经修饰的DKK2蛋白、对其编码的核酸、其制备方法及其用途与流程

本公开涉及一种添加有糖基或具有改善的结合亲和性的经修饰的dkk2蛋白、对其编码的核酸、其制备方法及其用途。
背景技术：
：糖基化是糖与蛋白质连接的工艺，主要分为n-糖基化和o-糖基化。糖基化由糖基转移酶催化，并已经报道有约200种糖基转移酶。糖的种类和结构可能会影响蛋白质折叠、稳定性、溶解度和对蛋白酶的敏感性、血清半衰期、抗原性、活性增加等。dkk2是wnt的阻遏蛋白，属于dickkopf家族，并已经报道作为wnt信号通路的抑制因子或刺激因子(wuw等,curr.biol.,10(24),第1611-1614页,2000)。dkk2可以包含两个特定的富含半胱氨酸的结构域(crd)，并包括不同长度的连接区域。特别地，dkk2在具有10个半胱氨酸氨基酸的dickkopf家族成员中高度保守半胱氨酸-2区域(krupnikve等,gene,238(2),第301-313页,1999)。dkk2是在动物细胞中因低表达效率而难以产生的蛋白质，因此使用dkk2的治疗剂的开发正在延迟。因此，需要能够维持结合亲和性或对其底物具有改善的结合亲和性并且还表现出增加的表达效率的dkk2。技术实现要素：问题的解决方案一方面，提供经修饰的dkk2多肽，其与野生型dkk2多肽的氨基酸序列相比，包含一个或多个额外糖基化位点。另一方面，提供编码经修饰的dkk2多肽的核酸。又另一方面，提供制备经修饰的dkk2多肽的方法。又另一方面，提供用于促进血管生成的药物组合物，所述组合物包含经修饰的dkk2多肽或编码经修饰的dkk2多肽的核酸、以及药学上可接受的载体。又另一方面，提供用于预防或治疗血管通透性相关疾病的药物组合物，所述组合物包括经修饰的dkk2多肽或编码经修饰的dkk2多肽的核酸、以及药学上可接受的载体。又另一方面，提供一种促进受试者的血管生成的方法，所述方法包括将经修饰的dkk2多肽或编码经修饰的dkk2多肽的核酸施用于所述受试者。又另一方面，提供一种预防或治疗受试者的血管通透性相关疾病的方法，所述方法包括将经修饰的dkk2多肽或编码经修饰的dkk2多肽的核酸施用于受试者。发明的有益效果根据一方面的经修饰的dkk2多肽、对其编码的核酸、其制备方法及其用途，可以有效制备具有额外糖基或对其底物lrp6具有改善的结合亲和性的经修饰的dkk2蛋白，从而被用于促进血管生成或用于预防或治疗血管通透性相关疾病。附图说明从以下结合附图对示例性实施方式的描述中，这些和/或其它方面将变得显而易见和更容易理解，其中：图1a和图1b分别是示出了含有各种标签的野生型dkk2蛋白和突变型dkk2蛋白的免疫印迹结果的图像，图1c是示出了含有fc标签的dkk2n-gly4和dkk2n-gly5蛋白的免疫印迹结果的图像，图1d是示出了纯化蛋白质的电泳结果的图像。图2a和图2b是示出了突变型dkk2蛋白对各200ng的mlrp6和hlrp6在490nm处的吸光度的图，图2c和图2d是示出了dkk2蛋白对各100ng的mlrp6和hlrp6在490nm处的吸光度的图；图3a和图3b是示出了在施用试验材料后第14天和第21天施用突变型dkk2蛋白质的视网膜的荧光强度(％)的图；以及图4是示出了伊文思蓝血管通透性(％)根据施用dkk2蛋白质的图。具体实施方式一方面，提供了与野生型dickkopf(dkk)2多肽的氨基酸序列相比包含一个或多个额外糖基化位点的经修饰的dkk2多肽。dkk2是指dickkopf-2蛋白，也称为dickkopf相关蛋白2、富含半胱氨酸的分泌蛋白2、crsp2、crispy2或crsp2蛋白。dkk2是一种在人类中由dkk2基因编码的蛋白质。该基因编码作为dickkopf家族成员的蛋白质。已知dkk2是一种分泌蛋白质，含有两个富含半胱氨酸的区域，通过与wnt信号通路的相互作用而参与胚胎发育。此外，它可以作为wnt/β-连环蛋白信号传导的激动剂或拮抗剂，这取决于细胞情况和辅因子kremen2的存在。dkk2可以与低密度脂蛋白受体相关蛋白6(lrp6)结合。野生型dkk2多肽可以是野生型dkk2前体多肽。野生型dkk2前体多肽可以是人类中具有基因库登录号为np_055236.1的氨基酸序列(序列号1)的多肽或小鼠中具有基因库登录号为np_064661.2的氨基酸序列的多肽。野生型dkk2前体多肽可以包括信号肽，并且信号肽可以通过共翻译修饰或翻译后修饰来切割。野生型dkk2多肽可以是成熟dkk2。成熟dkk2可以是包括通过从序列号1的氨基酸序列的n-末端去除位置1至33处的氨基酸序列(信号肽或前导肽)而得到的氨基酸序列的多肽，或包括序列号3的氨基酸序列的多肽。野生型dkk2前体多肽可以通过人类中具有基因库登录号为nm_014421的核苷酸序列(序列号4)的核酸以及小鼠中具有基因库登录号为nm_020265的核苷酸序列的核酸来编码。糖基化是指将糖基转移到蛋白质的反应。糖基化由糖基转移酶催化。糖基化可以是n-糖基化、o-糖基化、磷酸-丝氨酸糖基化、c-甘露糖基化、糖基磷脂酰肌醇化或其组合。n-糖基化是指糖分子与天冬酰胺的酰胺基团的连接。糖基化位点是指可以连接有糖分子或糖基的位点。例如，糖基化位点可以是asn-xaa-ser/thr的共有序列中的天冬酰胺残基(其是n-糖基化位点)。asn表示天冬酰胺，xaa表示除脯氨酸以外的氨基酸，ser表示丝氨酸，thr表示苏氨酸，ser/thr表示丝氨酸或苏氨酸。asn-xaa-ser/thr表示从n-末端起由天冬酰胺-氨基酸(不包括脯氨酸)-丝氨酸或氨基酸组成的多肽。糖分子与asn-xaa-ser/thr的天冬酰胺连接。野生型dkk2前体多肽可以从n-末端在位置36处包括一个糖基化位点。经修饰的dkk2多肽的糖基化位点可以通过将天门冬酰胺(asn，n)取代序列号3的氨基酸序列中选自由5i、31g、96p、110d、44p、2l、45c、57c、62q、63g、85p、6k、98t、101i、11g、121h、135p、138k、151l、152r、166f、187k、203g、211d、213t和214y组成的组中的一种或多种氨基酸而引入。数字表示从序列号3的氨基酸序列的n-末端起氨基酸所在的位置，并且字母字符表示氨基酸的1个字母代码。例如，‘5i’表示从序列号3的氨基酸序列的n-末端起位置5处的异亮氨酸。修饰可以是一个或多个氨基酸的取代。经修饰的dkk2多肽可以是dkk2多肽，其中通过引入糖基化位点而额外引入糖基。尽管经修饰的dkk2多肽可以在糖键、糖组成、糖基结构或其组合中变化，但糖基化发生在dkk2多肽的氨基酸序列的相同糖基化位点。作为结果，可以增加细胞内表达效率。例如，虽然经修饰的dkk2多肽可以在糖键(例如n-糖基化和o-糖基化)、糖组成(例如唾液酸化程度的差异)和糖基结构中根据宿主细胞(其中经修饰的dkk2多肽被表达)的种类而变化，但糖基化可以发生在dkk2多肽的相同糖基化位点。经修饰的dkk2多肽可以是包含一个或多个选自序列号5至30的氨基酸序列的多肽。标签可以进一步包含在经修饰的dkk2多肽的n-末端或c-末端。标签可以是连接到dkk2多肽的多肽，以促进表达、纯化、检测等。标签可以是例如fc(片段可结晶)区、多组氨酸肽或其组合。fc区可以是人fc区、小鼠fc区等。fc区可以是由序列号48的核苷酸序列编码的多肽。