Npas4基因过表达慢病毒的构建及其在SK-N-SH细胞的表达

Construction of Ientivirus Carrying Npas4 Gene and Identification of Its Expression in SK-N-SH Cells

研究旨在构建Npas4基因过表达慢病毒,为进一步深入探索Npas4基因的功能奠定基础。用人工合成大鼠Npas4基因c DNA片段,将其插入p CDH-CMV-MCS-EF1-cop GFP构建慢病毒表达质粒p CDH-Npas4。酶切、测序验证质粒后,将p CDHNpas4和辅助质粒共转染包装细胞293T,浓缩上清得病毒颗粒并测定病毒滴度。取病毒颗粒感染SK-N-SH细胞48 h,收集细胞采用Western blotting法检测Npas4蛋白的表达。p CDH-Npas4携载正确Npas4基因,将其包装293T细胞后能产生病毒。病毒滴度为1.05×109TU/m L。相比于转染GFP病毒对照组(GFP)和未转染对照组(control),Npas4重组慢病毒组(Npas4)的细胞Npas4蛋白表达显著增高。成功构建Npas4基因过表达的重组慢病毒载体p CDH-Npas4,并获得高效的重组慢病毒,能将外源Npas4基因导入SK-N-SH细胞,为进一步研究Npas4基因的相关功能奠定了基础。

The present study is aim to construct lentivirus containing GFP reporter gene driven by Npas4 gene and identify its expression in SK-N-SH cells. The sequence of Npas4 was obtained from Genbank and the c DNS sequences specifically targeting the Npas4 gene was synthetized and was subcloned into the lentiviral vector,p CDHCMV-MCS-EF1-cop GFP,to generate the lentiviral expression vector,p CDH-Npas4. The enzyme digestion and DNA sequencing analysis were employed to confirm the correction of the p CDH-Npas4. Following 293 T cells were transfected by the p CDH-Npas4 as well as the auxiliary packaging plasmids,the recombinant lentiviruses were produced. The supernatant was concentrated and the virus titer was tested. The recombinant lentiviruses were used for infecting the target cell line,SK-N-SH cells. The expression of Npas4 was determined by Western blotting. p CDHNpas4 carried the correct Npas4 gene. The recombinant lentiviruses carrying Npas4 gene could infect 293 T cells and the virus titer in the concentrated supernatant was 1. 05 × 10^9 TU/m L. After the SK-N-SH cells were infected by the recombinant lentivirus for 48 h,the expression level of Npas4 in the Npas4 group was significantly increased compared with that in the GFP group and the control group. The recombinant lentivirus carrying Npas4 was constructed successfully and the expression of Npas4 was increased in the infected SK-N-SH cells,which could improve the further research into the function of Npas4 gene in neuronal cells.