Expression of recombinant SARS-CoV-2 proteins in plants

引起COVID19大流行的严重急性呼吸综合征冠状病毒2型(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抗击该病毒。SARS-CoV-2编码的抗原蛋白可以在异源表达系统中以重组蛋白的形式表达。这些重组抗原蛋白可用于开发疫苗和诊断试剂盒，以识别对病毒有抗体的人。为了抗击全球大流行，需要一个低成本、高产量和可扩展的表达系统，以确保SARS-CoV-2重组蛋白能够在世界各地大量生产。这个负担能力问题在发展中国家尤为重要。植物表达系统为表达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，使SARS-CoV-2重组蛋白的产量非常高，并产生低碳足迹。该项目将确定适合开发植物疫苗的SARS-CoV-2抗原。来自SARS-CoV-2基因组电子筛查的抗原将被结合并展示在病毒样颗粒(VLP)上。这些VLP是由数百个拷贝的单一病毒蛋白自组装而成的。实际上，VLP是病毒的外衣，没有传染性核酸。每个VLP将展示多个SARS-CoV-2抗原，增强它们具有高度免疫原性的潜力。在叶绿体中的表达使重组蛋白的产量极高。SARS-CoV-2 VLP的蛋白质序列将使用Silico中的叶绿体密码子使用表重新翻译成DNA。由此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 VLP的叶绿体转化子将通过聚合酶链式反应和蛋白质印迹分析进行鉴定。这些技术被用来确认SARS-CoV-2 VLP的靶向转基因整合和浓度。在叶片中表达的SARS-CoV-2 VLP将被提纯，并用电子显微镜观察其结构。然后，将使用抗体和抗血清对纯化的SARS-CoV-2 VLP进行测试，以确定抗原的正确折叠和掺入。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。

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).