fc区可以是由序列号49的核苷酸序列组成的多肽。标签可以是本领域技术人员已知的。经修饰的dkk2多肽可以进一步包括n-末端处的信号肽。信号肽是指存在于大部分新合成的蛋白质的n-末端的肽(大约5-30个氨基酸长)，其注定分泌途径。信号肽可以是序列号1的氨基酸序列的n-末端起位置1至33处的氨基酸序列或包含序列号2的氨基酸序列的多肽。与野生型dkk2多肽相比，经修饰的dkk2多肽可以是具有额外糖基、lrp6结合亲和性的增加、或其组合的多肽。与作为对照组的野生型dkk2多肽相比，在经修饰的dkk2多肽中的额外糖基可以是糖基量的增加。由于额外糖基，与野生型dkk2多肽的量相比，经修饰的dkk2多肽的量可能在细胞中增加。与编码野生型dkk2多肽的转录物相比，编码经修饰的dkk2多肽的转录物的量可以在细胞中增加。多肽或转录物的量的增加可能意味着多肽的细胞内表达效率增加。与野生型dkk2多肽相比，经修饰的dkk2多肽对lrp6的结合亲和性可能增加。结合亲和性越高，解离常数(kd)可能越低。经修饰的dkk2多肽对于lrp6的解离常数可以是例如约0.1nm至约100nm、约1nm至约50nm、约2nm至约40nm、约3nm至约30nm、约4nm至约20nm、约5nm至约10nm、或约5.5nm。另一方面，提供一种编码经修饰的dkk2多肽的核酸。该经修饰的dkk2多肽与上述相同。核酸可以包括选自序列号43至47中的任一个核苷酸序列。核酸在编码经修饰的dkk2多肽的核酸的5'-末端或3'-末端处还可以包括编码标签的核苷酸序列。核酸在编码经修饰的dkk2多肽的核酸的5'-末端处还可以包括编码信号肽的核苷酸序列。核酸可以可操作地连接到基因表达调节元件，例如启动子、操纵子、增强子和/或转录终止子。另一方面，提供一种制备经修饰的dkk2多肽的方法，所述方法包括：在培养基的存在下培养用包含编码经修饰的dkk2多肽的核酸的载体引入的细胞，以获得培养产物；并从培养产物获得经修饰的dkk2多肽。该经修饰的dkk2多肽和编码经修饰的dkk2多肽的核酸与上述相同。细胞可以是真核细胞。例如，细胞可以是动物细胞。细胞可以是例如胚胎肾细胞、卵巢细胞、骨髓瘤细胞或视网膜衍生细胞。胚胎肾细胞可以是人胚胎肾(hek)293细胞或幼仓鼠肾(bhk)细胞。卵巢细胞可能是中国仓鼠卵巢细胞(cho)细胞。骨髓瘤细胞可以是ns0细胞或sp2/0细胞。视网膜衍生细胞可以是perc6细胞。根据细胞的种类，在糖键(例如n-糖基化和o-糖基化)、糖组成(例如唾液酸化程度的差异)和糖基结构中变化的dkk2多肽可以被表达。该方法还可以包括将包含编码经修饰的dkk2多肽的核酸的载体引入细胞。载体可以是例如质粒、病毒载体、粘粒或人造染色体。载体可以是包含启动子序列的表达载体。载体可以是能够在真核细胞中表达靶基因的载体。引入是指将核酸引入细胞。引入可以是例如通过转化、突变、转导、缀合或电穿孔引入。该方法包括在培养基的存在下培养引入的细胞以获得培养产物。培养基是指含有促进细胞存活或增殖所必需或有作用的成分的培养基。根据细胞种类，本领域技术人员可以选择培养基。可以使用本领域技术人员已知的方法进行孵育来进行培养。培养可以在例如约37℃的温度和5％co2条件下进行。该方法包括从培养产物得到经修饰的dkk2多肽。培养产物可以是不包括培养细胞的培养液或培养细胞。得到经修饰的dkk2多肽可以包括得到和裂解细胞，并纯化或过滤多肽。得到多肽的方法可以是本领域技术人员已知的。另一方面，提供一种用于促进血管生成的药物组合物，所述组合物包含经修饰的dkk2多肽或编码经修饰的dkk2多肽的核酸、以及药学上可接受的载体。该经修饰的dkk2多肽和编码经修饰的dkk2多肽的核酸与上述相同。血管生成是指形成新血管的过程。血管生成包括新血管从预先存在的血管生长的过程。血管生成是伤口愈合和肉芽组织以及生长和发育中的正常和重要的过程。此外，血管生成是肿瘤从休眠状态转变为恶性状态的基本步骤。促进可以在体外或体内实现。促进可能诱导具有缺血性血管疾病的受试者的新血管的形成或再生。药物组合物可以是用于预防或治疗缺血性血管疾病的药物组合物。缺血性血管疾病可以是例如烧伤、牛皮癣、溃疡、缺血、缺血性心脏病、缺血性脑血管疾病或勃起功能障碍。另一方面，提供一种用于预防或治疗血管通透性相关疾病的药物组合物，所述组合物包含经修饰的dkk2多肽或编码经修饰的dkk2多肽的核酸、以及药学上可接受的载体。该经修饰的dkk2多肽和编码经修饰的dkk2多肽的核酸与上述相同。血管通透性相关疾病是指具有由于血管通透性增加而导致身体流体进入周围组织的泄漏增加而引起的症状的疾病。血管通透性相关疾病可以是例如糖尿病性视网膜病变、糖尿病性黄斑水肿、视网膜静脉闭塞后的黄斑水肿、黄斑变性(例如新生血管(湿性)年龄相关性黄斑变性)、脉络膜新生血管形成、青光眼视网膜色素变性、视网膜病变早产儿(rop)、青光眼、角膜营养不良、视网膜疾病、斯塔格特氏病、常染色体显性药物、最佳黄斑营养不良、囊性黄斑水肿、缺血性视网膜病变、炎症诱导的视网膜退行性疾病、x连锁青少年视网膜病变、malattialeventinese(ml)或doyne蜂窝状视网膜营养不良症。如本文所用，术语“预防”是指通过施用药物组合物来抑制或延缓疾病发生的所有作用。术语“治疗”是指通过施用药物组合物有利于症状好转或改善的所有作用。药物组合物可以包括药学上可接受的载体。载体包括赋形剂、稀释剂或辅助物质。载体可以是例如选自乳糖、葡萄糖、蔗糖、山梨糖醇、甘露糖醇、木糖醇、赤藓糖醇、麦芽糖醇、淀粉、金合欢胶、藻酸盐、明胶、磷酸钙、硅酸钙、纤维素、甲基纤维素、聚乙烯基吡咯烷酮、水、生理盐水溶液、缓冲液例如pbs、羟基苯甲酸甲酯、羟基苯甲酸丙酯、滑石、硬脂酸镁和矿物油。组合物可以包括填料、抗凝集剂、润滑剂、润湿剂、风味剂、乳化剂、防腐剂等。药物组合物可以以任何制剂制备。组合物可以配制成口服剂型(例如粉剂、片剂、胶囊剂、糖浆剂、丸剂、颗粒剂)或肠胃外剂型(例如可注射制剂)。此外，组合物可以以全身剂型或局部剂型制备。药物组合物可以包括有效量的经修饰的dkk2多肽或编码经修饰的dkk2多肽的核酸。有效量可以根据本领域技术人员选择的细胞或受试者适当地选择。有效量可以根据疾病的严重程度、患者的年龄、体重、健康状况、性别和药物敏感性、给药时间、给药途径、排泄率、治疗期、和与组合物混合或共同给药的药物以及医疗领域中众所周知的其它因素来确定。基于药物组合物，有效量可以为约0.5μg至约2g、约1μg至约1g、约10μg至约500mg、约100μg至约100mg、约1mg至约50mg。药物组合物的给药剂量可以是例如每个成人约0.001mg/kg至约100mg/kg、约0.001mg/kg至约10mg/kg、约0.001mg/kg至约1mg/kg、约0.005mg/kg至约1mg/kg、约0.01mg/kg至约1mg/kg、或约0.1mg/kg至约1mg/kg，每天一次、每天数次、每周两次或三次、每月一次至四次、每年一至十二次。另一方面，提供一种促进受试者的血管生成的方法，所述方法包括将经修饰的dkk2多肽或编码经修饰的dkk2多肽的核酸施用于受试者。另一方面，提供一种预防或治疗受试者的血管通透性相关疾病的方法，所述方法包括将经修饰的dkk2多肽或编码经修饰的dkk2多肽的核酸施用于受试者。