腺病毒介导的THANK基因在SMMC-7721细胞中的表达

目的:构建THANK腺病毒载体,观察腺病毒感染后THANK在SMMC-7721细胞中的表达情况.方法:把THANK基因克隆入腺病毒穿梭载体pAdTrack-CMV中,与腺病毒骨架质粒pAdEasy-1共转染大肠杆菌BJ5183,获得腺病毒重组质粒,再将获取的腺病毒重组质粒转染入293细胞进行包装,获取THANK重组腺病毒,以不同感染复数(MOI)腺病毒感染SMMC-7721细胞,观察腺病毒对SMMC-7721细胞的感染情况.结果:成功地构建了THANK腺病毒,该腺病毒可高效介导THANK在SMMC-7721细胞中表达,基因转染后第5天,THANK表达高于35 ng/ml,且随时间延长增加.结论:腺病毒可诱导THANK基因在SMMC-7721细胞中高表达.

Expression of THANK gene in SMMC-7721 cell line mediated by THANK adenovirus

Objective:To construct a THANK adenovirus vector and observe expression of THANK in SMMC-7721 cell line after THANK adenovirus infection. Methods: Human THANK cDNA was cloned into shuttle vector pAdTrack-CMV, and the resultant plasmid was linearized and subsequently cotransformed into E.coli BJ5183 with an adenoviral backbone plasmid pAdEasy-1.Recombinants were then selected and the linearized recombinant plasmid was transfected into 293 cell lines. Finally, THANK recombinant adenovirus production was observed by fluorescent microscope and confirmed by PCR and Western blot analysis. Further THANK adenovirus were used to infect SMMC-7721 cells with different MOI. The expression of THANK in SMMC-7721 cells was observed by fluorescent microscope and by PCR and Western blot analysis. Results: A human THANK recombinant adenovirus vector was constructed and infected SMMC-7721 cell line with high efficiency. The expression of THANK in SMMC-7721 cells was over 35 ng/ml 5 d after transfection. Conclusion: Adenovirus can induce stable expression of THANK in SMMC-7721 cells.