CN112226424A - 一种用于治疗COVID-19的ACE2-Fc融合蛋白功能测试方法 - Google Patents
一种用于治疗COVID-19的ACE2-Fc融合蛋白功能测试方法 Download PDFInfo
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Abstract
本发明提供了一种用于治疗COVID‑19的ACE2‑Fc融合蛋白功能测试方法,具体包括以下内容:表达人野生型ACE2‑Fc融合蛋白:克隆了人野生型ACE的细胞外结构域(ECD)的DNA序列;将ACE2的ECD与人IgG1的Fc部分融合,延长可溶性ACE2的半衰期;将ACE2‑Fc融合蛋白在CHO细胞中进行表达,并使用蛋白A/G琼脂糖来纯化细胞培养上清液中的ACE2‑Fc融合蛋白;优化表达系统。该发明有效的证明了ACE2‑Fc融合蛋白可将阻止或阻止病毒进入细胞,减慢感染的进程,并使病毒暴露于细胞外,以被人类免疫系统识别和消除。
Description
技术领域
本发明涉及融合蛋白技术领域,具体为一种用于治疗COVID-19的ACE2-Fc融合蛋白功能测试方法。
背景技术
冠状病毒首先通过蛋白刺突与宿主细胞表面的靶受体结合,与细胞膜融合后病毒核衣壳进入细胞内进行后续复制。在SARS-CoV病例中,病毒粒子表面的S蛋白介导受体识别和膜融合。在病毒感染过程中,三聚体S蛋白被裂解成S1和S2亚基,S1亚基在向融合后构象转变的过程中被释放。S1包含受体结合域(RBD),后者直接与ACE2的肽酶域(PD)结合,而S2负责膜融合。鉴于SARS-CoV-2和SARS-CoV之间的高度序列同源性(70-80%),最近有研究表明SARS-CoV-2蛋白的胞外域与ACE2的PD结合,其解离常数(Kd)约为15nM。因此,针对SARS-CoV-2表面的S蛋白或宿主细胞上的ACE2是开发COVID-19生物制剂时需要考虑的重点。虽然中和抗体是一个非常有吸引力的选择,但开发针对S蛋白或ACE2的人源化抗体,使其具有足够高的亲和力和特异性来中和病毒可能需要数月甚至更长时间。而可溶性ACE2的开发是最为迅速和最有前途的策略之一,它可以与SARS-CoV-2高亲和度和高特异性结合,从而阻止病毒进入宿主细胞。
发明内容
本发明所解决的技术问题在于提供一种用于治疗COVID-19的ACE2-Fc融合蛋白功能测试方法,以解决上述背景技术中的存在的问题。
本发明所解决的技术问题采用以下技术方案来实现:一种用于治疗COVID-19的ACE2-Fc融合蛋白功能测试方法,具体包括以下内容:
步骤1、表达人野生型ACE2-Fc融合蛋白:
(1)克隆了人野生型ACE的细胞外结构域(ECD)的DNA序列;
(2)将ACE2的ECD与人IgG1的Fc部分融合,延长可溶性ACE2的半衰期;
(3)将ACE2-Fc融合蛋白在CHO细胞中进行表达,并使用蛋白A/G琼脂糖来纯化细胞培养上清液中的ACE2-Fc融合蛋白;
步骤2、体外测试ACE2-Fc融合蛋白对SARS-CoV-2 S1/RBD蛋白的结合功能和抑制功能:
(4)用10ug/ml的SARS-CoV-2RBD-His融合蛋白包被96孔板,再使用ACE2-Fc融合蛋白从5ug/ml开始以2倍系列稀释液进行滴定;
(5)记录滴定实验所得测试数据,并通过曲线拟合,用于计算EC50,从而通过ELISA来评估CHO细胞表达的ACE2-Fc融合蛋白与SARS-CoV-2 S1/RBD蛋白之间的结合亲和力;
(6)用10ug/ml的ACE2-Fc融合蛋白包被96孔板,ACE2-Fc融合蛋白在稀释液中滴定;将滴定数据进行曲线拟合,验证ACE2-Fc融合蛋白对S1/RBD-His与固定的ACE2蛋白结合的抑制活性;
步骤3、优化表达系统:
(7)对比大肠杆菌,酵母,植物和哺乳动物细胞(CHO细胞)在内的几种表达系统中测试了人ACE2-Fc蛋白的表达,选择酵母作为优化表达系统;
(8)将ACE2-Fc融合蛋白在酵母和CHO细胞中进行表达,并使用蛋白A/G琼脂糖来纯化细胞培养上清液中的ACE2-Fc融合蛋白;
(9)用10ug/ml的SARS-CoV-2 RBD-His融合蛋白包被96孔板,使用ACE2-Fc融合蛋白从5ug/ml开始以2倍系列稀释液进行滴定;
(10)记录滴定实验所得测试数据,并通过曲线拟合,用于计算EC50,从而测试从酵母和CHO细胞纯化的ACE2-Fc融合蛋白与SARS-CoV-2 S1/RBD蛋白之间的结合亲和力;