该经修饰的dkk2多肽、编码经修饰的dkk2多肽的核酸、血管生成、促进、血管通透性相关疾病、预防和治疗与上述相同。受试者可以是哺乳动物，例如人、牛、马、猪、狗、绵羊、山羊或猫。受试者可能是具有缺血性疾病或高可能性具有缺血性疾病的受试者。受试者可以是具有血管通透性相关疾病或高可能性具有血管通透性相关疾病的受试者。可以通过任何方式直接对受试者进行给药，例如口服、静脉内、肌肉内、透皮、粘膜、鼻内、气管内或皮下给药。给药可以是局部或全身给药。现在将详细参考示例性实施方式，其示例在附图中示出，其中相同的附图标记始终表示相同的元件。在这方面，本示例性实施方式可以具有不同的形式，并且不应被解释为限于本文所阐述的描述。因此，下面仅通过参考附图描述示例性实施方式来解释各方面。如本文所用，术语“和/或”包括一个或多个相关列出的项目的任何和所有组合。以下参照实施例对本发明进行更详细的说明。然而，这些实施例仅用于说明的目的，本发明的范围并不受这些实施例的限制。实施例1.n-糖基化突变型dkk2蛋白的制备和鉴定1.dkk2蛋白的n-糖基化位点的预测野生型dkk2蛋白的氨基酸序列(序列号3)从n-末端起在位置3处具有n-糖基化位点。为了人工诱导野生型dkk2蛋白中的n-糖基化，使用netnglyc1.0服务器(www.cbs.dtu.dk/services/netnglyc/)预测野生型dkk2蛋白的n-糖基化位点。从netnglyc1.0服务器预测了共26种dkk2蛋白的n-糖基化位点，结果如表1中所示。在表1中，氨基酸代表野生型dkk2蛋白的氨基酸(序列号3)和由其突变的天冬酰胺(asn，n)，数字表示从野生型dkk2蛋白的n-末端起突变氨基酸所在的位置。[表1]在26种dkk2蛋白的n-糖基化位点候选中，选择了在netnglyc1.0服务器中显示高n-糖基化潜能的5种突变型dkk2蛋白，并检测了蛋白质生产效率。所选择的5种突变型dkk2蛋白的n-糖基化潜能在下表2中给出。[表2]突变型dkk2蛋白n-糖基化的潜能dkk2n-gly10.7342dkk2n-gly20.7206dkk2n-gly30.7010dkk2n-gly40.7541dkk2n-gly50.73722.所选择的n-糖基化dkk2蛋白的生产效率的验证为了表达实施例1.1中选择的5种dkk2蛋白质，将其各自插入高效表达载体中，以通过瞬时表达系统检查蛋白质生产效率。(1)聚合酶链反应使用野生型dkk2天然形式(基因库登录号np_055236.1，序列号3)(由medpacto,inc.,korea提供)作为模板。设计用于dkk2n-糖基化的引物的核苷酸序列在表3中给出。[表3]对于聚合酶链反应，制备以下组成的混合物。100ng模板2.5单位pfudna聚合酶(spx16-rs500,solgentco.,ltd.)10pmol正向引物10pmol反向引物1μl10mmdntp5μl10xpfu聚合酶缓冲液50μl总体积将制备的混合物在95℃下孵育2分钟，然后重复30次循环，每次循环包括95℃下20秒、64℃下40秒、72℃下1分钟，然后在72℃下孵育10分钟。因此，得到如此扩增的突变型dkk2核酸。(2)扩增产物的克隆和宿主细胞中瞬时蛋白表达的检测将扩增的突变型dkk2核酸或野生型dkk2核酸在10单位的限制酶sfii(目录号r033s，enzynomics，korea)和1x反应缓冲液的存在下在50℃下孵育6小时。将反应产物在琼脂糖凝胶上电泳，使用凝胶纯化试剂盒(目录号1014876，qiagen，usa)纯化插入载体的核酸。将哺乳动物表达载体n293f-fc(atp-100，anrt)和纯化的核酸以1:3的重量比混合，随后在10单位的t4dna连接酶(目录号m001s，enzynomics，korea)和1x反应缓冲液的存在下在22℃下孵育4小时以上。将反应产物转化到大肠杆菌中，通过测序分析检查编码dkk2n-gly1、dkk2n-gly2、dkk2n-gly3、dkk2n-gly4或dkk2n-gly5(分别为序列号43至47)的核苷酸序列。选择包含引入有突变型dkk2核酸的n293f-fc载体的克隆。从选择的克隆得到引入有突变型dkk2核酸(mdkk2)的n293f-fc-mdkk2载体。fc核酸是编码人免疫球蛋白g1的fc片段的核酸(序列号48)。将哺乳动物hek293f悬浮细胞以5×105个细胞/ml的密度接种于自由式培养基(目录号1508027，invitrogen，usa)中，并在37℃和5％co2的条件下培养。培养细胞直到细胞的密度达到约1×106细胞/ml(约24小时)。将25μg引入有突变型dkk2核酸的n293f-fc载体、50μg聚乙烯亚胺(pei)(目录号23966，polysciences，usa)和600μlpmi培养基(目录号sh30027.01，hiclone，usa)混合，并在室温下孵育20分钟。向培养的hek293f细胞中加入反应产物，并在37℃和5％co2的条件下培养约5天。然后，为了得到由细胞分泌的水溶性蛋白质，从培养产物中除去细胞，仅收集不包括细胞的培养液。将200μl培养液进行10％sds-page，然后进行免疫印迹。由于得到的突变型dkk2蛋白和野生型dkk2蛋白在其n-末端包含fc-标签、his-标签或mfc-标签，使用以1：4000稀释的抗fc-hrp(目录号31414，pierce，usa)、以1：2000稀释的抗his-hrp(目录号a7058，sigma，usa)或以1：2000稀释的抗mfc(目录号31430，pierce，usa)。作为显色试剂，使用eclkit(目录号0034077geusa)以得到图像。图1a示出了含有各种标签的野生型dkk2蛋白的免疫印迹结果(暴露时间：1分钟，m：标记物(kda)，1：n-fc-dkk2，2：n-fc(igg4)-dkk2，3：n-his-dkk2，4：n-mfc-dkk2)。如图1a所示，即使暴露1分钟，也没有检测到野生型dkk2蛋白。因此，证实了野生型dkk2蛋白几乎不表达。图1b示出了包含各种标签的突变型dkk2蛋白的免疫印迹结果(暴露时间：1分钟(左)或1秒(右)，m：标记(kda)，1：n-mfc-dkk2-gly1，2：n-mfc-dkk2-gly2，3：n-mfc-dkk2-gly3，4：n-fc4-dkk2-gly1，5：n-his-dkk2-gly1，6：n-fc-dkk2-gly1，7：n-his-dkk2-gly2，8：n-fc4-dkk2-gly2，9：n-fc-dkk2-gly2，10：n-fc-dkk2-gly3，11：n-his-dkk2-gly3，12：n-fc4-dkk2-gly3)。如图1b所示，检测到包含fc标签的突变型dkk2蛋白，而几乎没有检测到包含fc以外的标签的突变型dkk2蛋白。因此，证实了含有fc标签的突变型dkk2蛋白显示出提高的表达效率。使用一次性开放柱纯化表达的蛋白质，并测量其浓度。由于含有fc标签的蛋白质显示出提高的表达效率，因此获得含有fc标签的dkk2n-gly4和dkk2n-gly5蛋白。将获得的蛋白质进行免疫印迹，结果示于图1c(曝光时间：1秒(左)或30秒(右)，m：标记(kda)，1：n-fc-dkk2-gly4，2：n-fc-dkk2-gly5)。