新月柄杆菌RsaA分泌系统用于大肠杆菌胞外递送重组蛋白的初步研究  

Preliminary Study on Exportation of Heterologous Recombinant Proteins in Escherichia coli Utilizing RsaA Secretion System of Caulobacter crescentus

作　　者：宁亚蕾[1] 周立雄[2] 肖斌[1] 张卫军[1] 曾浩[1] 毛旭虎[1] 邹全明[1] 

机构地区：[1]第三军医大学临床微生物及免疫学教研室暨重庆市生物制药工程技术研究中心,重庆400038 [2]重庆工学院生物工程学院,重庆400050

出　　处：《生物技术通讯》2008年第2期175-180,共6页Letters in Biotechnology

基　　金：重庆自然科学基金重点项目(2006BA5024)

摘　　要：目的:构建基于新月柄杆菌RsaA外运机制的以大肠杆菌为宿主的原核胞外分泌表达载体系统。方法:利用分子克隆手段,按RsaA分泌系统操纵子组织方式,将RsaA系统外运功能基因配合以异源调控序列克隆至pQE30骨架质粒。以绿色荧光蛋白(GFP)为报告分子、大肠杆菌M15为宿主菌,诱导表达后通过Western Blotting检测培养上清中GFP的表达。结果:获得了与设计完全一致的pQABPS载体,利用该载体系统,在培养上清中报告分子GFP的表达明显增加,且是通过特异的RsaA外运机制被分泌至胞外的,而非渗漏表达或简单的信号肽引导。结论:在大肠杆菌中重现了RsaA分泌系统的外运功能,为该系统在基因工程领域的应用研究打下了良好基础。Objective： To obtain a novel and universal extracellular secretion vector system in the host of Escherichia coli utilizing RsaA exportation mechanism of Caulobacter crescentus. Methods： DNA sequences needed were amplified by PCR including RsaA signal sequence, genes of the secretion apparatus, artificial regulation elements as well as the reporter GFP gene. These sequences were regrouped with reference to natural RsaA operen step by step and then were subcloned together into pQE30 vector to form the vector pQABPS. Western Blotting was conducted for assessing expression product of the recombinant GFP gene fused with RsaA signal sequence in the culture supernatant of E.coli M15 harboring the new-born vector. Results： The premeditated vector pQABPS was constructed and obtained successfully. Shown by Western Blot- ting, the expression of the recombinant GFP protein in the culture supernatant of the pQABPS vector-host system was enhanced significantly compared with the control. Conclusion： RsaA secretion system can be transplanted into E.coli successfully and thus provides an illuminating exemplar for future research on application of RsaA secretion system of C.crescentus in biotechnology.

关 键 词：新月柄杆菌 RsaA分泌系统 大肠杆菌 胞外分泌 表达载体 

分 类 号：Q78[生物学—分子生物学]