(11)从222.5nM开始,以5倍系列稀释度滴定CHO和酵母的ACE2-Fc融合蛋白,将来自SARS-CoV-2的RBD-His蛋白与滴定的ACE2-Fc进行孵育,然后添加到ACE2过表达的CHO细胞中;
(12)测量每个荧光峰的平均荧光单位(MFU),并针对CHO和酵母表达的ACE2-Fc浓度作图;对CHO和酵母表达的ACE2-Fc的竞争结合的实验数据进行最佳拟合,并用于计算IC50,用于验证CHO和酵母表达系统中ACE2-Fc蛋白的体外抑制作用。
优选的,所述(6)中的稀释液为2倍系列稀释液,且ACE2-Fc融合蛋起始浓度为5ug/ml。
优选的,所述(11)中将来自SARS-CoV-2的RBD-His蛋白与滴定的ACE2-Fc孵育0.5小时。
与已公开技术相比,本发明存在以下优点:本发明有效的证明了ACE2-Fc融合蛋白可以模拟上呼吸道和下呼吸道内衬的上皮细胞上的ACE2蛋白,当过量使用时,它将与游离状态的病毒S蛋白结合,可将阻止或阻止病毒进入细胞,减慢感染的进程,并使病毒暴露于细胞外,以被人类免疫系统识别和消除。
附图说明
图1为本发明中ACE2的ECD与人IgG1的Fc部分融合示意图;
图2为本发明中ELISA测量的CHO细胞表达的ACE2-Fc融合蛋白与固定的SARS-CoV-2 S1/RBS-His融合蛋白的结合实验数据及拟合曲线;
图3为本发明中ELISA测量的从CHO细胞纯化的ACE2-Fc融合蛋白与RBD-His蛋白的竞争与固定的ACE2-Fc融合蛋白的竞争实验数据及拟合曲线。
图4为本发明中ELISA测量的从酵母和CHO细胞纯化的ACE2-Fc融合蛋白与固定的SARS-CoV-2 S1/RBS-His融合蛋白结合实验数据及拟合曲线;
图5为本发明中从CHO和酵母表达系统纯化的ACE2-Fc在体外与RBD-His竞争实验数据及拟合曲线;
具体实施方式
为了使本发明的技术手段、创作特征、工作流程、使用方法达成目的与功效易于明白了解,下面将结合本发明实施例,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例
一种用于治疗COVID-19的ACE2-Fc融合蛋白功能测试方法,具体包括以下内容:
步骤1、表达人野生型ACE2-Fc融合蛋白:
(1)克隆了人野生型ACE的细胞外结构域(ECD)的DNA序列;
(2)将ACE2的ECD与人IgG1的Fc部分融合如图1所示,延长可溶性ACE2的半衰期;
(3)将ACE2-Fc融合蛋白在CHO细胞中进行表达,并使用蛋白A/G琼脂糖来纯化细胞培养上清液中的ACE2-Fc融合蛋白;
步骤2、体外测试ACE2-Fc融合蛋白对SARS-CoV-2 S1/RBD蛋白的结合功能和抑制功能:
(4)用10ug/ml的SARS-CoV-2RBD-His融合蛋白包被96孔板,再使用ACE2-Fc融合蛋白从5ug/ml开始以2倍系列稀释液进行滴定;
(5)记录滴定实验所得测试数据,并通过曲线拟合,用于计算EC50,从而通过ELISA来评估CHO细胞表达的ACE2-Fc融合蛋白与SARS-CoV-2 S1/RBD蛋白之间的结合亲和力,其结果如图2所示,结果表明,可溶性ACE2-Fc蛋白与S1/RBD-His具有高亲和力结合,其EC50为~36.18ng/ml(0.24nM);
(6)用10ug/ml的ACE2-Fc融合蛋白包被96孔板,ACE2-Fc融合蛋白在2倍系列稀释液中滴定,起始浓度为5ug/ml;将滴定数据进行曲线拟合,验证ACE2-Fc融合蛋白对S1/RBD-His与固定的ACE2蛋白结合的抑制活性,其结果如图3所示,结果表明,ACE2-Fc融合蛋白可以与固定的ACE2竞争RBD-His蛋白;
步骤3、优化表达系统:
(7)对比大肠杆菌,酵母,植物和哺乳动物细胞(CHO细胞)在内的几种表达系统中测试了人ACE2-Fc蛋白的表达,选择酵母作为优化表达系统;
(8)将ACE2-Fc融合蛋白在酵母和CHO细胞中进行表达,并使用蛋白A/G琼脂糖来纯化细胞培养上清液中的ACE2-Fc融合蛋白;
(9)用10ug/ml的SARS-CoV-2 RBD-His融合蛋白包被96孔板,使用ACE2-Fc融合蛋白从5ug/ml开始以2倍系列稀释液进行滴定;
(10)记录滴定实验所得测试数据,并通过曲线拟合,用于计算EC50,从而通过ELISA来测试从酵母和CHO细胞纯化的ACE2-Fc融合蛋白与SARS-CoV-2 S1/RBD蛋白之间的结合亲和力,其结果如图4所示,结果表明,酵母和CHO细胞中ACE2-Fc蛋白的结合亲和力非常相似,且酵母表达的ACE2-Fc和CHO细胞表达的ACE2-Fc的EC50值分别为55.57ng/ml(0.37nM)和36.18ng/ml(0.24nM);因此,从酵母表达和纯化的可溶性ACE2-Fc蛋白将用于以后的应用;同时该结果表明,在酵母中表达的ACE2-Fc蛋白与在CHO细胞中表达的ACE2-Fc蛋白表现出相似的结合活性;
