| 王森 赵毅 康海仙 丁伟斌 姚运红 胡新荣 王云 罗兵 朱伟.EBV融合基因Z2A 腺病毒的制备及其在诱导EBV阳性
细胞凋亡的应用[J].,2014,14(13):2426-2429 |
| EBV融合基因Z2A 腺病毒的制备及其在诱导EBV阳性
细胞凋亡的应用 |
| Construction of Recombinant Adenovirus Expressing EBV fusion gene Z2Aand its Application on Apoptosis of EBV+ Cells |
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| DOI： |
| 中文关键词: LMP2A BZLF1 融合基因 腺病毒载体 EBV |
| 英文关键词: LMP2A BZLF1 Fusion gene Adenoviral vector EBV |
| 基金项目:国家自然科学基金项目（81302244）；广东省自然科学基金博士启动项目（S2012040006311，S2012040006383）；
广东医学院科研基金项目（B2011004，B2011012） |
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| 中文摘要: |
| 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。 |
| 英文摘要: |
| 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. |
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