如图1c所示，证实了n-fc-dkk2-n-gly4比n-fc-dkk2-n-gly5显示出更高的表达效率。(3)蛋白质的大量生产和纯化如实施例1(2)中所述，选择用含有fc标签和突变性dkk2核酸的载体转化的细胞。将所选择的细胞在37℃和5％co2的条件下培养约5天。将除细胞外的培养液在室温下以4800rpm的速度离心20分钟以得到上清液。使用0.22μm过滤器(目录号pr02890，millipore，usa)进行过滤。制备装有蛋白a珠(目录号17-1279-03，gehealthcare，sweden)的5ml柱，并在4℃下将滤液以0.9ml/min的速率施加到珠子整夜。用100ml以上的pbs(磷酸缓冲盐水)(目录号71111，gibco，usa)洗涤珠子。此后，将0.1m甘氨酸-hcl(目录号g7126，sigma，usa)施加到珠子以洗脱6个级分。加入1mtris(目录号t-1503-5kg，sigma，usa)(ph9.0)以中和馏分。定量级分中的蛋白质，并收集含有蛋白质的级分。将级分施加到超滤离心管(目录号ufc805024，millipore，usa)，并根据制造商的说明书离心。将1xpbs加入浓缩物中并重复离心三次，以用pbs替代作为储存缓冲液。定量纯化的蛋白质。在40ml培养液中n-fc-dkk2-gly1的量为130μg，在40ml培养液中n-fc-dkk2-gly2的量为860μg，在40ml培养液中n-fc-dkk2-gly3的量为150μg，在40ml培养液中n-fc-dkk2-gly4的量为462μg，在40ml培养液中n-fc-dkk2-gly5的量为27μg。在引入有n-糖基化位点的突变型dkk2蛋白中，n-fc-dkk2-gly2显示出最高的表达效率，n-fc-dkk2-gly4显示出接着最高的表达效率。因此，与野生型dkk2蛋白相比，引入了n-糖基化位点的突变型dkk2蛋白质高度表达，n-fc-dkk2-gly2和n-fc-dkk2-gly4的表达效率比野生型dkk2分别高约80倍和约50倍。为了检查纯化的蛋白质的纯度，将3μg蛋白质通过10％sds-page电泳，结果示于图1d(m：标记物(kda)，1：n-fc-dkk2-gly1，2：n-fc-dkk2-gly2，3：n-fc-dkk2-gly3，4：n-fc-dkk2-gly4，5：n-fc-dkk2-gly5)。如图1d中所示，发现n-fc-dkk2-gly2和n-fc-dkk2-gly4的纯度相似。3.突变型dkk2蛋白的结合亲和性的检测由于已知dkk2蛋白与小鼠lrp6(mlrp6)和人lrp6(hlrp6)结合，因此检查了野生型dkk2和突变型dkk2蛋白对lrp6的结合亲和性。具体地，将200nghlrp6蛋白(目录号1505-lr-025，r&d，usa)或mlrp6蛋白(目录号2960-lr-025，r&d，usa)应用于elisa板(登录号439454，nunc，denmark)的每个孔，并在4℃下孵育过夜以用该蛋白质包覆该板。将200μl4％(w/v)脱脂乳(cat.no.232100，difco，france)/1xpbs施加到elisa板的孔中，并在室温下孵育约1小时以进行封闭。此后，从elisa板中除去封闭溶液。将每个纯化的dkk2蛋白和100μl1％(w/v)脱脂乳/1xpbs混合以制备100nm的dkk2蛋白，并将100nm的dkk2蛋白进行1/4系列稀释。将稀释的蛋白质施加到制备的elisa板上，并在室温下孵育约2小时。此后，将板用200μlpbst洗涤5次。将2μl抗人fc-hrp(目录号31413，thermo，usa)抗体与4ml含有1％(w/v)脱脂乳的pbs混合，并向elisa板的每个孔中加入200μl该混合物，并在室温下孵育1小时。然后，除去elisa板的二次抗体，并用200μlpbs将该板洗涤5次。将10μlh2o2溶液(目录号h1009-100ml，sigma，usa)、10mlpc缓冲液(5.1g柠檬酸一水合物，7.3g磷酸钠/l(ph5.0))和一片opd(目录号p8787-100tab，sigma，usa)混合以制备混合物，并将总体积为100μl的混合物加入到每个孔中。在室温下在黑暗中进行孵育10分钟，随后进行显色。此后，向每个孔中加入50μl终止缓冲液以终止显色。使用elisa读数器测量490nm处的吸光度，并根据测量的吸光度计算解离常数kd值(m)。图2a示出了突变型dkk2蛋白对200ngmlrp6在490nm处的吸光度，图2b示出了突变型dkk2蛋白对200nghlrp6在490nm处的吸光度(■：n-fc-dkk2-gly1，▲：n-fc-dkk2-gly2，▼：n-fc-dkk2-gly3，◆：n-fc-dkk2-gly4，●：n-fc-dkk2-gly5)。如图2a和2b中所示，n-fc-dkk2-gly4显示出对mlrp6和hlrp6的最大改善的结合亲和性。证实了n-fc-dkk2-gly2显示出最高的表达效率，但其对mlrp6和hlrp6的结合亲和性不高。因此，n-fc-dkk2-gly4被证实是表现出改善的表达效率和对mlrp6和hlrp6的高结合亲和性的突变型dkk2蛋白。为了比较在n-fc-dkk2-gly4与野生型dkk2(目录号6628-dk-010，r&d，usa)之间的对mlrp6和hlrp6的结合亲和性，以与上述相同的方式进行elisa，不同之处在于使用了100ng的lrp6。图2c示出了dkk2蛋白对100ngmlrp6在490nm处的吸光度，图2d示出了dkk2蛋白对100nghlrp6在490nm处的吸光度(■：n-fc-dkk2-gly4，▲：野生型dkk2)。从测量的吸光度计算出的解离常数(kd)和r2值在表4中给出。[表4]证实了n-fc-dkk2-gly4对hlrp6和mlrp6的结合亲和性比野生型dkk2高约5倍至约10倍。4.通过突变型dkk2蛋白的血管生成的检测为了检查突变型dkk2蛋白是否能够诱导血管生成，进行角膜袋检测(cpa)。c57bl6小鼠(雄性，9周龄，重21.81g-23.81g)购自orientbioinc.(korea)，然后在进行实验的动物设施中驯化约7天。为了向角膜施用药物，制备药物颗粒。具体地，将10g蔗糖八硫酸盐-铝络合物(s0652，sigma-aldrich)溶于100ml的pbs(磷酸盐缓冲液盐水)中以制备10％(w/v)硫糖铝溶液。将12g聚甲基丙烯酸2-羟基乙酯(p3932，sigma-aldrich)溶于100ml无水乙醇中以制备12％(w/v)聚hema溶液。将石蜡膜置于培养皿中并在紫外线下放置约15分钟。将5μl12％(w/v)聚hema溶液、1μl10％(w/v)硫糖铝溶液和4μl药物混合。将各0.2μl该混合物分配在石蜡膜上，并将分配的颗粒在室温下干燥约1-2小时。将干燥的颗粒在使用前储存在冰箱中。将适应的小鼠(雄性，10周龄，体重2123.43g-26.11g)分为2组(每组n＝5)，并通过腹膜内注射氯胺酮和赛拉嗪(rompuntm)，拜耳公司，德国)的4:1(v/v)混合物使其麻醉。大约10分钟后，将0.5％的普拉卡因滴入其角膜。