CN115400207A - Ace2改造蛋白及其应用 - Google Patents

Ace2改造蛋白及其应用 Download PDF

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CN115400207A
CN115400207A CN202110578921.6A CN202110578921A CN115400207A CN 115400207 A CN115400207 A CN 115400207A CN 202110578921 A CN202110578921 A CN 202110578921A CN 115400207 A CN115400207 A CN 115400207A
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高福
王奇慧
韩鹏程
仵丽丽
柴彦
马任义
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Abstract

本发明涉及基因工程技术领域。本发明提供一种ACE2改造蛋白在制备治疗和/或预防以ACE2为受体的SARS样冠状病毒引发疾病的药物中的应用或在制备预防以ACE2为受体的SARS样冠状病毒感染的疫苗中的应用。本发明的hACE2改造片段与hFc的改造蛋白为类抗体蛋白，能够广泛用于治疗由新冠病毒原始毒株及变异毒株引起的疾病，预期可以治疗由以ACE2为受体的新冠相关冠状病毒或SARS样冠状病毒引起的疾病。

Description

ACE2改造蛋白及其应用
技术领域：
本发明涉及基因工程技术领域，具体涉及针对新型冠状病毒具有中和效果的类抗体蛋白技术领域。
背景技术：
新冠肺炎疫情(COVID-19)对人类健康和全球公共卫生安全造成重大威胁，其病原体为SARS-CoV-2，是21世纪以来引起人类传染病疫情的第三种冠状病毒。目前，虽然已有多种针对SARS-CoV-2的疫苗和单克隆抗体药物获得紧急使用许可，但是多数研究表明它们对当下广泛传播的新冠病毒变异毒株(如英国株N501Y.V1、南非株N501Y.V2和巴西株N501Y.V3等)的保护效果降低。此外，大量研究数据表明蝙蝠等动物携带多种新冠相关冠状病毒或SARS样冠状病毒，如蝙蝠源RaTG13及穿山甲源GX/P2V/2017和GD/1/2019，目前的研究结果显示这三种病毒也可以利用血管紧张素转化酶2(ACE2)作为受体，提示它们存在感染人的潜力，因而亟需研发针对当下新冠变异毒株及未来可能出现的新的SARS样冠状病毒疫情的广谱药物或疫苗，这不仅是当前COVID-19疫情下，国家乃至全球的重大需求，也是关系到全球人类健康(One Health)的重要举措。
ACE2属于肾素-血管紧张素系统成员，通过调节血压及电解质的平衡维持心血管、肾脏及呼吸系统的功能，此外作为SARS-CoV、SARS-CoV-2等病毒的受体介导病毒入侵。研究发现，外源性ACE2重组蛋白(rACE2)可与内源性ACE2竞争结合SARS-CoV和SARS-CoV-2，进而抑制病毒感染。临床试验发现rACE2在健康人和严重呼吸窘迫综合症患者(ARDS)身上是安全的。这些研究结果表明，对于以ACE2为受体的冠状病毒而言，rACE2是一种潜在的大分子药物，因而设计开发高效rACE2具有潜在的临床应用价值。目前，虽有多款rACE2被报道用于抑制SARS-CoV、SARS-CoV-2感染，其中两款进入临床前准备阶段，一款进入临床Ⅱ期，但是它们对当下流行的新冠变异毒株的效果是未知的。
发明内容：
有鉴于此，本发明提供了一种ACE2改造蛋白在制备治疗和/或预防以ACE2为受体的SARS样冠状病毒引发疾病的药物中的应用或在制备预防以ACE2为受体的SARS样冠状病毒感染的疫苗中的应用。
在本发明的一些具体实施方案中，所述以ACE2为受体的SARS样冠状病毒为SARS-CoV-2原始毒株和/或SARS-CoV-2变异毒株和/或新冠相关冠状病毒等。
在本发明的一些具体实施方案中，所述的改造蛋白为hACE2-hFc-M4-1改造蛋白，其基因序列如序列列表SEQ ID No.5所示或氨基酸序列如序列列表SEQ ID No.6所示。
在本发明的一些具体实施方案中，所述的改造蛋白为hACE2-hFc-M4-2改造蛋白，其基因序列如序列列表SEQ ID No.7所示或氨基酸序列如序列列表SEQ ID No.8所示。
在本发明的一些具体实施方案中，所述的改造蛋白为hACE2-hFc-M5改造蛋白，其基因序列如序列列表SEQ ID No.9所示或氨基酸序列如序列列表SEQ ID No.10所示。
在本发明的一些具体实施方案中，所述的SARS-CoV-2原始毒株为SARS-CoV-2WT。
在本发明的一些具体实施方案中，所述的SARS-CoV-2变异毒株为英国株N501Y.V1、南非株N501Y.V2或巴西株N501Y.V3。
在本发明的一些具体实施方案中，所述的SARS-CoV-2变异毒株为水貂流行株Y453F、水貂流行株F486L或水貂流行株N501T。
本发明的hACE2改造片段与hFc的改造蛋白为类抗体蛋白，能够用于治疗由新冠病毒原始毒株及变异毒株引起的疾病，可以广泛应用于由以ACE2为受体的新冠相关冠状病毒或SARS样冠状病毒引起的疾病。
附图说明
图1.SARS-CoV-2RBD与人ACE2复合物结构图。
具体实施方式
基于此，发明人设计并筛选出了高效人ACE2重组蛋白，具有广谱中和SARS-CoV-2原始毒株及多种变异毒株的能力，同时排除了因ACE2自身的酶活效应带来的副作用，表明其可作为针对现有的或未来出现的新的新冠病毒变异毒株及将来可能出现的新的SARS样冠状病毒的一种潜在广谱大分子药物。
首先，根据SARS-CoV-2刺突蛋白受体结合区域(RBD)和人ACE2(hACE2)复合物结构(图1)，发现ACE2上参与RBD相互作用的氨基酸多为疏水性或带电氨基酸，因而发明人选取了一系列位点进行突变，以增强其疏水性或电性，同时，将其与人IgG1的Fc段(hFc)融合表达，最终形成一系列hACE2-hFc突变体。然后，通过测定这些hACE2-hFc突变体与SARS-CoV-2原始毒株(SARS-CoV-2WT)RBD的亲和力，筛选出亲和力增强的hACE2-hFc突变体，再将这些突变体进行组合，最终形成具有更强亲和力的组合突变体，包括T27F、K31Y、L79W和N330Y这四个位点。
此外，由于ACE2在调节血压及电解质的平衡中发挥着重要作用，为了排除过多的外源性ACE2蛋白可能导致的血压过度调节等副作用，根据文献报道，又将控制酶活性的关键氨基酸R273进行突变(R273Q)，以破坏其酶活功能，但不影响结合SARS-CoV-2，下文称为hACE2-hFc-M4-1(包括T27F、K31Y、N330Y和R273Q四个突变位点)、hACE2-hFc-M4-2(包括T27F、L79W、N330Y和R273Q四个突变位点)和hACE2-hFc-M5(包括T27F、K31Y、L79W、N330Y和R273Q五个突变位点)，它们的编码序列及氨基酸序列分别为SEQ ID No.5-10。
表达纯化获得hACE2-hFc-M4-1、hACE2-hFc-M4-2和hACE2-hFc-M5蛋白后，通过测定其与新冠病毒原始毒株及多种变异毒株RBD的亲和力，其中变异毒株包括英国株N501Y.V1、南非株N501Y.V2、巴西株N501Y.V3及三种水貂流行株Y453F、F486L和N501T株，相比于野生型hACE2-hFc(hACE2-hFc-WT)，发现hACE2-hFc-M4-1、hACE2-hFc-M4-2结合新冠病毒原始毒株及变异毒株RBD的能力均增强(表1)，并且二者结合SARS-CoV-2WT和水貂流行株Y453F株的能力相似，结合其他变异毒株的能力存在一定的差异，因而发明人又将hACE2-hFc-M4-1、hACE2-hFc-M4-2进行组合，形成hACE2-hFc-M5。
hACE2-hFc-M5结合新冠病毒原始毒株及变异毒株RBD是hACE2-hFc-WT的3.7～29.5倍，并且略高于hACE2-hFc-M4-1和hACE2-hFc-M4-2，其结合变异毒株(水貂流行株Y486L株除外)的能力高于SARS-CoV-2WT(表1)。
相比于hACE2-hFc-WT，hACE2-hFc-M4-1和hACE2-hFc-M4-2中和SARS-CoV-2原始毒株及变异毒株假病毒的能力均明显提高，分别提高8.3～104.1倍和6.5～44.6倍，尤其中和南非株501Y.V2和巴西株501Y.V3最为明显(表2)。hACE2-hFc-M5中和新冠病毒原始毒株及变异毒株假病毒是hACE2-hFc-WT的8.7～126倍，整体上高于或相似于hACE2-hFc-M4-1和hACE2-hFc-M4-2，尤其中和南非株501Y.V2和巴西株501Y.V3的能力进一步提高(表2)，这与亲和力的结果是相符的。
当新冠病毒侵染人体细胞时，本发明的hACE2改造蛋白优先识别并结合病毒，再加上融合了人IgG1 Fc段，从而形成了hACE2-hFc类抗体蛋白，进一步降低了半衰期，延长了中和病毒的能力。
本发明的三种hACE2改造片段的hFc融合蛋白特别适用于对新冠病毒突变株，例如英国株、巴西株、南非株及水貂流行株的治疗或预防，推测对以ACE2为受体的新冠相关冠状病毒或SARS样冠状病毒及未来出现的新的变异毒株也有作用，具有潜在的广泛应用价值。
本发明的hACE2改造蛋白可以通过本领域常规使用的方法得到。首先，将野生型hACE2氨基酸编码序列与hFc编码序列无缝连接构建至pCAGGS表达载体上，形成pCAGGS-hACE2-hFc，然后在此基础上进行氨基酸位点定点突变，进而得到hACE2改造蛋白的表达质粒，pCAGGS-hACE2-hFc-M4-1，pCAGGS-hACE2-hFc-M4-2，pCAGGS-hACE2-hFc-M5。
实施例1
hACE2-hFc改造蛋白的获得
首先，将编码野生型hACE2蛋白的基因序列(序列如序列表SEQ ID No.1所示)与编码人IgG1 Fc段蛋白基因(序列如序列表SEQ ID No.2所示)连接，将得到的序列人工合成(由苏州金唯智提供合成服务)，得到野生型hACE2-hFc融合蛋白基因(序列如序列表SEQ IDNo.3)，并将该融合蛋白基因克隆到pCAGGS真核细胞表达载体上，得到野生型hACE2-hFc融合蛋白的表达载体pCAGGS-hACE2-hFc，再通过氨基酸定点突变的方法将第27位氨基酸T突变成氨基酸F，第31位氨基酸K突变成氨基酸Y，第79位氨基酸L突变成氨基酸W，第330位氨基酸N突变成氨基酸Y，第273位氨基酸R突变成氨基酸Q，得到pCAGGS-hACE2-hFc-M4-1载体(包含突变位点T27F、K31Y、N330Y和R273Q，基因序列如序列表SEQ ID No.5所示)、pCAGGS-hACE2-hFc-M4-2载体(包含突变位点T27F、L79W、N330Y和R273Q，基因序列如序列表SEQ IDNo.7所示)、pCAGGS-hACE2-hFc-M5载体(包含突变位点T27F、K31Y、L79W、N330Y和R273Q，基因序列如序列表SEQ ID No.9所示)。
使用pCAGGS-hACE2-hFc载体和pCAGGS-hACE2-hFc-M4-1，pCAGGS-hACE2-hFc-M4-2，pCAGGS-hACE2-hFc-M5载体表达并纯化，分别得到了hACE2-hFc-WT融合蛋白(氨基酸序列如序列表SEQ ID No.4所示)，hACE2-hFc-M4-1改造蛋白(氨基酸序列如序列表SEQ ID No.6所示)，hACE2-hFc-M4-2改造蛋白(氨基酸序列如序列表SEQ ID No.8所示)，hACE2-hFc-M5改造蛋白(氨基酸序列如序列表SEQ ID No.10所示)。
氨基酸定点突变的方法：
通过设计引物将野生型hACE2基因特定位点氨基酸突变成目标氨基酸，然后经PCR扩增出突变后的目的片段，然后目的片段与线性化的载体pCAGGS进行同源重组，最终形成含有目的片段的环状载体，并经序列测序正确后，方可用于后续目的蛋白的表达。
突变位点包括：将第27位氨基酸T密码子(ACA)突变为氨基酸F密码子(TTT)，第31位氨基酸K密码子(AAG)突变成氨基酸Y密码子(TAC)，第79位氨基酸L密码子(CTT)突变成氨基酸W密码子(TGG)，第330位氨基酸N密码子(AAT)突变成氨基酸Y密码子(TAT)，第273位氨基酸R密码子(AGA)突变成氨基酸Q密码子(CAG)。
表达纯化的方法：
将重组好的目的质粒转染至HEK293F细胞中，细胞密度约为2×106/ml。转染后5-7天收集细胞上清，离心过滤，经蠕动泵将含有hACE2-hFc-WT，hACE2-hFc-M4-1，hACE2-hFc-M4-2，hACE2-hFc-M5改造蛋白的细胞上清在4℃环境下经过Protein A亲和层析柱，使改造蛋白与亲和层析柱充分结合。用结合缓冲液(20mM Na3HPO4，pH8.0)洗脱杂蛋白，用洗脱液(0.1M Glycine，pH3.0)洗脱改造蛋白，蛋白收集管里预先加入1M Tris-HCl(pH9.0)缓冲液，防止蛋白在过酸的环境下失活，最后经分子筛将蛋白换液至PBS缓冲液中。
