CENP-E蛋白动力结构域对细胞周期及染色体分离的影响

Effect of motor domain of CENP-E on cell cycle and separation of chromosome

目的:克隆含CENP-E蛋白动力结构域的真核表达载体,并观察其对染色体分离的影响.方法:采用RT-PCR、分子克隆技术获得含CENP-E动力结构域的真核表达载体pEG-FP-CENPE1(1～336位氨基酸),pEGFP-CENPET1(包括1～336和2078～2492位氨基酸),通过流式细胞术、染色体核型分析观察CENP-E蛋白动力结构域对染色体分离的影响.结果:成功构建出pEGFP-CENPE1,pEGFP-CENPET1融合质粒并在HEK293细胞中正确表达;流式细胞术和染色体核型分析结果表明:转染含CENP-E蛋白动力结构域的载体的细胞在用破坏纺锤体微管药物处理后不能保持于细胞分裂中期,细胞提前进入分裂后期;非整倍体细胞数目增多.结论:过度表达含CENP-E动力结构域不能使受诺考达唑处理的细胞保持于分裂中期,染色体分离出现错误,非整倍体数目细胞增多.

AIM： To construct a mammalian expression vector containing the motor domain of human CENP-E protein and to observe its influence on the separation of chromosomes. METHODS： cDNA sequences encoding the motor domain of CENP-E were amplified through RT-PCR and cloned into the enhanced GFP fluorescence protein （EGFP） expression vector. The effect of recombinant plasmids, namely pEGFP-CENPE1 （ 1 - 336aa） and pEGFP-CENPET1 （ 1 - 336 and 2078 - 2492aa） was observed respectively on the separation of chromosomes by FCM and karyotype analysis. RESULTS： Two recombinant vectors, namely pEGFP-CENPE1 and pEGFP-CENPET1 were constructed correctly and their expressions in HEK293 cells were confirmed respectively. Results from both FCM and karyotype analysis showed that the cells transfected with the plasmid expressing the motor domain of CENP-E could not maintain the midphase and exited mitosis ahead of schedule when treated with reagent destructing the tubulin. The abnormal chromosomal separation led to increased aneuploid cells. CONCLUSION： The over expression vectors containing the motor domain of CENP-E cannot maintain the midphase. Chromosome misseparation occurs and leads to aneuploid cells.