用vongraefe白内障刀，在显微镜下在角膜上制成一个微口袋。将制备的颗粒植入角膜微口袋(第1天)。为了防止感染，将土霉素眼药膏施用于眼睛。作为测试材料，使用n-fc-dkk2-gly4，每只小鼠施用475ng的n-fc-dkk2-gly4。作为阴性对照组，使用pbs。此后，在显微镜下检查角膜，通过图像分析仪观察血管生成和结构特征。在第5天，在阴性对照组中未发现血管生成，而在n-fc-dkk2-gly4处理组中检测到血管生成。计算n-fc-dkk2-gly4处理组中血管生成的面积(mm2)，结果在表5中给出。[表5]序号第1天第3天第5天第7天10.000.000.000.0020.000.000.000.0030.000.000.000.0040.000.000.020.0350.000.000.060.13如表5中所示，与阴性对照组相比，在n-fc-dkk2-gly4处理组中观察到血管生成。5.突变型dkk2蛋白的糖尿病性视网膜病变改善作用检查突变型dkk2蛋白是否能够改善糖尿病性视网膜病变的症状。栗鼠兔(雄性，10-11周龄，体重2.0kg-2.5kg)购自dreambioinc.(韩国)，然后在进行实验的动物设施中驯化约7天。将作为糖尿病诱导剂的四氧嘧啶一水合物(sigma-aldrichco.)施用于适应的兔子的耳静脉。在给药后第7天(第0天)测量血糖水平，选择具有血糖水平为300mg/dl的动物。将选择的兔子随机分配，使得每组的平均血糖水平平均分布。通过静脉注射zoletil50(virbac，法国)和赛拉嗪(rompuntm，bayerag，germany)使糖尿病诱导的兔子麻醉。作为测试材料，在第0天(施用四氧嘧啶后第7天)和第7天通过玻璃体内注射施用n-fc-dkk2-gly4(pbs中)，以总共10μl/眼的量进行两次给药，每天一次。作为正常对照组，注射pbs而不是四氧嘧啶。作为糖尿病对照组，施用四氧嘧啶，作为阳性对照组，在第0天以50μl/眼的量通过玻璃体内注射施用(bayerkorealtd.)一次。施用药物在下表6中给出。[表6]组动物数量施用材料一次剂量或施用频率备注17pbs0未处理27pbs0糖尿病诱导37eylea2000μg，一次糖尿病诱导47n-fc-dkk2-gly47μg，两次糖尿病诱导57n-fc-dkk2-gly420μg，两次糖尿病诱导67n-fc-dkk2-gly470μg，两次糖尿病诱导在第0天(测试材料给药开始的那天)、第14天和第21天，将散瞳剂(mydriacyl滴眼剂1％)滴入兔眼中，并用zoletil50(法国virbac)和赛拉嗪((rompuntm)，拜耳公司，德国)麻醉。此后，通过耳静脉注射2％(w/v)荧光素钠盐溶液(sigmaaldrich)，并使用眼底照相机(trc-50ix，topcon，japan)拍摄兔子的眼睛约2分钟或更短。通过荧光眼底图像评估视网膜和功效的检查。使用imagej软件(nih，bethesda，md)进行图像分析，以测量视网膜非血管区域的荧光强度，并基于组1(正常对照组)的平均值(100％)，计算每个受试者的测量值的相对水平(％)。计算出的荧光强度示于图3a和3b(图3a：第14天，图3b：第21天；*和***：与组1相比，分别为p<0.05和p<0.001；#、##和###：与组2相比，分别为p<0.05，p<0.01和p<0.001；$：与组3相比，p<0.05)。如图3a和3b中所示，n-fc-dkk2-gly4处理组根据给药剂量在视网膜的非血管区域中显示出显著低的荧光强度，表明n-fc-dkk2-gly4具有抑制视网膜非血管区域的血管渗漏的作用。此外，在第21天，用zoletil50和赛拉嗪麻醉兔子。通过耳静脉注射1％(v/v)伊文思蓝溶液(abcam)。约10分钟后，将兔子的眼睛切下并固定在10％(v/v)中性缓冲福尔马林溶液中。大约24小时后，将视网膜与眼睛分开，并将1ml蒸馏水施加到分离的视网膜上，然后涡旋约10分钟。将混合物在10000×g和室温下离心约10分钟。将0.3ml上清液转移到96孔微量培养板中，并使用versamax酶标仪(moleculardevice，usa)测量620nm处的吸光度。基于组1(正常对照组)的平均值(100％)，计算每个受试者的伊文思蓝血管通透性。计算的通透性示于图4(*和***：与组1相比，分别为p<0.05和p<0.001；##和###：与组2相比，分别为p<0.01和p<0.001；$$：与组3相比，p<0.01)。如图4中所示，n-fc-dkk2-gly4处理组根据给药剂量显示出显著低的血管通透性，表明n-fc-dkk2-gly4具有抑制视网膜血管通透性的作用。糖尿病性视网膜病变的特征在于视网膜血管生成(或新生血管形成)、玻璃体出血等。由于n-fc-dkk2-gly4抑制血管生成、玻璃体出血和视网膜血管通透性，证实了经修饰的dkk2多肽例如n-fc-dkk2-gly4对糖尿病性视网膜病变具有预防或治疗作用。应当理解，本文描述的示例性实施方式应仅在描述性意义上被考虑，而不是为了限制的目的。在其它示例性实施方式中，每个示例性实施方式中的特征或方面的描述通常被认为可用于其它类似特征或方面。虽然已经参考附图描述了一个或多个示例性实施方式，但是本领域普通技术人员将会理解，在不脱离由如下权利要求所定义的精神和范围的情况下，可以在形式和细节上进行各种改变。<110>株式会社麦迪帕克特<120>经修饰的dkk2蛋白、对其编码的核酸、其制备方法及其用途<130>px051158-pct<160>49<170>kopatentin2.0<210>1<211>259<212>prt<213>人工序列<220><223>人野生型dkk2前体<400>1metalaalaleumetargserlysaspsersercyscysleuleuleu151015leualaalavalleumetvalgluserserglnileglyserserarg202530alalysleuasnserilelysserserleuglyglygluthrprogly354045glnalaalaasnargseralaglymettyrglnglyleualaphegly505560glyserlyslysglylysasnleuglyglnalatyrprocysserser65707580asplysglucysgluvalglyargtyrcyshisserprohisglngly859095serseralacysmetvalc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yslysglyserhisglyleu180185190gluilepheglnargcysaspcysalalysasnleusercyslysval195200205trplysaspalathrtyrserserlysalaargleuhisvalcysgln210215220lysile225<210>28<211>226<212>prt<213>人工序列<220><223>dkk2n-gly24<400>28lysleuasnserilelysserserleuglyglygluthrproglygln151