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310C>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。
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.
出处
《西安交通大学学报（医学版）》
CAS
CSCD
北大核心
2015年第4期435-438,443,共5页
Journal of Xi’an Jiaotong University（Medical Sciences）
基金
国家自然科学基金资助项目(No.81170175)~~