实施例2
hACE2改造蛋白与新冠病毒突变株的RBD的亲和力
发明人利用表面等离子共振技术(SPR)，检测了SARS-CoV-2原始毒株(SARS-CoV-2WT)及变异毒株(英国株501Y.V1、南非株501Y.V2、巴西株501Y.V3、水貂流行453F株、水貂流行486L株、水貂流行N501T株)的RBD分别与hACE2-hFc-WT、hACE2-hFc-M4-1、hACE2-hFc-M4-2、hACE2-hFc-M5改造蛋白的亲和力。
仪器与材料：
RBD蛋白：SARS-CoV-2原始毒株、英国株501Y.V1、南非株501Y.V2、巴西株501Y.V3、水貂流行Y453F株、水貂流行F486L株、水貂流行N501T株的RBD蛋白均由申请人实验室表达纯化。
设备：BIAcore 8K，CM5芯片(GE Healthcare)
具体实验步骤如下：
a.芯片预处理：将识别human IgG1 Fc的二抗利用氨基偶联的方式固定在CM5芯片表面。CM5芯片共有8个通道，每个通道中包含两个Flow Cell(Fc 1和2)，Fc 2注射流动相蛋白样品，Fc 1作为对照。
b.进样：首先用运行缓冲液(20mM HEPES,150mM NaCl,0.005％(vol/vol)Tween20,pH 7.4)稀释样品，此实验样品含有不同突变株的RBD。
c.芯片再生：用再生缓冲液(10mM Glycine,pH 1.5)对芯片进行再生。
d.数据分析：用BIAcore 8K评价软件对数据进行分析，得出结合常数Ka，解离常数Kd及平衡解离常数KD。
表1.ACE2改造蛋白与野生型蛋白亲和力对比数据表
Figure BDA0003085468530000061
Figure BDA0003085468530000071
结果显示，与野生型hACE2-hFc(hACE2-hFc-WT)相比，发现三种hACE2改造蛋白(hACE2-hFc-M4-1、hACE2-hFc-M4-2和hACE2-hFc-M5)结合新冠病毒原始毒株及变异毒株RBD的能力均增强，尤其是hACE2-hFc-M5提高最为显著。
实施例3
hACE2-hFc改造蛋白的中和作用
测定了hACE2-hFc-WT，hACE2-hFc-M4-1、hACE2-hFc-M4-2、hACE2-hFc-M5改造蛋白在Vero E6细胞中对SARS-CoV-2原始毒株(SARS-CoV-2WT)及变异毒株(包括英国株N501Y.V1、南非株N501Y.V2、巴西株N501Y.V3及水貂流行株Y453F、水貂流行株F486L和水貂流行株N501T)假病毒的中和效果。
设置了Vero E6细胞(细胞阴性对照组)，Vero E6细胞+假病毒+培养基(无蛋白的阴性对照)，Vero E6细胞+假病毒+hACE2-hFc野生型蛋白(hACE2-hFc-WT组)、Vero E6细胞+假病毒+hACE2-hFc-M4-1蛋白(hACE2-hFc-M4-1组)、Vero E6细胞+假病毒+hACE2-hFc-M4-2蛋白(hACE2-hFc-M4-2组)、Vero E6细胞+假病毒+hACE2-hFc-M5蛋白(hACE2-hFc-5组)。
实验仪器和材料：
Vero E6细胞(申请人实验室保存)，hACE2-hFc-WT蛋白基因(由苏州金唯智合成，其编码序列为SEQ ID No.3)，hACE2-hFc-M4-1、hACE2-hFc-M4-2、hACE2-hFc-M5蛋白序列均为实施例1中得到的，SARS-CoV-2原始毒株及其变异毒株假病毒由申请人实验室包装获得。
蛋白原液配制：分别将hACE2-hFc-WT，hACE2-hFc-M4-1，hACE2-hFc-M4-2，hACE2-hFc-M5改造蛋白在超净工作台中经0.22μm无菌滤器过滤，用BCA法测定浓度，用含2％FBS的DMEM培养基配制成浓度60μg/ml的溶液。
改造蛋白的梯度稀释液的配制：用含2％FBS的DMEM培养基将各hACE2改造蛋白原液进行2倍倍比稀释，共稀释11个梯度，每个梯度8个复孔，每孔50μl。
假病毒稀释液：将SARS-CoV-2原始毒株及变异毒株假病毒液在Vero E6细胞上进行定量，将出现1000个FFU时的稀释度作为中和实验时的病毒用量。
具体步骤如下：
a.提前一天在96孔细胞培养板中铺Vero E6细胞，使第二天细胞汇合密度达到80-90％。
b.取上述各hACE2改造蛋白的梯度稀释液，在各孔中加入等体积(50μl)的上述假病毒稀释液，混匀后，在37℃孵育1h。
c.将96孔细胞培养板上清小心弃去，加入上述蛋白-病毒混合液(100μl/孔)，在培养箱中继续培养15h。
b.利用显微镜统计被感染了的细胞数，计算各浓度下的抑制率，再利用GraphPad计算出IC50。
表2.ACE2改造蛋白与野生型蛋白中和活性对比数据表
Figure BDA0003085468530000081
结果显示，相比于hACE2-hFc-WT，三种hACE2改造蛋白(hACE2-hFc-M4-1、hACE2-hFc-M4-2和hACE2-hFc-M5)中和SARS-CoV-2原始毒株及变异毒株假病毒的能力均明显提高，尤其对原始毒株、南非株501Y.V2、巴西株501Y.V3和水貂流行株F486L株提高的较为显著。
从整体上看，hACE2-hFc-M5的中和活性高于或相似于hACE2-hFc-M4-1和hACE2-hFc-M4-2，但是其中和南非株501Y.V2和巴西株501Y.V3的能力进一步提高，这与亲和力的结果也是相符的。
实施例4
hACE2-hFc改造蛋白在小鼠模型上的保护作用
将hACE2-hFc-WT、hACE2-hFc-M4-1、hACE2-hFc-M4-2、hACE2-hFc-M5改造蛋白注入小鼠模型体内，通过测定小鼠肺内病毒载量和肺组织HE染色评价ACE2改造蛋白治疗新冠病毒感染情况。
设置病毒+PBS组(阴性对照组)，病毒+hACE2-hFc-WT蛋白(hACE2-hFc-WT组)，病毒+hACE2-hFc-M4-1蛋白(hACE2-hFc-M4-1组)，病毒+hACE2-hFc-M4-2蛋白(hACE2-hFc-M4-2组)，病毒+hACE2-hFc-M5蛋白(hACE2-hFc-M5组)。
实验材料：
新冠病毒活病毒(中国科学院微生物所P3实验室)，BALB/c小鼠(购自维通利华实验动物公司)，hACE2-hFc-WT、hACE2-hFc-M4-1、hACE2-hFc-M4-2、hACE2-hFc-M5改造蛋白为实施例1中得到。
设备条件：中国科学院微生物所P3实验室
实验步骤如下：
a.注射新冠病毒：将5×105TCID50新冠病毒通过滴鼻感染的方式注入小鼠体内，每组5只。
b.注射ACE2改造蛋白：在攻毒后的第二天，将ACE2改造蛋白按10mg/kg剂量通过腹腔注射方式注入小鼠体内。
c.组织检测：在攻毒后的第五天，取小鼠肺组织，其中每组中的3只用于提取病毒RNA，测病毒载量，另外两只用于做组织切片，HE染色。
d.数据分析：根据肺组织中病毒载量和HE染色结果，判定ACE2改造蛋白保护小鼠抵抗新冠病毒感染情况。
本发明的hACE2改造片段与hFc的融合蛋白为类抗体蛋白，能够广泛用于治疗由新冠病毒原始毒株及变异毒株引起的疾病，可用于由以ACE2为受体的多数新冠相关冠状病毒或SARS样冠状病毒引起的疾病。
本发明的hACE2改造片段与hFc的融合蛋白特别适用于对新冠病毒变异毒株的治疗或预防，例如英国株、巴西株、南非株以及水貂流行株等，也可以广泛用于临床、流行病学调查及相应药物或疫苗的制备。
序列表
<110> 中国科学院微生物研究所
<120> ACE2改造蛋白及其应用
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2220
<212> DNA
<213> hACE2
<400> 1
atgtcaggct ctttctggct ccttctcagc cttgttgctg taactgctgc tcagtccacc 60
attgaggaac aggccaagac atttttggac aagtttaacc acgaagccga agacctgttc 120
tatcaaagtt cacttgcttc ttggaattat aacaccaata ttactgaaga gaatgtccaa 180
aacatgaata atgctgggga caaatggtct gcctttttaa aggaacagtc cacacttgcc 240
caaatgtatc cactacaaga aattcagaat ctcacagtca agcttcagct gcaggctctt 300
cagcaaaatg ggtcttcagt gctctcagaa gacaagagca aacggttgaa cacaattcta 360
aatacaatga gcaccatcta cagtactgga aaagtttgta acccagataa tccacaagaa 420
tgcttattac ttgaaccagg tttgaatgaa ataatggcaa acagtttaga ctacaatgag 480
aggctctggg cttgggaaag ctggagatct gaggtcggca agcagctgag gccattatat 540
gaagagtatg tggtcttgaa aaatgagatg gcaagagcaa atcattatga ggactatggg 600
gattattgga gaggagacta tgaagtaaat ggggtagatg gctatgacta cagccgcggc 660
cagttgattg aagatgtgga acataccttt gaagagatta aaccattata tgaacatctt 720
catgcctatg tgagggcaaa gttgatgaat gcctatcctt cctatatcag tccaattgga 780
tgcctccctg ctcatttgct tggtgatatg tggggtagat tttggacaaa tctgtactct 840
ttgacagttc cctttggaca gaaaccaaac atagatgtta ctgatgcaat ggtggaccag 900
gcctgggatg cacagagaat attcaaggag gccgagaagt tctttgtatc tgttggtctt 960
cctaatatga ctcaaggatt ctgggaaaat tccatgctaa cggacccagg aaatgttcag 1020
aaagcagtct gccatcccac agcttgggac ctggggaagg gcgacttcag gatccttatg 1080
tgcacaaagg tgacaatgga cgacttcctg acagctcatc atgagatggg gcatatccag 1140
tatgatatgg catatgctgc acaacctttt ctgctaagaa atggagctaa tgaaggattc 1200
catgaagctg ttggggaaat catgtcactt tctgcagcca cacctaagca tttaaaatcc 1260
attggtcttc tgtcacccga ttttcaagaa gacaatgaaa cagaaataaa cttcctgctc 1320
aaacaagcac tcacgattgt tgggactctg ccatttactt acatgttaga gaagtggagg 1380
tggatggtct ttaaagggga aattcccaaa gaccagtgga tgaaaaagtg gtgggagatg 1440
aagcgagaga tagttggggt ggtggaacct gtgccccatg atgaaacata ctgtgacccc 1500
gcatctctgt tccatgtttc taatgattac tcattcattc gatattacac aaggaccctt 1560
taccaattcc agtttcaaga agcactttgt caagcagcta aacatgaagg ccctctgcac 1620
aaatgtgaca tctcaaactc tacagaagct ggacagaaac tgttcaatat gctgaggctt 1680
ggaaaatcag aaccctggac cctagcattg gaaaatgttg taggagcaaa gaacatgaat 1740
gtaaggccac tgctcaacta ctttgagccc ttatttacct ggctgaaaga ccagaacaag 1800
