摘要
目的研究沙门菌基因突变株构建的方法,为利用基因工程技术对细菌进行遗传性状改造,深入研究沙门菌防治手段提供工具。方法选用不同感受态细菌培养时间、培养基、感受态细胞浓度和电击参数等条件,以小分子质粒pMDT-GFP电转化受试菌,通过计算转化效率确定优化的电转条件。在此基础上用大分子质粒pGBM151-spvB电转化受试菌,再以蔗糖诱导同源重组,筛选突变株。结果以含15 g/LNaCl的高渗LB培养基培养细菌至A600为0.3,制备浓度为1010cfu/ml的感受态细胞,在参数为2.5 kV、400Ω、50μF条件下,小质粒pMDT-GFP电转鼠伤寒沙门菌可获得高转化效率。以往难以转化的大质粒pGBM151-spvB在该条件下成功获得转化子,进而成功构建突变株。结论上述方法能用于高效构建沙门菌基因突变株。
Objective To investigate the method of Salmonella spp. mutant construction, optimizes the processing and provides the basis for further experiments in genetically modification. Methods Two different plasmid vectors DNAs pMDT-GFP and pGBM151-spvB were electrotransformed into S. typhimuri- um under various conditions including growth stage, medium, concentration of competent cells and pulse strength. The transformation efficiency changes were discovered and the optimized conditions were evalua- ted accordingly. Results The optimal electrotransformation effect can be obtained when A600 is O. 3, the medium is hypertonic LB contained 15g/L NaC1, concentration of competent cells is 1010cfu/ml and the electroporation parameters is 2.5 kV, 400Ω,50 μF. Conclusion This method can be used to efficiently of construct Salmonella spp. mutant .
出处
《苏州大学学报（医学版）》
CAS
2012年第6期764-768,共5页
Suzhou University Journal of Medical Science
基金
国家自然科学基金资助项目(30972768)
江苏省自然科学基金资助项目(L2134051511)
江苏省青蓝工程项目(SR13400211)
关键词
沙门菌
基因突变
转化
Salmonella spp.
gene mutation
transformation
