小鼠Egr-1基因调控序列的克隆及其辐射诱导特性的鉴定

Cloning of the Regulatory Sequence of Mouse Egr-1 Gene and Characteristics of Its Radiation-Inducible Property

Egr-1(early growth response-1)系-具有辐射诱导特性的转录因子.其调控序列被证实可通过与肿瘤杀伤基因相连接的方法而赋予后者以辐射诱导特性,因而被用于肿瘤的基因-放射治疗研究.为此,我们用PCR方法从BALB/c小鼠基因组DNA扩增出445bp长Egr-1基因调控序列,序列分析证实,除-392位一个碱基(A→G)相差外,其余均与文献报道一致,包括6个对辐射诱导特性起关键作用的CC(A+T)_6GG结构域-将克隆的Egr-1基因调控序列连入pCL3荧光素酶报告质粒,转染小鼠恶性黑色素瘤细胞(B16),转染细胞用^(60)Coγ-线进行2.5、5.0和10.0Gy不同剂量照射后,检测细胞裂解液中荧光素酶活性.与未照射细胞比较.照射后细胞荧光素酶活性明显提高,以2.5Gy剂量最为显著.提高约138%,表明Egr-1基因调控序列具有辐射后诱导下游基因表达的功能.

Egr-1 is a radiation-inducible transcription factor. It was demonstrated that the regulatory sequence in 5' flanking region of Egr-1 gene could confer the radiation inducibility upon a tumoricidal gene to which it was linked, and was consequently used in experiments on the gene-radiotherapy to tumor. As a first step to test the possibility, we have isolated the 445 bp-long regulatory sequence of Egr-1 gene from BALB/c mouse gcnomic DNA with PCR method. Sequence analysis confirmed that nucleotide sequence of this cloned fragment was identical with one reported previously with only one base difference in A to G substitution at residue - 392, and had six CC( A + T)6GG domains which were essential to the radiation-inducible property of Egr-1 gene. This fragment then was inserted into the Mlu I-Bgl II site of pGL3, a reporter vector containing luciferase gene. The constructs were used to transfect mouse malignant melanoma cells (B16) to characterize the regulatory function of this CC ( A + T)_(6)GG-rich sequence after exposure to r-radiation by a ^(60) Co source. The results indicate that the activity of luciferase in transfectant increases by 138% as compared with control levels, demonstrating that r-radiation inducibility of gene expression can be conferred by the regulatory sequence.