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ＤＮＡ损伤的检测对预防癌症和遗传病等非常重要。采用分子克隆技术，将报告基因—绿色荧光蛋白（ＧＦＰ）置于ＳＶ４０基本启动子调控下，构建成对照载体ｐＳＶ－ＧＦＰ。在ＳＶ４０基本启动子上游插入寡核苷酸Ｐ５３ＲＥ，构建成示踪载体ｐ５３ＲＥ－ＧＦＰ。转染ＮＩＨ３Ｔ３细胞，以ＧＦＰ示踪细胞内源性Ｐ５３的转录激活活性。紫外线照射或Ｈ２Ｏ２处理转化细胞使ＤＮＡ损伤，诱导细胞内源性Ｐ５３的表达。用激光扫描共聚焦成像系统（ＬＳＣＩＳ）对细胞进行红、绿、蓝三色光融合成像，并测定ＧＦＰ经４８８ｎｍ激发后发出的绿色荧光光密度，验证ＧＦＰ示踪Ｐ５３的特异性。ｐ５３ＲＥ－ＧＦＰ转化细胞３Ｔ３－ＲＥＧ经紫外线照射或Ｈ２Ｏ２处理后，ＧＦＰ的表达增高，处理后１ｈｒ光密度即达到最高水平，随后逐渐降低。血清“饥饿”—非ＤＮＡ损伤处理的３Ｔ３－ＲＥＧ细胞，以及经紫外和Ｈ２Ｏ２处理的对照载体ｐＳＶ－ＧＦＰ转化细胞３Ｔ３－ＳＶＧ，ＧＦＰ的表达无明显增强。实验表明：ＧＦＰ示踪内源性Ｐ５３转录激活活性用于检测ＤＮＡ损伤有很高的灵敏度和特异性，适宜推广应用。
The detection of DNA damage is very important for the prevention of cancer and genetic diseases. The reporter gene, green fluorescent protein (GFP), was placed under the control of the SV40 basic promoter using molecular cloning technology to construct a control vector pSV-GFP. The oligonucleotide P53RE was inserted upstream of the SV40 basic promoter and constructed as a tracer vector p53RE-GFP. NIH3T3 cells were transfected with GFP for the transcriptional activation of endogenous P53. UV irradiation or H2O2 treatment transforms cells to DNA damage and induces endogenous P53 expression. The cells were fused by red, green and blue light with laser scanning confocal imaging system (LSCIS), and the green fluorescence density of GFP after 488nm excitation was measured to verify the specificity of GFP tracing P53. The expression of GFP increased after treated with 3T3-REG of p53RE-GFP cells under ultraviolet light or H2O2 treatment. The optical density of p53RE-GFP reached the highest level at 1hr and then decreased gradually. Serum “starvation” - 3T3-REG cells not treated with DNA damage and 3T3-SVG cells transformed with UV and H2O2-treated control vector pSV-GFP showed no significant increase in GFP expression. Experiments show that: GFP tracing endogenous P53 transcriptional activation activity for the detection of DNA damage has a high sensitivity and specificity, suitable for the promotion and application.