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.
出处
《世界华人消化杂志》
CAS
北大核心
2012年第19期1720-1725,共6页
World Chinese Journal of Digestology