人Seipin基因真核表达质粒的构建及鉴定  

Construction and Characterization of Human Seipin Eukaryotic Expression Vector

作　　者：李道静[1] 童先宏[1] 

机构地区：[1]安徽医科大学附属省立医院生殖中心,合肥230001

出　　处：《临床输血与检验》2015年第2期111-113,共3页Journal of Clinical Transfusion and Laboratory Medicine

摘　　要：目的构建人Seipin基因的真核表达质粒并转染293T细胞后进行鉴定。方法采用PCR法从人睾丸组织中扩增人Seipin基因,通过BamHI/XhoI限制性酶切位点克隆入真核表达载体pEGFP-C1内,构建含人Seipin基因的真核表达质粒pEGFP-Seipin。运用真核转染、Western blot和免疫荧光实验的方法,鉴定Seipin在真核细胞293T中的表达。结果克隆的人Seipin基因,测序结果显示完全正确。瞬时转染真核细胞293T后,采用Western blot在预期的位置检测出目的条带,免疫荧光实验显示293T细胞中存在人Seipin基因的表达。结论成功构建了含人Seipin基因的真核表达质粒。Objective To construct and characterize an eukaryotic system for expression of human Seipin.MethodsThe human seipin gene was amplified by PCR from the template of testis tissue,and subcloned into pEGFP-C1 vector to construct a recombinant plasmid of pEGFP-C1-seipin.The expression of human Seipin in 293 Tcells was verified by Western blotting.Immunofluorescence assay was used to detect the expression efficiency of Seipin in host 293 Tcells.Results The inserted Seipin gene was confirmed by DNA sequencing.A high efficiency of expression of Seipin in 293 Tcells transfected with pEGFP-C1-seipin was identified by Western blotting.Conclusion An eukaryotic expression plasmid pEGFP-C1-seipin has been established successfully,which might provide a base for use of pEGFP-C1-seipin in the study of human reproduction.

关 键 词：Seipin基因 质粒 真核表达 免疫荧光 

分 类 号：R392.11[医药卫生—免疫学]