去泛素化酶USP10 RNAi慢病毒载体的构建及其对PC12细胞增殖的影响  

Construction and identification of lentiviral vector of RNA interference of USP10

作　　者：叶樱泽 张旭[1] 曾智[2] 古丽娟[1] YE Yingze;ZHANG Xu;ZENG Zhi;GU Lijuan(Central Laboratory, Wuhan 430060,Hubei,China;Dept.of Pathology,Renmin Hospital of Wuhan University, Wuhan 430060,Hubei,China)

机构地区：[1]武汉大学人民医院中心实验室,湖北武汉430060 [2]武汉大学人民医院病理科,湖北武汉430060

出　　处：《武汉大学学报（医学版）》2019年第1期6-10,16,共6页Medical Journal of Wuhan University

基　　金：国家自然科学基金资助项目(编号:81771283);国家自然科学基金青年项目(编号:81602535)

摘　　要：目的:构建大鼠去泛素化酶USP10siRNA重组慢病毒载体,为探讨USP10对神经元的调控作用提供体外模型。方法:设计并合成3对特异性大鼠USP10基因的RNAi靶序列,聚合酶链反应(PCR)扩增后,用AgeⅠ及EcoRⅠ双酶切及连接酶构建线性化的GV208慢病毒载体;转化感受态细胞,经Amp抗性筛选阳性克隆,PCR鉴定阳性克隆载体并测序;病毒包装并转染至293T细胞,观察绿色荧光蛋白(GFP)表达,荧光法检测滴度。用重组慢病毒感染PC12细胞,WesternBlot检测USP10蛋白的表达,CCK-8法检测PC12细胞增殖。结果:经测序鉴定成功构建了3对USP10RNAi慢病毒载体,感染PC12细胞后,USP10蛋白的表达显著被抑制,PC12细胞的增殖能力明显减弱。结论:成功构建能高效、稳定抑制USP10表达的PC12细胞株,并明显抑制PC12细胞增殖能力,为探讨USP10对神经元的调控作用提供基础。Objective: To construct a lentiviral vector of RNA interference(RNAi) of rat ubiquitination enzyme10(USP10), and then provide a cell model for exploring the regulation of USP10 on neurons.Methods: Three interfering sequences targeting USP10 were designed, amplification of USP10 gene fragment was performed by polymerase chain reaction(PCR). The GV208 lenti-virus vector was digested with Age Ⅰ and EcoR Ⅰ to linearize, then linked with the USP10 gene fragment by a ligase.The ligation products were transformed to competent cells, the positive clones were screened, and then identified by PCR follwed by RNA sequencing. Next, the lentiviral vectors carrying USP10 RNAi were packaged and transfected in 293 cells by using the GV208 Lentiviral Packaging Kit, and then the virus titer was measured by fluorescence method. Furthermore, the lentiviral particles were collected to infect PC12 cells, and the transfection efficiency was observed under fluorescence microscope.PC12 cells with stable USP10 down-regulation were screened with puromycin, and the expres-sion level of USP10 was detected by Western blotting. The proliferation of PC12 cells after the infection was assessed using CCK-8 assay. Results: RNA sequencing demonstrated successful construction of three USP10 RNAi lentivirus vectors. After infected PC12 cell, the stable cell line with USP10 knockdown was successfully screened and the proliferation of PC12 cells was also significantly decreased.Conclusion: PC12 cells line transfected by USP10 RNAi lentiviral vector is successfully constructed,which can effectively and stably inhibit the expression of USP10, and decrease proliferation of PC12 cells significantly, thus laying a foundation for further study on the regulation of USP10 on neurons

关 键 词：去泛素化酶(USP10) 慢病毒载体 核糖核酸干扰(RNAi) PC12细胞 

分 类 号：Q591.2[生物学—生物化学] R730.2[医药卫生—肿瘤]