WO2022032496A1 - 一种预防新型冠状病毒微颗粒的制备方法及应用 - Google Patents

一种预防新型冠状病毒微颗粒的制备方法及应用 Download PDF

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WO2022032496A1
WO2022032496A1 PCT/CN2020/108525 CN2020108525W WO2022032496A1 WO 2022032496 A1 WO2022032496 A1 WO 2022032496A1 CN 2020108525 W CN2020108525 W CN 2020108525W WO 2022032496 A1 WO2022032496 A1 WO 2022032496A1
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novel coronavirus
preparation
protein
microparticles
supernatant
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黄浩
金红林
卢利森
万超
郑俊武
郑成武
卫路
洪磊
金秀妍
刘群
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武汉圣润生物科技有限公司
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Definitions

  • the invention relates to the technical field of vaccines, in particular to a preparation method and application for preventing novel coronavirus microparticles.
  • the 2019 novel coronavirus pneumonia epidemic is defined by the World Health Organization as an international public health emergency.
  • the disease is a class of acute respiratory infectious diseases caused by the new coronavirus (SARS-CoV-2) infection.
  • SARS-CoV-2 new coronavirus
  • the main route of transmission is Respiratory droplet transmission, aerosol transmission and contact transmission, common symptoms are fever, cough, shortness of breath and dyspnea, severe infection may develop severe acute respiratory syndrome, renal failure and even death.
  • coronavirus The diameter of coronavirus is usually between 70-120nm, it is an enveloped positive-stranded single-stranded RNA virus, and its single-stranded non-segmental RNA genome length is between 26-32kb.
  • Coronaviruses belong to the Orthonaviridae subfamily of the family Coronaviridae, which includes 4 genera ( ⁇ , ⁇ , ⁇ and ⁇ ), usually only ⁇ and ⁇ genera have pathogenic effects on humans.
  • the new coronavirus is a beta coronavirus, and its spike (S) protein is a trimeric structure, in which each monomer has a site for binding to cellular receptors.
  • Recombinant adenovirus vaccine based on novel coronavirus spike protein (S);
  • mRNA messenger RNA vaccine based on S protein
  • recombinant adenovirus vaccines are easily cleared by the immune system, which affects their expression efficiency; mRNA vaccines are easily coated with special carriers to protect mRNAs from being degraded due to the poor stability of mRNA; and recombinant protein vaccines require cumbersome expression and The purification process is expensive and requires additional adjuvant to exert a strong effect.
  • Extracellular microparticles are important carriers for transmitting material information between cells. When cells are stimulated internally and externally, the cell membrane produces a phospholipid bilayer membrane structure with a diameter of 100-1000nm by budding. Extracellular microparticles can be loaded with a variety of biological information molecules, including proteins, mRNAs, and the like. Compared with traditional radiotherapy, which is one of the commonly used tumor treatment methods, studies have shown that radiotherapy can promote the release of extracellular microparticles by cells, and the microparticles released by radiotherapy-induced cells themselves have a strong function of activating the body's immune response.
  • the purpose of the present invention is to solve the need for complicated expression and purification steps in the preparation process of recombinant protein vaccine, and to provide a preparation method for preventing novel coronavirus microparticles and its application as a vaccine.
  • the present invention provides a preparation method for preventing novel coronavirus microparticles, comprising the following steps:
  • the mixture was centrifuged and the resulting microparticles were collected.
  • the step 1 is to synthesize an artificial gene based on the coding region of the novel coronavirus S protein, and insert it into the multi-cloning site of the plasmid vector to construct a core plasmid that can overexpress the S protein;
  • the process of packaging lentiviral particles uses psPAX2 plasmid and pMD2.G plasmid;
  • the tool cell includes one of a fibroblast cell line, a vascular endothelial cell line, and a human embryonic kidney cell line;
  • X-ray radiation dose is 2-20Gy
  • the X-ray radiation dose is 20Gy
  • the X-ray energy is 6MV
  • the supernatant is collected on the 2-7th day after continuous radiotherapy
  • the centrifugation operation described in the step 5 is: 1000g, centrifugation for 10min to obtain the first supernatant; the obtained first supernatant is centrifuged at 14000g for 2min to obtain the second supernatant; the obtained second supernatant is in 14000g, 60min centrifugation to obtain microparticles precipitate;
  • microparticle is a microvesicle structure carrying S protein, and the particle size of the microvesicle structure is 100-1000 nm.
  • Another object of the present invention is to provide a vaccine for preventing novel coronavirus, the vaccine comprising the microparticles obtained by the above microparticle preparation method.
