CN109071630B - 作为糖苷合酶的Endo-S2突变体、制备方法以及用于糖蛋白的糖工程化的用途 - Google Patents
作为糖苷合酶的Endo-S2突变体、制备方法以及用于糖蛋白的糖工程化的用途 Download PDFInfo
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Abstract
本发明提供了重组Endo‑S2突变体(被命名为Endo‑S2糖苷合酶)，其表现出降低的水解活性和增加的转糖基活性以用于合成糖蛋白，其中将所期望的糖链添加到岩藻糖基化或非岩藻糖基化的GlcNAc‑IgG受体。因而，本发明允许合成和重塑治疗性抗体，从而提供某些生物活性，如延长的体内半衰期、更小的免疫原性、增强的体内活性、提高的靶向能力、和/或递送治疗剂的能力。
Description
政府在本发明中的权利
本发明是在政府支持下在由国家卫生研究院(National Institutes of Health)授予的基金号R01 GM096973和R01 GM080374下作出的。政府享有本发明的某些权利。
相关申请的交叉引用
本申请要求2016年1月15日提交的共同未决的美国临时申请号62/279,087的优先权，该申请的内容在此通过引用并入本文用于所有目的。
技术领域
本发明涉及糖蛋白合成，并且更具体来说，涉及重组突变体Endo S2的用途，它是来自化脓性链球菌(Streptococcus pyogenes)的血清型M49的菌株NZ131的内切-β-N-乙酰氨基葡糖苷酶，具有转糖基活性和有限的水解活性，从而提供抗体的有效糖基化重塑。
背景技术
单克隆抗体(mAb)代表用于治疗癌症、炎症性病症、以及感染性疾病的一类主要的治疗性蛋白质[1-3]。令人信服的实验数据已经显示，糖基化对抗体的稳定性、生物功能、以及总体治疗功效可以具有深远的影响[4-7]。举例来说，Fc聚糖的核心岩藻糖基化可以显著降低抗体依赖性细胞毒性(ADCC)，并且具有低含量的核心岩藻糖基化的抗体已经在抗癌治疗中显示出提高的治疗功效[8,9]。另一方面，据报道，末端α-2,6-唾液酸化Fc糖型是静脉注射免疫球蛋白(IVIG)中的一种次要组分，负责IVIG的抗炎活性，如在动物模型中所证实[10-13]。然而，天然抗体和重组抗体通常作为异质糖型产生，所述异质糖型难以分离以进一步探测抗体的结构-活性关系。此外，对于市场上依靠ADCC作为治疗功效的主要机制的大部分抗癌mAb，最具活性的非岩藻糖基化糖型通常作为异质混合物中的少部分存在[8,9]。因此，可以引起产生结构明确、均质糖型的抗体的方法对于功能研究和开发更好的基于抗体的治疗剂来说是非常期望的。在尝试经由宿主糖基化途径工程化来控制糖基化[14-20]同时，一种化学酶促糖基化重塑方法已经作为获得均质抗体糖型的一种有前景的方法出现，所述方法包括完整抗体的内切糖苷酶催化的脱糖基化和后续的转糖基[21]。已显示，重组IgG-Fc结构域的Fc聚糖可以在适当的内切糖苷酶包括Endo-A和Endo-D催化下通过酶促脱糖基化-转糖基步骤来重塑，而不需要使蛋白质变性[22-24]。在2012年，出现了完整治疗性单克隆抗体和IVIG的糖基化重塑的第一个实例，这通过发现Endo-S的糖苷合酶突变体而得以实现，所述Endo-S是一种来自化脓性链球菌的内切糖苷酶[25]。在这种方法中，诸如利妥昔单抗(rituximab)的单克隆抗体的异质Fc聚糖由Endo S催化的脱糖基化去除以产生在糖基化位点处仅携带α-1,6-岩藻糖基化GlcNAc受体的抗体蛋白骨架。然后，通过Endo-S糖苷合酶突变体(Endo S-D233A或D233Q)以位点特异性和立体特异性方式将期望的N-聚糖转移到GlcNAc受体以重构均质糖型的抗体。Endo-S糖苷合酶突变体已经由几个研究小组用来合成抗体的不同均质糖型以用于结构和功能研究[26-31]。虽然Endo-S糖苷合酶能够转移双分支复杂型和修饰的Man3GlcNAc核心，但是这些突变体在转移高甘露糖型N-聚糖方面仅显示出很低的活性。最近，已经产生来自Endo-F3的糖苷合酶突变体，这是另一种GH18家族内切糖苷酶[32]。发现Endo-F3糖苷合酶如D165A突变体能够将三分支N-聚糖转移到完整抗体的Fc结构域，但是它们需要α1,6-岩藻糖基化GlcNAc部分作为转糖基的受体并且不能转移到非岩藻糖基化的GlcNAc受体。这些研究已经证实，迄今为止可获得的糖苷合酶仍然由于底物特异性而具有限制并且在转糖基效率方面也不同。
通过了解其他突变体的缺点，本文描述了扩大糖基化重塑策略的范围的努力，其中注意力转向来自血清型M49的化脓性链球菌的内切糖苷酶Endo-S2[33,34]。Endo-S2与来自相同细菌的Endo-S仅显示37％的序列同一性，并且在Fc脱糖基化中显示出比Endo-S更宽的聚糖底物特异性[34]。数据暗示与Endo-S相比，野生型Endo-S2可以水解来自抗体Fc结构域的各种不同类型的N-聚糖。但是，尚不清楚Endo-S2是否具有转糖基活性并且如果是的话，是否可以通过定点诱变产生Endo-S2糖苷合酶。
因此，将有利的是，评价是否可以从Endo-S2产生有效的糖苷合酶以及它们是否可能会在转糖基中具有与先前所报道的那些相比更宽的底物特异性。
发明内容
本发明提供了重组Endo-S2突变体(被命名为Endo-S2糖苷合酶)，其表现出降低的水解活性和增加的转糖基活性以用于合成糖蛋白，其中将所期望的糖链添加到岩藻糖基化或非岩藻糖基化的GlcNAc-IgG受体中。因而，本发明允许合成和重塑治疗性抗体，从而提供某些生物活性，如延长的体内半衰期、更小的免疫原性、增强的体内活性、提高的靶向能力、和/或递送治疗剂的能力。本发明的突变体Endo-S2糖苷合酶使得能够对治疗性抗体和其Fc片段进行糖基化重塑而增加不同聚糖如高甘露糖型和杂合型聚糖的数量，这是无法通过先前公开的Endo-S突变体实现的。
在一个方面，本发明提供了化脓性链球菌的血清型M49的菌株NZ131的内切-β-N-乙酰氨基葡糖苷酶(Endo-S2)(SEQ ID NO:1)的突变体的转糖基活性，其中所述突变体与其具有至少85％同源性并且对岩藻糖基化和非岩藻糖基化的GlcNAc-IgG受体都表现出转糖基活性，其中所述内切糖苷酶使得能够将低聚糖(呈活化的糖
唑啉的形式)整体转移到岩藻糖基化或非岩藻糖基化的GlcNAc-IgG以形成IgG的新的糖型。
在另一个方面，本发明提供了Endo-S2突变体蛋白，其相对于野生型Endo S2显示出显著提高的转糖基效率和减弱或消除的产物水解活性。突变体优选地包括位点特异性突变，包括Asp-184处的突变。所述突变体蛋白包括但不限于D184A(SEQ ID NO:2)、D184N(SEQID NO:3)、D184Q(SEQ ID NO:4)、D184R(SEQ ID NO:5)、D184C(SEQ ID NO:6)、D184M(SEQID NO:7)、D184E(SEQ ID NO:8)、D184G(SEQ ID NO:9)、D184H(SEQ ID NO:10)、D184I(SEQID NO:11)、D184L(SEQ ID NO:12)、D184K(SEQ ID NO:13)、D184F(SEQ ID NO:14)、D184P(SEQ ID NO:15)、D184S(SEQ ID NO:16)、D184T(SEQ ID NO.17)、D184W(SEQ ID NO:18)、D184Y(SEQ ID NO:19)、D184V(SEQ ID NO:20)或其片段，所述突变体蛋白包括催化结构域并且相对于野生型Endo-S2蛋白表现出增加的转糖基作用和降低的水解作用。优选地，所述突变体蛋白包括D184M(SEQ ID NO:7)、D184E(SEQ ID NO:8)以及D184Q(SEQ ID NO:4)。
值得注意的是，在D184位点处携带突变的任何Endo-S2片段和结构域都包括在本发明中，其中任何这样的结构域和片段可以与其他蛋白质融合，所述其他蛋白质包括但不限于CPD、Fc、MBP等。催化结构域和特异性突变位点对于酶活性是至关重要的，并且因此，酶上的其他位点可以被修饰或截短，如末端，而不会影响大部分的糖苷合酶活性。
在另一个方面，本发明提供了一种用于制备均质岩藻糖基化或非岩藻糖基化糖型的IgG抗体的化学酶促方法，所述方法包括：
a.提供选自以下的受体：核心岩藻糖基化的GlcNAc-IgG和非岩藻糖基化的GlcNAc-IgG或相应的IgG-Fc片段；以及
b.在化脓性链球菌Endo-S2 Asp-184突变体蛋白或其片段存在下使所述受体与包括活化低聚糖部分的供体底物反应，所述突变体蛋白或其片段包括催化结构域并且相对于野生型Endo-S2酶表现出增加的转糖基活性和降低的水解活性，以将所述活化的低聚糖部分转移到所述受体并且产生所述均质的岩藻糖基化或非岩藻糖基化的糖蛋白。
在又另一个方面，本发明提供了一种用于制备具有预定的低聚糖部分的核心岩藻糖基化IgG或IgG-Fc片段的方法，所述方法包括：
a.提供包含天冬酰胺连接的核心岩藻糖基化的N-乙酰葡糖胺(GlcNAc)残基的核心岩藻糖基化的受体蛋白；以及
b.在内切糖苷酶-S2 D184突变体蛋白或其片段存在下使所述核心岩藻糖基化的受体蛋白与活化的低聚糖供体发生酶促反应，所述突变体蛋白或其片段包括催化结构域并且相对于野生型Endo-S2酶表现出增加的转糖基活性和降低的水解活性，其中所述活化的低聚糖供体携带包含预定数量和类型的糖残基的低聚糖部分，其中所述低聚糖部分与所述受体蛋白共价连接，从而制备所述具有所述预定的低聚糖部分的核心岩藻糖基化的IgG或IgG-Fc片段。
在又另一个方面，本发明提供了一种活化的低聚糖部分，如聚糖或低聚糖
唑啉、糖基氟化物、糖基叠氮化物或芳基糖苷，其作为供体底物用于合成均质的核心岩藻糖基化的糖蛋白或非岩藻糖基化的糖蛋白。优选的是，所述活化的低聚糖部分是低聚糖
唑啉。
在另一个方面，本发明涉及一种用于制备均质的岩藻糖基化的糖蛋白或非岩藻糖基化的糖蛋白的化学酶促方法，所述方法包括：
a.提供选自以下的受体：核心岩藻糖基化或非岩藻糖基化的GlcNAc-IgG或IgG-Fc片段；以及
b.在化脓性链球菌Endo-S Asp-184突变体蛋白或其片段存在下使所述受体与供体底物反应，所述突变体蛋白或其片段包括催化结构域并且相对于野生型Endo-S2酶表现出增加的转糖基活性和降低的水解活性，其中所述供体底物包含具有限定数量和类型的糖残基和特定连接类型的预定低聚糖组分，从而提供所述均质的岩藻糖基化的糖蛋白或非岩藻糖基化的糖蛋白。
值得注意的是，化脓性链球菌Endo-S Asp-184突变体蛋白或其片段在氨基酸残基中可以包括另外的突变，但重要的是，携带D184突变。
在一个实施方式中，含有岩藻糖基化的GlcNAc的蛋白质是α-1-6-岩藻糖基-GlcNAc-蛋白。
在另一个方面，本发明涉及一种用具有预定的低聚糖组分的低聚糖进行IgG或IgG-Fc糖蛋白重塑的方法，所述预定的低聚糖组分具有限定数量和类型的糖残基并且具有特定的连接类型，所述方法包括：
a.提供包含Fc N-聚糖的岩藻糖基化的糖蛋白底物；
b.用内切酶处理所述岩藻糖基化的糖蛋白底物以水解所述N-聚糖中的两个核心GlcNAc残基之间的键以产生核心岩藻糖基化或非岩藻糖基化的GlcNAc-IgG或IgG-Fc片段；以及
c.在具有选自SEQ ID NO:2至SEQ ID NO:20的氨基酸序列的Endo-S2突变体或其片段存在下使所述低聚糖附接于所述Asn连接的GlcNAc部分，所述Endo-S2突变体或其片段包括催化结构域并且相对于野生型Endo-S2酶(SEQ ID NO:1)表现出增加的转糖基活性和降低的水解活性，从而添加预定的低聚糖组分。
在另一个方面，本发明涉及一种用具有预定的低聚糖组分的低聚糖进行岩藻糖基化或非岩藻糖基化的IgG或IgG-Fc片段重塑的方法，所述预定的低聚糖组分具有限定数量和类型的糖残基并且具有特定的连接类型，所述方法包括：
a.提供携带异质N-聚糖的从天然来源或重组来源中获得的岩藻糖基化或非岩藻糖基化的GlcNAc-IgG或IgG-Fc片段；
b.用内切酶(具有有效水解活性的野生型内切糖苷酶或突变型内切糖苷酶)处理所述天然或重组IgG或IgG-Fc片段以水解位于最靠近肽结构域处的两个GlcNAc残基之间的键，从而形成携带核心岩藻糖基化或非岩藻糖基化的GlcNAc-IgG或IgG-Fc片段的脱糖基化的蛋白质；以及
c.通过用化脓性链球菌Endo-S2Asp-184突变体酶或其片段进行转糖基来使所述预定的低聚糖与所述GlcNAc残基连接以重构天然β-1,4-糖苷键，所述突变体酶或其片段包括催化结构域并且相对于野生型Endo-S2酶表现出增加的转糖基活性和降低的水解活性，从而添加预定的低聚糖组分。
适用的低聚糖
唑啉包括但不限于高甘露糖型、杂合型、唾液酸聚糖
唑啉和复杂型N-聚糖以及它们的选择性修饰的衍生物，如具有特定标签的那些。优选地，利用二糖
唑啉、三糖
唑啉、四糖
唑啉、五糖
唑啉、六糖
唑啉、七糖
唑啉、八糖
唑啉、九糖
唑啉、十糖
唑啉、或十一糖
唑啉作为供体底物以用于高效化学酶促合成均质的核心岩藻糖基化或非岩藻糖基化的IgG抗体和IgG-Fc片段。
在又另一个方面，本发明涉及一种合成修饰的抗体或其片段的方法，所述方法包括：
a.使用携带Fc N-聚糖的天然存在的IgG抗体或重组抗体或Fc结构域作为前体；
b.使用内切糖苷酶，如野生型Endo-S2进行Fc脱糖基化以将所述Fc结构域脱糖基化以形成GlcNAc受体；其中所述GlcNAc部分位于所述抗体的Fc区域上并且所述GlcNAc部分是核心岩藻糖基化的或非岩藻糖基化的；以及
c.在选自Endo-S2突变体SEQ ID NO:2至SEQ ID NO:20或其片段的酶的催化下用具有预定数量的糖残基的低聚糖
唑啉或唾液酸聚糖
唑啉对所述抗体中的所述GlcNAc部分进行转糖基，所述酶包括催化结构域并且相对于野生型Endo-S2酶表现出增加的转糖基活性和降低的水解活性以形成具有预定数量的糖的修饰的抗体。
在又另一个方面，本发明提供了一种将表现出Fc唾液酸化糖型的静脉注射免疫球蛋白(IVIG)进行重塑的方法，所述方法包括：
a.提供携带Fc N-聚糖的IVIG；
b.使用包括Endo-S的内切糖苷酶将所述Fc N-聚糖进行Fc脱糖基化以形成GlcNAc受体；其中所述GlcNAc受体位于所述IVIG的Fc区域上并且所述GlcNAc受体是岩藻糖基化的或非岩藻糖基化的；以及
c.在选自Endo-S2突变体SEQ ID NO:2至SEQ ID NO:20或其片段的酶的催化下用具有预定数量的糖残基的唾液酸聚糖
唑啉对所述GlcNAc受体进行转糖基，所述酶包括催化结构域并且相对于野生型Endo-S2酶表现出增加的转糖基活性和降低的水解活性以形成唾液酸化的IVIG。
本发明的另一个方面提供了一种含有IVIG制备物的组合物，所述组合物包含至少90％的均质的唾液酸化的Fc糖型以增加抗炎活性，其中所述唾液酸化的Fc糖型是使用化脓性链球菌Endo-S2 Asp-184突变体与位于脱糖基化的IVIG的Fc区域上的GlcNAc部分和具有预定数量的糖残基的唾液酸聚糖
唑啉的组合而合成的。
在又另一个方面，本发明涉及一种合成均质的核心岩藻糖基化或非岩藻糖基化的IgG抗体或IgG-Fc片段的方法，所述方法包括：
a.提供天然或重组IgG抗体或IgG-Fc片段，其中所述重组IgG或IgG-Fc由典型的蛋白质表达系统产生，所述蛋白质表达系统包括但不限于酵母、昆虫、植物、以及任何哺乳动物表达系统；
b.通过选自Endo-H、Endo-A、Endo-S、Endo S2(WT)和/或Endo-F3的酶去除N-聚糖，以形成含有核心岩藻糖基化或非岩藻糖基化的GlcNAc的蛋白质；
c.提供具有在链中包含限定数量和类型的糖残基的所期望的低聚糖组分的糖
唑啉或唾液酸聚糖
唑啉；以及
d.使用选自化脓性链球菌Endo-S2Asp-184突变体酶或其片段的内切糖苷酶，用具有所期望的数量的糖残基的糖
唑啉或具有所期望的数量的糖残基和唾液酸残基的唾液酸聚糖
唑啉对含有岩藻糖基化或非岩藻糖基化的GlcNAc的蛋白质进行酶促转糖基，所述突变体酶或其片段相对于野生型Endo-S2酶表现出增加的转糖基活性和降低的水解活性，从而形成具有所期望的数量的糖残基和/或唾液酸的延伸结构的均质的核心岩藻糖基化或非岩藻糖基化的IgG抗体或IgG-Fc片段。
设想的是，具有预定低聚糖组分的低聚糖
唑啉或唾液酸聚糖
唑啉还可以包含另外的部分或标签，所述低聚糖组分具有限定数量和类型的糖残基，所述另外的部分或标签包括如用于治疗癌症、HIV或其他病毒的治疗剂或药物；激活细胞质膜上的受体的物质；影响细胞内化学的药剂；影响细胞物理学的药剂；基因；基因类似物；RNA；RNA类似物；DNA；DNA类似物；诸如CCR5或CD4的表面受体的氨基酸序列；对特定抗体具有亲和力的抗原结构；诸如gp120、gp41或gp160的受体配体的氨基酸序列；受体拮抗剂；受体阻断剂；酶；酶底物；酶抑制剂；酶调节剂；治疗性蛋白质；蛋白质类似物；代谢物；代谢物类似物；寡核苷酸；寡核苷酸类似物；抗原；抗原类似物；抗体或其片段；抗体类似物；对另外的受体具有反应性的与所述修饰的抗体不同的抗体；细菌；病毒；无机离子；金属离子；金属簇；聚合物；荧光化合物；以及其任何组合。
因而，本发明还提供了一种用于递送具有生物活性的药物或治疗剂以治疗病状的递送装置，所述递送装置包括：重塑的IgG或IgG-Fc片段，其具有预定的糖链或唾液酸聚糖、以及与末端糖残基或唾液酸附接的治疗剂或药物。
本发明设想了修饰与HIV相关的单克隆抗体，包括但不限于17b、48d、A32、C11、2G12、F240、IgG1b12、19e、X5、TNX-355以及F91，所有这些都是可商购获得的。
与癌症或其他疾病相关的其他抗体也可以被重塑以单独适于某些受体，从而增加生物活性，所述单克隆抗体可以包括但不限于西妥昔单抗(cetuximab)、利妥昔单抗、莫罗单抗(muromonab)-CD3、阿昔单抗(abciximab)、达利珠单抗(daclizumab)、巴利昔单抗(basiliximab)、帕利珠单抗(palivizumab)、英夫利昔单抗(infliximab)、曲妥珠单抗(trastuzumab)、吉妥珠单抗奥唑米星(gemtuzumab ozogamicin)、阿仑单抗(alemtuzumab)、替伊莫单抗(ibritumomab tiuxetan)、阿达木单抗(adalimumab)、奥马珠单抗(omalizumab)、托西莫单抗(tositumomab)、I-131托西莫单抗、依法利珠单抗(efalizumab)、贝伐单抗(bevacizumab)、帕尼单抗(panitumumab)、帕妥珠单抗(pertuzumab)、那他珠单抗(natalizumab)、依那西普(etanercept)、IGN101(Aphton公司)、伏洛昔单抗(volociximab)(百健艾迪公司(Biogen Idec)和PDL生物制药公司(PDLBioPharm))、抗CD80 mAb(百健艾迪公司)、抗CD23 mAb(百健艾迪公司)、CAT-3888(剑桥抗体技术公司(Cambridge Antibody Technology))、CDP-791(Imclone公司)、艾拉普妥珠单抗(eraptuzumab)(Immunomedics公司)、MDX-010(Medarex公司和BMS公司)、MDX-060(Medarex公司)、MDX-070(Medarex公司)、马妥珠单抗(matuzumab)(默克公司(Merck))、CP-675,206(辉瑞公司(Pfizer))、CAL(罗氏公司(Roche))、SGN-30(西雅图遗传学公司(Seattle Genetics))、扎木单抗(zanolimumab)(雪兰诺公司(Serono)和Genmab公司)、阿德木单抗(adecatumumab)(Sereno公司)、奥戈伏单抗(oregovomab)(联合医疗公司(UnitedTherapeutics))、尼妥珠单抗(nimotuzumab)(YM生物科技公司(YM Bioscience))、ABT-874(雅培实验室公司(Abbott Laboratories))、地诺单抗(denosumab)(安进公司(Amgen))、AM108(安进公司)、AMG 714(安进公司)、芳妥珠单抗(fontolizumab)(百健艾迪公司和PDL生物制药公司)、达利珠单抗(百健艾迪公司和PDL生物制药公司)、戈利木单抗(golimumab)(Centocor公司和先灵葆雅公司(Schering-Plough))、CNTO 1275(Centocor公司)、奥瑞珠单抗(ocrelizumab)(基因泰克公司(Genetech)和罗氏公司)、HuMax-CD20(Genmab公司)、贝利单抗(belimumab)(HGS公司和GSK公司)、依帕珠单抗(epratuzumab)(Immunomedics公司)、MLN1202(千年制药公司(Millennium Pharmaceuticals))、维西珠单抗(visilizumab)(PDL生物制药公司)、托珠单抗(tocilizumab)(罗氏公司)、奥克尔利珠单抗(ocrerlizumab)(罗氏公司)、聚乙二醇化赛妥珠单抗(certolizumab pegol)(UCB公司，以前的Celltech公司)、依库珠单抗(eculizumab)(亚力兄制药公司(AlexionPharmaceuticals))、培克珠单抗(pexelizumab)(亚力兄制药公司和宝洁公司(Procter&Gamble))、阿昔单抗(Centocor公司)、兰尼子木单抗(ranibizimumab)(基因泰克公司)、美泊利单抗(mepolizumab)(GSK公司)、TNX-355(Tanox公司)、或MYO-029(惠氏公司(Wyeth))。
本发明的再另一个方面涉及一种重塑最初包括异质糖链的抗体的方法，所述方法包括：
a.用内切糖苷酶从所述抗体中去除所述异质糖链以留下与原始糖基化位点连接的单个岩藻糖基化或非岩藻糖基化的GlcNAc部分；以及
b.通过内切糖苷酶催化的转糖基将具有至少一个标签的核心低聚糖或唾液酸聚糖
唑啉转移到所述岩藻糖基化或非岩藻糖基化的GlcNAc部分以产生标记抗体，其中所述内切糖苷酶选自Endo-S2突变体SEQ ID NO:2至SEQ ID NO:20或其片段，所述Endo-S2突变体或其片段包括催化结构域并且相对于野生型Endo-S2酶表现出增加的转糖基活性和降低的水解活性。
所述标签部分可以包括但不限于抗原、诸如用于癌症或HIV的治疗药物、毒素、荧光探针、生物素、PEG物质、脂质、或核苷酸。
在又另一个方面，本发明提供了抗体-药物结合物或ADC，其中所述抗体可以根据本发明进行修饰并且被设计成用于治疗癌症患者的靶向治疗。具体地，ADC含有两个部分：单克隆抗体和与所述抗体连接的少量的非常强效的细胞毒性药物。当ADC的抗体与靶细胞表面上的特定受体结合时，所述连接断裂并且ADC将致死毒素释放到所述细胞中。在本文随后描述具体的单克隆抗体和可能的细胞毒性药物。因此，本发明提供了一种修饰的抗体，其还包含另外的部分，所述部分包括用于治疗癌症的治疗剂，如趋化因子和/或细胞因子，从而形成抗体-药物结合物(ADC)。
在另一个方面，本发明提供了一种组合物，所述组合物包含选自以下的至少一种化脓性链球菌Endo-S2突变体：D184M(SEQ ID NO:7)和D184Q(SEQ ID No:4)。
在又另一个方面，本发明提供了一种具有预定低聚糖部分的核心岩藻糖基化或非岩藻糖基化的抗体或其Fc片段的基本上均质的制备物，其中所述基本上均质的制备物是通过上述方法中的任一种产生的。还提供了包含这样的均质制备物的组合物。
在另一个方面，本发明提供了一种治疗方法，所述治疗方法以足以在受治疗的受试者中调节生物活性的量使用具有所期望的糖基化状态和/或唾液酸化形式的重塑抗体。
在另一个方面，本发明提供了一种试剂盒，所述试剂盒包括至少一种化脓性链球菌Endo-S2突变体，所述突变体选自SEQ ID NO:2至20，并且优选地D184M(SEQ ID NO:7)、D184E(SEQ ID NO:8)以及D184Q(SEQ ID NO:4)。
根据随后的公开内容和权利要求书，本发明的其他方面、特征以及实施方式将更加完全地显而易见。
附图说明
图1示出了典型的IgG抗体和Fc N-聚糖的结构。a)显示功能区的人类IgG的α骨架结构(PDB代码1HZH)；b)与Fc结构域中的Asn-297相连的全长双分支复杂型N-聚糖的结构。
图2示出了Endo S2(SEQ ID NO:1)和Endo S(SEQ ID NO:21)的序列比对。标出了对于促进水解中
唑啉
离子形成来说关键的天冬氨酸残基(Endo-S的D233和Endo-S2的D184)以及催化性的一般酸/碱残基(Endo-S的E235和Endo-S2的E186)。
图3示出了用于将岩藻糖基化的利妥昔单抗糖基化重塑成均质的复杂、高甘露糖、以及杂合糖型的方案。
图4示出了利妥昔单抗的糖基化重塑的SDS-PAGE和ESI-MS分析。(A)SDS-PAGE分析：泳道1：市售的利妥昔单抗；泳道2：EndoS2脱糖基化的利妥昔单抗(1)；泳道3：来自EndoS2-D184Q催化的(1)与唾液酸复杂聚糖
唑啉(2)之间的反应的复杂糖型的转糖基产物(5)；泳道4：来自EndoS2-D184A催化的(1)和(3)的反应的Man9GlcNAc的转糖基产物(6)；泳道5：来自EndoS2-D184A催化的脱糖基化的利妥昔单抗(1)与杂合聚糖
唑啉(4)之间的反应的唾液酸杂合糖型的转糖基产物(7)；(B)市售的利妥昔单抗的轻链的ESI-MS(在解卷积之后)；(C)市售的利妥昔单抗的重链的ESI-MS(在解卷积之后)；(D)脱糖基化的利妥昔单抗(1)的ESI-MS。(E)转糖基复杂型产物(5)的ESI-MS。(F)转糖基Man9产物(6)的ESI-MS。(G)转糖基唾液酸杂合型产物(7)的ESI-MS。
图5示出了用于将利妥昔单抗酶促重塑成非岩藻糖基化的均质的复杂、高甘露糖以及杂合糖型的方案。
图6示出了将利妥昔单抗进行糖工程化成非岩藻糖基化的G2糖型的SDS-PAGE和ESI-MS分析。(A)SDS-PAGE分析：泳道1：市售的利妥昔单抗；泳道2：EndoS2脱糖基化的利妥昔单抗(1)；泳道3：脱岩藻糖基化产物(8)；泳道4：来自EndoS2-D184N催化反应的糖工程化的复杂糖型(9)；泳道5：来自EndoS2-D184N催化反应的糖工程化的Man9糖型(10)；泳道6：来自EndoS2-D184A催化反应的糖工程化的唾液酸杂合糖型(11)。(B)脱岩藻糖基化的利妥昔单抗(8)的重链的ESI-MS(在解卷积之后)。(C)糖工程化的复杂型利妥昔单抗(9)的重链的ESI-MS。(D)糖工程化的Man9利妥昔单抗(10)的重链的ESI-MS。(E)糖工程化的唾液酸杂合型利妥昔单抗(11)的重链的ESI-MS。
图7示出了EndoS2 D184的丙氨酸突变体、天冬酰胺突变体、谷氨酰胺突变体将复杂型聚糖
唑啉转糖基到岩藻糖基化的利妥昔单抗的活性的比较。
图8示出了使用利妥昔单抗作为底物，通过Endo-S2和它的突变体进行水解和转糖基的示意图。Fucα1,6GlcNAc-利妥昔单抗：在糖基化位点处携带核心岩藻糖基化的GlcNAc部分的脱糖基化的利妥昔单抗；SCT-利妥昔单抗：唾液酸复杂型糖型的利妥昔单抗。
图9示出了Endo-S2突变体对各种聚糖(HM、CT、以及杂合型)的底物特异性的评价。GlcNAc-利妥昔单抗：在Fc糖基化位点处仅携带第一GlcNAc部分的利妥昔单抗糖型；Fucα1,6GlcNAc-利妥昔单抗：在糖基化位点处携带核心岩藻糖基化的GlcNAc部分的脱糖基化的利妥昔单抗；SCT-利妥昔单抗：唾液酸复杂型糖型的利妥昔单抗；HM-利妥昔单抗：高甘露糖型糖型的利妥昔单抗；Hyb-利妥昔单抗：杂合型糖型的利妥昔单抗。
图10示出了使用Endo-S2 D184M对利妥昔单抗进行糖基化重塑的ESI-MS分析。A)市售利妥昔单抗的重链的ESI-MS(在解卷积之后)；B)Fucα1,6GlcNAc-利妥昔单抗(2)的重链的ESI-MS；C)和D)分别是转糖基产物(3)(SCT-利妥昔单抗)的重链和轻链的ESI-MS；E)和F)转糖基产物(8)(HM-利妥昔单抗)的重链和轻链的ESI-MS；G)和H)转糖基产物(6)(Hyb-利妥昔单抗)的重链和轻链的ESI-MS；I)和J)转糖基产物(11)(非岩藻糖基化的利妥昔单抗)的重链和轻链的ESI-MS。
图11示出了Endo-S2 D184M突变体对不同类型的聚糖的转糖基效率的比较。在Endo-S2 D184M(0.05mg/ml)的催化下，使用脱糖基化的利妥昔单抗(2)作为受体和不同类型的聚糖
唑啉作为供体底物进行转糖基反应。供体与受体的摩尔比是20:1。在此呈现的数据集代表两次独立实验。
图12示出了通过Endo-S2 D184Q、Endo-S2 D184M、以及Endo-S D233Q用SCT进行转糖基的比较。在0.05mg/ml的固定浓度的不同内切糖苷酶突变体的催化下，使用脱糖基化的利妥昔单抗(2)作为受体和SCT聚糖
唑啉(4)作为供体底物进行转糖基。供体与受体的摩尔比是20:1。在此呈现的数据集代表两次独立实验。
图13示出了使用一对Endo-S2酶(Endo-S2 WT和Endo-S2 D184M突变体)将赫赛汀(herceptin)(曲妥珠单抗)进行糖基化重塑并且A)市售曲妥珠单抗(赫赛汀)的重链的ESI-MS；B)脱糖基化的赫赛汀(12)的重链的ESI-MS；C)转糖基产物13(S2G2F-曲妥珠单抗)的重链的ESI-MS；D)转糖基产物(13)的轻链的ESI-MS；E)在PNG酶F催化的脱糖基化之后转糖基产物(13)的重链的ESI-MS。
图14示出了IVIG的Fc糖工程化以提高IVIG的抗炎活性。
具体实施方式
本发明提供了新型糖苷合酶EndoS2 Asp 184突变体，所述突变体显示出显著的转糖基效率，能够将复杂型、高甘露糖型、以及杂合型N-聚糖从活化的聚糖
唑啉转移到脱糖基化的完整抗体而没有产物水解。在本文已经发现，糖苷合酶EndoS2 Asp 184突变体有效作用于完整抗体的核心岩藻糖基化的和非岩藻糖基化的GlcNAc-Fc结构域以提供各种不同的限定的IgG糖型。如本文所述，Endo-S2确实具有强效的转糖基活性，并且系统定点诱变导致发现了几种糖苷合酶突变体，包括D184M和D184Q，所述糖苷合酶突变体显示出显著的转糖基活性而没有明显的产物水解活性。此外，在本文示出，Endo-S2糖苷合酶显示出显著放宽的底物特异性，从而能够转移三种主要类型(复杂型、高甘露糖型、以及杂合型)的N-聚糖以进行抗体糖基化重塑。此外，如本文进一步所述，已发现，Endo-S2糖苷合酶突变体对于转糖基一般具有比Endo-S突变体高得多的活性。描述了两种治疗性单克隆抗体利妥昔单抗和曲妥珠单抗(赫赛汀)的高效糖基化重塑。此外，将抗体和静脉注射免疫球蛋白转化成具有提高的抗炎活性的Fc完全唾液酸化的糖型。再此外，本发明提供了一种均质的非岩藻糖基化的糖型，其具有提高的ADCC活性和增强的FcγIIIa受体结合活性；以及叠氮基标记的糖型，其可以被进一步转化成其他糖型。
除非另外指明，否则本发明的实施将利用免疫学、分子生物学、微生物学、细胞生物学以及重组DNA的常规技术，这些技术在本领域的技术范围内。参见例如Sambrook等,MOLECULAR CLONING:A LABORATORY MANUAL(《分子克隆：实验室手册》),第2版(1989)；CURRENT PROTOCOLS IN MOLECULAR BIOLOGY(《最新分子生物学方案》)(F.M.Ausubel等编著(1987))；METHODS IN ENZYMOLOGY(《酶学方法》)丛书(学术出版公司(Academic Press,Inc.))：PCR 2:A PRACTICAL APPROACH(《PCR 2：实用方法》)(M.J.MacPherson,B.D.Hames以及G.R.Taylor编著(1995))；Harlow和Lane编著,(1988)ANTIBODIES,A LABORATORYMANUAL(《抗体：实验室手册》)；以及ANIMAL CELL CULTURE(《动物细胞培养》)(R.I.Freshney编著,(1987))。
应当了解的是，本文所述的本发明的方面包括“由……组成”和/或“基本上由……组成”方面。
定义
如本文说明书中所用，不带具体数量的指称可以意指一个或多个。如本文在一项或多项权利要求中所用，当结合词语“包含”使用时，不带具体数量的指称可以意指一个或多个。如本文所用的“另外的”可以意指至少第二个或更多。
如本文所用的“生物活性”指的是药效动力学特性和药代动力学特性，包括例如分子亲和性或所产生的生物化学或生理效应、受体亲和性或所产生的生物化学或生理效应、非受体亲和性或生物化学或生理效应、功效、生物利用度、吸收、分布、代谢、或消除。
如本文所用的“糖”指的是氧化或未氧化的含有碳水化合物的分子，包括但不限于单糖、二糖、三糖、低聚糖、或多糖，包括例如L-异构体或D-异构体的N-乙酰葡糖胺、甘露糖、半乳糖、N-乙酰神经氨酸(唾液酸)、葡萄糖、果糖、岩藻糖、山梨糖、鼠李糖、甘露庚酮糖、N-乙酰半乳糖胺、二羟基丙酮、木糖、木酮糖、阿拉伯糖、甘油醛、蔗糖、乳糖、麦芽糖、海藻糖、纤维二糖或其任何组合。糖还指天然、重组、合成和/或半合成产生的这样的分子。
如本文所用的“均质的”指的是核心岩藻糖基化的糖蛋白或非岩藻糖基化的糖蛋白，其中低聚糖组分包含至少75％，更优选地至少90％，并且最优选地至少95％的相同数量和类型的糖残基。
如本文所用的“蛋白质”或“糖蛋白”与术语肽和糖肽是可互换的。
如本文所用的“同源性”指的是在两个多肽之间具有显著同一性或相似性并且与参比多肽具有至少85％并且更优选地至少95％相似性的氨基酸序列。对于多肽，为获得序列之间的上述同源性百分比而进行比较的长度一般将是至少25个氨基酸，可选地至少50个氨基酸，更可能至少100个氨基酸，并且最可能200个氨基酸或更多个氨基酸。基本上同一或同源的多肽包括添加、截短、内部缺失或插入、保守取代和非保守取代、或其他修饰，它们位于氨基酸序列中不会破坏内切糖苷酶的功能的位置处。本领域技术人员将认识到众多可以被修饰或被其他化学上相似的残基取代而基本上不会改变活性的氨基酸。
如本文所用的“调节”指的是当比较本发明的糖基化工程抗体与非糖基化工程抗体时，如上文所定义的“生物活性”的提高或降低。
如本文所用的“免疫球蛋白分子”或“抗体”指的是含有特异性结合抗原的抗原结合位点或结合细胞受体的Fc区域的分子。在结构上，最简单的天然存在的抗体(例如IgG)包含四条多肽链，即两条重(H)链和两条轻(L)链，它们由二硫键相互连接。天然的免疫球蛋白代表一个大的分子家族，包括几种类型的分子，如IgD、IgG、IgA、IgM、以及IgE。所述术语还涵盖杂合抗体、或变更的抗体、以及其片段，包括但不限于Fab片段和Fc片段。
抗体可以使用如本文所述的常规技术片段化并且按照与对于全抗体所述的方式相同的方式筛选所述片段以加以利用。免疫球蛋白分子的Fab片段是多聚体蛋白质，其由免疫球蛋白分子的一部分组成，该部分含有共价偶联在一起并且能够与抗原特异性结合的免疫球蛋白重链和免疫球蛋白轻链的免疫活性部分。Fab片段和Fc片段可以通过使用本领域公知的方法，使用木瓜蛋白酶对基本上完整的免疫球蛋白分子进行蛋白水解消化来制备。然而，Fab片段或Fc片段也可以通过使用本领域已知的方法使免疫球蛋白重链和免疫球蛋白轻链的所需部分在合适的宿主细胞中表达来制备。
如本文所用，对于抗体，“基本上纯的”意指与在它的天然状态下伴随它的那些污染物或在获得抗体的过程中产生或使用的那些污染物分离的。该术语还包括具有单一糖基化状态的所期望的产物，无论该状态是在单个位点处还是在多个位点处包括糖基化。通常，当抗体占制备物中抗体的至少60重量％时，所述抗体是基本上纯的。举例来说，按重量计，制备物中的抗体中有至少约75％，在某些实施方式中至少约80％，在某些实施方式中至少约85％，在某些实施方式中至少约90％，在某些实施方式中至少约95％，并且最优选地至少约99％的所期望的抗体。基本上纯的抗体包括天然、重组、或合成产生的抗体。
如本文所用的“治疗有效量”指的是引起疾病或病状的症状的改善或减轻的量。
可用于作为标签与本发明的修饰的岩藻糖基化或非岩藻糖基化的糖蛋白并且更优选地抗体或其片段连接的抗原可以是外来抗原、内源抗原、其片段、或具有相同的功能活性的变体。
如本文所用的“内源抗原”指的是受体动物细胞或组织中天然存在的蛋白质或其部分，如细胞蛋白、免疫调节剂、或治疗剂。
如本文所用的“外来抗原”指的是蛋白质或其片段，它对于受体动物细胞或组织是外来的，包括但不限于病毒蛋白、寄生物蛋白、免疫调节剂、或治疗剂。
外来抗原可以是源自于病毒病原体和寄生物病原体的蛋白质、其一个或多个抗原片段。
可替选地，外来抗原可以由合成基因编码并且可以使用常规的重组DNA方法构建；所述合成基因可以表达源自于病毒病原体和寄生物病原体的抗原或其部分。这些病原体在人类、家畜或野生动物宿主中可以具有感染性。
外来抗原可以是由任何病毒病原体或寄生物病原体在进入它们的动物宿主中、在它们的动物宿主中定殖或复制之前或期间所表达的任何分子。
作为病毒抗原的来源的病毒病原体包括但不限于正粘病毒，如流感病毒(分类ID：59771)；逆转录病毒，如RSV、HTLV-1(分类ID：39015)以及HTLV-II(分类ID：11909)；疱疹病毒，如EBV(分类ID：10295)、CMV(分类ID：10358)或单纯疱疹病毒(ATCC编号：VR-1487)；慢病毒，如HIV-1(分类ID：12721)和HIV-2(分类ID：11709)；弹状病毒，如狂犬病毒；小核糖核酸病毒，如脊髓灰质炎病毒(分类ID：12080)；痘病毒，如牛痘(分类ID：10245)；轮状病毒(分类ID：10912)；以及细小病毒，如腺相关病毒1(分类ID：85106)。
