关键词: 帕金森病, DJ-1, PC12细胞, 慢病毒载体

Abstract: Objective To prepare gene overexpressing cell model of human wild-type DJ-1 and its L166P mutant, and to investigate the role of lentiviral vector in gene overexpressing cell model. Methods Wild type DJ-1 and L166P mutant DJ-1 lentiviral vector plasmids were constructed respectively. were constructed. After sequencing and comparing correctly, the plasmid was amplified and prepared and transfected into HEK293T cell line. Expression of WT DJ-1 and L166P mutant DJ-1 in cell lines were detected by fluorescence and Western blots.After determining the accurate expression of the target protein, a large amount of HEK293T cells were transfected and packaged to produce lentiviral particles. The PC12 cells were infected with the titer of virus supernatant. The fluorescence intensity of GFP and the expression of target protein were observed by fluorescence microscope and Western blots method, and the infection efficiency of the virus was determined.Results Lentiviral vectors carrying wild type DJ-1 and its mutants were successfully constructed. The virus vector can be transfected into HEK293T cells and the target protein can be correctly translated and expressed. It showed that the viral titers of LV-DJ-1 and LV-DJ-1/L166P were 2×109TU/ml and 2×108TU/ml, respectively. Virus supernatant can efficiently infect PC12 cells, and most cells can express target proteins. The protein expression levels of exogenous wild-type DJ-1 and L166P mutants were 315% and 285% of endogenous content,respectively.Conclution Lentivirus vector can infect cells efficiently, and it is a good way to prepare gene over expressing cell model. A cell model overexpressing DJ-1 or its L166P mutant was successfully prepared. The model can be used for subsequent DJ-1 function research.

Key words: Parkinson’s disease, DJ-1, PC12, lentivirus vector