【摘要】 目的:构建EB病毒基因LMP2A及BZLF1的融合基因(Z2A)重组腺病毒表达载体,探讨Z2A融合蛋白对EBV+细胞凋亡的作用。方法:利用AdEasy系统构建重组腺病毒载体pAd-Z2A,而后将之转染293细胞(人胚肾细胞)产生重组腺病毒rAd-Z2A。后者用于感染EBV阳性及阴性细胞,RT-PCR、Western-blotting检测Z2A的mRNA和蛋白表达,以及流式细胞术检测其对EBV阳性细胞凋亡的调控。结果:序列测定和酶切实验均证实,Z2A融合基因正确插入穿梭质粒,并成功获得重组腺病毒表达载体pAd-Z2A及重组腺病毒rAd-Z2A。感染rAd-Z2A的NEC靶细胞检测到Z2A的表达。流式细胞术检测发现,与EBV-细胞组及EBV+空白质粒转染组相比,接种rAd-Z2A的EBV+细胞组48h(P<0.05)凋亡细胞显著增多,72h时细胞近乎全部凋亡(P<0.01)。结论:重组腺病毒rAd-Z2A可有效感染EBV+及EBV-细胞,从而显著促进EBV+细胞凋亡而不影响EBV-细胞,为进一步特异性靶向EBV+肿瘤的基因治疗奠定基础。更多还原

【Abstract】 Objective: To construct recombinant adenovirus carrying EBV fusion gene Z2A(LMP2A/BZLF1) and to investigate its function on the apoptosis of EBV+ cells. Methods: The recombinant adenovirus vector carrying Z2A was constructed with AdEasy system. The resulting construct was linearized and then transfected to 293 cells to generate recombinant adenovirus rAd-Z2A. EBV+ and EBV- cells were infected with recombinant adenoviruses. The expression and its effect on EBV+ cells of target gene were tested by RT-PCR, Western-blotting, and FACS. Results: It was confirmed by sequencing identification and restrictive analysis that the recombinant adenovirus vector was constructed successfully. The recombinant adenovirus rAd-Z2A produced with 293 cells showed stable infectivity. Z2A expression was detected in EBV+ NEC cells infected by recombinant adenovirus rAd-Z2A. And the expression of Z2A promoted markedly the apoptosis of EBV+ cells, at 48h(P<0.05) and 72h(P<0.01) compared with that of EBV- cells and EBV+ cells infected with empty vector. Conclusion: The recombinant adenoviruses expressing Z2A can effectively infect EBV+ cells and promote the apoptosis of EBV+ cells without obvious effects on EBV- cells, which can be applied further to gene therapy of EBV infection-related tumors.更多还原