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Construction of Recombinant Adenovirus with siSPK1 Gene and Its Effect on Apoptosis of N2a Cells
Yang Yang1, Ma Rong2, Zhang Yuan1, Lü Bingjie3, Wang Min1, Sun Shenggang1, Li Gang1*
1Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China; 2Department of Pharmacology, Tongji Medical College, Huazhong University of Science and Technology,
Abstract: To construct recombinant adenovirus with siSPK1 gene using AdMax system， and to investigate the effect of SPK1 gene on the apoptosis of N2a cell， a SPK1-siRNA template DNA sequence， capable of forming a small hairpin structure， was designed. After renaturation， it was cloned into the vector pDC316-siRNA to construct the SPK1-siRNA expression vector pDC316-SPK1. After verification， the pDC316-SPK1 vector was co-transfected with pBHG lox ΔE1，3 Cre into HEK293 cells where they were packed as the recombinant adenovirus. Recombinant adenovirus was abundantly amplified and then virus titer was evaluated. The recombinant adenovirus was used to infect the N2a cells. Western blot was used to detect the SPK1 protein expression in N2a cells. Hoechst33258 staining was used to detect apoptosis. PCR and sequencing analyses showed that pDC316-SPK1 was constructed successfully. The titer of virus is 2.50E+10 PFU/mL. Western blot indicated that the expression of SPK1 protein was greatly inhibited after infection in N2a cells with recombinant adenovirus particles. Hoechst33258 staining indicated that N2a cell proliferation was decreased significantly by gene silencing (P<0.001). In conclusion， the recombinant adenovirus vector containing the SPK1-siRNA gene was successfully constructed， which can silence SPK1 gene and increase the apoptosis of N2a cells in vitro.
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