WO2022077968A1 - 基于SARS-CoV-2S蛋白RBD区域的靶向性外泌体及其制备方法 - Google Patents
基于SARS-CoV-2S蛋白RBD区域的靶向性外泌体及其制备方法 Download PDFInfo
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- the present invention provides a targeted exosome based on the RBD region of the SARS-CoV-2 S protein, which can efficiently and tissue-specifically deliver potential anti-SARS-CoV-2 drugs.
- the first object of the present invention is to provide a targeted exosome based on the RBD region of the SARS-CoV-2 S protein, the RBD-VSVG fusion protein is expressed on the targeted exosome, and the RBD-VSVG
- the fusion protein is the RBD (receptor binding domain) of the S protein (spike protein) of SARS-CoV-2 (severe acute respiratory syndrome type II coronavirus) replacing the extracellular part of VSVG (vesicular stomatitis virus glycoprotein G). area is obtained.
- amino acid sequence of the RBD-VSVG fusion protein is shown in SEQ ID NO.1.
- amino acid sequence of the signal peptide is shown in SEQ ID NO.2.
- the second object of the present invention is to provide the preparation method of the described targeting exosome, comprising the following steps:
- step S3 connect the RBD fragment of step S1 with the transmembrane region and the full-length gene fragment of the VSVG in step S2, and connect to the vector to obtain an expression vector;
- step S3 The expression vector in step S3 is transferred into a host cell, and the cell culture supernatant is collected after culturing, and the targeted exosomes are obtained by separation.
- the host cells are 293T cells or dendritic cells.
- the separation includes the following steps: centrifuging the cell culture supernatant at 8000-15000g for 20-40min to take the supernatant, filtering the supernatant through a micron membrane and ultracentrifuging at 80000-120000g for 60-80min, taking the precipitate , the precipitate was resuspended in buffer solution, and then ultracentrifuged at 80,000-120,000g for 60-80min, and the supernatant was removed to obtain the targeted exosomes.
- the third object of the present invention is to provide the application of the targeted exosomes in the preparation of drugs for the targeted treatment of COVID-19, the application is to combine the specific anti-SARS-CoV-2 functional biological The molecules are transferred into the targeted exosomes to obtain the targeted drug for treating COVID-19.
- the functional biomolecule is small interfering RNA, antibody or small molecule drug.
- Figure 2 is a transmission electron microscope image of normal cell exosomes and RBD-labeled exosomes
- Figure 3 is a graph of nanoparticle analysis of normal cell exosomes and RBD-labeled exosomes
- Figure 4 shows the immunoprecipitation results of normal cell exosomes and RBD-labeled exosomes
- FIG. 6 Humanized ACE2 mice were infected with SARS-CoV-2 pseudovirus with green fluorescent protein GFP by intranasal route. After 24 hours, normal cell exosomes (exo) carrying GFP siRNA were injected into the tail vein. -NC) and RBD-labeled exosomes (exo-RBD), after 48 hours, the GFP fluorescence intensity in mouse lung tissue was detected by tissue immunofluorescence, and it was found that RBD-labeled exosomes carrying GFP siRNA could significantly inhibit the first
- the expression of SARS-CoV-2 pseudovirus GFP indicates that RBD-labeled exosomes can be used as effective carriers to carry active antiviral drugs to target SARS-CoV-2 tropic tissues (lungs, etc.), thereby inhibiting the pathogenicity of the virus.
