WO2021253147A1 - 一种基于细胞介导的新冠肺炎疫苗的制备方法 - Google Patents

一种基于细胞介导的新冠肺炎疫苗的制备方法 Download PDF

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WO2021253147A1
WO2021253147A1 PCT/CN2020/096030 CN2020096030W WO2021253147A1 WO 2021253147 A1 WO2021253147 A1 WO 2021253147A1 CN 2020096030 W CN2020096030 W CN 2020096030W WO 2021253147 A1 WO2021253147 A1 WO 2021253147A1
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尹秀山
刘军花
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尹秀山
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Definitions

  • the present invention relates to the technical field of cells and genetic engineering, and specifically includes the construction of a new crown-specific vector presented by cells and a new crown pneumonia vaccine mediated by an improved version of stem cells.
  • Coronavirus disease 2019 (COVID-19) has spread all over the world, causing nearly millions of people worldwide to be infected. It has been called a "Pandemic” by the World Health Organization. The main pathogen causing this major epidemic is a new type of coronavirus-2 SARS coronavirus, referred to as "SARS-COV-2".
  • SARS-COV-2 coronavirus-2 SARS coronavirus
  • the typical symptoms of an infected person are headache, fever, cough, body aches and fatigue. People with mild illness generally have uncomfortable symptoms like influenza virus infection, while those with severe illness may cause partial obstruction of lung function or respiratory failure.
  • the virus was a single-stranded positive-stranded RNA (ssRNA+) virus coated with an envelope, which caused severe acute respiratory syndrome (SARS) in 2003 and the Middle East in 2012.
  • SARS severe acute respiratory syndrome
  • MERS Respiratory Syndrome-
  • the virus sequences found in bats have extremely high homology, and both belong to the beta subtype coronavirus.
  • the virus contains 4 main structural proteins, namely Spike (S) and membrane protein.
  • nucleocapsid protein Nucleocapsid, N
  • E Envelope
  • multiple non-structural proteins such as ORF1a/b, ORF3a, etc.
  • the virus needs to use its spike protein to tightly bind to the ACE2 (angiotensin-converting enzyme 2) protein on the surface of the human host cell.
  • ACE2 angiotensin-converting enzyme 2
  • the host cell nucleoplasmic component assists translation and packaging again. And release virus particles, and then adapt to and infect human cells.
  • inactivated virus vaccines refers to the injection of an inactivated virus to stimulate the body to produce an immune response.
  • this inactivated vaccine is easy to produce, it requires a large dose, a short immunization period, a single immunization route, and multiple immunization injections.
  • the adenovirus vector vaccine essentially refers to the modified adenovirus as an expression vector, using genetic engineering methods to insert part of the structural protein sequence of the new coronavirus to produce the key protein of the new coronavirus in the body, thereby stimulating the immune system to achieve the purpose of resisting the virus , Mainly based on S protein.
  • adenoviral vectors may produce immune resistance for some people, thereby limiting or weakening the effectiveness of the target vaccine.
  • the present invention proposes a new idea for developing a new coronavirus vaccine, that is, stem cells are used as the main mediator to induce the immune system to produce corresponding antibodies in vivo and establish long-term immune memory.
  • vaccine research and development platforms related to new pneumonia are mainly based on traditional inactivated or attenuated vaccines, nucleic acid vaccines, viral vector vaccines, and protein vaccines.
  • Many research and development units at home and abroad are conducting research and development on this basis.
  • the body may be repeatedly immunized many times, and some may even cause discomfort to the body. Therefore, a new crown vaccine development route based on a cell delivery platform has not yet been reported.
  • the present invention provides a new cell-based route for the research and development of new coronavirus vaccines.
  • the content mainly includes the following steps: First, the construction process of the new crown-specific S vector, M vector and N vector presented by the cells; second, the preparation process of the modified cells; third, the expression and expression in experimental animals The process of secreting soluble proteins to produce immune memory.
  • a cell-mediated COVID-19 vaccine refers to the use of cells as a new delivery platform to develop a COVID-19 vaccine, enabling stem cells carrying key viral proteins to enter the body to stimulate the body to produce effective immune resistance.
  • the development principle of the stem cell vaccine is similar to that of the nucleic acid vaccine, but the difference is that the stem cell vaccine does not directly use animal or human cells to produce part of the virus antigen protein. In short, it is a relatively safe new vaccine technology .
  • the COVID-19 adenovirus vector vaccine that has entered the second phase of clinical testing and the COVID-19 inactivated virus vaccine developed by Sinopharm Group are both vaccines in the traditional sense.
  • the inactivated virus vaccine refers to the high temperature.
  • This kind of vaccine is relatively simple to produce, but the large vaccination dose, short immune period, and the expansion of the virus are likely to cause a greater risk of re-contamination.
  • Adenovirus vector vaccines in principle, use the modified adenovirus as a mediator to insert nucleic acid sequences expressing the key proteins of SARS-COV-2 virus through genetic engineering methods, and inject them into the body to stimulate the immune system to achieve the purpose of resisting the virus.
  • S protein S protein.
  • the essence of the two is the specific treatment of the virus as the mediation vector.
  • adenoviral vectors may produce immune resistance for some people, thereby limiting or weakening the effectiveness of the target vaccine.
  • the present invention uses stem cells to present and secrete part of the key antigen proteins of the new coronavirus, one of which can accelerate the production of specific antibodies, and can effectively avoid the adverse body immune response after the virus vector vaccine is injected.
  • it can also realize the delivery of antigen proteins of multiple targets, such as S protein, M protein or N protein, so as to produce broad-spectrum and multi-effect antibodies.
  • the combination of stem cell therapy has a good clinical therapeutic significance, especially MSC.
  • the cells after injection will be eliminated by the body's immune system over time, so the stem cell-mediated new crown vaccine is safer in the human body .
  • the one-time immunization success rate of the traditional vaccine method will be greatly reduced.
  • the new crown vaccine that uses stem cells as a mediator will undergo protein glycosylation modification of the stem cell expression system, which can well simulate the process of real virus expression in the body with the help of the host.
  • the one-time immunization success rate Will greatly improve.
  • the cell-mediated new crown vaccine can detect the presence of a large number of antibodies in experimental animals within two weeks, which has a huge advantage over other types of vaccines in terms of timeliness.
  • Figure 1 is the preparation process and related analysis roadmap of a cell-mediated new coronary pneumonia vaccine involved in the present invention, including the preparation of cell-mediated modified vectors, the delivery process of related cells, immune injection and related immune antibody detection Analysis etc.
  • Figure 2 The results of the construction of the new crown-specific S vector, M vector and N vector presented by stem cells.
  • Figure 2A shows the insertion sites and related maps of the new crown-specific S sequence, M sequence and N sequence presented by stem cells;
  • Figure 2B, 2C Respectively, the fragments of S, M, and N genes after specific amplification (carrying specific restriction sites NotI, and Xbal);
  • Figure 2D shows the fragments of pc3.1 scaffold vector after the same NotI and Xbal digestion;
  • Figure 2E shows the amplified fragment of the M gene inserted by homologous recombination (carrying specific restriction sites NotI and Xbal).
