Expression of recombinant SARS-CoV-2 proteins in plants

重组 SARS-CoV-2 蛋白在植物中的表达

• 批准号: 2624970
• 金额: --
• 依托单位: University of Manchester
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2021
• 资助国家: 英国
• 起止时间: 2021 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2624970
• 关键词: Expression; recombinant; SARS; CoV; proteins

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) responsible for the COVID19 pandemic is a continuing threat to global health and prosperity. There is an urgent need to develop innovative vaccine technologies and diagnostic tests to combat the virus. Antigenic proteins encoded by SARS-CoV-2 can be expressed as recombinant proteins in heterologous expression systems. These recombinant antigenic proteins can be used to develop vaccines and diagnostic kits to identify people with antibodies against the virus. To combat the global pandemic, a low cost, high yield and scalable expression system is required to ensure that the SARS-CoV-2 recombinant proteins can be made in large amounts across the world. This affordability issue is particularly important in developing countries. Plant based expression systems provide the ideal vehicle for expressing SARS-CoV-2 recombinant proteins. This project will use a leaf-based expression system that enables extraordinarily high yields of SARS-CoV-2 recombinant proteins to be made with a low carbon footprint. This project will identify SARS-CoV-2 antigens that are suitable for development of a plant-based vaccine. Antigens, derived from in silico screening of the SARS-CoV-2 genome, will be incorporated and displayed on virus like particles (VLPs). These VLPs are formed by self-assembly of hundreds of copies of the single viral protein. In effect VLPs are the outer coating of the virus without the infectious nucleic acid. Each VLP will display multiple SARS-CoV-2 antigens, enhancing their potential to be highly immunogenic. Expression in leaf chloroplasts enables extraordinarily high yields of recombinant proteins. The protein sequences for SARS-CoV-2 VLPs will be back translated into DNA using a chloroplast codon usage table in silico. The resulting synthesized genes will be inserted into a transformation vector and transformed into chloroplasts. Chloroplast transformants expressing SARS-CoV-2 VLPs will be characterised by PCR and protein blot analyses. These techniques are used to confirm targeted transgene integration and concentration of SARS-CoV-2 VLPs. The SARS-CoV-2 VLPs expressed in leaves will be purified and their structures examined by electron microscopy. Purified SARS-CoV-2 VLPs will then be tested using antibodies and anti-sera to determine correct folding and incorporation of the antigens. The project combines expertise in plant expression systems (Day), vaccine development (Derrick) and visualisation of virus-like particles (Roseman).

导致covid - 19大流行的严重急性呼吸综合征冠状病毒2 （SARS-CoV-2）对全球健康和繁荣构成持续威胁。迫切需要开发创新的疫苗技术和诊断测试来对抗这种病毒。SARS-CoV-2编码的抗原蛋白可以在异源表达系统中作为重组蛋白表达。这些重组抗原蛋白可用于开发疫苗和诊断试剂盒，以识别具有抗病毒抗体的人。为了应对全球大流行，需要一种低成本、高产量、可扩展的表达系统，以确保在全球范围内大量生产SARS-CoV-2重组蛋白。这一负担能力问题在发展中国家尤为重要。基于植物的表达系统为表达SARS-CoV-2重组蛋白提供了理想的载体。该项目将使用一种基于叶子的表达系统，该系统可以在低碳足迹的情况下生产产量极高的SARS-CoV-2重组蛋白。该项目将确定适于开发植物性疫苗的SARS-CoV-2抗原。从SARS-CoV-2基因组的计算机筛选中获得的抗原将被整合并展示在病毒样颗粒（vlp）上。这些VLPs是由单个病毒蛋白的数百个拷贝的自组装形成的。实际上，VLPs是没有传染性核酸的病毒外壳。每个VLP将显示多种SARS-CoV-2抗原，增强其高度免疫原性的潜力。在叶片叶绿体中的表达使得重组蛋白的产量非常高。SARS-CoV-2 VLPs的蛋白质序列将使用叶绿体密码子使用表重新翻译成DNA。合成的基因将被插入转化载体并转化为叶绿体。表达SARS-CoV-2 VLPs的叶绿体转化体将通过PCR和蛋白印迹分析进行表征。这些技术用于确认SARS-CoV-2 VLPs的靶向转基因整合和浓度。对叶片中表达的SARS-CoV-2 VLPs进行纯化，并用电镜对其结构进行检测。然后将使用抗体和抗血清对纯化的SARS-CoV-2 VLPs进行测试，以确定抗原的正确折叠和结合。该项目结合了植物表达系统（Day）、疫苗开发（Derrick）和病毒样颗粒可视化（Roseman）方面的专业知识。