GGPPS基因干扰腺病毒载体的构建及鉴定

Construction and Identification of GGPPS Gene Interference Adenovirus

构建GGPPS基因干扰腺病毒质粒载体并加以鉴定.根据GGPPS基因序列设计GGPPS干扰序列引物,定向克隆至穿梭载体pshuttle-H1的BglⅡ和HindⅢ位点,PmeⅠ酶切线性化的pShuttle-H1-SiGGPPS干扰质粒,并与腺病毒载体(pAdEasy-1质粒)共同转化E.coli BJ5183感受态细菌,产生重组腺病毒载体.用PacⅠ酶切线性化的回收质粒,转染293A细胞包装腺病毒颗粒,在倒置显微镜下观察细胞CPE,用TCID50法测定病毒颗粒的浓度,并初步观察病毒感染PC12细胞对目的基因的干扰效率.经酶切鉴定、测序证实成功构建GGPPS基因干扰腺病毒载体.包装的腺病毒浓缩悬液滴度为1.995×107PFU/mL.GGPPS在人中具有保守性,该病毒能在人源的WRL-68细胞中成功表达,并且对GGPPS基因干扰效率达70%以上.

To construct the recombinant adenovirus vector expressing small RNA against human GGPPS gene, the interferce primer designed according to GGPPS gene sequence was cloned into pshuttle-H1 vector with Bgl Ⅱ and Hind Ⅲ restriction site. E. coli BJ5183 sensitive bacteria were cotransfected with vector lenealized by Pme I enzyme and adenovirus vector pAdEasy-1. The adenovirus was obtained in 293A cells transfected with linearized recombinant adenovirus plasmids. The titer of virus was measured based on the appearing of CPE and was determined by TC1D50 assay. The in- terference efficiency was caculated. The results obtained from DNA sequencing and digestion identification demonstrated that the adenovirus vectors tagerting to human GGPPS gene was constructed. The titer of concentrated virus was 1. 995 × l0^7 PFU/mL. The interference efficiency of endogenous GGPPS gene in WRL68 cells was above 70% as evidenced by western blot analysis. The interference adenovirus vector of human GGPPS gene is successfully constructed.