015alaalaasnargseralaglymettyrglnglyleualapheglygly202530serlyslysglylysasnleuglyglnalatyrprocysserserasp354045lysglucysgluvalglyargtyrcyshisserprohisglnglyser505560seralacysmetvalcysargarglyslyslysargcyshisargasp65707580glymetcyscysproserthrargcysasnasnglyilecysilepro859095valthrgluserileleuthrprohisileproalaleuaspglythr100105110arghisargaspargasnhisglyhistyrserasnhisaspleugly115120125trpglnasnleuglyargprohisthrlysmetserhisilelysgly130135140hisgluglyaspprocysleuargserseraspcysilegluglyphe145150155160cyscysalaarghisphetrpthrlysilecyslysprovalleuhis165170175glnglygluvalcysthrlysglnarglyslysglyserhisglyleu180185190gluilepheglnargcysaspcysalalysglyleusercyslysval195200205trplysasnalathrtyrserserlysalaargleuhisvalcysgln210215220lysile225<210>29<211>226<212>prt<213>人工序列<220><223>dkk2n-gly25<400>29lysleuasnserilelysserserleuglyglygluthrproglygln151015alaalaasnargseralaglymettyrglnglyleualapheglygly202530serlyslysglylysasnleuglyglnalatyrprocysserserasp354045lysglucysgluvalglyargtyrcyshisserprohisglnglyser505560seralacysmetvalcysargarglyslyslysargcyshisargasp65707580glymetcyscysproserthrargcysasnasnglyilecysilepro859095valthrgluserileleuthrprohisileproalaleuaspglythr100105110arghisargaspargasnhisglyhistyrserasnhisaspleugly115120125trpglnasnleuglyargprohisthrlysmetserhisilelysgly130135140hisgluglyaspprocysleuargserseraspcysilegluglyphe145150155160cyscysalaarghisphetrpthrlysilecyslysprovalleuhis165170175glnglygluvalcysthrlysglnarglyslysglyserhisglyleu180185190gluilepheglnargcysaspcysalalysglyleusercyslysval195200205trplysaspalaasntyrserserlysalaargleuhisvalcysgln210215220lysile225<210>30<211>226<212>prt<213>人工序列<220><223>dkk2n-gly26<400>30lysleuasnserilelysserserleuglyglygluthrproglygln151015alaalaasnargseralaglymettyrglnglyleualapheglygly202530serlyslysglylysasnleuglyglnalatyrprocysserserasp354045lysglucysgluvalglyargtyrcyshisserprohisglnglyser505560seralacysmetvalcysargarglyslyslysargcyshisargasp65707580glymetcyscysproserthrargcysasnasnglyilecysilepro859095valthrgluserileleuthrprohisileproalaleuaspglythr100105110arghisargaspargasnhisglyhistyrserasnhisaspleugly115120125trpglnasnleuglyargprohisthrlysmetserhisilelysgly130135140hisgluglyaspprocysleuargserseraspcysilegluglyphe145150155160cyscysalaarghisphetrpthrlysilecyslysprovalleuhis165170175glnglygluvalcysthrlysglnarglyslysglyserhisglyleu180185190gluilepheglnargcysaspcysalalysglyleusercyslysval195200205trplysaspalathrasnserserlysalaargleuhisvalcysgln210215220lysile225<210>31<211>40<212>dna<213>人工序列<220><223>dkk2n-gly1的正向引物<400>31ggcatgtgctgcaacagtacccgctgcaataatggcatct40<210>32<211>38<212>dna<213>人工序列<220><223>dkk2n-gly1的反向引物<400>32gcagcgggtactgttgcagcacatgccatctcggtggc38<210>33<211>42<212>dna<213>人工序列<220><223>dkk2n-gly2的正向引物<400>33aatctaggaagaaatcacactaagatgtcacatataaaaggg42<210>34<211>42<212>dna<213>人工序列<220><223>dkk2n-gly2的反向引物<400>34catcttagtgtgatttcttcctagattctgccatcccaagtc42<210>35<211>41<212>dna<213>人工序列<220><223>dkk2n-gly3的正向引物<400>35catcagggggaaaactgtaccaaacaacgcaagaagggttc41<210>36<211>40<212>dna<213>人工序列<220><223>dkk2n-gly3的反向引物<400>36ttgtttggtacagttttccccctgatggagcactggtttg