乙肝病毒X基因重组腺病毒载体的构建

目的为了探讨HBx基因与肝癌发生的关系，构建HBx基因重组腺病毒载体。方法应用pAdEasyTM腺病毒载体系统，首先从质粒pCDNA3．1-HBx中酶切得到HBx基因，插入腺病毒穿梭质粒pAdTrack—CMV中，得到pAdTrack—CMV—HBx，将其PmeI酶切线性化后，转到含腺病毒骨架质粒pAdEasy-1的BJ5183感受态细菌中，通过同源重组得到重组腺病毒质粒载体pAd—HBx，经PacⅠ酶切线性化后转染人胚肾293细胞，产生重组腺病毒颗粒Ad—HBx。结果、PCR和酶切鉴定证明重组腺病毒Ad—HBx构建成功，在转染的293细胞中可以观察到绿色荧光蛋白GFP。结论成功构建了携带HBx基因的重组腺病毒载体，为后续研究奠定了基础。

Construction of recombinant adenovirus vector carrying HBV X gene

： Objective To establish a recombinant adenovirus vector carrying HBx gene so as to explore the relationship between HBx gene and the pathogenesis of hepatocellular carcinoma. Methods By using AdEasyTM adenovirus vector system, a recombined shuttle vector was constructed through the clone of HBx gene into pAdTrack - CMV, then the vector pAdTrack - CMV - HBx was linearized using Pme I and transformed into BJ5183 cell with pAdEasy - 1 to generate recombined adenovirus plasmid pad - HBx through electroporation. Subsequently, the plasmid was linearized with Pac I and transfected into 293 cells to pack into recombined adenovirus Ad - HBx. Results PCR, DNA sequence and restriction endonuclease digestion analysis indicated that the recombined adenovirus vector Ad - HBx carrying HBx gene had been successfully constructed. Green fluorescent protein （GFP） was observed under the fluorescent microscope. Conclusion A recombinant adenovirus vector containing HBx gene was successfully constructed and laid basis for subsequent studies.