  • Figure 3 The best preparation method for transfection and the results of antibody detection in the immune serum of the stem cell N-type vaccine.
  • Figures 3A and 3B show the comparison of the ratio of different plasmids and transfection reagents to the transfection effect.
  • 3A means that cells are transfected with different ratios of plasmids and Lipofectamine®2000 reagent, and the density of cells carrying fluorescence is detected by flow cytometry.
  • the transfection ratios are 3 ⁇ g: 6 ⁇ l; 3 ⁇ g: 9 ⁇ l; 3 ⁇ g: 12 ⁇ l; 3 ⁇ g: 15 ⁇ l,
  • the transfection effects were 40.77%, 50.96%, 61.49%, 62.30%, respectively.
  • the most efficient transfection ratio is 3 ⁇ g: 15 ⁇ l.
  • Figures 3C and 3D show the detection of antibodies in the serum of immunized mice after the first and second injections.
  • N-SI 33 N gene-subcutaneous injection, number 33, male mice
  • N-MI 35 N gene-intramuscular injection, number 35, male mice
  • N-SI 41 N gene-subcutaneous injection, number 41, female mice
  • N-MI 43 N gene-intramuscular injection, number 43, female mice.
  • N-SI 30 N gene-subcutaneous injection, number 30, male mice
  • N-MI 28 N gene-intramuscular injection, number 28, male mice
  • N-SI 5&7 N gene-subcutaneous injection, numbers 5 and 7, female mice
  • N-MI 4&12 N gene-intramuscular injection, number 4 And 12, female mice
  • the specific content is to use part of the SARS-CoV-2 key protein as an epitope, first construct it into the pc3.1 eukaryotic expression vector, and then transfer it into a specific cell system , And then preliminarily formed a stem cell-mediated vaccine, which was injected into mice to detect immune response and antibody production.
  • the experimental reagents and consumables used in this article are: Q5® High-Fidelity DNA Polymerase (NEB #M0491), NotI-HF (NEB #R3189S), Xbal (NEB) #R0145V), Quick ligase (NEB #M2200S), Trelief TM 5a Chemically Competent Cell (TSINGKE TSC01), Endo-Free Plasmid Mini Kit II (OMEGA D6950-02), Lipofectamine 2000 (Invitrogen 11668-019), EasyGeno Recombinant Cloning Reagent Box (Tiangen Biochemical Technology (Beijing) Co., Ltd.
  • VI201 DMEM high glucose medium (Gibco C11995500BT), Opti-MEM I Reduced Serum Medium no phenol red (Gibco 11058021), PBS (Tianjin Haoyang TBD PB2004Y), penicillin/streptomycin solution 100X (Meilunbio MA0110), fetal bovine serum (Gibco), six-well plate and 10cm petri dish (Thermo Fisher SCIENTIFIC), 100um cell strainer (Corning FALCON 352360), 1ml disposable medical syringe (Jiangxi Hongda Medical Devices), Elisa reagent (source from Jilin University Immunology Research Group).
  • the specific culture conditions for the 293T cell line are that the cell line is cultured in DMEM high-sugar medium containing 10% FBS. All culture plates (dishes) containing cells involved in this experiment are placed at 37°C, 5% Cultivation is carried out in a CO2 normoxic incubator.
  • mice were selected as C57BL/6 strain mice (rats aged 6-8 weeks, provided by Liaoning Changsheng Biotechnology Co., Ltd.).
  • the S, M and N nucleic acid sequences were selected and sent to Suzhou Jinweizhi Biotechnology Company for gene synthesis, and the synthesized gene was inserted into the pUC57 vector.
  • the target gene is amplified by PCR (see primer table 2 for specific amplification primer sequences), and the target size band is recovered and purified, and then the pc3.1-Falg-XXX vector and the purified S and N fragments are restricted Endonuclease NotI-HF, Xbal was digested overnight in a 37°C water bath.
  • the target vector and the gene fragment containing the restriction site were recovered by the gel, ligated and transformed into Trelief TM 5a competent, picked a single colony and sequenced to verify the success of the construction.
  • the successfully constructed vectors are named pc3.1-Flag-SARS_COV_2-S and pc3.1-Flag-SARS_COV_2-N. The specific steps are as follows.
  • Q5 high-fidelity enzyme amplification system namely 5X Q5 Reaction Buffer 10ul, 10 mM dNTPs 1 ul, template puC57-S 2ul, 2.5ul each of pcDNA3.1-Flag-S F and R, 0.5ul Q5 enzyme, and add distilled water to 50ul.
  • the N fragment was amplified according to the composition and program settings in Table 3, and the amplified products S and N were recovered by agarose gel electrophoresis.
  • the band sizes were 3822bp and 1260bp, respectively ( The results are shown in Figures 2A, 2B and 2C).
  • Table 1 N amplification components and corresponding PCR programs.
  • Positive screening and obtaining monoclonal strains Take 200ul of the above-mentioned non-resistant culture for ampicillin resistance coating, and incubate overnight at 37°C. The next day, positive monoclonal colonies can be screened and randomly picked. Prepare 6 EP tubes containing ampicillin resistant medium in advance, pick the bacteria gently with a 10ul pipette tip and shake them quickly for 5 hours to perform PCR verification of the bacteria liquid. After the verification is correct, a small extraction of the plasmid can be carried out for sequencing verification. After the sequencing is correct, the transfection-grade plasmid is extracted and purified.
  • the M gene contains two restriction sites at the end of the vector, namely NotI and Xbal, we chose another method of homologous recombination to insert the M gene sequence.
  • the amplification system is 5X Q5 Reaction Buffer 10ul, 10 mM dNTPs 1 ul, template puC57-M 0.5ul, 2.5ul each of pcDNA3.1-Flag-M F and R, 0.5ul Q5 enzyme, and add distilled water to 50ul.
  • Pre-treatment of cell transfection Passage cells to a six-well plate, planting the same cell density in each well and ensuring that the cell density is about 80-90% during transfection.
  • mice of different genders are randomly divided into two groups according to the injection method (intravenous and subcutaneous two methods), and each group is divided into 2-3 parallel groups. Finally, each mouse was named by gene-injection method and male and female. At the same time, random ear tag processing was performed. The specific operation steps are as follows.
  • the Trypsin-EDTA method digests the cells transfected with the eukaryotic expression vector pc3.1-Flag-S, M, and N of the cell-mediated new coronary pneumonia vaccine, centrifuges, and takes the bottom cell aggregates and reconstitutes them with an appropriate amount of PBS solution. Hang. After counting with a hemocytometer, make a cell suspension of 200,000 cells/ ⁇ l, and place it on ice.
  • Immune serum antibody detection adopts the Elisa method for detection, in which the pre-coated board is provided by the immune research group of Jilin University.
  • the specific operation process is as follows.
  • washing liquid Discard the liquid in the hole, add the lotion, 300ul, repeat 5 times (use a one-time inverted button to shake the liquid in the hole to the trash can, and then use a 100ul gun to add washing liquid, which is prepared in advance.