aattcttttg tgggatggag taccgactgg agtccatatg cagaccaaag catcaaagtg 1860
aggataagcc taaaatcagc tcttggagat aaagcatatg aatggaacga caatgaaatg 1920
tacctgttcc gatcatctgt tgcatatgct atgaggcagt actttttaaa agtaaaaaat 1980
cagatgattc tttttgggga ggaggatgtg cgagtggcta atttgaaacc aagaatctcc 2040
tttaatttct ttgtcactgc acctaaaaat gtgtctgata tcattcctag aactgaagtt 2100
gaaaaggcca tcaggatgtc ccggagccgt atcaatgatg ctttccgtct gaatgacaac 2160
agcctagagt ttctggggat acagccaaca cttggacctc ctaaccagcc ccctgtttcc 2220
<210> 2
<211> 681
<212> DNA
<213> IgG1 Fc
<400> 2
gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 60
ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 120
tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 180
ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 240
cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 300
tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 360
gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 420
aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 480
tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 540
gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 600
aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 660
ctctccctgt ctccgggtaa a 681
<210> 3
<211> 2904
<212> DNA
<213> hACE2-hFc
<400> 3
atgtcaggct ctttctggct ccttctcagc cttgttgctg taactgctgc tcagtccacc 60
attgaggaac aggccaagac atttttggac aagtttaacc acgaagccga agacctgttc 120
tatcaaagtt cacttgcttc ttggaattat aacaccaata ttactgaaga gaatgtccaa 180
aacatgaata atgctgggga caaatggtct gcctttttaa aggaacagtc cacacttgcc 240
caaatgtatc cactacaaga aattcagaat ctcacagtca agcttcagct gcaggctctt 300
cagcaaaatg ggtcttcagt gctctcagaa gacaagagca aacggttgaa cacaattcta 360
aatacaatga gcaccatcta cagtactgga aaagtttgta acccagataa tccacaagaa 420
tgcttattac ttgaaccagg tttgaatgaa ataatggcaa acagtttaga ctacaatgag 480
aggctctggg cttgggaaag ctggagatct gaggtcggca agcagctgag gccattatat 540
gaagagtatg tggtcttgaa aaatgagatg gcaagagcaa atcattatga ggactatggg 600
gattattgga gaggagacta tgaagtaaat ggggtagatg gctatgacta cagccgcggc 660
cagttgattg aagatgtgga acataccttt gaagagatta aaccattata tgaacatctt 720
catgcctatg tgagggcaaa gttgatgaat gcctatcctt cctatatcag tccaattgga 780
tgcctccctg ctcatttgct tggtgatatg tggggtagat tttggacaaa tctgtactct 840
ttgacagttc cctttggaca gaaaccaaac atagatgtta ctgatgcaat ggtggaccag 900
gcctgggatg cacagagaat attcaaggag gccgagaagt tctttgtatc tgttggtctt 960
cctaatatga ctcaaggatt ctgggaaaat tccatgctaa cggacccagg aaatgttcag 1020
aaagcagtct gccatcccac agcttgggac ctggggaagg gcgacttcag gatccttatg 1080
tgcacaaagg tgacaatgga cgacttcctg acagctcatc atgagatggg gcatatccag 1140
tatgatatgg catatgctgc acaacctttt ctgctaagaa atggagctaa tgaaggattc 1200
catgaagctg ttggggaaat catgtcactt tctgcagcca cacctaagca tttaaaatcc 1260
attggtcttc tgtcacccga ttttcaagaa gacaatgaaa cagaaataaa cttcctgctc 1320
aaacaagcac tcacgattgt tgggactctg ccatttactt acatgttaga gaagtggagg 1380
tggatggtct ttaaagggga aattcccaaa gaccagtgga tgaaaaagtg gtgggagatg 1440
aagcgagaga tagttggggt ggtggaacct gtgccccatg atgaaacata ctgtgacccc 1500
gcatctctgt tccatgtttc taatgattac tcattcattc gatattacac aaggaccctt 1560
taccaattcc agtttcaaga agcactttgt caagcagcta aacatgaagg ccctctgcac 1620
aaatgtgaca tctcaaactc tacagaagct ggacagaaac tgttcaatat gctgaggctt 1680
ggaaaatcag aaccctggac cctagcattg gaaaatgttg taggagcaaa gaacatgaat 1740
gtaaggccac tgctcaacta ctttgagccc ttatttacct ggctgaaaga ccagaacaag 1800
aattcttttg tgggatggag taccgactgg agtccatatg cagaccaaag catcaaagtg 1860
aggataagcc taaaatcagc tcttggagat aaagcatatg aatggaacga caatgaaatg 1920
tacctgttcc gatcatctgt tgcatatgct atgaggcagt actttttaaa agtaaaaaat 1980
cagatgattc tttttgggga ggaggatgtg cgagtggcta atttgaaacc aagaatctcc 2040
tttaatttct ttgtcactgc acctaaaaat gtgtctgata tcattcctag aactgaagtt 2100
gaaaaggcca tcaggatgtc ccggagccgt atcaatgatg ctttccgtct gaatgacaac 2160
agcctagagt ttctggggat acagccaaca cttggacctc ctaaccagcc ccctgtttcc 2220
gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 2280
ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 2340
tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 2400
ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 2460
cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 2520
tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 2580
gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 2640
aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 2700
tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 2760
gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 2820
aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 2880
ctctccctgt ctccgggtaa atga 2904
<210> 4
<211> 967
<212> PRT
<213> hACE2-hFc-WT
<400> 4
Met Ser Gly Ser Phe Trp Leu Leu Leu Ser Leu Val Ala Val Thr Ala
1 5 10 15
Ala Gln Ser Thr Ile Glu Glu Gln Ala Lys Thr Phe Leu Asp Lys Phe
20 25 30
Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp
35 40 45
Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn
50 55 60
Ala Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala
65 70 75 80
Gln Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln
85 90 95
Leu Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys
100 105 110
Ser Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser
115 120 125
Thr Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu
130 135 140
Glu Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu
145 150 155 160
Arg Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu
165 170 175
Arg Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg
180 185 190
Ala Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu
195 200 205
