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目的构建NEP启动子缺失体-荧光素酶报告基因质粒,探查神经内肽酶(NEP)基因启动子中与人参皂苷Rb1影响NEP启动子活性有关的位点。方法用NEP基因5’上游启动区2.4 kb片段和荧光素酶报告基因载体pGL3-basic构建NEP启动子-荧光素酶报告基因质粒pGL3-nep2.4;用Erase-a-Base System对pGL3-nep2.4质粒2.4 kb DNA插入片段5’端进行缺失,构建NEP启动子缺失体-荧光素酶报告基因质粒;用Rb1处理NEP启动子缺失体转染的人神经母细胞瘤SH-SY5Y细胞,检测双荧光素酶活性,观察Rb1对NEP启动子活性的影响。结果成功构建了含NEP启动子DNA序列的重组质粒pGL3-nep2.4。荧光素酶活性检测显示,转染pGL3-nep2.4质粒的SH-SY5Y细胞荧光素酶活性是转染pGL3-basic的13.1倍(P<0.01)。Rb1处理可使转染pGL3-nep2.4质粒的SH-SY5Y细胞荧光素酶活性增加,其是对照组的2.9倍(P<0.01);细胞荧光素酶活性检测亦显示,NEP基因上游启动区-894~-857 bp(RegionⅠ)及-100~-82 bp(RegionⅣ)片段缺失后,启动子活性分别是缺失前的29%(P<0.01)和25%(P<0.01),-559~-534 bp(RegionⅡ)及-223~-179 bp(RegionⅢ)片段缺失后,启动子活性分别是缺失前的5.12倍(P<0.01)及1.81倍(P<0.01)。Rb1处理后,RegionⅠ、Ⅱ和Ⅲ缺失体质粒转染细胞荧光素酶活性均与对照组无统计学差异(P>0.05),RegionⅣ缺失质粒pGL3-226转染细胞荧光素酶活性也与对照组无明显差异(P>0.05),而含RegionⅣ质粒pGL3-244转染细胞荧光素酶活性则是对照组的1.61倍(P<0.01)。结论 NEP基因5’上游2.4 kb DNA片段有较强的启动子活性,Rb1能明显增加其活性。NEP基因2.4 kb DNA片段有2个正调控区(RegionⅠ和RegionⅣ)和2个负调控区(RegionⅡ和RegionⅢ),Rb1通过RegionⅣ发挥正调控作用。
OBJECTIVE: To construct a NEP promoter-luciferase reporter gene plasmid and investigate the relationship between NEP promoter and Ginsenoside Rb1 in NEP promoter. Methods The NEP promoter-luciferase reporter plasmid pGL3-nep2.4 was constructed by using the 2.4 kb fragment of the 5 ’upstream promoter region of the NEP gene and the luciferase reporter gene vector pGL3-basic. The pGL3-nep2 .4 plasmid DNA fragment of 2.4 kb was deleted at the 5 ’end to construct the NEP promoter deletion-luciferase reporter gene plasmid; the human neuroblastoma SH-SY5Y cells transfected with the NEP promoter deletion body were treated with Rb1 and detected Dual luciferase activity observed Rb1 NEP promoter activity. Results The recombinant plasmid pGL3-nep2.4 containing the NEP promoter DNA sequence was successfully constructed. The luciferase activity assay showed that the luciferase activity of SH-SY5Y cells transfected with pGL3-nep2.4 plasmid was 13.1 times that of pGL3-basic transfected cells (P <0.01). Rb1 treatment could increase the luciferase activity of SH-SY5Y cells transfected with pGL3-nep2.4 plasmid, which was 2.9 times that of the control group (P <0.01). The luciferase activity assay also showed that the upstream promoter region of NEP gene The promoter activity of -894 ~ -857 bp (Region Ⅰ) and -100 ~ -82 bp (Region Ⅳ) were 29% (P <0.01) and 25% (P <0.01) The promoter activity of -534 bp (Region Ⅱ) and -223 ~ -179 bp (Region Ⅲ) was 5.12-fold (P <0.01) and 1.81-fold (P <0.01) less than that before deletion, respectively. After Rb1 treatment, the luciferase activity of cells transfected with Region I, II and III deletion plasmids was not significantly different from that of the control group (P> 0.05). The luciferase activity of the plasmid pGL3-226 transfected with Region IV was also lower than that of the control group (P> 0.05). The luciferase activity of pGL3-244 transfected with RegionⅣ plasmid was 1.61 times that of the control group (P <0.01). Conclusion The 2.4 kb DNA fragment upstream of 5 ’of NEP gene has strong promoter activity, and Rb1 can significantly increase its activity. The 2.4 kb DNA fragment of NEP gene has two positive regulatory regions (Region I and Region IV) and two negative regulatory regions (Region II and Region III). Rb1 plays a positive regulatory role through Region IV.