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目的:构建表达乙肝病毒表面抗原(HBsAg)和e抗原(HBeAg)的非复制型重组腺病毒载体,并检测他能否在真核细胞中有效表达目的基因。方法:扩增乙肝病毒(HBV)前S_2/S基因和前C/C基因片段,分别亚克隆到腺病毒穿梭质粒pAdTrack-CMV上,与5型腺病毒骨架质粒pAdeasy-1共转染BJ5183细菌,经细菌内同源重组产生分别携带HBV S区和C区基因的重组腺病毒载体pAd-HBs和pAd-HBe,经脂质体法转化293细胞包装产生重组腺病毒Ad-HBs和Ad-HBe体,外转染293和Vero细胞,RT-PCR和ELISA法检测目的基因的表达。结果:构建了表达HBsAg和HBeAg基因的重组腺病毒,病毒滴度可达5×10~(12)pfu/L,并能在真核细胞中有效表达目的基因。结论:成功构建表达HBsAg和HBeAg的重组腺病毒载体,为进一步开展HBV基因治疗研究提供实验基础。
OBJECTIVE: To construct a non-replicating recombinant adenovirus vector expressing hepatitis B virus surface antigen (HBsAg) and e antigen (HBeAg) and test whether he can express the target gene in eukaryotic cells effectively. Methods: The pre-S 2 / S and pre-C / C gene fragments of hepatitis B virus (HBV) were amplified and subcloned into the adenovirus shuttle plasmid pAdTrack-CMV respectively. The recombinant adenovirus vector was co-transfected with the adenovirus backbone plasmid pAdeasy-1 into BJ5183 The recombinant adenovirus vectors pAd-HBs and pAd-HBe carrying the S and C regions of HBV were generated by homologous recombination in bacteria. The recombinant adenovirus Ad-HBs and Ad-HBe were transformed into 293 cells by lipofectamine The 293 and Vero cells were transfected in vitro and in vivo. The expression of the target gene was detected by RT-PCR and ELISA. Results: The recombinant adenovirus expressing HBsAg and HBeAg genes was constructed. The titer of the recombinant adenovirus was 5 × 10 ~ (12) pfu / L, and the target gene could be efficiently expressed in eukaryotic cells. Conclusion: The successful construction of recombinant adenovirus vector expressing HBsAg and HBeAg provides the experimental basis for further study of HBV gene therapy.