摘要
"跨界基因沉默"技术是一种全新的、跨原核界与真核界的小分子RNA干扰传递技术。该技术构建了一种大肠杆菌,在这种大肠杆菌中转录的短发夹RNA(short hairpin RNA,shRNA)能够引发真核细胞内特异性基因的RNA干扰(RNA interference,RNAi),所以此大肠杆菌又被称为"跨界基因沉默菌株"。"跨界基因沉默菌株"具有RNA干扰传递功能,主要得益于"跨界干扰质粒"(trans-kingdom RNAi plasmid,TRIP)。TRIP质粒主要包含inv基因、hly基因以及能够转录shRNA的基因片段。hly基因表达的listeriolysin O蛋白可以帮助细菌逃离真核细胞的吞噬体,但listeriolysin O蛋白是一种重要的毒力因子,对宿主细胞具有一定的损害。为了克服这种缺陷,在hly基因上设计了△PEST和G486D两种减毒突变。同时,为了使减毒后的TRIP质粒能够在"跨界基因沉默菌株"中稳定存在,进一步利用Red/ET同源重组将该质粒整合进大肠杆菌的基因组中。实验结果表明,减毒、整合后的"跨界基因沉默菌株"能更好地发挥RNAi作用。该技术的完善将推进工程菌的研发和利用。
Trans-kingdom RNA interference （RNAi） technology is an all-new small molecule RNA interference technology across prokaryotic and eukaryotic kingdom. This technology aims to rebuild a kind of engineered Escherichia coli to produce a short hairpin RNA （shRNA） targeting a mammalian gene inducing trans- kingdom RNAi. The Escherichia coli was named as ＂trans-kingdom RNAi bacterium＂. The special function of the Escherichia coli is mainly attributed to trans-kingdom RNAi plasmid（TRIP）, which contains gene inv, gene hly and transcribing shRNA gene. Listeriolysin O, expressed by hly, is able to assist bacteria to run away from the phagosome of eukaryotic cells. However, listeriolysin O is also an important virulence factor, causing certain damages to host cells. To conquer this drawback,△PEST and G486D, two attenuated mutations, had been imposed on hly. Meanwhile, to make sure the attenuated TRIP could stably exist, the plasmid was integrated into genome of the Escherichia coli through Red/ET homologous recombination. After attenuation and integration, the trans-kingdom RNAi bacteria became more favorable for RNAi. The improvement of this technology will make a difference for the development and utilization of genetically engineered bacteria.
出处
《生命科学研究》
CAS
CSCD
2016年第3期208-213,共6页
Life Science Research
基金
湖南省科技厅重点项目(2014FJ2006)
长沙市科技局重点项目(K1205221-31)