Val Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu
210 215 220
Asp Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu
225 230 235 240
His Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile
245 250 255
Ser Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly
260 265 270
Arg Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys
275 280 285
Pro Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala
290 295 300
Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu
305 310 315 320
Pro Asn Met Thr Gln Gly Phe Trp Glu Asn Ser Met Leu Thr Asp Pro
325 330 335
Gly Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly
340 345 350
Lys Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp
355 360 365
Phe Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala
370 375 380
Tyr Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe
385 390 395 400
His Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys
405 410 415
His Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn
420 425 430
Glu Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly
435 440 445
Thr Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe
450 455 460
Lys Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met
465 470 475 480
Lys Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr
485 490 495
Tyr Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe
500 505 510
Ile Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala
515 520 525
Leu Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile
530 535 540
Ser Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu
545 550 555 560
Gly Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala
565 570 575
Lys Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe
580 585 590
Thr Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr
595 600 605
Asp Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu
610 615 620
Lys Ser Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met
625 630 635 640
Tyr Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu
645 650 655
Lys Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val
660 665 670
Ala Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro
675 680 685
Lys Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile
690 695 700
Arg Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn
705 710 715 720
Ser Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn Gln
725 730 735
Pro Pro Val Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
740 745 750
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
755 760 765
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
770 775 780
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
785 790 795 800
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
805 810 815
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
820 825 830
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
835 840 845
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
850 855 860
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
865 870 875 880
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
885 890 895
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
900 905 910
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
915 920 925
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
930 935 940
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
945 950 955 960
Leu Ser Leu Ser Pro Gly Lys
965
<210> 5
<211> 2904
<212> DNA
<213> hACE2-hFc-M4-1
<400> 5
atgtcaggct ctttctggct ccttctcagc cttgttgctg taactgctgc tcagtccacc 60
attgaggaac aggccaagtt ttttttggac tactttaacc acgaagccga agacctgttc 120
tatcaaagtt cacttgcttc ttggaattat aacaccaata ttactgaaga gaatgtccaa 180
aacatgaata atgctgggga caaatggtct gcctttttaa aggaacagtc cacacttgcc 240
caaatgtatc cactacaaga aattcagaat ctcacagtca agcttcagct gcaggctctt 300
cagcaaaatg ggtcttcagt gctctcagaa gacaagagca aacggttgaa cacaattcta 360
aatacaatga gcaccatcta cagtactgga aaagtttgta acccagataa tccacaagaa 420
tgcttattac ttgaaccagg tttgaatgaa ataatggcaa acagtttaga ctacaatgag 480
aggctctggg cttgggaaag ctggagatct gaggtcggca agcagctgag gccattatat 540
gaagagtatg tggtcttgaa aaatgagatg gcaagagcaa atcattatga ggactatggg 600
gattattgga gaggagacta tgaagtaaat ggggtagatg gctatgacta cagccgcggc 660
cagttgattg aagatgtgga acataccttt gaagagatta aaccattata tgaacatctt 720
catgcctatg tgagggcaaa gttgatgaat gcctatcctt cctatatcag tccaattgga 780
tgcctccctg ctcatttgct tggtgatatg tggggtcagt tttggacaaa tctgtactct 840
ttgacagttc cctttggaca gaaaccaaac atagatgtta ctgatgcaat ggtggaccag 900
gcctgggatg cacagagaat attcaaggag gccgagaagt tctttgtatc tgttggtctt 960
cctaatatga ctcaaggatt ctgggaatat tccatgctaa cggacccagg aaatgttcag 1020
aaagcagtct gccatcccac agcttgggac ctggggaagg gcgacttcag gatccttatg 1080
tgcacaaagg tgacaatgga cgacttcctg acagctcatc atgagatggg gcatatccag 1140
tatgatatgg catatgctgc acaacctttt ctgctaagaa atggagctaa tgaaggattc 1200
catgaagctg ttggggaaat catgtcactt tctgcagcca cacctaagca tttaaaatcc 1260
attggtcttc tgtcacccga ttttcaagaa gacaatgaaa cagaaataaa cttcctgctc 1320
aaacaagcac tcacgattgt tgggactctg ccatttactt acatgttaga gaagtggagg 1380
tggatggtct ttaaagggga aattcccaaa gaccagtgga tgaaaaagtg gtgggagatg 1440
aagcgagaga tagttggggt ggtggaacct gtgccccatg atgaaacata ctgtgacccc 1500
gcatctctgt tccatgtttc taatgattac tcattcattc gatattacac aaggaccctt 1560
taccaattcc agtttcaaga agcactttgt caagcagcta aacatgaagg ccctctgcac 1620
aaatgtgaca tctcaaactc tacagaagct ggacagaaac tgttcaatat gctgaggctt 1680
ggaaaatcag aaccctggac cctagcattg gaaaatgttg taggagcaaa gaacatgaat 1740
gtaaggccac tgctcaacta ctttgagccc ttatttacct ggctgaaaga ccagaacaag 1800
