流感病毒RNA聚合酶PA亚基重组质粒pQE32-PA-2转化效率的实验研究

The Experimental Investigation of the Transformative Efficiency on Recombining the Molecule of Genes of the RNA Polymerase PA Subunit in Influenza Virus

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。

Objective To establish an efficient and swift transformative system of the recombinant plasmid pQE32-PA-2.Methods The different effects of the transformative condition which included CaCl2 concertration(0?25?50?75?100?150mmol/l),of the various transformative time(7?15?30?45?60?75?90?105?120min),of the various heat shock temperature(0?30? 37?42?50 ℃)and of the incubative time(0?0.5?1?1.5?2hr)on the transformative recombinant plasmid pQE32-PA-2 were investigated in this paper.pQE32-PA-2 was a recombinant molecule of genes of the RNA polymerase PAsubunit in influenza virus with the vector pQE32.The host cell was E.coliDH5α.Results The effects of different concentration of CaCl2 and the heat shock temperature on the transformative frequency own significant differences, and the effects of the transformative time are also obvious,but the effect of incubative time after transformation is insignificant.Conclusions The best condition for pQE32-PA-2 transformation in this experiment prepares the competent host cells with 75mmol/lCaCl2,transforming for 30 minutes,heat shocking after transformation in 42℃ for 90 seconds,and then following the direct selection onAmp+LB plate.