辐射诱导表达载体pEgr-p16的构建及其体外蛋白表达的研究

Construction of expression vector pEgr-p16 and determination of p16 in vitro expression induced by ionizing radiation

目的构建辐射诱导表达载体pEgrp16并研究其体外稳定转染联合60Coγ射线照射对人宫颈癌HeLa细胞p16蛋白表达变化的影响。方法利用双酶切、粘端连接的方法构建了含有辐射诱导特性的早期生长反应因子Egr1和p16的pEgrp16的质粒载体,以脂质体介导的方法,将重组载体导入人HeLa细胞,采用免疫细胞化学和流式细胞术的方法检测照射转染后的HeLa细胞p16蛋白表达的变化。结果经全自动测序证明辐射诱导表达载体pEgrp16构建正确;稳定转染可见有明显的p16表达增强;5Gy以内p16蛋白表达呈剂量依赖性的增加,在2Gy照射后2hp16蛋白表达水平即开始增高,4h达到最高,12h趋于正常水平。结论本研究成功构建了辐射诱导表达载体pEgrp16,在HeLa细胞中蛋白表达明显增强。

Objective In this study, a recombinant plasmid pEgr-p16 was constructed, and the protein expression of p16 in HeLa cells was determined after the cells were transfected in combination with ^60 Co γ-rays irradiation. Methods The pEgr-p16 recombinant plasmid containing radio-sensitive promoter Egr-1 and p16 encoding sequence was constructed by double-enzyme cleavage and sticky end ligation techniques, pEgr-p16 plasmid was packed with lipofectamine to transfect human cervix epithelial carcinoma HeLa cell line, and p16 protein level was detected by immunocytochemistry and FCM. Results The plasmid pEgr-p16 was constructed correctly as confirmed by sequencing in both directions. The expression level of p16 protein was increased significantly. The level of p16 expression was dose-dependently increased below 5 Gy irradiation. The p16 level began to increase at 2 h after 2 Gy irradiation and there was a significant increase at 4 h, then gradually decreased to the control level at 12 h. Conclusion The expression vector pEgr-p16 which can be induced by ionizing radiation has been constructed successfully in this study. The expression level of p16 protein in HeLa cells increases markedly.