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目的 :构建DPC4基因RNA干扰慢病毒载体及其基因沉默效应。方法 :针对DPC4基因序列,利用网站设计程序按照RNA干扰序列设计原则,设计多个RNA干扰靶点序列,根据设计经验和设计软件进行评估测定,选择最佳的动力学参数靶点进入后续实验流程;生工生物合成含干扰序列的双链DNA oligo,其两端含酶切位点粘端,直接连入酶切后的RNA干扰载体上。将连接好的产物转入制备好的细菌感受态细胞,对长出的克隆进行酶切鉴定,挑选出阳性克隆测序,进行测序比对后,鉴定阳性克隆即为构建成功的目的基因RNA干扰慢病毒载体。结果:经过Western blot方法检测和测序证实,成功构建了DPC4 shRNA慢病毒载体LVshSmad4,并成功制备了DPC4shRNA慢病毒,3株病毒感染细胞后均具有有效的基因沉默,其中SH1序列最为显著。结论 :DPC4基因RNA干扰靶点的成功设计和RNA干扰靶点慢病毒载体制备,而且具有显著的基因沉默效果。
Objective: To construct RNA interference lentivirus vector of DPC4 gene and its gene silencing effect. Methods: Aiming at the sequence of DPC4 gene, a series of RNA interference target sequences were designed according to the design principles of RNA interference sequence using web site design program. According to the design experience and design software, the target sequence of DPC4 gene was selected for evaluation. ; Biosynthesis double-stranded DNA oligo containing interfering sequence, both ends of the enzyme-containing site sticky ends, directly connected to the digested RNA interference vector. The connected product is transferred to the prepared bacterial competent cells, and the clones are identified by restriction enzyme digestion. The positive clones are selected and sequenced. After sequencing and comparison, the positive clones are identified as the target genes for the successful construction of RNA interference slow Viral vector. Results: After confirmed by Western blot and sequencing, DshRNA4 shRNA lentiviral vector LVshSmad4 was successfully constructed and DPC4 shRNA lentivirus was successfully prepared. All the 3 strains of virus infected cells had effective gene silencing, of which SH1 sequence was the most significant. Conclusion: The successful design of RNA interference target of DPC4 gene and the preparation of RNA interference target lentiviral vector, and has significant gene silencing effect.