MEK高表达细胞膜层析柱制备及应用

Preparation and application of MEK high expression cell membrane chromatography column

[目的]通过制备MEK高表达细胞膜色谱层析柱,建立MEK潜在抑制剂筛选方法和体系。[方法]以非小细胞肺癌细胞系A549为材料,提取总RNA,RT-PCR体外扩增mek基因,随后克隆至p EGFP-C1质粒中,脂质体转染A549细胞,G418筛选,获得稳定转染细胞株,以此稳定转染细胞株为对象,构建MEK+/+的细胞膜层析柱。最后,以MEK抑制剂AZD-6244为受测体,流动相为磷酸盐缓冲液,变性洗脱液为p H 3. 3的磷酸盐缓冲液。所得所有组分采用高效液相检测,检测条件为:Dikma C18柱(150 mm×4. 6 mm,5μm);流动相为甲醇:5 mmol/L磷酸盐缓冲液(60∶40);检测波长为203 nm;柱温为30℃;流速为1. 0 m L/min。[结果]所构建的p EGFP-C1-MEK质粒经酶切、测序鉴定,结果正确;Western Blot检测稳定转染细胞株中MEK蛋白含量较A549细胞大幅提高;高效液相检测表明AZD6244在MEK+的A549细胞膜上有保留,表明所建MEK+A549细胞膜层析柱可应用于MEK抑制剂的筛选和检测。[结论]所构建的MEK高表达细胞膜层析柱具有成为MEK潜在抑制剂筛选的潜在有效方法。

[Objective]To build the novel MEK high expression cell membrane chromatography column.[Method]the full length of mek gene was amplificated from A549 cells using RT-PCR,then was cloned to plasmid pEGFP-C1.Plasmid pEGFP-C1-MEK was transfected to A549 cells with liposomes.On order to obtaining the stable transfected cells,G418 was used to screen the transfected cells.Then,the MEK+/+cell membrane chromatography column was constructed using the stable transfected cells.At last,AZD-6244,a MEK inhibitor,was used as the testing,the PBS as mobile phase,and pH 3.3 PBS as denatured eluent.All the components were detected by HPLC:dikma C18 column(150 mm*4.6 mm,5μm);mobile phase:methanol:5 mmol/L phosphate buffer(60∶40);detection wavelength:203 nm;column temperature:30℃;flow rate:1.0 ml/min.[Result]The results of restriction enzyme digestion and sequencing showed that the constructed pEGFP-C1-MEK plasmid were correct.Western Blot showed that the MEK protein content in stable transfected cells was significantly higher than that in A549 cells.The HPLC results showed that AZD-6244 was retained on the membrane of MEK overexpression A549 cells,indicated that the membrane chromatography column could be used to screen MEK receptors.[Conclusion]MEK high expression cell membrane chromatography column is expected to be an effective method for screening MEK potential inhibitors.