Tet-on控制的Luciferase和SupF突变报告基因质粒的构建及其在顺铂致突变作用中的应用研究

Construction of bidirectional Tet-responsive promoter plamid and application in the research of cisplatin mediated mutation

目的研究TCR在顺铂导致的细胞内DNA损伤和突变过程中的分子机制,并为进一步研究转录偶联修复与突变发生的分子机制提供重要分子生物学依据。方法首先将SupF突变报告基因克隆到带有双向真核启动子的含有Tet调控元件TRE的表达载体pBI-L的多克隆位点(MCS)上,获得pTCR-1,再将SV40ori元件插入pTCR-1载体,最终获得pTCR-2质粒。酶切和DNA测序证实后,用顺铂将pTCR-2进行体外DNA损伤后瞬时转染至Tet-on293细胞,在Dox诱导下培养48h,从细胞内提取质粒,纯化后转化SY204菌株,蓝白斑筛选挑取白色突变斑,计算突变频率并进行DNA测序检测突变频谱。结果经酶切鉴定和测序分析,插入片段长度和序列正确。在Dox诱导下取得了SupF报告基因在转录偶联修复途径中的突变频率与频谱。结论重组的pTCR-2质粒具有在真核细胞中Tet启动的转录活性,应用于顺铂致突变研究中,使转录条件下的突变情况能够得以反映。

Objective For the research of the molecular mechanism of TCR process, we constructed a bidirectional Tet-responsive promoter plasmid including two report gene luciferase and SupF gene. This plasmid was used to detect the mutation frequency and spectra mediated by cisplatin in the TCR pathway. Methods SupF gene was cloned into a Tet-responsive plamid PBI-L that contains a bidirectional Tet-responsive promoter, and this subclone was named pTCR-1. Then SV40 ori fragment obtained form pEGFP-1 plasmid was ligated with pTCR-1, and named pTCR-2. The pTCR-2 plasmid was exposed to cisplatin in vitro and transfected into Tet-on 293 cell lines, then cultured in the media containing DOX for 48 h. The mutation frequency and spectra of supF gene were finally detected. Results The restriction digestion and sequence analysis demonstrated that the length of pTCR-2 plasmid and the SupF gene sequence were correct. The mutation frequency and spectra were ob- tained at the present of DOX. Conclusion lished and works as expected. It can be used A new Tet-responsive promoter plasmid has been successfully estab-to analyze the mutation frequency and spectra induced by cisplatin.