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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 study the method of constructing Salmonella gene mutants and to provide a tool for the further research on the means of Salmonella control by gene engineering technology. Methods Using different conditions of bacterial culture time, medium, competent cell concentration and electric shock parameters of different competent bacteria, electrotransformation of tested bacteria with small molecule plasmid pMDT-GFP was used to determine the optimal electrotransport conditions by calculating the transformation efficiency. On the basis of this, the recombinant plasmid pGBM151-spvB was used to transform the tested bacteria, then sucrose was used to induce homologous recombination and the mutants were screened. Results Bacteria were cultivated in a high osmotic LB medium containing 15 g / L NaCl for 0.3 times. The competent cells were prepared at the concentration of 1010 cfu / ml. Under the conditions of 2.5 kV, 400 Ω and 50 μF, the small plasmid pMDT-GFP mice Salmonella typhi can achieve high conversion efficiency. In the past, the plasmid pGBM151-spvB, which was difficult to transform, was successfully transformed into transformant under the conditions, and then the mutant was successfully constructed. Conclusion The above method can be used to efficiently construct Salmonella mutants.