Sirt3基因过表达慢病毒载体的构建及其在人神经母细胞瘤 SH-SY5Y细胞中的表达

Objective To construct the lentiviral vector overexpressing the Sirt3 gene,establish the SH-SY5Y cell lines overexpressing Sirt3 gene through stably infecting human blastoma SH-SY5Y cells,and provide a new method for study on the function of Sirt3 in Parkinson's disease cell models. Methods According to the Sirt3 gene nucleotide sequence provided by GenBank database,the specific primers to human Sirt3 gene were designed and synthesized. The Sirt3 gene cDNA fragment was amplified,and it was cloned into lentiviral vector Ubi-MCS-3FLAG-SV40-puromycin to construct the lentiviral vector plasmid. The positive clones were identified by polymerase chain reaction and sequencing analysis. The recombinant plasmids and helper plasmids pHelperl. 0 and pHelper2. 0 were cotransfected into the 293T cells. The cell supernatant riched in lentivirus particles was collected and the titer of virus was detected. The SH-SY5Y cells in logarithmic growth phase were randomly divided into the blank control group, negative control group and Sirt3 group. The SH-SY5Y cells in the blank control group were only added culture medium, the SH-SY5Y cells in the negative control group were transfected with negative control virus, and the SH-SY5Y cells in the Sirt3 group were transfected with Sirt3 gene overexpression lentivirus, then the stably transfected cells were obtained by using puromycin selection. The relative expression level of Sirt3 mRNA and protein in each group were detected by real-time polymerase chain reaction and Western blotting. Results The Sirt3 gene fragment was successfully amplified, about 1 242 bp in size. Sequencing analysis showed that Sirt3 gene sequence in overexpression lentivirus was similar to that in GenBank. The titer of the lentivirus was 1. 2 x 1012 TU • L . The relative expression level of Sirt3 mRNA and protein in the Sirt3 group were significantly higher than those in the blank control group and negative control group ( P < 0. 01 ) , respectively. And there was no statistical difference in the relative expression level of Sirt3 mRNA and protein between the blank control group and the negative control group ( P > 0. 05 ). Conclusion The lentivirus vector with Sirt3 gene was successfully constructed, and the SH-SY5Y cell lines stably overexpressing Sirt3 gene were selected.