小鼠Calb_2基因重组腺病毒载体的构建与鉴定  

Construction and Idenfication of Recombinant Adenovirus Vectors for Mouse Calb_2 Expression

作　　者：王菁[1] 罗建[1] 刘珊[1] 代晓南[1] 高莉[1] 高超[1] 刘嘉茵[1] 崔毓桂[1] 

机构地区：[1]南京医科大学第一附属医院生殖医学中心,210029

出　　处：《国际生殖健康／计划生育杂志》2012年第4期261-264,I0001,共5页Journal of International Reproductive Health/Family Planning

基　　金：国家自然科学基金(81170559)

摘　　要：目的:为了体外研究Calretinin(Calb2)基因在生殖系统、神经系统、视觉传导中的作用,构建小鼠Calb2基因表达重组腺病毒载体。方法:用反转录聚合酶链反应(RT-PCR)方法,以小鼠Calb2cDNA为模板,扩增Calb2基因。亚克隆后,将Calb2基因片段克隆至腺病毒穿梭质粒pAdTrack-CMV上,构建穿梭质粒pAdTrack-Calb2。将穿梭质粒pAdtrack-Calb2转化至BJ-5183感受态细菌中,与腺病毒骨架质粒pAdEasy-1同源重组,获得重组质粒AdCMV-Calb2。重组质粒AdCMV-Calb2转染AD-293细胞,进行病毒包装和扩增,通过绿色荧光蛋白(GFP)报告基因观察重组病毒的产生。同时,构建对照腺病毒载体AdCMV-GFP。用获得的重组腺病毒感染AD-293细胞,通过观察GFP检测感染效率、蛋白质印迹方法检测Calb2基因的表达。结果:测序和酶切鉴定重组腺病毒质粒构建正确;经AD-293细胞包装后可观察到GFP表达;获得的重组腺病毒载体体外感染AD-293细胞,观察到GFP表达;经蛋白质印迹方法检测,与未感染腺病毒载体组(Mock)和感染对照腺病毒载体组(AdCMV)相比,Calb2基因腺病毒载体(AdCMV-Calb2)感染组CALB2蛋白水平显著增高(P<0.05)。结论:成功构建了小鼠Calb2基因重组腺病毒载体,并在AD-293细胞超表达,为课题组研究Calb2基因在生殖相关疾病的生理与病理生理作用奠定了基础。Objective： To construct the recombinant adenovirus vector of Calretinin （Calb2） gene for over-expression of Calb2 gene,as useful tools to study the effects of Calb2 on the regulation of reproductive endocrinology, central nerve system, and visual conduction. Methods：The cDNA sequence of Calb2 was cloned by the reverse transcriptive polymerase chain reaction （RT-PCR）. A Calb2 gene fragment was sub-cloned into adenovirus shuttle plasmid pAdTrack-CMV to construct the shuttle plasmid pAdTrack-Calb2. Then it was transformed into BJ5183 cells with the adenoviral backbone pAdEasy-1 to obtain the homologous recombinant AdCMV-Calb2. The recombinant AdCMV-Calb2 was further packaged and amplificated in AD293 cells. The expression of Calb2 protein in AD293 cells was detected by Western blotting. Results：The Calb2 gene recombinant adenovirus vectors,AdCMV-Calb2,were constructed successfully by endonulease digestion and sequencing. The AD293 cells infected with AdCMV-Calb2 significantly expressed GFP protein. The expression of CALB2 protein was significantly up-regulated in AD293 cells infected with AdCMV-Calb2 plasmids. Conclusions： The recombinant adenovirus vectors of AdCMV-Calb2 were successfully constructed and their expression was identified in AD293 ,which has provided a laboratory basis to study the function of Calb2 gene.

关 键 词：Calb2基因 腺病毒科 遗传载体 泌尿生殖系统 

分 类 号：R346[医药卫生—基础医学]