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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 further study the role of LCN2 gene in acute kidney injury, cloning the coding sequence of LCN2, constructing luciferase reporter gene vector containing LCN2 gene and expressing it in renal tubular epithelial cell line HK-2. Methods Reverse transcription-polymerase chain reaction (RT-PCR) was performed using RNA extracted from HK-2 cell line as a template. The amplified product was digested with HindIII and SmaI and cloned into the dual luciferase reporter gene vector PGL3-Basic The constructed plasmid PGL3-Basic / LCN2 was introduced into HK-2 by non-liposome method. Ampicillin was selected and cultured, and its expression was confirmed by dual-luciferase. Results The PCR and sequencing results showed that the amplified LCN2 promoter sequence was correct. The restriction enzyme digestion confirmed that the recombinant luciferase reporter gene vector was successfully constructed. The luciferase activity of HK-2 cells transfected with PGL3-Basic / LCN2 plasmid was significantly higher than that of empty vector (P <0.000 1). Conclusion The coding sequence of LCN2 was successfully cloned and its luciferase reporter gene vector PGL3-Basic / LCN2 was constructed. It is helpful to further study the mechanism of LCN2 gene in acute renal injury.