【摘要】 目的:构建GFP-Plk1同义突变表达载体及其稳定转染细胞系。方法:设计Polo样激酶1(Plk1)si RNA序列及相对应的同义突变引物,并利用二次PCR方法扩增Plk1基因,定向克隆到p Rex-EGFP-IRES-Hygro载体中,构建p Rex-EGFP-r Plk1-IRES-Hygro表达载体;利用逆转录病毒感染的方法,构建He La/GFP-r Plk1稳定细胞系;利用免疫印迹及激光共聚焦显微镜,验证Plk1 si RNA的干扰效果及稳定细胞系的构建。结果:双酶切鉴定和测序结果表明构建的p Rex-EGFP-r Plk1-IRES-Hygro正确;免疫印迹实验证明Plk1 si RNA序列可以有效抑制He La/GFP-r Plk1细胞中内源性Plk1蛋白的表达,但不能干扰掉外源GFP-r Plk1蛋白;在荧光共聚焦显微镜下,观察到有丝分裂的前中期和末期,GFP-r Plk1分别定位于着丝粒和中间体上。结论:构建了Plk1同义突变表达载体p Rex-EGFP-r Plk1-IRES-Hygro和He La/GFP-r Plk1稳定细胞系,为下一步研究Plk1在有丝分裂期的调控机制提供了模型。更多还原

【Abstract】 Objective: To construct GFP-Plk1 synonymous mutation expression vector and stably transfected cells.Methods: The si RNA and synonymous mutation primers targeting Polo-like kinase 1(Plk1) were designed. Plk1 gene was amplified by two steps PCR and then inserted into vector p Rex-EGFP-IRES-Hygro to construct p Rex-EGFP-r Plk1-IRES-Hygro plasmid. He La/GFP-r Plk1 stably transfected cells were constructed through retroviral in-fection, immunbloting and confocal laser scanning microscopy were used to verify Plk1 si RNA interference and sta-bility of constructed cells. Results: Double enzymes digestion and sequencing results showed that the constructedvector p Rex-EGFP-r Plk1-IRES-Hygro was correct. Immunoblotting results demonstrated that Plk1 si RNA could ef-fectively inhibit the expression of endogenous Plk1 protein, but not exogenous GFP-r Plk1 protein. Fluorescence con-focal microscopy images showed that during prometaphase and telophase, GFP- r Plk1 was respectively located inthe kinetochore and midbody. Conclusion: Anti-si RNA GFP-Plk1 synonymous mutation expression vector p Rex-EGFP-r Plk1-IRES-Hygro and He La/GFP-r Plk1 stable cells were constructed, which will contribute to the furtherresearch on the regulation of Plk1 in mitosis.更多还原