大鼠Eap1-shRNA慢病毒干扰载体的构建及细胞水平阻断Eap1对Kiss1表达的影响

Construction of sh RNA lentivirus vector targeting rat Eap1 gene and Eap1 effects on Kiss1 gene at cellular level

目的·构建大鼠Eap1-shRNA慢病毒干扰载体,观察在细胞阻断Eap1基因表达对Kiss1基因表达的影响。方法·针对大鼠Eap1基因mRNA序列设计4条shRNA干扰序列及1条阴性对照序列,序列合成后连接至LV3质粒构建重组慢病毒质粒载体。经鉴定正确后,与包装质粒pGag/Pol、pRev、p VSV-G共同转染293T细胞进行慢病毒包装并测定病毒滴度。将构建好的慢病毒感染NRK-52E细胞,感染72 h后收集细胞,采用real-time PCR及Western blotting技术检测Eap1基因mRNA和蛋白表达水平的变化。筛选出干扰效果最佳的干扰慢病毒再次感染NRK-52E细胞(设置空白对照和阴性对照),感染72 h后检测Kiss1 mRNA表达水平的变化。结果·NRK-52E细胞感染72 h后,阴性对照慢病毒与空白对照组相比,Eap1 mRNA及蛋白的表达量均无明显差异,而各干扰病毒组Eap1 mRNA及蛋白的表达量均显著降低,且以慢病毒Eap1-shRNA4干扰效果最佳。阻断NRK-52E细胞Eap1基因表达后,与对照组相比,其Kiss1 mRNA表达水平明显增高。结论·成功构建大鼠Eap1-shRNA慢病毒表达载体;阻断NRK-52E细胞Eap1基因表达后,Kiss1表达水平下降。

Objective · To construct effective shRNA-lentiviral vector targeting rat Eapl gene and explore the effect of Eapl on Kiss1 at cellular level. Methods. Four shRNA sequences targeting rat Eapl mRNA and one negative control sequence were designed and synthesized, and then recombined with lentiviral vectors. After DNA sequencing identification, the shRNA recombinant vectors were co-transfected into 293T cells with pGag/Pol, pRev, pVSV-G to packaging lentiviral particles and the virus titers were determined. NRK-52E cells were transfected with all the four lentiviral-Eapl-shRNAs and the negative control lentivirus, and cells were harvested after 72 h. Real-time PCR and Western blotting was performed to detect the change of the mRNA and protein level ofEapl gene. The most effective LV-Eapl-shRNA was used to infect NRK-52E cells [non-lentivirus and Eapl-shRNA（-） lentivirus as control groups], and then the level of Kiss1 mRNA expression was detected after infection for 72 h. Results. Seventy-two hours after transfection, the Eapl mRNA and protein expression between blank group and LV-shRNA（-） group showed no significant difference; while Eapl expression in all LV-Eapl-shRNA groups reduced significantly in comparing to the control groups. In addition, the lentiviral-Eapl-shRNA4 was the most effective in RNA interference. After blocking Eapl expression in NRK-52E cells, the Kiss1 mRNA level was significantly increased comparing to the control groups. Conclusion. The effective Eapl-shRNA lentiviral vector is established successfully. The Kissl gene expression decreases after blocking Eapl in NRK-52E cell line.