病毒抗原的实例包括但不限于人类免疫缺陷病毒抗原Nef(国家过敏和传染病研究所(National Institute of Allergy and Infectious Disease)HIV储存库目录号：183；基因库登录号：AF238278)、Gag、Env(国家过敏和传染病研究所HIV储存库目录号：2433；基因库登录号：U39362)、Tat(国家过敏和传染病研究所HIV储存库目录号：827；基因库登录号：M13137)、Rev(国家过敏和传染病研究所HIV储存库目录号：2088；基因库登录号：L14572)、Pol(国家过敏和传染病研究所HIV储存库目录号：238；基因库登录号：AJ237568)以及gp120的T细胞和B细胞表位；乙型肝炎表面抗原(基因库登录号：AF043578)；轮状病毒抗原，如VP4(基因库登录号：AJ293721)和VP7(基因库登录号：AY003871)；流感病毒抗原，如血凝素(基因库登录号：AJ404627)；核蛋白(基因库登录号：AJ289872)；以及单纯疱疹病毒抗原，如胸苷激酶(基因库登录号：AB047378)。
作为细菌抗原的来源的细菌病原体包括但不限于分枝杆菌属菌种(Mycobacterium spp.)、幽门螺杆菌(Helicobacter pylori)、沙门氏菌属菌种(Salmonella spp.)、志贺氏菌属菌种(Shigella spp.)、大肠杆菌(E.coli)、立克次氏体属菌种(Rickettsia spp.)、李斯特菌属菌种(Listeria spp.)、肺炎军团菌(Legionellapneumoniae)、假单胞菌属菌种(Pseudomonas spp.)、弧菌属菌种(Vibrio spp.)、以及伯氏疏螺旋体(Borellia burgdorferi)。
细菌病原体的保护性抗原的实例包括产肠毒素性大肠杆菌的菌体抗原，如CFA/I菌毛抗原和热不稳定毒素的无毒性B亚基；百日咳博德特氏菌(Bordetella pertussis)的百日咳杆菌粘附素、百日咳博德特氏菌的腺苷酸环化酶-溶血素、破伤风梭菌(Clostridiumtetani)的破伤风毒素的片段C、伯氏疏螺旋体的OspA、普氏立克次氏体(Rickettsiaprowazekii)和斑疹伤寒立克次氏体(Rickettsia typhi)的保护性类结晶表面层蛋白、单核细胞增生性李斯特菌(Listeria monocytogenes)的李斯特菌溶胞素(也被称为“Llo”和“Hly”)和/或超氧化物歧化酶(也被称为“SOD”和“p60”)；幽门螺杆菌(Helicobacterpylori)的尿素酶、以及炭疽芽孢杆菌(Bacillus anthrax)的致死毒素的受体结合结构域和/或保护性抗原。
来自生物武器或病原体的抗原的实例包括但不限于天花、炭疽、兔热病、瘟疫、李斯特菌、布氏杆菌病、肝炎、牛痘、分枝杆菌、柯萨奇病毒(coxsackievirus)、结核病、疟疾、埃立克体病以及细菌性脑膜炎。
作为寄生物抗原的来源的寄生物病原体包括但不限于疟原虫属物种(Plasmodiumspp.)，如恶性疟原虫(Plasmodium falciparum)(ATCC编号：30145)；锥虫属物种(Trypanosome spp.)，如克氏锥虫(Trypanosoma cruzi)(ATCC编号：50797)；贾第鞭毛虫属物种(Giardia spp.)，如肠贾第鞭毛虫(Giardia intestinalis)(ATCC编号：30888D)；牛蜱属物种(Boophilus spp.)；巴贝虫属物种(Babesia spp.)，如田鼠巴贝虫(Babesiamicroti)(ATCC编号：30221)；内阿米巴属物种(Entamoeba spp.)，如溶组织内阿米巴(Entamoeba histolytica)(ATCC编号：30015)；艾美球虫属物种(Eimeria spp.)，如巨型艾美球虫(Eimeria maxima)(ATCC编号：40357)；利什曼虫属物种(Leishmania spp.)(分类ID：38568)；裂体吸虫属物种(Schistosome spp.)，如曼森氏裂体吸虫(Schistosomamansoni)(基因库登录号：AZ301495)；布鲁格氏丝虫属物种(Brugia spp.)，如马来布鲁格氏丝虫(Brugia malayi)(基因库登录号：BE352806)；片吸虫属物种(Fascida spp.)，如肝片吸虫(Fasciola hepatica)(基因库登录号：AF286903)；恶丝虫属物种(Dirofilariaspp.)，如犬恶丝虫(Dirofilaria immitis)(基因库登录号：AF008300)；吴策线虫属物种(Wuchereria spp.)，如班氏吴策线虫(Wuchereria bancrofti)(基因库登录号：AF250996)；以及盘尾丝虫属物种(Onchocerca spp)，如旋盘尾丝虫(Onchocercavolvulus)(基因库登录号：BE588251)。
寄生物抗原的实例包括但不限于疟原虫属物种的红细胞前期抗原，如恶性疟原虫(基因库登录号：M22982)、间日疟原虫(P vivax)(基因库登录号：M20670)的环子孢子抗原；疟原虫属物种的肝期抗原，如肝期抗原1(被称为LSA-1；基因库登录号：AF086802)；疟原虫属物种的裂殖子期抗原，如裂殖子表面抗原-1(也被称为MSA-1或MSP-1；基因库登录号：AF199410)；溶组织内阿米巴的表面抗原，如半乳糖特异性凝集素(基因库登录号：M59850)或富含丝氨酸的溶组织内阿米巴蛋白；利什曼虫属物种的表面蛋白，如硕大利什曼虫(Leishmania major)的63kDa糖蛋白(gp63)(基因库登录号：Y00647)或硕大利什曼虫的46kDa糖蛋白(gp46)；马来布鲁格氏丝虫的副肌球蛋白(基因库登录号：U77590)；曼森氏裂体吸虫的磷酸丙糖异构酶(基因库登录号：W06781)；蛇形毛圆线虫(Trichostrongyluscolubriformis)的分泌球蛋白样蛋白(基因库登录号：M63263)；肝片吸虫(基因库登录号：M77682)、牛裂体吸虫(Schistosoma bovis)(基因库登录号：M77682)、日本裂体吸虫(S.japonicum)(基因库登录号：U58012)的谷胱甘肽-S转移酶；以及牛裂体吸虫和日本裂体吸虫的KLH(Bashir等,同上)。
肿瘤特异性抗原的实例包括前列腺特异性抗原(PSA)、TAG-72以及CEA；人类酪氨酸酶(基因库登录号：M27160)；酪氨酸酶相关蛋白(也被称为TRP；基因库登录号：AJ132933)；以及肿瘤特异性肽抗原。
移植抗原的实例包括T细胞上的CD3分子和组织相容性抗原，如HLA A、HLA B、HLAC、HLA DR以及HLA。
自身免疫抗原的实例包括IASβ链，其可用于针对自身免疫性脑脊髓炎的治疗性疫苗中(基因库登录号：D88762)；谷氨酸脱羧酶，其可用于针对胰岛素依赖性1型糖尿病的治疗性疫苗中(基因库登录号：NM013445)；促甲状腺激素受体(TSHr)，其可用于针对格雷夫斯病(Grave's disease)的治疗性疫苗中(基因库登录号：NM000369)；以及酪氨酸酶相关蛋白1，其可用于针对白癜风的治疗性疫苗中(基因库登录号：NM000550)。
可以用于构建标记抗体或其片段的HIV药物包括但不限于抗病毒剂，如核苷RT抑制剂、CCR5抑制剂/拮抗剂、病毒进入抑制剂以及它们的功能性类似物。确切地说，抗病毒剂可以是核苷RT抑制剂，如齐多夫定(Zidovudine)(ZDV、AZT)、拉米夫定(Lamivudine)(3TC)、司他夫定(Stavudine)(d4T)、去羟肌苷(Didanosine)(ddl)、扎西他滨(Zalcitabine)(ddC)、阿巴卡韦(Abacavir)(ABC)、艾莫瑞凡(Emirivine)(FTC)、替诺福韦(Tenofovir)(TDF)、地拉韦定(Delaviradine)(DLV)、依法韦仑(Efavirenz)(EFV)、奈韦拉平(Nevirapine)(NVP)、沙奎那韦(Saquinavir)(SQV)、利托那韦(Ritonavir)(RTV)、茚地那韦(Indinavir)(IDV)、奈非那韦(Nelfinavir)(NFV)、安普那韦(Amprenavir)(APV)、洛匹那韦(Lopinavir)(LPV)、阿扎那韦(Atazanavir)、可比韦(Combivir)(ZDV/3TC)、克力芝(Kaletra)(RTV/LPV)、三协唯(Trizivir)(ZDV/3TC/ABC)；
CCR5抑制剂/拮抗剂，如SCH-C、SCH-D、PRO 140、TAK 779、TAK-220、RANTES类似物、AK602、UK-427、857、单克隆抗体；以及病毒进入抑制剂，如福泽昂(Fuzeon)(T-20)(恩夫韦地(enfuvirtide))、NB-2、NB-64、T-649、T-1249、SCH-C、SCH-D、PRO 140、TAK 779、TAK-220、RANTES类似物、AK602、UK-427、857；以及其功能性类似物或等同物。
设想的是，许多不同的岩藻糖基化的糖蛋白和非岩藻糖基化的糖蛋白可以根据本发明的方法修饰或用作治疗剂以用于与末端糖结合，包括但不限于促肾上腺皮质激素(ACTH)；促肾上腺皮质激素衍生物类(例如依比拉肽(ebiratide))；血管紧张素；血管紧张素II；天冬酰胺酶；心房钠尿肽类；心房钠利尿肽类；杆菌肽；β-内啡肽类；凝血因子VII、VIII以及IX；血胸腺因子(FTS)；血胸腺因子衍生物类；铃蟾素；骨形态发生因子(BMP)；骨形态发生蛋白；缓激肽；雨蛙素(caerulein)；降钙素基因相关多肽(CGRP)；降钙素类；CCK-8；细胞生长因子类(例如EGF；TGF-α；TGF-β；PDGF；酸性FGF；碱性FGF)；雨蛙肽(cerulein)；趋化因子类；胆囊收缩素；胆囊收缩素-8；胆囊收缩素-肠促胰酶素(CCK-PZ)；粘菌素；集落刺激因子类(例如CSF；GCSF；GMCSF；MCSF)；促肾上腺皮质激素释放因子(CRF)；细胞因子类；去氨加压素；达诺非(dinorphin)；二肽；歧化酶；强啡肽；章鱼素；内啡肽类；内皮素(endothelin)；内皮素拮抗肽类；内皮素类(endotherin)；脑啡肽类；脑啡肽衍生物类；表皮生长因子(EGF)；促红细胞生成素(EPO)；促卵泡激素(FSH)；甘丙肽；肠抑胃肽；胃泌素释放多肽(GRP)；胃泌素类；G-CSF；胰高血糖素；谷胱甘肽过氧化物酶；glutathio-过氧化物酶；促性腺激素类(例如人绒毛膜促性腺激素以及其α亚基和β亚基)；短杆菌肽；短杆菌肽类；生长因子(EGF)；生长激素释放因子(GRF)；生长激素类；激素释放激素(LHRH)；人心房钠尿多肽(h-ANP)；人胎盘催乳素；胰岛素；胰岛素样生长因子类(IGF-I；IGF-II)；干扰素；干扰素类(例如α-干扰素、β-干扰素以及γ-干扰素)；白细胞介素类(例如1；2；3；4；5；6；7；8；9；10；11以及12)；肠多肽(VIP)；激肽释放酶；京都啡肽；促黄体素释放素；促黄体激素(LH)；促黄体激素释放激素(LH-RH)；氯化溶菌酶；黑色素细胞刺激激素(MSH)；促黑激素；蜂毒肽；促胃动素；胞壁酰；胞壁酰二肽；神经生长因子(NGF)；神经营养因子类(例如NT-3；NT-4；CNTF；GDNF；BDNF)；神经肽Y；神经降压素；催产素；胰抑制素；胰多肽；肠促胰酶素；甲状旁腺激素(PTH)；五肽胃泌素；多肽YY；垂体腺苷酸环化酶激活多肽类(PACAP)；血小板衍生生长因子；多粘菌素B；催乳素；蛋白质合成刺激多肽；PTH相关蛋白；松弛素；肾素；分泌素；血清胸腺因子；生长调节素类；生长抑素衍生物类；超氧化物歧化酶；他夫素(taftsin)；四肽胃泌素；促血小板生成素(TPO)；胸腺体液因子(THF)；胸腺生成素；胸腺素；胸腺刺激素；甲状腺激素释放激素；促甲状腺激素(TSH)；促甲状腺素释放激素(TRH)；胰蛋白酶；促吞噬素；肿瘤生长因子(TGF-α)；肿瘤坏死因子(TNF)；短杆菌酪肽；尿抑胃素；尿激酶；血管活性肠多肽；以及血管加压素。
岩藻糖基化的糖蛋白和非岩藻糖基化的糖蛋白是重要的生物分子类别，它们在许多生物事件中起至关重要的作用，如细胞粘附、肿瘤转移、病原体感染、以及免疫应答。如本文先前所示，岩藻糖基化的糖蛋白或非岩藻糖基化的糖蛋白的结构研究和功能研究中的主要问题是它们的结构微观异质性。天然和重组的岩藻糖基化的糖蛋白或非岩藻糖基化的糖蛋白通常是作为仅在侧链低聚糖的结构方面不同的糖型的混合物而产生。
可以对重塑的糖蛋白，如抗体进行任何进一步的结构修饰，这些结构修饰是必要的或所期望的，包括但不限于糖基转移、和选择性连接(例如点击化学、施陶丁格反应(Staudinger reaction)等)以引入另外的官能团或标签。官能团可以是任何合适的类型，包括但不限于毒素、特殊抗原(如α-Gal)、放射性物质、光敏性物质、PEG等。糖蛋白可以在糖蛋白的叠氮化物官能团与带有令人感兴趣的官能部分的炔烃的“点击化学”环加成反应中发生催化反应。叠氮基官能团和炔烃官能团可以在对应的连接组分中转换，并且糖蛋白可以被炔基官能团官能化并且与包括令人感兴趣的部分的叠氮化物官能化的化合物反应。还将领会到，其他连接对也可以被设计用于点击化学反应。
根据本文所述的方法产生的岩藻糖基化和非岩藻糖基化的糖蛋白可以用于诊断和治疗。在市场上和/或目前在临床试验中使用的治疗性蛋白质中有约三分之二是糖蛋白。然而，天然和重组糖蛋白的不同糖型中的结构异质性是开发基于糖蛋白的药物的主要障碍，这是因为不同的糖型可能具有不同的生物活性并且在表达期间控制糖基化以获得均质的糖型是非常难的。先前发现一类内切糖苷酶的转糖基活性代表了糖基化工程化领域中的一个重大进步以提高糖蛋白的治疗和诊断潜能，并且本发明的Endo-S2突变体能够对岩藻糖基化和非岩藻糖基化的天然和重组糖蛋白进行转糖基而没有不利的水解情况。
通过以下非限制性实施例更充分展示本发明的特征和优势。
实施例
Endo-S2糖苷合酶突变体的产生以及它们用于完整单克隆抗体利妥昔单抗的糖基化重塑的用途
糖苷合酶先前已经通过负责在水解期间促进
唑啉
离子中间体形成的GH85家族中关键天冬酰胺(Asn)或GH18家族中天冬氨酸(Asp)残基的定点诱变，由几种GH85内切糖苷酶(ENG酶)制成，所述GH85内切糖苷酶包括EndoA、EndoM、EndoD以及GH18内切糖苷酶EndoS。[36,38]Endo-S2是属于糖苷水解酶家族18(GH18)的内切糖苷酶[33]，它与最近被证实具有转糖基活性的EndoS、EndoF1、EndoF2、以及EndoF3在同一GH家族中。基于如下假设，即EndoS2催化的水解也通过涉及形成
唑啉
离子中间体的底物辅助机制进行，如由其他GH18内切糖苷酶如EndoS所示，通过鉴定负责促进
唑啉
离子形成的残基并且使其突变来产生源自Endo-S2的潜在的糖苷合酶。先前对Endo-S的结构和诱变研究已经显示，位置233处的天冬氨酸残基(D233)负责促进
唑啉形成并且E235残基是用于催化水解的一般酸/碱[47,48]。EndoS2与EndoS的序列比对(图2)使得鉴定出EndoS中用于催化的两个关键残基：负责促进
唑啉
离子形成的D184残基(对应于EndoS中的D233)和作为聚糖水解中一般酸/碱残基的E186残基(相当于EndoS的E235)，如图2中所示。因此，在假设D184是经由底物辅助机制在水解中促进
唑啉
离子中间体形成的关键残基的情况下，通过Endo-S2(SEQ ID NO:1)的定点诱变产生十九种特定的突变体D184A-Y(SEQ ID NO:2-20)。使这些突变体以及野生型EndoS2在大肠杆菌中以高收率(20mg/L-30mg/L)作为CPD融合蛋白表达并且通过Ni-NTA亲和色谱法纯化。
利妥昔单抗是一种治疗性单克隆抗体，其被用作模型mAb以研究酶的脱糖基化活性和潜在的转糖基活性。市售的利妥昔单抗的主要Fc聚糖是核心岩藻糖基化的双分支复杂型低聚糖，其携带0个-2个半乳糖部分，分别被命名为G0F糖型、G1F糖型以及G2F糖型，如图4C中所示。用EndoS2-CPD融合蛋白(在此，被称为野生型EndoS2或EndoS2)处理利妥昔单抗引起快速的脱糖基化，得到脱糖基化的利妥昔单抗，其在糖基化位点(N297)处带有岩藻糖基化的GlcNAc二糖部分(Fucα1,6GlcNAc)。这些结果确认了野生型EndoS2对完整IgG的显著的Fc聚糖水解活性，这暗示了它可用于mAb的糖基化重塑的第一步骤中。然后使用脱糖基化的利妥昔单抗作为受体以及复杂、高甘露糖、和杂合聚糖
唑啉作为供体底物来研究Endo-S2突变体的转糖基潜能，如图3中所示。通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)和液相色谱质谱(LC-MS)分析来监测糖基化重塑过程，如图4中所示。在还原条件下，利妥昔单抗的重链和轻链分别在约50KDa和约25KDa处出现(泳道1，在图4A中)。在用野生型EndoS2脱糖基化之后，重链作为单一条带在约48KDa处出现，这表明利妥昔单抗中的两个N-聚糖(每条重链各一个)被去除(泳道2，在图4A中)。将脱糖基化的利妥昔单抗(1)和唾液酸复杂型聚糖
唑啉(2)(供体/受体，20:1，摩尔比)与突变体EndoS2-D184Q一起温育得到转糖基产物(5)(图4E)，其重链作为比脱糖基化的利妥昔单抗(1)的重链更大约2KDa的单一条带出现(图4A，泳道3)。该结果表明新的N-聚糖与Fc重链中的每一条连接。有趣的是，在1小时温育内实现了完整抗体的Fc结构域的基本上定量的转糖基。
通过LC-MS分析对转糖基进行进一步表征。在LC-MS条件下分离利妥昔单抗的重链和轻链。轻链MS数据的解卷积给出了23 039的质量(图4B)，这与利妥昔单抗轻链的计算质量(M＝23 042Da)相一致。[47]重链的MS数据的解卷积给出了三种不同m/z物质，即50508、50669、以及50829，如图4C中所示，它们分别与以下重链糖型的理论质量有良好的一致性：G0F，M＝50 515Da；G1F，M＝50 677Da；以及G2F，M＝50 839Da。[47]脱糖基化的利妥昔单抗(1)的重链的解卷积的电喷雾电离质谱(ESI-MS)在49 411处显示出单一物质，如图4D中的图表c中所示，这与携带Fucα1,6GlcNAc二糖部分的重链(计算值，M＝49 420Da)良好匹配。在糖基化重塑之后，从具有复杂聚糖的转糖基产物(5)的重链观测到51 414处的单峰，向利妥昔单抗的脱糖基化的重链增添了2003Da，如图4E中的图表中所示。该结果表明来自相应糖
唑啉(2)的唾液酸聚糖与重链连接。
除了唾液酸化的复杂型N-聚糖
唑啉(2)之外，EndoS2突变体还同样有效地使用高甘露糖Man9GlcNAc核心
唑啉(3)和唾液酸杂合型
唑啉(4)来进行利妥昔单抗糖工程化，从而分别引起相应的均质糖型(6)和(7)的形成(图4F和图4G)。转糖基产物(6)的重链的解卷积的ESI-MS在51074处显示出单一物质，如图4F中所示，其与携带Man9GlcNAc2聚糖的利妥昔单抗重链的计算分子质量(M＝51082Da)良好匹配。类似地，转糖基产物(7)的重链的解卷积的ESI-MS在51080处显示出单一物质，如图4G中所示，其与携带N3Man3GlcNAc2聚糖的利妥昔单抗重链的计算分子质量(M＝50190Da)有良好的一致性。本文所述的结果代表了关于经由通过EndoS2和基于EndoS2的糖苷合酶的组合使用而实现的高效脱糖基化-再糖基化方案将全尺寸的天然高甘露糖型(Man9)和唾液酸杂合型N-聚糖整体转移到Fc结构域中对完整IgG单克隆抗体进行糖基化重塑的首次报道。在完成转糖基之后，可以通过简单的蛋白A亲和色谱法将产物纯化，得到明确限定的均质糖型。值得注意的是，已经报道了通过使用EndoS/基于EndoS的糖苷合酶将双分支复杂型N-聚糖转移到Fc结构域中来对完整利妥昔单抗进行糖工程化。然而，该系统在将高甘露糖型和杂合型聚糖转移到抗体中的方面是低效的。EndoS2/EndoS2糖苷合酶系统的开发显著扩大了抗体的化学酶促糖工程化的聚糖种类的范围。
将利妥昔单抗进行糖工程化以提供非岩藻糖基化的复杂、高甘露糖、以及杂合糖型
对于抗癌治疗，非岩藻糖基化的IgG糖型是所期望的，这是因为先前已经证实具有低岩藻糖含量的Fc N-聚糖的mAb显示出增强的体外ADCC活性和增强的体内抗癌功效，特别是对于携带FcγIIIa受体的低亲和力F158等位基因的那些患者来说[8,39,40,49]。然而，没有α岩藻糖苷酶可用于去除完整利妥昔单抗中的α1,6-岩藻糖。α-1,6-岩藻糖部分可能被Fc结构域和/或复杂N-聚糖遮挡，从而使得α-岩藻糖苷酶无法接近。理论上，在脱糖基化后，所得利妥昔单抗的Fuc(α1,6)GlcNAc糖型可能更容易被α-岩藻糖苷酶接近。实际上，在用EndoS2脱糖基化之后，可以通过与来自干酪乳杆菌(Lactobacillus casei)的α-1,6-岩藻糖苷酶一起过夜温育来去除大部分的α-1,6-岩藻糖部分以得到含有GlcNAc的利妥昔单抗(8)。使用LC-MS确认结果(图6B)。随后，确定糖苷合酶EndoS2-D184A、EndoS-D184Q、或EndoS-D184N也有效识别(8)中的非岩藻糖基化的GlcNAc以用复杂
唑啉(2)、高甘露糖
唑啉(3)或杂合
唑啉(4)进行转糖基而以基本上定量的转化率提供相对均质的非岩藻糖基化的复杂型(9)、高甘露糖型(10)以及杂合型(11)糖型(图5)。通过SDS-PAGE和LC-MS分析确认糖工程化的产物(9、10、11)的身份和纯度，如图6中所示。脱岩藻糖基化的利妥昔单抗(8)在49266处显示出主峰(图6B)，这确认了岩藻糖的去除(GlcNAc-利妥昔单抗的重链的计算值，M＝49274Da)。复杂型聚糖的转糖基产物(9)的重链的解卷积的ESI-MS在51268处作为主要物质出现(图6C)，这与携带唾液酸化的双分支复杂型N-聚糖Sia2Gal2GlcNAc2Man3GlcNAc2的利妥昔单抗重链的计算分子质量(M＝51276Da)良好匹配。类似地，高甘露糖型聚糖(10)和杂合型聚糖(11)的转糖基产物的重链的LC-MS分析分别在50930(图6D)和50939(图6E)处作为主要物质出现，这与计算分子质量(50936Da和50944Da)良好匹配。在转移杂合聚糖
唑啉时，约33％的起始材料未被转移。在优化反应条件(增加EndoS2突变体浓度、添加更多的
唑啉等)的情况下，应当能够推动反应直到完成。似乎岩藻糖基化的利妥昔单抗(1)是比非岩藻糖基化的利妥昔单抗(8)更优选的受体。在比较研究中，还发现突变体D184A、D184N以及D184Q对岩藻糖基化的GlcNAc-利妥昔单抗(1)具有比对非岩藻糖基化的受体(8)更快的转糖基反应(数据未示)。综上所述，这些实验结果揭示了一种从可商购获得的单克隆抗体制备非岩藻糖基化的复杂型、高甘露糖型以及杂合型均质(或相对均质)糖型的组合酶促方法。所得的非岩藻糖基化的利妥昔单抗预期获得改进的ADCC和CDC效应功能，如先前的研究所表明。[8,42,49]
EndoS2 D184的不同突变体的转糖基活性的比较
比较EndoS2-D184A、D184N、以及D184Q将唾液酸双分支复杂聚糖
唑啉(2)转移到岩藻糖基化的GlcNAc-利妥昔单抗(1)的活性。使用相同的反应条件，天冬酰胺(N)突变体在5分钟内将超过30％的
唑啉转移到受体中，而丙氨酸(A)突变体和谷氨酰胺(Q)突变体转移少于20％(图7)。似乎N突变体在转移复杂型
唑啉方面比A突变体和Q突变体更具活性。
Endo-S2的克隆、表达以及表征
将编码Endo-S2(SEQ ID NO:1的44-843)的cDNA序列(Endo-S2基因(ndoS2)的基因库登录号是ACI61688)克隆到pET22b-CPD载体中，其向表达的蛋白质的C末端添加霍乱弧菌(Vibrio cholerae)MARTX毒素的半胱氨酸蛋白酶结构域(CPD)和10×组氨酸标签[35](SEQID NO 22)。最近报道，Endo-F3和它的突变体的高水平可溶性表达可以使用这种载体实现[32]。在可替选方案中，编码Endo-S2的cDNA序列可以包括(SEQ ID NO:1的氨基酸1和44-843)。遵循相似的方法，Endo-S2在大肠杆菌中成功表达并且容易使用固定化金属离子亲和色谱法(IMAC)纯化而以超过20mg/L的收率获得可溶性酶。重组Endo-S2显示出高水解活性，如由它将市售的利妥昔单抗快速脱糖基化所证实，所述脱糖基化是通过LC-MS分析来监测的。
从Endo-S2产生糖苷合酶突变体
通过在关键残基处进行定点突变来产生来自GH85家族和GH18家族的内切糖苷酶的糖苷合酶突变体，所述关键残基负责在水解期间促进
唑啉
离子中间体的形成，这是以底物辅助机制进行的。这些包括GH85家族的内切糖苷酶Endo-A(Asn171)[36]、Endo-M(Asn175)[37,38]、以及Endo-D(Asn322)[24]的关键天冬酰胺残基、或GH18家族内切糖苷酶Endo-S(Asp233)[25]和Endo-F3(Asp-165)[32]的关键天冬氨酸残基。Endo-S2和Endo-S的序列比对揭示，Endo-S2的Asp-184是相当于Endo-S的Asp233的对促进水解中
唑啉
离子形成来说必要的残基(图2)。为了从Endo-S2产生有效的糖苷合酶突变体，在包含44至843的残基的截短表达蛋白中使用定点诱变用其他19个天然氨基酸残基系统地置换Asp-184。值得注意的是，设想非天然蛋白质也可以用于取代。按照与对于野生型酶所示的方式相同的方式，也使所得的19种D184突变体在pET22bCPD载体中作为可溶性蛋白质表达并且使用固定化金属离子亲和色谱法纯化。突变体酶的表达产生与野生型酶的表达相当的收率(15mg/L-20mg/L)。
值得注意的是，设想非天然蛋白质也可以用于取代。可以由翻译系统使用的非天然氨基酸的实例包括：酪氨酸氨基酸的非天然类似物；谷氨酰胺氨基酸的非天然类似物；苯丙氨酸氨基酸的非天然类似物；丝氨酸氨基酸的非天然类似物；苏氨酸氨基酸的非天然类似物；烷基、芳基、酰基、叠氮基、氰基、卤代、肼、酰肼、羟基、烯基、炔基、醚、硫醇、磺酰基、硒代、酯、硫代酸、硼酸酯、有机硼酸酯、磷酸、膦酰基、膦、杂环、烯酮、亚胺、醛、羟胺、酮、或氨基取代的氨基酸、或其任何组合。
对19种突变体和WT的水解活性和转糖基活性的比较研究
通过遵循如图8中所示的方案来评估对完整抗体中的Fc N-聚糖的水解活性和使用聚糖
唑啉的转糖基活性。结果表明大部分的D184残基处的突变体对Fc N-聚糖产生显著降低或完全减弱的水解活性。在它们当中，D184F、D184H、D184K、D184R、以及D184W突变体完全缺乏水解活性，而发现包括D184C、D184E、D184G、D184N、D184S、D184Y在内的几种其他突变体仍保留显著的水解活性(表1)。另一方面，对转糖基的评价表明当使用脱糖基化的利妥昔单抗作为受体和双分支复杂型聚糖
唑啉作为供体底物时，几乎所有的突变体都具有转糖基活性，但是所述活性在不同的突变体之间显著不同(图8、表2)。其中，发现D184C、D184M、D184G、D184E、D184Y、D184S、以及D184A是最具活性的突变体。然而，D184C、D184G、D184E、D184Y、D184S、以及D184A也显示出显著的残留水解活性。最令人感兴趣的突变体是D184M，它仅保留很低的水解活性，但是显示出非常高的转糖基活性(仅次于D184C)，这使得它成为被选用于糖基化重塑的最佳的糖苷合酶突变体之一。
Endo-S2糖苷合酶在转糖基中显示出非常宽的底物特异性
利妥昔单抗是一种治疗性单克隆抗体，将其用作模型来研究Endo-S2 D184突变体的转糖基活性。市售利妥昔单抗的主要Fc聚糖是核心岩藻糖基化的双分支复杂型低聚糖，其携带0个-2个半乳糖部分，分别被命名为G0F糖型、G1F糖型以及G2F糖型。一般的糖基化重塑方法示于图9中并且通过LC-MS分析来评估反应产物(图10)。用野生型Endo-S2处理利妥昔单抗(1)引起利妥昔单抗的完全脱糖基化，如由市售利妥昔单抗(图10A)中存在的糖型混合物(G0F、G1F以及G2F)转化成利妥昔单抗的Fucα1,6GlcNAc糖型(2)(图10B)所证实。通过蛋白A亲和色谱法将Fucα1,6GlcNAc-利妥昔单抗(2)从野生型内切糖苷酶和释放的聚糖中纯化出来并且用作转糖基反应中的受体。发现EndoS2 D184M能够有效地将唾液酸化的双分支复杂型(SCT)N聚糖从相应的聚糖
唑啉(4)转移到Fucα1,6GlcNAc-利妥昔单抗受体(2)中以形成利妥昔单抗的S2G2F糖型(3)。使用20摩尔当量(即每个单体Fc结构域10摩尔当量)的聚糖
唑啉可以容易地推动反应完成。通过LC-MS分析估计反应收率超过95％，因为几乎没有检测到起始材料，这确认了转糖基的完成。携带SCT N-聚糖的转糖基产物(3)的LC-MS分析揭示，(3)的重链分别在51412处作为单一物质出现(解卷积数据)，这与携带SCT N-聚糖(具有核心岩藻糖)的重链的计算分子质量(M＝51421Da)有良好的一致性(图10C)。
除了复杂型N-聚糖之外，还使用高甘露糖型(HM)Man9GlcNAc
唑啉(7)和唾液酸杂合型(Hyb)Neu5AcGalGlcNAcMan5GlcNAc
唑啉(5)在转糖基反应中作为供体底物来进一步测试Endo-S2的特异性。所述反应分别引起相应的均质糖型(8)和(6)的形成(图10E、10G)。转糖基产物(8)的重链的解卷积的ESI-MS在51074处显示出单一物质，如图10E中所示，这与携带Man9GlcNAc2聚糖的利妥昔单抗重链的计算分子质量(M＝51081Da)良好匹配。类似地，转糖基产物(6)的重链的解卷积的ESI-MS在51082处显示出单一物质，如图10G中所示，这与携带Neu5AcGalGlcNAcMan5GlcNAc2聚糖的利妥昔单抗重链的计算分子质量(M＝51090Da)有良好的一致性。在相同的条件下，发现先前报道的Endo-S突变体包括Endo-S的D233A突变体和D233Q突变体[25]，在高甘露糖型和杂合型N-聚糖的情况下仅显示出很低的转糖基活性，尽管它们可以有效地转移双分支复杂型N-聚糖。此外，最近报道的Endo-F3的D165A突变体[32]不能转移高甘露糖型或杂合型N聚糖，但是可以对双分支和三分支复杂型糖起作用。因此，这些Endo-S2衍生的突变体代表了可以有效地将高甘露糖型和杂合型N-聚糖转移到完整抗体中的核心岩藻糖基化的GlcNAc受体中的首批糖苷合酶。应当提到的是，Endo-A突变体(N171A和N171Q)可以将高甘露糖型N-聚糖转移到GlcNAcFc结构域中，但是它们不能使用核心岩藻糖基化的GlcNAc-Fc作为受体[22,23,36]。这些研究还显示，野生型Endo-S2和Endo-S2糖苷合酶突变体的组合使用提供了从单一前体开始到各种均质糖型的抗体的特别有效的糖基化重塑方法。
用于制备非岩藻糖基化的糖型的基于Endo-S2的糖基化重塑
对于抗癌治疗，非岩藻糖基化的IgG糖型是所期望的，这是因为先前已经证实具有Fc糖基化的低岩藻糖含量的mAb显示出增强的体外ADCC活性和增强的体内抗癌功效，特别是对于携带FcγIIIa受体的低亲和力F158等位基因的那些患者来说[8,9,39,40]。为了测试Endo-S2是否可以对非岩藻糖基化的IgG进行糖基化，将Fucα1,6GlcNAc-利妥昔单抗(2)与来自干酪乳杆菌的重组α1,6-岩藻糖苷酶[41]一起温育以得到没有核心岩藻糖的GlcNAc-利妥昔单抗(9)。使用唾液酸双分支复杂型(CT)N-聚糖
唑啉(10)进行Endo-S2D184M催化的GlcNAc-利妥昔单抗(9)的转糖基。发现D184M突变体可以有效地将N-聚糖转移到抗体中的GlcNAc受体中而以基本上定量的转化率得到完全半乳糖基化和非岩藻糖基化的糖型(11)。转糖基产物(11)的重链的解卷积的ESI-MS在50684处显示出单一物质，如图10I中所示，这与携带完全半乳糖基化的双分支复杂型N-聚糖而没有核心岩藻糖的利妥昔单抗重链的计算分子质量(M＝50693Da)有良好的一致性。除了通过LC-MS分析结合酶促转化确认重链的位点特异性糖基化之外，转糖基产物(3、6、8、11)的轻链也在23034处作为单一物质出现，这与没有任何修饰的利妥昔单抗的轻链的计算分子质量(M＝23039Da)匹配(图10D、图10F、图10H、图10J)。这些结果表明在Endo-S2催化的糖基化重塑过程期间，除了Fc结构域处GlcNAc受体处转移N聚糖的连接之外，重链和轻链上没有发生非酶促修饰。应当指出的是，利妥昔单抗的完全半乳糖基化且非岩藻糖基化的糖型(11)先前被证实对FcγIIIA受体具有的亲和力是市售的利妥昔单抗的至少20倍，这是显著增强的ADCC的指示[25]。
Endo-S2 D184M突变体对不同的N-聚糖底物的转糖基效率的比较
为了对Endo-S2 D184M的N-聚糖底物偏好进行进一步表征，分别使用复杂型(SCT)、高甘露糖型(HM)、以及杂合型(Hyb)的N-聚糖
唑啉进行三个平行的转糖基反应。通过对在多个时间点获取的反应等分试样进行LC-MS分析来监测反应进展并且结果汇总于图11中。在相同的反应条件下，使用SCT
唑啉(4)进行的转糖基反应在20分钟内完成而得到S2G2F-利妥昔单抗(3)，而使用HM
唑啉(7)和Hyb
唑啉(5)进行的转糖基要慢得多。这些结果表明相对于高甘露糖型和杂合型N-聚糖，Endo-S2 D184M偏好复杂型N-聚糖，尽管它具有显著放松的N-聚糖特异性。
典型的Endo-S2和Endo-S突变体的转糖基效率的比较
先前产生了Endo-S糖苷合酶突变体D233Q和D233A，它们有效地用复杂型N-聚糖
唑啉对利妥昔单抗进行转糖基[25]。EndoS D233突变体最近已经被用于产生均质的单克隆抗体以用于结构研究和功能研究[26-31]。为了比较来自Endo-S2和Endo-S的糖苷合酶突变体的转糖基效率，选择Endo-S D233Q突变体，相当的Endo-S2 D184Q突变体和Endo-S2D184M突变体来催化三个平行的转糖基反应。通过LC-MS分析来监测转糖基反应的时间过程并且汇总于图12中。在反应条件下，Endo-S2的D184M突变体显示出显著强效的转糖基活性，并且在10分钟内达到了聚糖转移的完成。另一种Endo-S2突变体也可以顺利地转移聚糖并且在1小时内达到完成。然而，相应的Endo-S突变体(D233Q)效率低得多，在相同的条件下在1小时达到约10％的转糖基(图12)。如单独的实验中所证实，需要多得多(10倍)的Endo-SD233Q突变体和更大量过量的聚糖
唑啉来实现相同水平的由Endo-S2 D184Q突变体催化的转糖基，并且Endo-S2的D184M突变体甚至比D184Q突变体更加有效。这些研究表明新发现的Endo-S2 D184突变体对于抗体糖基化重塑来说在反应效率和底物多样性的广度这两个方面优于先前报道的Endo-S突变体。
使用一对Endo-S2酶对曲妥珠单抗(赫赛汀)进行糖基化重塑
为了证实所观测到的Endo-S2和其突变体的酶特性一般地适用于抗体糖基化重塑，进行另一种单克隆抗体的糖基化重塑，所述单克隆抗体是曲妥珠单抗(赫赛汀)，其广泛用于治疗乳腺癌。通过合成曲妥珠单抗的唾液酸化的糖型来评估糖基化重塑(图13)。用野生型Endo-S2处理曲妥珠单抗引起完全脱糖基化，如由市售曲妥珠单抗(图13A)中存在的糖型混合物(G0F、G1F、以及G2F)转化成曲妥珠单抗的Fucα1,6GlcNAc-糖型(12)(图13B)所证实。将脱糖基化的曲妥珠单抗通过蛋白A亲和色谱法从内切糖苷酶和释放的聚糖中纯化出来并且用作转糖基反应中的受体。在Endo-S2 D184M突变体存在下将Fucα1,6GlcNAc-曲妥珠单抗(12)与供体底物SCT-
唑啉(4)一起温育引起脱糖基化的曲妥珠单抗(12)快速转化成完全糖基化的曲妥珠单抗(13)。所述反应是基本上定量的以得到单一转糖基产物(13)。转糖基产物(13)的LC-MS分析揭示，(13)的重链在51500Da处作为单一物质出现(解卷积数据)(图13C)，这表明单一唾液酸化的N-聚糖连接在重链上。另一方面，转糖基产物(13)的轻链在23438处作为单一物质出现，这与没有任何另外的修饰的曲妥珠单抗的轻链有良好的一致性(图13D)。
为了确认N-聚糖特异性连接到Fc结构域的Asn-297 N-糖基化位点，而不是可能通过如最近的出版物[30]中所涉及的非酶促反应而发生的连接到多肽骨架的其他位点，用PNG酶F处理转糖基产物(13)并且通过质谱分析研究蛋白质部分。PNG酶F具有高度特异性并且只有当N聚糖在N-糖蛋白中保守的糖基化位点处以N-糖基酰胺键连接到Asn侧链时，才可以释放N聚糖。PNG酶F处理的转糖基产物(13)的重链的LC-MS分析得到了49150的单一物质(解卷积数据)，其对应于没有任何另外的修饰的重链的多肽骨架(图13E)。综上所述，这些结果清楚地表明单一唾液酸化的双分支N-聚糖与抗体重链结合，并且完整N-聚糖连接在保守的N-糖基化位点处而没有抗体的任何非酶促糖化。由Endo-S2糖苷合酶突变体催化的高效转化允许少得多的反应时间和使用更少量过量的聚糖
唑啉来实现定量转化。
本文描述了源自于化脓性链球菌血清型M49的内切糖苷酶(Endo-S2)的一类新的糖苷合酶的发现，这些糖苷合酶在抗体糖基化重塑方面显示出比先前报道的糖苷合酶如源自于Endo-A、Endo-M、Endo-S、以及Endo-F3的那些更宽的底物特异性和强效得多的转糖基活性。这些发现是通过在关键残基D184处进行系统诱变，结合对所得的19种突变体的水解活性和转糖基活性的比较分析来实现的。实验数据还揭示了所述19种突变体之间水解活性和转糖基活性两方面的显著差异(表1和表2)，这在没有该比较研究的情况下将难以预测。鉴定出几种值得注意的突变体，包括D184M突变体和D184Q突变体，它们对于糖基化重塑显示出高的转糖基活性，但是仅保留很低的水解活性。
