摘要
目的构建含乙型肝炎病毒(hepatitis B virus,HBV)X基因的真核表达载体,为探讨HBV X基因与慢性乙型肝炎及肝癌发生的关系提供实验依据。方法用PCR方法扩增含EcoRⅠ和HindⅢ酶切位点的X基因序列,对pcDNA3.1(+)载体及X基因PCR产物双酶切,用连接酶将两者连接并转化大肠杆菌DH5α,筛选阳性克隆,并对其进行双酶切和测序鉴定,构建HBV X基因真核表达载体pcDNA3.1(+)-HBx。用梭华-SofastTM基因转染试剂将其质粒转染THP-1巨噬细胞,细胞裂解液经SDS-PAGE及转硝酸纤维素膜后做Western blotting,鉴定目的蛋白。结果双酶切pcDNA3.1(+)-HBx后,琼脂糖电泳可分别见到大小约为5.4 kb和465 bp片断,说明X亚克隆入pcDNA3.1(+),经序列测定其含有完整的X基因片段;Western blotting结果显示pcDNA3.1(+)-HBx能在THP-1巨噬细胞中表达X蛋白。结论成功构建了真核表达载体pcDNA3.1(+)-HBx,并能在THP-1巨噬细胞中表达X蛋白。
Abstract：Objective This study was designed to construct and express the eukaryotic expression vector of HBV X gene for exploring the contribution of X gene to the chronic hepatitis and the hepatocellular. Methods X gene with EcoR Ⅰand Ⅲ endoenzyme sites was obtained by using PCR, and subcloned into pcDNA3.1（ ＋ ） vector. After identified by restrictive enzymes digestion and sequencing, reconstructed plasmid was transfected into THP- 1 cells. Expression of X were assayed in THP- 1 cell lysate by Western - blotting. Results The target gene X fragment about 465 bp was obtained. In THP - 1 cell, the pcDNA3.1 （ ＋ ） - HBx plasmid expressed X protein with molecular weight of 17 kDa by Western - blotting. Conclusion Eukaryotic expression vector pcDNA3.1 （ ＋ ） - HBx was constructed successfully and X protein was expressed in THP - 1 cell.
出处
《南华大学学报（医学版）》
2006年第3期320-323,共4页
Journal of Nanhua University(Medical Edition)
基金
湖南省卫生厅资助课题(编号:B2005084)
