大鼠galectin-9基因重组腺病毒载体的构建和鉴定  

Construction and Identification of Recombinant Adenovirus Vector of Rat Galectin-9 Gene

作　　者：李来邦[1] 李立[1] 冉江华[1] 赵永恒[1] 张升宁[1] 刘静[1] 李铸[1] 蒋益舟[1] 

机构地区：[1]昆明市第一人民医院暨昆明医学院附属甘美医院肝胆胰一科,云南省器官移植研究所肝移植研究中心,云南昆明650011

出　　处：《中国普外基础与临床杂志》2011年第2期131-136,共6页Chinese Journal of Bases and Clinics In General Surgery

基　　金：云南省科技厅社会发展项目基金资助(项目编号:2007CA007)~~

摘　　要：目的克隆大鼠galectin-9基因全长cDNA,构建含大鼠galectin-9基因的重组腺病毒载体,并予以鉴定。方法从大鼠的肝脏组织中用RT-PCR的方法克隆扩增大鼠galectin-9基因,再定向插入到带NotⅠ和HindⅢ酶切的pDC316-GFP穿梭质粒中,获得穿梭质粒pDC316-GFP-galectin-9。经PCR、NotⅠ和HindⅢ酶切及测序鉴定后,用脂质体将穿梭质粒pDC316-GFP-galectin-9与腺病毒骨架质粒pBHGlox△E1.3Cre共转染HEK-293细胞。经位点特异性重组获得含目的基因的重组腺病毒Ad5-galectin-9,行PCR鉴定,经HEK-293细胞扩增及纯化制备高滴度病毒液,用细胞培养方法测定病毒TCID50滴度。结果 PCR、酶切及测序证实穿梭质粒构建正确,PCR鉴定证实大鼠galectin-9基因重组腺病毒载体构建正确,病毒的感染滴度为1.4×109U/ml。结论成功构建了含大鼠galectin-9基因的重组腺病毒表达载体,为进一步研究大鼠galectin-9基因的功能奠定了基础。Objective To clone full-length cDNA of rat galectin-9 and construct recombinant adenovirus granule containing rat galectin-9 gene.Methods The galectin-9 gene was amplified by RT-PCR from rat liver tissue and inserted orientationally into plasmid pDC316-GFP digested by restriction endonucleases NotⅠ and HindⅢ.The recombinant pDC316-GFP-galectin-9 shuttle plasmid was identified by PCR,restriction endonuclease digestion and sequencing,and then co-transfected with rescue plasmid pBHGlox△E1.3Cre into HEK-293 cells by liposome reagent.Recombinant adenovirus vector containing rat galectin-9 gene（Ad5-galectin-9） was generated by site-specific recombination and confirmed by PCR,and then Ad5-galectin-9 was propagated in HEK-293 cells and purified.The infectious titer of viral stock was determined by TCID50 assay.Results Construction of pDC316-GFP-galectin-9 shuttle plasmid was confirmed to be correct by PCR,restriction endonuclease digestion and sequencing.Construction of recombinant adenovirus Ad5-galectin-9 was confirmed to be correct by PCR.The infective titer of Ad5-galectin-9 was 1.4×109 U/ml.Conclusion Recombinant adenovirus vector containing rat galectin-9 gene（Ad5-galectin-9） is successfully constructed,which provides the foundation of further research on the function of galectin-9 gene.

关 键 词：大鼠 GALECTIN-9 基因克隆 腺病毒载体 RT-PCR 

分 类 号：R341[医药卫生—基础医学]