基于新月柄杆菌RsaA外运机制的EspA及EspA-IL-24融合蛋白胞外分泌表达研究

目的：实现大肠杆菌分泌蛋白（Esp）A及EspA与白细胞介素（IL）-24融合蛋白的胞外分泌表达，进一步验证基于新月柄杆菌RsaA外运机制的原核胞外分泌表达载体系统的有效性和通用性，并改造优化该系统。方法：利用分子克隆手段，按RsaA分泌系统操纵子组织方式，将获得的RsaA系统元件编码序列和异源调控序列克隆至pQE30骨架质粒，构建新的胞外分泌表达质粒pQABP2S；以大肠杆菌为宿主菌诱导表达EspA及EspA-IL-24融合蛋白，并通过Westernblot检测目标蛋白在培养上清中的表达。结果：获得了新的胞外分泌表达载体pQABP2S；与对照相比，该载体宿主系统培养上清中目标蛋白EspA及EspA-IL-24的表达量明显增加。结论：在大肠杆菌中通过RsaA分泌系统可实现分子大小不同的EspA及EspA-IL-24融合蛋白的特异性分泌表达，进一步证实该分泌表达策略的有效性和通用性；调整调控序列以优化分泌系统的尝试，为此类基因工程技术平台的开发提供了借鉴。

Study on Extracellular Expression of EspA and EspA-IL-24 Proteins in Escherichia coli Following RsaA Exportation Mechanism of Caulobacter crescentus

Objective： To realize extracellular expression of E.coli secreted protein（Esp） A and EspA-IL-24 proteins in E, coli and to further verify the potency of the established prokaryotic secretion expression vector-host system following RsaA exportation mechanism of Caulobacter crescentus in delivery of diverse heterogeneous proteins as well as to attempt optimizing the system by adjusting key regulation elements. Methods： Genes and DNA fragments needed were obtained by DNA cloning techniques and then regrouped with reference to natural rsaA operon step by step by DNA manipulation on the basis of pQE30 vector to form the new secretion expression vector pQABP2S carrying gene espA or espA-ii-24 respectively. Western Blot was conducted for assessing expression of EspA or EspA-IL-24 fused with RsaA signal sequence in the culture superoatant of E.coli M15 harboring individual newborn expression vector. Results： The premeditated vector pQABP2S was constructed and obtained successfully. Shown by Western Blot, expression of EspA and EspA-IL-24 proteins in the culture superoatant of pQABP2S/E.coli M15 system were enhanced significantly compared with the control respectively. Conclusion： Extracellular expression of heterogeneous EspA or EspA-IL-24 protein in E.coli can be realized by utilizing RsaA secretion system and hereby the potency of established prokaryotic secretion expression vector-host system in biotechnology is further verified. It is also an illuminating attempt that optimizing the system by adjusting key regulation elements.