EBV即刻早期基因BZLF1腺病毒载体的构建和表达

发布时间：2018-07-23 15:02

【摘要】：目的　构建EBV即刻早期基因BZLF1的非复制型重组腺病毒载体,293细胞包装腺病毒,并检测BZLF1在293细胞中的表达,为进一步研究腺病毒介导BZLF1进行EBV相关肿瘤的基因治疗奠定基础。方法 RT-PCR扩增EBV BZLF1基因片段,经双酶切后亚克隆到腺病毒穿梭质粒pAdTrack-CMV上,用TSS两步法与腺病毒骨架质粒pAdEasy-1共转染大肠杆菌BJ5183,经细菌内同源重组产生携带BZLF1基因的重组腺病毒载体pAd-BZ,脂质体法转染人胚肾293细胞,包装产生重组腺病毒vAd-BZ。采用RT-PCR和Western blotting检测BZLF1基因在293细胞中的表达。结果通过PCR、序列测定、双酶切等技术鉴定BZLF1基因已正确插入穿梭质粒中,并与病毒骨架质粒pAdEasy-1重组,成功构建了表达BZLF1基因的重组腺病毒。荧光显微镜观察到绿色荧光,证实在293细胞中包装出vAd-BZ,PCR和Western结果表明转染pAd-BZ的293细胞中有BZLF1基因的表达。结论成功构建了表达EBV BZLF1重组腺病毒vAd-BZ,为进一步探讨BZLF1基因表达诱导EBV阳性肿瘤细胞中潜伏感染状态病毒进入裂解期,进而杀伤EBV阳性肿瘤细胞奠定了实验基础。
[Abstract]:Objective to construct the non-replicating recombinant adenovirus vector of EBV immediate early gene BZLF1, and to detect the expression of BZLF1 in 293cells, so as to lay a foundation for the further study of adenovirus-mediated BZLF1 gene therapy for EBV related tumors. Methods the EBV BZLF1 gene fragment was amplified by RT-PCR and subcloned into adenovirus shuttle plasmid pAdTrack-CMV after double enzyme digestion. E. coli BJ5183 was co-transfected by TSS two-step method with adenovirus skeleton plasmid pAdEasy-1. Recombinant adenovirus vector pAd-BZwith BZLF1 gene was produced by homologous recombination in bacteria. The recombinant adenovirus vAd-BZwas packaged and transfected into human embryonic kidney 293 cells by liposome method. The expression of BZLF1 gene in 293 cells was detected by RT-PCR and Western blotting. Results the BZLF1 gene was correctly inserted into the shuttle plasmid by PCR sequencing and double enzyme digestion. The recombinant adenovirus expressing BZLF1 gene was successfully constructed by recombination with the viral skeleton plasmid pAdEasy-1. Green fluorescence was observed by fluorescence microscope. The results of vAd-BZN PCR and Western showed that the BZLF1 gene was expressed in 293 cells transfected with pAd-BZ. Conclusion the expression of EBV BZLF1 recombinant adenovirus vAd-BZwas successfully constructed, which laid an experimental foundation for further study on the induction of latent infection virus in EBV positive tumor cells into lytic phase by BZLF1 gene expression and the killing of EBV positive tumor cells.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R346

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