  • the washing solution operation time is between 30-60s, and the plate wall is gently shaken).
  • Termination Add 50ul of termination solution to each well, mix well, and detect the OD value or scan the color contrast image at the 450nm wavelength of the microplate reader within 15 minutes.
  • mice The sera of immunized mice were analyzed by the Elisa detection method. The results are shown in Figure 3C and Figure 3D: After testing the mouse serum two weeks later and the mouse serum four weeks later, almost all immunized mice can effectively Antibodies were detected. Among them, about 50% of mice showed strong positive expression of antibodies, especially the N-gene modified cell vaccines. Since the data statistics table in FIG. 3E is only an illustration of the results after one injection, the cellular neo-coronary pneumonia vaccine prepared by the present invention has better immune efficacy.

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Abstract

提供了一种可基于细胞介导的新冠肺炎疫苗及其制备方法，其步骤包含：由干细胞呈递的新冠特异抗原载体的构建，以及与干细胞修饰和组装。通过免疫小鼠两周后，约有50％小鼠体内抗体呈现强阳性表达，尤以N基因修饰的干细胞疫苗最为显著。

Description

一种基于细胞介导的新冠肺炎疫苗的制备方法 技术领域
本发明涉及细胞以及基因工程技术领域，具体包括有由细胞呈递的新冠特异性载体构建，以及一种基于经改良版干细胞介导的新冠肺炎疫苗。
 背景技术
2019新冠肺炎疫情（Coronavirus disease 2019，COVID-19）现已遍及世界各地，导致了全球近数百万人感染。现已被世界卫生组织成为“大流行（Pandemic）”。引起此次重大疫情的主要病原体为一种新型冠状病毒-2型SARS冠状病毒，简称“SARS-COV-2”。感染者的典型症状表现为头痛，发热，咳嗽，身体酸痛乏力等。轻症者普遍出现似流感病毒感染的不适症状，而重症者会使肺部功能局部受阻，或呼吸衰竭等。通过从新冠肺炎病人体内分离得到的病毒进行核酸测序，发现该病毒为由包膜包被的单股正链 RNA （ssRNA+）病毒，与引起2003年严重急性呼吸综合征（SARS）、2012年中东呼吸综合征-（MERS）以及蝙蝠体内所发现的病毒序列具有极高的同源性，均属beta亚型冠状病毒。随着该病毒的核酸、蛋白结构特征的不断地揭示，该病毒包含有4个主要的结构蛋白，即纤突蛋白 (Spike，S)、膜蛋白 (Membrane，M)、核衣壳蛋白 (Nucleocapsid，N)及包膜蛋白(Envelope，E) 和多个非结构蛋白，如ORF1a/b，ORF3a等。在感染与免疫过程中，该病毒需借助自身的spike蛋白与人体宿主细胞表面的ACE2（angiotensin-converting enzyme 2）蛋白紧密结合，一旦病毒RNA释放进入细胞，宿主细胞核质组分辅助再次翻译、包装并释放病毒颗粒，进而适应并感染人类细胞。但关于该病毒的最原始的溯源问题、跨物种传播以及中间宿主媒介等仍未有明确的研究结果。
面对突如其来的疫情，除了前期的核酸检测（RT-PCR、RT-LAMP等）、抗体检测（如胶体金法，酶联免疫法），影像学监测以及新一代检测手段，如张锋团队的CRISPR-SHERLOCK技术等诊断工作以外，针对新冠病毒相关的疫苗研发也是疫情防控的核心工作。截至目前为止，世界上暂无针对性治疗该病毒的特效药物，而且相关的疫苗研发也是医学上现存在的棘手问题。
目前，世界上关于新冠病毒的疫苗研发的方向有多种，包括灭活病毒疫苗，核酸疫苗，病毒载体疫苗，蛋白疫苗等。在国内，现有报道的腺病毒载体疫苗进入临床二期测试阶段，以及国药集团现研发的灭活疫苗。而这两种疫苗均利用传统的疫苗研发手段来获得的相应疫苗，其灭活病毒疫苗是指注射一种经灭活的病毒，刺激机体产生免疫反应。这种灭活疫苗虽生产简便，但需接种剂量大、免疫期较短、免疫途径单一，且需多次进行免疫注射。其次，腺病毒载体疫苗，实质上是指经改良后的腺病毒为表达载体利用基因工程手段插入新冠病毒的部分结构蛋白序列在体内产生新冠病毒的关键蛋白，进而刺激免疫系统达到抵抗病毒的目的，主要是以S蛋白为主。同时腺病毒载体对于一些人来说可能会产生免疫抗性，进而限制或削弱目的疫苗的有效性。本文中，结合干细胞在治疗方面的优越性，尤以MSC为主。基于以上观点，本发明提出一种新的研发新冠疫苗的思路，即以干细胞为主要介导载体，进行体内诱导免疫系统产生相应抗体及建立长期的免疫记忆。
 技术问题
目前对于新型肺炎相关的疫苗研发平台主要以传统的灭活或减活疫苗，核酸类疫苗，病毒载体疫苗以及蛋白类疫苗等，国内外的不少研发单位均在以此为基点研发进行中。而对于某些新冠疫苗可能会多次反复免疫机体，甚至有些会引起机体的不适症状等，那么，一种以细胞递送平台为主的新冠疫苗研发路线目前还未见相关报道。
 技术解决方案
本发明提供了一种新的以细胞为主的用于新冠病毒疫苗研发路线。其内容主要包括以下几个步骤：其一，由细胞呈递的新冠特异性S载体、M载体以及N载体的构建过程；其二，被修饰的细胞的制备过程；其三，实验动物体内表达和分泌可溶性蛋白产生免疫记忆的过程。
 有益效果
“一种由细胞介导的新冠疫苗”指的是以细胞作为一种新的递送平台来研发新冠疫苗，实现携带病毒关键蛋白的干细胞进入体内刺激机体产生有效的免疫抵抗效力。从原理上讲，该干细胞疫苗的研发原理与核酸疫苗较为相似，但不同的是干细胞疫苗并非直接利用动物或人体细胞产生部分病毒抗原蛋白，总言之，它是一种较为安全的新型疫苗技术。
现阶段进入临床二期测试的COVID-19腺病毒载体疫苗和国药集团研发的COVID-19灭活病毒疫苗均为传统意义上的疫苗，从原理上来说，其灭活病毒疫苗是指已经过高温或化学灭活的病毒，来刺激机体产生免疫反应。这种疫苗在生产上较为简便，但接种剂量大、免疫期较短，以及病毒的扩大化问题，易引起再次污染的风险较大。腺病毒载体疫苗，在原理上，利用经改良后的腺病毒为介导载体通过基因工程手段插入可表达SARS-COV-2病毒关键蛋白的核酸序列，注入体内以刺激免疫系统达到抵抗病毒的目的，现主要是以S蛋白为主。二者的实质是经特定处理的病毒作为介导载体。同时腺病毒载体可能对于某些人来说会产生免疫抗性，进而限制或削弱目的疫苗的有效性。
本发明与现有技术相比，借助干细胞呈递并分泌新冠病毒的部分关键抗原蛋白，其一可以加速特异性抗体的产生，可有效避免病毒载体疫苗注射后引发不良的机体免疫反应。同时也可实现多个靶点的抗原蛋白的传递，比如S蛋白，M蛋白或N蛋白等，从而产生广谱多效的抗体。
其二，结合干细胞治疗的良好的临床治疗意义，尤以MSC为主，同时随着时间，注射后的细胞会被不断机体免疫系统消除掉，那么以干细胞为介导新冠疫苗在人体内较为安全。
其三，现已有研究表明新冠病毒表面蛋白的大部分是经过糖基化修饰作用的，占比约有50%以上，那么，一般情况下传统疫苗方式的一次性免疫成功率会大大降低，而以干细胞为介导载体的新冠疫苗会经过干细胞表达系统的蛋白糖基化修饰作用，可很好地模拟真实病毒在体内的借助宿主表达的过程，较传统疫苗相比，一次性免疫成功率会大大提高。
其四，由细胞介导的新冠疫苗在两周的时间内就可以在实验动物体内检测到大量抗体的存在，较其他类型疫苗在时效上存在巨大的优势。
 附图说明
图1为本发明中涉及的一种基于细胞介导的新冠肺炎疫苗的制备流程以及相关分析路线图，包括细胞介导修饰载体的制备，有关细胞的递送过程，免疫注射以及相关的免疫抗体检测分析等。
