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目的构建Smad3基因RNAi慢病毒载体。方法针对已经筛选确定的Smad3基因RNAi有效靶序列,合成靶序列的Oligo DNA,退火形成双链DNA,与经AgeⅠ和EcoRⅠ双酶切后的pGCSIL-GFP载体连接产生GC-shSmad3慢病毒载体,PCR筛选阳性克隆,测序鉴定。用GC-shSmad3、pHelper 1.0载体和pHelper 2.0载体共转染包装细胞293T细胞,包装产生慢病毒,以293T细胞GFP蛋白的表达水平测定病毒滴度。结果PCR和测序证实,构建出了Smad3 shRNA的慢病毒载体GC-shSmad3。包装慢病毒,浓缩病毒悬液的滴度为3×108TU/ml。结论成功构建Smad3基因RNAi慢病毒载体。
Objective To construct Smad3 RNAi lentiviral vector. Methods Targeted RNAi effective target sequences of Smad3 gene were screened and synthesized. Oligo DNA of the target sequence was synthesized and double-stranded DNA was annealed to generate the GC-shSmad3 lentiviral vector. The PCR products were ligated with the pGCSIL-GFP vector digested with AgeⅠand EcoRⅠ. Positive clones were screened and sequenced. The 293T cells were co-transfected with GC-shSmad3, pHelper 1.0 vector and pHelper 2.0 vector to produce lentivirus, and the virus titer was determined by the expression level of GFP protein in 293T cells. Results PCR and sequencing confirmed that lentiviral vector GC-shSmad3 of Smad3 shRNA was constructed. The lentivirus was packaged and the titer of the concentrated virus suspension was 3 × 10 8 TU / ml. Conclusion Smad3 gene RNAi lentiviral vector was successfully constructed.