摘要
目的:制备携带Bclxl基因的重组腺相关病毒,并鉴定其感染细胞有效性。方法:抽提BALB/c小鼠脾脏总RNA,通过RT-PCR获得cDNA,然后设计引物通过PCR获得Bclxl基因的编码序列。将得到的Bclxl编码序列插入腺相关病毒质粒pAAV-MCS中的多克隆位点,从而构建出重组腺相关病毒质粒pAAV-Bclxl。将质粒转染细胞后使用Western Blot和流式分析检测Bclxl蛋白的表达。进一步包装制备了携带Bclxl基因的重组腺相关病毒颗粒,用其感染HT1080细胞后用流式细胞仪分析了感染前后细胞中Bclxl基因的表达情况。结果:质粒转染后细胞中Bclxl蛋白的表达水平显著高于转染前,病毒感染后细胞中Bclxl蛋白的表达水平显著高于感染前。结论:携带Bclxl基因的重组腺相关病毒颗粒包装成功,能高效感染细胞,为后续的遗传改造树突细胞打下了基础。
Objective：To construct a recombinant adeno-associated virus containing Bclxl gene,and to detect its infection capability.Methods：Splenic total RNA were extracted from BALB/c mice,and cDNA were obtained by RT-PCR.Bclxl-encoded sequence was obtained by a further PCR from the cDNA and then inserted into the multiple clone sites of an adeno-associated viral plasmid pAAV-Bclxl to construct an adeno-associated viral plasmid pAAV-Bclxl.Bclxl protein was detected in pAAV-Bclxl-transfected cells by western blot and FACS analysis.Further,the recombinant adeno-associated virus containing Bclxl was assembled,and used to infect HT1080 cells.FACS analysis was used to detect Bclxl expression in infected cells.Results：The results indicate that the Bclxl expression level in the transfected or infected cells is significantly higher than that in the control cells.Conclusion：The recombinant adeno-associated virus containing Bclxl was successfully constructed,which laid a foundation for using Bclxl to genetically modify dendritic cells.
出处
《现代生物医学进展》
CAS
2010年第1期34-36,49,共4页
Progress in Modern Biomedicine
基金
国家863课题(2006AA02A102)
国家973课题(2007CB948103和2007CB947901)