图2干细胞呈递的新冠特异性S载体、M载体以及N载体的构建结果图，图2A 为干细胞呈递的新冠特异性S序列、M序列以及N序列的插入位点以及相关图谱；图2B，2C分别为S，M和N基因经特异性扩增后所得片段（携带有特异酶切位点NotI,和Xbal）；图2D为pc3.1支架载体经同样的NotI和Xbal酶切后所得片段；图2E为以同源重组方式插入的M基因的扩增片段（携带有特异酶切位点NotI和Xbal）。
图3 最佳转染制备方式及干细胞N型疫苗的免疫血清中的抗体检测结果图，图3A 和3B为不同的质粒与转染试剂比例条件对比后转染效果。3A为细胞转染有不同比例的质粒与Lipofectamine®2000试剂，并通过流式细胞仪检测携带荧光的细胞密度，转染比例分别为3µg：6µl；3µg：9µl；3µg：12µl；3µg：15µl，转染效果为分别为40.77%、50.96%、61.49%、62.30%。其中，效率最高的转染比例为3µg：15µl。
图3C和3D为第一次和第二次注射后免疫小鼠血清中的抗体检测，其中，第一批次中，N-SI 33（N基因-皮下注射，编号33，雄性小鼠）；N-MI 35（N基因-肌肉注射，编号35，雄性小鼠）；N-SI 41（N基因-皮下注射，编号41，雌性小鼠）；N-MI 43（N基因-肌肉注射，编号43，雌性小鼠）。第二批次中，N-SI 30（N基因-皮下注射，编号30，雄性小鼠）、N-MI 28（N基因-肌肉注射，编号28，雄性小鼠）、N-SI 5&7（N基因-皮下注射，编号5和7，雌性小鼠）、N-MI 4&12（N基因-肌肉注射，编号4和12，雌性小鼠），实验结果表明，表明该细胞介导的新冠疫苗可以诱导小鼠产生抗体，且在免疫小鼠血清检测中以肌肉注射的小鼠呈现的抗体阳性率最高。
 本发明的最佳实施方式
本说明中的由细胞呈递的新冠特异性S、M和N载体准确构建及鉴定均为最优的步骤，同时基于细胞介导的新冠肺炎疫苗的制备过程相关的细胞转染条件和注射用量均为最优条件。具体的实施步骤参见本发明的实施方式。
 本发明的实施方式
1.下面结合具体实施方案对本文进行进一步阐述，具体内容为利用SARS-CoV-2部分关键蛋白作为抗原表位，先构建到pc3.1真核表达载体中，再转入特定的细胞体系中，进而初步形成由干细胞介导的疫苗，经注射到小鼠体内来检测免疫反应及抗体产生情况。
2.本文中所使用的实验试剂和耗材为：Q5® High-Fidelity DNA Polymerase (NEB #M0491)，NotI-HF(NEB #R3189S)，Xbal(NEB #R0145V)，Quick ligase(NEB #M2200S)，Trelief TM 5a Chemically Competent Cell(TSINGKE TSC01)，Endo-Free Plasmid Mini Kit II (OMEGA D6950-02)，Lipofectamine 2000 (Invitrogen 11668-019)， EasyGeno 重组克隆试剂盒（天根生化科技(北京)有限公司 VI201）， DMEM高糖培养基 (Gibco C11995500BT)，Opti-MEM I Reduced Serum Medium no phenol red (Gibco 11058021)，PBS( 天津灏洋TBD PB2004Y)，青霉素/链霉素溶液100X（Meilunbio MA0110），胎牛血清（Gibco），六孔板以及10cm培养皿（Thermo Fisher SCIENTIFIC），100um cell strainer（Corning FALCON 352360），1ml一次性医用注射器（江西洪达医疗器械），Elisa试剂（来源吉林大学免疫课题组）。
3.  细胞系和小鼠品系。
293T细胞系 具体的培养条件为该细胞系在含10% FBS的DMEM高糖培养基中培养，本实验中涉及的所有含细胞的培养板（皿）置于含37℃，5% CO2的常氧培养箱中进行培养。
实验小鼠选择为C57BL/6品系小鼠（鼠龄为6-8周，由辽宁长生生物技术故分有限公司提供）。
4.  实施项 1   由细胞呈递的新冠特异性 S 、 N 载体构建及鉴定。
参考NCBI中所注释的SARS-CoV-2病毒基因组序列，选取S、M和N核酸序列送至苏州金唯智生物技术公司进行基因合成，合成的基因插入在pUC57载体上。首先，通过PCR扩增目的基因(具体扩增引物序列参见引物表格2)，并基回收提纯目的大小条带，然后将pc3.1-Falg-XXX载体和提纯后的S、N片段用限制性核酸内切酶NotI-HF、 Xbal于37℃水浴锅中进行过夜酶切。第二天，胶回收目的载体和含有酶切位点的基因片段，连接转化至Trelief TM 5a 感受态中，挑单克隆菌落并进行测序验证构建成功情况。最后，将构建成功的载体命名为pc3.1-Flag-SARS_COV_2-S和pc3.1-Flag-SARS_COV_2-N，具体步骤如下。
（1）扩增目的片段S和N：以puC57-S为扩增模板，加入Q5高保真酶扩增体系，即5X Q5 Reaction Buffer 10ul，10 mM dNTPs 1 ul，模板puC57-S 2ul，pcDNA3.1-Flag-S F和R各2.5ul，Q5酶0.5ul，并补蒸馏水至50ul。在PCR仪中98℃--30s预变性；98 ℃--10s，56 ℃--30s, 72 ℃--2min循环35次；72 ℃最后延伸5min，4℃保存。同理，以puC57-N为扩增模板，按照表3的成分及程序设置进行扩增N片段，并进行琼脂糖凝胶电泳回收扩增产物S和N，条带大小分别为3822bp和1260bp（结果见图2A，2B和2C）。
表1：N扩增成分及相应PCR程序。
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。
（2）目的产物和待插入的骨架载体pc3.1-flag-xxx酶切处理：将回收的S和N片段中分别加入0.2ul的NotI-HF、Xbal以及相应的2ul 10x Cutsmart Buffer溶液形成完整的20ul酶切体系，37℃过夜孵育，同时，作为骨架的pc3.1载体也进行同样位点的酶切并电泳回收大片段，条带大小为5400bp（图2D）。
（3）待酶切完毕后，利用Omega的DNA gel recovery kit 进行回收，并进一步利用Narodrop2000测定所得3个产物浓度。
（4）连接转化 取S片段6ul和线性化骨架载体pc3.1-flag-xxx 3ul，加入quick ligase 1ul及相应2X Ligation buffer 10ul，室温连接15min。然后取50 ul Trelief TM 5a 感受态细胞冰上解冻，加入5ul连接产物冰上继续静置30min。接着42℃热激90s，再次冰上孵育5min。最后，加入无抗性LB培养液600ul于250-300rpm摇床中摇菌60min。
（5）阳性筛选并获取单克隆株  取200ul上述无抗性培养物进行氨苄抗性涂板操作，过夜37℃孵育，第二天，可筛选并随机挑取阳性单克隆菌落。事先准备含有氨苄抗性培养基的EP管6个，用10ul枪头轻轻挑取菌落后快速摇5hr，进行菌液PCR验证。验证无误后，可进行小提质粒，进行测序验证。待测序正确后进行转染级别的质粒中提纯化。
表2. 有关细胞疫苗系列的载体构建过程中所涉及的引物序列。
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。
实施项 2 基于由细胞呈递的新冠特异性 M 载体的构建及鉴定。
 由于M基因内部含有载体末端的两个酶切位点，即NotI和Xbal，故我们选用另一种同源重组的方式插入M基因序列。首先，利用合成的M基因序列（见附件2）为模板，仍然利用Q5高保真酶PCR体系进行扩增，扩增体系为5X Q5 Reaction Buffer 10ul，10 mM dNTPs 1 ul，模板puC57-M 0.5ul，pcDNA3.1-Flag-M F和R各2.5ul，Q5酶0.5ul，并补蒸馏水至50ul。在PCR仪中98℃--30s预变性；98 ℃--10s，60 ℃--30s, 72 ℃--2min循环35次；72 ℃最后延伸5min，4℃保存。电泳并胶回收目的片段大小为666bp(如图2E)，无需进行酶切处理，直接将M特异性扩增产物与实施项1提及的线性化骨架载体pc3.1-flag-xxx借助EasyGeno 重组克隆试剂盒进行连接，后续的常规转化和阳性菌筛选处理同实施项1所述一致。成功获取接入M基因的克隆菌株，其携带的载体命名为pcDNA3.1-flag-SARS_COV_2_M(INfusion)。
实施项 3 细胞系转染效率的优化实验。
细胞转染前处理：传代细胞至六孔板中，每孔种植细胞密度一致且保证转染时细胞密度在80-90%左右。
转染操作：（1） 准备一个新的EP管将14ug质粒溶解至700ul无血清的培养基中（折合浓度c=20ng/ul）。
