大鼠miR-1慢病毒表达载体的构建与鉴定

Construction and identification of recombinant lentiviral vector encoding miR-1 of rat

目的构建并鉴定大鼠miR-1慢病毒过表达系统。方法采用EcoRⅠ酶双酶切将目的基因从合成载体上酶切后连接入EcoRⅠ线性化慢病毒载体,连接产物转化后进行阳性克隆鉴定。成功构建的miR-1重组质粒与慢病毒辅助包装质粒及Lipofectamine 2000共转染293T细胞,产生慢病毒浓缩液并标定病毒滴度。重组慢病毒载体转染大鼠骨髓间充质干细胞(MSCs),观察转染效率及miR-1表达水平。结果测序结果显示目的基因与Genebank序列完全一致;孔稀释法检测病毒滴度为3×108 TU/μL;以感染复数(MOI)50感染大鼠MSCs,感染效率达90%以上,聚合酶链反应显示miR-1高水平表达。结论成功构建大鼠miR-1慢病毒表达载体并可高效转染大鼠MSCs,为进一步研究miR-1基因的相关功能提供了合适的载体。

Objective To construct and identify lentiviral vector system over-expressing miR-1 gene of rat.Methods The miR-1 gene was digested from synthesised vehicle by EcoR I restriction endonuclease twice,and linked with linearized lentiviral vector system.Then the product was transformed into competent cells.Recombinant lentivector plasmids and the other two helping plasmids were co-transfected into 293T cells by Lipofectamine 2000,and cell culture supernatant was collected after 48 hours.The virus supernatant was concentrated and tittered.In order to observe the transfection efficiency and the expression of miR-1,the vector was transfected into rat mesenchymal stem cells（MSCs） following by cytoflurimetric analysis of GFP positive cells and qPCR.Results The miR-1 gene sequence was consistent with the sequence reported in genebank;the titer of miR-1 gene virus was 3×108TU/μL（TU,transduction unit）.The best transfection efficiency was up to 90% and the expression levels which determined by qPCR were quite higher than those of control group when multiply of infection（MOI）was 50.Conclusion The construction of over-expression lentiviral system of rat miR-1 gene may support the further study of function of miR-1 gene as a potential over-expression technique.