Asp Ser Leu Arg Val Ser Cys Ala
245 250 255
Ala Ser Gly Arg Asp Phe Ser Asp Tyr Val Met Gly Trp Phe Arg Gln
260 265 270
Ala Pro Gly Lys Gly Leu Glu Phe Val Ala Ala Ile Ser Arg Asn Gly
275 280 285
Leu Thr Thr Arg Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser
290 295 300
Arg Asp Asn Ser Lys Asn Met Leu Tyr Leu Gln Met Asn Ser Leu Arg
305 310 315 320
Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Val Asn Ser Ala Gly Thr
325 330 335
Tyr Val Ser Pro Arg Ser Arg Glu Tyr Asp Tyr Trp Gly Gln Gly Thr
340 345 350
Leu Val Thr Val Ser Ser Glu Ile Lys Arg Thr Val Ala Ala Pro Ser
355 360 365
Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala
370 375 380
Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val
385 390 395 400
Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser
405 410 415
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr
420 425 430
Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys
435 440 445
Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn
450 455 460
Arg Gly Glu Cys
465
<210> 25
<211> 375
<212> DNA
<213> Llama
<400> 25
caggtaaagc tggaggagtc tgggggagga ttggtgcagg ctggggactc tctgagagtc 60
tcctgtgcag cctctggacg cgacttcagt gattatgtca tgggctggtt ccgccaggct 120
ccagggaagg agcgtgagtt tgtagcagct attagcagga atggtcttac gactcgctat 180
gcagactccg tgaagggccg atttaccatc tccagagaca atgacaaaaa catggtgtac 240
ctgcaaatga acagcctgaa acctgaggac acggccgttt attactgtgc agtaaattcg 300
gccgggacat acgttagtcc ccgctcgaga gagtatgact actggggcca ggggacccag 360
gtcaccgtct cctca 375
<210> 26
<211> 369
<212> DNA
<213> Llama
<400> 26
caggtgcagc tggtggagtc tgggggagga ttggtgcagg ctgggggctc tctgaccctc 60
tcctgtgcag cctctggagg caccttcagt agctatgcca tgggctggtt ccgccaggct 120
ccagggaagg agcgtgagtt tgtagcagct attagcgggc gtagttctat aagaaactat 180
gatgactccg tgaagggccg attcgccatc tccagagaca gcgccaagaa cacggtgtat 240
ctgcaaatga acagcctgaa acctgaggac acggccgttt attattgtgc agcagatacg 300
gtattccggt cgtttgttgt tggcaacgtt aaagaatggg gtcaggggac ccaggtcacc 360
gtctcctca 369
<210> 27
<211> 375
<212> DNA
<213> Artifical sequence
<400> 27
caggtcaagt tggaagagag tggaggcggc ctcgtccagc ctggcgatag tctccgggtc 60
agttgtgccg catccggacg cgactttagc gactacgtca tgggatggtt tagacaggcc 120
cccggaaagg gcttagagtt tgtcgccgca atcagtagga atggactgac aacacggtac 180
gccgattccg tgaagggacg gttcaccatt tcgagggata acgacaagaa tatgctctac 240
ctccagatga atagcctccg ggctgaggat acagcagtgt actactgcgc cgtcaattcc 300
gctggaacat acgtcagtcc tcgttcacgc gagtacgatt actggggcca gggaacccag 360
gtgaccgtgt cttct 375
<210> 28
<211> 375
<212> DNA
<213> Artifical sequence
<400> 28
caggtcaagt tggtcgagag tggaggcggc ctcgtccagc ctggcgatag tctccgggtc 60
agttgtgccg catccggacg cgactttagc gactacgtca tgggatggtt tagacaggcc 120
cccggaaagg gcttagagtt tgtcgccgca atcagtagga atggactgac aacacggtac 180
gccgattccg tgaagggacg gttcaccatt tcgagggata actccaagaa tatgctctac 240
ctccagatga atagcctccg ggctgaggat acagcagtgt actactgcgc cgtcaattcc 300
gctggaacat acgtcagtcc tcgttcacgc gagtacgatt actggggcca gggaaccttg 360
gtgaccgtgt cttct 375
<210> 29
<211> 375
<212> DNA
<213> Artifical sequence
<400> 29
gaggtccagt tggtcgagag tggaggcggc ctcgtccagc ctggcgatag tctccggctc 60
agttgtgccg catccggacg cgactttagc gactacgtca tgggatggtt tagacaggcc 120
cccggaaagg gcttagagtt tgtcgccgca atcagtagga atggactgac aacacggtac 180
gccgattccg tgaagggacg gttcaccatt tcgagggata actccaagaa tatgctctac 240
ctccagatga atagcctccg ggctgaggat acagcagtgt actactgcgc cgtcaattcc 300
gctggaacat acgtcagtcc tcgttcacgc gagtacgatt actggggcca gggaaccttg 360
gtgaccgtgt cttct 375
<210> 30
<211> 375
<212> DNA
<213> Artifical sequence
<400> 30
caggtccagt tggtcgagag tggaggcggc ctcgtccagc ctggcgatag tctccggctc 60
agttgtgccg catccggacg cgactttagc gactacgtca tgggatggtt tagacaggcc 120
cccggaaagg gcttagagtt tgtcgccgca atcagtagga atggactgac aacacggtac 180
