HBx基因重组腺病毒载体构建及其在SMMC-7721细胞中的表达

目的:利用pAdEasy1腺病毒载体系统构建人HBx基因重组腺病毒,感染肝癌细胞株SMMC7721使HBx基因有效表达。方法:自真核表达载体pcDNA3.1(-)HBx中获得HBx基因,插入腺病毒穿梭质粒pAdTrackCMV中构建pAdTrackCMVHBx,然后经PmeⅠ酶切线性化,电转化到含腺病毒骨架质粒pAdEasy1的BJ5183感受态细菌中。挑选和鉴定正确的同源重组质粒,然后将线性化的重组质粒转染293N细胞,产生重组病毒颗粒,并进一步感染SMMC7721细胞株。结果:经限制性内切酶酶切和基因测序鉴定,证实pAdTrackCMVpAdEasy1HBx重组成功,借助荧光显微镜可以观察到绿色荧光蛋白GFP在293N和SMMC7721细胞株中表达。结论:成功构建了携带HBx基因的重组腺病毒载体,并在SMMC7721细胞中使HBx基因有效表达,为后续研究奠定了基础。

Construction of recombinant adenovirus vector containing HBx gene and its expression in SMMC-7721 cell line

OBJECTIVE:To construct a recombinant adenovirus vector mediated HBx expression in SMMC-7721 cell line. METHODS:HBx gene was obtained from pcDNA3.1(-)-HBx by enzyme digestion, and then inserted into adenovirus shuttle plasmid pAdTrack-CMV to generate a recombinant plasmid pAdTrack-CMV-HBx. After linearized by PmeI, the recombinant was transformed into E. coli BJ5183 cells containing adenovirus backbone plasmid pAdEasy-1 by electroporation. The homologous recombinant was identified, linearized, and then transfected into 293N cells to package the adenovirus, followed by infection of SMMC-7721 cell line. RESULTS: Recombinant plasmid pAdTrack-CMV- pAdEasy-1-HBx was successfully constructed, which was confirmed by restriction enzyme digestion and gene sequencing. The high expression of green fluorescence protein expression in 293N and SMMC-7721 cell lines was found under fluorescent microscope. CONCLUSIONS: The successful construction of the recombinant adenoviral vector containing HBx gene and the effective expression of HBx gene in SMMC-7721 cell line has laid a foundation for further study of its anticancer function.