突变体的水解活性和转糖基活性的比较揭示了几个有趣的特征。首先，显示出高转糖基活性的大部分突变体，包括D184C突变体、D184G突变体、D184E突变体、D184Y突变体、D184S突变体、以及D184A突变体，也具有相对高的残留水解活性。一个例外是D184M突变体，它显示出显著的转糖基活性，但是仅保留残余的水解活性，这使得它成为用于糖基化重塑的最有效的糖苷合酶。其次，D184残基被具有带正电荷的侧链(K、R、H)或大体积疏水侧链(I、L、F、W)的氨基酸置换的大部分的突变体显示出非常低的转糖基活性和水解活性。但是，有趣的是，在所有突变体中，D184Y突变体保留最高的水解活性，并且还具有相对高的转糖基活性。
另一个重要的发现是发现Endo-S2衍生的糖苷合酶突变体的底物特异性比先前报道的糖苷合酶宽得多。发现所鉴定出的两种值得注意的Endo-S2糖苷合酶D184M突变体和D184Q突变体能够在抗体糖基化重塑中有效地转移所有三种主要类型的N-聚糖，包括高甘露糖型、复杂型、以及杂合型。此外，Endo-S2糖苷合酶可以识别Fc结构域处的核心岩藻糖基化的GlcNAc或非岩藻糖基化的GlcNAc部分这两者作为转糖基的受体。这些发现显著地扩大了糖基化重塑策略的范围。举例来说，先前报道的Endo-S和Endo-F3对复杂型N-聚糖具有特异性并且不能有效地转移高甘露糖型和杂合型N-聚糖，并且Endo-F3仅对核心岩藻糖基化的GlcNAc受体有效[32]。典型的Endo-S2和Endo-S糖苷合酶突变体的转糖基活性的直接比较揭示了Endo-S2突变体一般比相应的Endo-S突变体具有高得多的活性。通过估计初始速率，Endo-S2的D184Q突变体的活性是Endo-S的相应D233Q突变体的至少10倍，并且最好的Endo-S2突变体D184M据估计在将脱糖基化的利妥昔单抗糖基化方面是Endo-S D233Q突变体的100倍(图12)。最终，除了重塑利妥昔单抗之外，通过使用一对Endo-S2(野生型和D184M突变体)酶将曲妥珠单抗(赫赛汀)高效糖基化重塑以得到单一均质糖型而没有发生副反应(图13)也展示了新发现的糖苷合酶的能力。预期还显示出显著放宽的底物特异性的这些高效的糖苷合酶将广泛应用于产生各种均质糖型的抗体以用于结构和功能研究，以及用于开发更有效的基于抗体的治疗剂。
图14示出了使用本发明以提供所期望的均质糖型的抗体。IVIG广泛用于治疗类风湿性关节炎，但是通常需要高剂量，如(1g/kg-1.5g/kg)。然而，如图14中所示，均质糖型提供了降低剂量、增强功效以及减少副作用的益处。这样的作用归因于由于使用本发明的Endo S2突变体而产生的具有大于95％唾液酸化的重塑IVIG。糖基化的这样的改进适用于糖蛋白激素、细胞因子(IL-2、干扰素等)以及酶替代疗法(溶酶体病)。
实验程序
材料：单克隆抗体利妥昔单抗和曲妥珠单抗(赫赛汀)是来自基因泰克公司(Genetech Inc)(South San Francisco,CA)的产品。唾液酸和唾液酸聚糖复杂型
唑啉是遵循先前报道的程序合成的[42]。高甘露糖型(HM)聚糖(Man9GlcNAc)是通过先前描述的程序从大豆粉制备的[43]。杂合型(Hyb)聚糖(Neu5AcGalGlcNAcMan5GlcNAc)的合成是通过在β-1,2-GlcNAc转移酶(GnT1)[44]、β-1,4-半乳糖基转移酶以及α-2,6-唾液酸转移酶[46]的催化下对Man5GlcNAc[43]进行顺序酶促糖基化[45]来实现的。HM和杂合聚糖
唑啉是遵循先前描述的一锅法转化程序来合成的[42]。遵循我们先前的程序将来自化脓性链球菌的Endo-S D233Q过表达并且纯化[25]。
重组Endo-S2的定点诱变以及表达和纯化
将编码来自化脓性链球菌NZ131(血清型M49)的Endo-S2的氨基酸44-843的cDNA通过PCR扩增并且克隆到pCPDLasso载体(pET22b-CPD衍生物)中(35)，其中CPD(霍乱弧菌MARTX毒素的半胱氨酸蛋白酶结构域和组氨酸标签)的序列示于SEQ ID NO:22中。对于Asp-184残基的饱和诱变，使用正向引物5'-CGTAAATTCGTGCTCAATNNNAATATCTAGTCCATCGACACCACGATCAGTT-3'(SEQ ID NO:23)和反向引物5'-AACTGATCGTGGTGTCGATGGACTAGATATTNNNATTGAGCACGAATTTACG-3'(SEQ ID NO:24)。通过DNA测序确认突变。将含有突变的Endo-S2基因的质粒转化到大肠杆菌BL21(DE3)中。对于同时产生20种Endo-S2 D184变体，将转化体培养在补充有100μg/mL羧苄西林(carbenicillin)的20mL 2xYT肉汤培养基中。将培养物在37℃培养直到细胞达到0.8-1.0的OD600为止。然后，在20℃将0.5mM的异丙基β-D-1-硫代吡喃半乳糖苷(IPTG)添加到培养物中以诱导蛋白质过量产生。在24小时之后，通过离心收获细胞。通过细菌细胞裂解缓冲液(Gold Biotechnology公司)，遵循制造商的说明书来裂解细胞沉淀物。用NiNTA旋转柱(Qiagen)纯化10×组氨酸(His10)标记的EndoS2/CPD融合蛋白。用Amicon超滤(10kDa，Millipore)，利用离心渗滤将纯化的EndoS2蛋白脱盐到PBS(pH 7.4)中。通过SDS-PAGE确认蛋白质纯度并且在NanoDrop 2000c上，使用280nm处的吸光度测量浓度。对于所选择的Endo-S2变体的大规模纯化，使用1L培养物。将细胞裂解物施加到HisTrapHP柱(GE)并且用含0.5M NaCl和20mM咪唑的PBS(pH 7.4)洗涤。用0mM-250mM咪唑梯度的PBS缓冲液洗脱结合的His标记的蛋白。汇集含有Endo-S2蛋白的洗脱级分，浓缩并且进一步经由HiPrep 16/60Sephacryl S-200HR柱(GE)通过尺寸排阻色谱法进行纯化。
IgG的液相色谱质谱(LC-ESI-MS)
在Exactive Plus Orbitrap(Thermo Scientific)上进行LC-MS分析。对于完整抗体，使用Waters XBridgeTM BEH300 C4柱(3.5μm，2.1mm×50mm)用含有0.1％甲酸的5％-90％MeCN的线性梯度以0.4毫升/分钟的流速在9分钟内进行分析。为了分析抗体轻链和重链，将IgG抗体用50mM TCEP处理并且在37℃加热20分钟，然后用Agilent Poroshell300SB-C8柱(5μm，75mm×1mm)进行LC-MS分析。在60℃进行分析，用含有0.1％甲酸的25％-35％MeCN的线性梯度以0.40毫升/分钟的流速在6分钟内洗脱。以相同方式对PNG酶F处理的抗体糖型进行LC-MS分析，但是包括在TCEP处理之前与PNG酶F一起温育3小时。使用MagTran(Amgen)将原始数据解卷积。
通过野生型Endo-S2将利妥昔单抗脱糖基化以得到(Fucα1,6)GlcNAc-利妥昔单抗
在37℃将市售的利妥昔单抗在它的原始缓冲液中与野生型Endo-S2以500:1(按重量计)的抗体与酶的比率温育1小时。LC-MS分析表明重链上N-聚糖的完全切割。通过蛋白A色谱法将脱糖基化的利妥昔单抗纯化。LC-MS：(Fucα1,6)GlcNAc-利妥昔单抗(2)的重链的计算值，M＝49420Da；实测值(m/z)：49412(解卷积数据)。
通过细菌α-岩藻糖苷酶将(Fucα1,6)GlcNAc-利妥昔单抗脱岩藻糖基化
在37℃将(Fucα1,6)GlcNAc-利妥昔单抗(2)于Tris-HCl缓冲液(50mM，pH 7.4)中的溶液与来自干酪乳杆菌的α-岩藻糖苷酶AlfC以50:1的抗体与酶的比率温育。在温育16小时之后，LC-MS监测指示(Fucα1,6)GlcNAc利妥昔单抗(2)完全脱岩藻糖基化以得到产物GlcNAc-利妥昔单抗(9)。通过蛋白A色谱法将脱岩藻糖基化的利妥昔单抗纯化。LC-MS：携带GlcNAc部分的GlcNAc-利妥昔单抗(9)的重链的计算值，M＝49274Da；实测值(m/z)：49265(解卷积数据)。
酶测定
在30℃用纯的唾液酸复杂型(S2G2F)利妥昔单抗3(10μg，7.0μM)作为底物在PBS缓冲液(pH 7.4，10μl)中对每一种Endo-S2变体(0.1μg)的水解活性进行测定。将每种反应混合物的等分试样在0.1％甲酸中稀释以停止反应并且通过LC-MS分析。在使用MagTran将原始数据解卷积并且对相应的MS峰进行积分之后，定量底物和水解产物的相对量。如下测定用合成糖
唑啉进行的转糖基反应：将(Fucα1,6)GlcNAc-利妥昔单抗(100μg，69μM)、SCTox(1.38mM，20当量)与0.1μg的每种Endo-S2变体在30℃在PBS(pH 7.4，10μl)中温育。如上述水解测定中那样停止反应并且分析。将每种突变体的实验重复至少两次以确保结果的一致性。
通过Endo-S2 D184M用SCT-ox、Hyb-ox以及HM-ox对Fuc1,6GlcNAc利妥昔单抗进行转糖基：3、6、8的合成
在30℃将Fuc1,6GlcNAc-利妥昔单抗(1mg，69μM)(2)和SCTox(1.38mM，20当量)(4)的溶液与Endo-S2 D184M(5μg)在100μl的100mM Tris缓冲液(pH 7.4)中温育15分钟。LC-MS分析指示转糖基反应的完成。使用蛋白A色谱法将产物(3)纯化。LC-MS：携带完全唾液酸化的双分支N-聚糖的(3)的重链的计算值，M＝51421Da；实测值(m/z)：51412(解卷积数据)。
在30℃将Fuc1,6GlcNAc-利妥昔单抗(1mg，69μM)(2)和Hybox(1.38mM，20当量)(5)的溶液与Endo-S2 D184M(5μg)在100μl的100mM Tris缓冲液(pH 7.4)中温育30分钟。然后，再添加10当量的Hybox(5)并且通过等分试样的LC-MS监测反应。当LC-MS分析指示转糖基反应接近完成时，使用蛋白A色谱法将产物(6)纯化。LC-MS：携带唾液酸化杂合型N聚糖的(5)的重链的计算值，M＝51090Da；实测值(m/z)：51082(解卷积数据)。
在30℃将Fuc1,6GlcNAc-利妥昔单抗(1mg，69μM)(2)和HMox(1.38mM，20当量)(7)的溶液与Endo-S2 D184M(5μg)在100μl的100mM Tris缓冲液(pH 7.4)中温育30分钟。然后，再添加10当量的HMox(7)并且通过等分试样的LC-MS监测反应。当LC-MS分析指示转糖基反应完成时，使用蛋白A色谱法将产物(8)纯化。LC-MS：携带高甘露糖型(Man9GlcNAc2)N-聚糖的(12)的重链的计算值，M＝51081Da；实测值(m/z)：51074(解卷积数据)。
通过Endo-S2 D184M用CT-ox对GlcNAc-利妥昔单抗进行转糖基：11的合成
在30℃将GlcNAc-利妥昔单抗(1mg，69μM)(9)和CTox(1.38mM，20当量)(10)的溶液与Endo-S2 D184M(5μg)在100μl的100mM Tris缓冲液(pH 7.4)中温育30分钟。LC-MS分析指示转糖基反应完成。使用蛋白A色谱法将产物(11)纯化。LC-MS：携带双分支N-聚糖的(11)的重链的计算值，M＝50693Da；实测值(m/z)：50684(解卷积数据)。
使用SCT
唑啉、HM
唑啉以及Hyb
唑啉作为供体底物比较Endo-S2 D184M突变体的转糖基活性
在三个单独的反应中，在30℃将Fuc1,6GlcNAc-利妥昔单抗(0.2mg，69μM)(2)连同SCTox(4)、Hyb-ox(5)或HM-ox(7)(1.38mM，20当量)一起与Endo-S2 D184M(1μg)在20μl的100mM Tris缓冲液(pH 7.4)中温育。在几个时间点取出反应的等分试样(0.5μl)并且在0.1％甲酸中稀释以停止反应。通过LC-MS分析所有等分试样并且由解卷积数据计算转糖基％。
使用SCT
唑啉作为供体底物比较Endo-S2 D184M突变体、D184Q突变体和Endo-SD233Q突变体的转糖基活性
在三个单独的反应中，在30℃将Fuc1,6GlcNAc-利妥昔单抗(0.2mg，69μΜ)(2)和SCTox(4)(1.38mM，20当量)与Endo-S2 D184M、Endo-S2 D184Q或Endo-S D233Q(1μg)在20μl的100mM Tris缓冲液(pH 7.4)中温育。在几个时间点取出反应的等分试样(0.5μl)并且在0.1％甲酸中稀释以停止反应。通过LC-MS分析所有等分试样并且由解卷积数据计算转糖基％。
通过Endo-S2 D184M用SCT-ox对Fuc1,6GlcNAc曲妥珠单抗进行转糖基
将市售的曲妥珠单抗(冻干粉末)溶解在水中并且遵循用于利妥昔单抗的相同方法用野生型Endo-S2脱糖基化。对于转糖基，在30℃将Fuc1,6GlcNAc-曲妥珠单抗(12)(1mg，69μM)和SCTox(4)(1.38mM，20当量)的溶液与Endo-S2 D184M(5μg)在100μl的100mM Tris缓冲液(pH 7.4)中温育15分钟。LC-MS分析指示转糖基反应完成。使用蛋白A色谱法将产物(13)纯化。
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表1：使用利妥昔单抗的合成复杂糖型作为底物比较Endo-S2 D184突变体的特异性水解活性。
表2：使用唾液酸聚糖
唑啉作为供体底物和脱糖基化的利妥昔单抗作为受体底物比较Endo-S2 D184突变体的特异性转糖基活性。
序列表
<110> 马里兰大学帕克分校（University of Maryland, College Park）
<120> 作为糖苷合酶的Endo-S2突变体、制备方法以及用于糖蛋白的糖工程化的用途
<130> 035412.305 PCT
<150> 62/279087
<151> 2016-01-15
<160> 24
<170> PatentIn version 3.5
<210> 1
<211> 843
<212> PRT
<213> 化脓性链球菌（Streptococcus pyogenes）
<400> 1
Met Asp Lys His Leu Leu Val Lys Arg Thr Leu Gly Cys Val Cys Ala
1 5 10 15
Ala Thr Leu Met Gly Ala Ala Leu Ala Thr His His Asp Ser Leu Asn
20 25 30
Thr Val Lys Ala Glu Glu Lys Thr Val Gln Thr Gly Lys Thr Asp Gln
35 40 45
Gln Val Gly Ala Lys Leu Val Gln Glu Ile Arg Glu Gly Lys Arg Gly
50 55 60
Pro Leu Tyr Ala Gly Tyr Phe Arg Thr Trp His Asp Arg Ala Ser Thr
65 70 75 80
Gly Ile Asp Gly Lys Gln Gln His Pro Glu Asn Thr Met Ala Glu Val
85 90 95
Pro Lys Glu Val Asp Ile Leu Phe Val Phe His Asp His Thr Ala Ser
100 105 110
Asp Ser Pro Phe Trp Ser Glu Leu Lys Asp Ser Tyr Val His Lys Leu
115 120 125
His Gln Gln Gly Thr Ala Leu Val Gln Thr Ile Gly Val Asn Glu Leu
130 135 140
Asn Gly Arg Thr Gly Leu Ser Lys Asp Tyr Pro Asp Thr Pro Glu Gly
145 150 155 160
Asn Lys Ala Leu Ala Ala Ala Ile Val Lys Ala Phe Val Thr Asp Arg
165 170 175
Gly Val Asp Gly Leu Asp Ile Asp Ile Glu His Glu Phe Thr Asn Lys
180 185 190
Arg Thr Pro Glu Glu Asp Ala Arg Ala Leu Asn Val Phe Lys Glu Ile
195 200 205
Ala Gln Leu Ile Gly Lys Asn Gly Ser Asp Lys Ser Lys Leu Leu Ile
210 215 220
Met Asp Thr Thr Leu Ser Val Glu Asn Asn Pro Ile Phe Lys Gly Ile
225 230 235 240
Ala Glu Asp Leu Asp Tyr Leu Leu Arg Gln Tyr Tyr Gly Ser Gln Gly
245 250 255
Gly Glu Ala Glu Val Asp Thr Ile Asn Ser Asp Trp Asn Gln Tyr Gln
260 265 270
Asn Tyr Ile Asp Ala Ser Gln Phe Met Ile Gly Phe Ser Phe Phe Glu
275 280 285
Glu Ser Ala Ser Lys Gly Asn Leu Trp Phe Asp Val Asn Glu Tyr Asp
290 295 300
Pro Asn Asn Pro Glu Lys Gly Lys Asp Ile Glu Gly Thr Arg Ala Lys
305 310 315 320
Lys Tyr Ala Glu Trp Gln Pro Ser Thr Gly Gly Leu Lys Ala Gly Ile
325 330 335
Phe Ser Tyr Ala Ile Asp Arg Asp Gly Val Ala His Val Pro Ser Thr
340 345 350
Tyr Lys Asn Arg Thr Ser Thr Asn Leu Gln Arg His Glu Val Asp Asn
355 360 365
Ile Ser His Thr Asp Tyr Thr Val Ser Arg Lys Leu Lys Thr Leu Met
370 375 380
Thr Glu Asp Lys Arg Tyr Asp Val Ile Asp Gln Lys Asp Ile Pro Asp
385 390 395 400
Pro Ala Leu Arg Glu Gln Ile Ile Gln Gln Val Gly Gln Tyr Lys Gly
405 410 415
Asp Leu Glu Arg Tyr Asn Lys Thr Leu Val Leu Thr Gly Asp Lys Ile
420 425 430
Gln Asn Leu Lys Gly Leu Glu Lys Leu Ser Lys Leu Gln Lys Leu Glu
435 440 445
Leu Arg Gln Leu Ser Asn Val Lys Glu Ile Thr Pro Glu Leu Leu Pro
450 455 460
Glu Ser Met Lys Lys Asp Ala Glu Leu Val Met Val Gly Met Thr Gly
465 470 475 480
Leu Glu Lys Leu Asn Leu Ser Gly Leu Asn Arg Gln Thr Leu Asp Gly
485 490 495
Ile Asp Val Asn Ser Ile Thr His Leu Thr Ser Phe Asp Ile Ser His
500 505 510
Asn Ser Leu Asp Leu Ser Glu Lys Ser Glu Asp Arg Lys Leu Leu Met
515 520 525
Thr Leu Met Glu Gln Val Ser Asn His Gln Lys Ile Thr Val Lys Asn
530 535 540
Thr Ala Phe Glu Asn Gln Lys Pro Lys Gly Tyr Tyr Pro Gln Thr Tyr
545 550 555 560
Asp Thr Lys Glu Gly His Tyr Asp Val Asp Asn Ala Glu His Asp Ile
565 570 575
Leu Thr Asp Phe Val Phe Gly Thr Val Thr Lys Arg Asn Thr Phe Ile
580 585 590
Gly Asp Glu Glu Ala Phe Ala Ile Tyr Lys Glu Gly Ala Val Asp Gly
595 600 605
Arg Gln Tyr Val Ser Lys Asp Tyr Thr Tyr Glu Ala Phe Arg Lys Asp
610 615 620
Tyr Lys Gly Tyr Lys Val His Leu Thr Ala Ser Asn Leu Gly Glu Thr
625 630 635 640
Val Thr Ser Lys Val Thr Ala Thr Thr Asp Glu Thr Tyr Leu Val Asp
645 650 655
Val Ser Asp Gly Glu Lys Val Val His His Met Lys Leu Asn Ile Gly
660 665 670
Ser Gly Ala Ile Met Met Glu Asn Leu Ala Lys Gly Ala Lys Val Ile
675 680 685
Gly Thr Ser Gly Asp Phe Glu Gln Ala Lys Lys Ile Phe Asp Gly Glu
690 695 700
Lys Ser Asp Arg Phe Phe Thr Trp Gly Gln Thr Asn Trp Ile Ala Phe
705 710 715 720
Asp Leu Gly Glu Ile Asn Leu Ala Lys Glu Trp Arg Leu Phe Asn Ala
725 730 735
Glu Thr Asn Thr Glu Ile Lys Thr Asp Ser Ser Leu Asn Val Ala Lys
740 745 750
Gly Arg Leu Gln Ile Leu Lys Asp Thr Thr Ile Asp Leu Glu Lys Met
755 760 765
Asp Ile Lys Asn Arg Lys Glu Tyr Leu Ser Asn Asp Glu Asn Trp Thr
770 775 780
Asp Val Ala Gln Met Asp Asp Ala Lys Ala Ile Phe Asn Ser Lys Leu
785 790 795 800
Ser Asn Val Leu Ser Arg Tyr Trp Arg Phe Cys Val Asp Gly Gly Ala
805 810 815
Ser Ser Tyr Tyr Pro Gln Tyr Thr Glu Leu Gln Ile Leu Gly Gln Arg
820 825 830
Leu Ser Asn Asp Val Ala Asn Thr Leu Lys Asp
835 840
<210> 2
<211> 843
<212> PRT
<213> 人工序列
<220>
<223> 合成的构建物
<400> 2
Met Asp Lys His Leu Leu Val Lys Arg Thr Leu Gly Cys Val Cys Ala
1 5 10 15
Ala Thr Leu Met Gly Ala Ala Leu Ala Thr His His Asp Ser Leu Asn
20 25 30
Thr Val Lys Ala Glu Glu Lys Thr Val Gln Thr Gly Lys Thr Asp Gln
35 40 45
Gln Val Gly Ala Lys Leu Val Gln Glu Ile Arg Glu Gly Lys Arg Gly
50 55 60
Pro Leu Tyr Ala Gly Tyr Phe Arg Thr Trp His Asp Arg Ala Ser Thr
65 70 75 80
Gly Ile Asp Gly Lys Gln Gln His Pro Glu Asn Thr Met Ala Glu Val
85 90 95
Pro Lys Glu Val Asp Ile Leu Phe Val Phe His Asp His Thr Ala Ser
100 105 110
Asp Ser Pro Phe Trp Ser Glu Leu Lys Asp Ser Tyr Val His Lys Leu
115 120 125
His Gln Gln Gly Thr Ala Leu Val Gln Thr Ile Gly Val Asn Glu Leu
130 135 140
Asn Gly Arg Thr Gly Leu Ser Lys Asp Tyr Pro Asp Thr Pro Glu Gly
145 150 155 160
Asn Lys Ala Leu Ala Ala Ala Ile Val Lys Ala Phe Val Thr Asp Arg
165 170 175
Gly Val Asp Gly Leu Asp Ile Ala Ile Glu His Glu Phe Thr Asn Lys
180 185 190
Arg Thr Pro Glu Glu Asp Ala Arg Ala Leu Asn Val Phe Lys Glu Ile
195 200 205
Ala Gln Leu Ile Gly Lys Asn Gly Ser Asp Lys Ser Lys Leu Leu Ile
210 215 220
Met Asp Thr Thr Leu Ser Val Glu Asn Asn Pro Ile Phe Lys Gly Ile
225 230 235 240
Ala Glu Asp Leu Asp Tyr Leu Leu Arg Gln Tyr Tyr Gly Ser Gln Gly
245 250 255
Gly Glu Ala Glu Val Asp Thr Ile Asn Ser Asp Trp Asn Gln Tyr Gln
260 265 270
Asn Tyr Ile Asp Ala Ser Gln Phe Met Ile Gly Phe Ser Phe Phe Glu
275 280 285
Glu Ser Ala Ser Lys Gly Asn Leu Trp Phe Asp Val Asn Glu Tyr Asp
290 295 300
Pro Asn Asn Pro Glu Lys Gly Lys Asp Ile Glu Gly Thr Arg Ala Lys
305 310 315 320
Lys Tyr Ala Glu Trp Gln Pro Ser Thr Gly Gly Leu Lys Ala Gly Ile
325 330 335
Phe Ser Tyr Ala Ile Asp Arg Asp Gly Val Ala His Val Pro Ser Thr
340 345 350
Tyr Lys Asn Arg Thr Ser Thr Asn Leu Gln Arg His Glu Val Asp Asn
355 360 365
Ile Ser His Thr Asp Tyr Thr Val Ser Arg Lys Leu Lys Thr Leu Met
370 375 380
Thr Glu Asp Lys Arg Tyr Asp Val Ile Asp Gln Lys Asp Ile Pro Asp
385 390 395 400
Pro Ala Leu Arg Glu Gln Ile Ile Gln Gln Val Gly Gln Tyr Lys Gly
405 410 415
Asp Leu Glu Arg Tyr Asn Lys Thr Leu Val Leu Thr Gly Asp Lys Ile
420 425 430
Gln Asn Leu Lys Gly Leu Glu Lys Leu Ser Lys Leu Gln Lys Leu Glu
435 440 445
Leu Arg Gln Leu Ser Asn Val Lys Glu Ile Thr Pro Glu Leu Leu Pro
450 455 460
Glu Ser Met Lys Lys Asp Ala Glu Leu Val Met Val Gly Met Thr Gly
465 470 475 480
Leu Glu Lys Leu Asn Leu Ser Gly Leu Asn Arg Gln Thr Leu Asp Gly
485 490 495
Ile Asp Val Asn Ser Ile Thr His Leu Thr Ser Phe Asp Ile Ser His
500 505 510
Asn Ser Leu Asp Leu Ser Glu Lys Ser Glu Asp Arg Lys Leu Leu Met
515 520 525
Thr Leu Met Glu Gln Val Ser Asn His Gln Lys Ile Thr Val Lys Asn
530 535 540
Thr Ala Phe Glu Asn Gln Lys Pro Lys Gly Tyr Tyr Pro Gln Thr Tyr
545 550 555 560
Asp Thr Lys Glu Gly His Tyr Asp Val Asp Asn Ala Glu His Asp Ile
565 570 575
Leu Thr Asp Phe Val Phe Gly Thr Val Thr Lys Arg Asn Thr Phe Ile
580 585 590
Gly Asp Glu Glu Ala Phe Ala Ile Tyr Lys Glu Gly Ala Val Asp Gly
595 600 605
Arg Gln Tyr Val Ser Lys Asp Tyr Thr Tyr Glu Ala Phe Arg Lys Asp
610 615 620
Tyr Lys Gly Tyr Lys Val His Leu Thr Ala Ser Asn Leu Gly Glu Thr
625 630 635 640
Val Thr Ser Lys Val Thr Ala Thr Thr Asp Glu Thr Tyr Leu Val Asp
645 650 655
Val Ser Asp Gly Glu Lys Val Val His His Met Lys Leu Asn Ile Gly
660 665 670
Ser Gly Ala Ile Met Met Glu Asn Leu Ala Lys Gly Ala Lys Val Ile
675 680 685
Gly Thr Ser Gly Asp Phe Glu Gln Ala Lys Lys Ile Phe Asp Gly Glu
690 695 700
Lys Ser Asp Arg Phe Phe Thr Trp Gly Gln Thr Asn Trp Ile Ala Phe
705 710 715 720
Asp Leu Gly Glu Ile Asn Leu Ala Lys Glu Trp Arg Leu Phe Asn Ala
725 730 735
Glu Thr Asn Thr Glu Ile Lys Thr Asp Ser Ser Leu Asn Val Ala Lys
740 745 750
Gly Arg Leu Gln Ile Leu Lys Asp Thr Thr Ile Asp Leu Glu Lys Met
755 760 765
Asp Ile Lys Asn Arg Lys Glu Tyr Leu Ser Asn Asp Glu Asn Trp Thr
770 775 780
Asp Val Ala Gln Met Asp Asp Ala Lys Ala Ile Phe Asn Ser Lys Leu
785 790 795 800
Ser Asn Val Leu Ser Arg Tyr Trp Arg Phe Cys Val Asp Gly Gly Ala
805 810 815
Ser Ser Tyr Tyr Pro Gln Tyr Thr Glu Leu Gln Ile Leu Gly Gln Arg
820 825 830
Leu Ser Asn Asp Val Ala Asn Thr Leu Lys Asp
835 840
<210> 3
<211> 843
<212> PRT
<213> 人工序列
<220>
<223> 合成的构建物
<400> 3
Met Asp Lys His Leu Leu Val Lys Arg Thr Leu Gly Cys Val Cys Ala
1 5 10 15
Ala Thr Leu Met Gly Ala Ala Leu Ala Thr His His Asp Ser Leu Asn
20 25 30
Thr Val Lys Ala Glu Glu Lys Thr Val Gln Thr Gly Lys Thr Asp Gln
35 40 45
Gln Val Gly Ala Lys Leu Val Gln Glu Ile Arg Glu Gly Lys Arg Gly
50 55 60
Pro Leu Tyr Ala Gly Tyr Phe Arg Thr Trp His Asp Arg Ala Ser Thr
65 70 75 80
Gly Ile Asp Gly Lys Gln Gln His Pro Glu Asn Thr Met Ala Glu Val
85 90 95
Pro Lys Glu Val Asp Ile Leu Phe Val Phe His Asp His Thr Ala Ser
100 105 110
Asp Ser Pro Phe Trp Ser Glu Leu Lys Asp Ser Tyr Val His Lys Leu
115 120 125
His Gln Gln Gly Thr Ala Leu Val Gln Thr Ile Gly Val Asn Glu Leu
130 135 140
Asn Gly Arg Thr Gly Leu Ser Lys Asp Tyr Pro Asp Thr Pro Glu Gly
145 150 155 160
Asn Lys Ala Leu Ala Ala Ala Ile Val Lys Ala Phe Val Thr Asp Arg
165 170 175
Gly Val Asp Gly Leu Asp Ile Asn Ile Glu His Glu Phe Thr Asn Lys
180 185 190
Arg Thr Pro Glu Glu Asp Ala Arg Ala Leu Asn Val Phe Lys Glu Ile
195 200 205
Ala Gln Leu Ile Gly Lys Asn Gly Ser Asp Lys Ser Lys Leu Leu Ile
210 215 220
Met Asp Thr Thr Leu Ser Val Glu Asn Asn Pro Ile Phe Lys Gly Ile
225 230 235 240
Ala Glu Asp Leu Asp Tyr Leu Leu Arg Gln Tyr Tyr Gly Ser Gln Gly
245 250 255
Gly Glu Ala Glu Val Asp Thr Ile Asn Ser Asp Trp Asn Gln Tyr Gln
260 265 270
Asn Tyr Ile Asp Ala Ser Gln Phe Met Ile Gly Phe Ser Phe Phe Glu
275 280 285
Glu Ser Ala Ser Lys Gly Asn Leu Trp Phe Asp Val Asn Glu Tyr Asp
290 295 300
Pro Asn Asn Pro Glu Lys Gly Lys Asp Ile Glu Gly Thr Arg Ala Lys
305 310 315 320
Lys Tyr Ala Glu Trp Gln Pro Ser Thr Gly Gly Leu Lys Ala Gly Ile
325 330 335
Phe Ser Tyr Ala Ile Asp Arg Asp Gly Val Ala His Val Pro Ser Thr
340 345 350
Tyr Lys Asn Arg Thr Ser Thr Asn Leu Gln Arg His Glu Val Asp Asn
355 360 365
