| LIU Jian-xiang,SU Xu.Construction of expression vector pEgr-p16 and determination of p16 in vitro expression induced by ionizing radiation[J].Chinese Journal of Radiological Medicine and Protection,2005,25(5):416-419 |
| Construction of expression vector pEgr-p16 and determination of p16 in vitro expression induced by ionizing radiation |
| Received:January 18, 2005 |
| DOI： |
| KeyWords:Human suppressor gene p16 pEgr-16 gene Expression vector induced by ionization radiation Gene combined with radiation therapy |
| FundProject:中国科学院近代物理研究所重离子加速器国家实验室课题基金资助 |
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| Abstract:: |
| 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 60Co 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. |
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