人载脂蛋白AⅠ重组腺病毒载体的构建及其在小鼠体内的表达  

Construction and Expression of Recombinant Adenovirus Vector Containing Human Apolipoprotein AⅠ Gene

作　　者：李悦[1] 董继斌[1] 吴满平[1] 

机构地区：[1]复旦大学药学院生物化学与分子生物学教研室,上海市200032

出　　处：《中国动脉硬化杂志》2008年第3期201-204,共4页Chinese Journal of Arteriosclerosis

摘　　要：目的构建人载脂蛋白AⅠ基因的重组腺病毒载体,探讨人载脂蛋白AⅠ在小鼠体内的表达,建立人载脂蛋白AⅠ过表达小鼠模型。方法采用基因工程技术从重组质粒pDNR-LIB-apoAⅠ-PA获得人载脂蛋白AⅠ基因,插入穿梭质粒pshuttle CMV后,在细菌内与pAdEasy-1进行同源重组,经脂质体转染HEK293细胞,在细胞内包装获得腺病毒颗粒AdV-AⅠ,并进行大量扩增和纯化。纯化的腺病毒经尾静脉注射入BALB/c小鼠体内,通过双向琼脂免疫扩散反应、免疫比浊法及免疫印迹法检测人载脂蛋白AⅠ在小鼠体内的表达。结果成功构建了重组腺病毒载体,纯化后的病毒滴度达4.0×1013pfu/L。重组腺病毒载体能在小鼠体内高表达人载脂蛋白AⅠ,且在注射1天后表达高达740±110 mg/L。结论成功构建了重组腺病毒载体,并能介导外源基因人载脂蛋白AⅠ在小鼠体内的表达,建立了人载脂蛋白AⅠ过表达小鼠模型,为进一步研究载脂蛋白AⅠ的抗内毒素功能奠定了基础。Aim To construct recombinant adenovirus vector containing human apoliproprotein AⅠ （apoAⅠ ） geneand to explore the expression of human apoA Ⅰ in mice and establish human apoA Ⅰ overexpressing mice medel. Methods Full length apoA Ⅰ cDNA was obtained from recombinant plasmid pDNR-LIB-apoA Ⅰ -PA via digestion with Sal Ⅰ and HindⅢ and was subcloned into the pShutth vector pshuttle CMV to construct a shuttle plasmid pshuttle CMV-A Ⅰ. Then it was co-transformed into E. cob BJ5183 with adenovirus genomic plasmid pAdEasy-1 to achieve homologous recombination pAd CMV-A Ⅰ . The DNA of identified recombinant plasmid was transfected into HEK293 cells via liposome mediation to package and amplify adenovirus AdV-A Ⅰ . BALB/c mice was administrated by purified adenovirus, then human apoAⅠ protein in the serum of mice was detected by double immunodiffusion, immunolngical turbidity kit and Western blotting. Results The titer of ptLrified virus was 4.0 × 10^13 pfu/L. The over-expression of human apoA Ⅰ was observed after administration of BALB/c mice with adenovirus particles. Human apoAⅠ concentration in mice serum reached 740±110mg/L at 1 day after administration of adenovirus particles. Conclusion The recombinant adenovirus AdV-AⅠ was successfully constructed and human apoAⅠ was detected in the serum of mice. The mice model of overexpressing human apoA Ⅰ was established. This result lays the foundation for flatter study to investigate the beneficial effects of apoA Ⅰ on endotoxemia.

关 键 词：分子生物学 载脂蛋白AⅠ 腺病毒载体 基因表达 脂多糖 

分 类 号：Q7[生物学—分子生物学]