gccgattccg tgaagggacg gttcaccatt tcgagggata actccaagaa tatgctctac 240
ctccagatga atagcctccg ggctgaggat acagcagtgt actactgcgc cgtcaattcc 300
gctggaacat acgtcagtcc tcgttcacgc gagtacgatt actggggcca gggaaccttg 360
gtgaccgtgt cttct 375
<210> 31
<211> 375
<212> DNA
<213> Artifical sequence
<400> 31
gaggtccagt tggtcgagag tggaggcggc ctcgtccagc ctggcgatag tctccgggtc 60
agttgtgccg catccggacg cgactttagc gactacgtca tgggatgggt tagacaggcc 120
cccggaaagg gcttagagtt tgtcgccgca atcagtagga atggactgac aacacggtac 180
gccgattccg tgaagggacg gttcaccatt tcgagggata actccaagaa tatgctctac 240
ctccagatga atagcctccg ggctgaggat acagcagtgt actactgcgc cgtcaattcc 300
gctggaacat acgtcagtcc tcgttcacgc gagtacgatt actggggcca gggaaccttg 360
gtgaccgtgt cttct 375
<210> 32
<211> 369
<212> DNA
<213> Artifical sequence
<400> 32
caggtgcagc tcgtcgagtc tggcggcgga ctggtgcagc ccggcggcag tctcactctc 60
agttgcgccg catccggcgg aacattttct tcttacgcta tgggctggtt tagacaggca 120
cctggaaagg gactggagtt tgtcgctgcc attagtggca gaagtagcat tcgcaactac 180
gatgatagcg tgaagggacg gttcgctatt agtagggata gcgctaagaa tactctgtac 240
ctccagatga actccctgcg cgcagaggat accgctgtct actactgcgc cgccgatacc 300
gtgttcagat cctttgtggt cggcaatgtg aaggagtggg gacagggaac tcaggtcact 360
gtgagttct 369
<210> 33
<211> 369
<212> DNA
<213> Artifical sequence
<400> 33
caggtgcagc tcgtcgagtc tggcggcgga ctggtgcagc ccggcggcag tctcactctc 60
agttgcgccg catccggcgg aacattttct tcttacgcta tgggctggtt tagacaggca 120
cctggaaagg gactggagtt tgtcgctgcc attagtggca gaagtagcat tcgcaactac 180
gatgatagcg tgaagggacg gttcgctatt agtagggata actctaagaa tactgtgtac 240
ctccagatga actccctgcg cgcagaggat accgctgtct actactgcgc cgccgatacc 300
gtgttcagat cctttgtggt cggcaatgtg aaggagtggg gacagggaac tctcgtcact 360
gtgagttct 369
<210> 34
<211> 369
<212> DNA
<213> Artifical sequence
<400> 34
gaggtgcagc tcgtcgagtc tggcggcgga ctggtgcagc ccggcggcag tctccgtctc 60
agttgcgccg catccggcgg aacattttct tcttacgcta tgggctggtt tagacaggca 120
cctggaaagg gactggagtt tgtcgctgcc attagtggca gaagtagcat tcgcaactac 180
gatgatagcg tgaagggacg gttcgctatt agtagggata actctaagaa tactctgtac 240
ctccagatga actccctgcg cgcagaggat accgctgtct actactgcgc cgccgatacc 300
gtgttcagat cctttgtggt cggcaatgtg aaggagtggg gacagggaac tctcgtcact 360
gtgagttct 369
<210> 35
<211> 369
<212> DNA
<213> Artifical sequence
<400> 35
caggtgcagc tcgtcgagtc tggcggcgga ctggtgcagc ccggcggcag tctccgtctc 60
agttgcgccg catccggcgg aacattttct tcttacgcta tgggctggtt tagacaggca 120
cctggaaagg gactggagtt tgtcgctgcc attagtggca gaagtagcat tcgcaactac 180
gatgatagcg tgaagggacg gttcgctatt agtagggata actctaagaa tactctgtac 240
ctccagatga actccctgcg cgcagaggat accgctgtct actactgcgc cgccgatacc 300
gtgttcagat cctttgtggt cggcaatgtg aaggagtggg gacagggaac tctcgtcact 360
gtgagttct 369
<210> 36
<211> 369
<212> DNA
<213> Artifical sequence
<400> 36
gaggtgcagc tcgtcgagtc tggcggcgga ctggtgcagc ccggcggcag tctccgtctc 60
agttgcgccg catccggcgg aacattttct tcttacgcta tgggctgggt tagacaggca 120
cctggaaagg gactggagtt tgtcgctgcc attagtggca gaagtagcat tcgcaactac 180
gatgatagcg tgaagggacg gttcgctatt agtagggata actctaagaa tactctgtac 240
ctccagatga actccctgcg cgcagaggat accgctgtct actactgcgc cgccgatacc 300
gtgttcagat cctttgtggt cggcaatgtg aaggagtggg gacagggaac tctcgtcact 360
gtgagttct 369
<210> 37
<211> 1515
<212> DNA
<213> Artifical sequence
<400> 37
atggagaccg acacactgct gctgtgggtc ctgctgctgt gggtgccagg ctccaccggc 60
caggtcaagt tggaagagag tggaggcggc ctcgtccagc ctggcgatag tctccgggtc 120
agttgtgccg catccggacg cgactttagc gactacgtca tgggatggtt tagacaggcc 180
cccggaaagg gcttagagtt tgtcgccgca atcagtagga atggactgac aacacggtac 240
gccgattccg tgaagggacg gttcaccatt tcgagggata acgacaagaa tatgctctac 300
ctccagatga atagcctccg ggctgaggat acagcagtgt actactgcgc cgtcaattcc 360
gctggaacat acgtcagtcc tcgttcacgc gagtacgatt actggggcca gggaacccag 420
gtgaccgtgt cttctggcgg aggcggaagt ggcggaggcg gatctcaggt gcagctcgtc 480
