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目的构建靶向人ERG基因的siRNA重组慢病毒表达载体。方法针对人ERG基因的mRAN序列,设计特异性干扰序列,两端引入酶切位点,人工合成OligoDNA,退火后与双酶切的pLVX-shRNA2慢病毒载体质粒连接,构建慢病毒载体质粒pLVX-shRNA2-ERGsiRNA,通过测序鉴定连接正确后,以293T细胞包装得到荷载ERGsiRNA基因的慢病毒;根据293T细胞表达绿色荧光蛋白水平计算病毒滴度;病毒感染前列腺癌VCaP细胞株,通过实时荧光定量PCR检测ERGmRNA的表达。结果测序结果表明,靶向人ERG基因的siRNA慢病毒表达载体构建成功,包装后的慢病毒滴度为1.1×108TU/ml。感染VCaP细胞后,实验组和对照组相比ERGmRNA的表达水平显著降低,siRNA对ERG表达的干扰效率为87.46%。结论成功构建靶向人ERG基因的siRNA慢病毒表达载体。
Objective To construct siRNA recombinant lentivirus vector targeting human ERG gene. Methods Specific mRAN sequences of human ERG gene were designed and specific interference sequences were designed. OligoDNA was synthesized by enzyme digestion at both ends. After annealed, pLVX-shRNA2 lentiviral vector plasmid was ligated to construct lentiviral plasmid pLVX- shRNA2-ERGsiRNA. After sequencing and sequencing, the lentivirus carrying ERGsiRNA gene was packaged with 293T cells. The virus titer was calculated based on the green fluorescence protein level of 293T cells. The virus was infected into VCaP cells by real-time fluorescence quantitative PCR ERG mRNA expression. Results The sequencing results showed that siRNA lentiviral vector targeting human ERG gene was successfully constructed and the titer of packaged lentivirus was 1.1 × 108TU / ml. Compared with the control group, the expression of ERGmRNA in VCaP cells was significantly decreased, and the interference efficiency of siRNA on ERG expression was 87.46%. Conclusion siRNA lentiviral vector targeting human ERG gene was successfully constructed.