针对丝聚合蛋白基因RNA干扰重组慢病毒载体的构建及筛选

Construction of RNA interference lentiviral vector with human FLG gene

目的构建丝聚合蛋白（FLG）基因RNA干扰（RNA interference,RNAi）慢病毒表达载体。方法设计针对人FLG基因的mRNA干扰靶序列,合成相应双链DNA,通过BamH I和EcoRI酶切后的PGLV/H1/GFP载体连接产生shRNA载体,并与pHelper 1.0、pHelper 2.0两种载体共转染293T细胞培养,获得重组慢病毒载体后,转染目的细胞,实现针对FLG基因的RNA干扰。结果经鉴定该实验的慢病毒载体,对人正常皮肤细胞HACAT干扰效果达75%-95%,以RNAi3的沉默效应最佳。结论重组的慢病毒载体为有关FLG基因的进一步研究奠定良好实验基础,并能广泛用于体内基因治疗及基因功能研究。

[ Objective ] To construct the lentiviral RNA interference （RNAi） vectors of the filaggrin （FLG） gene. [Methods] RNAi target sequences were designed according to the FLG gene sequences, then the double stranded DNA were synthesized, which was connected PGLV/H1 /GFP vector to constructed lentiviral vector with BamH Ⅰ and EcoR Ⅰ digested. 293T cells were co-transfected with pHelper 1.0, pHelper 2.0 and the plasmids to produce lentiviral particles, and the recombinant lentiviruses were transfected into HACAT cells to interfere FLG. [Results] It was approved that interference effect on the HACAT cells was 75% 95% reduction and RNAi3 had the best effect of gene silence. [ Conclusion ] The shRNA vector of the FLG gene established the experimental foundation for further research and might be widely used in the study of gene therapy and function.