aattcttttg tgggatggag taccgactgg agtccatatg cagaccaaag catcaaagtg 1860
aggataagcc taaaatcagc tcttggagat aaagcatatg aatggaacga caatgaaatg 1920
tacctgttcc gatcatctgt tgcatatgct atgaggcagt actttttaaa agtaaaaaat 1980
cagatgattc tttttgggga ggaggatgtg cgagtggcta atttgaaacc aagaatctcc 2040
tttaatttct ttgtcactgc acctaaaaat gtgtctgata tcattcctag aactgaagtt 2100
gaaaaggcca tcaggatgtc ccggagccgt atcaatgatg ctttccgtct gaatgacaac 2160
agcctagagt ttctggggat acagccaaca cttggacctc ctaaccagcc ccctgtttcc 2220
gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 2280
ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 2340
tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 2400
ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 2460
cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 2520
tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 2580
gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 2640
aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 2700
tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 2760
gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 2820
aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 2880
ctctccctgt ctccgggtaa atga 2904
<210> 6
<211> 967
<212> PRT
<213> hACE2-hFc-M4-1
<400> 6
Met Ser Gly Ser Phe Trp Leu Leu Leu Ser Leu Val Ala Val Thr Ala
1 5 10 15
Ala Gln Ser Thr Ile Glu Glu Gln Ala Lys Phe Phe Leu Asp Tyr Phe
20 25 30
Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp
35 40 45
Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn
50 55 60
Ala Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Leu Ala
65 70 75 80
Gln Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln
85 90 95
Leu Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys
100 105 110
Ser Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser
115 120 125
Thr Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu
130 135 140
Glu Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu
145 150 155 160
Arg Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu
165 170 175
Arg Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg
180 185 190
Ala Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu
195 200 205
Val Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu
210 215 220
Asp Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu
225 230 235 240
His Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile
245 250 255
Ser Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly
260 265 270
Gln Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys
275 280 285
Pro Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala
290 295 300
Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu
305 310 315 320
Pro Asn Met Thr Gln Gly Phe Trp Glu Tyr Ser Met Leu Thr Asp Pro
325 330 335
Gly Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly
340 345 350
Lys Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp
355 360 365
Phe Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala
370 375 380
Tyr Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe
385 390 395 400
His Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys
405 410 415
His Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn
420 425 430
Glu Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly
435 440 445
Thr Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe
450 455 460
Lys Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met
465 470 475 480
Lys Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr
485 490 495
Tyr Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe
500 505 510
Ile Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala
515 520 525
Leu Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile
530 535 540
Ser Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu
545 550 555 560
Gly Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala
565 570 575
Lys Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe
580 585 590
Thr Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr
595 600 605
Asp Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu
610 615 620
Lys Ser Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met
625 630 635 640
Tyr Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu
645 650 655
Lys Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val
660 665 670
Ala Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro
675 680 685
Lys Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile
690 695 700
Arg Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn
705 710 715 720
Ser Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn Gln
725 730 735
Pro Pro Val Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
740 745 750
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
755 760 765
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
770 775 780
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
785 790 795 800
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
805 810 815
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
820 825 830
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
835 840 845
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
850 855 860
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
865 870 875 880
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
885 890 895
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
900 905 910
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
915 920 925
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
930 935 940
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
945 950 955 960
Leu Ser Leu Ser Pro Gly Lys
965
<210> 7
<211> 2904
<212> DNA
<213> hACE2-hFc-M4-2
<400> 7