gagtctggcg gcggactggt gcagcccggc ggcagtctca ctctcagttg cgccgcatcc 540
ggcggaacat tttcttctta cgctatgggc tggtttagac aggcacctgg aaagggactg 600
gagtttgtcg ctgccattag tggcagaagt agcattcgca actacgatga tagcgtgaag 660
ggacggttcg ctattagtag ggatagcgct aagaatactc tgtacctcca gatgaactcc 720
ctgcgcgcag aggataccgc tgtctactac tgcgccgccg ataccgtgtt cagatccttt 780
gtggtcggca atgtgaagga gtggggacag ggaactcagg tcactgtgag ttctgataag 840
acccacactt gtcctccttg ccccgctcct gagctgctcg gcggcccatc tgtgtttctg 900
tttccaccaa agccaaagga tcagctcatg attagtagaa cacccgaggt gacatgcgtc 960
gtggttgatg tgagccacga agatcccgag gtcaagttta attggtacgt tgatggcgtg 1020
gaggtgcaca acgcaaagac caagccacgc gaggagcagt acaatagcac ttaccgggtg 1080
gtgagcgtgc tgaccgtgct gcaccaggat tggctcaatg gaaaggagta caagtgtaaa 1140
gtctctaata aggctctgcc cgcacctatt gaaaaaacta ttagtaaggc taagggccag 1200
cctagagagc cccaggtcta cacactgcca ccatctcgcg aggagatgac caagaatcag 1260
gtgtccctga catgtctcgt caagggcttt taccctagcg atattgccgt cgagtgggag 1320
agcaacggac agcctgagaa taattacaag acaaccccac ctgtgctcga ttccgacggc 1380
agcttcttcc tgtactctaa gctcacagtc gataagtcca gatggcagca gggcaatgtg 1440
ttttcttgta gtgtgctgca cgaggcactc cacaatcact acacacagaa gtccctgtcc 1500
ctcagtcccg gctaa 1515
<210> 38
<211> 1515
<212> DNA
<213> Artifical sequence
<400> 38
atggagaccg acacactgct gctgtgggtc ctgctgctgt gggtgccagg ctccaccggc 60
caggtcaagt tggtcgagag tggaggcggc ctcgtccagc ctggcgatag tctccgggtc 120
agttgtgccg catccggacg cgactttagc gactacgtca tgggatggtt tagacaggcc 180
cccggaaagg gcttagagtt tgtcgccgca atcagtagga atggactgac aacacggtac 240
gccgattccg tgaagggacg gttcaccatt tcgagggata actccaagaa tatgctctac 300
ctccagatga atagcctccg ggctgaggat acagcagtgt actactgcgc cgtcaattcc 360
gctggaacat acgtcagtcc tcgttcacgc gagtacgatt actggggcca gggaaccttg 420
gtgaccgtgt cttctggcgg aggcggaagt ggcggaggcg gatctcaggt gcagctcgtc 480
gagtctggcg gcggactggt gcagcccggc ggcagtctca ctctcagttg cgccgcatcc 540
ggcggaacat tttcttctta cgctatgggc tggtttagac aggcacctgg aaagggactg 600
gagtttgtcg ctgccattag tggcagaagt agcattcgca actacgatga tagcgtgaag 660
ggacggttcg ctattagtag ggataactct aagaatactg tgtacctcca gatgaactcc 720
ctgcgcgcag aggataccgc tgtctactac tgcgccgccg ataccgtgtt cagatccttt 780
gtggtcggca atgtgaagga gtggggacag ggaactctcg tcactgtgag ttctgataag 840
acccacactt gtcctccttg ccccgctcct gagctgctcg gcggcccatc tgtgtttctg 900
tttccaccaa agccaaagga tcagctcatg attagtagaa cacccgaggt gacatgcgtc 960
gtggttgatg tgagccacga agatcccgag gtcaagttta attggtacgt tgatggcgtg 1020
gaggtgcaca acgcaaagac caagccacgc gaggagcagt acaatagcac ttaccgggtg 1080
gtgagcgtgc tgaccgtgct gcaccaggat tggctcaatg gaaaggagta caagtgtaaa 1140
gtctctaata aggctctgcc cgcacctatt gaaaaaacta ttagtaaggc taagggccag 1200
cctagagagc cccaggtcta cacactgcca ccatctcgcg aggagatgac caagaatcag 1260
gtgtccctga catgtctcgt caagggcttt taccctagcg atattgccgt cgagtgggag 1320
agcaacggac agcctgagaa taattacaag acaaccccac ctgtgctcga ttccgacggc 1380
agcttcttcc tgtactctaa gctcacagtc gataagtcca gatggcagca gggcaatgtg 1440
ttttcttgta gtgtgctgca cgaggcactc cacaatcact acacacagaa gtccctgtcc 1500
ctcagtcccg gctaa 1515
<210> 39
<211> 1515
<212> DNA
<213> Artifical sequence
<400> 39
atggagaccg acacactgct gctgtgggtc ctgctgctgt gggtgccagg ctccaccggc 60
caggtgcagc tcgtcgagtc tggcggcgga ctggtgcagc ccggcggcag tctcactctc 120
agttgcgccg catccggcgg aacattttct tcttacgcta tgggctggtt tagacaggca 180
cctggaaagg gactggagtt tgtcgctgcc attagtggca gaagtagcat tcgcaactac 240
gatgatagcg tgaagggacg gttcgctatt agtagggata gcgctaagaa tactctgtac 300
ctccagatga actccctgcg cgcagaggat accgctgtct actactgcgc cgccgatacc 360
gtgttcagat cctttgtggt cggcaatgtg aaggagtggg gacagggaac tcaggtcact 420
gtgagttctg gcggaggcgg aagtggcgga ggcggatctc aggtcaagtt ggaagagagt 480
ggaggcggcc tcgtccagcc tggcgatagt ctccgggtca gttgtgccgc atccggacgc 540
gactttagcg actacgtcat gggatggttt agacaggccc ccggaaaggg cttagagttt 600
