小鼠 Egr-1启动子的克隆及其辐射诱导特性的研究

Study on cloning of the Egr-1 promoter and its radiation-inducible property

目的 :扩增 Egr- 1启动子并构建 Egr- PGL重组质粒 ,探讨不同剂量 X射线诱导被转染的NIH3T3细胞荧光素酶表达活性。方法 :利用 PCR方法扩增 Egr- 1p与 PGL3- E表达载体连接 ,用发光仪测定荧光素酶与底物反应的发光值 ,分析 X射线诱导荧光素酶表达。结果 :对 PCR产物进行测序 ,序列基本正确 ;电泳检测重组质粒 ,有正确的特异性片段 ;重组质粒转染小鼠 NIH3T3细胞鉴定 Egr- 1p的辐射调节特性 ,发现照射组发光值均高于假照组 ,75m Gy照射组的表达量约为假照组的 5倍 ,2、4、6、8和 10 Gy照射组的表达量分别约为假照组的 5～ 7.5倍。结论 :扩增得到的 Egr- 1启动子在不同剂量 X射线照射下均具有诱导下游基因表达增强作用 ;低剂量照射诱导 Egr- 1启动子下游基因表达增强作用的发现可能在肿瘤基因治疗中更具有实用价值。

? Objective:Egr-1 promoter was amplificated and Egr-PGL plasmid was constructed to study the expression of luciferase in transfected NIH3T3 cells after different doses of X-ray irradiation.Methods:Egr-1p was amlificated by PCR and inserted into PGL3-E vector.The expression of luciferase induced by X-ray was studied by counting the light of luciferase and stubstrate.Results:Egr-1 cDNA was obtained by PCR and was sequenced.The results indicated the sequence was almost correct.The Egr-1p was connected with PGL3 vector and was detected by electrophoresis.The constructs were used to transfect mouse NIH3T3 cells to characterize the regulatory function of Egr-1p after exposure to X-ray irradiation.The results indicate that the expression of luciferase of all groups irradiated is highter than that of 0 Gy group.The expression of groups irradiated is about 5-7.5 times greater than that of 0 Gy group.Conclusion:Egr-1pobtained can induce the expression of its downstream gene after different doses of X ray irradiation.Low dose irradiation also can do it and it is may be more important in tumor gene therapy. 〔