CN112439058A - 基于外泌体为载体的重组新型冠状病毒纳米疫苗方法 - Google Patents

基于外泌体为载体的重组新型冠状病毒纳米疫苗方法 Download PDF

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CN112439058A
CN112439058A CN202011336893.9A CN202011336893A CN112439058A CN 112439058 A CN112439058 A CN 112439058A CN 202011336893 A CN202011336893 A CN 202011336893A CN 112439058 A CN112439058 A CN 112439058A
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段莉
梁宇杰
徐晓
徐丽梅
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Shenzhen Second Peoples Hospital
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Abstract

本发明涉及疫苗技术领域，具体为基于外泌体为载体的重组新型冠状病毒纳米疫苗方法，包括以下步骤：S1：表达载体构建，构建含有编码2019新型冠状病毒S蛋白胞外区域(1‑1195)的与VSV‑G基因的融合多核苷酸的穿梭质粒载体；将步骤S1：所构建好的穿梭质粒载体转染入供体细胞AAV‑293；S3：利用无血清细胞培养基进行体外扩增培养步骤S2所述供体细胞；本发明的有益效果是：通过合理的利用纳米级外泌体的优势，以外泌体为媒介物加载和增强抗原蛋白质的体内递送。本发明技术方案所生产的纳米级疫苗能够很好的刺激人体的免疫系统识别并产生免疫性，具有广泛的市场应用前景。

Description

基于外泌体为载体的重组新型冠状病毒纳米疫苗方法
技术领域
本发明涉及疫苗技术领域，具体为基于外泌体为载体的重组新型冠状病毒纳米疫苗方法。
背景技术
COVID-19是种新型冠状病毒与先前确定的冠状病毒(例如，严重急性呼吸系统综合症冠状病毒(SARS-CoV)和中东呼吸综合症冠状病毒(MERS-CoV))相比，COVID-19的致死率要低得多，但传播力很强。到目前为止，该病毒已传播到213个国家以上，感染了13241,000多人，并夺走了575,000多条生命(Worldometer，7月14日)。
SARS-CoV-2的病毒粒子为平均直径为125nm的球体形式，具有基于脂质的病毒包膜和正链单链RNA基因组。SARS-CoV-2的病毒颗粒具有四种结构蛋白：刺突(S)，膜(M)，包膜(E)和核衣壳(N)蛋白，其中S蛋白在病毒与附着中起关键作用。COVID-19的症状通常与流感相似，似乎始于发烧，继而出现干咳和疲劳，但只有5％的确诊病例报告了流鼻涕，打喷嚏或咽喉痛。大约一周后，病情恶化将导致呼吸急促，约20％的患者需要住院治疗。对于老年人和患有慢性疾病的患者，早期症状可能发展为肺炎，并伴有胸闷，胸痛和呼吸急促。也有一些病例几乎没有症状，暴露于病毒后最多需要14天才会出现，但即使是无症状的人也可能会脱落病毒感染其他人患病。迅速增加的COVID-19感染及死亡人数为全球敲响了生命的警钟。目前，全球正在努力制止其扩散。
目前，正在开展多种药物研究，但尚无针对COVID-19的抗病毒治疗方法。纳米技术为预防，诊断和治疗COVID-19的各种棘手问题提供创新的解决方案方面并具有广阔的应用前景，其中一些已经推进到在临床试验中。对于疫苗研发是一项重要的管线，疫苗过程研发有很多不确定因素。我国当前主要科研攻关组布局了病毒的灭活疫苗、核酸疫苗、重组蛋白疫苗、腺病毒载体疫苗、减毒流感病毒载体疫苗这样5条技术路线。
但是现有的疫苗研发和制备过程中没有利用纳米级别的外泌体来制备疫苗的优势，忽略了纳米级外泌体存在的作用，同时没有合理的纳米级疫苗的制备方法。
发明内容
本发明的目的在于提供基于外泌体为载体的重组新型冠状病毒纳米疫苗方法，以解决背景技术中提到的问题以及达到背景技术中所提到的目的。
为实现上述目的，本发明提供如下技术方案：基于外泌体为载体的重组新型冠状病毒纳米疫苗方法，包括以下步骤：
S1：表达载体构建，构建含有编码2019新型冠状病毒S蛋白胞外区域(1-1195)的与VSV-G基因的融合多核苷酸的穿梭质粒载体；
S2：将步骤S1：所构建好的穿梭质粒载体转染入供体细胞AAV-293；
S3：利用无血清细胞培养基进行体外扩增培养步骤S2所述供体细胞；
S4：步骤S3中所述供体细胞中AAV-293在培养4-5天后收获从所分泌释放到细胞上清；
S5：对步聚S4中所收集的上清进行重组外泌体纯化，而后即可获得该纳米疫苗。
优选的，步骤S1中的S蛋白会通过VSVG连接方式插入到外泌体膜表面。
优选的，步骤S1中提到的载体VSV-G在AAV-293细胞中真核表达。
优选的，步骤S5中制的纳米疫苗可以制备成滴鼻剂、喷雾剂和肌肉注射剂。
优选的，步骤S5中制的纳米疫苗在预防新型冠状病毒中得到应用。
优选的，所述S蛋白与VSV-G融合表达形成重组外泌体纳米疫苗。
与现有技术相比，本发明的有益效果是：
通过合理的利用纳米级外泌体的优势，以外泌体为媒介物加载和增强抗原蛋白质的体内递送。同时，并且加上合理的制备手段，能够让纳米级的疫苗发挥最大的优势。
本发明提到的纳米疫苗的优点是：安全、高效、可规模化生产。本发明技术方案所生产的纳米级疫苗能够很好的刺激人体的免疫系统识别并产生免疫性，具有广泛的市场应用前景。
具体实施方式
下面将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。
本发明提供一种技术方案：基于外泌体为载体的重组新型冠状病毒纳米疫苗方法，包括以下步骤：
S1：表达载体构建，构建含有编码2019新型冠状病毒S蛋白胞外区域(1-1195)的与VSV-G基因的融合多核苷酸的穿梭质粒载体；
S2：将步骤S1：所构建好的穿梭质粒载体转染入供体细胞AAV-293；
S3：利用无血清细胞培养基进行体外扩增培养步骤S2供体细胞；
S4：步骤S3中供体细胞中AAV-293在培养4-5天后收获从所分泌释放到细胞上清；
S5：对步聚S4中所收集的上清进行重组外泌体纯化，而后即可获得该纳米疫苗。
步骤S1中的S蛋白会通过VSVG连接方式插入到外泌体膜表面。
步骤S1中提到的载体VSV-G在AAV-293细胞中真核表达。
步骤S5中制的纳米疫苗可以制备成滴鼻剂、喷雾剂和肌肉注射剂。
步骤S5中制的纳米疫苗在预防新型冠状病毒中得到应用。
S蛋白与VSV-G融合表达形成重组外泌体纳米疫苗。
纳米级别的外泌体是在人体中循环的天然纳米囊泡，具有将各种蛋白质转运到受体细胞的内在能力，一种高效递送载体。本发明以外泌体进行抗原蛋白质展示和递送，将其作为治疗剂向哺乳动物细胞的细胞内递送的活载体通过融合水泡性口炎病毒糖蛋白(VSVG)，将S蛋白货物装载并展示到外泌体上，又可以通过VSVG提高其传递能力,以外泌体为媒介物加载和增强抗原蛋白质的体内递送。
本发明是综合了重组蛋白疫苗和纳米疫苗的优势，开发出基于外泌体纳米材料的COVID-19疫苗，把新型冠状病毒的Spike蛋白的胞外域区域(1-1195)作为基因工程重组亚单位疫苗。通过基因工程方法，在真核细胞内大量生产新冠病毒最有可能作为抗原的S蛋白，通过S蛋白与VSVG融合表达插入到外泌体膜表面，通过细胞分泌出的纳米级别的外泌体上就携带上S蛋白，注射机体上就能刺激机体产生抗体。
通过本发明的制备方法，即，本发明的疫苗方案相当于不生产完整病毒，而是单独生产很多新冠病毒的关键部件“S蛋白”，形成类似病毒结构的纳米结构展示S蛋白，将其刺激人体的免疫系统识别并产生免疫性。
尽管已经示出和描述了本发明的实施例，对于本领域的普通技术人员而言，可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由所附权利要求及其等同物限定。

