长双歧杆菌NCC2705高效转化系统的建立及GFP表达

目的:建立长双歧杆菌NCC2705高效稳定的电转化系统,并以其为宿主表达绿色荧光蛋白(GFP),构建一个稳定的带报告基因的表达载体。方法:从双歧杆菌克隆载体pDG7中切取其在双歧杆菌中复制所必需的pMB1复制子插入质粒pUC19的多克隆位点区,并将gfp基因在双歧杆菌中进行表达;设计双歧杆菌电击转化体系,研究在不同电击电压条件下的电转效率。结果:首先构建了大肠杆菌-长双歧杆菌穿梭质粒pUB1和带有gfp基因的表达质粒pUB2,用电击法将之转化至双歧杆菌,当细菌生长到D600nm约为0.5时制备感受态细胞,在电容25μF、电阻200Ω、电压12.5 kV/cm的电击条件下进行完整质粒转化,可得到较高的转化率。结论:构建了以长双歧杆菌NCC2705为宿主的高效稳定的表达系统,为进一步研究益生菌的分子生物学和功能特性提供了基础。

Establishment of an Efficient Transformation System and Expression of GFP in Bifidobacterium Longum NCC2705

Objective： To establish an efficient electro-transformation system of Bifidobacterium longum NCC2705,and express GFP to test if this system was stable with a convenient report gene.Methods： The pMB1 replicon which is vital for the plasmid pDG7 replication in B.longum was cloned into pUC19 to construct an E.coli-B.longum shuttle vector pUB1.Then,the gfp gene was cloned into pUB1 to create pUB2.Study on electro-transformation efficient by using different electro-transformation system.Results： At first,an E.coli-B.longum shuttle vector pUB1 and pUB2 with gfp gene were constructed.After being tested correct,the plasmid pUB2 was electro-trans-formed into NCC2705 to express gfp gene in B.longum.A higher efficient could be gotten when parameters of electro-transformation were 25 μF,200 Ω and 12.5 kV / cm.Conclusion： Our results of an efficient electro-trans-formation system of B.longum NCC2705 will lay a foundation for further investigation on the molecular biology and function of probiotic B.longum NCC2705.