LCN2基因启动子区荧光素酶报告基因载体的构建及鉴定  

Construction and identification of luciferase reporter gene vector containing LCN2 promoter

作　　者：崔丽艳[1] 杨硕[1] 张捷[1] 

机构地区：[1]北京大学第三医院检验科,北京100191

出　　处：《中国误诊学杂志》2011年第21期5045-5047,共3页Chinese Journal of Misdiagnostics

基　　金：北京大学第三医院中青年骨干基金资助(72528-01)

摘　　要：目的为进一步研究LCN2(Lipocalin 2)基因在急性肾损伤中的作用,克隆LCN2的编码序列,构建含LCN2基因的荧光素酶报告基因载体,并在肾小管上皮细胞系HK-2中表达。方法以HK-2细胞系提取的RNA为模板进行反转录-聚合酶链反应(RT-PCR),扩增产物用HindⅢ和SmaI双酶切后克隆到双荧光素酶报告基因载体PGL3-Basic中,用非脂质体法将构建质粒PGL3-Basic/LCN2导入HK-2中,氨苄青霉素选择培养,经双荧光素酶鉴定其表达。结果PCR和测序结果表明扩增的LCN2启动子序列正确,酶切检测证实重组荧光素酶报告基因载体构建成功。转染PGL3-Basic/LCN2质粒的HK-2细胞荧光素酶活性比转染空载体的明显增加(P<0.000 1)。结论成功克隆了LCN2的编码序列,构建了其荧光素酶报告基因载体PGL3-Basic/LCN2,有助于进一步研究LCN2基因在急性肾损伤中的作用机制。Objective To clone the coding sequence of LCN2 gene,establish LCN2 containing luciferase reporter system and further express LCN2 in renal tubular epithelial cell strain HK-2.Methods Reverse transcription polymerase chain reaction（RT-PCR） was performed based on the RNA module extracted from human renal tubular epithelial cell strain HK-2.The product for amplification was cloned to the oriented reporter gene vector PGL3-Basic after being cleaved by HindⅢ and SmaI,and was identified according to both the recombinant plasmid PGL3-Basic/LCN2 cleaved by restriction endonuclease and DNA sequencing.The recombined vector was transfected into HK-2 cells using the non lysosomal method,and the activity of the luriferase was determined after cells,selected by Amp for culture,and was identified using dual luriferase detection.Results PCR and sequencing results indicated that the amplified sequence was correct.The results of restriction enzyme digestion indicated that the recombinant vector PGL3-Basic/LCN2 was successfully constructed.The luciferase reporter system showed that the transcription activation of PGL3-Basic/LCN2 was significantly higher than PGL3-Basic（P0.000 1）.Conclusion The coding sequence of human LCN2 gene is successfully cloned in this study.The establishment of luciferase reporter gene vector PGL3-Basic/LCN2 may be helpful to the understanding of mechanism by which it influences the acute kidney injury in further studies.

关 键 词：急相蛋白质类/遗传学 原癌基因蛋白质类/遗传学 肾/损伤 

分 类 号：R692[医药卫生—泌尿科学]