人GJB6基因表达载体的构建及鉴定  

Construction of GJB6-DsRed fusion protein vectors

作　　者：李丽娜[1] 张延平[1] 孙玉蕊[1] 张宗林[1] 王戈[1] 张晓强[1] 冯冲[2] 潘登科[2] 

机构地区：[1]解放军第309医院耳鼻喉科,北京100091 [2]中国农业科学院北京畜牧兽医研究所,北京100094

出　　处：《中华耳科学杂志》2010年第4期470-473,共4页Chinese Journal of Otology

基　　金：军队"十一五"归国人员课题(06H048);国家自然科学基金面上项目青年基金(30700936);解放军第309医院课题(LCB08MS12;309-09-03)

摘　　要：目的构建人野生型Cx30与红色荧光蛋白DsRed的融合蛋白表达载体,为揭示Cx30突变患者发病机制提供实验依据。方法用PCR法扩增GJB6基因,将PCR产物与T载体连接,用双切酶酶切pEASY-GJB6与载体DsRed-N1,连接回收后的片断,构建野生型Cx30编码序列与PDsRed2表达载体,测序鉴定序列正确性。将GJB6-DsRed用脂质体转染HEK293细胞,荧光显微镜观察表达的融合蛋白。结果 GJB6-DsRed在HEK293细胞中高效表达,表达主要位于细胞膜中。结论成功构建了人野生型Cx30与红色荧光蛋白DsRed的融合蛋白表达载体,为进一步研究非综合征性聋的致聋机制奠定了基础。Objective To construct the wide-type human Cx30 and DsRed fusion protein vector as a tool for studying mechanism of mutations in the GJB6 gene causing nonsyndromic hearing loss.Methods Wide-type GJB6 gene was amplified by PCR.The PCR products were inserted into the T-vector.After cutting by restriction enzymes EcoR I/Sac II,GJB6 was inserted into DsRed2-N1 vector.Sequencing was used to verify the validity of the fusion vectors.HEK293 cells were transfected with the recombinant DNA samples by the liposome complex method.Results The recombined plasmids were highly expressed in HEK293 cells.Red fluorescence singals were distributed mainly in the cell membrane.Conclusion Wide-type human GJB6 gene and DsRed fusion protein vector is constructed successfully.It may provide a useful tool to explore the reasons of nonsyndromic hearing loss caused by GJB6 and GJB2.

关 键 词：缝隙连接蛋白30 红色荧光蛋白 融合蛋白表达载体 

分 类 号：R349.6[医药卫生—基础医学]