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目的构建BC047440基因RNAi慢病毒干扰载体,并检测其对人肝癌细胞系HepG2细胞中BC047440基因的沉默效果。方法针对已经筛选确定的BC047440基因RNAi有效靶序列,合成BC047440基因特异性shRNA序列,退火形成双链DNA,与经HpaⅠ和XhoⅠ双酶切后的pFU-GW-iRNA载体连接产生pFU-GW-shBC047440慢病毒载体,PCR、DNA测序鉴定阳性克隆。用脂质体转染法将pFU-GW-shBC047440、pHelper 1.0载体和pHelper 2.0载体共转染293T细胞,产生具备感染能力的慢病毒。以293T细胞中绿色荧光蛋白的表达水平测定病毒滴度,并测定其对人肝癌细胞株HepG2细胞最适感染复数(MOI)。以最适MOI感染HepG2细胞,分BC047440-shRNA组、control-shRNA组和HepG2组;RT-PCR和Western blot检测各组细胞BC047440 mRNA及蛋白的表达差异。结果经PCR鉴定,成功构建BC047440基因RNAi慢病毒载体。测定慢病毒滴度为5×108TU/ml,其在HepG2细胞中最适MOI为20;RT-PCR和Western blot检测结果分别显示BC047440-shRNA组中BC047440 mRNA及BC047440蛋白表达较control-shRNA组和HepG2组明显降低,control-shRNA组与HepG2组间无明显差异。结论成功构建BC047440特异性RNAi慢病毒载体,感染人肝癌细胞系HepG2细胞后,实现了对HepG2细胞中BC047440基因的有效沉默。
Objective To construct the RNAi lentivirus vector of BC047440 gene and test its silencing effect on BC047440 gene in HepG2 cell line. Methods The specific siRNA sequence of BC047440 gene was screened and the specific shRNA sequence of BC047440 gene was synthesized and annealed to form double-stranded DNA. The double-stranded DNA was ligated with pFU-GW-iRNA vector digested with HpaI and XhoI to generate pFU-GW-shBC047440 Lentiviral vectors, PCR, DNA sequencing identified positive clones. 293T cells were cotransfected with pFU-GW-shBC047440, pHelper 1.0 vector and pHelper 2.0 vector by lipofection method to produce infectious lentivirus. The virus titer was determined by the expression level of green fluorescent protein in 293T cells and the optimal multiplicity of infection (MOI) for HepG2 cells was determined. HepG2 cells were infected with the optimal MOI, and were divided into BC047440-shRNA group, control-shRNA group and HepG2 group. The expression of BC047440 mRNA and protein in each group were detected by RT-PCR and Western blot. Results The BC047440 gene RNAi lentiviral vector was successfully constructed by PCR. The titer of lentivirus was 5 × 108TU / ml and the optimum MOI was 20 in HepG2 cells. The results of RT-PCR and Western blot showed that the expression of BC047440 mRNA and BC047440 in BC047440-shRNA group were significantly higher than that in control-shRNA group and HepG2 group was significantly reduced, control-shRNA group and HepG2 group no significant difference. Conclusion The BC047440 specific RNAi lentiviral vector was constructed successfully and infected with human hepatocellular carcinoma cell line HepG2. The effective silencing of BC047440 gene in HepG2 cells was achieved.