  • the vaccine may be loaded with an adjuvant for immunization.
  • the vaccine achieves the purpose of preventing new coronavirus infection by activating B lymphocytes to produce anti-new coronavirus antibodies.
  • the vaccine can produce a large number of specific antibodies against the new coronavirus without adding immune adjuvants;
  • radiotherapy microparticles loaded with the S protein are prepared, thereby obtaining a novel coronavirus vaccine that can activate the body to produce a large number of novel coronavirus neutralizing antibodies, and avoid S protein. Protein enrichment and purification process to improve vaccine yield and reduce production cost.
  • the novel coronavirus prevention microparticles obtained by the present invention have a simple preparation process, a short cycle, and have good biosafety and biocompatibility.
  • Fig. 1 is the electron micrograph of the prevention novel coronavirus microparticle of the present invention
  • Fig. 2 is the particle size diagram of the prevention novel coronavirus microparticle of the present invention.
  • Fig. 3 is the detection result of S protein content of stably transformed fibroblasts
  • Figure 4 shows the protein quantification results of different radiotherapy doses inducing tool cells to produce radiotherapy microparticles
  • Fig. 5 is the detection result of the S protein content of the prevention of novel coronavirus microparticles according to the present invention.
  • Fig. 7 is the detection result of anti-novel coronavirus antibody content produced in mice by the cell lysate overexpressing S protein and the vaccine microparticles of the present invention
  • Fig. 8 is a graph showing the weight statistics of mice during the administration of the prevention of novel coronavirus microparticles according to the present invention.
  • the cells will secrete microparticles rich in the S protein of the novel coronavirus.
  • the resulting microparticles can induce the body to produce antibodies against the novel coronavirus.
  • the storage conditions for preventing novel coronavirus microparticles are 4°C and within 7 days.
  • the microparticle structure of the prevention of novel coronavirus according to the present invention is shown in Figure 1, and the particle size is shown in Figure 2.
  • Human embryonic kidney 293T cells were used in 10% FBS (fetal bovine serum) medium in a 10mm ⁇ 10mm culture dish, and when the cell density in the dish reached about 50%, 10 ml of fresh medium was replaced. Take 2 sterile, RNase-free EP tubes, add 1.5 mL of Opti-MEM optimized medium to one of them, and add 30 ⁇ L of PEI transfection reagent into it. After gently pipetting and mixing with the pipette tip, let it stand at room temperature for 5 min.
  • FBS fetal bovine serum
  • FBS fetal bovine serum
  • FBS fetal bovine serum
  • the dye enhancer polybrene was used for two consecutive days of infection. Screening with puromycin-containing medium (1:1000) to obtain tool cells overexpressing S protein (tool cells can be normal tissue cell lines such as fibroblast cell line, vascular endothelial cell line, human embryonic kidney cell line, etc.) , in order to avoid the risk of malignant transformation of cell lines such as tumor cells as a starting point).
  • the quantitative lysate of stably transduced fibroblasts overexpressing S protein was mixed with 1/4 volume of 5 ⁇ SDS loading buffer, and heated at 100°C for 10min.
  • Prepare 12% separating gel and 5% stacking gel according to the formula add protein samples to the loading wells, and add an equal volume of 1 ⁇ SDS loading buffer to the edge wells.
  • the stacking gel was kept at a constant voltage of 80V, and when the protein markers were separated, the voltage was adjusted to a constant voltage of 120V.
  • the electrophoresis runs until bromophenol blue is at the bottom of the separating gel, the electrophoresis is terminated. Pour the transfer solution into the iron pan and place the transfer clip.
  • the stably transformed fibroblasts were cultured with 10% FBS (fetal bovine serum) medium in a 10mm ⁇ 10mm petri dish. When the number of cells in the dish reached about 5 ⁇ 10 6 , an energy of 6MV and a dose of 20Gy were administered.
  • Radiotherapy change the medium on the first day after radiotherapy, add 20ml of medium containing 10% FBS, and collect on the third day (the time to collect microparticles is generally selected from the 2nd to the 7th day after radiotherapy, which can be selected according to the situation.
  • Example: Select day 3 All liquids in the petri dish were extracted by gradient centrifugation.
  • mice were subcutaneously injected with 50 ⁇ L of PBS (blank group), microparticles (5 mg/kg) extracted from ordinary fibroblasts on the 3rd day after 20Gy radiotherapy (control group), and fibroblasts overexpressing S protein on the 3rd day after 20Gy radiotherapy.