gtcgccgcaa tcagtaggaa tggactgaca acacggtacg ccgattccgt gaagggacgg 660
ttcaccattt cgagggataa cgacaagaat atgctctacc tccagatgaa tagcctccgg 720
gctgaggata cagcagtgta ctactgcgcc gtcaattccg ctggaacata cgtcagtcct 780
cgttcacgcg agtacgatta ctggggccag ggaacccagg tgaccgtgtc ttctgataag 840
acccacactt gtcctccttg ccccgctcct gagctgctcg gcggcccatc tgtgtttctg 900
tttccaccaa agccaaagga tcagctcatg attagtagaa cacccgaggt gacatgcgtc 960
gtggttgatg tgagccacga agatcccgag gtcaagttta attggtacgt tgatggcgtg 1020
gaggtgcaca acgcaaagac caagccacgc gaggagcagt acaatagcac ttaccgggtg 1080
gtgagcgtgc tgaccgtgct gcaccaggat tggctcaatg gaaaggagta caagtgtaaa 1140
gtctctaata aggctctgcc cgcacctatt gaaaaaacta ttagtaaggc taagggccag 1200
cctagagagc cccaggtcta cacactgcca ccatctcgcg aggagatgac caagaatcag 1260
gtgtccctga catgtctcgt caagggcttt taccctagcg atattgccgt cgagtgggag 1320
agcaacggac agcctgagaa taattacaag acaaccccac ctgtgctcga ttccgacggc 1380
agcttcttcc tgtactctaa gctcacagtc gataagtcca gatggcagca gggcaatgtg 1440
ttttcttgta gtgtgctgca cgaggcactc cacaatcact acacacagaa gtccctgtcc 1500
ctcagtcccg gctaa 1515
<210> 40
<211> 1515
<212> DNA
<213> Artifical sequence
<400> 40
atggagaccg acacactgct gctgtgggtc ctgctgctgt gggtgccagg ctccaccggc 60
caggtgcagc tcgtcgagtc tggcggcgga ctggtgcagc ccggcggcag tctcactctc 120
agttgcgccg catccggcgg aacattttct tcttacgcta tgggctggtt tagacaggca 180
cctggaaagg gactggagtt tgtcgctgcc attagtggca gaagtagcat tcgcaactac 240
gatgatagcg tgaagggacg gttcgctatt agtagggata actctaagaa tactgtgtac 300
ctccagatga actccctgcg cgcagaggat accgctgtct actactgcgc cgccgatacc 360
gtgttcagat cctttgtggt cggcaatgtg aaggagtggg gacagggaac tctcgtcact 420
gtgagttctg gcggaggcgg aagtggcgga ggcggatctc aggtcaagtt ggtcgagagt 480
ggaggcggcc tcgtccagcc tggcgatagt ctccgggtca gttgtgccgc atccggacgc 540
gactttagcg actacgtcat gggatggttt agacaggccc ccggaaaggg cttagagttt 600
gtcgccgcaa tcagtaggaa tggactgaca acacggtacg ccgattccgt gaagggacgg 660
ttcaccattt cgagggataa ctccaagaat atgctctacc tccagatgaa tagcctccgg 720
gctgaggata cagcagtgta ctactgcgcc gtcaattccg ctggaacata cgtcagtcct 780
cgttcacgcg agtacgatta ctggggccag ggaaccttgg tgaccgtgtc ttctgataag 840
acccacactt gtcctccttg ccccgctcct gagctgctcg gcggcccatc tgtgtttctg 900
tttccaccaa agccaaagga tcagctcatg attagtagaa cacccgaggt gacatgcgtc 960
gtggttgatg tgagccacga agatcccgag gtcaagttta attggtacgt tgatggcgtg 1020
gaggtgcaca acgcaaagac caagccacgc gaggagcagt acaatagcac ttaccgggtg 1080
gtgagcgtgc tgaccgtgct gcaccaggat tggctcaatg gaaaggagta caagtgtaaa 1140
gtctctaata aggctctgcc cgcacctatt gaaaaaacta ttagtaaggc taagggccag 1200
cctagagagc cccaggtcta cacactgcca ccatctcgcg aggagatgac caagaatcag 1260
gtgtccctga catgtctcgt caagggcttt taccctagcg atattgccgt cgagtgggag 1320
agcaacggac agcctgagaa taattacaag acaaccccac ctgtgctcga ttccgacggc 1380
agcttcttcc tgtactctaa gctcacagtc gataagtcca gatggcagca gggcaatgtg 1440
ttttcttgta gtgtgctgca cgaggcactc cacaatcact acacacagaa gtccctgtcc 1500
ctcagtcccg gctaa 1515
<210> 41
<211> 1515
<212> DNA
<213> Artifical sequence
<400> 41
atggagaccg acacactgct gctgtgggtc ctgctgctgt gggtgccagg ctccaccggc 60
caggtcaagt tggaagagag tggaggcggc ctcgtccagc ctggcgatag tctccgggtc 120
agttgtgccg catccggacg cgactttagc gactacgtca tgggatggtt tagacaggcc 180
cccggaaagg gcttagagtt tgtcgccgca atcagtagga atggactgac aacacggtac 240
gccgattccg tgaagggacg gttcaccatt tcgagggata acgacaagaa tatgctctac 300
ctccagatga atagcctccg ggctgaggat acagcagtgt actactgcgc cgtcaattcc 360
gctggaacat acgtcagtcc tcgttcacgc gagtacgatt actggggcca gggaacccag 420
gtgaccgtgt cttctggcgg aggcggaagt ggcggaggcg gatctcaggt gcagctcgtc 480
gagtctggcg gcggactggt gcagcccggc ggcagtctca ctctcagttg cgccgcatcc 540
ggcggaacat tttcttctta cgctatgggc tggtttagac aggcacctgg aaagggactg 600
gagtttgtcg ctgccattag tggcagaagt agcattcgca actacgatga tagcgtgaag 660