Ile Ser His Thr Asp Tyr Thr Val Ser Arg Lys Leu Lys Thr Leu Met
370 375 380
Thr Glu Asp Lys Arg Tyr Asp Val Ile Asp Gln Lys Asp Ile Pro Asp
385 390 395 400
Pro Ala Leu Arg Glu Gln Ile Ile Gln Gln Val Gly Gln Tyr Lys Gly
405 410 415
Asp Leu Glu Arg Tyr Asn Lys Thr Leu Val Leu Thr Gly Asp Lys Ile
420 425 430
Gln Asn Leu Lys Gly Leu Glu Lys Leu Ser Lys Leu Gln Lys Leu Glu
435 440 445
Leu Arg Gln Leu Ser Asn Val Lys Glu Ile Thr Pro Glu Leu Leu Pro
450 455 460
Glu Ser Met Lys Lys Asp Ala Glu Leu Val Met Val Gly Met Thr Gly
465 470 475 480
Leu Glu Lys Leu Asn Leu Ser Gly Leu Asn Arg Gln Thr Leu Asp Gly
485 490 495
Ile Asp Val Asn Ser Ile Thr His Leu Thr Ser Phe Asp Ile Ser His
500 505 510
Asn Ser Leu Asp Leu Ser Glu Lys Ser Glu Asp Arg Lys Leu Leu Met
515 520 525
Thr Leu Met Glu Gln Val Ser Asn His Gln Lys Ile Thr Val Lys Asn
530 535 540
Thr Ala Phe Glu Asn Gln Lys Pro Lys Gly Tyr Tyr Pro Gln Thr Tyr
545 550 555 560
Asp Thr Lys Glu Gly His Tyr Asp Val Asp Asn Ala Glu His Asp Ile
565 570 575
Leu Thr Asp Phe Val Phe Gly Thr Val Thr Lys Arg Asn Thr Phe Ile
580 585 590
Gly Asp Glu Glu Ala Phe Ala Ile Tyr Lys Glu Gly Ala Val Asp Gly
595 600 605
Arg Gln Tyr Val Ser Lys Asp Tyr Thr Tyr Glu Ala Phe Arg Lys Asp
610 615 620
Tyr Lys Gly Tyr Lys Val His Leu Thr Ala Ser Asn Leu Gly Glu Thr
625 630 635 640
Val Thr Ser Lys Val Thr Ala Thr Thr Asp Glu Thr Tyr Leu Val Asp
645 650 655
Val Ser Asp Gly Glu Lys Val Val His His Met Lys Leu Asn Ile Gly
660 665 670
Ser Gly Ala Ile Met Met Glu Asn Leu Ala Lys Gly Ala Lys Val Ile
675 680 685
Gly Thr Ser Gly Asp Phe Glu Gln Ala Lys Lys Ile Phe Asp Gly Glu
690 695 700
Lys Ser Asp Arg Phe Phe Thr Trp Gly Gln Thr Asn Trp Ile Ala Phe
705 710 715 720
Asp Leu Gly Glu Ile Asn Leu Ala Lys Glu Trp Arg Leu Phe Asn Ala
725 730 735
Glu Thr Asn Thr Glu Ile Lys Thr Asp Ser Ser Leu Asn Val Ala Lys
740 745 750
Gly Arg Leu Gln Ile Leu Lys Asp Thr Thr Ile Asp Leu Glu Lys Met
755 760 765
Asp Ile Lys Asn Arg Lys Glu Tyr Leu Ser Asn Asp Glu Asn Trp Thr
770 775 780
Asp Val Ala Gln Met Asp Asp Ala Lys Ala Ile Phe Asn Ser Lys Leu
785 790 795 800
Ser Asn Val Leu Ser Arg Tyr Trp Arg Phe Cys Val Asp Gly Gly Ala
805 810 815
Ser Ser Tyr Tyr Pro Gln Tyr Thr Glu Leu Gln Ile Leu Gly Gln Arg
820 825 830
Leu Ser Asn Asp Val Ala Asn Thr Leu Lys Asp
835 840
<210> 4
<211> 843
<212> PRT
<213> 人工序列
<220>
<223> 合成的构建物
<400> 4
Met Asp Lys His Leu Leu Val Lys Arg Thr Leu Gly Cys Val Cys Ala
1 5 10 15
Ala Thr Leu Met Gly Ala Ala Leu Ala Thr His His Asp Ser Leu Asn
20 25 30
Thr Val Lys Ala Glu Glu Lys Thr Val Gln Thr Gly Lys Thr Asp Gln
35 40 45
Gln Val Gly Ala Lys Leu Val Gln Glu Ile Arg Glu Gly Lys Arg Gly
50 55 60
Pro Leu Tyr Ala Gly Tyr Phe Arg Thr Trp His Asp Arg Ala Ser Thr
65 70 75 80
Gly Ile Asp Gly Lys Gln Gln His Pro Glu Asn Thr Met Ala Glu Val
85 90 95
Pro Lys Glu Val Asp Ile Leu Phe Val Phe His Asp His Thr Ala Ser
100 105 110
Asp Ser Pro Phe Trp Ser Glu Leu Lys Asp Ser Tyr Val His Lys Leu
115 120 125
His Gln Gln Gly Thr Ala Leu Val Gln Thr Ile Gly Val Asn Glu Leu
130 135 140
Asn Gly Arg Thr Gly Leu Ser Lys Asp Tyr Pro Asp Thr Pro Glu Gly
145 150 155 160
Asn Lys Ala Leu Ala Ala Ala Ile Val Lys Ala Phe Val Thr Asp Arg
165 170 175
Gly Val Asp Gly Leu Asp Ile Gln Ile Glu His Glu Phe Thr Asn Lys
180 185 190
Arg Thr Pro Glu Glu Asp Ala Arg Ala Leu Asn Val Phe Lys Glu Ile
195 200 205
Ala Gln Leu Ile Gly Lys Asn Gly Ser Asp Lys Ser Lys Leu Leu Ile
210 215 220
Met Asp Thr Thr Leu Ser Val Glu Asn Asn Pro Ile Phe Lys Gly Ile
225 230 235 240
Ala Glu Asp Leu Asp Tyr Leu Leu Arg Gln Tyr Tyr Gly Ser Gln Gly
245 250 255
Gly Glu Ala Glu Val Asp Thr Ile Asn Ser Asp Trp Asn Gln Tyr Gln
260 265 270
Asn Tyr Ile Asp Ala Ser Gln Phe Met Ile Gly Phe Ser Phe Phe Glu
275 280 285
Glu Ser Ala Ser Lys Gly Asn Leu Trp Phe Asp Val Asn Glu Tyr Asp
290 295 300
Pro Asn Asn Pro Glu Lys Gly Lys Asp Ile Glu Gly Thr Arg Ala Lys
305 310 315 320
Lys Tyr Ala Glu Trp Gln Pro Ser Thr Gly Gly Leu Lys Ala Gly Ile
325 330 335
Phe Ser Tyr Ala Ile Asp Arg Asp Gly Val Ala His Val Pro Ser Thr
340 345 350
Tyr Lys Asn Arg Thr Ser Thr Asn Leu Gln Arg His Glu Val Asp Asn
355 360 365
Ile Ser His Thr Asp Tyr Thr Val Ser Arg Lys Leu Lys Thr Leu Met
370 375 380
Thr Glu Asp Lys Arg Tyr Asp Val Ile Asp Gln Lys Asp Ile Pro Asp
385 390 395 400
Pro Ala Leu Arg Glu Gln Ile Ile Gln Gln Val Gly Gln Tyr Lys Gly
405 410 415
Asp Leu Glu Arg Tyr Asn Lys Thr Leu Val Leu Thr Gly Asp Lys Ile
420 425 430
Gln Asn Leu Lys Gly Leu Glu Lys Leu Ser Lys Leu Gln Lys Leu Glu
435 440 445
Leu Arg Gln Leu Ser Asn Val Lys Glu Ile Thr Pro Glu Leu Leu Pro
450 455 460
Glu Ser Met Lys Lys Asp Ala Glu Leu Val Met Val Gly Met Thr Gly
465 470 475 480
Leu Glu Lys Leu Asn Leu Ser Gly Leu Asn Arg Gln Thr Leu Asp Gly
485 490 495
Ile Asp Val Asn Ser Ile Thr His Leu Thr Ser Phe Asp Ile Ser His
500 505 510
Asn Ser Leu Asp Leu Ser Glu Lys Ser Glu Asp Arg Lys Leu Leu Met
515 520 525
Thr Leu Met Glu Gln Val Ser Asn His Gln Lys Ile Thr Val Lys Asn
530 535 540
Thr Ala Phe Glu Asn Gln Lys Pro Lys Gly Tyr Tyr Pro Gln Thr Tyr
545 550 555 560
Asp Thr Lys Glu Gly His Tyr Asp Val Asp Asn Ala Glu His Asp Ile
565 570 575
Leu Thr Asp Phe Val Phe Gly Thr Val Thr Lys Arg Asn Thr Phe Ile
580 585 590
Gly Asp Glu Glu Ala Phe Ala Ile Tyr Lys Glu Gly Ala Val Asp Gly
595 600 605
Arg Gln Tyr Val Ser Lys Asp Tyr Thr Tyr Glu Ala Phe Arg Lys Asp
610 615 620
Tyr Lys Gly Tyr Lys Val His Leu Thr Ala Ser Asn Leu Gly Glu Thr
625 630 635 640
Val Thr Ser Lys Val Thr Ala Thr Thr Asp Glu Thr Tyr Leu Val Asp
645 650 655
Val Ser Asp Gly Glu Lys Val Val His His Met Lys Leu Asn Ile Gly
660 665 670
Ser Gly Ala Ile Met Met Glu Asn Leu Ala Lys Gly Ala Lys Val Ile
675 680 685
Gly Thr Ser Gly Asp Phe Glu Gln Ala Lys Lys Ile Phe Asp Gly Glu
690 695 700
Lys Ser Asp Arg Phe Phe Thr Trp Gly Gln Thr Asn Trp Ile Ala Phe
705 710 715 720
Asp Leu Gly Glu Ile Asn Leu Ala Lys Glu Trp Arg Leu Phe Asn Ala
725 730 735
Glu Thr Asn Thr Glu Ile Lys Thr Asp Ser Ser Leu Asn Val Ala Lys
740 745 750
Gly Arg Leu Gln Ile Leu Lys Asp Thr Thr Ile Asp Leu Glu Lys Met
755 760 765
Asp Ile Lys Asn Arg Lys Glu Tyr Leu Ser Asn Asp Glu Asn Trp Thr
770 775 780
Asp Val Ala Gln Met Asp Asp Ala Lys Ala Ile Phe Asn Ser Lys Leu
785 790 795 800
Ser Asn Val Leu Ser Arg Tyr Trp Arg Phe Cys Val Asp Gly Gly Ala
805 810 815
Ser Ser Tyr Tyr Pro Gln Tyr Thr Glu Leu Gln Ile Leu Gly Gln Arg
820 825 830
Leu Ser Asn Asp Val Ala Asn Thr Leu Lys Asp
835 840
<210> 5
<211> 843
<212> PRT
<213> 人工序列
<220>
<223> 合成的构建物
<400> 5
Met Asp Lys His Leu Leu Val Lys Arg Thr Leu Gly Cys Val Cys Ala
1 5 10 15
Ala Thr Leu Met Gly Ala Ala Leu Ala Thr His His Asp Ser Leu Asn
20 25 30
Thr Val Lys Ala Glu Glu Lys Thr Val Gln Thr Gly Lys Thr Asp Gln
35 40 45
Gln Val Gly Ala Lys Leu Val Gln Glu Ile Arg Glu Gly Lys Arg Gly
50 55 60
Pro Leu Tyr Ala Gly Tyr Phe Arg Thr Trp His Asp Arg Ala Ser Thr
65 70 75 80
Gly Ile Asp Gly Lys Gln Gln His Pro Glu Asn Thr Met Ala Glu Val
85 90 95
Pro Lys Glu Val Asp Ile Leu Phe Val Phe His Asp His Thr Ala Ser
100 105 110
Asp Ser Pro Phe Trp Ser Glu Leu Lys Asp Ser Tyr Val His Lys Leu
115 120 125
His Gln Gln Gly Thr Ala Leu Val Gln Thr Ile Gly Val Asn Glu Leu
130 135 140
Asn Gly Arg Thr Gly Leu Ser Lys Asp Tyr Pro Asp Thr Pro Glu Gly
145 150 155 160
Asn Lys Ala Leu Ala Ala Ala Ile Val Lys Ala Phe Val Thr Asp Arg
165 170 175
Gly Val Asp Gly Leu Asp Ile Arg Ile Glu His Glu Phe Thr Asn Lys
180 185 190
Arg Thr Pro Glu Glu Asp Ala Arg Ala Leu Asn Val Phe Lys Glu Ile
195 200 205
Ala Gln Leu Ile Gly Lys Asn Gly Ser Asp Lys Ser Lys Leu Leu Ile
210 215 220
Met Asp Thr Thr Leu Ser Val Glu Asn Asn Pro Ile Phe Lys Gly Ile
225 230 235 240
Ala Glu Asp Leu Asp Tyr Leu Leu Arg Gln Tyr Tyr Gly Ser Gln Gly
245 250 255
Gly Glu Ala Glu Val Asp Thr Ile Asn Ser Asp Trp Asn Gln Tyr Gln
260 265 270
Asn Tyr Ile Asp Ala Ser Gln Phe Met Ile Gly Phe Ser Phe Phe Glu
275 280 285
Glu Ser Ala Ser Lys Gly Asn Leu Trp Phe Asp Val Asn Glu Tyr Asp
290 295 300
Pro Asn Asn Pro Glu Lys Gly Lys Asp Ile Glu Gly Thr Arg Ala Lys
305 310 315 320
Lys Tyr Ala Glu Trp Gln Pro Ser Thr Gly Gly Leu Lys Ala Gly Ile
325 330 335
Phe Ser Tyr Ala Ile Asp Arg Asp Gly Val Ala His Val Pro Ser Thr
340 345 350
Tyr Lys Asn Arg Thr Ser Thr Asn Leu Gln Arg His Glu Val Asp Asn
355 360 365
Ile Ser His Thr Asp Tyr Thr Val Ser Arg Lys Leu Lys Thr Leu Met
370 375 380
Thr Glu Asp Lys Arg Tyr Asp Val Ile Asp Gln Lys Asp Ile Pro Asp
385 390 395 400
Pro Ala Leu Arg Glu Gln Ile Ile Gln Gln Val Gly Gln Tyr Lys Gly
405 410 415
Asp Leu Glu Arg Tyr Asn Lys Thr Leu Val Leu Thr Gly Asp Lys Ile
420 425 430
Gln Asn Leu Lys Gly Leu Glu Lys Leu Ser Lys Leu Gln Lys Leu Glu
435 440 445
Leu Arg Gln Leu Ser Asn Val Lys Glu Ile Thr Pro Glu Leu Leu Pro
450 455 460
Glu Ser Met Lys Lys Asp Ala Glu Leu Val Met Val Gly Met Thr Gly
465 470 475 480
Leu Glu Lys Leu Asn Leu Ser Gly Leu Asn Arg Gln Thr Leu Asp Gly
485 490 495
Ile Asp Val Asn Ser Ile Thr His Leu Thr Ser Phe Asp Ile Ser His
500 505 510
Asn Ser Leu Asp Leu Ser Glu Lys Ser Glu Asp Arg Lys Leu Leu Met
515 520 525
Thr Leu Met Glu Gln Val Ser Asn His Gln Lys Ile Thr Val Lys Asn
530 535 540
Thr Ala Phe Glu Asn Gln Lys Pro Lys Gly Tyr Tyr Pro Gln Thr Tyr
545 550 555 560
Asp Thr Lys Glu Gly His Tyr Asp Val Asp Asn Ala Glu His Asp Ile
565 570 575
Leu Thr Asp Phe Val Phe Gly Thr Val Thr Lys Arg Asn Thr Phe Ile
580 585 590
Gly Asp Glu Glu Ala Phe Ala Ile Tyr Lys Glu Gly Ala Val Asp Gly
595 600 605
Arg Gln Tyr Val Ser Lys Asp Tyr Thr Tyr Glu Ala Phe Arg Lys Asp
610 615 620
Tyr Lys Gly Tyr Lys Val His Leu Thr Ala Ser Asn Leu Gly Glu Thr
625 630 635 640
Val Thr Ser Lys Val Thr Ala Thr Thr Asp Glu Thr Tyr Leu Val Asp
645 650 655
Val Ser Asp Gly Glu Lys Val Val His His Met Lys Leu Asn Ile Gly
660 665 670
Ser Gly Ala Ile Met Met Glu Asn Leu Ala Lys Gly Ala Lys Val Ile
675 680 685
Gly Thr Ser Gly Asp Phe Glu Gln Ala Lys Lys Ile Phe Asp Gly Glu
690 695 700
Lys Ser Asp Arg Phe Phe Thr Trp Gly Gln Thr Asn Trp Ile Ala Phe
705 710 715 720
Asp Leu Gly Glu Ile Asn Leu Ala Lys Glu Trp Arg Leu Phe Asn Ala
725 730 735
Glu Thr Asn Thr Glu Ile Lys Thr Asp Ser Ser Leu Asn Val Ala Lys
740 745 750
Gly Arg Leu Gln Ile Leu Lys Asp Thr Thr Ile Asp Leu Glu Lys Met
755 760 765
Asp Ile Lys Asn Arg Lys Glu Tyr Leu Ser Asn Asp Glu Asn Trp Thr
770 775 780
Asp Val Ala Gln Met Asp Asp Ala Lys Ala Ile Phe Asn Ser Lys Leu
785 790 795 800
Ser Asn Val Leu Ser Arg Tyr Trp Arg Phe Cys Val Asp Gly Gly Ala
805 810 815
Ser Ser Tyr Tyr Pro Gln Tyr Thr Glu Leu Gln Ile Leu Gly Gln Arg
820 825 830
Leu Ser Asn Asp Val Ala Asn Thr Leu Lys Asp
835 840
<210> 6
<211> 843
<212> PRT
<213> 人工序列
<220>
<223> 合成的构建物
<400> 6
Met Asp Lys His Leu Leu Val Lys Arg Thr Leu Gly Cys Val Cys Ala
1 5 10 15
Ala Thr Leu Met Gly Ala Ala Leu Ala Thr His His Asp Ser Leu Asn
20 25 30
Thr Val Lys Ala Glu Glu Lys Thr Val Gln Thr Gly Lys Thr Asp Gln
35 40 45
Gln Val Gly Ala Lys Leu Val Gln Glu Ile Arg Glu Gly Lys Arg Gly
50 55 60
Pro Leu Tyr Ala Gly Tyr Phe Arg Thr Trp His Asp Arg Ala Ser Thr
65 70 75 80
Gly Ile Asp Gly Lys Gln Gln His Pro Glu Asn Thr Met Ala Glu Val
85 90 95
Pro Lys Glu Val Asp Ile Leu Phe Val Phe His Asp His Thr Ala Ser
100 105 110
Asp Ser Pro Phe Trp Ser Glu Leu Lys Asp Ser Tyr Val His Lys Leu
115 120 125
His Gln Gln Gly Thr Ala Leu Val Gln Thr Ile Gly Val Asn Glu Leu
130 135 140
Asn Gly Arg Thr Gly Leu Ser Lys Asp Tyr Pro Asp Thr Pro Glu Gly
145 150 155 160
Asn Lys Ala Leu Ala Ala Ala Ile Val Lys Ala Phe Val Thr Asp Arg
165 170 175
Gly Val Asp Gly Leu Asp Ile Cys Ile Glu His Glu Phe Thr Asn Lys
180 185 190
Arg Thr Pro Glu Glu Asp Ala Arg Ala Leu Asn Val Phe Lys Glu Ile
195 200 205
Ala Gln Leu Ile Gly Lys Asn Gly Ser Asp Lys Ser Lys Leu Leu Ile
210 215 220
Met Asp Thr Thr Leu Ser Val Glu Asn Asn Pro Ile Phe Lys Gly Ile
225 230 235 240
Ala Glu Asp Leu Asp Tyr Leu Leu Arg Gln Tyr Tyr Gly Ser Gln Gly
245 250 255
Gly Glu Ala Glu Val Asp Thr Ile Asn Ser Asp Trp Asn Gln Tyr Gln
260 265 270
Asn Tyr Ile Asp Ala Ser Gln Phe Met Ile Gly Phe Ser Phe Phe Glu
275 280 285
Glu Ser Ala Ser Lys Gly Asn Leu Trp Phe Asp Val Asn Glu Tyr Asp
290 295 300
Pro Asn Asn Pro Glu Lys Gly Lys Asp Ile Glu Gly Thr Arg Ala Lys
305 310 315 320
Lys Tyr Ala Glu Trp Gln Pro Ser Thr Gly Gly Leu Lys Ala Gly Ile
325 330 335
Phe Ser Tyr Ala Ile Asp Arg Asp Gly Val Ala His Val Pro Ser Thr
340 345 350
Tyr Lys Asn Arg Thr Ser Thr Asn Leu Gln Arg His Glu Val Asp Asn
355 360 365
Ile Ser His Thr Asp Tyr Thr Val Ser Arg Lys Leu Lys Thr Leu Met
370 375 380
Thr Glu Asp Lys Arg Tyr Asp Val Ile Asp Gln Lys Asp Ile Pro Asp
385 390 395 400
Pro Ala Leu Arg Glu Gln Ile Ile Gln Gln Val Gly Gln Tyr Lys Gly
405 410 415
Asp Leu Glu Arg Tyr Asn Lys Thr Leu Val Leu Thr Gly Asp Lys Ile
420 425 430
Gln Asn Leu Lys Gly Leu Glu Lys Leu Ser Lys Leu Gln Lys Leu Glu
435 440 445
Leu Arg Gln Leu Ser Asn Val Lys Glu Ile Thr Pro Glu Leu Leu Pro
450 455 460
Glu Ser Met Lys Lys Asp Ala Glu Leu Val Met Val Gly Met Thr Gly
465 470 475 480
Leu Glu Lys Leu Asn Leu Ser Gly Leu Asn Arg Gln Thr Leu Asp Gly
485 490 495
Ile Asp Val Asn Ser Ile Thr His Leu Thr Ser Phe Asp Ile Ser His
500 505 510
Asn Ser Leu Asp Leu Ser Glu Lys Ser Glu Asp Arg Lys Leu Leu Met
515 520 525
Thr Leu Met Glu Gln Val Ser Asn His Gln Lys Ile Thr Val Lys Asn
530 535 540
Thr Ala Phe Glu Asn Gln Lys Pro Lys Gly Tyr Tyr Pro Gln Thr Tyr
545 550 555 560
Asp Thr Lys Glu Gly His Tyr Asp Val Asp Asn Ala Glu His Asp Ile
565 570 575
Leu Thr Asp Phe Val Phe Gly Thr Val Thr Lys Arg Asn Thr Phe Ile
580 585 590
Gly Asp Glu Glu Ala Phe Ala Ile Tyr Lys Glu Gly Ala Val Asp Gly
595 600 605
Arg Gln Tyr Val Ser Lys Asp Tyr Thr Tyr Glu Ala Phe Arg Lys Asp
610 615 620
Tyr Lys Gly Tyr Lys Val His Leu Thr Ala Ser Asn Leu Gly Glu Thr
625 630 635 640
Val Thr Ser Lys Val Thr Ala Thr Thr Asp Glu Thr Tyr Leu Val Asp
645 650 655
Val Ser Asp Gly Glu Lys Val Val His His Met Lys Leu Asn Ile Gly
660 665 670
Ser Gly Ala Ile Met Met Glu Asn Leu Ala Lys Gly Ala Lys Val Ile
675 680 685
Gly Thr Ser Gly Asp Phe Glu Gln Ala Lys Lys Ile Phe Asp Gly Glu
690 695 700
Lys Ser Asp Arg Phe Phe Thr Trp Gly Gln Thr Asn Trp Ile Ala Phe
705 710 715 720
Asp Leu Gly Glu Ile Asn Leu Ala Lys Glu Trp Arg Leu Phe Asn Ala
725 730 735
Glu Thr Asn Thr Glu Ile Lys Thr Asp Ser Ser Leu Asn Val Ala Lys
740 745 750
Gly Arg Leu Gln Ile Leu Lys Asp Thr Thr Ile Asp Leu Glu Lys Met
755 760 765
Asp Ile Lys Asn Arg Lys Glu Tyr Leu Ser Asn Asp Glu Asn Trp Thr
770 775 780
Asp Val Ala Gln Met Asp Asp Ala Lys Ala Ile Phe Asn Ser Lys Leu
785 790 795 800
Ser Asn Val Leu Ser Arg Tyr Trp Arg Phe Cys Val Asp Gly Gly Ala
805 810 815
Ser Ser Tyr Tyr Pro Gln Tyr Thr Glu Leu Gln Ile Leu Gly Gln Arg
820 825 830
Leu Ser Asn Asp Val Ala Asn Thr Leu Lys Asp
835 840
<210> 7
<211> 843
<212> PRT
<213> 人工序列
<220>
<223> 合成的构建物
<400> 7
Met Asp Lys His Leu Leu Val Lys Arg Thr Leu Gly Cys Val Cys Ala
1 5 10 15
Ala Thr Leu Met Gly Ala Ala Leu Ala Thr His His Asp Ser Leu Asn
20 25 30
Thr Val Lys Ala Glu Glu Lys Thr Val Gln Thr Gly Lys Thr Asp Gln
35 40 45
Gln Val Gly Ala Lys Leu Val Gln Glu Ile Arg Glu Gly Lys Arg Gly
50 55 60
Pro Leu Tyr Ala Gly Tyr Phe Arg Thr Trp His Asp Arg Ala Ser Thr
65 70 75 80
Gly Ile Asp Gly Lys Gln Gln His Pro Glu Asn Thr Met Ala Glu Val
85 90 95
Pro Lys Glu Val Asp Ile Leu Phe Val Phe His Asp His Thr Ala Ser
100 105 110
Asp Ser Pro Phe Trp Ser Glu Leu Lys Asp Ser Tyr Val His Lys Leu
115 120 125
His Gln Gln Gly Thr Ala Leu Val Gln Thr Ile Gly Val Asn Glu Leu
130 135 140
Asn Gly Arg Thr Gly Leu Ser Lys Asp Tyr Pro Asp Thr Pro Glu Gly
145 150 155 160
Asn Lys Ala Leu Ala Ala Ala Ile Val Lys Ala Phe Val Thr Asp Arg
165 170 175
Gly Val Asp Gly Leu Asp Ile Met Ile Glu His Glu Phe Thr Asn Lys
180 185 190
Arg Thr Pro Glu Glu Asp Ala Arg Ala Leu Asn Val Phe Lys Glu Ile
195 200 205
Ala Gln Leu Ile Gly Lys Asn Gly Ser Asp Lys Ser Lys Leu Leu Ile
210 215 220
Met Asp Thr Thr Leu Ser Val Glu Asn Asn Pro Ile Phe Lys Gly Ile
225 230 235 240
Ala Glu Asp Leu Asp Tyr Leu Leu Arg Gln Tyr Tyr Gly Ser Gln Gly
245 250 255
Gly Glu Ala Glu Val Asp Thr Ile Asn Ser Asp Trp Asn Gln Tyr Gln
260 265 270
Asn Tyr Ile Asp Ala Ser Gln Phe Met Ile Gly Phe Ser Phe Phe Glu
275 280 285
Glu Ser Ala Ser Lys Gly Asn Leu Trp Phe Asp Val Asn Glu Tyr Asp
290 295 300
Pro Asn Asn Pro Glu Lys Gly Lys Asp Ile Glu Gly Thr Arg Ala Lys
305 310 315 320
Lys Tyr Ala Glu Trp Gln Pro Ser Thr Gly Gly Leu Lys Ala Gly Ile
325 330 335
