人miR-378慢病毒表达载体的构建及鉴定

Reconstruction and identification of recombinant lentiviral vector expressing has-miR378

目的构建人miR-378慢病毒表达载体。方法酶切法从已构建的质粒获得pri-miR-378,克隆于含绿色荧光蛋白(GFP)编码基因的PGCSIL载体,转化感受态细胞,产生重组慢病毒质粒PGCSIL-miR-378,并进行PCR及测序鉴定。将PGCSIL-miR-378、pHelper 1.0和pHelper 2.0三种质粒DNA共转染293T细胞,包装产生慢病毒,以293T细胞GFP蛋白的表达水平用逐孔稀释滴度法测定病毒滴度。结果成功构建miR-378的慢病毒表达载体,测序证实所插入基因序列完全正确。荧光显微镜检测证实PGCSIL-miR-378携有正确的miR-378基因,并能在293T细胞中表达。检测病毒滴度为8×108 TU/ml。结论成功建立了miR-378慢病毒表达系统。

Objective To construct a recombinant lentiviral vector expressing human miR378. Methods A fulllength gene sequence of premiR378 was obtained by restriction endonuclease digestion and inserted into linear PGCSIL vector which contained coding gene of green fluorescent protein（GFP）, and then was transferred into competent cells detected by PCR amplification for sequencing analysis. The resulting recombinant lentiviral vector expressing miR378 was called PGCSIL miR378 PGCSILmiR378, pHelper 1.0 and pHelper 2.0 were cotransfected：into cell 293T by lipofectamine 2000 and were packed by lentivirus. The concentration of virus was measured with viral biology titer according to the expression level of GFP in 293T cells. Results Recombinant lentiviral vector expressing miR378 was successfully established and was confirmed by PCR and DNA sequencing. The right miR378 gene in constructed PGCSILmiR378 lentiviral vector was detected by both immunofluorescence microscopy and expressed in 293T cells. The detected titer of concentrated virus was 8 X 108 TU/ml. Conclusion The recombinant lentiviral vector PGCSILmiR378 has been successfully constructed.