（2）再准备4个新的EP管，参考 Lipofectamine 2000试剂盒推荐的四种浓度（6，9，12，15ul）以确定新的细胞系最佳用量比。将6，9，12，15ul的Lipofectamine ® Reagent稀释至分别装有150ul 无血清的培养基(DMEM ® Medium)的EP管中。
（3）转染期间，将稀释后的质粒溶液轻轻地打入到含有Lipofectamine 2000的EP管中，轻轻振荡混合，即150ul（含3ug）+150ul，室温孵育5min-10min。
（4）取出待转染的六孔板，进行换液处理，加入1.5ml新鲜的培养基。然后将已孵育的质粒-lipo2000 混合液300ul加入六孔板的一个孔里（即3.0 ug/per well），摇匀后，移回37℃培养箱孵育。
（5）转染后48hr采用流式法检测转染效率。
最佳转染比例确定实验结果如附图3A 所示，结果表明，针对人胚胎肾细胞(293T细胞)而言，当质粒与Lipofectamine 2000比例为3ug：15ul时，转染效率最佳，效率指标可高达60-70%左右。因此，后续的转染步骤均以最佳比例为基础进行。
实施项 4 由细胞介导的新冠肺炎疫苗的初步制备。
准备4个EP管，里面加入150ul 无血清的培养基，分别加入3ug pcDNA3.1-flag-2019_nCOV_S，pcDNA3.1-flag-2019_nCOV_N，pcDNA3.1-flag-SARS_COV_2_M(INfusion)，以及三者混合体（1：1：1），混匀。
 再准备4个新的EP管，标记同前4个管子一致，里面均加入15ul Lipofectamine 2000试剂，轻微混匀后，将含有质粒的溶液对应地转入含Lipofectamine 2000的EP管中。静置10min。
 取出待转染的六孔板，换入1.5ml新鲜的培养基。然后将已孵育的质粒-lipo2000 混合液300ul分散式滴加到六孔板的一个孔里，摇匀后，移回37℃培养箱孵育。48hr后进行收集细胞。
实施项 5 免疫注射实验动物步骤。
本文中的动物注射实验采用黑色C57BL/6 系小鼠，将不同性别小鼠按照注射方式（静脉和皮下两种方式）随机分为两组，每组又分为2-3个平行小组。最终，每只小鼠均以基因-注射方式以及雌雄来命名，同时也进行随机挂耳标号处理，具体操作步骤如下。
（1）采用0.25% Trypsin-EDTA的方法将转染有由细胞介导的新冠肺炎疫苗的真核表达载体pc3.1-Flag-S、M和N的细胞进行消化，离心，取底部细胞聚集物用适量PBS溶液重悬。采用血球计数板计数后，制成20万个细胞/微升的细胞悬液，冰上静置。
（2）取鼠龄为6-8周C57BL/6小鼠，采用肌肉和皮下注射两种方式进行注射。选取小鼠的右后侧大腿部位，用75%酒精消毒后，用1ml注射器吸取50ul（含100万干细胞）已修饰的干细胞悬液；而皮下注射选择侧腹部或头颈部注射同样数量的已修饰的干细胞悬液。最终登记方式：性别-基因名称-注射方式-标号。
实施项 6 由细胞介导的疫苗注射后的免疫动物血清中抗体检测。
免疫血清抗体检测采用Elisa方法进行检测，其中预包被板子由吉林大学的免疫课题组提供，具体操作流程如下。
（1）加样：将预包被板子固定于板架上，设置阴性对照2-3孔，空白对照2孔，实验样本组首先在每孔中加入样本稀释液100ul（空白除外），再加入待测样本血清5ul，震荡均匀。37℃恒温培养箱中孵育60 min左右。
（2）洗涤：弃去孔内液体，加入洗液，300ul，重复5次（采用一次性倒扣甩孔内液体至垃圾桶中，再利用100ul排枪加洗涤液，其中洗涤液事先配好放在一个类似pcr管架的盒子中。洗液操作时间30-60s之间，轻轻振荡包被板壁）。
（3）加二抗：每孔加入二抗（含辣根过氧化物的羊抗鼠单克隆抗体）100ul（1:10000稀释），置于37℃恒温培养箱中孵育30 min左右。
（4）洗涤：弃去孔内液体，加入洗液，300ul，重复5次。
（5）显色：每孔加入底物缓冲液50ul,再加入底物液50ul 混匀，37℃烘箱孵育15min。
（6）终止：每孔加入终止液50ul，混匀，15min内，在酶标仪的450nm波长下检测OD值或进行颜色对比图像扫描。
通过Elisa检测方法对免疫小鼠的血清进行分析，结果如附图3C和图3D所示：经过检测两周后的小鼠血清和四周后的小鼠血清，几乎所有免疫小鼠均能有效地检测到抗体，其中，约有50%小鼠体内抗体呈现强阳性表达，尤以N基因修饰的细胞疫苗最为显著。由于图3E的的数据统计表仅为一次注射后的结果说明，因此，本发明所制备的细胞新冠肺炎疫苗具有较好的免疫效力。
此外，以上阐述仅为本发明的优选实施项，对于本技术领域的科研人员，凡是以此实验所述的构思框架为基而进行的改进或补充，包括针对其他病毒，细菌及病原体的细胞疫苗，不局限于干细胞，也应视为本发明的保护范围。
 序列表自由内容
由细胞呈递的新冠特异性 S 载体、 M 载体以及 N 载体的完整图谱。
>pc3.1-Flag-SARS_COV_2-N。
gacggatcgggagatctcccgatcccctatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatctgctccctgcttgtgtgttggaggtcgctgagtagtgcgcgagcaaaatttaagctacaacaaggcaaggcttgaccgacaattgcatgaagaatctgcttagggttaggcgttttgcgctgcttcgcgatgtacgggccagatatacgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaactagagaacccactgcttactggcttatcgaaattaatacgactcactatagggagacccagctagcgtttaaacttaagcttggtaccgccatggactacaaggacgacgacgacaaggggcggccgccctctgataatggaccccaaaatcagcgaaatgcaccccgcattacgtttggtggaccctcagattcaactggcagtaaccagaatggagaacgcagtggggcgcgatcaaaacaacgtcggccccaaggtttacccaataatactgcgtcttggttcaccgctctcactcaacatggcaaggaagaccttaaattccctcgaggacaaggcgttccaattaacaccaatagcagtccagatgaccaaattggctactaccgaagagctaccagacgaattcgtggtggtgacggtaaaatgaaagatctcagtccaagatggtatttctactacctaggaactgggccagaagctggacttccctatggtgctaacaaagacggcatcatatgggttgcaactgagggagccttgaatacaccaaaagatcacattggcacccgcaatcctgctaacaatgctgcaatcgtgctacaacttcctcaaggaacaacattgccaaaaggcttctacgcagaagggagcagaggcggcagtcaagcctcttctcgttcctcatcacgtagtcgcaacagttcaagaaattcaactccaggcagcagtaggggaacttctcctgctagaatggctggcaatggcggtgatgctgctcttgctttgctgctgcttgacagattgaaccagcttgagagcaaaatgtctggtaaaggccaacaacaacaaggccaaactgtcactaagaaatctgctgctgaggcttctaagaagcctcggcaaaaacgtactgccactaaagcatacaatgtaacacaagctttcggcagacgtggtccagaacaaacccaaggaaattttggggaccaggaactaatcagacaaggaactgattacaaacattggccgcaaattgcacaatttgcccccagcgcttcagcgttcttcggaatgtcgcgcattggcatggaagtcacaccttcgggaacgtggttgacctacacaggtgccatcaaattggatgacaaagatccaaatttcaaagatcaagtcattttgctgaataagcatattgacgcatacaaaacattcccaccaacagagcctaaaaaggacaaaaagaagaaggctgatgaaactcaagccttaccgcagagacagaagaaacagcaaactgtgactcttcttcctgctgcagatttggatgatttctccaaacaattgcaacaatccatgagcagtgctgactcaactcaggcctaatctagagggcccgtttaaacccgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggcttctgaggcggaaagaaccagctggggctctagggggtatccccacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattaattctgtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctctgcctctgagctattccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaaaagctcccgggagcttgtatatccattttcggatctgatcaagagacaggatgaggatcgtttcgcatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgagcgggactctggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatttcgattccaccgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgccggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccaccccaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctgtataccgtcgacctctagctagagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtc。
>pc3.1-Flag-SARS_COV_2-S。
gacggatcgggagatctcccgatcccctatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatctgctccctgcttgtgtgttggaggtcgctgagtagtgcgcgagcaaaatttaagctacaacaaggcaaggcttgaccgacaattgcatgaagaatctgcttagggttaggcgttttgcgctgcttcgcgatgtacgggccagatatacgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaactagagaacccactgcttactggcttatcgaaattaatacgactcactatagggagacccagctagcgtttaaacttaagcttggtaccgccatggactacaaggacgacgacgacaaggggcggccgccctttgtttttcttgttttattgccactagtctctagtcagtgtgttaatcttacaaccagaactcaattaccccctgcatacactaattctttcacacgtggtgtttattaccctgacaaagttttcagatcctcagttttacattcaactcaggacttgttcttacctttcttttccaatgttacttggttccatgctatacatgtctctgggaccaatggtactaagaggtttgataaccctgtcctaccatttaatgatggtgtttattttgcttccactgagaagtctaacataataagaggctggatttttggtactactttagattcgaagacccagtccctacttattgttaataacgctactaatgttgttattaaagtctgtgaatttcaattttgtaatgatccatttttgggtgtttattaccacaaaaacaacaaaagttggatggaaagtgagttcagagtttattctagtgcgaataattgcacttttgaatatgtctctcagccttttcttatggaccttgaaggaaaacagggtaatttcaaaaatcttagggaatttgtgtttaagaatattgatggttattttaaaatatattctaagcacacgcctattaatttagtgcgtgatctccctcagggtttttcggctttagaaccattggtagatttgccaataggtattaacatcactaggtttcaaactttacttgctttacatagaagttatttgactcctggtgattcttcttcaggttggacagctggtgctgcagcttattatgtgggttatcttcaacctaggacttttctattaaaatataatgaaaatggaaccattacagatgctgtagactgtgcacttgaccctctctcagaaacaaagtgtacgttgaaatccttcactgtagaaaaaggaatctatcaaacttctaactttagagtccaaccaacagaatctattgttagatttcctaatattacaaacttgtgcccttttggtgaagtttttaacgccaccagatttgcatctgtttatgcttggaacaggaagagaatcagcaactgtgttgctgattattctgtcctatataattccgcatcattttccacttttaagtgttatggagtgtctcctactaaattaaatgatctctgctttactaatgtctatgcagattcatttgtaattagaggtgatgaagtcagacaaatcgctccagggcaaactggaaagattgctgattataattataaattaccagatgattttacaggctgcgttatagcttggaattctaacaatcttgattctaaggttggtggtaattataattacctgtatagattgtttaggaagtctaatctcaaaccttttgagagagatatttcaactgaaatctatcaggccggtagcacaccttgtaatggtgttgaaggttttaattgttactttcctttacaatcatatggtttccaacccactaatggtgttggttaccaaccatacagagtagtagtactttcttttgaacttctacatgcaccagcaactgtttgtggacctaaaaagtctactaatttggttaaaaacaaatgtgtcaatttcaacttcaatggtttaacaggcacaggtgttcttactgagtctaacaaaaagtttctgcctttccaacaatttggcagagacattgctgacactactgatgctgtccgtgatccacagacacttgagattcttgacattacaccatgttcttttggtggtgtcagtgttataacaccaggaacaaatacttctaaccaggttgctgttctttatcaggatgttaactgcacagaagtccctgttgctattcatgcagatcaacttactcctacttggcgtgtttattctacaggttctaatgtttttcaaacacgtgcaggctgtttaataggggctgaacatgtcaacaactcatatgagtgtgacatacccattggtgcaggtatatgcgctagttatcagactcagactaattctcctcggcgggcacgtagtgtagctagtcaatccatcattgcctacactatgtcacttggtgcagaaaattcagttgcttactctaataactctattgccatacccacaaattttactattagtgttaccacagaaattctaccagtgtctatgaccaagacatcagtagattgtacaatgtacatttgtggtgattcaactgaatgcagcaatcttttgttgcaatatggcagtttttgtacacaattaaaccgtgctttaactggaatagctgttgaacaagacaaaaacacccaagaagtttttgcacaagtcaaacaaatttacaaaacaccaccaattaaagattttggtggttttaatttttcacaaatattaccagatccatcaaaaccaagcaagaggtcatttattgaagatctacttttcaacaaagtgacacttgcagatgctggcttcatcaaacaatatggtgattgccttggtgatattgctgctagagacctcatttgtgcacaaaagtttaacggccttactgttttgccacctttgctcacagatgaaatgattgctcaatacacttctgcactgttagcgggtacaatcacttctggttggacctttggtgcaggtgctgcattacaaataccatttgctatgcaaatggcttataggtttaatggtattggagttacacagaatgttctctatgagaaccaaaaattgattgccaaccaatttaatagtgctattggcaaaattcaagactcactttcttccacagcaagtgcacttggaaaacttcaagatgtggtcaaccaaaatgcacaagctttaaacacgcttgttaaacaacttagctccaattttggtgcaatttcaagtgttttaaatgatatcctttcacgtcttgacaaagttgaggctgaagtgcaaattgataggttgatcacaggcagacttcaaagtttgcagacatatgtgactcaacaattaattagagctgcagaaatcagagcttctgctaatcttgctgctactaaaatgtcagagtgtgtacttggacaatcaaaaagagttgatttttgtggaaagggctatcatcttatgtccttccctcagtcagcacctcatggtgtagtcttcttgcatgtgacttatgtccctgcacaagaaaagaacttcacaactgctcctgccatttgtcatgatggaaaagcacactttcctcgtgaaggtgtctttgtttcaaatggcacacactggtttgtaacacaaaggaatttttatgaaccacaaatcattactacagacaacacatttgtgtctggtaactgtgatgttgtaataggaattgtcaacaacacagtttatgatcctttgcaacctgaattagactcattcaaggaggagttagataaatattttaagaatcatacatcaccagatgttgatttaggtgacatctctggcattaatgcttcagttgtaaacattcaaaaagaaattgaccgcctcaatgaggttgccaagaatttaaatgaatctctcatcgatctccaagaacttggaaagtatgagcagtatataaaatggccatggtacatttggctaggttttatagctggcttgattgccatagtaatggtgacaattatgctttgctgtatgaccagttgctgtagttgtctcaagggctgttgttcttgtggatcctgctgcaaatttgatgaagacgactctgagccagtgctcaaaggagtcaaattacattacacataagctctagagggcccgtttaaacccgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggcttctgaggcggaaagaaccagctggggctctagggggtatccccacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattaattctgtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctctgcctctgagctattccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaaaagctcccgggagcttgtatatccattttcggatctgatcaagagacaggatgaggatcgtttcgcatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgagcgggactctggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatttcgattccaccgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgccggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccaccccaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctgtataccgtcgacctctagctagagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtc。
>pcDNA3.1-Flag-SARS_COV_2_M(INfusion)。
gacggatcgggagatctcccgatcccctatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatctgctccctgcttgtgtgttggaggtcgctgagtagtgcgcgagcaaaatttaagctacaacaaggcaaggcttgaccgacaattgcatgaagaatctgcttagggttaggcgttttgcgctgcttcgcgatgtacgggccagatatacgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaactagagaacccactgcttactggcttatcgaaattaatacgactcactatagggagacccagctagcgtttaaacttaagcttggtaccgccatggactacaaggacgacgacgacaaggggcggccgcaagcagattccaacggtactattaccgttgaagagcttaaaaagctccttgaacaatggaacctagtaataggtttcctattccttacatggatttgtcttctacaatttgcctatgccaacaggaataggtttttgtatataattaagttaattttcctctggctgttatggccagtaactttagcttgttttgtgcttgctgctgtttacagaataaattggatcaccggtggaattgctatcgcaatggcttgtcttgtaggcttgatgtggctcagctacttcattgcttctttcagactgtttgcgcgtacgcgttccatgtggtcattcaatccagaaactaacattcttctcaacgtgccactccatggcactattctgaccagaccgcttctagaaagtgaactcgtaatcggagctgtgatccttcgtggacatcttcgtattgctggacaccatctaggacgctgtgacatcaaggacctgcctaaagaaatcactgttgctacatcacgaacgctttcttattacaaattgggagcttcgcagcgtgtagcaggtgactcaggttttgctgcatacagtcgctacaggattggcaactataaattaaacacagaccattccagtagcagtgacaatattgctttgcttgtacagtaatctagagggcccgtttaaacccgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggcttctgaggcggaaagaaccagctggggctctagggggtatccccacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattaattctgtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctctgcctctgagctattccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaaaagctcccgggagcttgtatatccattttcggatctgatcaagagacaggatgaggatcgtttcgcatgattgaacaagatggattgcacgcaggttctccggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcaggggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactgcaggacgaggcagcgcggctatcgtggctggccacgacgggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtcatctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctcgcgccagccgaactgttcgccaggctcaaggcgcgcatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaatatcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtgatattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgccttcttgacgagttcttctgagcgggactctggggttcgaaatgaccgaccaagcgacgcccaacctgccatcacgagatttcgattccaccgccgccttctatgaaaggttgggcttcggaatcgttttccgggacgccggctggatgatcctccagcgcggggatctcatgctggagttcttcgcccaccccaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctgtataccgtcgacctctagctagagcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtc。

Claims (4)

  1. 一种基于细胞介导的新冠肺炎疫苗，其特征在于：它包含被用于修饰干细胞呈递的新冠特异抗原载体的构建，以及干细胞修饰和组装；所述的新冠特异抗原载体为常用的真核瞬时表达的pc3.1载体；所述的干细胞修饰为将表达新冠的抗原蛋白的载体转染至干细胞中，这里的干细胞包括人胚胎肾细胞，和来源于人脐带的间充质干细胞。
  2. 如权利要求1所述的细胞介导的新冠肺炎疫苗的制备方法，其特征在于：包括以下几个关键步骤：其一，制备可用于干细胞呈递的新冠特异抗原载体，包括S蛋白，M蛋白，和N蛋白；其二，制备干细胞介导的新冠肺炎疫苗。
  3. 根据权利要求2所述的由细胞呈递的新冠特异抗原载体的制备方法，其特征在于：步骤一的具体方法为：以权利要求1所述的真核瞬时表达的pc3.1载体为支架，在合适部位插入附录中所述的核酸序列，即S基因，M基因和N基因，其中S和N利用相同酶切位点接入pc3.1载体中，而M基因是根据另一种同源重组原理接入pc3.1载体中，根据每个基因的核酸序列的起始和尾部序列设计3对有效的克隆引物，具体引物为：
    S基因的前引物S-FP: 5’-GGGCGGCCGCCCTTTGTTTTTCTTGTTTTATTGC-3’；
    S基因的后引物S-RP: 5’-CCCTCTAGAGCTTATGTGTAATGTAATTTGACTC-3’；
    N基因的前引物N-FP:  5’-GGGCGGCCGCCCTCTGATAATGGACCCCA-3’；
    N 基因的前引物S-RP: 5’-CCCTCTAGATTAGGCCTGAGTTGAGTC-3’；
    M基因的前引物M-FP:  5’-GACGACAAGGGGCGGCCGCAAGCAGATTCCAACGGTACTAT-3’；
    M基因的前引物S-RP: 5’-TTTAAACGGGCCCTCTAGATTACTGTACAAGCAAAGCAAT-3’。
  4. 根据权利要求2所述的由干细胞呈递的新冠特异抗原载体的制备方法，其特征在于：步骤二的具体方法为：将步骤一所得的可用于干细胞呈递的新冠特异抗原载体通过脂质体转染的方式导入至人胚胎肾细胞，和来源于人脐带的间充质干细胞，按照最佳转染效率比例，即脂质体体积与步骤一所得载体用量比为15ul:3ug，进行转染至权利要求1所述的干细胞中；再在转染后的36hr-48hr，回收经修饰后的干细胞沉淀，要求在2-6 ℃下，500 g离心5min回收，最终以该沉淀加入适量的体积的不含钙、镁离子的PBS缓冲液进行重悬，即为所述干细胞介导的新冠肺炎疫苗。
PCT/CN2020/096030 2020-06-15 2020-06-15 一种基于细胞介导的新冠肺炎疫苗的制备方法 WO2021253147A1 (zh)

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Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1990042A (zh) * 2005-05-31 2007-07-04 中国人民解放军第三军医大学第一附属医院 抗sars冠状病毒细胞疫苗及应用
CN103230600A (zh) * 2013-04-08 2013-08-07 四川大学 HBx修饰的抗肝癌全细胞疫苗及其制备方法和用途
CN103820483A (zh) * 2014-02-14 2014-05-28 中国福利会国际和平妇幼保健院 表达α-gal表位的卵巢癌干细胞疫苗及其制备方法
CN110951756A (zh) * 2020-02-23 2020-04-03 广州恩宝生物医药科技有限公司 表达SARS-CoV-2病毒抗原肽的核酸序列及其应用
CN110997711A (zh) * 2017-06-08 2020-04-10 布里格姆妇女医院 用于鉴定表位的方法和组合物
CN111218458A (zh) * 2020-02-27 2020-06-02 珠海丽凡达生物技术有限公司 编码SARS-CoV-2病毒抗原的mRNA和疫苗及疫苗的制备方法
CN111217919A (zh) * 2020-03-04 2020-06-02 中山大学 一种基于火球菌铁蛋白的新型冠状病毒s蛋白双区域亚单位纳米疫苗
CN111228475A (zh) * 2020-02-21 2020-06-05 赛诺（深圳）生物医药研究有限公司 用于预防新型冠状病毒的生物制品
CN111330002A (zh) * 2020-03-09 2020-06-26 北京鼎成肽源生物技术有限公司 靶向冠状病毒的通用dc细胞疫苗及其制备方法和应用

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1990042A (zh) * 2005-05-31 2007-07-04 中国人民解放军第三军医大学第一附属医院 抗sars冠状病毒细胞疫苗及应用
CN103230600A (zh) * 2013-04-08 2013-08-07 四川大学 HBx修饰的抗肝癌全细胞疫苗及其制备方法和用途
CN103820483A (zh) * 2014-02-14 2014-05-28 中国福利会国际和平妇幼保健院 表达α-gal表位的卵巢癌干细胞疫苗及其制备方法
CN110997711A (zh) * 2017-06-08 2020-04-10 布里格姆妇女医院 用于鉴定表位的方法和组合物
CN111228475A (zh) * 2020-02-21 2020-06-05 赛诺（深圳）生物医药研究有限公司 用于预防新型冠状病毒的生物制品
CN110951756A (zh) * 2020-02-23 2020-04-03 广州恩宝生物医药科技有限公司 表达SARS-CoV-2病毒抗原肽的核酸序列及其应用
CN111218458A (zh) * 2020-02-27 2020-06-02 珠海丽凡达生物技术有限公司 编码SARS-CoV-2病毒抗原的mRNA和疫苗及疫苗的制备方法
CN111217919A (zh) * 2020-03-04 2020-06-02 中山大学 一种基于火球菌铁蛋白的新型冠状病毒s蛋白双区域亚单位纳米疫苗
CN111330002A (zh) * 2020-03-09 2020-06-26 北京鼎成肽源生物技术有限公司 靶向冠状病毒的通用dc细胞疫苗及其制备方法和应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SUZANNE L TOMCHUCK; NORTON ELIZABETH B; GARRY ROBERT F; BUNNELL BRUCE A; MORRIS CINDY A; FREYTAG LUCY C; CLEMENTS JOHN D: "Mesenchymal stem cells as a novel vaccine platform", FRONTIERS IN CELLULAR AND INFECTION MICROBIOLOGY, vol. 2, 16 November 2012 (2012-11-16), pages 1 - 8, XP055286040, DOI: 10.3389/fcimb.2012.00140 *

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