作　　者： ; ; ; ; ;

机构地区： 广东省农业科学院

出　　处： 《华南农业大学学报》 2005年第3期117-119,共3页

摘　　要： 应用DNA重组技术构建了表达绿色荧光蛋白(GFP)的重组乳酸杆菌.将pGFP2质粒中的绿色荧光蛋白基因切下,克隆进表达载体质粒pThioHisB的NcoⅠ和SacⅠ位点之间,构建成重组表达质粒pThioGFP,然后转化大肠杆菌BL21.从BL21中提取重组质粒,用电击法转化乳酸杆菌Lac1001,当细菌处于D600nm为0.6的生长期时,使用PEB作为电击缓冲液,对100μL体积的细胞悬液在电容25μF,电阻200Ω,电压2.2kV的电击条件进行完整质粒转化可得到较高的转化率.电击转化后Lac1001于LB培养基上呈绿色,在荧光透射仪下观察,整个菌体发绿色荧光.构建的重组乳酸杆菌能够稳定表达绿色荧光蛋白. A recombinant Lactobacillus expressing green fluorescent protein (GFP) gene was constructed using recombinant DNA techniques. The GFP gene was excised from plasmid pGFP2 and subcloned into expression vector pThioHisB as an NcoⅠ/SacⅠ fragment.The resulting recombinant plasmid was named as pGhisGFP and then transformed into the BL21 strain of E. coli. The recombinant plasmid pThioGFP was extracted from the BL21, and then transformed into Lactobacillus by electroporation. The optimum parameters for electroporation were:D_(600 nm) 0.6, voltage 2.2 kV, buffer PEB, 100 μL cell volume.The recombinant bacterial grew on LB plate as green colonies, and the results showed that the GFP gene was expressed stably in the host bacterial (Lac1001).

关 键 词： 绿色荧光蛋白 重组乳酸杆菌 质粒构建 电击

领　　域： [农业科学] [农业科学] [农业科学]