表达人NEP基因重组腺病毒载体的构建及其降Aβ作用的研究  

Construction of recombinant adenovirus vector expressing neprilysin and effect on reducing the Aβ's deposition of recombinant adenovirus

作　　者：张静[1] 段珊[1] 杨玲玲[1] 崔行[1] 

机构地区：[1]山东大学医学院生物化学与分子生物学研究所,济南250012

出　　处：《山东大学学报（医学版）》2009年第10期23-27,31,共6页Journal of Shandong University:Health Sciences

基　　金：高等学校博士点科研基金资助项目(20050422045)

摘　　要：目的构建携带人全长NEP基因的重组腺病毒载体,包装扩增获得高滴度病毒,检测其对损伤细胞的降β-淀粉样肽(Aβ)作用。方法构建含NEP基因的腺病毒穿梭载体pAdTrack-nep,并与骨架载体pAdEasy-l在细菌内重组为pAd-NEP,经293细胞包装为增殖缺陷性腺病毒,感染人神经母细胞瘤SK-N-SH细胞。采用RT-PCR和Westernblot法分析检测NEP的表达水平及感染内源性损伤的SK-N-SH细胞(sweAPP-SK),采用Elisa法初步检测NEP对Aβ的降解程度。结果成功构建重组腺病毒AD-NEP和对照病毒AD-GFP,且重组腺病毒能感染人神经母细胞瘤SK-N-SH细胞并检测到目的基因的高表达。Elisa结果显示,NEP高表达有明显减轻Aβ在细胞外累积的作用。结论利用细菌内质粒同源重组法可快速简捷地制备表达外源基因的腺病毒,感染AD-NEP的神经细胞通过NEP高表达可以明显减轻Aβ累积。Objective To construct the recombinant adenovirus vector carrying the human gene neprilysin（NEP）,obtain a high titer virus by packing and increasing,and explore the reducing of Aβ＇s deposition to the damaged cells.Methods A shuttle plasmid pAdeasy-l was constructed in bacteria and pAd-NEP was generated.The replication-deficient adenovirus was packed and propagated in the cell 293.Then the person＇s nerve metrocyte lump cell SK-N-SH cells were infected and NEP expression was examined by RT-PCR and Western blot.The endogenous damaged SK-N-SH cells were infected and the degeneration to Aβ was examined.Results Ad-NEP and control adenovirus Ad-GFP were successfully constructed.The recombinant adenovirus can efficiently infect the person＇s nerve metrocyte lump cell SK-N-SH cells and can be used to examine high expression of NEP.The result of Elisa showed that high expression of NEP can reduce the extracellular deposition of Aβ.Conclusion Homologous recombination in bacteria is a simple and convenient strategy to generate adenovirus expressing exogenous genes.The nerve cells infected by AD-NEP may reduce the deposition of Aβ through high expression of NEP.

关 键 词：内肽酶类 腺病毒 人 淀粉样Β蛋白 阿尔茨海默病 

分 类 号：Q78[生物学—分子生物学] R741.02[医药卫生—神经病学与精神病学]