pCMV-tag2B-HBX质粒的构建和表达

Construction and expression detection of plasmid expressing pCMV-tag2B-HBx gene

目的构建乙型肝炎病毒X基因(HBX)的真核表达质粒,诱导并鉴定其表达。方法将克隆获得的HBX全长cDNA片段插入带有报告基因的真核表达质粒pCMV-tag2B,构建HBX的重组表达载体pCMV-tag2B-HBX,经DNA测序证明HBX的cDNA片段的插入方向和其全长cDNA的碱基顺序,将重组质粒转染HepG2细胞系,诱导其表达,用Western Blot进行鉴定。结果构建的重组表达载体pCMV-tag2B-HBX经双酶切表明有相应大小的DNA片断,序列分析证实为正确的有完整读码框的X基因,用脂质体转染法可将pC-MV-tag2B-HBX导入HepG2细胞并成功表达,目的蛋白经Western Blot鉴定具有生物学活性。结论成功构建了HBX真核表达质粒pC-MV-tag2B-HBX,重组质粒能表达具有生物学活性的HBX蛋白,为研究HBX的功能打下了基础。

Objective To construct the eukaryotic expression plasmid of HBV-X gene. Methods The eukaryotic recombinant vector of HBV-X gene pCMV-tag2B-HBx was constructed with full HBV-X cDNA isolated from pEcob6 vector and transfected into hepatocellular carcinoma cell line HepG2 by using FuGENE^TM6 Transfection Reagent. DNA sequencing confirmed the cDNA sequence and the orientation of the insert. To determine the expression of HBx gene, Western Blot was performed,and the results showed that HepG2 cells transfected by the recombinant CMV- tag2B-HBX could express HBX gene effectively. Results DNA fragments with corresponding size were demonstated in the contracted recombinant plasmid and contracted X gene were identified through restriction endonucleases digestion, DNA sequence analysis displayed that the correct and en- tire opening reading frame of HBV X gene was inserted. Through lipofection ,pCMV-tag2B-HBX could be transferred into HepG2 and expressed successfully,target protein had biologic activity identified by Western bot. Conclusions Eukaryotic expression recombinant CMV-tag2B-HBX which could express HBX protein with biological activity can be constructed successfully ,it might be helpful for further research about HBX＇s founction.