趋化因子受体4基因重组腺病毒载体的构建及鉴定

Construction and identification of recombinant adenovirus vector containing CXC chemokine receptor 4 gene

背景:目前研究发现,基质细胞衍生因子1/趋化因子受体4轴具有介导骨髓间充质干细胞定向迁移的作用。目的:构建小鼠趋化因子受体4基因重组腺病毒载体。方法:从C57BL/6小鼠中提取总RNA,以RT-PCR方法获得小鼠趋化因子受体4基因全长1080bp的完整编码序列,通过穿梭质粒pAdTrack-CMV,将目的片段克隆入AdEasy-1腺病毒DNA中,获得重组腺病毒DNA,通过脂质体转染293细胞,经包装扩增后,获得重组腺病毒pAdTrack-CMV-CXCR4,并感染293细胞,PCR鉴定、Western blot检测蛋白表达。结果与结论:含趋化因子受体4基因的重组腺病毒pAdTrack-CMV-CXCR4构建成功,经PCR鉴定鉴定重组病毒中含有趋化因子受体4cDNA全长,经序列测定表明趋化因子受体4cDNA序列正确无丢失和错配现象,病毒滴度高,Western blot检测感染后293细胞趋化因子受体4的蛋白表达较未感染293细胞明显增加。

BACKGROUND：Recent studies have demonstrated that stromal cell-derived factor-1（SDF-1）/CXC chemokine receptor 4（CXCR4） is capable of promoting the migration of bone marrow mesenchymal stem cells（BMSCs）.OBJECTIVE：To construct recombinant adenovirus vector containing CXCR4 gene and provide the basis for further research of CXCR4 gene for application.METHODS：The total RNA of mouse was extracted and CXCR4 gene which is 1080 bp in size was amplified by RT-PCR.Fragment containing CXCR4 was cloned into the shuttle vector pAdTrack-CMV that carried a green fluorescence protein（GFP） gene to generate a recombinant plasmid pAdTrack-CMV-CXCR4.The recombinant adenovirus pAdEasy-CMV-CXCR4 was transferred into HEK293 cells for packaging and amplification.The viral titer was determined and the insert of CXCR4 gene was verified by PCR method and western blot.RESULTS AND CONCLUSION：The recombinant adenovirus vector（pAdEasy-CMV-CXCR4） was successfully constructed and the sequence was corrected by PCR and sequencing,which possesses high titers.Western blot showed CXCR4 protein expression in infected HEK293 cells was markedly higher than that in uninfected HEK293 cells.