摘要
载脂蛋白B mRNA编辑酶催化多肽(apolipoprotein B mRNA-editing enzyme catalytic-polypeptide,APOBEC)家族成员,是近年来发现的具有抗病毒作用的天然免疫分子,对HIV和HBV等多种病毒具有抑制作用,为研究APOBEC的抗病毒作用,对APOBEC-3F和-3G进行克隆、表达及亚细胞定位分析.HBV主要的生物合成发生于细胞核中,利用核定位信号(NLS)及二联核定位信号(B-NLS)与APOBEC-3F和-3G融合表达,以进一步了解APOBEC-3F和-3G的亚细胞定位及功能.利用RT-PCR从植物凝集素(PHA)刺激的人外周血淋巴细胞RNA中,对APOBEC-3F和-3G进行克隆,利用HindⅢ和KpnⅠ双酶切插入到pcFlag载体中,脂质体转染MDCK细胞后利用免疫荧光检测APOBEC重组蛋白的亚细胞定位.电泳结果表明,RT-PCR扩增得到APOBEC-3F和-3G基因,双酶切鉴定结果表明,APOBEC真核表达载体构建成功,基因序列经DNA测序证实.免疫荧光结果表明,转染表达后的APOBEC-3F和-3G均定位于细胞胞浆中.APOBEC-3F和-3G可以在HepG2.2.15细胞中显著抑制HBV的复制.可以有效转运绿色荧光蛋白(GFP)入核的NLS及B-NLS,不能将APOBEC-3F和-3G有效地转运至核中.成功地对APOBEC家族中的两个重要成员APOBEC-3F和-3G进行了克隆、表达及亚细胞定位研究,为进一步利用APOBEC家族蛋白开发抗HIV及HBV药物提供基础.
APOBEC （apolipoprotein B mRNA-editing enzyme catalytic-polypeptide） family members were reported as innate immune molecules with anti-viral activity for many viruses, such as HW and HBV. In order to understand the function of APOBEC, the APOBEC-3F and -3G were cloned, expressed, and the sub-cellular localization of them was detected. The genes of APBEC-3F and -3G were cloned from PHA-stimulated PMBC and expressed in the MDCK cell by transfection. The sub-cellular localization of APOBEC-3F and -3G were detected by immunofluorescence. APOBEC-3F and -3G were cloned by RT-PCR and confirmed by DNA sequencing. The immunofluorescence indicated APOBEC-3F and -3G were located in the cytosal. APOBEC-3F and -3G could inhibit HBV replication effectively in HepG2.2.15 cell. APOBEC-3F and -3G could not be trans-located into nuclear by nuclear location signal （NLS） or bi-NLS （B-NLS）. These results will help the future research on the function of APOBEC.
出处
《生物化学与生物物理进展》
SCIE
CAS
CSCD
北大核心
2009年第3期331-338,共8页
Progress In Biochemistry and Biophysics
基金
国家自然科学基金(30571674
30771988)
广东省自然科学基金(05008347
04020239)资助项目.~~
