中文摘要：

目的：构建重组表达HBx的腺病毒,并通过感染细胞建立HBx高表达的细胞模型。方法：首先将HBx基因插入腺病毒穿梭质粒pAdTrack中,限制性酶切鉴定正确后将其用PmeⅠ酶切线性化,转到BJ5183感受态细菌进行同源重组,重组后的腺病毒载体pAd-HBx经PacⅠ酶切线性化后,转染到QBI-293A细胞中,根据EGFP的表达判断重组腺病毒的包装情况。RT-PCR鉴定重组腺病毒的HBx基因表达。根据GFP的表达检测病毒滴度和感染效率。将病毒感染人肝癌HepG2细胞,48h后收集裂解细胞,Westernblot检测HBx蛋白的表达。结果：重组腺病毒载体pAd-HBx经酶切鉴定确认构建成功。将pAd-HBx转染到QBI-293A细胞,荧光检测提示病毒获得成功包装,RT-PCR检测到细胞中HBx基因的表达。重组病毒感染人肝癌HepG2细胞,Westernblot检测到HBx蛋白表达。结论：成功构建了携带HBx基因的重组腺病毒,感染肝癌HepG2细胞后检测到了HBx蛋白的表达,提示建立了HBx高表达的细胞模型,为后续的HBx的功能研究奠定了基础。

英文摘要：

Aim：To construct recombinant HBx-expressed adenovirus, and establish a cell culture model with high- level HBx expression by infection. Methods：HBx gene was cloned into the shuttle vector pAdTrack. After confirmed by re- striction digestion, the resultant plasmid was linearized by digestion with Pme I and was subsequently transformed into competent E. coli cells BJ5183 for homologous recombination. The recombination vector pAd-HBx was linearized with Pac I and transfected into QBI-293A cells. The packing of adenovirus was determined by the expression of EGFP. The expres- sion of HBx gene in recombinant adenovirus was identified by RT-PCR. Viral titer and infection efficiency of adenovirus were detected according to the expression of GFP. HepG2 ceils were infected by recombinant adenovirus. Forty-eight hours later, cells were collected and lysed to detect the expression of HBx protein by Western blot. Results：Restriction digestion showed that the recombinant adenovirus vector pAd-HBx was successfully constructed. After pAd-HBx transfected into QBI- 293A cells, fluorescence detection indicated that the adenovirus was successfully packed. The expression of HBx gene was detected by RT-PCR. After infection to the hepatoma HepG2 cells, the expression of HBx protein was detected by Western blot. Conclusion： The recombinant adenovirus expressing HBx gene has been successfully constructed. After infecting HepG2 cell,the HBx protein expression is detected, suggesting cell culture model with high-level expression of HBx is es- tablished.