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目的研究单独表达无功能的无义突变体L539fs/47在HEK293细胞中杂合状态下对野生型HERG电流的作用。方法用脂质体转染法将野生型HERG及突变体HERG-L539fs/47分别转染HEK293细胞36h后,RT-PCR检测HERG mRNA表达,免疫荧光及免疫印迹检测HERG蛋白定位及表达量,全细胞膜片钳检测IHERG电流密度。结果野生组HERG的mRNA表达量高于L539fs/47突变组(1.066±0.612 vs.0.254±0.397,P<0.01)。免疫荧光检测发现野生型HERG蛋白多于突变体,野生型HERG蛋白主要分布在细胞膜上;而HERG突变体蛋白大部分滞留胞质。免疫印迹显示:不同于野生型HERG135ku及155ku 2个条带,突变体仅60ku 1个条带。全细胞膜片钳检测WT+MT组IHERG较WT组下降55.23%(22.03±2.62 vs.49.20±2.31pApF,P<0.01)。结论 HEK293细胞中杂合状态下截断突变体L539fs/47对野生型HERG电流的不完全负显性抑制作用。
AIM To investigate the effect of nonsense mutant L539fs / 47 expressing wild-type alone on heterozygous HERG currents in heterozygous HEK293 cells. Methods HERG mRNA expression was detected by RT-PCR after transfection of wild-type HERG and mutant HERG-L539fs / 47 into HEK293 cells by lipofectamine. The localization and expression of HERG protein were detected by immunofluorescence and immunoblotting Cell patch clamp detection of IHERG current density. Results The mRNA expression level of HERG in wild group was higher than that in L539fs / 47 mutant group (1.066 ± 0.612 vs.0.254 ± 0.397, P <0.01). Immunofluorescence showed that there were more wild type HERG proteins than mutants, while wild type HERG proteins were mainly distributed on the cell membrane. Most HERG mutant proteins retained cytoplasm. Western blotting showed that, unlike the wild-type HERG135ku and 155ku two bands, the mutant only had 60ku 1 band. The IHERG of whole cell patch clamp in WT + MT group decreased by 55.23% (22.03 ± 2.62 vs.49.20 ± 2.31pApF, P <0.01) compared with WT group. Conclusion The incomplete negative control of wild-type HERG current by truncated mutant L539fs / 47 in HEK293 cells was observed.