DPC4 RNAi慢病毒载体的构建及其基因沉默效应

目的：构建DPC4基因tLNA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IKNA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1KNA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DPC4shRNA慢病毒栽体LVshSmad4，并成功制备了DPC4 shRNA慢病毒，3株病毒感染细胞后均具有有效的基因沉默，其中SHI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。

Construction of Lentiviral Vector of DPC4 RNA Interference and Its Silencing Effects

Objective： To construct and design lentiviral shRNA targeting DPC4 gene and gene silencing. Methods： Web-based program was used to analyze the DPC4 sequence for the shRNA target sites and to design RNA interference sequence which fits the designing principles. Based on the designing software evaluation, the sequences with the best dynamic parameters were selected for synthesis of DNA oligonucleotide with sense-loop-antisense structure and restriction enzyme sticky ends. DNA oligos were PAGE purified and inserted into the shRNA lentiviral vector cutted with restriction enzymes. The product was transformed into the competent cells. The positive constructs were further confirmed by sequencing. In addition, the Western blot was prepared from the infected 293T cells for analysis of the knowdown effect of the shRNA lentivirus. Results： LVshDPC4 vectors were confirmed by restriction analysis and DNA sequencing. The Ientiviral particles were prepared and showed effective infection capacity and knockdown effects. Conclusion： Lentiviral shRNA targeting DPC4 gene is successfully constructed and can effectively knock down DPC4 expression.