SCN5A基因L812Q突变体细胞模型的建立与细胞定位

Construction of cell model of the SCN5A gene-L812Q mutation and its celluar localization

目的建立SCN5A基因L812Q突变体细胞模型,并细胞定位。方法应用重叠PCR的方法构建SCN5A基因L812Q突变;以p MD19-T载体克隆SCN5A基因L812Q突变体;SCN5A-L812Q通过EcoRⅠ和ACCⅠ限制性内切酶位点插入表达载体质粒p EGFP-C1-h H1中,测序鉴定;将构建好的p EGFP-C1-h H1-L812Q突变型质粒、p EGFP-C1-h H1野生型质粒瞬时转染进入HEK293细胞,通过免疫荧光检测L812Q突变的细胞定位;将p EGFP-C1-h H1、p EGFP-C1-h H1-L812Q和p EGFP-C1-h H1+p EGFP-C1-h H1-L812Q分别转染进入HEK293细胞,用Western blot方法分析L812Q突变体的表达。结果测序结果显示p EGFP-C1-h H1-L812Q表达载体构建成功。荧光检测验证,野生型SCN5A主要定位在细胞膜上,而突变型定位在细胞质中。通过Western blot分析发现,突变型通道蛋白在细胞质中的表达量高于野生型。结论本研究成功构建了SCN5A基因突变载体p EGFP-C1-h H1-L812Q,证明SCN5A基因L812Q突变型主要定位在细胞质中。

Objective To establish the cell model of the SCN5A gene-L812Q mutation and to explore its cell localization. Methods The overlapping derivative PCR was used to construct the L812 Q mutation of SCN5 A gene,and the L812 Q of SCN5 A gene was cloned into p MD19-T simple vector. Furthermore,L812 Q was inserted into the p EGFP-C1-h H1 vector between the EcoRⅠ and ACCⅠ restriction enzyme sites and then verified by sequencing. The p EGFP-C1-hH1-L812Q and pEGFP-C1-hH1 vector were transfected into HEK293 cells respectively,the cell location of L812 Q mutation was verified using immunofluorescence assay. The pEGFP-C1-hH1,pEGFP-C1-hH1-L812Q and pEGFP-C1-hH1＋pEGFP-C1-hH1- L812Q vector were transfected into HEK293 cells respectively,and then the expression of L812Q was determined by Western blot. Results The p EGFP-C1-hH1-L812Q expression vector was successfully constructed. Wild type SCN5 A was mainly located on the cell membrane and mutant type SCN5 A was located in the cytoplasm. Western blot results showed that the expression level of L812 Q mutation was higher than that of wild type in cytoplasm. Conclusion The p EGFP-C1-hH1-L812Q expression vector is successfully constructed. The L812 Q mutant type of SCN5 A gene is located in the cytoplasm.