ggacggttcg ctattagtag ggatagcgct aagaatactc tgtacctcca gatgaactcc 720
ctgcgcgcag aggataccgc tgtctactac tgcgccgccg ataccgtgtt cagatccttt 780
gtggtcggca atgtgaagga gtggggacag ggaactcagg tcactgtgag ttctgataag 840
acccacactt gtcctccttg ccccgctcct gagctgctcg gcggcccatc tgtgtttctg 900
tttccaccaa agccaaagga tcagctcatg attagtagaa cacccgaggt gacatgcgtc 960
gtggttgatg tgagccacga agatcccgag gtcaagttta attggtacgt tgatggcgtg 1020
gaggtgcaca acgcaaagac caagccacgc gaggagcagt acaatagcac ttaccgggtg 1080
gtgagcgtgc tgaccgtgct gcaccaggat tggctcaatg gaaaggagta caagtgtaaa 1140
gtctctaata aggctctgcc cgcacctatt gaaaaaacta ttagtaaggc taagggccag 1200
cctagagagc cccaggtcta cacactgcca ccatctcgcg aggagatgac caagaatcag 1260
gtgtccctga catgtctcgt caagggcttt taccctagcg atattgccgt cgagtgggag 1320
agcaacggac agcctgagaa taattacaag acaaccccac ctgtgctcga ttccgacggc 1380
agcttcttcc tgtactctaa gctcacagtc gataagtcca gatggcagca gggcaatgtg 1440
ttttcttgta gtgtgctgca cgaggcactc cacaatcact acacacagaa gtccctgtcc 1500
ctcagtcccg gctaa 1515
<210> 42
<211> 1527
<212> DNA
<213> Artifical sequence
<400> 42
atggagaccg acacactgct gctgtgggtc ctgctgctgt gggtgccagg ctccaccggc 60
caggtcaagt tggtcgagag tggaggcggc ctcgtccagc ctggcgatag tctccgggtc 120
agttgtgccg catccggacg cgactttagc gactacgtca tgggatggtt tagacaggcc 180
cccggaaagg gcttagagtt tgtcgccgca atcagtagga atggactgac aacacggtac 240
gccgattccg tgaagggacg gttcaccatt tcgagggata actccaagaa tatgctctac 300
ctccagatga atagcctccg ggctgaggat acagcagtgt actactgcgc cgtcaattcc 360
gctggaacat acgtcagtcc tcgttcacgc gagtacgatt actggggcca gggaaccttg 420
gtgaccgtgt cttctgataa gacccacact tgtcctcctt gccccgctcc tgagctgctc 480
ggcggcccat ctgtgtttct gtttccacca aagccaaagg atcagctcat gattagtaga 540
acacccgagg tgacatgcgt cgtggttgat gtgagccacg aagatcccga ggtcaagttt 600
aattggtacg ttgatggcgt ggaggtgcac aacgcaaaga ccaagccacg cgaggagcag 660
tacaatagca cttaccgggt ggtgagcgtg ctgaccgtgc tgcaccagga ttggctcaat 720
ggaaaggagt acaagtgtaa agtctctaat aaggctctgc ccgcacctat tgaaaaaact 780
attagtaagg ctaagggcca gcctagagag ccccaggtct acacactgcc accatctcgc 840
gaggagatga ccaagaatca ggtgtccctg acatgtctcg tcaagggctt ttaccctagc 900
gatattgccg tcgagtggga gagcaacgga cagcctgaga ataattacaa gacaacccca 960
cctgtgctcg attccgacgg cagcttcttc ctgtactcta agctcacagt cgataagtcc 1020
agatggcagc agggcaatgt gttttcttgt agtgtgctgc acgaggcact ccacaatcac 1080
tacacacaga agtccctgtc cctcagtccc ggaggcggcg gaagtggcgg aggcggaagt 1140
ggcggaggcg gatctcaggt gcagctcgtc gagtctggcg gcggactggt gcagcccggc 1200
ggcagtctca ctctcagttg cgccgcatcc ggcggaacat tttcttctta cgctatgggc 1260
tggtttagac aggcacctgg aaagggactg gagtttgtcg ctgccattag tggcagaagt 1320
agcattcgca actacgatga tagcgtgaag ggacggttcg ctattagtag ggataactct 1380
aagaatactg tgtacctcca gatgaactcc ctgcgcgcag aggataccgc tgtctactac 1440
tgcgccgccg ataccgtgtt cagatccttt gtggtcggca atgtgaagga gtggggacag 1500
ggaactctcg tcactgtgag ttcttaa 1527
<210> 43
<211> 1527
<212> DNA
<213> Artifical sequence
<400> 43
atggagaccg acacactgct gctgtgggtc ctgctgctgt gggtgccagg ctccaccggc 60
caggtgcagc tcgtcgagtc tggcggcgga ctggtgcagc ccggcggcag tctcactctc 120
agttgcgccg catccggcgg aacattttct tcttacgcta tgggctggtt tagacaggca 180
cctggaaagg gactggagtt tgtcgctgcc attagtggca gaagtagcat tcgcaactac 240
gatgatagcg tgaagggacg gttcgctatt agtagggata gcgctaagaa tactctgtac 300
ctccagatga actccctgcg cgcagaggat accgctgtct actactgcgc cgccgatacc 360
gtgttcagat cctttgtggt cggcaatgtg aaggagtggg gacagggaac tcaggtcact 420
gtgagttctg ataagaccca cacttgtcct ccttgccccg ctcctgagct gctcggcggc 480
ccatctgtgt ttctgtttcc accaaagcca aaggatcagc tcatgattag tagaacaccc 540
gaggtgacat gcgtcgtggt tgatgtgagc cacgaagatc ccgaggtcaa gtttaattgg 600
tacgttgatg gcgtggaggt gcacaacgca aagaccaagc cacgcgagga gcagtacaat 660
agcacttacc gggtggtgag cgtgctgacc gtgctgcacc aggattggct caatggaaag 720
gagtacaagt gtaaagtctc taataaggct ctgcccgcac ctattgaaaa aactattagt 780