  • Microparticles (5 mg/kg) extracted on 3 days (vaccine group) were injected at a frequency of 1/week for 3 consecutive weeks.
  • Anti-S protein antibody content in mouse serum was detected by ELISA on the 15th day after the first injection.
  • Figure 6 compared with the blank group and the control group, the mice in the vaccine group produced anti-S protein antibodies in vivo.
  • mice in the vaccine microparticle group produced more anti-S protein antibodies compared to the tool cell lysate containing the S protein.
  • the body weight of the mice in the vaccine group was not significantly different from the other two groups, indicating that the microparticles that can prevent the infection of the new coronavirus have no obvious side effects.
  • the virus-infected microparticles prepared through the above steps are produced by the genetically engineered tool cells subjected to radiation treatment.
  • the principle is that radiotherapy can promote the release of microparticles of phospholipid bilayer structure with a diameter of 100-1000nm from the cell membrane in the form of budding. Because it carries the S protein of the new coronavirus, and the microparticles themselves have the function of targeting immune cells. , with immune activation function, can promote the occurrence of immune response.
  • novel coronavirus infection microparticles have a significant immune activation effect, which can promote the body's B lymphocytes to produce anti-nCoV antibodies to prevent novel coronavirus infection. From the subcutaneous inoculation of new coronavirus microparticles in mice, it can be seen that the new coronavirus microparticles have good biosafety and biocompatibility, and can be used alone or loaded with immune adjuvants to further activate the body's immune system to enhance its prevention. The function of preventing new coronavirus infection.

Abstract

一种预防新型冠状病毒微颗粒制备方法及应用，所述制备方法包括以下步骤：1)构建过表达刺突(S)蛋白的重组质粒；2)将重组质粒包装成慢病毒颗粒；3)用慢病毒颗粒对成纤维细胞进行感染，得到表达S蛋白的工具性细胞；4)将得到的工具性细胞及培养液进行X线照射，收集放疗后的上清液，获得所需微颗粒和凋亡的工具性细胞碎片混合物；5)将得到的混合物进行离心、浓缩、纯化后，即得疫苗。该方法利用了放疗诱导工具细胞释放的微颗粒作为载体，可在过表达新型冠状病毒S蛋白的情况下，直接激活机体抗病毒免疫，产生抗新型冠状病毒的中和抗体。

