大鼠PAX2基因RNA干扰慢病毒载体的构建及沉默效果

Construction of lentiviral vector of shRNA specific for rat PAX2 and the silencing effect

目的构建PAX2特异性shRNA干扰慢病毒载体并观察其对大鼠肾小管上皮细胞(NRK52E)中PAX2基因的沉默效果。方法采用PAX2基因RNAi靶点序列合成靶序列的Oligo DNA,退火形成双链DNA,与pGCSIL-GFP载体连接产生shRNA慢病毒载体,经PCR筛选阳性克隆进行DNA测序鉴定。用脂质体转染法将质粒共转染293T细胞,将包装产生的慢病毒颗粒感染NRK52E细胞,RT-PCR、Western印迹检测PAX2 mRNA和蛋白的表达。结果 PCR与DNA测序证实合成的含PAX2 shRNA慢病毒载体寡核苷酸链插入正确。经RNA干扰后的靶细胞PAX2 mRNA及蛋白表达水平明显降低。结论成功构建人PAX2基因RNA干扰慢病毒载体,为以PAX2基因为靶点的梗阻性肾病基因治疗研究奠定了基础。

Objective To observe the influence of lentiviral vector-mediated RNA interference on expression of rat PAX 2 gene in rat normal renal tubular epithelial cells (NRK52E).Methods The effective RNA interference sequence targeting PAX 2 gene was screened and determined according to previous study.The complementary DNA containing both sense and antisense Oligo DNA of the targeting sequence was designed,synthesized and cloned into pGCSIL-GFP vector to construct a lentiviral vector which expressed PAX 2 shRNA,subsequently confirmed by PCR and DNA sequencing analysis.293 T cells were co-transfected with the plasmids using liposome transfection methods ,and packaged to produce lentiviral particles.The recombinant lentiviruses were transfected into NRK52 E cells, and the PAX2 mRNA and protein expression were examined by RT-PCR and Western blot .The results were compared with those of the non-transfected and blank vector trans-fected NRK52E cells.Results PCR analysis and DNA sequencing confirmed that the PAX 2 shRNA sequences were successfully inserted in-to the lentiviral vectors.After transfection with PAX2 shRNA,the PAX2 expression in NRK52E cells was significantly inhibited at both mR-NA and protein levels compared with that in non-transfected and empty vector transfected NRK 52E cells.Conclusions The lentiviral RNAi vectors targeting PAX2 gene have been successfully constructed ,and can effectively inhibit the expression of PAX 2 gene in NRK52E cells.It paves a way for PAX2-targeted gene therapy of obstructive nephropathy.