DH结构域缺失对SGEF在293T细胞中定位的影响  

Effect of DH Domain Deletionon on SGEF Subcellular Localization in 293T Cell

作　　者：王洪涛[1] 俞岚[1] 施庆国[1] 李山虎[1] 钱晓龙[1] 周建光[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100850

出　　处：《生物技术通讯》2011年第2期163-167,共5页Letters in Biotechnology

基　　金：国家自然科学基金(30770834;30870961);国家高技术研究发展计划(2008AA02Z123)

摘　　要：目的:通过构建带EGFP标签的SGEF基因DH结构域缺失的真核表达载体pEGFP-C1-SGEF-△DH并使其在293T细胞表达,观察DH结构域缺失后SGEF在293T细胞中的定位。方法:利用重叠PCR技术在pcDNA3.1-SGEF质粒上扩增缺失DH结构域的SGEF基因,然后将PCR产物亚克隆到真核表达载体pEGFP-C1上,对阳性克隆进行双酶切和测序鉴定,利用脂质体转染方法转染293T细胞,并用Western印迹和细胞免疫荧光技术对重组质粒pEGFP-C1-SGEF-△DH在293T细胞中的表达及其蛋白定位进行分析。结果:双酶切和测序鉴定表明,pEGFP-C1-SGEF-△DH真核表达质粒构建成功,转染实验发现该质粒能够在293T细胞中表达,表达产物主要定位在细胞核内。结论:构建了带EGFP标签的人SGEF基因DH结构域缺失的真核表达载体,该载体能够在哺乳动物细胞293T中表达,表达产物定位于细胞核,为进一步研究SGEF基因DH结构域的细胞生物学功能提供了一个重要的工具。Objective：To detect the subcellular localization of Src homology 3 domain-containing guanine nucleotide exchange factor（SGEF） without Dbl homology domain（DH） domain in 293T cell line by constructing eukaryotic expression vectors of SGEF gene without DH domain labeled with EGFP and transfecting 293T cells with it.Methods：The SGEF gene without DH domain sequence was amplified with the vector pcDNA3.1-SGEF as template by overlap extension PCR,and inserted into vector pEGFP-C1 to construct the eukaryotic expression vector pEGFP-C1-SGEF-△DH.The 293T cells were transfected with the vector pEGFP-C1-SGEF-△DH,which had been identified by double digestion with restriction endonuelease,followed by sequencing.After culturing for a certain time,the expression of EGFP-SGEF-△DH was detected with Western blotting and the localization was detected with immunofluorescence.Results：The results of identification by double digestion with restriction endonuelease and sequencing indicated that the vector pEGFP-C1-SGEF-△DH was correctly constructed.The transfection experiments and Western blotting suggested that the EGFP-SGEF-△DH gene carried by vector pEGFP-C1-SGEF-△DH could express in 293T cells.The protein EGFP-SGEF-△DH mainly accumulated in the area of the nucleus.Conclusion：The eukaryotic expression vector of human SGEF gene without DH domain labeled with EGFP sequence has been successfully constructed.It has been observed that the constructed vector may be expressed in the area of the nucleus of the 293T cells.Thus,the present work has provided an important tool for further study on the SGEF function inside cells.

关 键 词：SGEF DH结构域 载体构建 重叠PCR 基因表达 蛋白定位 

分 类 号：Q78[生物学—分子生物学]