Description

一种预防新型冠状病毒微颗粒的制备方法及应用 【技术领域】
本发明涉及疫苗技术领域，具体涉及一种预防新型冠状病毒微颗粒的制备方法及应用。
【背景技术】
2019年新型冠状病毒肺炎疫情被世界卫生组织定义为国际突发公共卫生事件，该疾病是由新型冠状病毒(SARS-CoV-2)感染引起的一类急性呼吸道类传染病，其主要传播途径为呼吸道飞沫传播、气溶胶传播和接触传播，常见的症状有发热、咳嗽、气促和呼吸困难等，严重感染者可能出现严重急性呼吸综合征、肾衰竭甚至死亡。
冠状病毒的直径通常在70-120nm之间，为含包膜的正链单股RNA病毒，其单股非节段性RNA基因组长度在26-32kb之间。冠状病毒为冠状病毒科正冠状病毒亚科，该亚科包括4个属(α、β、γ和δ)，通常仅α、β属对人类具有致病作用。新型冠状病毒为β属冠状病毒，其刺突(S)蛋白为三聚体结构，其中每个单体都有与细胞受体结合的位点。研究表明，S蛋白上的受体结构域可与人受体蛋白血管紧张素转化酶2(ACE2)相结合从而入侵细胞。由于感染新型冠状病毒后的康复患者体内缺乏免疫记忆反应，其中和抗体只能维持较短时间，这导致治愈患者还可能发生二次感染。因此，探索安全有效的疫苗来刺激机体产生免疫应答，形成抗病毒抗体和免疫记忆反应是战胜疫情最重要的措施之一。
目前，新型冠状病毒疫苗研究主要集中在以下几个方向：
1、基于新型冠状病毒刺突蛋白(S)的重组腺病毒疫苗；
2、基于S蛋白的信使RNA(mRNA)疫苗；
3、基于S蛋白的重组蛋白疫苗。
其中重组腺病毒疫苗容易被免疫系统清除，影响其表达效率；mRNA疫苗由于mRNA本身稳定性较差，易被需要特殊载体包被以保护mRNA不被降解；而重组蛋白疫苗由于需要繁琐的表达和纯化过程，其成本昂贵，且需额外添加佐剂后才能发挥较强的效果。
胞外微颗粒是细胞间传递物质信息的重要载体，在细胞受到内外刺激时，细胞膜通过出芽的方式产生直径为100-1000nm的磷脂双层膜结构。胞外微颗粒可负载多种生物信息分子，包括蛋白质、mRNA等。相比于传统的放疗是常用的肿瘤治疗手段之一，研究表明，放疗可以促进细胞释放胞外微颗粒，且放疗诱导细胞释放的微颗粒本身具备较强激活机体免疫反应的功能。
但目前尚未有使用放疗诱导手段结合S蛋白过表达重组细胞载体促进胞外微颗粒制备疫苗研究的先例。因此有必要提供一种新型冠状病毒疫苗的制备方法，利用放疗诱导工具细胞释放的微颗粒作为载体，通过的免疫激活效应，在过表达新型冠状病毒S蛋白的情况下，直接激活机体抗病毒免疫。并通过验证通过放疗诱导工具细胞释放的含S蛋白的疫苗微颗粒产生新型冠状病毒抗体的性能，从而避免体外制备重组蛋白的纯化过程，促进新型冠状病毒疫苗的研究和产业化应用。
【发明内容】
本发明的目的在于解决重组蛋白疫苗制备过程中由于需要繁琐的表达和纯化步骤，提供了一种预防新型冠状病毒微颗粒的制备方法及作为疫苗的应用。
为解决上述技术问题，本发明提供了一种预防新型冠状病毒微颗粒的制备方法，包括以下步骤：
1.构建过表达S蛋白的重组质粒；
2.将重组质粒包装成慢病毒颗粒；
3.用慢病毒颗粒对工具性细胞进行感染，得到过表达S蛋白的工具性细胞；
4.将得到的工具性细胞及培养液进行X线照射，收集放疗后的上清液，经浓缩、纯化，获得所需微颗粒和凋亡的工具性细胞碎片混合物；
5.将混合物进行离心，收集所得微颗粒。
进一步地，所述步骤1为，基于新型冠状病毒S蛋白质编码区合成人工基因，并插入质粒载体的多克隆位点中，以构建可过表达S蛋白的核心质粒；
进一步地，所述步骤2中，包装慢病毒颗粒的过程使用psPAX2质粒和pMD2.G质粒；
进一步地，所述步骤3中工具性细胞包括成纤维细胞系、血管内皮细胞系、人胚肾细胞系中的一种；
进一步地，所述步骤4中X线放射剂量为2-20Gy；
进一步地，所述步骤4中X线放射剂量为20Gy，X线能量为6MV，持续放疗后的第2-7天收集上清液；
进一步地，所述步骤5中所述离心操作为：1000g，离心10min得第一上清液；所得第一上清液于14000g，离心2min得第二上清液；所得第二上清液于14000g，60min离心得微颗粒沉淀；
进一步地，所述微颗粒为携载S蛋白的微囊泡结构，所述微囊泡结构的粒径为100-1000nm。
本发明的另一个目的在于，提供了一种预防新型冠状病毒的疫苗，所述疫苗包含以上微颗粒制备方法获得的微颗粒。
进一步地，所述疫苗可以负载免疫的佐剂。
相比现有技术，本发明的有益效果为：
1.该疫苗通过激活B淋巴细胞产生抗新型冠状病毒抗体以实现预防新型冠状病毒感染的目的，该疫苗不添加免疫佐剂，即可产生大量针对新型冠状病毒的特异性抗体；
2.本发明通过制备过表达S蛋白的工具细胞，经X线放疗，制备负载S蛋白的放疗微颗粒，由此获得可激活机体产生大量新型冠状病毒中和抗体的新型冠状病毒疫苗，避免S蛋白的富集和纯化过程，提高疫苗产率并降低生产成本。
3.本发明所得的预防新型冠状病毒微颗粒制备工艺简单，周期短，具备良好的生物安全性和生物相容性。
