小鼠FGF-21 siRNA腺病毒载体的构建与鉴定

Construction and identification of FGF-21 siRNA recombinant adenovirus vector

目的构建含FGF-21 siRNA基因的重组腺病毒载体并鉴定。方法首先用BamHⅠ+HindⅢ双酶切本课题组前期构建的质粒pSi-lencer1.0-shFGF-21,得到0.3 bp的目的片段,并将目的片段亚克隆入穿梭质粒(pShuttle-shFGF-21,),用I-CeuI和I-SceI双酶切处理pAdxsi组腺病载体及pShuttle-shFGF-21,连接,转化DH5α感受态细胞,筛选、鉴定、测序后,最后在293细胞内包装扩增为重组腺病毒。结果设计并构建了小鼠FGF-21基因特异性siRNA腺病毒载体,并经酶切和测序鉴定。空斑形成实验测得腺病毒滴度为1×1010PFU/ml。结论成功构建了小鼠FGF-21基因特异性siRNA腺病毒载体。

Objective To construct the RNA interference adenovirus expression vector specific for fibroblast growth factor-21 （ FGF- 21 ） gene. Methods Firstly, the plasmid pSilencer1. 0-shFGF-21 developed a vector-based system in earlier studies was digested by BamH Ⅰ ＋ Hind Ⅲ for getting the fragment. The adenovirus vector plasmid, pAd.shFGF-21 was constructed according to a two-step transformation protocol. The newly constructed plasmid was transfected into 293 packaging ceils to grow adenovirus, which was further multiplied and purified. Results The recombinant adenoviral plasmid pAd-shFGF-21 was successfully constructed. The titer of the purified pAd-shFGF-21 was 1× 10^10 PFU/mL. Conclusions The RNA interference adenovirus expression vector specific for FGF-21 gene is established successfully.