粪肠球菌内源性质粒的序列分析及其穿梭载体的构建

为了对粪肠球菌EXW27的内源性质粒pXW进行DNA序列的测定和分析，并基于其最小复制子构建大肠杆菌-粪肠球菌穿梭载体。本研究从具有优良益生特性的粪肠球菌EXW27中分离得到了内源性质粒pXW，对其进行了DNA序列的测定及分析，然后利用质粒pXW的复制子构建大肠杆菌-乳酸菌穿梭载体，并研究大肠杆菌-乳酸菌穿梭载体的宿主范围、转化效率和稳定性。结果表明该质粒大小为8617 bp，GC含量为33.29%，包含8个ORF，推定为θ型复制质粒。质粒pXW在粪肠球菌EXW27中拷贝数最高可达32.09±0.93，表明质粒pXW属于高拷贝数质粒。本研究确定了质粒pXW的最小复制子，并基于该复制子构建了大肠杆菌-粪肠球菌穿梭载体pXWM1。该穿梭载体具有较宽的宿主范围，可成功转化至不同类型乳酸菌，转化效率介于1.96×102~8.96×104 CFU/μg （质粒DNA）之间，质粒丢失率介于28.54%~54.17%之间。本研究成功构建了一个具有宽宿主范围、高转化效率以及高稳定性的大肠杆菌-粪肠球菌穿梭载体，为乳酸菌基因操作提供了新工具。

Sequence Analysis of an Endogenous Plasmid from Enterococcus faecalis and the Construction of Shuttle Vectors

The aim of this study was to determine and analyze the DNA sequence of the endogenous plasmid pXW from Enterococcus faecalis EXW27, and to construct an Escherichia coli/Enterococcus faecalis shuttle vector based on its minimal replicon. The endogenous plasmid pXW was isolated from E. faecalis EXW27 with good probiotic properties. The DNA sequence of pXW was determined and analyzed, and then the replicon of plasmid pXW was used to construct an Escherichia coli/Enterococcus faecalis shuttle vector. The host range, transformation efficiency and stability of the Escherichia coli/Enterococcus faecalis shuttle vector were also studied. The results showed that plasmid pXW was 8617 bp in size and its GC content was 33.29%. It contained 8 ORFs and was assumed to be θ-type replicating plasmid. The copy number of plasmid pXW in E. faecalis EXW27 was up to 32.09±0.93, indicating that pXW was a high copy number plasmid. In this study, the minimal replicon of plasmid pXW was determined, and an Escherichia coli/Enterococcus faecalis shuttle vector was constructed based on this replicon. The shuttle vector had a wide host range and was successfully transformed into different types of lactic acid bacteria. The transformation efficiency was between 1.96×102~8.96×104 CFU/μg (plasmid DNA), and the plasmid loss rate was between 28.54% and 54.17%. In this study, we successfully constructed an Escherichia coli/Enterococcus faecalis shuttle vector with wide host range, high transformation efficiency and high stability, which provides a new tool for gene manipulation of lactic acid bacteria.