中国人常见GJB2基因突变片段TA克隆载体构建  

Construction of TA cloning vector carrying common GJB2 mutations in the Chinese population

作　　者：张元丁[1,2] 张延平[1] 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喉科,北京100091 [2]吉林大学第一医院耳鼻咽喉一头颈外科,长春130021

出　　处：《中国听力语言康复科学杂志》2009年第1期13-16,共4页Chinese Scientific Journal of Hearing and Speech Rehabilitation

基　　金：基金项目：全军十一五项目归国人员课题（项目编号：06H048）;国家自然科学基金面上项目青年基金（项目编号：30700936）

摘　　要：目的应用TA克隆技术构建含中国人常见GJB2基因突变片段的载体，为构建突变绿色荧光蛋白融合载体提供基础。方法首先利用定点诱变法在体外构建235delC．299-300delAT和176del16bp绿色荧光蛋白非融合载体，以此为模板利用PCR扩增突变基因片段，然后将PCR扩增产物克隆到TA载体上，用限制性内切酶法和测序法鉴定重组质粒序列正确性。结果用限制性内切酶法和测序方法证实扩增片段为目的片段。结论成功地构建了235delC、299-300delAT和176del16bp突变基因片段TA克隆载体，为构建突变绿色荧光蛋白融合载体、进一步研究突变致聋机制奠定了基础。Objective To construct vectors of common GJB2 gene mutations in the Chinese population with the TA cloning technique, and to provide mutant DNA for making EGFP（enhanced green fluorescent protein） fusion protein vectors. Methods Non-fusion protein vectors of 235delC, 299-300delAT and 176del16bp were first made by site-directed mutagenesis in vitro. Then the three mutant gene fragments were amplified by PCR. The PCR products were cloned into TA cloning vectors. The restriction enzyme and sequencing were used to verify the validity of the mutations. Results The TA cloning vectors were identified to have 235delC, 299-300delAT and 176del16bp by restriction enzyme analysis and sequencing. Conclusion The TA cloning vectors with common GJB2 gene mutations in the Chinese population were constructed successfully, and this can be used for the construction of EGFP fusion protein vectors and further research on the mechanism of non-syndromic hearing loss.

关 键 词：缝隙连接蛋白26 突变 TA克隆载体 

分 类 号：R764.5[医药卫生—耳鼻咽喉科]