【附图说明】
为了更清楚地说明本发明实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本发明的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。
图1为本发明预防新型冠状病毒微颗粒的电镜图；
图2为本发明预防新型冠状病毒微颗粒的粒径图；
图3为稳转成纤维细胞S蛋白含量检测结果；
图4为不同放疗剂量诱导工具细胞产生放疗微颗粒的蛋白定量结果；
图5为本发明预防新型冠状病毒微颗粒S蛋白含量检测结果；
图6为本发明预防新型冠状病毒微颗粒使小鼠体内产生的抗新型冠状病毒抗体含量检测结果；
图7为过表达S蛋白的细胞裂解液与本发明中的疫苗微颗粒使小鼠体内产生的抗新型冠状病毒抗体含量检测结果；
图8为本发明预防新型冠状病毒微颗粒给药过程中小鼠的体重统计图。
【具体实施方式】
以下实例用于说明本发明，但不限制本发明的范围。在不背离本发明精神和实质的前提下，对本发明的方法、步骤或条件所作的修改或替换，均属于本发明的范围。
本发明在使用X射线照射基因工程改造的成纤维细胞后，细胞将分泌富含新型冠状病毒S蛋白的微颗粒。所得到的微颗粒可诱发机体产出抗新型冠状病毒抗体。本发明中，预防新型冠状病毒微颗粒储存条件为4℃和7天以内。本发明所述的预防新型冠状病毒微颗粒结构如图1所示，粒径大小如图2所示。
以下是本发明的实施例：
1、构建重组质粒并包装成慢病毒颗粒感染工具细胞
在Pubmed网站确定新型冠状病毒S蛋白质编码区基因序列：atgtttgtttttcttgttttattgccactagtctctagtcagtgtgttaatcttacaaccagaactcaattaccccctgcatacactaattctttcacacgtggtgtttattaccctgacaaagttttcagatcctcagttttacattcaactcaggacttgttcttacctttcttttccaatgttacttggttccatgctatacatgtctctgggaccaatggtactaagaggtttgataaccctgtcctaccatttaatgatggtgtttattttgcttccactgagaagtctaacataataagaggctggatttttggtactactttagattcgaagacccagtccctacttattgttaataacgctactaatgttgttattaaagtctgtgaatttcaattttgtaatgatccatttttgggtgtttattaccacaaaaacaacaaaagttggatggaaagtgagttcagagtttattctagtgcgaataattgcacttttgaatatgtctctcagccttttcttatggaccttgaaggaaaacagggtaatttcaaaaatcttagggaatttgtgtttaagaatattgatggttattttaaaatatattctaagcacacgcctattaatttagtgcgtgatctccctcagggtttttcggctttagaaccattggtagatttgccaataggtattaacatcactaggtttcaaactttacttgctttacatagaagttatttgactcctggtgattcttcttcaggttggacagctggtgctgcagcttattatgtgggttatcttcaacctaggacttttctattaaaatataatgaaaatggaaccattacagatgctgtagactgtgcacttgaccctctctcagaaacaaagtgtacgttgaaatccttcactgtagaaaaaggaatctatcaaacttctaactttaga gtccaaccaacagaatctattgttagatttcctaatattacaaacttgtgcccttttggtgaagtttttaacgccaccagatttgcatctgtttatgcttggaacaggaagagaatcagcaactgtgttgctgattattctgtcctatataattccgcatcattttccacttttaagtgttatggagtgtctcctactaaattaaatgatctctgctttactaatgtctatgcagattcatttgtaattagaggtgatgaagtcagacaaatcgctccagggcaaactggaaagattgctgattataattataaattaccagatgattttacaggctgcgttatagcttggaattctaacaatcttgattctaaggttggtggtaattataattacctgtatagattgtttaggaagtctaatctcaaaccttttgagagagatatttcaactgaaatctatcaggccggtagcacaccttgtaatggtgttgaaggttttaattgttactttcctttacaatcatatggtttccaacccactaatggtgttggttaccaaccatacagagtagtagtactttcttttgaacttctacatgcaccagcaactgtttgtggacctaaaaagtctactaatttggttaaaaacaaatgtgtcaatttcaacttcaatggtttaacaggcacaggtgttcttactgagtctaacaaaaagtttctgcctttccaacaatttggcagagacattgctgacactactgatgctgtccgtgatccacagacacttgagattcttgacattacaccatgttcttttggtggtgtcagtgttataacaccaggaacaaatacttctaaccaggttgctgttctttatcaggatgttaactgcacagaagtccctgttgctattcatgcagatcaacttactcctacttggcgtgtttattctacaggttctaatgtttttcaaacacgtgcaggctgtttaataggggctgaacatgtcaacaactcatatgagtgtgacatacccattggtgcaggtatatgcgctagttatcagactcagactaattctcctcggcgggcacgtagtgtagctagtcaatccatcattgcctacactatgtcacttggtgcagaaaattcagttgcttactctaataactctattgccatacccacaaattttactattagtgttaccacagaaattctaccagtgtctatgaccaagacatcagtagattgtacaatgtacatttgtggtgattcaactgaatgcagcaatcttttgttgcaatatggcagtttttgtacacaattaaaccgtgctttaactggaatagctgttgaacaagacaaaaacacccaagaagtttttgcacaagtcaaacaaatttacaaaacaccaccaattaaagattttggtggttttaatttttcacaaatattaccagatccatcaaaaccaagcaagaggtcatttattgaagatctacttttcaacaaagtgacacttgcagatgctggcttcatcaaacaatatggtgattgccttggtgatattgctgctagagacctcatttgtgcacaaaagtttaacggccttactgttttgccacctttgctcacagatgaaatgattgctcaatacacttctgcactgttagcgggtacaatcacttctggttggacctttggtgcaggtgctgcattacaaataccatttgctatgcaaatggcttataggtttaatggtattggagttacacagaatgttctctatgagaaccaaaaattgattgccaaccaatttaatagtgctattggcaaaattcaagactcactttcttccacagcaagtgcacttggaaaacttcaagatgtggtcaaccaaaatgcacaagctttaaacacgcttgttaaacaacttagctccaattttggtgcaatttcaagtg ttttaaatgatatcctttcacgtcttgacaaagttgaggctgaagtgcaaattgataggttgatcacaggcagacttcaaagtttgcagacatatgtgactcaacaattaattagagctgcagaaatcagagcttctgctaatcttgctgctactaaaatgtcagagtgtgtacttggacaatcaaaaagagttgatttttgtggaaagggctatcatcttatgtccttccctcagtcagcacctcatggtgtagtcttcttgcatgtgacttatgtccctgcacaagaaaagaacttcacaactgctcctgccatttgtcatgatggaaaagcacactttcctcgtgaaggtgtctttgtttcaaatggcacacactggtttgtaacacaaaggaatttttatgaaccacaaatcattactacagacaacacatttgtgtctggtaactgtgatgttgtaataggaattgtcaacaacacagtttatgatcctttgcaacctgaattagactcattcaaggaggagttagataaatattttaagaatcatacatcaccagatgttgatttaggtgacatctctggcattaatgcttcagttgtaaacattcaaaaagaaattgaccgcctcaatgaggttgccaagaatttaaatgaatctctcatcgatctccaagaacttggaaagtatgagcagtatataaaatggccatggtacatttggctaggttttatagctggcttgattgccatagtaatggtgacaattatgctttgctgtatgaccagttgctgtagttgtctcaagggctgttgttcttgtggatcctgctgcaaatttgatgaagacgactctgagccagtgctcaaaggagtcaaattacattacacataa，在体外将病毒S蛋白对应的基因序列合成出来，并将其插入质粒载体的多克隆位点中，以构建可过表达S蛋白的核心质粒。