Phe Ser Tyr Ala Ile Asp Arg Asp Gly Val Ala His Val Pro Ser Thr
340 345 350
Tyr Lys Asn Arg Thr Ser Thr Asn Leu Gln Arg His Glu Val Asp Asn
355 360 365
Ile Ser His Thr Asp Tyr Thr Val Ser Arg Lys Leu Lys Thr Leu Met
370 375 380
Thr Glu Asp Lys Arg Tyr Asp Val Ile Asp Gln Lys Asp Ile Pro Asp
385 390 395 400
Pro Ala Leu Arg Glu Gln Ile Ile Gln Gln Val Gly Gln Tyr Lys Gly
405 410 415
Asp Leu Glu Arg Tyr Asn Lys Thr Leu Val Leu Thr Gly Asp Lys Ile
420 425 430
Gln Asn Leu Lys Gly Leu Glu Lys Leu Ser Lys Leu Gln Lys Leu Glu
435 440 445
Leu Arg Gln Leu Ser Asn Val Lys Glu Ile Thr Pro Glu Leu Leu Pro
450 455 460
Glu Ser Met Lys Lys Asp Ala Glu Leu Val Met Val Gly Met Thr Gly
465 470 475 480
Leu Glu Lys Leu Asn Leu Ser Gly Leu Asn Arg Gln Thr Leu Asp Gly
485 490 495
Ile Asp Val Asn Ser Ile Thr His Leu Thr Ser Phe Asp Ile Ser His
500 505 510
Asn Ser Leu Asp Leu Ser Glu Lys Ser Glu Asp Arg Lys Leu Leu Met
515 520 525
Thr Leu Met Glu Gln Val Ser Asn His Gln Lys Ile Thr Val Lys Asn
530 535 540
Thr Ala Phe Glu Asn Gln Lys Pro Lys Gly Tyr Tyr Pro Gln Thr Tyr
545 550 555 560
Asp Thr Lys Glu Gly His Tyr Asp Val Asp Asn Ala Glu His Asp Ile
565 570 575
Leu Thr Asp Phe Val Phe Gly Thr Val Thr Lys Arg Asn Thr Phe Ile
580 585 590
Gly Asp Glu Glu Ala Phe Ala Ile Tyr Lys Glu Gly Ala Val Asp Gly
595 600 605
Arg Gln Tyr Val Ser Lys Asp Tyr Thr Tyr Glu Ala Phe Arg Lys Asp
610 615 620
Tyr Lys Gly Tyr Lys Val His Leu Thr Ala Ser Asn Leu Gly Glu Thr
625 630 635 640
Val Thr Ser Lys Val Thr Ala Thr Thr Asp Glu Thr Tyr Leu Val Asp
645 650 655
Val Ser Asp Gly Glu Lys Val Val His His Met Lys Leu Asn Ile Gly
660 665 670
Ser Gly Ala Ile Met Met Glu Asn Leu Ala Lys Gly Ala Lys Val Ile
675 680 685
Gly Thr Ser Gly Asp Phe Glu Gln Ala Lys Lys Ile Phe Asp Gly Glu
690 695 700
Lys Ser Asp Arg Phe Phe Thr Trp Gly Gln Thr Asn Trp Ile Ala Phe
705 710 715 720
Asp Leu Gly Glu Ile Asn Leu Ala Lys Glu Trp Arg Leu Phe Asn Ala
725 730 735
Glu Thr Asn Thr Glu Ile Lys Thr Asp Ser Ser Leu Asn Val Ala Lys
740 745 750
Gly Arg Leu Gln Ile Leu Lys Asp Thr Thr Ile Asp Leu Glu Lys Met
755 760 765
Asp Ile Lys Asn Arg Lys Glu Tyr Leu Ser Asn Asp Glu Asn Trp Thr
770 775 780
Asp Val Ala Gln Met Asp Asp Ala Lys Ala Ile Phe Asn Ser Lys Leu
785 790 795 800
Ser Asn Val Leu Ser Arg Tyr Trp Arg Phe Cys Val Asp Gly Gly Ala
805 810 815
Ser Ser Tyr Tyr Pro Gln Tyr Thr Glu Leu Gln Ile Leu Gly Gln Arg
820 825 830
Leu Ser Asn Asp Val Ala Asn Thr Leu Lys Asp
835 840
<210> 8
<211> 843
<212> PRT
<213> 人工序列
<220>
<223> 合成的构建物
<400> 8
Met Asp Lys His Leu Leu Val Lys Arg Thr Leu Gly Cys Val Cys Ala
1 5 10 15
Ala Thr Leu Met Gly Ala Ala Leu Ala Thr His His Asp Ser Leu Asn
20 25 30
Thr Val Lys Ala Glu Glu Lys Thr Val Gln Thr Gly Lys Thr Asp Gln
35 40 45
Gln Val Gly Ala Lys Leu Val Gln Glu Ile Arg Glu Gly Lys Arg Gly
50 55 60
Pro Leu Tyr Ala Gly Tyr Phe Arg Thr Trp His Asp Arg Ala Ser Thr
65 70 75 80
Gly Ile Asp Gly Lys Gln Gln His Pro Glu Asn Thr Met Ala Glu Val
85 90 95
Pro Lys Glu Val Asp Ile Leu Phe Val Phe His Asp His Thr Ala Ser
100 105 110
Asp Ser Pro Phe Trp Ser Glu Leu Lys Asp Ser Tyr Val His Lys Leu
115 120 125
His Gln Gln Gly Thr Ala Leu Val Gln Thr Ile Gly Val Asn Glu Leu
130 135 140
Asn Gly Arg Thr Gly Leu Ser Lys Asp Tyr Pro Asp Thr Pro Glu Gly
145 150 155 160
Asn Lys Ala Leu Ala Ala Ala Ile Val Lys Ala Phe Val Thr Asp Arg
165 170 175
Gly Val Asp Gly Leu Asp Ile Glu Ile Glu His Glu Phe Thr Asn Lys
180 185 190
Arg Thr Pro Glu Glu Asp Ala Arg Ala Leu Asn Val Phe Lys Glu Ile
195 200 205
Ala Gln Leu Ile Gly Lys Asn Gly Ser Asp Lys Ser Lys Leu Leu Ile
210 215 220
Met Asp Thr Thr Leu Ser Val Glu Asn Asn Pro Ile Phe Lys Gly Ile
225 230 235 240
Ala Glu Asp Leu Asp Tyr Leu Leu Arg Gln Tyr Tyr Gly Ser Gln Gly
245 250 255
Gly Glu Ala Glu Val Asp Thr Ile Asn Ser Asp Trp Asn Gln Tyr Gln
260 265 270
Asn Tyr Ile Asp Ala Ser Gln Phe Met Ile Gly Phe Ser Phe Phe Glu
275 280 285
Glu Ser Ala Ser Lys Gly Asn Leu Trp Phe Asp Val Asn Glu Tyr Asp
290 295 300
Pro Asn Asn Pro Glu Lys Gly Lys Asp Ile Glu Gly Thr Arg Ala Lys
305 310 315 320
Lys Tyr Ala Glu Trp Gln Pro Ser Thr Gly Gly Leu Lys Ala Gly Ile
325 330 335
Phe Ser Tyr Ala Ile Asp Arg Asp Gly Val Ala His Val Pro Ser Thr
340 345 350
Tyr Lys Asn Arg Thr Ser Thr Asn Leu Gln Arg His Glu Val Asp Asn
355 360 365
Ile Ser His Thr Asp Tyr Thr Val Ser Arg Lys Leu Lys Thr Leu Met
370 375 380
Thr Glu Asp Lys Arg Tyr Asp Val Ile Asp Gln Lys Asp Ile Pro Asp
385 390 395 400
Pro Ala Leu Arg Glu Gln Ile Ile Gln Gln Val Gly Gln Tyr Lys Gly
405 410 415
Asp Leu Glu Arg Tyr Asn Lys Thr Leu Val Leu Thr Gly Asp Lys Ile
420 425 430
Gln Asn Leu Lys Gly Leu Glu Lys Leu Ser Lys Leu Gln Lys Leu Glu
435 440 445
Leu Arg Gln Leu Ser Asn Val Lys Glu Ile Thr Pro Glu Leu Leu Pro
450 455 460
Glu Ser Met Lys Lys Asp Ala Glu Leu Val Met Val Gly Met Thr Gly
465 470 475 480
Leu Glu Lys Leu Asn Leu Ser Gly Leu Asn Arg Gln Thr Leu Asp Gly
485 490 495
Ile Asp Val Asn Ser Ile Thr His Leu Thr Ser Phe Asp Ile Ser His
500 505 510
Asn Ser Leu Asp Leu Ser Glu Lys Ser Glu Asp Arg Lys Leu Leu Met
515 520 525
Thr Leu Met Glu Gln Val Ser Asn His Gln Lys Ile Thr Val Lys Asn
530 535 540
Thr Ala Phe Glu Asn Gln Lys Pro Lys Gly Tyr Tyr Pro Gln Thr Tyr
545 550 555 560
Asp Thr Lys Glu Gly His Tyr Asp Val Asp Asn Ala Glu His Asp Ile
565 570 575
Leu Thr Asp Phe Val Phe Gly Thr Val Thr Lys Arg Asn Thr Phe Ile
580 585 590
Gly Asp Glu Glu Ala Phe Ala Ile Tyr Lys Glu Gly Ala Val Asp Gly
595 600 605
Arg Gln Tyr Val Ser Lys Asp Tyr Thr Tyr Glu Ala Phe Arg Lys Asp
610 615 620
Tyr Lys Gly Tyr Lys Val His Leu Thr Ala Ser Asn Leu Gly Glu Thr
625 630 635 640
Val Thr Ser Lys Val Thr Ala Thr Thr Asp Glu Thr Tyr Leu Val Asp
645 650 655
Val Ser Asp Gly Glu Lys Val Val His His Met Lys Leu Asn Ile Gly
660 665 670
Ser Gly Ala Ile Met Met Glu Asn Leu Ala Lys Gly Ala Lys Val Ile
675 680 685
Gly Thr Ser Gly Asp Phe Glu Gln Ala Lys Lys Ile Phe Asp Gly Glu
690 695 700
Lys Ser Asp Arg Phe Phe Thr Trp Gly Gln Thr Asn Trp Ile Ala Phe
705 710 715 720
Asp Leu Gly Glu Ile Asn Leu Ala Lys Glu Trp Arg Leu Phe Asn Ala
725 730 735
Glu Thr Asn Thr Glu Ile Lys Thr Asp Ser Ser Leu Asn Val Ala Lys
740 745 750
Gly Arg Leu Gln Ile Leu Lys Asp Thr Thr Ile Asp Leu Glu Lys Met
755 760 765
Asp Ile Lys Asn Arg Lys Glu Tyr Leu Ser Asn Asp Glu Asn Trp Thr
770 775 780
Asp Val Ala Gln Met Asp Asp Ala Lys Ala Ile Phe Asn Ser Lys Leu
785 790 795 800
Ser Asn Val Leu Ser Arg Tyr Trp Arg Phe Cys Val Asp Gly Gly Ala
805 810 815
Ser Ser Tyr Tyr Pro Gln Tyr Thr Glu Leu Gln Ile Leu Gly Gln Arg
820 825 830
Leu Ser Asn Asp Val Ala Asn Thr Leu Lys Asp
835 840
<210> 9
<211> 843
<212> PRT
<213> 人工序列
<220>
<223> 合成的构建物
<400> 9
Met Asp Lys His Leu Leu Val Lys Arg Thr Leu Gly Cys Val Cys Ala
1 5 10 15
Ala Thr Leu Met Gly Ala Ala Leu Ala Thr His His Asp Ser Leu Asn
20 25 30
Thr Val Lys Ala Glu Glu Lys Thr Val Gln Thr Gly Lys Thr Asp Gln
35 40 45
Gln Val Gly Ala Lys Leu Val Gln Glu Ile Arg Glu Gly Lys Arg Gly
50 55 60
Pro Leu Tyr Ala Gly Tyr Phe Arg Thr Trp His Asp Arg Ala Ser Thr
65 70 75 80
Gly Ile Asp Gly Lys Gln Gln His Pro Glu Asn Thr Met Ala Glu Val
85 90 95
Pro Lys Glu Val Asp Ile Leu Phe Val Phe His Asp His Thr Ala Ser
100 105 110
Asp Ser Pro Phe Trp Ser Glu Leu Lys Asp Ser Tyr Val His Lys Leu
115 120 125
His Gln Gln Gly Thr Ala Leu Val Gln Thr Ile Gly Val Asn Glu Leu
130 135 140
Asn Gly Arg Thr Gly Leu Ser Lys Asp Tyr Pro Asp Thr Pro Glu Gly
145 150 155 160
Asn Lys Ala Leu Ala Ala Ala Ile Val Lys Ala Phe Val Thr Asp Arg
165 170 175
Gly Val Asp Gly Leu Asp Ile Gly Ile Glu His Glu Phe Thr Asn Lys
180 185 190
Arg Thr Pro Glu Glu Asp Ala Arg Ala Leu Asn Val Phe Lys Glu Ile
195 200 205
Ala Gln Leu Ile Gly Lys Asn Gly Ser Asp Lys Ser Lys Leu Leu Ile
210 215 220
Met Asp Thr Thr Leu Ser Val Glu Asn Asn Pro Ile Phe Lys Gly Ile
225 230 235 240
Ala Glu Asp Leu Asp Tyr Leu Leu Arg Gln Tyr Tyr Gly Ser Gln Gly
245 250 255
Gly Glu Ala Glu Val Asp Thr Ile Asn Ser Asp Trp Asn Gln Tyr Gln
260 265 270
Asn Tyr Ile Asp Ala Ser Gln Phe Met Ile Gly Phe Ser Phe Phe Glu
275 280 285
Glu Ser Ala Ser Lys Gly Asn Leu Trp Phe Asp Val Asn Glu Tyr Asp
290 295 300
Pro Asn Asn Pro Glu Lys Gly Lys Asp Ile Glu Gly Thr Arg Ala Lys
305 310 315 320
Lys Tyr Ala Glu Trp Gln Pro Ser Thr Gly Gly Leu Lys Ala Gly Ile
325 330 335
Phe Ser Tyr Ala Ile Asp Arg Asp Gly Val Ala His Val Pro Ser Thr
340 345 350
Tyr Lys Asn Arg Thr Ser Thr Asn Leu Gln Arg His Glu Val Asp Asn
355 360 365
Ile Ser His Thr Asp Tyr Thr Val Ser Arg Lys Leu Lys Thr Leu Met
370 375 380
Thr Glu Asp Lys Arg Tyr Asp Val Ile Asp Gln Lys Asp Ile Pro Asp
385 390 395 400
Pro Ala Leu Arg Glu Gln Ile Ile Gln Gln Val Gly Gln Tyr Lys Gly
405 410 415
Asp Leu Glu Arg Tyr Asn Lys Thr Leu Val Leu Thr Gly Asp Lys Ile
420 425 430
Gln Asn Leu Lys Gly Leu Glu Lys Leu Ser Lys Leu Gln Lys Leu Glu
435 440 445
Leu Arg Gln Leu Ser Asn Val Lys Glu Ile Thr Pro Glu Leu Leu Pro
450 455 460
Glu Ser Met Lys Lys Asp Ala Glu Leu Val Met Val Gly Met Thr Gly
465 470 475 480
Leu Glu Lys Leu Asn Leu Ser Gly Leu Asn Arg Gln Thr Leu Asp Gly
485 490 495
Ile Asp Val Asn Ser Ile Thr His Leu Thr Ser Phe Asp Ile Ser His
500 505 510
Asn Ser Leu Asp Leu Ser Glu Lys Ser Glu Asp Arg Lys Leu Leu Met
515 520 525
Thr Leu Met Glu Gln Val Ser Asn His Gln Lys Ile Thr Val Lys Asn
530 535 540
Thr Ala Phe Glu Asn Gln Lys Pro Lys Gly Tyr Tyr Pro Gln Thr Tyr
545 550 555 560
Asp Thr Lys Glu Gly His Tyr Asp Val Asp Asn Ala Glu His Asp Ile
565 570 575
Leu Thr Asp Phe Val Phe Gly Thr Val Thr Lys Arg Asn Thr Phe Ile
580 585 590
Gly Asp Glu Glu Ala Phe Ala Ile Tyr Lys Glu Gly Ala Val Asp Gly
595 600 605
Arg Gln Tyr Val Ser Lys Asp Tyr Thr Tyr Glu Ala Phe Arg Lys Asp
610 615 620
Tyr Lys Gly Tyr Lys Val His Leu Thr Ala Ser Asn Leu Gly Glu Thr
625 630 635 640
Val Thr Ser Lys Val Thr Ala Thr Thr Asp Glu Thr Tyr Leu Val Asp
645 650 655
Val Ser Asp Gly Glu Lys Val Val His His Met Lys Leu Asn Ile Gly
660 665 670
Ser Gly Ala Ile Met Met Glu Asn Leu Ala Lys Gly Ala Lys Val Ile
675 680 685
Gly Thr Ser Gly Asp Phe Glu Gln Ala Lys Lys Ile Phe Asp Gly Glu
690 695 700
Lys Ser Asp Arg Phe Phe Thr Trp Gly Gln Thr Asn Trp Ile Ala Phe
705 710 715 720
Asp Leu Gly Glu Ile Asn Leu Ala Lys Glu Trp Arg Leu Phe Asn Ala
725 730 735
Glu Thr Asn Thr Glu Ile Lys Thr Asp Ser Ser Leu Asn Val Ala Lys
740 745 750
Gly Arg Leu Gln Ile Leu Lys Asp Thr Thr Ile Asp Leu Glu Lys Met
755 760 765
Asp Ile Lys Asn Arg Lys Glu Tyr Leu Ser Asn Asp Glu Asn Trp Thr
770 775 780
Asp Val Ala Gln Met Asp Asp Ala Lys Ala Ile Phe Asn Ser Lys Leu
785 790 795 800
Ser Asn Val Leu Ser Arg Tyr Trp Arg Phe Cys Val Asp Gly Gly Ala
805 810 815
Ser Ser Tyr Tyr Pro Gln Tyr Thr Glu Leu Gln Ile Leu Gly Gln Arg
820 825 830
Leu Ser Asn Asp Val Ala Asn Thr Leu Lys Asp
835 840
<210> 10
<211> 843
<212> PRT
<213> 人工序列
<220>
<223> 合成的构建物
<400> 10
Met Asp Lys His Leu Leu Val Lys Arg Thr Leu Gly Cys Val Cys Ala
1 5 10 15
Ala Thr Leu Met Gly Ala Ala Leu Ala Thr His His Asp Ser Leu Asn
20 25 30
Thr Val Lys Ala Glu Glu Lys Thr Val Gln Thr Gly Lys Thr Asp Gln
35 40 45
Gln Val Gly Ala Lys Leu Val Gln Glu Ile Arg Glu Gly Lys Arg Gly
50 55 60
Pro Leu Tyr Ala Gly Tyr Phe Arg Thr Trp His Asp Arg Ala Ser Thr
65 70 75 80
Gly Ile Asp Gly Lys Gln Gln His Pro Glu Asn Thr Met Ala Glu Val
85 90 95
Pro Lys Glu Val Asp Ile Leu Phe Val Phe His Asp His Thr Ala Ser
100 105 110
Asp Ser Pro Phe Trp Ser Glu Leu Lys Asp Ser Tyr Val His Lys Leu
115 120 125
His Gln Gln Gly Thr Ala Leu Val Gln Thr Ile Gly Val Asn Glu Leu
130 135 140
Asn Gly Arg Thr Gly Leu Ser Lys Asp Tyr Pro Asp Thr Pro Glu Gly
145 150 155 160
Asn Lys Ala Leu Ala Ala Ala Ile Val Lys Ala Phe Val Thr Asp Arg
165 170 175
Gly Val Asp Gly Leu Asp Ile His Ile Glu His Glu Phe Thr Asn Lys
180 185 190
Arg Thr Pro Glu Glu Asp Ala Arg Ala Leu Asn Val Phe Lys Glu Ile
195 200 205
Ala Gln Leu Ile Gly Lys Asn Gly Ser Asp Lys Ser Lys Leu Leu Ile
210 215 220
Met Asp Thr Thr Leu Ser Val Glu Asn Asn Pro Ile Phe Lys Gly Ile
225 230 235 240
Ala Glu Asp Leu Asp Tyr Leu Leu Arg Gln Tyr Tyr Gly Ser Gln Gly
245 250 255
Gly Glu Ala Glu Val Asp Thr Ile Asn Ser Asp Trp Asn Gln Tyr Gln
260 265 270
Asn Tyr Ile Asp Ala Ser Gln Phe Met Ile Gly Phe Ser Phe Phe Glu
275 280 285
Glu Ser Ala Ser Lys Gly Asn Leu Trp Phe Asp Val Asn Glu Tyr Asp
290 295 300
Pro Asn Asn Pro Glu Lys Gly Lys Asp Ile Glu Gly Thr Arg Ala Lys
305 310 315 320
Lys Tyr Ala Glu Trp Gln Pro Ser Thr Gly Gly Leu Lys Ala Gly Ile
325 330 335
Phe Ser Tyr Ala Ile Asp Arg Asp Gly Val Ala His Val Pro Ser Thr
340 345 350
Tyr Lys Asn Arg Thr Ser Thr Asn Leu Gln Arg His Glu Val Asp Asn
355 360 365
Ile Ser His Thr Asp Tyr Thr Val Ser Arg Lys Leu Lys Thr Leu Met
370 375 380
Thr Glu Asp Lys Arg Tyr Asp Val Ile Asp Gln Lys Asp Ile Pro Asp
385 390 395 400
Pro Ala Leu Arg Glu Gln Ile Ile Gln Gln Val Gly Gln Tyr Lys Gly
405 410 415
Asp Leu Glu Arg Tyr Asn Lys Thr Leu Val Leu Thr Gly Asp Lys Ile
420 425 430
Gln Asn Leu Lys Gly Leu Glu Lys Leu Ser Lys Leu Gln Lys Leu Glu
435 440 445
Leu Arg Gln Leu Ser Asn Val Lys Glu Ile Thr Pro Glu Leu Leu Pro
450 455 460
Glu Ser Met Lys Lys Asp Ala Glu Leu Val Met Val Gly Met Thr Gly
465 470 475 480
Leu Glu Lys Leu Asn Leu Ser Gly Leu Asn Arg Gln Thr Leu Asp Gly
485 490 495
Ile Asp Val Asn Ser Ile Thr His Leu Thr Ser Phe Asp Ile Ser His
500 505 510
Asn Ser Leu Asp Leu Ser Glu Lys Ser Glu Asp Arg Lys Leu Leu Met
515 520 525
Thr Leu Met Glu Gln Val Ser Asn His Gln Lys Ile Thr Val Lys Asn
530 535 540
Thr Ala Phe Glu Asn Gln Lys Pro Lys Gly Tyr Tyr Pro Gln Thr Tyr
545 550 555 560
Asp Thr Lys Glu Gly His Tyr Asp Val Asp Asn Ala Glu His Asp Ile
565 570 575
Leu Thr Asp Phe Val Phe Gly Thr Val Thr Lys Arg Asn Thr Phe Ile
580 585 590
Gly Asp Glu Glu Ala Phe Ala Ile Tyr Lys Glu Gly Ala Val Asp Gly
595 600 605
Arg Gln Tyr Val Ser Lys Asp Tyr Thr Tyr Glu Ala Phe Arg Lys Asp
610 615 620
Tyr Lys Gly Tyr Lys Val His Leu Thr Ala Ser Asn Leu Gly Glu Thr
625 630 635 640
Val Thr Ser Lys Val Thr Ala Thr Thr Asp Glu Thr Tyr Leu Val Asp
645 650 655
Val Ser Asp Gly Glu Lys Val Val His His Met Lys Leu Asn Ile Gly
660 665 670
Ser Gly Ala Ile Met Met Glu Asn Leu Ala Lys Gly Ala Lys Val Ile
675 680 685
Gly Thr Ser Gly Asp Phe Glu Gln Ala Lys Lys Ile Phe Asp Gly Glu
690 695 700
Lys Ser Asp Arg Phe Phe Thr Trp Gly Gln Thr Asn Trp Ile Ala Phe
705 710 715 720
Asp Leu Gly Glu Ile Asn Leu Ala Lys Glu Trp Arg Leu Phe Asn Ala
725 730 735
Glu Thr Asn Thr Glu Ile Lys Thr Asp Ser Ser Leu Asn Val Ala Lys
740 745 750
Gly Arg Leu Gln Ile Leu Lys Asp Thr Thr Ile Asp Leu Glu Lys Met
755 760 765
Asp Ile Lys Asn Arg Lys Glu Tyr Leu Ser Asn Asp Glu Asn Trp Thr
770 775 780
Asp Val Ala Gln Met Asp Asp Ala Lys Ala Ile Phe Asn Ser Lys Leu
785 790 795 800
Ser Asn Val Leu Ser Arg Tyr Trp Arg Phe Cys Val Asp Gly Gly Ala
805 810 815
Ser Ser Tyr Tyr Pro Gln Tyr Thr Glu Leu Gln Ile Leu Gly Gln Arg
820 825 830
Leu Ser Asn Asp Val Ala Asn Thr Leu Lys Asp
835 840
<210> 11
<211> 843
<212> PRT
<213> 人工序列
<220>
<223> 合成的构建物
<400> 11
Met Asp Lys His Leu Leu Val Lys Arg Thr Leu Gly Cys Val Cys Ala
1 5 10 15
Ala Thr Leu Met Gly Ala Ala Leu Ala Thr His His Asp Ser Leu Asn
20 25 30
Thr Val Lys Ala Glu Glu Lys Thr Val Gln Thr Gly Lys Thr Asp Gln
35 40 45
Gln Val Gly Ala Lys Leu Val Gln Glu Ile Arg Glu Gly Lys Arg Gly
50 55 60
Pro Leu Tyr Ala Gly Tyr Phe Arg Thr Trp His Asp Arg Ala Ser Thr
65 70 75 80
Gly Ile Asp Gly Lys Gln Gln His Pro Glu Asn Thr Met Ala Glu Val
85 90 95
Pro Lys Glu Val Asp Ile Leu Phe Val Phe His Asp His Thr Ala Ser
100 105 110
Asp Ser Pro Phe Trp Ser Glu Leu Lys Asp Ser Tyr Val His Lys Leu
115 120 125
His Gln Gln Gly Thr Ala Leu Val Gln Thr Ile Gly Val Asn Glu Leu
130 135 140
Asn Gly Arg Thr Gly Leu Ser Lys Asp Tyr Pro Asp Thr Pro Glu Gly
145 150 155 160
Asn Lys Ala Leu Ala Ala Ala Ile Val Lys Ala Phe Val Thr Asp Arg
165 170 175
Gly Val Asp Gly Leu Asp Ile Ile Ile Glu His Glu Phe Thr Asn Lys
180 185 190
Arg Thr Pro Glu Glu Asp Ala Arg Ala Leu Asn Val Phe Lys Glu Ile
195 200 205
Ala Gln Leu Ile Gly Lys Asn Gly Ser Asp Lys Ser Lys Leu Leu Ile
210 215 220
Met Asp Thr Thr Leu Ser Val Glu Asn Asn Pro Ile Phe Lys Gly Ile
225 230 235 240
Ala Glu Asp Leu Asp Tyr Leu Leu Arg Gln Tyr Tyr Gly Ser Gln Gly
245 250 255
Gly Glu Ala Glu Val Asp Thr Ile Asn Ser Asp Trp Asn Gln Tyr Gln
260 265 270
Asn Tyr Ile Asp Ala Ser Gln Phe Met Ile Gly Phe Ser Phe Phe Glu
275 280 285
Glu Ser Ala Ser Lys Gly Asn Leu Trp Phe Asp Val Asn Glu Tyr Asp
290 295 300