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Abstract
提供了一种基于SARS-CoV-2 S蛋白RBD区域的靶向性外泌体及其制备方法。该靶向性外泌体上表达RBD-VSVG融合蛋白，该RBD-VSVG融合蛋白为SARS-CoV-2 S蛋白的RBD替换VSVG的胞外区域得到。利用该靶向性外泌体包裹SARS-CoV-2 siRNA，从而实现在组织器官中特异性抑制病毒复制。
Description
本发明涉及一种基于SARS-CoV-2S蛋白RBD区域的靶向性外泌体及其制备方法，属于生物医药技术领域。
严重急性呼吸系统综合征II型冠状病毒(SARS-CoV-2)是2019冠状病毒病(COVID-19)的病因，死亡率一直处于上升态势，成为全球重大公共健康问题。到目前为止，还没有特异性的药物或疫苗得到正式批准。结构分析和病理观察确认SARS-CoV-2病毒进入组织器官是通过与宿主细胞上的血管紧张素转换酶2(ACE-2)结合，病毒进入细胞取决于特异性识别ACE2的SARS-CoV-2刺突蛋白(S)的受体结合域(RBD)。目前认为阻断RBD和ACE2结合是开发疫苗、中和抗体和小分子药物对抗COVID-19的主要潜在策略。
外泌体是由多种细胞类型分泌的天然运输纳米囊泡(约30-100nm)。目前已知外泌体能够将特定的功能性生物分子(如核酸，包括质粒DNA和小干扰RNA、抗体及小分子药物)输送到受体细胞或组织器官，发挥治疗特定疾病的能力。然而，已有研究表明，大多数静脉注射的外泌体被肝脏所吸收代谢，因此，需要通过对外泌体改造，使其具备靶向治疗能力，将外源性治疗药物递送到体内特定的组织或细胞。目前为止，并没有特异性运输治疗COVID-19药物的靶向载体，本项发明通过对外泌体的改造，使其具备靶向SARS-CoV-2嗜性的组织器官，为运输相关特异性抗病毒药物提供靶向载体，实现对SARS-CoV-2感染的 精准治疗。
发明内容
为解决上述问题，本发明提供一种基于SARS-CoV-2S蛋白RBD区域的靶向性外泌体，能够高效、组织特异性的递送抗SARS-CoV-2潜在药物。
本发明的第一个目的是提供一种基于SARS-CoV-2S蛋白RBD区域的靶向性外泌体，所述靶向性外泌体上表达RBD-VSVG融合蛋白，所述的RBD-VSVG融合蛋白为SARS-CoV-2(严重急性呼吸系统综合征II型冠状病毒)S蛋白(刺突蛋白)的RBD(受体结合域)替换VSVG(水泡性口炎病毒糖蛋白G)的胞外区域得到。
进一步地，所述的RBD-VSVG融合蛋白的氨基酸序列如SEQ ID NO.1所示。
进一步地，所述的RBD-VSVG融合蛋白的氮端设有信号肽。
进一步地，所述的信号肽的氨基酸序列如SEQ ID NO.2所示。
本发明的第二个目的是提供所述的靶向性外泌体的制备方法，包括如下步骤：
S1、以含有SARS-CoV-2的样本cDNA为模板，PCR扩增获得RBD片段；
S2、体外合成VSVG的跨膜区及胞内区全长基因片段；
S3、将S1步骤的RBD片段和S2步骤的VSVG的跨膜区及胞内区全长基因片段进行连接，并连接到载体上得到表达载体；
S4、将S3步骤的表达载体转入宿主细胞中，培养后收集细胞培养上清，分离得到所述的靶向性外泌体。
进一步地，所述的宿主细胞为293T细胞或树突状细胞。
进一步地，所述的载体是pCMV载体。
进一步地，所述的分离包括如下步骤：将细胞培养上清在8000-15000g离心20-40min取上清液，将上清液经过微米膜过滤后在80000-120000g超速离心60-80min，取沉淀，将沉淀采用缓冲溶液重悬，再在80000-120000g超速离心60-80min，去除上清，得到所述的靶向性外泌体。
本发明的第三个目的是提供所述的靶向性外泌体在制备靶向治疗COVID-19的药物中的应用，所述的应用是将特异性抗SARS-CoV-2的功能性生物分子转入到所述的靶向性外泌体中，得到所述的靶向治疗COVID-19的药物。
