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目的构建醌型二氢生物碟呤还原酶(quinonoid dihydropteridine reductase,QDPR)敲低慢病毒模型,检测其对肾小管上皮NRK-52E细胞QDPR基因的敲低情况。方法设计合成QDPR的干涉shRNA序列,并重组入GP2慢病毒载体质粒,测序检验重组质粒是否转入该序列。三质粒共转染获得干涉慢病毒,将其感染NRK-52E细胞,新霉素筛选抗性细胞,用实时定量PCR、western blot和QDPR酶活性测量检测干涉效果。结果重组入GP2的序列与设计序列一致,病毒转染NRK-52E细胞使其获得了新霉素抗性,QDPR基因表达、蛋白含量及酶活性显著降低。结论本研究合成的QDPR基因敲低慢病毒能使NRK-52E细胞QDPR基因的mRNA转录、蛋白浓度及活性降低,为后续QDPR基因在糖尿病肾病中的作用研究提供了模型。
OBJECTIVE: To construct a quercetin-type dihydropteridine reductase (QDPR) knock-down lentivirus model to detect the knockdown of QDPR gene in renal tubular epithelial cells of NRK-52E cells. Methods Interfering shRNA sequences of QDPR were designed and synthesized. The GP2 lentiviral vector plasmid was recombined and sequenced to determine if the recombinant plasmid was transfected into this sequence. Three plasmids were co-transfected into lentivirus to infect NRK-52E cells. Neomycin was screened for the resistant cells. The interference effect was detected by real-time PCR, western blot and QDPR enzyme activity assay. Results The sequence of recombinant GP2 was identical with the designed sequence. The virus was transfected into NRK-52E cells to obtain neomycin resistance, and the QDPR gene expression, protein content and enzyme activity were significantly decreased. Conclusions The knockdown of lentivirus with QDPR gene in this study can reduce mRNA transcription, protein concentration and activity of QDPR gene in NRK-52E cells, which provides a model for further research on the role of QDPR gene in diabetic nephropathy.