携带siSPK1重组腺病毒的构建及其对N2a细胞凋亡的影响

Construction of Recombinant Adenovirus with siSPK1 Gene and Its Effect on Apoptosis of N2a Cells

应用AdMax腺病毒载体系统构建携带siSPK1基因的重组腺病毒,进一步研究SPK1基因对N2a细胞凋亡的影响。设计可形成小发夹结构的SPK1-siRNA模板cDNA序列,克隆至质粒pDC316-siRNA,构建SPK1-siRNA穿梭质粒pDC316-SPK1,经鉴定正确后,将pDC316-SPK1与辅助包装质粒(pBHG loxΔE1,3 Cre)共转染至HEK293细胞,包装纯化并扩增重组腺病毒颗粒,终点稀释法测定病毒滴度;病毒感染N2a细胞,Western blot法检测SPK1蛋白的表达;Hoechst33258染色检测SPK1基因对N2a细胞凋亡的影响。酶切后PCR分析、测序鉴定表明,pDC316-SPK1构建成功,病毒纯化后滴度为2.50E+10 PFU/mL;重组病毒可在蛋白水平抑制SPK1的表达;SPK1基因抑制后,N2a细胞凋亡增加(P<0.001)。上述结果表明,含有SPK1-siRNA的重组腺病毒构建成功,且具有抑制SPK1蛋白表达的功能,该基因沉默后,N2a细胞凋亡增加。

To construct recombinant adenovirus with siSPK1 gene using AdMax system, and to investigate the effect ofSPK1 gene on the apoptosis of N2a cell, a SPKI-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 SPKI-siRNA expression vector pDC316-SPK1. After verification, the pDC316-SPK1 vector was co-transfected with pBHG lox AE1,3 Cre into HEK293 cells where they were packed as the recombinant adenovirus. Recombi- nant 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-SPK! 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 SPKI-siRNA gene was successfully constructed, which can silence SPK1 gene and increase the apoptosis of N2a cells in vitro.