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目的 将甲型流感病毒RNA聚合酶PA亚基的基因工程重组质粒pQE3 2 -PA - 2转化入受体菌 ,并优化转化条件 ,以建立其高效、快速的转化体系。方法 以大肠杆菌DH5α菌株为受体菌 ,研究了氯化钙溶液浓度 ( 0、2 5、5 0、75、10 0、15 0mmol/L) ,热休克温度 ( 0、3 0、3 7、42、5 0℃ ) ,转化时间 ( 7、15、3 0、45、60、75、90、10 5、12 0min)及转化后的温浴时间 ( 0、0 .5、1、1.5、2hr)对基因工程重组质粒pQE3 2 -PA - 2转化效率的影响。 结果 氯化钙溶液浓度及热休克温度对转化效率的影响极为显著 ,转化时间在一定范围内对转化效率有明显的影响 ,而转化后的温浴时间对转化效率的影响不明显。结论 在本实验条件下 ,以 75mmol/L氯化钙制备感受态细胞 ,转化 3 0分钟 ,经 42℃热休克作用 90秒后直接涂平板选择转化子 ,可获得高效、快速的转化效果
Objective To transform the recombinant plasmid pQE3 2 -PA - 2 of PA subunit of influenza A virus RNA polymerase into recipient bacteria and optimize the transformation conditions to establish its efficient and rapid transformation system. Methods The Escherichia coli DH5α strain was used as the recipient strain to study the effects of calcium chloride solution concentration (0,25,5 0,75,10 0,15 0 mmol / L), heat shock temperature (0,3 0,3 7, (40 ° C), conversion time (7,15,3 0,45,60,75,90,10 5,12 0min), and incubation time after transformation (0,0.5,1,1.5,2hr) On genetically engineered recombinant plasmid pQE3 2 -PA - 2 conversion efficiency. Results The effects of calcium chloride solution concentration and heat shock temperature on the conversion efficiency were significant. The conversion time had a significant effect on conversion efficiency within a certain range, while the effect of conversion time on the conversion efficiency was insignificant. Conclusion In this experiment, competent cells were prepared with 75mmol / L calcium chloride and transformed for 30 minutes. After heat shock at 42 ° C for 90 seconds, transformants were plated directly to obtain high efficiency and rapid transformation