atgtcaggct ctttctggct ccttctcagc cttgttgctg taactgctgc tcagtccacc 60
attgaggaac aggccaagtt ttttttggac aagtttaacc acgaagccga agacctgttc 120
tatcaaagtt cacttgcttc ttggaattat aacaccaata ttactgaaga gaatgtccaa 180
aacatgaata atgctgggga caaatggtct gcctttttaa aggaacagtc cacatgggcc 240
caaatgtatc cactacaaga aattcagaat ctcacagtca agcttcagct gcaggctctt 300
cagcaaaatg ggtcttcagt gctctcagaa gacaagagca aacggttgaa cacaattcta 360
aatacaatga gcaccatcta cagtactgga aaagtttgta acccagataa tccacaagaa 420
tgcttattac ttgaaccagg tttgaatgaa ataatggcaa acagtttaga ctacaatgag 480
aggctctggg cttgggaaag ctggagatct gaggtcggca agcagctgag gccattatat 540
gaagagtatg tggtcttgaa aaatgagatg gcaagagcaa atcattatga ggactatggg 600
gattattgga gaggagacta tgaagtaaat ggggtagatg gctatgacta cagccgcggc 660
cagttgattg aagatgtgga acataccttt gaagagatta aaccattata tgaacatctt 720
catgcctatg tgagggcaaa gttgatgaat gcctatcctt cctatatcag tccaattgga 780
tgcctccctg ctcatttgct tggtgatatg tggggtcagt tttggacaaa tctgtactct 840
ttgacagttc cctttggaca gaaaccaaac atagatgtta ctgatgcaat ggtggaccag 900
gcctgggatg cacagagaat attcaaggag gccgagaagt tctttgtatc tgttggtctt 960
cctaatatga ctcaaggatt ctgggaatat tccatgctaa cggacccagg aaatgttcag 1020
aaagcagtct gccatcccac agcttgggac ctggggaagg gcgacttcag gatccttatg 1080
tgcacaaagg tgacaatgga cgacttcctg acagctcatc atgagatggg gcatatccag 1140
tatgatatgg catatgctgc acaacctttt ctgctaagaa atggagctaa tgaaggattc 1200
catgaagctg ttggggaaat catgtcactt tctgcagcca cacctaagca tttaaaatcc 1260
attggtcttc tgtcacccga ttttcaagaa gacaatgaaa cagaaataaa cttcctgctc 1320
aaacaagcac tcacgattgt tgggactctg ccatttactt acatgttaga gaagtggagg 1380
tggatggtct ttaaagggga aattcccaaa gaccagtgga tgaaaaagtg gtgggagatg 1440
aagcgagaga tagttggggt ggtggaacct gtgccccatg atgaaacata ctgtgacccc 1500
gcatctctgt tccatgtttc taatgattac tcattcattc gatattacac aaggaccctt 1560
taccaattcc agtttcaaga agcactttgt caagcagcta aacatgaagg ccctctgcac 1620
aaatgtgaca tctcaaactc tacagaagct ggacagaaac tgttcaatat gctgaggctt 1680
ggaaaatcag aaccctggac cctagcattg gaaaatgttg taggagcaaa gaacatgaat 1740
gtaaggccac tgctcaacta ctttgagccc ttatttacct ggctgaaaga ccagaacaag 1800
aattcttttg tgggatggag taccgactgg agtccatatg cagaccaaag catcaaagtg 1860
aggataagcc taaaatcagc tcttggagat aaagcatatg aatggaacga caatgaaatg 1920
tacctgttcc gatcatctgt tgcatatgct atgaggcagt actttttaaa agtaaaaaat 1980
cagatgattc tttttgggga ggaggatgtg cgagtggcta atttgaaacc aagaatctcc 2040
tttaatttct ttgtcactgc acctaaaaat gtgtctgata tcattcctag aactgaagtt 2100
gaaaaggcca tcaggatgtc ccggagccgt atcaatgatg ctttccgtct gaatgacaac 2160
agcctagagt ttctggggat acagccaaca cttggacctc ctaaccagcc ccctgtttcc 2220
gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 2280
ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 2340
tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 2400
ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 2460
cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 2520
tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 2580
gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 2640
aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 2700
tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 2760
gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 2820
aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 2880
ctctccctgt ctccgggtaa atga 2904
<210> 8
<211> 967
<212> PRT
<213> hACE2-hFc-M4-2
<400> 8
Met Ser Gly Ser Phe Trp Leu Leu Leu Ser Leu Val Ala Val Thr Ala
1 5 10 15
Ala Gln Ser Thr Ile Glu Glu Gln Ala Lys Phe Phe Leu Asp Lys Phe
20 25 30
Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp
35 40 45
Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn
50 55 60
Ala Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Trp Ala
65 70 75 80
Gln Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln
85 90 95
Leu Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys
100 105 110
Ser Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser
115 120 125
Thr Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu
130 135 140
Glu Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu
145 150 155 160
Arg Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu
165 170 175
Arg Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg
180 185 190
Ala Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu
195 200 205
Val Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu
210 215 220
Asp Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu
225 230 235 240
His Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile
245 250 255
Ser Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly
260 265 270
Gln Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys
275 280 285
Pro Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala
290 295 300
Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu
305 310 315 320
Pro Asn Met Thr Gln Gly Phe Trp Glu Tyr Ser Met Leu Thr Asp Pro
325 330 335
Gly Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly
340 345 350
Lys Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp
355 360 365
Phe Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala
370 375 380
Tyr Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe
385 390 395 400
His Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys
405 410 415
His Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn
420 425 430
Glu Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly
435 440 445
Thr Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe
450 455 460
Lys Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met
465 470 475 480
Lys Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr
485 490 495