在10mm×10mm的培养皿中用10％FBS(胎牛血清)的培养基人胚肾293T细胞，待皿中细胞密度约达50％时，更换10毫升新鲜培养基。取2个无菌、无RNase的EP管，其中一个加入Opti-MEM优化培养基1.5mL，向内加入30μL转染试剂PEI，枪头轻轻吹打混匀后，室温静置5min。另一个EP管中同样加入Opti-MEM优化培养基1.5mL，向内加入核心质粒6μg、psPAX2质粒4.5μg和pMD2.G质粒1.5μg，轻轻吹匀。将有转染试剂的EP管中液体加入有质粒的EP管中，轻轻吹匀，室温静置20min。将上述混合静置后的液体加入到293T细胞中，轻轻摇匀，放入培养箱中培养。24h后更换13mL培养基，继续培养48h后，将细胞上清用0.45μm过滤。在10mm×10mm的培养皿中用10％FBS(胎牛血清)的培养基L929细胞，待皿中细胞密度约50％时，更换过滤所得上清5mL和新鲜培养基5mL，同时加入10μL基因转染增强剂polybrene， 连续感染两天。用含有嘌呤霉素的培养基(1∶1000)进行筛选，得到过表达S蛋白的工具细胞(工具性细胞可为成纤维细胞系、血管内皮细胞系、人胚肾细胞系等正常组织细胞系，以考虑避免肿瘤细胞等细胞系存在的恶性转化风险为出发点)。
2、过表达S蛋白的稳转成纤维细胞验证
将定量的过表达S蛋白的稳转成纤维细胞裂解液与1/4裂解液体积的5×SDS loading buffer混匀，100℃加热10min。按配方制备12％分离胶和5％浓缩胶，在上样孔中加入蛋白样品，边缘孔加等体积的1×SDS上样缓冲液。电泳时，浓缩胶80V恒压，待蛋白marker分开时，调电压至120V恒压。当电泳跑至溴酚蓝于分离胶底部时，结束电泳。把转膜液倒入铁盘中，并放入转膜夹。撬开玻璃板，用切胶板将所需目的蛋白根据marker的位置横切下来，置于黑胶面滤纸上，将用甲醇浸泡过的PVDF膜覆盖于胶上，夹紧夹子。将夹子放入转膜槽中，把铁盘中的转膜液倒入转膜槽里，夹子黑面对槽的黑面，把转膜槽放入装冰的泡沫盒中，选择200mA恒流转膜。2h后，将PVDF膜取出，用5％脱脂牛奶封闭液室温摇床上封闭1h。用1×TBST洗涤3遍，每遍10min，按照一定的稀释比用一抗稀释液配置一抗液，膜的蛋白面与抗体接触，放入4℃冰箱过夜。将膜在摇床上用1×TBST洗涤3遍，每遍10min，再与HRP标记的二抗(用5％脱脂牛奶稀释成1∶5000)室温摇床孵育1h。将膜在摇床上用1×TBST洗涤3遍，每遍10min，配置ECL显影液(ECLA∶ECL B＝1∶1)，将ECL显影液滴在膜上，用曝光仪曝光。如图3所示，稳转成纤维细胞中含有S蛋白。
3、构建并BCA定量可预防新型冠状病毒感染微颗粒以及保存方法
在10mm×10mm的培养皿中用10％FBS(胎牛血清)的培养基培养稳转成纤维细胞，待皿中细胞达到约5×10 6个时，，给予能量为6MV，20Gy的剂量进 行放疗，放疗后的第一天换液，加入20ml含10％FBS的培养基，第3天收集(收集微颗粒的时间一般会选取放疗后的第2-7天，可根据情况选择，本实施例选择第3天)培养皿内所有液体采用梯度离心法提取微颗粒。取放疗后的细胞培养基1000g离心10min后取上清，再将上清14000g，离心2min去除碎片后弃沉淀，最后将上清14000g、4℃离心60min，弃上清，沉淀即是微颗粒，将沉淀再用生理盐水洗两遍，1mlPBS(即磷酸缓冲盐)溶液重悬后4℃保存，取100μl液体离心后，加入适量蛋白裂解液，冰上充分裂解30min后，12000g离心30min，取上清加入BCA定量液进行蛋白定量。
如图4所示，采用不同放疗剂量处理的工具细胞，不难看出，从给与2Gy剂量开始，其释放的疫苗微颗粒总蛋白的含量随放疗剂量递增，到20Gy时微颗粒释放达到高峰，到达30Gy时微颗粒释放变化不大，因此选择2-20Gy是比较理想的选择，最理想为20Gy。
4、可预防新型冠状病毒感染的微颗粒S蛋白含量检测
