慢病毒介导的重组人α1-抗胰蛋白酶在小鼠体内的表达  

Lentivirus-mediated expression of human 1-antitrypsin in mice

作　　者：欧海龙[1] 雷霆雯[1] 李红梅[1] 王筑婷[1] 莫晓川[1] 

机构地区：[1]贵阳医学院生物化学与分子生物学教研室,贵州省贵阳市550004

出　　处：《世界华人消化杂志》2012年第19期1720-1725,共6页World Chinese Journal of Digestology

摘　　要：目的:构建重组人α1-抗胰蛋白酶(hAAT)因子的慢病毒表达载体,并通过体外细胞水平和小鼠体内分析其表达情况.方法:通过RT-PCR的方法扩增出hAAT基因的编码序列,并构建重组慢病毒质粒;经体外包装后,感染鼠成纤维细胞及注射小鼠.荧光显微镜下观察GFP的表达情况,同时对收获的细胞及感染小鼠的肝脏或血浆进行Western blot、ELISA检测.结果:获得重组hAAT因子的慢病毒表达质粒pLVX-ser;包装后得到8×106TU/mL滴度的慢病毒颗粒.通过荧光显微镜下观察,显示重组hAAT因子在成纤维细胞中正常表达;对小鼠尾静脉注射病毒之后,进行hAAT因子检测,Western blot结果说明hAAT因子在小鼠体内成功表达;通过ELISA检测发现hAAT在小鼠体内的表达平均达190g/L左右,而且在慢病毒的介导下hAAT在小鼠中的表达可持续3mo以上.结论:重组慢病毒载体可高效、持续表达hAAT因子,为通过基因工程生产重组hAAT因子以及为α1-AT缺乏症的基因治疗奠定基础.AIM： To construct a recombinant lentiviral vec- tor carrying the human α1-antitrypsin （hAAT） gene, then express the hAAT in to fibroblasts and mice. METHODS： The coding sequence of the hAAT gene was amplified by RT-PCR and ligated into a lentiviral vector to construct a recombinant lentiviral vector （pLVX-ser）. Lentiviral particles were packaged in vitro and used to infect fibro- blasts and mice. GFP expression was detected by fluorescence microscopy. The supernatants of in- fected cells and liver samples from infected mice were used to detect the expression of hAAT by Western blot and ELISA, RESULTS： The recombinant hAAT lentiviral vector pLVX-ser was successfully constructed. The titer of lentiviral particles reached 8×10^6 TU/mL after viral packaging. Fluorescence microscopic analysis showed that hAAT was suc- cessfully expressed in fibroblasts. Western blot analysis suggested that hAAT was expressed well in mice, and ELISA assay showed that the mean expression level amounted to 190 μg/L. The expression of hAAT in mice could even last for several months. CONCLUSION： The recombinant lentiviral vec- tor carrying the hAAT gene allows efficient and persistent expression of hAAT in mice, which paves the way to producing hAAT in industry and gene therapy for AATD disease.

关 键 词：Α1-抗胰蛋白酶 α1-AT缺乏症 慢病毒 

分 类 号：R346[医药卫生—基础医学]