Tyr Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe
500 505 510
Ile Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala
515 520 525
Leu Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile
530 535 540
Ser Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu
545 550 555 560
Gly Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala
565 570 575
Lys Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe
580 585 590
Thr Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr
595 600 605
Asp Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu
610 615 620
Lys Ser Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met
625 630 635 640
Tyr Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu
645 650 655
Lys Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val
660 665 670
Ala Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro
675 680 685
Lys Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile
690 695 700
Arg Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn
705 710 715 720
Ser Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn Gln
725 730 735
Pro Pro Val Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
740 745 750
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
755 760 765
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
770 775 780
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
785 790 795 800
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
805 810 815
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
820 825 830
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
835 840 845
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
850 855 860
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
865 870 875 880
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
885 890 895
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
900 905 910
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
915 920 925
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
930 935 940
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
945 950 955 960
Leu Ser Leu Ser Pro Gly Lys
965
<210> 9
<211> 2904
<212> DNA
<213> hACE2-hFc-M5
<400> 9
atgtcaggct ctttctggct ccttctcagc cttgttgctg taactgctgc tcagtccacc 60
attgaggaac aggccaagtt ttttttggac tactttaacc acgaagccga agacctgttc 120
tatcaaagtt cacttgcttc ttggaattat aacaccaata ttactgaaga gaatgtccaa 180
aacatgaata atgctgggga caaatggtct gcctttttaa aggaacagtc cacatgggcc 240
caaatgtatc cactacaaga aattcagaat ctcacagtca agcttcagct gcaggctctt 300
cagcaaaatg ggtcttcagt gctctcagaa gacaagagca aacggttgaa cacaattcta 360
aatacaatga gcaccatcta cagtactgga aaagtttgta acccagataa tccacaagaa 420
tgcttattac ttgaaccagg tttgaatgaa ataatggcaa acagtttaga ctacaatgag 480
aggctctggg cttgggaaag ctggagatct gaggtcggca agcagctgag gccattatat 540
gaagagtatg tggtcttgaa aaatgagatg gcaagagcaa atcattatga ggactatggg 600
gattattgga gaggagacta tgaagtaaat ggggtagatg gctatgacta cagccgcggc 660
cagttgattg aagatgtgga acataccttt gaagagatta aaccattata tgaacatctt 720
catgcctatg tgagggcaaa gttgatgaat gcctatcctt cctatatcag tccaattgga 780
tgcctccctg ctcatttgct tggtgatatg tggggtcagt tttggacaaa tctgtactct 840
ttgacagttc cctttggaca gaaaccaaac atagatgtta ctgatgcaat ggtggaccag 900
gcctgggatg cacagagaat attcaaggag gccgagaagt tctttgtatc tgttggtctt 960
cctaatatga ctcaaggatt ctgggaatat tccatgctaa cggacccagg aaatgttcag 1020
aaagcagtct gccatcccac agcttgggac ctggggaagg gcgacttcag gatccttatg 1080
tgcacaaagg tgacaatgga cgacttcctg acagctcatc atgagatggg gcatatccag 1140
tatgatatgg catatgctgc acaacctttt ctgctaagaa atggagctaa tgaaggattc 1200
catgaagctg ttggggaaat catgtcactt tctgcagcca cacctaagca tttaaaatcc 1260
attggtcttc tgtcacccga ttttcaagaa gacaatgaaa cagaaataaa cttcctgctc 1320
aaacaagcac tcacgattgt tgggactctg ccatttactt acatgttaga gaagtggagg 1380
tggatggtct ttaaagggga aattcccaaa gaccagtgga tgaaaaagtg gtgggagatg 1440
aagcgagaga tagttggggt ggtggaacct gtgccccatg atgaaacata ctgtgacccc 1500
gcatctctgt tccatgtttc taatgattac tcattcattc gatattacac aaggaccctt 1560
taccaattcc agtttcaaga agcactttgt caagcagcta aacatgaagg ccctctgcac 1620
aaatgtgaca tctcaaactc tacagaagct ggacagaaac tgttcaatat gctgaggctt 1680
ggaaaatcag aaccctggac cctagcattg gaaaatgttg taggagcaaa gaacatgaat 1740
gtaaggccac tgctcaacta ctttgagccc ttatttacct ggctgaaaga ccagaacaag 1800
aattcttttg tgggatggag taccgactgg agtccatatg cagaccaaag catcaaagtg 1860
aggataagcc taaaatcagc tcttggagat aaagcatatg aatggaacga caatgaaatg 1920
tacctgttcc gatcatctgt tgcatatgct atgaggcagt actttttaaa agtaaaaaat 1980
cagatgattc tttttgggga ggaggatgtg cgagtggcta atttgaaacc aagaatctcc 2040
tttaatttct ttgtcactgc acctaaaaat gtgtctgata tcattcctag aactgaagtt 2100
gaaaaggcca tcaggatgtc ccggagccgt atcaatgatg ctttccgtct gaatgacaac 2160
agcctagagt ttctggggat acagccaaca cttggacctc ctaaccagcc ccctgtttcc 2220
gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 2280
ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 2340
tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 2400
ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 2460
cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 2520
tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 2580
gggcagcccc gagaaccaca ggtgtacacc ctgcccccat cccgggatga gctgaccaag 2640
aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 2700
tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 2760
gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 2820
aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 2880
ctctccctgt ctccgggtaa atga 2904
<210> 10
<211> 967
<212> PRT
<213> hACE2-hFc-M5
<400> 10
Met Ser Gly Ser Phe Trp Leu Leu Leu Ser Leu Val Ala Val Thr Ala
1 5 10 15