Pro Asn Asn Pro Glu Lys Gly Lys Asp Ile Glu Gly Thr Arg Ala Lys
305 310 315 320
Lys Tyr Ala Glu Trp Gln Pro Ser Thr Gly Gly Leu Lys Ala Gly Ile
325 330 335
Phe Ser Tyr Ala Ile Asp Arg Asp Gly Val Ala His Val Pro Ser Thr
340 345 350
Tyr Lys Asn Arg Thr Ser Thr Asn Leu Gln Arg His Glu Val Asp Asn
355 360 365
Ile Ser His Thr Asp Tyr Thr Val Ser Arg Lys Leu Lys Thr Leu Met
370 375 380
Thr Glu Asp Lys Arg Tyr Asp Val Ile Asp Gln Lys Asp Ile Pro Asp
385 390 395 400
Pro Ala Leu Arg Glu Gln Ile Ile Gln Gln Val Gly Gln Tyr Lys Gly
405 410 415
Asp Leu Glu Arg Tyr Asn Lys Thr Leu Val Leu Thr Gly Asp Lys Ile
420 425 430
Gln Asn Leu Lys Gly Leu Glu Lys Leu Ser Lys Leu Gln Lys Leu Glu
435 440 445
Leu Arg Gln Leu Ser Asn Val Lys Glu Ile Thr Pro Glu Leu Leu Pro
450 455 460
Glu Ser Met Lys Lys Asp Ala Glu Leu Val Met Val Gly Met Thr Gly
465 470 475 480
Leu Glu Lys Leu Asn Leu Ser Gly Leu Asn Arg Gln Thr Leu Asp Gly
485 490 495
Ile Asp Val Asn Ser Ile Thr His Leu Thr Ser Phe Asp Ile Ser His
500 505 510
Asn Ser Leu Asp Leu Ser Glu Lys Ser Glu Asp Arg Lys Leu Leu Met
515 520 525
Thr Leu Met Glu Gln Val Ser Asn His Gln Lys Ile Thr Val Lys Asn
530 535 540
Thr Ala Phe Glu Asn Gln Lys Pro Lys Gly Tyr Tyr Pro Gln Thr Tyr
545 550 555 560
Asp Thr Lys Glu Gly His Tyr Asp Val Asp Asn Ala Glu His Asp Ile
565 570 575
Leu Thr Asp Phe Val Phe Gly Thr Val Thr Lys Arg Asn Thr Phe Ile
580 585 590
Gly Asp Glu Glu Ala Phe Ala Ile Tyr Lys Glu Gly Ala Val Asp Gly
595 600 605
Arg Gln Tyr Val Ser Lys Asp Tyr Thr Tyr Glu Ala Phe Arg Lys Asp
610 615 620
Tyr Lys Gly Tyr Lys Val His Leu Thr Ala Ser Asn Leu Gly Glu Thr
625 630 635 640
Val Thr Ser Lys Val Thr Ala Thr Thr Asp Glu Thr Tyr Leu Val Asp
645 650 655
Val Ser Asp Gly Glu Lys Val Val His His Met Lys Leu Asn Ile Gly
660 665 670
Ser Gly Ala Ile Met Met Glu Asn Leu Ala Lys Gly Ala Lys Val Ile
675 680 685
Gly Thr Ser Gly Asp Phe Glu Gln Ala Lys Lys Ile Phe Asp Gly Glu
690 695 700
Lys Ser Asp Arg Phe Phe Thr Trp Gly Gln Thr Asn Trp Ile Ala Phe
705 710 715 720
Asp Leu Gly Glu Ile Asn Leu Ala Lys Glu Trp Arg Leu Phe Asn Ala
725 730 735
Glu Thr Asn Thr Glu Ile Lys Thr Asp Ser Ser Leu Asn Val Ala Lys
740 745 750
Gly Arg Leu Gln Ile Leu Lys Asp Thr Thr Ile Asp Leu Glu Lys Met
755 760 765
Asp Ile Lys Asn Arg Lys Glu Tyr Leu Ser Asn Asp Glu Asn Trp Thr
770 775 780
Asp Val Ala Gln Met Asp Asp Ala Lys Ala Ile Phe Asn Ser Lys Leu
785 790 795 800
Ser Asn Val Leu Ser Arg Tyr Trp Arg Phe Cys Val Asp Gly Gly Ala
805 810 815
Ser Ser Tyr Tyr Pro Gln Tyr Thr Glu Leu Gln Ile Leu Gly Gln Arg
820 825 830
Leu Ser Asn Asp Val Ala Asn Thr Leu Lys Asp
835 840
<210> 12
<211> 843
<212> PRT
<213> 人工序列
<220>
<223> 合成的构建物
<400> 12
Met Asp Lys His Leu Leu Val Lys Arg Thr Leu Gly Cys Val Cys Ala
1 5 10 15
Ala Thr Leu Met Gly Ala Ala Leu Ala Thr His His Asp Ser Leu Asn
20 25 30
Thr Val Lys Ala Glu Glu Lys Thr Val Gln Thr Gly Lys Thr Asp Gln
35 40 45
Gln Val Gly Ala Lys Leu Val Gln Glu Ile Arg Glu Gly Lys Arg Gly
50 55 60
Pro Leu Tyr Ala Gly Tyr Phe Arg Thr Trp His Asp Arg Ala Ser Thr
65 70 75 80
Gly Ile Asp Gly Lys Gln Gln His Pro Glu Asn Thr Met Ala Glu Val
85 90 95
Pro Lys Glu Val Asp Ile Leu Phe Val Phe His Asp His Thr Ala Ser
100 105 110
Asp Ser Pro Phe Trp Ser Glu Leu Lys Asp Ser Tyr Val His Lys Leu
115 120 125
His Gln Gln Gly Thr Ala Leu Val Gln Thr Ile Gly Val Asn Glu Leu
130 135 140
Asn Gly Arg Thr Gly Leu Ser Lys Asp Tyr Pro Asp Thr Pro Glu Gly
145 150 155 160
Asn Lys Ala Leu Ala Ala Ala Ile Val Lys Ala Phe Val Thr Asp Arg
165 170 175
Gly Val Asp Gly Leu Asp Ile Leu Ile Glu His Glu Phe Thr Asn Lys
180 185 190
Arg Thr Pro Glu Glu Asp Ala Arg Ala Leu Asn Val Phe Lys Glu Ile
195 200 205
Ala Gln Leu Ile Gly Lys Asn Gly Ser Asp Lys Ser Lys Leu Leu Ile
210 215 220
Met Asp Thr Thr Leu Ser Val Glu Asn Asn Pro Ile Phe Lys Gly Ile
225 230 235 240
Ala Glu Asp Leu Asp Tyr Leu Leu Arg Gln Tyr Tyr Gly Ser Gln Gly
245 250 255
Gly Glu Ala Glu Val Asp Thr Ile Asn Ser Asp Trp Asn Gln Tyr Gln
260 265 270
Asn Tyr Ile Asp Ala Ser Gln Phe Met Ile Gly Phe Ser Phe Phe Glu
275 280 285
Glu Ser Ala Ser Lys Gly Asn Leu Trp Phe Asp Val Asn Glu Tyr Asp
290 295 300
Pro Asn Asn Pro Glu Lys Gly Lys Asp Ile Glu Gly Thr Arg Ala Lys
305 310 315 320
Lys Tyr Ala Glu Trp Gln Pro Ser Thr Gly Gly Leu Lys Ala Gly Ile
325 330 335
Phe Ser Tyr Ala Ile Asp Arg Asp Gly Val Ala His Val Pro Ser Thr
340 345 350
Tyr Lys Asn Arg Thr Ser Thr Asn Leu Gln Arg His Glu Val Asp Asn
355 360 365
Ile Ser His Thr Asp Tyr Thr Val Ser Arg Lys Leu Lys Thr Leu Met
370 375 380
Thr Glu Asp Lys Arg Tyr Asp Val Ile Asp Gln Lys Asp Ile Pro Asp
385 390 395 400
Pro Ala Leu Arg Glu Gln Ile Ile Gln Gln Val Gly Gln Tyr Lys Gly
405 410 415
Asp Leu Glu Arg Tyr Asn Lys Thr Leu Val Leu Thr Gly Asp Lys Ile
420 425 430
Gln Asn Leu Lys Gly Leu Glu Lys Leu Ser Lys Leu Gln Lys Leu Glu
435 440 445
Leu Arg Gln Leu Ser Asn Val Lys Glu Ile Thr Pro Glu Leu Leu Pro
450 455 460
Glu Ser Met Lys Lys Asp Ala Glu Leu Val Met Val Gly Met Thr Gly
465 470 475 480
Leu Glu Lys Leu Asn Leu Ser Gly Leu Asn Arg Gln Thr Leu Asp Gly
485 490 495
Ile Asp Val Asn Ser Ile Thr His Leu Thr Ser Phe Asp Ile Ser His
500 505 510
Asn Ser Leu Asp Leu Ser Glu Lys Ser Glu Asp Arg Lys Leu Leu Met
515 520 525
Thr Leu Met Glu Gln Val Ser Asn His Gln Lys Ile Thr Val Lys Asn
530 535 540
Thr Ala Phe Glu Asn Gln Lys Pro Lys Gly Tyr Tyr Pro Gln Thr Tyr
545 550 555 560
Asp Thr Lys Glu Gly His Tyr Asp Val Asp Asn Ala Glu His Asp Ile
565 570 575
Leu Thr Asp Phe Val Phe Gly Thr Val Thr Lys Arg Asn Thr Phe Ile
580 585 590
Gly Asp Glu Glu Ala Phe Ala Ile Tyr Lys Glu Gly Ala Val Asp Gly
595 600 605
Arg Gln Tyr Val Ser Lys Asp Tyr Thr Tyr Glu Ala Phe Arg Lys Asp
610 615 620
Tyr Lys Gly Tyr Lys Val His Leu Thr Ala Ser Asn Leu Gly Glu Thr
625 630 635 640
Val Thr Ser Lys Val Thr Ala Thr Thr Asp Glu Thr Tyr Leu Val Asp
645 650 655
Val Ser Asp Gly Glu Lys Val Val His His Met Lys Leu Asn Ile Gly
660 665 670
Ser Gly Ala Ile Met Met Glu Asn Leu Ala Lys Gly Ala Lys Val Ile
675 680 685
Gly Thr Ser Gly Asp Phe Glu Gln Ala Lys Lys Ile Phe Asp Gly Glu
690 695 700
Lys Ser Asp Arg Phe Phe Thr Trp Gly Gln Thr Asn Trp Ile Ala Phe
705 710 715 720
Asp Leu Gly Glu Ile Asn Leu Ala Lys Glu Trp Arg Leu Phe Asn Ala
725 730 735
Glu Thr Asn Thr Glu Ile Lys Thr Asp Ser Ser Leu Asn Val Ala Lys
740 745 750
Gly Arg Leu Gln Ile Leu Lys Asp Thr Thr Ile Asp Leu Glu Lys Met
755 760 765
Asp Ile Lys Asn Arg Lys Glu Tyr Leu Ser Asn Asp Glu Asn Trp Thr
770 775 780
Asp Val Ala Gln Met Asp Asp Ala Lys Ala Ile Phe Asn Ser Lys Leu
785 790 795 800
Ser Asn Val Leu Ser Arg Tyr Trp Arg Phe Cys Val Asp Gly Gly Ala
805 810 815
Ser Ser Tyr Tyr Pro Gln Tyr Thr Glu Leu Gln Ile Leu Gly Gln Arg
820 825 830
Leu Ser Asn Asp Val Ala Asn Thr Leu Lys Asp
835 840
<210> 13
<211> 843
<212> PRT
<213> 人工序列
<220>
<223> 合成的构建物
<400> 13
Met Asp Lys His Leu Leu Val Lys Arg Thr Leu Gly Cys Val Cys Ala
1 5 10 15
Ala Thr Leu Met Gly Ala Ala Leu Ala Thr His His Asp Ser Leu Asn
20 25 30
Thr Val Lys Ala Glu Glu Lys Thr Val Gln Thr Gly Lys Thr Asp Gln
35 40 45
Gln Val Gly Ala Lys Leu Val Gln Glu Ile Arg Glu Gly Lys Arg Gly
50 55 60
Pro Leu Tyr Ala Gly Tyr Phe Arg Thr Trp His Asp Arg Ala Ser Thr
65 70 75 80
Gly Ile Asp Gly Lys Gln Gln His Pro Glu Asn Thr Met Ala Glu Val
85 90 95
Pro Lys Glu Val Asp Ile Leu Phe Val Phe His Asp His Thr Ala Ser
100 105 110
Asp Ser Pro Phe Trp Ser Glu Leu Lys Asp Ser Tyr Val His Lys Leu
115 120 125
His Gln Gln Gly Thr Ala Leu Val Gln Thr Ile Gly Val Asn Glu Leu
130 135 140
Asn Gly Arg Thr Gly Leu Ser Lys Asp Tyr Pro Asp Thr Pro Glu Gly
145 150 155 160
Asn Lys Ala Leu Ala Ala Ala Ile Val Lys Ala Phe Val Thr Asp Arg
165 170 175
Gly Val Asp Gly Leu Asp Ile Lys Ile Glu His Glu Phe Thr Asn Lys
180 185 190
Arg Thr Pro Glu Glu Asp Ala Arg Ala Leu Asn Val Phe Lys Glu Ile
195 200 205
Ala Gln Leu Ile Gly Lys Asn Gly Ser Asp Lys Ser Lys Leu Leu Ile
210 215 220
Met Asp Thr Thr Leu Ser Val Glu Asn Asn Pro Ile Phe Lys Gly Ile
225 230 235 240
Ala Glu Asp Leu Asp Tyr Leu Leu Arg Gln Tyr Tyr Gly Ser Gln Gly
245 250 255
Gly Glu Ala Glu Val Asp Thr Ile Asn Ser Asp Trp Asn Gln Tyr Gln
260 265 270
Asn Tyr Ile Asp Ala Ser Gln Phe Met Ile Gly Phe Ser Phe Phe Glu
275 280 285
Glu Ser Ala Ser Lys Gly Asn Leu Trp Phe Asp Val Asn Glu Tyr Asp
290 295 300
Pro Asn Asn Pro Glu Lys Gly Lys Asp Ile Glu Gly Thr Arg Ala Lys
305 310 315 320
Lys Tyr Ala Glu Trp Gln Pro Ser Thr Gly Gly Leu Lys Ala Gly Ile
325 330 335
Phe Ser Tyr Ala Ile Asp Arg Asp Gly Val Ala His Val Pro Ser Thr
340 345 350
Tyr Lys Asn Arg Thr Ser Thr Asn Leu Gln Arg His Glu Val Asp Asn
355 360 365
Ile Ser His Thr Asp Tyr Thr Val Ser Arg Lys Leu Lys Thr Leu Met
370 375 380
Thr Glu Asp Lys Arg Tyr Asp Val Ile Asp Gln Lys Asp Ile Pro Asp
385 390 395 400
Pro Ala Leu Arg Glu Gln Ile Ile Gln Gln Val Gly Gln Tyr Lys Gly
405 410 415
Asp Leu Glu Arg Tyr Asn Lys Thr Leu Val Leu Thr Gly Asp Lys Ile
420 425 430
Gln Asn Leu Lys Gly Leu Glu Lys Leu Ser Lys Leu Gln Lys Leu Glu
435 440 445
Leu Arg Gln Leu Ser Asn Val Lys Glu Ile Thr Pro Glu Leu Leu Pro
450 455 460
Glu Ser Met Lys Lys Asp Ala Glu Leu Val Met Val Gly Met Thr Gly
465 470 475 480
Leu Glu Lys Leu Asn Leu Ser Gly Leu Asn Arg Gln Thr Leu Asp Gly
485 490 495
Ile Asp Val Asn Ser Ile Thr His Leu Thr Ser Phe Asp Ile Ser His
500 505 510
Asn Ser Leu Asp Leu Ser Glu Lys Ser Glu Asp Arg Lys Leu Leu Met
515 520 525
Thr Leu Met Glu Gln Val Ser Asn His Gln Lys Ile Thr Val Lys Asn
530 535 540
Thr Ala Phe Glu Asn Gln Lys Pro Lys Gly Tyr Tyr Pro Gln Thr Tyr
545 550 555 560
Asp Thr Lys Glu Gly His Tyr Asp Val Asp Asn Ala Glu His Asp Ile
565 570 575
Leu Thr Asp Phe Val Phe Gly Thr Val Thr Lys Arg Asn Thr Phe Ile
580 585 590
Gly Asp Glu Glu Ala Phe Ala Ile Tyr Lys Glu Gly Ala Val Asp Gly
595 600 605
Arg Gln Tyr Val Ser Lys Asp Tyr Thr Tyr Glu Ala Phe Arg Lys Asp
610 615 620
Tyr Lys Gly Tyr Lys Val His Leu Thr Ala Ser Asn Leu Gly Glu Thr
625 630 635 640
Val Thr Ser Lys Val Thr Ala Thr Thr Asp Glu Thr Tyr Leu Val Asp
645 650 655
Val Ser Asp Gly Glu Lys Val Val His His Met Lys Leu Asn Ile Gly
660 665 670
Ser Gly Ala Ile Met Met Glu Asn Leu Ala Lys Gly Ala Lys Val Ile
675 680 685
Gly Thr Ser Gly Asp Phe Glu Gln Ala Lys Lys Ile Phe Asp Gly Glu
690 695 700
Lys Ser Asp Arg Phe Phe Thr Trp Gly Gln Thr Asn Trp Ile Ala Phe
705 710 715 720
Asp Leu Gly Glu Ile Asn Leu Ala Lys Glu Trp Arg Leu Phe Asn Ala
725 730 735
Glu Thr Asn Thr Glu Ile Lys Thr Asp Ser Ser Leu Asn Val Ala Lys
740 745 750
Gly Arg Leu Gln Ile Leu Lys Asp Thr Thr Ile Asp Leu Glu Lys Met
755 760 765
Asp Ile Lys Asn Arg Lys Glu Tyr Leu Ser Asn Asp Glu Asn Trp Thr
770 775 780
Asp Val Ala Gln Met Asp Asp Ala Lys Ala Ile Phe Asn Ser Lys Leu
785 790 795 800
Ser Asn Val Leu Ser Arg Tyr Trp Arg Phe Cys Val Asp Gly Gly Ala
805 810 815
Ser Ser Tyr Tyr Pro Gln Tyr Thr Glu Leu Gln Ile Leu Gly Gln Arg
820 825 830
Leu Ser Asn Asp Val Ala Asn Thr Leu Lys Asp
835 840
<210> 14
<211> 843
<212> PRT
<213> 人工序列
<220>
<223> 合成的构建物
<400> 14
Met Asp Lys His Leu Leu Val Lys Arg Thr Leu Gly Cys Val Cys Ala
1 5 10 15
Ala Thr Leu Met Gly Ala Ala Leu Ala Thr His His Asp Ser Leu Asn
20 25 30
Thr Val Lys Ala Glu Glu Lys Thr Val Gln Thr Gly Lys Thr Asp Gln
35 40 45
Gln Val Gly Ala Lys Leu Val Gln Glu Ile Arg Glu Gly Lys Arg Gly
50 55 60
Pro Leu Tyr Ala Gly Tyr Phe Arg Thr Trp His Asp Arg Ala Ser Thr
65 70 75 80
Gly Ile Asp Gly Lys Gln Gln His Pro Glu Asn Thr Met Ala Glu Val
85 90 95
Pro Lys Glu Val Asp Ile Leu Phe Val Phe His Asp His Thr Ala Ser
100 105 110
Asp Ser Pro Phe Trp Ser Glu Leu Lys Asp Ser Tyr Val His Lys Leu
115 120 125
His Gln Gln Gly Thr Ala Leu Val Gln Thr Ile Gly Val Asn Glu Leu
130 135 140
Asn Gly Arg Thr Gly Leu Ser Lys Asp Tyr Pro Asp Thr Pro Glu Gly
145 150 155 160
Asn Lys Ala Leu Ala Ala Ala Ile Val Lys Ala Phe Val Thr Asp Arg
165 170 175
Gly Val Asp Gly Leu Asp Ile Phe Ile Glu His Glu Phe Thr Asn Lys
180 185 190
Arg Thr Pro Glu Glu Asp Ala Arg Ala Leu Asn Val Phe Lys Glu Ile
195 200 205
Ala Gln Leu Ile Gly Lys Asn Gly Ser Asp Lys Ser Lys Leu Leu Ile
210 215 220
Met Asp Thr Thr Leu Ser Val Glu Asn Asn Pro Ile Phe Lys Gly Ile
225 230 235 240
Ala Glu Asp Leu Asp Tyr Leu Leu Arg Gln Tyr Tyr Gly Ser Gln Gly
245 250 255
Gly Glu Ala Glu Val Asp Thr Ile Asn Ser Asp Trp Asn Gln Tyr Gln
260 265 270
Asn Tyr Ile Asp Ala Ser Gln Phe Met Ile Gly Phe Ser Phe Phe Glu
275 280 285
Glu Ser Ala Ser Lys Gly Asn Leu Trp Phe Asp Val Asn Glu Tyr Asp
290 295 300
Pro Asn Asn Pro Glu Lys Gly Lys Asp Ile Glu Gly Thr Arg Ala Lys
305 310 315 320
Lys Tyr Ala Glu Trp Gln Pro Ser Thr Gly Gly Leu Lys Ala Gly Ile
325 330 335
Phe Ser Tyr Ala Ile Asp Arg Asp Gly Val Ala His Val Pro Ser Thr
340 345 350
Tyr Lys Asn Arg Thr Ser Thr Asn Leu Gln Arg His Glu Val Asp Asn
355 360 365
Ile Ser His Thr Asp Tyr Thr Val Ser Arg Lys Leu Lys Thr Leu Met
370 375 380
Thr Glu Asp Lys Arg Tyr Asp Val Ile Asp Gln Lys Asp Ile Pro Asp
385 390 395 400
Pro Ala Leu Arg Glu Gln Ile Ile Gln Gln Val Gly Gln Tyr Lys Gly
405 410 415
Asp Leu Glu Arg Tyr Asn Lys Thr Leu Val Leu Thr Gly Asp Lys Ile
420 425 430
Gln Asn Leu Lys Gly Leu Glu Lys Leu Ser Lys Leu Gln Lys Leu Glu
435 440 445
Leu Arg Gln Leu Ser Asn Val Lys Glu Ile Thr Pro Glu Leu Leu Pro
450 455 460
Glu Ser Met Lys Lys Asp Ala Glu Leu Val Met Val Gly Met Thr Gly
465 470 475 480
Leu Glu Lys Leu Asn Leu Ser Gly Leu Asn Arg Gln Thr Leu Asp Gly
485 490 495
Ile Asp Val Asn Ser Ile Thr His Leu Thr Ser Phe Asp Ile Ser His
500 505 510
Asn Ser Leu Asp Leu Ser Glu Lys Ser Glu Asp Arg Lys Leu Leu Met
515 520 525
Thr Leu Met Glu Gln Val Ser Asn His Gln Lys Ile Thr Val Lys Asn
530 535 540
Thr Ala Phe Glu Asn Gln Lys Pro Lys Gly Tyr Tyr Pro Gln Thr Tyr
545 550 555 560
Asp Thr Lys Glu Gly His Tyr Asp Val Asp Asn Ala Glu His Asp Ile
565 570 575
Leu Thr Asp Phe Val Phe Gly Thr Val Thr Lys Arg Asn Thr Phe Ile
580 585 590
Gly Asp Glu Glu Ala Phe Ala Ile Tyr Lys Glu Gly Ala Val Asp Gly
595 600 605
Arg Gln Tyr Val Ser Lys Asp Tyr Thr Tyr Glu Ala Phe Arg Lys Asp
610 615 620
Tyr Lys Gly Tyr Lys Val His Leu Thr Ala Ser Asn Leu Gly Glu Thr
625 630 635 640
Val Thr Ser Lys Val Thr Ala Thr Thr Asp Glu Thr Tyr Leu Val Asp
645 650 655
Val Ser Asp Gly Glu Lys Val Val His His Met Lys Leu Asn Ile Gly
660 665 670
Ser Gly Ala Ile Met Met Glu Asn Leu Ala Lys Gly Ala Lys Val Ile
675 680 685
Gly Thr Ser Gly Asp Phe Glu Gln Ala Lys Lys Ile Phe Asp Gly Glu
690 695 700
Lys Ser Asp Arg Phe Phe Thr Trp Gly Gln Thr Asn Trp Ile Ala Phe
705 710 715 720
Asp Leu Gly Glu Ile Asn Leu Ala Lys Glu Trp Arg Leu Phe Asn Ala
725 730 735
Glu Thr Asn Thr Glu Ile Lys Thr Asp Ser Ser Leu Asn Val Ala Lys
740 745 750
Gly Arg Leu Gln Ile Leu Lys Asp Thr Thr Ile Asp Leu Glu Lys Met
755 760 765
Asp Ile Lys Asn Arg Lys Glu Tyr Leu Ser Asn Asp Glu Asn Trp Thr
770 775 780
Asp Val Ala Gln Met Asp Asp Ala Lys Ala Ile Phe Asn Ser Lys Leu
785 790 795 800
Ser Asn Val Leu Ser Arg Tyr Trp Arg Phe Cys Val Asp Gly Gly Ala
805 810 815
Ser Ser Tyr Tyr Pro Gln Tyr Thr Glu Leu Gln Ile Leu Gly Gln Arg
820 825 830
Leu Ser Asn Asp Val Ala Asn Thr Leu Lys Asp
835 840
<210> 15
<211> 843
<212> PRT
<213> 人工序列
<220>
<223> 合成的构建物
<400> 15
Met Asp Lys His Leu Leu Val Lys Arg Thr Leu Gly Cys Val Cys Ala
1 5 10 15
Ala Thr Leu Met Gly Ala Ala Leu Ala Thr His His Asp Ser Leu Asn
20 25 30
Thr Val Lys Ala Glu Glu Lys Thr Val Gln Thr Gly Lys Thr Asp Gln
35 40 45
Gln Val Gly Ala Lys Leu Val Gln Glu Ile Arg Glu Gly Lys Arg Gly
50 55 60
Pro Leu Tyr Ala Gly Tyr Phe Arg Thr Trp His Asp Arg Ala Ser Thr
65 70 75 80
Gly Ile Asp Gly Lys Gln Gln His Pro Glu Asn Thr Met Ala Glu Val
85 90 95
Pro Lys Glu Val Asp Ile Leu Phe Val Phe His Asp His Thr Ala Ser
100 105 110
Asp Ser Pro Phe Trp Ser Glu Leu Lys Asp Ser Tyr Val His Lys Leu
115 120 125
His Gln Gln Gly Thr Ala Leu Val Gln Thr Ile Gly Val Asn Glu Leu
130 135 140
Asn Gly Arg Thr Gly Leu Ser Lys Asp Tyr Pro Asp Thr Pro Glu Gly
145 150 155 160
Asn Lys Ala Leu Ala Ala Ala Ile Val Lys Ala Phe Val Thr Asp Arg
165 170 175
Gly Val Asp Gly Leu Asp Ile Pro Ile Glu His Glu Phe Thr Asn Lys
180 185 190
Arg Thr Pro Glu Glu Asp Ala Arg Ala Leu Asn Val Phe Lys Glu Ile
195 200 205
Ala Gln Leu Ile Gly Lys Asn Gly Ser Asp Lys Ser Lys Leu Leu Ile
210 215 220
Met Asp Thr Thr Leu Ser Val Glu Asn Asn Pro Ile Phe Lys Gly Ile
225 230 235 240
Ala Glu Asp Leu Asp Tyr Leu Leu Arg Gln Tyr Tyr Gly Ser Gln Gly
245 250 255
Gly Glu Ala Glu Val Asp Thr Ile Asn Ser Asp Trp Asn Gln Tyr Gln
260 265 270
