APE1基因RNAi慢病毒载体的构建与鉴定

目的构建APE1基因慢病毒载体,为其后续的体内外实验研究提供基础。方法应用基因工程技术,筛选出两条针对APE1基因的RNAi靶序列KD1、KD2,分别与pGCIL-GFP载体连接,经转化筛选鉴定后,包装产生慢病毒颗粒并测定病毒滴度,分别命名为LV-APE1-shRNA1、LV-APE1-shRNA2,两种病毒颗粒分别感染MHCC97-H细胞,设为感染LV-APE1-shRNA1、LV-APE1-shRNA2细胞组,同时设未感染病毒组和感染空载体细胞组。Real-time PCR和Western blot检测MHCC97-H细胞中APE1基因的mRNA和蛋白的表达。结果 PCR及测序结果与预期结果一致,LV-APE1-shRNA1、LV-APE1-shRNA2的病毒滴度为4×10^8TU/mL和7×10^8TU/mL。与未感染病毒组和感染空载体细胞组相比,2组慢病毒组APE1基因mRNA和蛋白的表达均明显下降,LV-APE1-shRNA1、LV-APE1-shRNA2对APE1基因的mRNA表达的抑制率分别为75%,90%,对APE1蛋白表达的抑制率达90%,95%。结论成功构建高效阻断APE1基因表达的RNAi慢病毒表达载体,为应用RNAi进一步研究APE1基因在肝癌中的作用机制和基因治疗奠定基础。

Construction and Identification of a Lentiviral Vector For RNA Interference of APE1 Gene

Objective To construct a lentiviral vector targeting APE1 gene for further experiments in vivo and in vitro.Methods Gene engineering technique was used to screen 2 RNA interference sequences targeting APE1 gene KD1and KD2.The sequences were separately cloned into the pGCIL-GFP vector,and subsequently confirmed by PCR and DNA sequencing analysis.The two kinds of recombinant 1entiviruses were named LV-APE1-shRNA1 and LV-APE1-shRNA2,and non-transfected and empty vector transfected groups were designed.Then they were injected into MHCC97-H cells,the APE1 mRNA and protein expression were examined by real-time PCR and Western blot.Results PCR and DNA sequencing demonstrated that the sequences were correctly inserted.The titer of virus was 4×10^8 Tu/mL and 7×10^8 Tu/mL.After transfection with LV-APE1-shRN A1 and LV-APE1-shRNA2,when compared with non-transfected and empty vector transfected groups APE1 mRNA expression in MHCC97-H cells was inhibited mRNA by 75%,90% respectively and protein levels by 90%,95% respectively.Conclusion The high eficient lentiviral RNAi vector of APE1 was constructed successfully as the basis for further studying the mechanism and gene thempy in hepatocellular carcinoma（HCC）.