摘要
目的用AdMax载体系统构建人SCL基因重组腺病毒载体,为研究SCL基因对ICC样细胞功能恢复奠定基础。方法采用PCR方法从含人SCL基因质粒中扩增SCL基因,连接到腺病毒穿梭质粒的多克隆位点上,构建重组穿梭质粒pDC315-EGFP/SCL,在脂质体介导下与腺病毒辅助大质粒pBHGlox(delta)E1、3Cre共转染293细胞,包装产生复制缺陷型重组腺病毒pDC315-SCL经HEK293细胞扩增,纯化后测定病毒滴度。结果 PCR结果和Western blotting检测证实pDC315-SCL重组腺病毒载体构建成功,滴度达到1×1010PFU/mL。结论 AdMax载体系统可成功构建pDC315-SCL重组腺病毒载体。
[Objective] Human SCL gene were constructed into recombinant adenovirus expression vector by AdMax vector system in order to research SCL gene function in ICC-like cells. [Methods] SCL gene was amplified from plasmid with SCL gene by PCR and inserted to the polyclonal site of adenovirus shuttle plasmid pDC315-EGFP. HEK 293 cells were co-transfected with the consu＇ucted recombinant shuttle plasmid pDC315-EGFP/SCL and large adenovirus helper plasmid pBHGlox （delta） El, 3Cre in mediation of tiposome. The obtained replication-defective recombinant adenovirus pDC315-SCL was propagated in HEK 293 cells, purified pDC315-SCL plasmid and determined for virus titer. [Results] PCR analysis and Western Blot in- spection confirmed that the human SCL gene was successfully inserted into the adenovirus vector. The titer of the recombinant adenovirus was l×l0^10 PFU/mL. [Conclusions] Recombinant adenovirus containing human SCL gene was successfully constructed by AdMax vector system.
出处
《中国现代医学杂志》
CAS
CSCD
北大核心
2011年第17期1949-1952,共4页
China Journal of Modern Medicine
基金
国家自然科学基金(No:30860281)
关键词
人SCL基因
重组腺病毒载体
转染
human SCL gene
recombinant advenovirus vector
transfection