进一步地，所述的功能性生物分子为小干扰RNA、抗体或小分子药物。
本发明的有益效果：
本发明构建了能够高效、组织特异性的递送抗SARS-CoV-2潜在药物的靶向外泌体；利用靶向外泌体包裹SARS-CoV-2siRNA，从而实现在组织器官中特异性抑制病毒复制。
在小鼠动物模型中，尾静脉注射外泌体包裹SARS-CoV-2siRNA，能够显著抑制小鼠肺组织中的病毒复制量，减轻病毒感染引发的肺炎等症状。
图1为利用SARS-CoV-2S蛋白的RBD区域替换水泡性口炎病毒糖蛋白G(VSVG)的细胞示意图；
图2为正常细胞外泌体与RBD标记的外泌体的透射电镜图；
图3为正常细胞外泌体与RBD标记的外泌体的纳米颗粒分析图；
图4为正常细胞外泌体与RBD标记的外泌体的免疫沉淀结果；
图5为正常细胞外泌体与RBD标记的外泌体的靶向富集效果图；
图6为正常细胞外泌体与RBD标记的外泌体携带活性抗病毒药物抑制病毒效果图。
下面结合具体实施例对本发明作进一步说明，以使本领域的技术人员可以更好地理解本发明并能予以实施，但所举实施例不作为对本发明的限定。
实施例1：
1、设计RBD的引物(F：5’-ATGTTTCCTAATATTACAAACTTGTGCC-3’，SEQ ID NO.3；R:5’-TTATGCTGGTGCATGTAGAAGTTCA-3’，SEQ ID NO.4)，以COVID-19患者咽拭子样本cDNA为模板，运用TaKaRa PCR试剂盒扩增获得RBD完整片段，2％的琼脂糖电泳，Axygen切胶回收试剂盒进行DNA产物纯化；体外合成VSVG跨膜区及胞内区全长DNA，通过T4酶连插入到pCMV载体中，构建成pCMV-VSVG载体，测序验证序列；
2、将步骤1中PCR纯化回收后的RBD基因与pCMV-VSVG载体进行T4酶连，16℃反应过夜，构建成pCMV-RBD-VSVG载体，测序验证；通过载体转化，涂板，挑单克隆，进行扩大培养，并通过Axygen质粒提取试剂盒提取pCMV-RBD-VSVG质粒载体；
图1是利用SARS-CoV-2S蛋白的RBD区域替换水泡性口炎病毒糖蛋白G(VSVG)的胞外区域，形成RBD与VSVG融合载体，转染进入细胞表达后，所产生的外泌体带有RBD-VSVG融合蛋白表达，其中胞外区为RBD蛋白，跨膜区及胞内区为VSVG蛋白的跨膜区及胞内区。CD9和CD63为外泌体特异性标记蛋白。
3、293T细胞铺板(100mm皿)，密度约60-70％，将步骤2中的pCMV-RBD-VSVG重组载体通过PEI转染(质粒与PEI质量比为1：3)细胞，6小时后，用无外泌体培养基培养换液，继续培养48小时后，收集细胞培养上清；
4、将收集的上清液首先在10000g，4度离心30分钟，去掉细胞碎片，将离心后的上清液通过0.22微米膜过滤后，在超速离心机(德国Beckman)以 100,000g，4度，离心70分钟，小心去掉上清液，加入适量PBS，将外泌体沉淀吹打悬浮混匀，再以100,000g，4度，70分钟离心，弃上清，用适量的PBS重悬，获得靶向外泌体，通过电镜技术和纳米粒径仪检测外泌体大小；
图2、图3分别通过透射电镜(TEM)和纳米颗粒分析(NTA)所提取的外泌体，与正常细胞分泌的外泌体(exo-NC)对比，RBD标记的外泌体(exo-RBD)在体积大小方面没有显著性差异，说明RBD的标记并没有影响到外泌体正常物理形态和特征。
图4通过外泌体免疫沉淀技术，用偶联RBD抗体的磁珠分别与正常细胞外泌体(exo-NC)和RBD标记的外泌体(exo-RBD)共孵育，通过磁力架分离，western blot结果揭示RBD蛋白是表达在外泌体外膜上。