Asn Tyr Ile Asp Ala Ser Gln Phe Met Ile Gly Phe Ser Phe Phe Glu
275 280 285
Glu Ser Ala Ser Lys Gly Asn Leu Trp Phe Asp Val Asn Glu Tyr Asp
290 295 300
Pro Asn Asn Pro Glu Lys Gly Lys Asp Ile Glu Gly Thr Arg Ala Lys
305 310 315 320
Lys Tyr Ala Glu Trp Gln Pro Ser Thr Gly Gly Leu Lys Ala Gly Ile
325 330 335
Phe Ser Tyr Ala Ile Asp Arg Asp Gly Val Ala His Val Pro Ser Thr
340 345 350
Tyr Lys Asn Arg Thr Ser Thr Asn Leu Gln Arg His Glu Val Asp Asn
355 360 365
Ile Ser His Thr Asp Tyr Thr Val Ser Arg Lys Leu Lys Thr Leu Met
370 375 380
Thr Glu Asp Lys Arg Tyr Asp Val Ile Asp Gln Lys Asp Ile Pro Asp
385 390 395 400
Pro Ala Leu Arg Glu Gln Ile Ile Gln Gln Val Gly Gln Tyr Lys Gly
405 410 415
Asp Leu Glu Arg Tyr Asn Lys Thr Leu Val Leu Thr Gly Asp Lys Ile
420 425 430
Gln Asn Leu Lys Gly Leu Glu Lys Leu Ser Lys Leu Gln Lys Leu Glu
435 440 445
Leu Arg Gln Leu Ser Asn Val Lys Glu Ile Thr Pro Glu Leu Leu Pro
450 455 460
Glu Ser Met Lys Lys Asp Ala Glu Leu Val Met Val Gly Met Thr Gly
465 470 475 480
Leu Glu Lys Leu Asn Leu Ser Gly Leu Asn Arg Gln Thr Leu Asp Gly
485 490 495
Ile Asp Val Asn Ser Ile Thr His Leu Thr Ser Phe Asp Ile Ser His
500 505 510
Asn Ser Leu Asp Leu Ser Glu Lys Ser Glu Asp Arg Lys Leu Leu Met
515 520 525
Thr Leu Met Glu Gln Val Ser Asn His Gln Lys Ile Thr Val Lys Asn
530 535 540
Thr Ala Phe Glu Asn Gln Lys Pro Lys Gly Tyr Tyr Pro Gln Thr Tyr
545 550 555 560
Asp Thr Lys Glu Gly His Tyr Asp Val Asp Asn Ala Glu His Asp Ile
565 570 575
Leu Thr Asp Phe Val Phe Gly Thr Val Thr Lys Arg Asn Thr Phe Ile
580 585 590
Gly Asp Glu Glu Ala Phe Ala Ile Tyr Lys Glu Gly Ala Val Asp Gly
595 600 605
Arg Gln Tyr Val Ser Lys Asp Tyr Thr Tyr Glu Ala Phe Arg Lys Asp
610 615 620
Tyr Lys Gly Tyr Lys Val His Leu Thr Ala Ser Asn Leu Gly Glu Thr
625 630 635 640
Val Thr Ser Lys Val Thr Ala Thr Thr Asp Glu Thr Tyr Leu Val Asp
645 650 655
Val Ser Asp Gly Glu Lys Val Val His His Met Lys Leu Asn Ile Gly
660 665 670
Ser Gly Ala Ile Met Met Glu Asn Leu Ala Lys Gly Ala Lys Val Ile
675 680 685
Gly Thr Ser Gly Asp Phe Glu Gln Ala Lys Lys Ile Phe Asp Gly Glu
690 695 700
Lys Ser Asp Arg Phe Phe Thr Trp Gly Gln Thr Asn Trp Ile Ala Phe
705 710 715 720
Asp Leu Gly Glu Ile Asn Leu Ala Lys Glu Trp Arg Leu Phe Asn Ala
725 730 735
Glu Thr Asn Thr Glu Ile Lys Thr Asp Ser Ser Leu Asn Val Ala Lys
740 745 750
Gly Arg Leu Gln Ile Leu Lys Asp Thr Thr Ile Asp Leu Glu Lys Met
755 760 765
Asp Ile Lys Asn Arg Lys Glu Tyr Leu Ser Asn Asp Glu Asn Trp Thr
770 775 780
Asp Val Ala Gln Met Asp Asp Ala Lys Ala Ile Phe Asn Ser Lys Leu
785 790 795 800
Ser Asn Val Leu Ser Arg Tyr Trp Arg Phe Cys Val Asp Gly Gly Ala
805 810 815
Ser Ser Tyr Tyr Pro Gln Tyr Thr Glu Leu Gln Ile Leu Gly Gln Arg
820 825 830
Leu Ser Asn Asp Val Ala Asn Thr Leu Lys Asp
835 840
<210> 16
<211> 843
<212> PRT
<213> 人工序列
<220>
<223> 合成的构建物
<400> 16
Met Asp Lys His Leu Leu Val Lys Arg Thr Leu Gly Cys Val Cys Ala
1 5 10 15
Ala Thr Leu Met Gly Ala Ala Leu Ala Thr His His Asp Ser Leu Asn
20 25 30
Thr Val Lys Ala Glu Glu Lys Thr Val Gln Thr Gly Lys Thr Asp Gln
35 40 45
Gln Val Gly Ala Lys Leu Val Gln Glu Ile Arg Glu Gly Lys Arg Gly
50 55 60
Pro Leu Tyr Ala Gly Tyr Phe Arg Thr Trp His Asp Arg Ala Ser Thr
65 70 75 80
Gly Ile Asp Gly Lys Gln Gln His Pro Glu Asn Thr Met Ala Glu Val
85 90 95
Pro Lys Glu Val Asp Ile Leu Phe Val Phe His Asp His Thr Ala Ser
100 105 110
Asp Ser Pro Phe Trp Ser Glu Leu Lys Asp Ser Tyr Val His Lys Leu
115 120 125
His Gln Gln Gly Thr Ala Leu Val Gln Thr Ile Gly Val Asn Glu Leu
130 135 140
Asn Gly Arg Thr Gly Leu Ser Lys Asp Tyr Pro Asp Thr Pro Glu Gly
145 150 155 160
Asn Lys Ala Leu Ala Ala Ala Ile Val Lys Ala Phe Val Thr Asp Arg
165 170 175
Gly Val Asp Gly Leu Asp Ile Ser Ile Glu His Glu Phe Thr Asn Lys
180 185 190
Arg Thr Pro Glu Glu Asp Ala Arg Ala Leu Asn Val Phe Lys Glu Ile
195 200 205
Ala Gln Leu Ile Gly Lys Asn Gly Ser Asp Lys Ser Lys Leu Leu Ile
210 215 220
Met Asp Thr Thr Leu Ser Val Glu Asn Asn Pro Ile Phe Lys Gly Ile
225 230 235 240
Ala Glu Asp Leu Asp Tyr Leu Leu Arg Gln Tyr Tyr Gly Ser Gln Gly
245 250 255
Gly Glu Ala Glu Val Asp Thr Ile Asn Ser Asp Trp Asn Gln Tyr Gln
260 265 270
Asn Tyr Ile Asp Ala Ser Gln Phe Met Ile Gly Phe Ser Phe Phe Glu
275 280 285
Glu Ser Ala Ser Lys Gly Asn Leu Trp Phe Asp Val Asn Glu Tyr Asp
290 295 300
Pro Asn Asn Pro Glu Lys Gly Lys Asp Ile Glu Gly Thr Arg Ala Lys
305 310 315 320
Lys Tyr Ala Glu Trp Gln Pro Ser Thr Gly Gly Leu Lys Ala Gly Ile
325 330 335
Phe Ser Tyr Ala Ile Asp Arg Asp Gly Val Ala His Val Pro Ser Thr
340 345 350
Tyr Lys Asn Arg Thr Ser Thr Asn Leu Gln Arg His Glu Val Asp Asn
355 360 365
Ile Ser His Thr Asp Tyr Thr Val Ser Arg Lys Leu Lys Thr Leu Met
370 375 380
Thr Glu Asp Lys Arg Tyr Asp Val Ile Asp Gln Lys Asp Ile Pro Asp
385 390 395 400
Pro Ala Leu Arg Glu Gln Ile Ile Gln Gln Val Gly Gln Tyr Lys Gly
405 410 415
Asp Leu Glu Arg Tyr Asn Lys Thr Leu Val Leu Thr Gly Asp Lys Ile
420 425 430
Gln Asn Leu Lys Gly Leu Glu Lys Leu Ser Lys Leu Gln Lys Leu Glu
435 440 445
Leu Arg Gln Leu Ser Asn Val Lys Glu Ile Thr Pro Glu Leu Leu Pro
450 455 460
Glu Ser Met Lys Lys Asp Ala Glu Leu Val Met Val Gly Met Thr Gly
465 470 475 480
Leu Glu Lys Leu Asn Leu Ser Gly Leu Asn Arg Gln Thr Leu Asp Gly
485 490 495
Ile Asp Val Asn Ser Ile Thr His Leu Thr Ser Phe Asp Ile Ser His
500 505 510
Asn Ser Leu Asp Leu Ser Glu Lys Ser Glu Asp Arg Lys Leu Leu Met
515 520 525
Thr Leu Met Glu Gln Val Ser Asn His Gln Lys Ile Thr Val Lys Asn
530 535 540
Thr Ala Phe Glu Asn Gln Lys Pro Lys Gly Tyr Tyr Pro Gln Thr Tyr
545 550 555 560
Asp Thr Lys Glu Gly His Tyr Asp Val Asp Asn Ala Glu His Asp Ile
565 570 575
Leu Thr Asp Phe Val Phe Gly Thr Val Thr Lys Arg Asn Thr Phe Ile
580 585 590
Gly Asp Glu Glu Ala Phe Ala Ile Tyr Lys Glu Gly Ala Val Asp Gly
595 600 605
Arg Gln Tyr Val Ser Lys Asp Tyr Thr Tyr Glu Ala Phe Arg Lys Asp
610 615 620
Tyr Lys Gly Tyr Lys Val His Leu Thr Ala Ser Asn Leu Gly Glu Thr
625 630 635 640
Val Thr Ser Lys Val Thr Ala Thr Thr Asp Glu Thr Tyr Leu Val Asp
645 650 655
Val Ser Asp Gly Glu Lys Val Val His His Met Lys Leu Asn Ile Gly
660 665 670
Ser Gly Ala Ile Met Met Glu Asn Leu Ala Lys Gly Ala Lys Val Ile
675 680 685
Gly Thr Ser Gly Asp Phe Glu Gln Ala Lys Lys Ile Phe Asp Gly Glu
690 695 700
Lys Ser Asp Arg Phe Phe Thr Trp Gly Gln Thr Asn Trp Ile Ala Phe
705 710 715 720
Asp Leu Gly Glu Ile Asn Leu Ala Lys Glu Trp Arg Leu Phe Asn Ala
725 730 735
Glu Thr Asn Thr Glu Ile Lys Thr Asp Ser Ser Leu Asn Val Ala Lys
740 745 750
Gly Arg Leu Gln Ile Leu Lys Asp Thr Thr Ile Asp Leu Glu Lys Met
755 760 765
Asp Ile Lys Asn Arg Lys Glu Tyr Leu Ser Asn Asp Glu Asn Trp Thr
770 775 780
Asp Val Ala Gln Met Asp Asp Ala Lys Ala Ile Phe Asn Ser Lys Leu
785 790 795 800
Ser Asn Val Leu Ser Arg Tyr Trp Arg Phe Cys Val Asp Gly Gly Ala
805 810 815
Ser Ser Tyr Tyr Pro Gln Tyr Thr Glu Leu Gln Ile Leu Gly Gln Arg
820 825 830
Leu Ser Asn Asp Val Ala Asn Thr Leu Lys Asp
835 840
<210> 17
<211> 843
<212> PRT
<213> 人工序列
<220>
<223> 合成的构建物
<400> 17
Met Asp Lys His Leu Leu Val Lys Arg Thr Leu Gly Cys Val Cys Ala
1 5 10 15
Ala Thr Leu Met Gly Ala Ala Leu Ala Thr His His Asp Ser Leu Asn
20 25 30
Thr Val Lys Ala Glu Glu Lys Thr Val Gln Thr Gly Lys Thr Asp Gln
35 40 45
Gln Val Gly Ala Lys Leu Val Gln Glu Ile Arg Glu Gly Lys Arg Gly
50 55 60
Pro Leu Tyr Ala Gly Tyr Phe Arg Thr Trp His Asp Arg Ala Ser Thr
65 70 75 80
Gly Ile Asp Gly Lys Gln Gln His Pro Glu Asn Thr Met Ala Glu Val
85 90 95
Pro Lys Glu Val Asp Ile Leu Phe Val Phe His Asp His Thr Ala Ser
100 105 110
Asp Ser Pro Phe Trp Ser Glu Leu Lys Asp Ser Tyr Val His Lys Leu
115 120 125
His Gln Gln Gly Thr Ala Leu Val Gln Thr Ile Gly Val Asn Glu Leu
130 135 140
Asn Gly Arg Thr Gly Leu Ser Lys Asp Tyr Pro Asp Thr Pro Glu Gly
145 150 155 160
Asn Lys Ala Leu Ala Ala Ala Ile Val Lys Ala Phe Val Thr Asp Arg
165 170 175
Gly Val Asp Gly Leu Asp Ile Thr Ile Glu His Glu Phe Thr Asn Lys
180 185 190
Arg Thr Pro Glu Glu Asp Ala Arg Ala Leu Asn Val Phe Lys Glu Ile
195 200 205
Ala Gln Leu Ile Gly Lys Asn Gly Ser Asp Lys Ser Lys Leu Leu Ile
210 215 220
Met Asp Thr Thr Leu Ser Val Glu Asn Asn Pro Ile Phe Lys Gly Ile
225 230 235 240
Ala Glu Asp Leu Asp Tyr Leu Leu Arg Gln Tyr Tyr Gly Ser Gln Gly
245 250 255
Gly Glu Ala Glu Val Asp Thr Ile Asn Ser Asp Trp Asn Gln Tyr Gln
260 265 270
Asn Tyr Ile Asp Ala Ser Gln Phe Met Ile Gly Phe Ser Phe Phe Glu
275 280 285
Glu Ser Ala Ser Lys Gly Asn Leu Trp Phe Asp Val Asn Glu Tyr Asp
290 295 300
Pro Asn Asn Pro Glu Lys Gly Lys Asp Ile Glu Gly Thr Arg Ala Lys
305 310 315 320
Lys Tyr Ala Glu Trp Gln Pro Ser Thr Gly Gly Leu Lys Ala Gly Ile
325 330 335
Phe Ser Tyr Ala Ile Asp Arg Asp Gly Val Ala His Val Pro Ser Thr
340 345 350
Tyr Lys Asn Arg Thr Ser Thr Asn Leu Gln Arg His Glu Val Asp Asn
355 360 365
Ile Ser His Thr Asp Tyr Thr Val Ser Arg Lys Leu Lys Thr Leu Met
370 375 380
Thr Glu Asp Lys Arg Tyr Asp Val Ile Asp Gln Lys Asp Ile Pro Asp
385 390 395 400
Pro Ala Leu Arg Glu Gln Ile Ile Gln Gln Val Gly Gln Tyr Lys Gly
405 410 415
Asp Leu Glu Arg Tyr Asn Lys Thr Leu Val Leu Thr Gly Asp Lys Ile
420 425 430
Gln Asn Leu Lys Gly Leu Glu Lys Leu Ser Lys Leu Gln Lys Leu Glu
435 440 445
Leu Arg Gln Leu Ser Asn Val Lys Glu Ile Thr Pro Glu Leu Leu Pro
450 455 460
Glu Ser Met Lys Lys Asp Ala Glu Leu Val Met Val Gly Met Thr Gly
465 470 475 480
Leu Glu Lys Leu Asn Leu Ser Gly Leu Asn Arg Gln Thr Leu Asp Gly
485 490 495
Ile Asp Val Asn Ser Ile Thr His Leu Thr Ser Phe Asp Ile Ser His
500 505 510
Asn Ser Leu Asp Leu Ser Glu Lys Ser Glu Asp Arg Lys Leu Leu Met
515 520 525
Thr Leu Met Glu Gln Val Ser Asn His Gln Lys Ile Thr Val Lys Asn
530 535 540
Thr Ala Phe Glu Asn Gln Lys Pro Lys Gly Tyr Tyr Pro Gln Thr Tyr
545 550 555 560
Asp Thr Lys Glu Gly His Tyr Asp Val Asp Asn Ala Glu His Asp Ile
565 570 575
Leu Thr Asp Phe Val Phe Gly Thr Val Thr Lys Arg Asn Thr Phe Ile
580 585 590
Gly Asp Glu Glu Ala Phe Ala Ile Tyr Lys Glu Gly Ala Val Asp Gly
595 600 605
Arg Gln Tyr Val Ser Lys Asp Tyr Thr Tyr Glu Ala Phe Arg Lys Asp
610 615 620
Tyr Lys Gly Tyr Lys Val His Leu Thr Ala Ser Asn Leu Gly Glu Thr
625 630 635 640
Val Thr Ser Lys Val Thr Ala Thr Thr Asp Glu Thr Tyr Leu Val Asp
645 650 655
Val Ser Asp Gly Glu Lys Val Val His His Met Lys Leu Asn Ile Gly
660 665 670
Ser Gly Ala Ile Met Met Glu Asn Leu Ala Lys Gly Ala Lys Val Ile
675 680 685
Gly Thr Ser Gly Asp Phe Glu Gln Ala Lys Lys Ile Phe Asp Gly Glu
690 695 700
Lys Ser Asp Arg Phe Phe Thr Trp Gly Gln Thr Asn Trp Ile Ala Phe
705 710 715 720
Asp Leu Gly Glu Ile Asn Leu Ala Lys Glu Trp Arg Leu Phe Asn Ala
725 730 735
Glu Thr Asn Thr Glu Ile Lys Thr Asp Ser Ser Leu Asn Val Ala Lys
740 745 750
Gly Arg Leu Gln Ile Leu Lys Asp Thr Thr Ile Asp Leu Glu Lys Met
755 760 765
Asp Ile Lys Asn Arg Lys Glu Tyr Leu Ser Asn Asp Glu Asn Trp Thr
770 775 780
Asp Val Ala Gln Met Asp Asp Ala Lys Ala Ile Phe Asn Ser Lys Leu
785 790 795 800
Ser Asn Val Leu Ser Arg Tyr Trp Arg Phe Cys Val Asp Gly Gly Ala
805 810 815
Ser Ser Tyr Tyr Pro Gln Tyr Thr Glu Leu Gln Ile Leu Gly Gln Arg
820 825 830
Leu Ser Asn Asp Val Ala Asn Thr Leu Lys Asp
835 840
<210> 18
<211> 843
<212> PRT
<213> 人工序列
<220>
<223> 合成的构建物
<400> 18
Met Asp Lys His Leu Leu Val Lys Arg Thr Leu Gly Cys Val Cys Ala
1 5 10 15
Ala Thr Leu Met Gly Ala Ala Leu Ala Thr His His Asp Ser Leu Asn
20 25 30
Thr Val Lys Ala Glu Glu Lys Thr Val Gln Thr Gly Lys Thr Asp Gln
35 40 45
Gln Val Gly Ala Lys Leu Val Gln Glu Ile Arg Glu Gly Lys Arg Gly
50 55 60
Pro Leu Tyr Ala Gly Tyr Phe Arg Thr Trp His Asp Arg Ala Ser Thr
65 70 75 80
Gly Ile Asp Gly Lys Gln Gln His Pro Glu Asn Thr Met Ala Glu Val
85 90 95
Pro Lys Glu Val Asp Ile Leu Phe Val Phe His Asp His Thr Ala Ser
100 105 110
Asp Ser Pro Phe Trp Ser Glu Leu Lys Asp Ser Tyr Val His Lys Leu
115 120 125
His Gln Gln Gly Thr Ala Leu Val Gln Thr Ile Gly Val Asn Glu Leu
130 135 140
Asn Gly Arg Thr Gly Leu Ser Lys Asp Tyr Pro Asp Thr Pro Glu Gly
145 150 155 160
Asn Lys Ala Leu Ala Ala Ala Ile Val Lys Ala Phe Val Thr Asp Arg
165 170 175
Gly Val Asp Gly Leu Asp Ile Trp Ile Glu His Glu Phe Thr Asn Lys
180 185 190
Arg Thr Pro Glu Glu Asp Ala Arg Ala Leu Asn Val Phe Lys Glu Ile
195 200 205
Ala Gln Leu Ile Gly Lys Asn Gly Ser Asp Lys Ser Lys Leu Leu Ile
210 215 220
Met Asp Thr Thr Leu Ser Val Glu Asn Asn Pro Ile Phe Lys Gly Ile
225 230 235 240
Ala Glu Asp Leu Asp Tyr Leu Leu Arg Gln Tyr Tyr Gly Ser Gln Gly
245 250 255
Gly Glu Ala Glu Val Asp Thr Ile Asn Ser Asp Trp Asn Gln Tyr Gln
260 265 270
Asn Tyr Ile Asp Ala Ser Gln Phe Met Ile Gly Phe Ser Phe Phe Glu
275 280 285
Glu Ser Ala Ser Lys Gly Asn Leu Trp Phe Asp Val Asn Glu Tyr Asp
290 295 300
Pro Asn Asn Pro Glu Lys Gly Lys Asp Ile Glu Gly Thr Arg Ala Lys
305 310 315 320
Lys Tyr Ala Glu Trp Gln Pro Ser Thr Gly Gly Leu Lys Ala Gly Ile
325 330 335
Phe Ser Tyr Ala Ile Asp Arg Asp Gly Val Ala His Val Pro Ser Thr
340 345 350
Tyr Lys Asn Arg Thr Ser Thr Asn Leu Gln Arg His Glu Val Asp Asn
355 360 365
Ile Ser His Thr Asp Tyr Thr Val Ser Arg Lys Leu Lys Thr Leu Met
370 375 380
Thr Glu Asp Lys Arg Tyr Asp Val Ile Asp Gln Lys Asp Ile Pro Asp
385 390 395 400
Pro Ala Leu Arg Glu Gln Ile Ile Gln Gln Val Gly Gln Tyr Lys Gly
405 410 415
Asp Leu Glu Arg Tyr Asn Lys Thr Leu Val Leu Thr Gly Asp Lys Ile
420 425 430
Gln Asn Leu Lys Gly Leu Glu Lys Leu Ser Lys Leu Gln Lys Leu Glu
435 440 445
Leu Arg Gln Leu Ser Asn Val Lys Glu Ile Thr Pro Glu Leu Leu Pro
450 455 460
Glu Ser Met Lys Lys Asp Ala Glu Leu Val Met Val Gly Met Thr Gly
465 470 475 480
Leu Glu Lys Leu Asn Leu Ser Gly Leu Asn Arg Gln Thr Leu Asp Gly
485 490 495
Ile Asp Val Asn Ser Ile Thr His Leu Thr Ser Phe Asp Ile Ser His
500 505 510
Asn Ser Leu Asp Leu Ser Glu Lys Ser Glu Asp Arg Lys Leu Leu Met
515 520 525
Thr Leu Met Glu Gln Val Ser Asn His Gln Lys Ile Thr Val Lys Asn
530 535 540
Thr Ala Phe Glu Asn Gln Lys Pro Lys Gly Tyr Tyr Pro Gln Thr Tyr
545 550 555 560
Asp Thr Lys Glu Gly His Tyr Asp Val Asp Asn Ala Glu His Asp Ile
565 570 575
Leu Thr Asp Phe Val Phe Gly Thr Val Thr Lys Arg Asn Thr Phe Ile
580 585 590
Gly Asp Glu Glu Ala Phe Ala Ile Tyr Lys Glu Gly Ala Val Asp Gly
595 600 605
Arg Gln Tyr Val Ser Lys Asp Tyr Thr Tyr Glu Ala Phe Arg Lys Asp
610 615 620
Tyr Lys Gly Tyr Lys Val His Leu Thr Ala Ser Asn Leu Gly Glu Thr
625 630 635 640
Val Thr Ser Lys Val Thr Ala Thr Thr Asp Glu Thr Tyr Leu Val Asp
645 650 655
Val Ser Asp Gly Glu Lys Val Val His His Met Lys Leu Asn Ile Gly
660 665 670
Ser Gly Ala Ile Met Met Glu Asn Leu Ala Lys Gly Ala Lys Val Ile
675 680 685
Gly Thr Ser Gly Asp Phe Glu Gln Ala Lys Lys Ile Phe Asp Gly Glu
690 695 700
Lys Ser Asp Arg Phe Phe Thr Trp Gly Gln Thr Asn Trp Ile Ala Phe
705 710 715 720
Asp Leu Gly Glu Ile Asn Leu Ala Lys Glu Trp Arg Leu Phe Asn Ala
725 730 735
Glu Thr Asn Thr Glu Ile Lys Thr Asp Ser Ser Leu Asn Val Ala Lys
740 745 750
Gly Arg Leu Gln Ile Leu Lys Asp Thr Thr Ile Asp Leu Glu Lys Met
755 760 765
Asp Ile Lys Asn Arg Lys Glu Tyr Leu Ser Asn Asp Glu Asn Trp Thr
770 775 780
Asp Val Ala Gln Met Asp Asp Ala Lys Ala Ile Phe Asn Ser Lys Leu
785 790 795 800
Ser Asn Val Leu Ser Arg Tyr Trp Arg Phe Cys Val Asp Gly Gly Ala
805 810 815
Ser Ser Tyr Tyr Pro Gln Tyr Thr Glu Leu Gln Ile Leu Gly Gln Arg
820 825 830
Leu Ser Asn Asp Val Ala Asn Thr Leu Lys Asp
835 840
<210> 19
<211> 843
<212> PRT
<213> 人工序列
<220>
<223> 合成的构建物
<400> 19
Met Asp Lys His Leu Leu Val Lys Arg Thr Leu Gly Cys Val Cys Ala
1 5 10 15
Ala Thr Leu Met Gly Ala Ala Leu Ala Thr His His Asp Ser Leu Asn
20 25 30
Thr Val Lys Ala Glu Glu Lys Thr Val Gln Thr Gly Lys Thr Asp Gln
35 40 45
Gln Val Gly Ala Lys Leu Val Gln Glu Ile Arg Glu Gly Lys Arg Gly
50 55 60
Pro Leu Tyr Ala Gly Tyr Phe Arg Thr Trp His Asp Arg Ala Ser Thr
65 70 75 80
Gly Ile Asp Gly Lys Gln Gln His Pro Glu Asn Thr Met Ala Glu Val
85 90 95
Pro Lys Glu Val Asp Ile Leu Phe Val Phe His Asp His Thr Ala Ser
100 105 110
Asp Ser Pro Phe Trp Ser Glu Leu Lys Asp Ser Tyr Val His Lys Leu
115 120 125
His Gln Gln Gly Thr Ala Leu Val Gln Thr Ile Gly Val Asn Glu Leu
130 135 140
Asn Gly Arg Thr Gly Leu Ser Lys Asp Tyr Pro Asp Thr Pro Glu Gly
145 150 155 160
Asn Lys Ala Leu Ala Ala Ala Ile Val Lys Ala Phe Val Thr Asp Arg
165 170 175
Gly Val Asp Gly Leu Asp Ile Tyr Ile Glu His Glu Phe Thr Asn Lys
180 185 190
Arg Thr Pro Glu Glu Asp Ala Arg Ala Leu Asn Val Phe Lys Glu Ile
195 200 205
Ala Gln Leu Ile Gly Lys Asn Gly Ser Asp Lys Ser Lys Leu Leu Ile
210 215 220
Met Asp Thr Thr Leu Ser Val Glu Asn Asn Pro Ile Phe Lys Gly Ile