aaggctaagg gccagcctag agagccccag gtctacacac tgccaccatc tcgcgaggag 840
atgaccaaga atcaggtgtc cctgacatgt ctcgtcaagg gcttttaccc tagcgatatt 900
gccgtcgagt gggagagcaa cggacagcct gagaataatt acaagacaac cccacctgtg 960
ctcgattccg acggcagctt cttcctgtac tctaagctca cagtcgataa gtccagatgg 1020
cagcagggca atgtgttttc ttgtagtgtg ctgcacgagg cactccacaa tcactacaca 1080
cagaagtccc tgtccctcag tcccggaggc ggcggaagtg gcggaggcgg aagtggcgga 1140
ggcggatctc aggtcaagtt ggaagagagt ggaggcggcc tcgtccagcc tggcgatagt 1200
ctccgggtca gttgtgccgc atccggacgc gactttagcg actacgtcat gggatggttt 1260
agacaggccc ccggaaaggg cttagagttt gtcgccgcaa tcagtaggaa tggactgaca 1320
acacggtacg ccgattccgt gaagggacgg ttcaccattt cgagggataa cgacaagaat 1380
atgctctacc tccagatgaa tagcctccgg gctgaggata cagcagtgta ctactgcgcc 1440
gtcaattccg ctggaacata cgtcagtcct cgttcacgcg agtacgatta ctggggccag 1500
ggaacccagg tgaccgtgtc ttcttaa 1527
<210> 44
<211> 1527
<212> DNA
<213> Artifical sequence
<400> 44
atggagaccg acacactgct gctgtgggtc ctgctgctgt gggtgccagg ctccaccggc 60
caggtgcagc tcgtcgagtc tggcggcgga ctggtgcagc ccggcggcag tctcactctc 120
agttgcgccg catccggcgg aacattttct tcttacgcta tgggctggtt tagacaggca 180
cctggaaagg gactggagtt tgtcgctgcc attagtggca gaagtagcat tcgcaactac 240
gatgatagcg tgaagggacg gttcgctatt agtagggata actctaagaa tactgtgtac 300
ctccagatga actccctgcg cgcagaggat accgctgtct actactgcgc cgccgatacc 360
gtgttcagat cctttgtggt cggcaatgtg aaggagtggg gacagggaac tctcgtcact 420
gtgagttctg ataagaccca cacttgtcct ccttgccccg ctcctgagct gctcggcggc 480
ccatctgtgt ttctgtttcc accaaagcca aaggatcagc tcatgattag tagaacaccc 540
gaggtgacat gcgtcgtggt tgatgtgagc cacgaagatc ccgaggtcaa gtttaattgg 600
tacgttgatg gcgtggaggt gcacaacgca aagaccaagc cacgcgagga gcagtacaat 660
agcacttacc gggtggtgag cgtgctgacc gtgctgcacc aggattggct caatggaaag 720
gagtacaagt gtaaagtctc taataaggct ctgcccgcac ctattgaaaa aactattagt 780
aaggctaagg gccagcctag agagccccag gtctacacac tgccaccatc tcgcgaggag 840
atgaccaaga atcaggtgtc cctgacatgt ctcgtcaagg gcttttaccc tagcgatatt 900
gccgtcgagt gggagagcaa cggacagcct gagaataatt acaagacaac cccacctgtg 960
ctcgattccg acggcagctt cttcctgtac tctaagctca cagtcgataa gtccagatgg 1020
cagcagggca atgtgttttc ttgtagtgtg ctgcacgagg cactccacaa tcactacaca 1080
cagaagtccc tgtccctcag tcccggaggc ggcggaagtg gcggaggcgg aagtggcgga 1140
ggcggatctc aggtcaagtt ggtcgagagt ggaggcggcc tcgtccagcc tggcgatagt 1200
ctccgggtca gttgtgccgc atccggacgc gactttagcg actacgtcat gggatggttt 1260
agacaggccc ccggaaaggg cttagagttt gtcgccgcaa tcagtaggaa tggactgaca 1320
acacggtacg ccgattccgt gaagggacgg ttcaccattt cgagggataa ctccaagaat 1380
atgctctacc tccagatgaa tagcctccgg gctgaggata cagcagtgta ctactgcgcc 1440
gtcaattccg ctggaacata cgtcagtcct cgttcacgcg agtacgatta ctggggccag 1500
ggaaccttgg tgaccgtgtc ttcttaa 1527
<210> 45
<211> 1524
<212> DNA
<213> Artifical sequence
<400> 45
atggagaccg acacactgct gctgtgggtc ctgctgctgt gggtgccagg ctccaccggc 60
caggtcaagt tggaagagag tggaggcggc ctcgtccagc ctggcgatag tctccgggtc 120
agttgtgccg catccggacg cgactttagc gactacgtca tgggatggtt tagacaggcc 180
cccggaaagg gcttagagtt tgtcgccgca atcagtagga atggactgac aacacggtac 240
gccgattccg tgaagggacg gttcaccatt tcgagggata acgacaagaa tatgctctac 300
ctccagatga atagcctccg ggctgaggat acagcagtgt actactgcgc cgtcaattcc 360
gctggaacat acgtcagtcc tcgttcacgc gagtacgatt actggggcca gggaacccag 420
gtgaccgtgt ccagcaccaa gggccccagc gtcttcccac tggctccttc ctctaaaagc 480
actagcggag ggaccgcagc cctgggctgt ctggtgaaag actacttccc cgagcccgtg 540
accgtctcct ggaactctgg agccctgacc tccggggtgc acacctttcc cgccgtgctg 600
cagtcttctg gactgtactc cctgtcctcc gtcgtgactg tgcccagctc ctccctggga 660
actcagacat acatctgcaa cgtgaaccac aagccttcca acacaaaggt ggacaagaga 720
gtcgagccca agagctgtga taagacccat catcatcatc atcatatgga gaccgacacc 780
ctcctcctgt gggtgctgct gctgtgggtg cccggctcta ccggccaggt gcagctcgtc 840
