CAJ | 학술논문

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；West-ern 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。
목적：구건병험증 Brugada 종합정상관납통도 SCN5a 기인 R104W 돌변체。방법채용일보법 PCR 돌변기술,이 pEGFP-SCN5a 위모판,체외정점유변구건 pEGFP-SCN5a-R104W 돌변체,진행기인측서,병용 Lipofectamine TM3000지질체전염법전입 HEK293세포,통과 Western blotting 화막편겸기록검측단백질표체화전류가이험증。결과측서결과현시 SCN5a-R104W 돌변체기인서렬상제310 C>T,기타서렬감기여야생형상비미발생개변；West-ern blotting 결과현시 SCN5a-R104W 돌변체단백표체량교야생형 SCN5a 강저；형광현미경하현시 SCN5a-R104W 돌변체단백위우포질내；SCN5a-R104W 돌변체전염후막편겸기록미능검측도납전류,차 R104W 돌변체대야생형통도유부현성억제작용。결론성공구건병험증 SCN5a 기인 R104W 돌변체。
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.