抑制内源蛋白酶对病毒介导表达的外源蛋白在植物中积累的影响

内源蛋白酶胞内水解外源重组蛋白致使其积累量低是植物病毒表达平台向商业化迈进的关键“瓶颈”问题。抑制重组蛋白胞内降解相关蛋白酶是打破桎梏的关键。然而，共表达蛋白酶抑制剂（PIs）原位保护胞内重组蛋白的策略存在着竞争抑制效应；利用小RNA沉默内源蛋白酶基因的研究鲜有报道。鉴于此，拟以rPA为研究对象，构建其病毒表达载体和PIs植物表达载体；通过测定共接种叶片内重组蛋白的积累，分析竞争抑制效应在PIs中的普遍性，揭示共表达体系中PIs相对丰度与竞争抑制效应的关系，提出弱化PIs共表达竞争抑制的策略。同时，依据重组蛋白胞内降解相关蛋白酶基因序列，设计、构建内源蛋白酶基因amiRNA表达载体，筛选高沉默效率的amiRNA；探索内含子表达蛋白酶基因amiRNA的技术关键，构建单个转录元件同时表达外源重组蛋白和内源蛋白酶基因多元amiRNA的新系统，为推动植物病毒表达系统的商业化进程奠定理论和实践基础。

As a novel expression system, plant virus RNA-based vectors allow for the rapid, high level, transient expression of proteins such as biopharmaceuticals in whole plants. A major limiting factor for a broader application of this expression system for the production of valuable recombinant proteins is the low yield of intact recombinant proteins. These low yields are at least in part due to the action of endogenous plant proteases on the foreign recombinant proteins. However, the strategy of safeguarding the target protein against degradation in situ by companion protease inhibitors (PIs) may have competitive effects. Moreover, the studies, which involved the usage of small RNAs to silence the protease genes in cells has also been rarely reported. For these reasons, viral RNA-based expression vectors carrying rPA gene will be constructed. Simultaneously, plant binary expression vectors carrying protease inhibitors gene also will be constructed and co-inoculated into the leaves of Nicotiana benthamiana with viral expression vectors expressing rPA. By measuring the expression of recombinant proteins in co-inoculated leaves, the prevalence of competitive effects of co-expression of PIs and the effects of relative abundance of PIs on the accumulation of recombinant proteins will be analyzed. And then strategies to weaken the competitive effects due to co-expression of PIs will be proposed. Meanwhile, amiRNA will be designed according to the protease gene sequences which have a direct relation to the degradation of the recombinant protein. The amiRNA expression vector will be constructed and will be used to screen the most efficient one for silencing protease gene in cells. Then we will further explore the key of technology for intron-based expression of the protease gene amiRNA. Furthermore, a novel system, with a single transcription element, will be established for simultaneously expressing multiple amiRNAs of protease genes and recombinant proteins in the same cell. Finally, the feasibility of increasing the accumulation of exogenous recombinant proteins mediated by viral vectors in plants by tackling the hydrolysis of endogenous proteases will be clarified, which will lay a foundation for promoting the rational utilization and commercialization of plant viral expression systems.