gagtctggcg gcggactggt gcagcccggc ggcagtctca ctctcagttg cgccgcatcc 900
ggcggaacat tttcttctta cgctatgggc tggtttagac aggcacctgg aaagggactg 960
gagtttgtcg ctgccattag tggcagaagt agcattcgca actacgatga tagcgtgaag 1020
ggacggttcg ctattagtag ggatagcgct aagaatactc tgtacctcca gatgaactcc 1080
ctgcgcgcag aggataccgc tgtctactac tgcgccgccg ataccgtgtt cagatccttt 1140
gtggtcggca atgtgaagga gtggggacag ggaactcagg tcactgtgag ttctgagatc 1200
aagcggaccg tggccgcccc atccgtgttc attttcccac cttccgacga gcagctgaag 1260
tctggcaccg ccagcgtggt gtgcctgctg aacaacttct acccccgcga ggccaaggtg 1320
cagtggaagg tggacaacgc cctgcagagc ggcaacagcc aggagagcgt gaccgagcag 1380
gactccaagg acagcaccta cagcctgagc agcaccctga ccctgagcaa ggccgactac 1440
gagaagcaca aggtgtacgc ctgcgaggtg acccaccagg gactgtctag ccccgtgacc 1500
aagagcttca accggggcga gtgc 1524
<210> 46
<211> 1524
<212> DNA
<213> Artifical sequence
<400> 46
atggagaccg acacactgct gctgtgggtc ctgctgctgt gggtgccagg ctccaccggc 60
caggtcaagt tggtcgagag tggaggcggc ctcgtccagc ctggcgatag tctccgggtc 120
agttgtgccg catccggacg cgactttagc gactacgtca tgggatggtt tagacaggcc 180
cccggaaagg gcttagagtt tgtcgccgca atcagtagga atggactgac aacacggtac 240
gccgattccg tgaagggacg gttcaccatt tcgagggata actccaagaa tatgctctac 300
ctccagatga atagcctccg ggctgaggat acagcagtgt actactgcgc cgtcaattcc 360
gctggaacat acgtcagtcc tcgttcacgc gagtacgatt actggggcca gggaaccttg 420
gtgaccgtgt cttctaccaa gggccccagc gtcttcccac tggctccttc ctctaaaagc 480
actagcggag ggaccgcagc cctgggctgt ctggtgaaag actacttccc cgagcccgtg 540
accgtctcct ggaactctgg agccctgacc tccggggtgc acacctttcc cgccgtgctg 600
cagtcttctg gactgtactc cctgtcctcc gtcgtgactg tgcccagctc ctccctggga 660
actcagacat acatctgcaa cgtgaaccac aagccttcca acacaaaggt ggacaagaga 720
gtcgagccca agagctgtga taagacccat catcatcatc atcatatgga gaccgacacc 780
ctcctcctgt gggtgctgct gctgtgggtg cccggctcta ccggccaggt gcagctcgtc 840
gagtctggcg gcggactggt gcagcccggc ggcagtctca ctctcagttg cgccgcatcc 900
ggcggaacat tttcttctta cgctatgggc tggtttagac aggcacctgg aaagggactg 960
gagtttgtcg ctgccattag tggcagaagt agcattcgca actacgatga tagcgtgaag 1020
ggacggttcg ctattagtag ggataactct aagaatactg tgtacctcca gatgaactcc 1080
ctgcgcgcag aggataccgc tgtctactac tgcgccgccg ataccgtgtt cagatccttt 1140
gtggtcggca atgtgaagga gtggggacag ggaactctcg tcactgtgag ttctgagatc 1200
aagcggaccg tggccgcccc atccgtgttc attttcccac cttccgacga gcagctgaag 1260
tctggcaccg ccagcgtggt gtgcctgctg aacaacttct acccccgcga ggccaaggtg 1320
cagtggaagg tggacaacgc cctgcagagc ggcaacagcc aggagagcgt gaccgagcag 1380
gactccaagg acagcaccta cagcctgagc agcaccctga ccctgagcaa ggccgactac 1440
gagaagcaca aggtgtacgc ctgcgaggtg acccaccagg gactgtctag ccccgtgacc 1500
aagagcttca accggggcga gtgc 1524
<210> 47
<211> 1524
<212> DNA
<213> Artifical sequence
<400> 47
atggagaccg acacactgct gctgtgggtc ctgctgctgt gggtgccagg ctccaccggc 60
caggtgcagc tcgtcgagtc tggcggcgga ctggtgcagc ccggcggcag tctcactctc 120
agttgcgccg catccggcgg aacattttct tcttacgcta tgggctggtt tagacaggca 180
cctggaaagg gactggagtt tgtcgctgcc attagtggca gaagtagcat tcgcaactac 240
gatgatagcg tgaagggacg gttcgctatt agtagggata gcgctaagaa tactctgtac 300
ctccagatga actccctgcg cgcagaggat accgctgtct actactgcgc cgccgatacc 360
gtgttcagat cctttgtggt cggcaatgtg aaggagtggg gacagggaac tcaggtcact 420
gtgagttcta ccaagggccc cagcgtcttc ccactggctc cttcctctaa aagcactagc 480
ggagggaccg cagccctggg ctgtctggtg aaagactact tccccgagcc cgtgaccgtc 540
tcctggaact ctggagccct gacctccggg gtgcacacct ttcccgccgt gctgcagtct 600
tctggactgt actccctgtc ctccgtcgtg actgtgccca gctcctccct gggaactcag 660
acatacatct gcaacgtgaa ccacaagcct tccaacacaa aggtggacaa gagagtcgag 720
cccaagagct gtgataagac ccatcatcat catcatcata tggagaccga caccctcctc 780
ctgtgggtgc tgctgctgtg ggtgcccggc tctaccggcc aggtcaagtt ggaagagagt 840
ggaggcggcc tcgtccagcc tggcgatagt ctccgggtca gttgtgccgc atccggacgc 900
gactttagcg actacgtcat gggatggttt agacaggccc ccggaaaggg cttagagttt 960