Claims (6)

1.基于外泌体为载体的重组新型冠状病毒纳米疫苗方法，其特征在于包括以下步骤：
S1：表达载体构建，构建含有编码2019新型冠状病毒S蛋白胞外区域(1-1195)的与VSV-G基因的融合多核苷酸的穿梭质粒载体；
S2：将步骤S1：所构建好的穿梭质粒载体转染入供体细胞AAV-293；
S3：利用无血清细胞培养基进行体外扩增培养步骤S2所述供体细胞；
S4：步骤S3中所述供体细胞中AAV-293在培养4-5天后收获从所分泌释放到细胞上清；
S5：对步聚S4中所收集的上清进行重组外泌体纯化，而后即可获得该纳米疫苗。
2.根据权利要求1所述的基于外泌体为载体的重组新型冠状病毒纳米疫苗方法，其特征在于：步骤S1中的S蛋白会通过VSVG连接方式插入到外泌体膜表面。
3.根据权利要求1所述的基于外泌体为载体的重组新型冠状病毒纳米疫苗方法，其特征在于：步骤S1中提到的载体VSV-G在AAV-293细胞中真核表达。
4.根据权利要求1所述的基于外泌体为载体的重组新型冠状病毒纳米疫苗方法，其特征在于：步骤S5中制的纳米疫苗可以制备成滴鼻剂、喷雾剂和肌肉注射剂。
5.根据权利要求1所述的基于外泌体为载体的重组新型冠状病毒纳米疫苗方法，其特征在于：步骤S5中制的纳米疫苗在预防新型冠状病毒中得到应用。
6.根据权利要求1所述的基于外泌体为载体的重组新型冠状病毒纳米疫苗方法，其特征在于：所述S蛋白与VSV-G融合表达形成重组外泌体纳米疫苗。
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