Tat-p21融合蛋白的表达及其在A549细胞中的活性研究

Expression of Tat-p21 fusion protein and its activity in A549 cells

在本研究中,通过分子克隆的手段,将编码Tat跨膜转运结构域的CDS与人野生型p21蛋白的CDS相连接,并通过原核表达系统表达纯化相应蛋白;然后用该蛋白按浓度梯度:100,200,400,800,1000nmol/L与A549细胞共同孵育,各浓度在10,60,120,240min终止反应,通过Western Blotting检测进入细胞的p21蛋白量,以此确定最佳的转导条件;接着检测了转导进入A549细胞的Tat-p21融合蛋白在细胞中稳定存在的时间.然后通过绘制细胞生长曲线的方法比较了转导Tat-p21的细胞与负对照实验组生长速度的区别.结果发现,融合Tat跨膜结构域的p21蛋白能够高效进入A549细胞,并在细胞内发挥相应生物学功能,使细胞的生长受到抑制,增殖减慢;而作为负对照的p21蛋白则无相应功能.

In this study, by using a molecular cloning method, a recombinant protein Tat-p21 was constructed and expressed in vitro and purified. Incubated with A549 cells in a concentration series of 100, 200,400,800,1000 nmol/L. Each group of cells was harvested 10, 60, 120, 240 minutes after and tested by Western Blotting to find out the best condition for transduction. Assay testing the fusion protein＇ s period of time in stability after transduction into A549 was later conducted. A comparison of proliferation between the A549 transducted and not was brought out by cells counting and depicted in cell growth curves.