225 230 235 240
Ala Glu Asp Leu Asp Tyr Leu Leu Arg Gln Tyr Tyr Gly Ser Gln Gly
245 250 255
Gly Glu Ala Glu Val Asp Thr Ile Asn Ser Asp Trp Asn Gln Tyr Gln
260 265 270
Asn Tyr Ile Asp Ala Ser Gln Phe Met Ile Gly Phe Ser Phe Phe Glu
275 280 285
Glu Ser Ala Ser Lys Gly Asn Leu Trp Phe Asp Val Asn Glu Tyr Asp
290 295 300
Pro Asn Asn Pro Glu Lys Gly Lys Asp Ile Glu Gly Thr Arg Ala Lys
305 310 315 320
Lys Tyr Ala Glu Trp Gln Pro Ser Thr Gly Gly Leu Lys Ala Gly Ile
325 330 335
Phe Ser Tyr Ala Ile Asp Arg Asp Gly Val Ala His Val Pro Ser Thr
340 345 350
Tyr Lys Asn Arg Thr Ser Thr Asn Leu Gln Arg His Glu Val Asp Asn
355 360 365
Ile Ser His Thr Asp Tyr Thr Val Ser Arg Lys Leu Lys Thr Leu Met
370 375 380
Thr Glu Asp Lys Arg Tyr Asp Val Ile Asp Gln Lys Asp Ile Pro Asp
385 390 395 400
Pro Ala Leu Arg Glu Gln Ile Ile Gln Gln Val Gly Gln Tyr Lys Gly
405 410 415
Asp Leu Glu Arg Tyr Asn Lys Thr Leu Val Leu Thr Gly Asp Lys Ile
420 425 430
Gln Asn Leu Lys Gly Leu Glu Lys Leu Ser Lys Leu Gln Lys Leu Glu
435 440 445
Leu Arg Gln Leu Ser Asn Val Lys Glu Ile Thr Pro Glu Leu Leu Pro
450 455 460
Glu Ser Met Lys Lys Asp Ala Glu Leu Val Met Val Gly Met Thr Gly
465 470 475 480
Leu Glu Lys Leu Asn Leu Ser Gly Leu Asn Arg Gln Thr Leu Asp Gly
485 490 495
Ile Asp Val Asn Ser Ile Thr His Leu Thr Ser Phe Asp Ile Ser His
500 505 510
Asn Ser Leu Asp Leu Ser Glu Lys Ser Glu Asp Arg Lys Leu Leu Met
515 520 525
Thr Leu Met Glu Gln Val Ser Asn His Gln Lys Ile Thr Val Lys Asn
530 535 540
Thr Ala Phe Glu Asn Gln Lys Pro Lys Gly Tyr Tyr Pro Gln Thr Tyr
545 550 555 560
Asp Thr Lys Glu Gly His Tyr Asp Val Asp Asn Ala Glu His Asp Ile
565 570 575
Leu Thr Asp Phe Val Phe Gly Thr Val Thr Lys Arg Asn Thr Phe Ile
580 585 590
Gly Asp Glu Glu Ala Phe Ala Ile Tyr Lys Glu Gly Ala Val Asp Gly
595 600 605
Arg Gln Tyr Val Ser Lys Asp Tyr Thr Tyr Glu Ala Phe Arg Lys Asp
610 615 620
Tyr Lys Gly Tyr Lys Val His Leu Thr Ala Ser Asn Leu Gly Glu Thr
625 630 635 640
Val Thr Ser Lys Val Thr Ala Thr Thr Asp Glu Thr Tyr Leu Val Asp
645 650 655
Val Ser Asp Gly Glu Lys Val Val His His Met Lys Leu Asn Ile Gly
660 665 670
Ser Gly Ala Ile Met Met Glu Asn Leu Ala Lys Gly Ala Lys Val Ile
675 680 685
Gly Thr Ser Gly Asp Phe Glu Gln Ala Lys Lys Ile Phe Asp Gly Glu
690 695 700
Lys Ser Asp Arg Phe Phe Thr Trp Gly Gln Thr Asn Trp Ile Ala Phe
705 710 715 720
Asp Leu Gly Glu Ile Asn Leu Ala Lys Glu Trp Arg Leu Phe Asn Ala
725 730 735
Glu Thr Asn Thr Glu Ile Lys Thr Asp Ser Ser Leu Asn Val Ala Lys
740 745 750
Gly Arg Leu Gln Ile Leu Lys Asp Thr Thr Ile Asp Leu Glu Lys Met
755 760 765
Asp Ile Lys Asn Arg Lys Glu Tyr Leu Ser Asn Asp Glu Asn Trp Thr
770 775 780
Asp Val Ala Gln Met Asp Asp Ala Lys Ala Ile Phe Asn Ser Lys Leu
785 790 795 800
Ser Asn Val Leu Ser Arg Tyr Trp Arg Phe Cys Val Asp Gly Gly Ala
805 810 815
Ser Ser Tyr Tyr Pro Gln Tyr Thr Glu Leu Gln Ile Leu Gly Gln Arg
820 825 830
Leu Ser Asn Asp Val Ala Asn Thr Leu Lys Asp
835 840
<210> 20
<211> 843
<212> PRT
<213> 人工序列
<220>
<223> 合成的构建物
<400> 20
Met Asp Lys His Leu Leu Val Lys Arg Thr Leu Gly Cys Val Cys Ala
1 5 10 15
Ala Thr Leu Met Gly Ala Ala Leu Ala Thr His His Asp Ser Leu Asn
20 25 30
Thr Val Lys Ala Glu Glu Lys Thr Val Gln Thr Gly Lys Thr Asp Gln
35 40 45
Gln Val Gly Ala Lys Leu Val Gln Glu Ile Arg Glu Gly Lys Arg Gly
50 55 60
Pro Leu Tyr Ala Gly Tyr Phe Arg Thr Trp His Asp Arg Ala Ser Thr
65 70 75 80
Gly Ile Asp Gly Lys Gln Gln His Pro Glu Asn Thr Met Ala Glu Val
85 90 95
Pro Lys Glu Val Asp Ile Leu Phe Val Phe His Asp His Thr Ala Ser
100 105 110
Asp Ser Pro Phe Trp Ser Glu Leu Lys Asp Ser Tyr Val His Lys Leu
115 120 125
His Gln Gln Gly Thr Ala Leu Val Gln Thr Ile Gly Val Asn Glu Leu
130 135 140
Asn Gly Arg Thr Gly Leu Ser Lys Asp Tyr Pro Asp Thr Pro Glu Gly
145 150 155 160
Asn Lys Ala Leu Ala Ala Ala Ile Val Lys Ala Phe Val Thr Asp Arg
165 170 175
Gly Val Asp Gly Leu Asp Ile Val Ile Glu His Glu Phe Thr Asn Lys
180 185 190
Arg Thr Pro Glu Glu Asp Ala Arg Ala Leu Asn Val Phe Lys Glu Ile
195 200 205
Ala Gln Leu Ile Gly Lys Asn Gly Ser Asp Lys Ser Lys Leu Leu Ile
210 215 220
Met Asp Thr Thr Leu Ser Val Glu Asn Asn Pro Ile Phe Lys Gly Ile
225 230 235 240
Ala Glu Asp Leu Asp Tyr Leu Leu Arg Gln Tyr Tyr Gly Ser Gln Gly
245 250 255
Gly Glu Ala Glu Val Asp Thr Ile Asn Ser Asp Trp Asn Gln Tyr Gln
260 265 270
Asn Tyr Ile Asp Ala Ser Gln Phe Met Ile Gly Phe Ser Phe Phe Glu
275 280 285
Glu Ser Ala Ser Lys Gly Asn Leu Trp Phe Asp Val Asn Glu Tyr Asp
290 295 300
Pro Asn Asn Pro Glu Lys Gly Lys Asp Ile Glu Gly Thr Arg Ala Lys
305 310 315 320
Lys Tyr Ala Glu Trp Gln Pro Ser Thr Gly Gly Leu Lys Ala Gly Ile
325 330 335
Phe Ser Tyr Ala Ile Asp Arg Asp Gly Val Ala His Val Pro Ser Thr
340 345 350
Tyr Lys Asn Arg Thr Ser Thr Asn Leu Gln Arg His Glu Val Asp Asn
355 360 365
Ile Ser His Thr Asp Tyr Thr Val Ser Arg Lys Leu Lys Thr Leu Met
370 375 380
Thr Glu Asp Lys Arg Tyr Asp Val Ile Asp Gln Lys Asp Ile Pro Asp
385 390 395 400
Pro Ala Leu Arg Glu Gln Ile Ile Gln Gln Val Gly Gln Tyr Lys Gly
405 410 415
Asp Leu Glu Arg Tyr Asn Lys Thr Leu Val Leu Thr Gly Asp Lys Ile
420 425 430
Gln Asn Leu Lys Gly Leu Glu Lys Leu Ser Lys Leu Gln Lys Leu Glu
435 440 445
Leu Arg Gln Leu Ser Asn Val Lys Glu Ile Thr Pro Glu Leu Leu Pro
450 455 460
Glu Ser Met Lys Lys Asp Ala Glu Leu Val Met Val Gly Met Thr Gly
465 470 475 480
Leu Glu Lys Leu Asn Leu Ser Gly Leu Asn Arg Gln Thr Leu Asp Gly
485 490 495
Ile Asp Val Asn Ser Ile Thr His Leu Thr Ser Phe Asp Ile Ser His
500 505 510
Asn Ser Leu Asp Leu Ser Glu Lys Ser Glu Asp Arg Lys Leu Leu Met
515 520 525
Thr Leu Met Glu Gln Val Ser Asn His Gln Lys Ile Thr Val Lys Asn
530 535 540
Thr Ala Phe Glu Asn Gln Lys Pro Lys Gly Tyr Tyr Pro Gln Thr Tyr
545 550 555 560
Asp Thr Lys Glu Gly His Tyr Asp Val Asp Asn Ala Glu His Asp Ile
565 570 575
Leu Thr Asp Phe Val Phe Gly Thr Val Thr Lys Arg Asn Thr Phe Ile
580 585 590
Gly Asp Glu Glu Ala Phe Ala Ile Tyr Lys Glu Gly Ala Val Asp Gly
595 600 605
Arg Gln Tyr Val Ser Lys Asp Tyr Thr Tyr Glu Ala Phe Arg Lys Asp
610 615 620
Tyr Lys Gly Tyr Lys Val His Leu Thr Ala Ser Asn Leu Gly Glu Thr
625 630 635 640
Val Thr Ser Lys Val Thr Ala Thr Thr Asp Glu Thr Tyr Leu Val Asp
645 650 655
Val Ser Asp Gly Glu Lys Val Val His His Met Lys Leu Asn Ile Gly
660 665 670
Ser Gly Ala Ile Met Met Glu Asn Leu Ala Lys Gly Ala Lys Val Ile
675 680 685
Gly Thr Ser Gly Asp Phe Glu Gln Ala Lys Lys Ile Phe Asp Gly Glu
690 695 700
Lys Ser Asp Arg Phe Phe Thr Trp Gly Gln Thr Asn Trp Ile Ala Phe
705 710 715 720
Asp Leu Gly Glu Ile Asn Leu Ala Lys Glu Trp Arg Leu Phe Asn Ala
725 730 735
Glu Thr Asn Thr Glu Ile Lys Thr Asp Ser Ser Leu Asn Val Ala Lys
740 745 750
Gly Arg Leu Gln Ile Leu Lys Asp Thr Thr Ile Asp Leu Glu Lys Met
755 760 765
Asp Ile Lys Asn Arg Lys Glu Tyr Leu Ser Asn Asp Glu Asn Trp Thr
770 775 780
Asp Val Ala Gln Met Asp Asp Ala Lys Ala Ile Phe Asn Ser Lys Leu
785 790 795 800
Ser Asn Val Leu Ser Arg Tyr Trp Arg Phe Cys Val Asp Gly Gly Ala
805 810 815
Ser Ser Tyr Tyr Pro Gln Tyr Thr Glu Leu Gln Ile Leu Gly Gln Arg
820 825 830
Leu Ser Asn Asp Val Ala Asn Thr Leu Lys Asp
835 840
<210> 21
<211> 995
<212> PRT
<213> 化脓性链球菌（Streptococcus pyogenes）
<400> 21
Met Asp Lys His Leu Leu Val Lys Arg Thr Leu Gly Cys Val Cys Ala
1 5 10 15
Ala Thr Leu Met Gly Ala Ala Leu Ala Thr His His Asp Ser Leu Asn
20 25 30
Thr Val Lys Ala Glu Glu Lys Thr Val Gln Val Gln Lys Gly Leu Pro
35 40 45
Ser Ile Asp Ser Leu His Tyr Leu Ser Glu Asn Ser Lys Lys Glu Phe
50 55 60
Lys Glu Glu Leu Ser Lys Ala Gly Gln Glu Ser Gln Lys Val Lys Glu
65 70 75 80
Ile Leu Ala Lys Ala Gln Gln Ala Asp Lys Gln Ala Gln Glu Leu Ala
85 90 95
Lys Met Lys Ile Pro Glu Lys Ile Pro Met Lys Pro Leu His Gly Pro
100 105 110
Leu Tyr Gly Gly Tyr Phe Arg Thr Trp His Asp Lys Thr Ser Asp Pro
115 120 125
Thr Glu Lys Asp Lys Val Asn Ser Met Gly Glu Leu Pro Lys Glu Val
130 135 140
Asp Leu Ala Phe Ile Phe His Asp Trp Thr Lys Asp Tyr Ser Leu Phe
145 150 155 160
Trp Lys Glu Leu Ala Thr Lys His Val Pro Lys Leu Asn Lys Gln Gly
165 170 175
Thr Arg Val Ile Arg Thr Ile Pro Trp Arg Phe Leu Ala Gly Gly Asp
180 185 190
Asn Ser Gly Ile Ala Glu Asp Thr Ser Lys Tyr Pro Asn Thr Pro Glu
195 200 205
Gly Asn Lys Ala Leu Ala Lys Ala Ile Val Asp Glu Tyr Val Tyr Lys
210 215 220
Tyr Asn Leu Asp Gly Leu Asp Val Asp Val Glu His Asp Ser Ile Pro
225 230 235 240
Lys Val Asp Lys Lys Glu Asp Thr Ala Gly Val Glu Arg Ser Ile Gln
245 250 255
Val Phe Glu Glu Ile Gly Lys Leu Ile Gly Pro Lys Gly Val Asp Lys
260 265 270
Ser Arg Leu Phe Ile Met Asp Ser Thr Tyr Met Ala Asp Lys Asn Pro
275 280 285
Leu Ile Glu Arg Gly Ala Pro Tyr Ile Asn Leu Leu Leu Val Gln Val
290 295 300
Tyr Gly Ser Gln Gly Glu Lys Gly Gly Trp Glu Pro Val Ser Asn Arg
305 310 315 320
Pro Glu Lys Thr Met Glu Glu Arg Trp Gln Gly Tyr Ser Lys Tyr Ile
325 330 335
Arg Pro Glu Gln Tyr Met Ile Gly Phe Ser Phe Tyr Glu Glu Asn Ala
340 345 350
Gln Glu Gly Asn Leu Trp Tyr Asp Ile Asn Ser Arg Lys Asp Glu Asp
355 360 365
Lys Ala Asn Gly Ile Asn Thr Asp Ile Thr Gly Thr Arg Ala Glu Arg
370 375 380
Tyr Ala Arg Trp Gln Pro Lys Thr Gly Gly Val Lys Gly Gly Ile Phe
385 390 395 400
Ser Tyr Ala Ile Asp Arg Asp Gly Val Ala His Gln Pro Lys Lys Tyr
405 410 415
Ala Lys Gln Lys Glu Phe Lys Asp Ala Thr Asp Asn Ile Phe His Ser
420 425 430
Asp Tyr Ser Val Ser Lys Ala Leu Lys Thr Val Met Leu Lys Asp Lys
435 440 445
Ser Tyr Asp Leu Ile Asp Glu Lys Asp Phe Pro Asp Lys Ala Leu Arg
450 455 460
Glu Ala Val Met Ala Gln Val Gly Thr Arg Lys Gly Asp Leu Glu Arg
465 470 475 480
Phe Asn Gly Thr Leu Arg Leu Asp Asn Pro Ala Ile Gln Ser Leu Glu
485 490 495
Gly Leu Asn Lys Phe Lys Lys Leu Ala Gln Leu Asp Leu Ile Gly Leu
500 505 510
Ser Arg Ile Thr Lys Leu Asp Arg Ser Val Leu Pro Ala Asn Met Lys
515 520 525
Pro Gly Lys Asp Thr Leu Glu Thr Val Leu Glu Thr Tyr Lys Lys Asp
530 535 540
Asn Lys Glu Glu Pro Ala Thr Ile Pro Pro Val Ser Leu Lys Val Ser
545 550 555 560
Gly Leu Thr Gly Leu Lys Glu Leu Asp Leu Ser Gly Phe Asp Arg Glu
565 570 575
Thr Leu Ala Gly Leu Asp Ala Ala Thr Leu Thr Ser Leu Glu Lys Val
580 585 590
Asp Ile Ser Gly Asn Lys Leu Asp Leu Ala Pro Gly Thr Glu Asn Arg
595 600 605
Gln Ile Phe Asp Thr Met Leu Ser Thr Ile Ser Asn His Val Gly Ser
610 615 620
Asn Glu Gln Thr Val Lys Phe Asp Lys Gln Lys Pro Thr Gly His Tyr
625 630 635 640
Pro Asp Thr Tyr Gly Lys Thr Ser Leu Arg Leu Pro Val Ala Asn Glu
645 650 655
Lys Val Asp Leu Gln Ser Gln Leu Leu Phe Gly Thr Val Thr Asn Gln
660 665 670
Gly Thr Leu Ile Asn Ser Glu Ala Asp Tyr Lys Ala Tyr Gln Asn His
675 680 685
Lys Ile Ala Gly Arg Ser Phe Val Asp Ser Asn Tyr His Tyr Asn Asn
690 695 700
Phe Lys Val Ser Tyr Glu Asn Tyr Thr Val Lys Val Thr Asp Ser Thr
705 710 715 720
Leu Gly Thr Thr Thr Asp Lys Thr Leu Ala Thr Asp Lys Glu Glu Thr
725 730 735
Tyr Lys Val Asp Phe Phe Ser Pro Ala Asp Lys Thr Lys Ala Val His
740 745 750
Thr Ala Lys Val Ile Val Gly Asp Glu Lys Thr Met Met Val Asn Leu
755 760 765
Ala Glu Gly Ala Thr Val Ile Gly Gly Ser Ala Asp Pro Val Asn Ala
770 775 780
Arg Lys Val Phe Asp Gly Gln Leu Gly Ser Glu Thr Asp Asn Ile Ser
785 790 795 800
Leu Gly Trp Asp Ser Lys Gln Ser Ile Ile Phe Lys Leu Lys Glu Asp
805 810 815
Gly Leu Ile Lys His Trp Arg Phe Phe Asn Asp Ser Ala Arg Asn Pro
820 825 830
Glu Thr Thr Asn Lys Pro Ile Gln Glu Ala Ser Leu Gln Ile Phe Asn
835 840 845
Ile Lys Asp Tyr Asn Leu Asp Asn Leu Leu Glu Asn Pro Asn Lys Phe
850 855 860
Asp Asp Glu Lys Tyr Trp Ile Thr Val Asp Thr Tyr Ser Ala Gln Gly
865 870 875 880
Glu Arg Ala Thr Ala Phe Ser Asn Thr Leu Asn Asn Ile Thr Ser Lys
885 890 895
Tyr Trp Arg Val Val Phe Asp Thr Lys Gly Asp Arg Tyr Ser Ser Pro
900 905 910
Val Val Pro Glu Leu Gln Ile Leu Gly Tyr Pro Leu Pro Asn Ala Asp
915 920 925
Thr Ile Met Lys Thr Val Thr Thr Ala Lys Glu Leu Ser Gln Gln Lys
930 935 940
Asp Lys Phe Ser Gln Lys Met Leu Asp Glu Leu Lys Ile Lys Glu Met
945 950 955 960
Ala Leu Glu Thr Ser Leu Asn Ser Lys Ile Phe Asp Val Thr Ala Ile
965 970 975
Asn Ala Asn Ala Gly Val Leu Lys Asp Cys Ile Glu Lys Arg Gln Leu
980 985 990
Leu Lys Lys
995
<210> 22
<211> 231
<212> PRT
<213> 人工序列
<220>
<223> 合成的构建物
<400> 22
Leu Gly Ser Gly Lys Ile Leu His Asn Gln Asn Val Asn Ser Trp Gly
1 5 10 15
Pro Ile Thr Val Thr Pro Thr Thr Asp Gly Gly Glu Thr Arg Phe Asp
20 25 30
Gly Gln Ile Ile Val Gln Met Glu Asn Asp Pro Val Val Ala Lys Ala
35 40 45
Ala Ala Asn Leu Ala Gly Lys His Ala Glu Ser Ser Val Val Val Gln
50 55 60
Leu Asp Ser Asp Gly Asn Tyr Arg Val Val Tyr Gly Asp Pro Ser Lys
65 70 75 80
Leu Asp Gly Lys Leu Arg Trp Gln Leu Val Gly His Gly Arg Asp His
85 90 95
Ser Glu Thr Asn Asn Thr Arg Leu Ser Gly Tyr Ser Ala Asp Glu Leu
100 105 110
Ala Val Lys Leu Ala Lys Phe Gln Gln Ser Phe Asn Gln Ala Glu Asn
115 120 125
Ile Asn Asn Lys Pro Asp His Ile Ser Ile Val Gly Cys Ser Leu Val
130 135 140
Ser Asp Asp Lys Gln Lys Gly Phe Gly His Gln Phe Ile Asn Ala Met
145 150 155 160
Asp Ala Asn Gly Leu Arg Val Asp Val Ser Val Arg Ser Ser Glu Leu
165 170 175
Ala Val Asp Glu Ala Gly Arg Lys His Thr Lys Asp Ala Asn Gly Asp
180 185 190
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Claims (16)
1.一种制备具有预定的低聚糖部分的岩藻糖基化或非岩藻糖基化的糖蛋白的方法，所述方法包括：
提供岩藻糖基化或非岩藻糖基化的受体蛋白，所述岩藻糖基化或非岩藻糖基化的受体蛋白包括岩藻糖基化的N-乙酰葡糖胺(GlcNAc)受体-蛋白或非岩藻糖基化的GlcNAc受体-蛋白；以及
使用血清型M49化脓性链球菌(Streptococcus pyogenes)内切糖苷酶-S2(Endo-S2)Asp184突变体酶使所述岩藻糖基化或非岩藻糖基化的GlcNAc受体-蛋白和活化的低聚糖供体发生酶促反应，所述突变体酶包括催化结构域并且相对于如SEQ ID NO:1所示的野生型Endo-S2酶表现出增加的转糖基活性和降低的水解活性，其中所述Endo-S2 Asp184突变体酶是如SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、或SEQ ID NO:9所示的突变体酶，其中所述活化的低聚糖供体是合成低聚糖
唑啉或含有
唑啉和包含预定数量和类型的糖残基的低聚糖部分的天然N-聚糖，其中经由酶促反应，所述活化的低聚糖部分与所述岩藻糖基化或非岩藻糖基化的GlcNAc受体-蛋白共价连接，从而制备所述具有预定的低聚糖部分的岩藻糖基化或非岩藻糖基化的糖蛋白。
2.权利要求1的方法，其中所述岩藻糖基化或非岩藻糖基化的受体蛋白是抗体或其含有Fc的片段。
3.权利要求2的方法，其中所述岩藻糖基化或非岩藻糖基化的受体蛋白是静脉注射免疫球蛋白(IVIG)。
4.权利要求1-3任一项的方法，其中所述活化的低聚糖供体是合成低聚糖
唑啉或天然N-聚糖，其中所述天然N-聚糖包含复杂、高甘露糖、或杂合
唑啉。
5.权利要求1-3任一项的方法，其中所述合成低聚糖
唑啉是二糖
唑啉、三糖
唑啉、四糖
唑啉、五糖
唑啉、六糖
唑啉、七糖
唑啉、八糖
唑啉、九糖
唑啉、十糖
唑啉或十一糖
唑啉。
6.权利要求1-3任一项的方法，其中所述活化的低聚糖供体还包含另外的生物活性剂或标签。
7.权利要求6的方法，其中所述另外的生物活性剂或标签是药物、毒素、荧光探针、生物素、PEG、脂质、或多肽。
8.权利要求1-3任一项的方法，其中所述岩藻糖基化的受体蛋白是α-1-6-岩藻糖基-GlcNAc-蛋白。
9.权利要求1-3任一项的方法，其中所述岩藻糖基化或非岩藻糖基化的糖蛋白是选自以下的单克隆抗体：17b、48d、A32、C11、2G12、F240、IgG1b12、19e、X5、TNX-355、西妥昔单抗、利妥昔单抗、莫罗单抗-CD3、阿昔单抗、达利珠单抗、巴利昔单抗、帕利珠单抗、英夫利昔单抗、曲妥珠单抗、吉妥珠单抗奥唑米星、阿仑单抗、替伊莫单抗、阿达木单抗、奥马珠单抗、托西莫单抗、I-131托西莫单抗、依法利珠单抗、贝伐单抗、帕尼单抗、帕妥珠单抗、那他珠单抗、依那西普、IGN101、伏洛昔单抗、抗CD80 mAb、抗CD23 mAb、CAT-3888、CDP-791、艾拉普妥珠单抗、MDX-010、MDX-060、MDX-070、马妥珠单抗、CP-675,206、CAL、SGN-30、扎木单抗、阿德木单抗、奥戈伏单抗、尼妥珠单抗、ABT-874、地诺单抗、AM 108、AMG 714、芳妥珠单抗、达利珠单抗、戈利木单抗、CNTO 1275、奥瑞珠单抗、HuMax-CD20、贝利单抗、依帕珠单抗、MLN1202、维西珠单抗、托珠单抗、奥克尔利珠单抗、聚乙二醇化赛妥珠单抗、依库珠单抗、培克珠单抗、阿昔单抗、兰尼子木单抗、美泊利单抗以及MYO-029。
10.权利要求2的方法，其中所述抗体还包含选自以下的另外的部分：用于治疗癌症的治疗剂、用于HIV的治疗剂、毒素、对另外的受体具有反应性的与所述抗体不同的抗体、抗原、趋化因子以及细胞因子。
11.一种合成均质的岩藻糖基化或非岩藻糖基化的糖蛋白的方法，所述方法包括：
(a)提供包含N-乙酰葡糖胺(GlcNAc)部分的天然糖蛋白或重组糖蛋白；
(b)通过选自Endo-H、Endo-F3、Endo S和Endo-A的酶去除异质N-聚糖以形成均质的含有岩藻糖基化或非岩藻糖基化的GlcNAc的蛋白；
(c)提供含有
唑啉的低聚糖，所述低聚糖具有低聚糖组分，所述低聚糖组分包含具有限定数量和类型的糖残基的N-聚糖；以及
(d)使用血清型M49化脓性链球菌Endo-S2 Asp184突变体蛋白，用所述含有
唑啉的低聚糖对所述含有岩藻糖基化或非岩藻糖基化的GlcNAc的蛋白进行酶促转糖基，其中所述Endo-S2 Asp184突变体蛋白是如SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:6、SEQ ID NO:9、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:16或SEQ ID NO:17所示的突变体蛋白，所述突变体蛋白包括催化结构域并且相对于如SEQ ID NO:1所示的野生型Endo-S2酶表现出增加的酶促转糖基效率，从而形成具有所述限定数量和类型的糖残基的均质的岩藻糖基化或非岩藻糖基化的糖蛋白。
12.权利要求11的方法，其中所述含有
唑啉的低聚糖是二糖
唑啉、三糖
唑啉、四糖
唑啉、五糖
唑啉、六糖
唑啉、七糖
唑啉、八糖
唑啉、九糖
唑啉、十糖
唑啉、或十一糖
唑啉。
13.权利要求11或12的方法，其中所述含有岩藻糖基化的GlcNAc的蛋白是α-1-6-岩藻糖基-GlcNAc-蛋白。
14.一种合成修饰的抗体或其Fc片段的方法，所述方法包括：
提供包含岩藻糖基化或非岩藻糖基化的N-乙酰葡糖胺(GlcNAc)部分的具有Fc区域的抗体或Fc片段以形成GlcNAc-蛋白受体；其中所述岩藻糖基化或非岩藻糖基化的N-乙酰葡糖胺(GlcNAc)部分位于所述抗体的Fc区域上；以及
在化脓性链球菌内切糖苷酶-S2(Endo-S2)Asp184突变体酶的催化活性下使具有预定数量的糖的低聚糖
唑啉与GlcNAc-蛋白受体进行转糖基，所述突变体酶包括催化结构域并且相对于如SEQ ID NO:1所示的野生型Endo-S2酶表现出增加的转糖基活性和降低的水解活性，以形成具有所述预定数量的糖的修饰的抗体或Fc片段，其中所述Endo-S2 Asp184突变体酶是如SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、和SEQ ID NO:9所示的突变体酶。
15.权利要求14的方法，其中所述修饰的抗体还包含至少一种选自以下的另外的部分：用于治疗癌症的治疗剂、用于HIV的治疗剂、毒素、对另外的受体具有反应性的与所述修饰的抗体不同的抗体、抗原、治疗性多肽、趋化因子和细胞因子，其附接于所述低聚糖
唑啉。
16.一种合成静脉注射免疫球蛋白(IVIG)制备物的方法，所述制备物表现出Fc唾液酸化的糖型，所述方法包括：
提供携带Fc N-聚糖的IVIG；
使用选自Endo-H、Endo-F3、Endo-S和Endo-A的酶将所述Fc N-聚糖脱糖基化以形成GlcNAc受体部分，其中所述GlcNAc受体部分位于所述IVIG的Fc区域上并且所述GlcNAc受体部分是岩藻糖基化的或非岩藻糖基化的；以及
在血清型M49化脓性链球菌内切糖苷酶-S2(Endo-S2)Asp184突变体酶的催化活性下用具有预定数量的糖残基的唾液酸聚糖
唑啉对所述IVIG中的所述GlcNAc受体部分进行转糖基，其中所述Endo-S2Asp184突变体酶是如SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、或SEQ ID NO:9所示的突变体酶，并且相对于如SEQ ID NO:1所示的野生型Endo-S2酶表现出增加的转糖基活性和降低的水解活性，以形成唾液酸化的IVIG。
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