40<210>37<211>39<212>dna<213>人工序列<220><223>dkk2n-gly3的正向引物<400>37atcccggctctgaatggtactcggcacagagatcgaaac39<210>38<211>39<212>dna<213>人工序列<220><223>dkk2n-gly4的的反向引物<400>38gtgccgagtaccattcagagccgggatgtgaggggttaa39<210>39<211>42<212>dna<213>人工序列<220><223>dkk2n-gly5的正向引物<400>39gggcaggcctacaattgtagcagtgataaggagtgtgaagtt42<210>40<211>39<212>dna<213>人工序列<220><223>dkk2n-gly5的反向引物<400>40atcactgctacaattgtaggcctgccccaggtttttgcc39<210>41<211>29<212>dna<213>人工序列<220><223>载体的正向引物<400>41accggtggtaccgccaccatgggatggag29<210>42<211>29<212>dna<213>人工序列<220><223>载体的反向引物<400>42ggatttatacaaggaggagaaaatgaaag29<210>43<211>678<212>dna<213>人工序列<220><223>编码dkk2n-gly1多肽的核酸<400>43aaactcaactccaacaagtcctctctgggcggggagacgcctggtcaggccgccaatcga60tctgcgggcatgtaccaaggactggcattcggcggcagtaagaagggcaaaaacctgggg120caggcctacccttgtagcagtgataaggagtgtgaagttgggaggtattgccacagtccc180caccaaggatcatcggcctgcatggtgtgtcggagaaaaaagaagcgctgccaccgagat240ggcatgtgctgccccagtacccgctgcaataatggcatctgtatcccagttactgaaagc300atcttaacccctcacatcccggctctggatggtactcggcacagagatcgaaaccacggt360cattactcaaaccatgacttgggatggcagaatctaggaagaccacacactaagatgtca420catataaaagggcatgaaggagacccctgcctacgatcatcagactgcattgaagggttt480tgctgtgctcgtcatttctggaccaaaatctgcaaaccagtgctccatcagggggaagtc540tgtaccaaacaacgcaagaagggttctcatgggctggaaattttccagcgttgcgactgt600gcgaagggcctgtcttgcaaagtatggaaagatgccacctactcctccaaagccagactc660catgtgtgtcagaaaatt678<210>44<211>681<212>dna<213>人工序列<220><223>编码dkk2n-gly2多肽的核酸<400>44aaactcaactccatcaagtcctctctgggcggggagacgcctggtcaggccgccaatcga60tctgcgggcatgtaccaaggactggcattcaatggcagtaagaagggcaaaaacctgggg120caggcctacccttgtagcagtgataaggagtgtgaagttgggaggtattgccacagtccc180caccaaggatcatcggcctgcatggtgtgtcggagaaaaaagaagcgctgccaccgagat240ggcatgtgctgccccagtacccgctgcaataatggcatctgtatcccagttactgaaagc300atcttaacccctcacatcccggctctggatggaattactcggcacagagatcgaaaccac360ggtcattactcaaaccatgacttgggatggcagaatctaggaagaccacacactaagatg420tcacatataaaagggcatgaaggagacccctgcctacgatcatcagactgcattgaaggg480ttttgctgtgctcgtcatttctggaccaaaatctgcaaaccagtgctccatcagggggaa540gtctgtaccaaacaacgcaagaagggttctcatgggctggaaattttccagcgttgcgac600tgtgcgaagggcctgtcttgcaaagtatggaaagatgccacctactcctccaaagccaga660ctccatgtgtgtcagaaaatt681<210>45<211>678<212>dna<213>人工序列<220><223>编码dkk2n-gly3多肽的核酸<400>45aaactcaactccatcaagtcctctctgggcggggagacgcctggtcaggccgccaatcga60tctgcgggcatgtaccaaggactggcattcggcggcagtaagaagggcaaaaacctgggg120caggcctacccttgtagcagtgataaggagtgtgaagttgggaggtattgccacagtccc180caccaaggatcatcggcctgcatggtgtgtcggagaaaaaagaagcgctgccaccgagat240ggcatgtgctgccccagtacccgctgcaataatggcatctgtatcaatgttactgaaagc300atcttaacccctcacatcccggctctggatggtactcggcacagagatcgaaaccacggt360cattactcaaaccatgacttgggatggcagaatctaggaagaccacacactaagatgtca420catataaaagggcatgaaggagacccctgcctacgatcatcagactgcattgaagggttt480tgctgtgctcgtcatttctggaccaaaatctgcaaaccagtgctccatcagggggaagtc540tgtaccaaacaacgcaagaagggttctcatgggctggaaattttccagcgttgcgactgt600gcgaagggcctgtcttgcaaagtatggaaagatgccacctactcctccaaagccagactc660catgtgtgtcagaaaatt678<210>46<211>678<212>dna<213>人工序列<220><223>编码dkk2n-gly4多肽的核酸<400>46aaactcaactccatcaagtcctctctgggcggggagacgcctggtcaggccgccaatcga60tctgcgggcatgtaccaaggactggcattcggcggcagtaagaagggcaaaaacctgggg120caggcctacccttgtagcagtgataaggagtgtgaagttgggaggtattgccacagtccc180caccaaggatcatcggcctgcatggtgtgtcggagaaaaaagaagcgctgccaccgagat240ggcatgtgctgccccagtacccgctgcaataatggcatctgtatcccagttactgaaagc300atcttaacccctcacatcccggctctgaatggtactcggcacagagatcgaaaccacggt360cattactcaaaccatgacttgggatggcagaatctaggaagaccacacactaagatgtca420catataaaagggcatgaaggagacccctgcctacgatcatcagactgcattgaagggttt480tgctgtgctcgtcatttctggaccaaaatctgcaaaccagtgctccatcagggggaagtc540tgtaccaaacaacgcaagaagggttctcatgggctggaaattttccagcgttgcgactgt600gcgaagggcctgtcttgcaaagtatggaaagatgccacctactcctccaaagccagactc660catgtgtgtcagaaaatt678<210>47<211>678<212>dna<213>人工序列<220><223>编码dkk2n-gly5多肽的核酸<400>47aaactcaactccatcaagtcctctctgggcggggagacgcctggtcaggccgccaatcga60tctgcgggcatgtaccaaggactggcattcggcggcagtaagaagggcaaaaacctgggg120caggcctacaattgtagcagtgataaggagtgtgaagttgggaggtattgccacagtccc180caccaaggatcatcggcctgcatggtgtgtcggagaaaaaagaagcgctgccaccgagat240ggcatgtgctgccccagtacccgctgcaataatggcatctgtatcccagttactgaaagc300atcttaacccctcacatcccggctctggatggtactcggcacagagatcgaaaccacggt360cattactcaaaccatgacttgggatggcagaatctaggaagaccacacactaagatgtca420catataaaagggcatgaaggagacccctgcctacgatcatcagactgcattgaagggttt480tgctgtgctcgtcatttctggaccaaaatctgcaaaccagtgctccatcagggggaagtc540tgtaccaaacaacgcaagaagggttctcatgggctggaaattttccagcgttgcgactgt600gcgaagggcctgtcttgcaaagtatggaaagatgccacctactcctccaaagccagactc660catgtgtgtcagaaaatt678<210>48<211>696<212>dna<213>人工序列<220><223>编码人免疫球蛋白g1的fc区的核酸<400>48gagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctg60gggggaccgtcagtcttcctctttcccccaaaacccaaggacaccctcatgatctcccgg120acccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttc180aactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcag240tacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaat300ggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaacc360atctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgg420gatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagc480gacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcct540cccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagc600aggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccac660tacacgcagaagagcctctccctgtctccgggtaaa696<210>49<211>232<212>prt<213>人工序列<220><223>免疫球蛋白g1的fc区<400>49gluprolyssercysasplysthrhisthrcysproprocysproala151015progluleuleuglyglyproservalpheleupheproprolyspro202530lysaspthrleumetileserargthrprogluvalthrcysvalval354045valaspvalserhisgluaspprogluvallyspheasntrptyrval505560aspglyvalgluvalhisasnalalysthrlysproarggluglugln65707580tyrasnserthrtyrargvalvalservalleuthrvalleuhisgln859095asptrpleuasnglylysglutyrlyscyslysvalserasnlysala100105110leuproalaproileglulysthrileserlysalalysglyglnpro115120125arggluproglnvaltyrthrleuproproserargaspgluleuthr130135140lysasnglnvalserleuthrcysleuvallysglyphetyrproser145150155160aspilealavalglutrpgluserasnglyglnprogluasnasntyr165170175lysthrthrproprovalleuaspseraspglyserphepheleutyr180185190serlysleuthrvalasplysserargtrpglnglnglyasnvalphe195200205sercysservalmethisglualaleuhisasnhistyrthrglnlys210215220serleuserleuserproglylys225230当前第1页12