SARS亚基疫苗-突刺蛋白受体结合区RBD在烟草叶绿体中的高效表达

High-efficiency expression of a receptor-binding domain of SARS-CoV spike protein in tobacco chloroplasts

摘要:

叶绿体表达系统为植物源重组药用蛋白和亚基疫苗的生产提供了一个有效的途径。为验证SARS亚基疫苗在叶绿体中表达的可行性，以及为植物源SARS亚基疫苗的生产提供一套高效、低成本的技术平台，本研究将人工优化合成的SARS-CoV突刺蛋白 (S蛋白) 受体结合区序列RBD与载体分子CTB融合基因导入烟草叶绿体基因组中。PCR和Southern杂交分析表明，外源融合基因已整合到烟草叶绿体基因组中，并获得同质化。Western杂交分析表明，重组融合蛋白CTB-RBD在叶绿体转基因烟草中获得表达，且主要以可溶性单体形式存在。ELISA分析表明，在不同生长阶段、不同生长部位和不同时间点烟草叶片中，重组融合蛋白CTB-RBD的表达水平呈现明显的变化。重组蛋白在成熟叶片中的表达水平最高可以达到10.2% TSP。本研究通过SARS亚基疫苗RBD在烟草叶绿体中的高效表达，有望为植物源SARS亚基疫苗的生产以及SARS血清抗体的检测提供一个有效的技术平台。

Abstract:

Chloroplast-based expression system is promising for the hyper-expression of plant-derived recombinant therapeutic proteins and vaccines. To verify the feasibility of obtaining high-level expression of the SARS subunit vaccine and to provide a suitable plant-derived vaccine production platform against the severe acute respiratory syndrome coronavirus (SARS-CoV), a 193-amino acid fragment of SARS CoV spike protein receptor-binding domain (RBD), fused with the peptide vector cholera toxin B subunit (CTB), was expressed in tobacco chloroplasts. Codon-optimized CTB-RBD sequence was integrated into the chloroplast genome and homoplasmy was obtained, as confirmed by PCR and Southern blot analysis. Western blot showed expression of the recombinant fusion protein mostly in soluble monomeric form. Quantification of the recombinant fusion protein CTB-RBD was conducted by ELISA analysis from the transplastomic leaves at different developmental stages, attachment positions and time points in a day and the different expression levels of the CTB-RBD were observed with the highest expression of 10.2% total soluble protein obtained from mature transplastomic leaves. Taken together, our results demonstrate the feasibility of highly expressing SARS subunit vaccine RBD, indicating its potential in subsequent development of a plant-derived recombinant subunit vaccine and reagents production for antibody detection in SARS serological tests.