(11)从222.5nM开始,以5倍系列稀释度滴定CHO和酵母的ACE2-Fc融合蛋白,将来自SARS-CoV-2的RBD-His蛋白与滴定的ACE2-Fc孵育0.5小时,然后添加到ACE2过表达的CHO细胞中;
(12)测量每个荧光峰的平均荧光单位(MFU),并针对CHO和酵母表达的ACE2-Fc浓度作图;对CHO和酵母表达的ACE2-Fc的竞争结合的实验数据进行最佳拟合,并用于计算IC50,其结果如图5所示,从44.5nM开始,两种ACE2-Fc蛋白都显示了对SARS-Cov-2 S/RBD-His蛋白与细胞表面过表达的ACE2蛋白结合具有抑制作用,且来自CHO和酵母的ACE2-Fc蛋白对于RBD-His结合的IC50值分别为98.9nM和90.2nM。
以上显示和描述了本发明的基本原理、主要特征及本发明的优点。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,在不脱离本发明精神和范围的前提下,本发明还会有各种变化和改进,这些变化和改进都落入要求保护的本发明范围内。本发明的要求保护范围由所附的权利要求书及其等效物界定。
Claims (3)
1.一种用于治疗COVID-19的ACE2-Fc融合蛋白功能测试方法,其特征在于:具体包括以下内容,
步骤1、表达人野生型ACE2-Fc融合蛋白:
(1)克隆了人野生型ACE的细胞外结构域(ECD)的DNA序列;
(2)将ACE2的ECD与人IgG1的Fc部分融合,延长可溶性ACE2的半衰期;
(3)将ACE2-Fc融合蛋白在CHO细胞中进行表达,并使用蛋白A/G琼脂糖来纯化细胞培养上清液中的ACE2-Fc融合蛋白;
步骤2、体外测试ACE2-Fc融合蛋白对SARS-CoV-2S1/RBD蛋白的结合功能和抑制功能:
(4)用10ug/ml的SARS-CoV-2RBD-His融合蛋白包被96孔板,再使用ACE2-Fc融合蛋白从5ug/ml开始以2倍系列稀释液进行滴定;
(5)记录滴定实验所得测试数据,并通过曲线拟合,用于计算EC50,从而通过ELISA来评估CHO细胞表达的ACE2-Fc融合蛋白与SARS-CoV-2S1/RBD蛋白之间的结合亲和力;
(6)用10ug/ml的ACE2-Fc融合蛋白包被96孔板,ACE2-Fc融合蛋白在稀释液中滴定;将滴定数据进行曲线拟合,验证ACE2-Fc融合蛋白对S1/RBD-His与固定的ACE2蛋白结合的抑制活性;
步骤3、优化表达系统:
(7)对比大肠杆菌,酵母,植物和哺乳动物细胞(CHO细胞)在内的几种表达系统中测试了人ACE2-Fc蛋白的表达,选择酵母作为优化表达系统;
(8)将ACE2-Fc融合蛋白在酵母和CHO细胞中进行表达,并使用蛋白A/G琼脂糖来纯化细胞培养上清液中的ACE2-Fc融合蛋白;
(9)用10ug/ml的SARS-CoV-2RBD-His融合蛋白包被96孔板,使用ACE2-Fc融合蛋白从5ug/ml开始以2倍系列稀释液进行滴定;
(10)记录滴定实验所得测试数据,并通过曲线拟合,用于计算EC50,从而测试从酵母和CHO细胞纯化的ACE2-Fc融合蛋白与SARS-CoV-2S1/RBD蛋白之间的结合亲和力;
(11)从222.5nM开始,以5倍系列稀释度滴定CHO和酵母的ACE2-Fc融合蛋白,将来自SARS-CoV-2的RBD-His蛋白与滴定的ACE2-Fc进行孵育,然后添加到ACE2过表达的CHO细胞中;
(12)测量每个荧光峰的平均荧光单位(MFU),并针对CHO和酵母表达的ACE2-Fc浓度作图;对CHO和酵母表达的ACE2-Fc的竞争结合的实验数据进行最佳拟合,并用于计算IC50,用于验证CHO和酵母表达系统中ACE2-Fc蛋白的体外抑制作用。
2.根据权利要求1所述的一种用于治疗COVID-19的ACE2-Fc融合蛋白功能测试方法,其特征在于:所述(6)中的稀释液为2倍系列稀释液,且ACE2-Fc融合蛋起始浓度为5ug/ml。
3.根据权利要求1所述的一种用于治疗COVID-19的ACE2-Fc融合蛋白功能测试方法,其特征在于:所述(11)中将来自SARS-CoV-2的RBD-His蛋白与滴定的ACE2-Fc孵育0.5小时。
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