Ala Gln Ser Thr Ile Glu Glu Gln Ala Lys Phe Phe Leu Asp Tyr Phe
20 25 30
Asn His Glu Ala Glu Asp Leu Phe Tyr Gln Ser Ser Leu Ala Ser Trp
35 40 45
Asn Tyr Asn Thr Asn Ile Thr Glu Glu Asn Val Gln Asn Met Asn Asn
50 55 60
Ala Gly Asp Lys Trp Ser Ala Phe Leu Lys Glu Gln Ser Thr Trp Ala
65 70 75 80
Gln Met Tyr Pro Leu Gln Glu Ile Gln Asn Leu Thr Val Lys Leu Gln
85 90 95
Leu Gln Ala Leu Gln Gln Asn Gly Ser Ser Val Leu Ser Glu Asp Lys
100 105 110
Ser Lys Arg Leu Asn Thr Ile Leu Asn Thr Met Ser Thr Ile Tyr Ser
115 120 125
Thr Gly Lys Val Cys Asn Pro Asp Asn Pro Gln Glu Cys Leu Leu Leu
130 135 140
Glu Pro Gly Leu Asn Glu Ile Met Ala Asn Ser Leu Asp Tyr Asn Glu
145 150 155 160
Arg Leu Trp Ala Trp Glu Ser Trp Arg Ser Glu Val Gly Lys Gln Leu
165 170 175
Arg Pro Leu Tyr Glu Glu Tyr Val Val Leu Lys Asn Glu Met Ala Arg
180 185 190
Ala Asn His Tyr Glu Asp Tyr Gly Asp Tyr Trp Arg Gly Asp Tyr Glu
195 200 205
Val Asn Gly Val Asp Gly Tyr Asp Tyr Ser Arg Gly Gln Leu Ile Glu
210 215 220
Asp Val Glu His Thr Phe Glu Glu Ile Lys Pro Leu Tyr Glu His Leu
225 230 235 240
His Ala Tyr Val Arg Ala Lys Leu Met Asn Ala Tyr Pro Ser Tyr Ile
245 250 255
Ser Pro Ile Gly Cys Leu Pro Ala His Leu Leu Gly Asp Met Trp Gly
260 265 270
Gln Phe Trp Thr Asn Leu Tyr Ser Leu Thr Val Pro Phe Gly Gln Lys
275 280 285
Pro Asn Ile Asp Val Thr Asp Ala Met Val Asp Gln Ala Trp Asp Ala
290 295 300
Gln Arg Ile Phe Lys Glu Ala Glu Lys Phe Phe Val Ser Val Gly Leu
305 310 315 320
Pro Asn Met Thr Gln Gly Phe Trp Glu Tyr Ser Met Leu Thr Asp Pro
325 330 335
Gly Asn Val Gln Lys Ala Val Cys His Pro Thr Ala Trp Asp Leu Gly
340 345 350
Lys Gly Asp Phe Arg Ile Leu Met Cys Thr Lys Val Thr Met Asp Asp
355 360 365
Phe Leu Thr Ala His His Glu Met Gly His Ile Gln Tyr Asp Met Ala
370 375 380
Tyr Ala Ala Gln Pro Phe Leu Leu Arg Asn Gly Ala Asn Glu Gly Phe
385 390 395 400
His Glu Ala Val Gly Glu Ile Met Ser Leu Ser Ala Ala Thr Pro Lys
405 410 415
His Leu Lys Ser Ile Gly Leu Leu Ser Pro Asp Phe Gln Glu Asp Asn
420 425 430
Glu Thr Glu Ile Asn Phe Leu Leu Lys Gln Ala Leu Thr Ile Val Gly
435 440 445
Thr Leu Pro Phe Thr Tyr Met Leu Glu Lys Trp Arg Trp Met Val Phe
450 455 460
Lys Gly Glu Ile Pro Lys Asp Gln Trp Met Lys Lys Trp Trp Glu Met
465 470 475 480
Lys Arg Glu Ile Val Gly Val Val Glu Pro Val Pro His Asp Glu Thr
485 490 495
Tyr Cys Asp Pro Ala Ser Leu Phe His Val Ser Asn Asp Tyr Ser Phe
500 505 510
Ile Arg Tyr Tyr Thr Arg Thr Leu Tyr Gln Phe Gln Phe Gln Glu Ala
515 520 525
Leu Cys Gln Ala Ala Lys His Glu Gly Pro Leu His Lys Cys Asp Ile
530 535 540
Ser Asn Ser Thr Glu Ala Gly Gln Lys Leu Phe Asn Met Leu Arg Leu
545 550 555 560
Gly Lys Ser Glu Pro Trp Thr Leu Ala Leu Glu Asn Val Val Gly Ala
565 570 575
Lys Asn Met Asn Val Arg Pro Leu Leu Asn Tyr Phe Glu Pro Leu Phe
580 585 590
Thr Trp Leu Lys Asp Gln Asn Lys Asn Ser Phe Val Gly Trp Ser Thr
595 600 605
Asp Trp Ser Pro Tyr Ala Asp Gln Ser Ile Lys Val Arg Ile Ser Leu
610 615 620
Lys Ser Ala Leu Gly Asp Lys Ala Tyr Glu Trp Asn Asp Asn Glu Met
625 630 635 640
Tyr Leu Phe Arg Ser Ser Val Ala Tyr Ala Met Arg Gln Tyr Phe Leu
645 650 655
Lys Val Lys Asn Gln Met Ile Leu Phe Gly Glu Glu Asp Val Arg Val
660 665 670
Ala Asn Leu Lys Pro Arg Ile Ser Phe Asn Phe Phe Val Thr Ala Pro
675 680 685
Lys Asn Val Ser Asp Ile Ile Pro Arg Thr Glu Val Glu Lys Ala Ile
690 695 700
Arg Met Ser Arg Ser Arg Ile Asn Asp Ala Phe Arg Leu Asn Asp Asn
705 710 715 720
Ser Leu Glu Phe Leu Gly Ile Gln Pro Thr Leu Gly Pro Pro Asn Gln
725 730 735
Pro Pro Val Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
740 745 750
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
755 760 765
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
770 775 780
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
785 790 795 800
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
805 810 815
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
820 825 830
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
835 840 845
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
850 855 860
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
865 870 875 880
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
885 890 895
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
900 905 910
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
915 920 925
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
930 935 940
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
945 950 955 960
Leu Ser Leu Ser Pro Gly Lys
965

Claims (8)

1.ACE2改造蛋白在制备治疗和/或预防以ACE2为受体的SARS样冠状病毒引发疾病的药物中的应用或在制备预防以ACE2为受体的SARS样冠状病毒感染的疫苗中的应用。
2.依据权利要求1所述的ACE2改造蛋白，其特征在于，所述以ACE2为受体的SARS样冠状病毒为SARS-CoV-2原始毒株和/或SARS-CoV-2变异毒株和/或新冠相关冠状病毒等。
3.依据权利要求1或2的ACE2改造蛋白，其特征在于，所述的ACE2改造蛋白为hACE2-hFc-M4-1改造蛋白，其基因序列如序列列表SEQ ID No.5所示或氨基酸序列如序列列表SEQID No.6所示。
4.依据权利要求1或2的ACE2改造蛋白，其特征在于，所述的ACE2改造蛋白为hACE2-hFc-M4-2改造蛋白，其基因序列如序列列表SEQ ID No.7所示或氨基酸序列如序列列表SEQID No.8所示。
5.依据权利要求1或2的ACE2改造蛋白，其特征在于，所述的ACE2改造蛋白为hACE2-hFc-M5改造蛋白，其基因序列如序列列表SEQ ID No.9所示或氨基酸序列如序列列表SEQID No.10所示。
6.依据权利要求1或2的ACE2改造蛋白，其特征在于，所述的SARS-CoV-2原始毒株为SARS-CoV-2WT。
7.依据权利要求1或2的ACE2改造蛋白，其特征在于，所述的SARS-CoV-2变异毒株为英国株N501Y.V1、南非株N501Y.V2或巴西株N501Y.V3等变异毒株。
8.依据权利要求1或2的ACE2改造蛋白，其特征在于，所述的SARS-CoV-2变异毒株为水貂流行株Y453F、水貂流行株F486L或水貂流行株N501T。
CN202110578921.6A 2021-05-26 2021-05-26 Ace2改造蛋白及其应用 Pending CN115400207A (zh)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113897346A (zh) * 2021-09-16 2022-01-07 四川大学 能够提高与SARS-CoV-2 S蛋白亲和力的ACE2突变组合及应用

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113897346A (zh) * 2021-09-16 2022-01-07 四川大学 能够提高与SARS-CoV-2 S蛋白亲和力的ACE2突变组合及应用

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