中国人常见GJB2基因突变表达载体的构建及鉴定  

Construction of GJB2 mutations common in Chinese EGFP fusion protein vectors

作　　者：张延平[1] 张元丁[2] 李丽娜[1] 马磊[1] 孙玉蕊[1] 张宗霖[1] 刘金伟[1] 邓惠严[1] 祝威[2] 

机构地区：[1]解放军总医院第二附属医院耳鼻咽喉科,北京100091 [2]吉林大学第一医院耳鼻咽喉-头颈外科

出　　处：《临床耳鼻咽喉头颈外科杂志》2009年第16期724-727,共4页Journal of Clinical Otorhinolaryngology Head And Neck Surgery

基　　金：全军十一五项目归国人员课题(No:06H048);国家自然科学基金面上项目青年基金(No:30700936)资助

摘　　要：目的:构建中国人常见GJB2基因突变235de1C、299-300delAT和176de116bp与增强型绿色荧光蛋白(EGFP)的融合蛋白表达载体,寻找体外研究GJB2基因缺失突变致聋机制的有效途径。方法:用体外定点突变法构建235de1C、299-300delAT和176de116bp突变全序列与EGFP表达载体,以此为模板PCR扩增突变有效表达序列,将PCR产物连接到pMD19-T载体中,EcoRI/BamHI双酶切克隆载体,测序鉴定序列正确性后,将酶切产物插入pEGFP-N1载体中,脂质体转染HEK293细胞,荧光显微镜观察表达的融合蛋白。结果:GJB2基因突变235de1C、299-300delAT和176de116bp在HEK293细胞中高效表达,表达主要位于细胞质中。结论:成功构建了中国人常见GJB2基因突变235de1C、299-300delAT和176de116bp与EGFP的融合蛋白表达载体,为进一步研究其致聋机制奠定了基础。Objective：To construct GJB2 gene mutaitons common in Chinese EGFP fusion protein vectors, and to search for better way to study the mechanism of deletion mutaitons in GJB2 gene. Method： Non-fusion protein vectors of 235delC, 299-300 del AT and 176 del 16 bp were first made by point mutaiton methods in vitro. Then expression part of the upper 3 mutations were amplified by PCR and the PCR products were cloned into TA clo- ning vector. After cutting by restriction enzymes EcoRI/BamHI, three deletion mutaions were inserted into pEG- FP-N1 vector. Sequencing was used to verify the validity of the fusion protein vectors. HEK293 cells were transfected with the recombinant DNA samples by the liposome complex method. Result：The recombined plasmids were highly expressed in HEK293 cells. Green fluorescence singals were distributed uniformly in cytoplasm. Conclusion： GJB2 mutations common in Chinese EGFP fusion protein vectors were constructed successfully. It may provide a better way to explore the reasons of nonsyndromic hearing loss common in Chinese.

关 键 词：缝隙连接蛋白26 突变 绿色荧光蛋白 

分 类 号：R764.43[医药卫生—耳鼻咽喉科]