将定量的微颗粒裂解液与1/4裂解液体积的5×SDS loading buffer混匀，100℃加热10min。按配方制备12％分离胶和5％浓缩胶，在上样孔中加入蛋白样品，边缘孔加等体积的1×SDS上样缓冲液。电泳时，浓缩胶80V恒压，待蛋白marker分开时，调电压至120V恒压。当电泳跑至溴酚蓝于分离胶底部时，结束电泳。把转膜液倒入铁盘中，并放入转膜夹。撬开玻璃板，用切胶板将所需目的蛋白根据marker的位置横切下来，置于黑胶面滤纸上，将用甲醇浸泡过的PVDF膜覆盖于胶上，夹紧夹子。将夹子放入转膜槽中，把铁盘中的转膜液倒入转膜槽里，夹子黑面对槽的黑面，把转膜槽放入装冰的泡沫盒中，选择200mA恒流转膜。2h后，将PVDF膜取出，用5％脱脂牛奶封闭液室温摇床上封闭1h。用1×TBST洗涤3遍，每遍10min，按照一定的稀释比用一抗稀释液配 置一抗液，膜的蛋白面与抗体接触，放入4℃冰箱过夜。将膜在摇床上用1×TBST洗涤3遍，每遍10min，再与HRP标记的二抗(用5％脱脂牛奶稀释成1∶5000)室温摇床孵育1h。将膜在摇床上用1×TBST洗涤3遍，每遍10min，配置ECL显影液(ECLA∶ECL B＝1∶1)，将ECL显影液滴在膜上，用曝光仪曝光。如图5所示，可预防新型冠状病毒感染的微颗粒中含有S蛋白。
5、小鼠皮下预防新型冠状病毒微颗粒接种
在C57小鼠皮下分别注射50μL的PBS(空白组)、普通成纤维细胞20Gy放疗后第3天提取的微颗粒(5mg/kg)(对照组)和过表达S蛋白成纤维细胞20Gy放疗后第3天提取的微颗粒(5mg/kg)(疫苗组)，以1次/周的频率连续注射3周。第一次注射后第15天用ELISA检测小鼠血清中抗S蛋白抗体含量。如图6所示，与空白组和对照组相比，疫苗组小鼠体内产生了抗S蛋白抗体。如图7所示，与含S蛋白的工具细胞裂解液相比，疫苗微颗粒组小鼠产生更多抗S蛋白抗体。如图8所示，疫苗组小鼠体重与另外两组无明显差别，说明可预防新型冠状病毒感染的微颗粒无明显毒副作用。
通过以上步骤制备得到的病毒感染微颗粒，由基因工程改造的工具细胞受放射线处理所产生。其原理是放疗可促进细胞膜以出芽的方式向胞外释放直径为100-1000nm的磷脂双分子层结构的微颗粒，由于其携带了新冠病毒S蛋白，且微颗粒本身存在对免疫细胞靶向功能，具备免疫激活功能，可促进免疫反应的发生。
该新型冠状病毒感染微颗粒，具有显著的免疫激活作用，可促进机体B淋巴细胞产生抗新型冠状病毒抗体以实现预防新型冠状病毒感染的作用。从小鼠皮下预防新型冠状病毒微颗粒接种的情况看出，该新型冠状病毒微颗粒生物安 全性和生物相容性好，可单独使用，也可负载免疫佐剂以进一步激活机体免疫系统增强其预防防新型冠状病毒感染的功能。
本发明并不仅仅限于说明书和实施方式中所描述，因此对于熟悉领域的人员而言可容易地实现另外的优点和改进，故在不背离权利要求及等同范围所限定的一般概念的精神和范围的情况下，本发明并不限于特定的细节、代表性的方案和这里示出与描述的图示示例。

Claims (10)

  1. 一种预防新型冠状病毒微颗粒的制备方法，其特征在于，包括以下步骤：
    S1.构建过表达S蛋白的重组质粒；
    S2.将重组质粒包装成慢病毒颗粒；
    S3.用慢病毒颗粒对工具性细胞进行感染，得到过表达S蛋白的工具性细胞；
    S4.将得到的工具性细胞及培养液进行X线照射，收集放疗后的上清液，获得所需微颗粒和凋亡的工具性细胞碎片混合物；
    S5.将混合物进行离心，收集沉淀即得预防新型冠状病毒微颗粒。
  2. 根据权利要求1所述的预防新型冠状病毒微颗粒的制备方法，其特征在于，所述步骤S1为，基于新型冠状病毒S蛋白质编码区合成人工基因，并插入质粒载体的多克隆位点中，以构建可过表达S蛋白的核心质粒。
  3. 根据权利要求2所述的预防新型冠状病毒微颗粒的制备方法，其特征在于，所述步骤S2中，包装慢病毒颗粒的过程使用psPAX2质粒和pMD2.G质粒。
  4. 根据权利要求1所述的预防新型冠状病毒微颗粒的制备方法，其特征在于，所述步骤S3中工具性细胞包括成纤维细胞系、血管内皮细胞系、人胚肾细胞系中的一种。
  5. 根据权利要求1所述的预防新型冠状病毒微颗粒的制备方法，其特征在于，所述步骤S4中X线放射剂量为2-20Gy。
  6. 根据权利要求1所述的预防新型冠状病毒微颗粒的制备方法，其特征在于，所述步骤S4中X线放射剂量为20Gy，X线能量为6MV，放疗后的第2-7天收集上清液。
  7. 根据权利要求1所述的预防新型冠状病毒微颗粒的制备方法，其特征在于，所述步骤S5中所述离心操作为：1000g，离心10min得第一上清液；所得 第一上清液于14000g，离心2min得第二上清液；所得第二上清液于14000g，60min离心得微颗粒沉淀。
  8. 根据权利要求1所述预防新型冠状病毒微颗粒的制备方法，其特征在于，所述疫苗为微囊泡结构，所述微囊泡结构的粒径为100-1000nm。
  9. 一种新型冠状病毒疫苗，其特征在于，包含权利要求1-8任意一项所述预制备方法获得的微颗粒。
  10. 根据权利要求9所述的新型冠状病毒疫苗，其特征在于，所述疫苗负载有免疫的佐剂。
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