gtcgccgcaa tcagtaggaa tggactgaca acacggtacg ccgattccgt gaagggacgg 1020
ttcaccattt cgagggataa cgacaagaat atgctctacc tccagatgaa tagcctccgg 1080
gctgaggata cagcagtgta ctactgcgcc gtcaattccg ctggaacata cgtcagtcct 1140
cgttcacgcg agtacgatta ctggggccag ggaacccagg tgaccgtgtc cagcgagatc 1200
aagcggaccg tggccgcccc atccgtgttc attttcccac cttccgacga gcagctgaag 1260
tctggcaccg ccagcgtggt gtgcctgctg aacaacttct acccccgcga ggccaaggtg 1320
cagtggaagg tggacaacgc cctgcagagc ggcaacagcc aggagagcgt gaccgagcag 1380
gactccaagg acagcaccta cagcctgagc agcaccctga ccctgagcaa ggccgactac 1440
gagaagcaca aggtgtacgc ctgcgaggtg acccaccagg gactgtctag ccccgtgacc 1500
aagagcttca accggggcga gtgc 1524
<210> 48
<211> 1524
<212> DNA
<213> Artifical sequence
<400> 48
atggagaccg acacactgct gctgtgggtc ctgctgctgt gggtgccagg ctccaccggc 60
caggtgcagc tcgtcgagtc tggcggcgga ctggtgcagc ccggcggcag tctcactctc 120
agttgcgccg catccggcgg aacattttct tcttacgcta tgggctggtt tagacaggca 180
cctggaaagg gactggagtt tgtcgctgcc attagtggca gaagtagcat tcgcaactac 240
gatgatagcg tgaagggacg gttcgctatt agtagggata actctaagaa tactgtgtac 300
ctccagatga actccctgcg cgcagaggat accgctgtct actactgcgc cgccgatacc 360
gtgttcagat cctttgtggt cggcaatgtg aaggagtggg gacagggaac tctcgtcact 420
gtgagttcta ccaagggccc cagcgtcttc ccactggctc cttcctctaa aagcactagc 480
ggagggaccg cagccctggg ctgtctggtg aaagactact tccccgagcc cgtgaccgtc 540
tcctggaact ctggagccct gacctccggg gtgcacacct ttcccgccgt gctgcagtct 600
tctggactgt actccctgtc ctccgtcgtg actgtgccca gctcctccct gggaactcag 660
acatacatct gcaacgtgaa ccacaagcct tccaacacaa aggtggacaa gagagtcgag 720
cccaagagct gtgataagac ccatcatcat catcatcata tggagaccga caccctcctc 780
ctgtgggtgc tgctgctgtg ggtgcccggc tctaccggcc aggtcaagtt ggtcgagagt 840
ggaggcggcc tcgtccagcc tggcgatagt ctccgggtca gttgtgccgc atccggacgc 900
gactttagcg actacgtcat gggatggttt agacaggccc ccggaaaggg cttagagttt 960
gtcgccgcaa tcagtaggaa tggactgaca acacggtacg ccgattccgt gaagggacgg 1020
ttcaccattt cgagggataa ctccaagaat atgctctacc tccagatgaa tagcctccgg 1080
gctgaggata cagcagtgta ctactgcgcc gtcaattccg ctggaacata cgtcagtcct 1140
cgttcacgcg agtacgatta ctggggccag ggaaccttgg tgaccgtgtc ttctgagatc 1200
aagcggaccg tggccgcccc atccgtgttc attttcccac cttccgacga gcagctgaag 1260
tctggcaccg ccagcgtggt gtgcctgctg aacaacttct acccccgcga ggccaaggtg 1320
cagtggaagg tggacaacgc cctgcagagc ggcaacagcc aggagagcgt gaccgagcag 1380
gactccaagg acagcaccta cagcctgagc agcaccctga ccctgagcaa ggccgactac 1440
gagaagcaca aggtgtacgc ctgcgaggtg acccaccagg gactgtctag ccccgtgacc 1500
aagagcttca accggggcga gtgc 1524

Claims (12)

1.抗EGFR人源化的单域抗体，其特征在于，所述单域抗体的氨基酸序列为SEQ ID NO.3所示。
2.权利要求1所述的单域抗体的编码基因，其特征在于，所述编码基因的核苷酸序列为SEQ ID NO.27所示。
3.由权利要求1的抗EGFR人源化的单域抗体以及另一种抗EGFR人源化的单域抗体与Fc蛋白融合得到的Fc融合蛋白，其特征在于，所述的Fc融合蛋白的氨基酸序列选自SEQ IDNO.13、SEQ ID NO.15、SEQ ID NO.17或SEQ ID NO.19中所示的任何一种氨基酸序列。
4.权利要求3所述的融合蛋白的编码基因，其特征在于，所述编码基因的核苷酸序列选自SEQ ID NO.37、SEQ ID NO.39、SEQ ID NO.41、或SEQ ID NO.43 所示的任何一种多核苷酸序列。
5.权利要求1的抗EGFR人源化的单域抗体与人IgG-CH1和人CLκ构建得到的重链Fab蛋白，其特征在于，所述重链Fab蛋白的氨基酸序列选自SEQ ID NO.21或SEQ ID NO.23所示的任何一种氨基酸序列。
6.权利要求5所述的重链Fab蛋白的编码基因，其特征在于，所述编码基因的核苷酸序列为SEQ ID NO.45或SEQ ID NO.47所示的任何一种多核苷酸序列。
7.含有权利要求2所述单域抗体的编码基因、权利要求4所述的Fc融合蛋白的编码基因或权利要求6所述重链Fab蛋白的编码基因的表达载体。
8.按照权利要求7所述的表达载体，其特征在于，所述表达载体为原核细胞表达载体或真核细胞表达载体。
9.权利要求1的抗EGFR人源化的单域抗体、权利要求3的Fc融合蛋白或权利要求5所述的重链Fab蛋白在制备检测EGFR的试剂或药物或制备治疗EGFR表达异常相关疾病药物中的应用。
10.权利要求2所述的单域抗体的编码基因、权利要求4所述的Fc融合蛋白的编码基因或权利要求6所述的重链Fab蛋白的编码基因在制备检测EGFR的试剂或制备治疗EGFR表达异常相关疾病药物中的应用。
11.一种检测EGFR的试剂，其特征在于，所述试剂以权利要求1所述的抗EGFR人源化的单域抗体、权利要求要求3所述的Fc融合蛋白或权利要求5所述的重链Fab蛋白为活性成分制备而成；其中，所述的抗EGFR人源化的单域抗体、所述的Fc融合蛋白或所述的重链Fab蛋白与细胞毒性剂、酶相、放射性同位素或化学发光化合物中的一种或多种相连。
12.一种治疗EGFR表达异常相关疾病的药物，其特征在于，所述药物以权利要求1所述的抗EGFR人源化的单域抗体、权利要求要求3所述的Fc融合蛋白或权利要求5所述的重链Fab蛋白为活性成分制备而成；其中，所述的抗EGFR人源化的单域抗体、所述的Fc融合蛋白或所述的重链Fab蛋白与细胞毒性剂、酶相、放射性同位素或化学发光化合物中的一种或多种相连。
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