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目的建立稳定敲低UBC9蛋白表达的293T细胞株,检测其对雌激素受体β(ERβ)类泛素化修饰水平的调节作用。方法构建4条针对人UBC9基因的慢病毒短发夹RNA(shRNA)的干扰载体,将其与慢病毒包装辅助质粒共转染293T细胞后,收集病毒上清,感染293T细胞,经嘌呤霉素(puromycin)筛选后获得稳定表达慢病毒介导UBC9 shRNA的混合细胞集落,分别用实时(real-time)PCR和Western印迹方法检测UBC9的表达情况,瞬时转染方法检测UBC9表达水平对ERβ类泛素化修饰水平的影响。结果建立了稳定敲低UBC9的293T细胞株,UBC9降低能够抑制ERβ类泛素化修饰水平。结论 UBC9在ERβ类泛素化修饰反应中发挥重要作用。
OBJECTIVE: To establish a 293T cell line stably knocking down the expression of UBC9 protein and to investigate its regulation on ubiquitination level of estrogen receptor β (ERβ). Methods Four shRNA lentiviral vectors targeting human UBC9 gene were constructed and co-transfected with lentivirus packaging helper plasmids into 293T cells. The virus supernatants were collected and infected into 293T cells. After puromycin The mixed cell colonies stably expressing lentivirus-mediated UBC9 shRNA were obtained by puromycin screening. The expression of UBC9 was detected by real-time PCR and Western blotting respectively. The expression of UBC9 was detected by transient transfection method. The effect of the level of chemical modification. Results The 293T cell line stably knocked down UBC9 was established. The decrease of UBC9 could inhibit the ERβ ubiquitination level. Conclusion UBC9 plays an important role in ERβ ubiquitination modification.