为了检测细胞周期性蛋白激酶CDK1与CDK2干扰对CBRH-7919细胞周期的影响,构建了CDK1和CDK2特异性sh RNA慢病毒沉默表达载体,三质粒共转染293FT细胞产生病毒颗粒,收集浓缩后感染CBRH-7919细胞,荧光显微镜下观察了细胞形态,实时定量荧光PCR和聚丙烯酰胺凝胶电泳检测了细胞中CDK1和CDK2 m RNA和蛋白质表达水平的变化,MTT法和流式细胞仪分别检测了细胞增殖和细胞周期的变化情况.结果表明:成功构建了CDK1与CDK2特异性sh RNA慢病毒表达载体,干扰CDK1导致细胞G2/M期的阻滞,细胞增殖明显降低,细胞碎片增多;而干扰CDK2后细胞仍正常生长.
[Objective] The paper was to improve the preparation efficacy of Taq DNA polymerase. [Method] Ni column was used to purify Taq DNA polymerase carrying with 6xHis tag,and recombined vector. Using the thermal-resistant characteristics of Taq DNA polymerase,the crude extract was treated at 75 ℃ for 1 h,and the activity of prepared enzyme solution was verified by PCR test. [Result] The recombinant pET-32A-Taq could highly express in BL21 （DE3） host bacteria and remove hybrid protein by thermal denaturation. The enzyme preparation with the activity further higher than purchased Taq DNA polymerase was obtained. [Conclusion] Taq DNA polymerase prepared by thermal purification method is simple with low cost,and can meet the needs of a large number of conventional PCR amplification.
