Restin基因启动子的克隆及其在HeLa细胞中的转录活性

Molecular cloning of restin gene promoter and its transcriptional activities in HeLa cells

目的:扩增细胞静息因子(restin)基因启动子并构建该启动子的荧光报告系统,探讨其对全反式维甲酸(all-transretinoicacid,ATRA)刺激的反应.方法:①对restin基因上游约2000bp基因组序列进行计算机生物信息学分析.②利用PCR技术扩增restin基因上游序列,将其克隆入pG5-luc中,插入荧光素酶报告基因上游,取代5个GAL-4结合位点及腺病毒启动子,构建Rp-luc质粒.③将构建的质粒转染入He-La细胞,ATRA诱导检测该质粒中荧光素酶的表达.结果:酶切及测序结果均证实成功克隆了restin基因上游序列,已将该序列插入到荧光素酶基因的上游;ATRA诱导转入HeLa细胞中的真核表达载体Rp-luc,荧光素酶表达明显增加.结论:成功构建了含有restin基因启动子序列的荧光报告系统,为进一步研究restin基因功能及其转录调控奠定了基础.

AIM： To clone the promoter and construct its luciferase report vector of our newly found gene rcstin and to analyze its response to the stimulation with all-trans-retinoic acid （ATRA）. METHODS： Approximate 2000 bp up from the ATG of gene rcstin was bioinformatics analyzed with computer. The fragment was generated by polymerase chain reaction and cloned the upstream of luciferase report gone into plasmid pG5-1uc, replacing the five GAL-4 binding sites and adenovints promoter to generate the plasmid Rp-luc. Rp-luc was transfected into HcLa cells and luciferase activities were measured with or without the treatment of ATRA at indicated time, RESULTS： We successfully cloned and identified the promoter of the gene restin and constructed its luciferase report plasmid. Obvious response to ATRA was found when transfected into HeLa cells and a high expression of luciferase was detected when induced with ATRA. CONCLUSION： The luciferase report system of restin gene promoter we have constructed can be applied for further study of the function and transcriptional regulation of restin.