5、设计针对SARS-CoV-2的siRNA并合成，通过电穿孔仪(Bio-Rad)把针对SARS-CoV-2基因组的特异性干扰RNA(siRNA)电转到所获得的靶向外泌体中，使靶向外泌体包裹siRNA进行运载，使用比例为外泌体：siRNA质量比1：1；在超速离心机(德国Beckman)以100,000g，4度，离心70分钟，去除多余的siRNA后，PBS悬浮外泌体沉淀；
6、以人源化ACE2(SARS-CoV-2特异性受体)小鼠为模型，通过尾静脉注射靶向外泌体，150ug/只，从而实现siRNA靶向递送到SARS-CoV-2嗜性的组织器官，感染病毒复制。
图5：将正常细胞外泌体(exo-NC)和RBD标记的外泌体(exo-RBD)分别通过DiD亲脂性染料标记荧光，通过尾静脉注射后人源化ACE2小鼠，以24h为时间间隔，连续观察96h，结果发现exo-RBD能够显著靶向富集在小鼠肺组织，心脏以及肾组织中，且持续时间不低于96h，而未标记RBD蛋白的正常外泌体exo-NC未在上述组织中富集。
图6：通过滴鼻途径，使人源化ACE2小鼠感染带有绿色荧光蛋白GFP的SARS-CoV-2假病毒，24h后，尾静脉分别注射携带有GFP siRNA的正常细胞 外泌体(exo-NC)和RBD标记的外泌体(exo-RBD)，48h后，通过组织免疫荧光检测小鼠肺组织中GFP荧光强度，结果发现携带有GFP siRNA的RBD标记的外泌体能够先显著抑制SARS-CoV-2假病毒GFP的表达，说明RBD标记的外泌体能够作为有效载体，携带活性抗病毒药物靶向SARS-CoV-2嗜性组织(肺等)，从而抑制病毒致病性。
以上所述实施例仅是为充分说明本发明而所举的较佳的实施例，本发明的保护范围不限于此。本技术领域的技术人员在本发明基础上所作的等同替代或变换，均在本发明的保护范围之内。本发明的保护范围以权利要求书为准。
Claims (10)
- 一种基于SARS-CoV-2 S蛋白RBD区域的靶向性外泌体，其特征在于，所述的靶向性外泌体上表达RBD-VSVG融合蛋白，所述的RBD-VSVG融合蛋白为SARS-CoV-2 S蛋白的RBD替换VSVG的胞外区域得到。
- 根据权利要求1所述的靶向性外泌体，其特征在于，所述的RBD-VSVG融合蛋白的氨基酸序列如SEQ ID NO.1所示。
- 根据权利要求2所述的靶向性外泌体，其特征在于，所述的RBD-VSVG融合蛋白的氮端设有信号肽。
- 根据权利要求3所述的靶向性外泌体，其特征在于，所述的信号肽的氨基酸序列如SEQ ID NO.2所示。
- 一种权利要求1-4任一项所述的靶向性外泌体的制备方法，其特征在于，包括如下步骤：S1、以含有SARS-CoV-2的样本cDNA为模板，PCR扩增获得RBD片段；S2、体外合成VSVG的跨膜区及胞内区全长基因片段；S3、将S1步骤的RBD片段和S2步骤的VSVG的跨膜区及胞内区全长基因片段连接到载体上得到表达载体；S4、将S3步骤的表达载体转入宿主细胞中，培养后收集细胞培养上清，分离得到所述的靶向性外泌体。
- 根据权利要求5所述的制备方法，其特征在于，所述的宿主细胞为293T细胞或树突状细胞。
- 根据权利要求5所述的制备方法，其特征在于，所述的载体是pCMV载体。
- 根据权利要求5所述的制备方法，其特征在于，所述的分离包括如下步 骤：将细胞培养上清在8000-15000g离心20-40min取上清液，将上清液经过微米膜过滤后在80000-120000g超速离心60-80min，取沉淀，将沉淀采用缓冲溶液重悬，再在80000-120000g超速离心60-80min，去除上清，得到所述的外泌体。
- 权利要求1-4任一项所述的靶向性外泌体在制备靶向治疗COVID-19的药物中的应用，所述的应用是将特异性抗SARS-CoV-2的功能性生物分子转入到所述的靶向性外泌体中，得到所述的靶向治疗COVID-19的药物。
- 根据权利要求9所述的应用，其特征在于，所述的功能性生物分子为小干扰RNA、抗体或小分子药物。
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