利用重叠延伸PCR构建红色荧光蛋白布鲁氏菌表达载体

Construction of Red Fluorescent Protein Expression Vector of Brucella Using Overlapping Extension PCR Method

为了构建能够在布鲁氏杆菌宿主细胞和宿主个体中稳定表达红色荧光蛋白的载体,试验通过PCR分别获得核糖体结合位点-红色荧光蛋白(RBS-Red)与布鲁氏菌特异DNA(BDNA)片段,利用重叠延伸PCR获得RBS-Red-BDNA重叠片段;将重叠片段插入pLB载体,利用SmaⅠ与SacⅡ对重组pLB载体和pMC-221质粒进行双酶切后连接目的片段,连接产物转化大肠杆菌DH5α感受态细胞;提取的质粒电转布鲁氏菌16M菌株感受态细胞,将电转后的16M-pMC-Red菌株涂布于含有氯霉素抗性的布鲁氏菌培养基,倒置荧光显微镜下观察菌株颜色;16M-pMC-Red菌株侵染小鼠巨噬细胞,制成细胞爬片,激光共聚焦荧光显微镜下观察小鼠巨噬细胞,鉴定红色荧光蛋白表达情况。结果显示,利用重叠延伸PCR技术成功改造了红色荧光蛋白布鲁氏菌双启动子表达载体;将改造获得的质粒电转进布鲁氏杆菌16M菌株,菌株荧光鉴定能够稳定表达红色荧光蛋白;电转成功的16M-pMCRed菌株侵染小鼠巨噬细胞后,可以稳定表达红色荧光蛋白。试验构建的发红光质粒pMC-Red可以与发绿光质粒pMC-221联用,为不同种株布鲁氏菌之间的联合研究奠定了基础,也为布鲁氏菌病检测提供了一个以荧光检测为指标的新策略。

To construct red fluorescent protein expression vector of Brucella host cells and individuals,RBS-Red and BDNA(Brucella DNA)fragments were obtained by PCR,and the RBS-Red-BDNA overlapping fragments were obtained by overlapping extension PCR method in this experiment.The overlapped fragments were inserted into pLB vector,the recombinant pLB vector and pMC-221 plasmid were digested by SmaⅠand SacⅡdouble enzyme,the ligation fragments were transformed into E.coli DH5α,the extract plasmid was electroporated into Brucella 16M competent cell,16M-pMC-Red strain was coated with Brucella culture medium containing chloramphenicol resistance,and observed the strain color under inverted fluorescence microscope.The 16M-pMC-Red strain was used to infect the mouse macrophages and make the cell crawling slices,observation of mouse macrophages under laser confocal fluorescence microscopy and identification of red fluorescent expression.The results showed that the double promoter expression vector of red fluorescent protein in Brucella was successfully transformed by overlapping extension PCR method,the transformed plasmid was electroporated into Brucella strain 16M,and the strain could stably express the red fluorescent protein.The 16M-pMC-Red strain in mouse macrophages could stably express the red fluorescent protein.The red light-producing plasmid pMC-Red could be used together with the green light-emitting plasmid pMC-221,which laid a foundation for the research of different strains of Brucella,and also provided a new strategy using fluorescence detection as an indicator for the detection of brucellosis.