%0 Journal Article
%T 钠通道SCN5a突变体R104W的构建及验证<br>Construction and verification of R104W mutant of SCN5a sodium channel
%A 胡培静
%A 杜
%A 媛
%A 王
%A 娅
%A 王亭忠
%A 王娟娟
%A 陈春燕
%A 马爱群
%J 西安交通大学学报(医学版)
%D 2015
%R 10.7652/jdyxb201504002
%X 摘要：目的 构建并验证Brugada综合征相关钠通道SCN5a基因R104W突变体。方法 采用一步法PCR突变技术，以pEGFP-SCN5a为模板，体外定点诱变构建pEGFP-SCN5a-R104W突变体，进行基因测序，并用Lipofectamine TM3000脂质体转染法转入HEK293细胞，通过Western blotting和膜片钳记录检测蛋白质表达和电流加以验证。结果 测序结果显示SCN5a-R104W突变体基因序列上第310 C>T，其他序列碱基与野生型相比未发生改变；Western blotting结果显示SCN5a-R104W突变体蛋白表达量较野生型SCN5a降低；荧光显微镜下显示SCN5a-R104W突变体蛋白位于胞质内；SCN5a-R104W突变体转染后膜片钳记录未能检测到钠电流，且R104W突变体对野生型通道有负显性抑制作用。结论 成功构建并验证SCN5a基因R104W突变体。<br>ABSTRACT: Objective To construct and verify the R104W mutant of SCN5a channel. Methods The SCN5a-R104W mutant was constructed by rapid site-directed mutagenesis, and the expected mutation was confirmed by direct sequencing. The mutant DNA was transfected into HEK293 cells using Lipofectamine TM3000. Function of the SCN5a-R104W mutant was tested by Western blot analysis and whole-cell patch clamp recording. Results Sequencing results showed that the base on 310 was changed from C to T of SCN5a-R104W mutant DNA. Protein expression of SCN5a-R104W mutant was lower than that of wild-type SCN5a (SCN5a-WT) channel. SCN5a-WT channels were expressed on the cell surface and SCN5a-R104W channels were mainly expressed in the cytoplasm. Patch clamping result showed that no sodium current was recorded from the cells expressing SCN5a-R104W mutant channel, and SCN5a-R104W exerted dominant-negative effect on SCN5a-WT channel. Conclusion Trafficking deficient SCN5a-R104W mutant channel was successfully constructed and verified
%K 钠通道
%K R104W突变
%K Brugada综合征
%K 定点诱变&lt
%K br&gt
%K sodium channel
%K R104W mutant
%K Brugada syndrome
%K site-directed mutagenesis
%U http://yxxb.xjtu.edu.cn//oa/darticle.aspx?type=view&id=201504002