携EGFP的人Synoviolin基因慢病毒表达载体的构建与鉴定  

Construction and identification of lentiviral expression vector containing human Synoviolin gene and enhanced green fluorescent protein gene

作　　者：陈钢[1] 张绍祥[1] 岳娟娟[3] 杨星[2] 张正治[1] 

机构地区：[1]第三军医大学基础医学部中心实验室,重庆400038 [2]解放军第324医院肝胆外科,重庆400020 [3]检验系药理学教研室

出　　处：《免疫学杂志》2010年第5期448-451,共4页Immunological Journal

基　　金：国家自然科学基金(30672122)

摘　　要：目的构建携EGFP的人Synoviolin基因慢病毒表达载体。方法应用基因重组手段,SalⅠ/NotⅠ双酶切质粒pIRES2-EGFP-syno,得到含EGFP和人Synoviolin的基因片段,将其亚克隆至入门载体pENTR1A的多克隆位点内得到入门质粒pENTR1A-syno-egfp。采用LR重组酶将pENTR1A-syno-egfp和目的载体pLenti4/TO/V5-DEST进行重组反应,形成慢病毒表达载体pLenti4/TO/V5-DEST-syno-egfp。将pLenti4/TO/V5-DEST-syno-egfp与包装质粒混合,利用脂质体共转染293FT细胞,包装产生慢病毒,以293FT细胞GFP蛋白的表达水平测定病毒滴度。结果PCR,酶切及测序结果表明慢病毒表达载体pLenti4/TO/V5-DEST-syno-egfp构建成功,转染后的293FT细胞在荧光显微镜下观察可见强绿色荧光。包装的慢病毒原液滴度为1×10^8TU/ml。结论成功构建了EGFP和Synoviolin基因共表达的慢病毒表达载体,为Synoviolin基因的功能研究提供了高效稳定的转基因技术平台。In this paper,we aimed to construct a lentiviral expression vector containing the EGFP-Synoviolin gene,for further study on the function of Synoviolin gene.The plasmid plRES2-EGFP-syno was digested by SalⅠ/NotⅠto obtain Synoviolin-EGFP cDNA fragment,then the fragment was subcloned into the multiple sites of pENTR1A for constructing the entry plasmid pENTR1A-syno-egfp. After LR reaction between pENTR1A-syno-egfp and pLenti4/TO/V5-DEST,the pLenti4/TO/V5-DEST-syno-egfp was obtained, and then confirmed by PCR,DNA sequencing,and restriction enzyme digestion analysis.Consequently,the recombinant vector and lentiviral packaging system were cotransfected into 293FT cells through lipofectamine,while the titer of virus was tested according to the expression level of GFP.The expression of EGFP could be seen by confocal fluorescent microscope;the green fluorescence was observed in the 293FT cells under fluorescence microscope after transfection;and the titer of virus was 1×10~8 TU/ml.The results above indicate that the lentiviral expression vector with Synoviolin and EGFP is successfully constructed,which provides an effective and stable transgenic technology platform for the function study on Synoviolin.

关 键 词：Synoviolin 绿色荧光蛋白 慢病毒表达载体 

分 类 号：Q78[生物学—分子生物学]