SCN3A 基因突变型表达载体的构建及在 HEK 293 细胞中的表达

目的 体外构建含有SCN3A基因mRNA片段的质粒pCMV₆-XL₄-SCN3A的突变体N302S(c.905A>G),为进一步的功能研究奠定实验基础。方法设计带突变位点的引物,以野生型质粒为模板进行定点诱变,构建突变质粒。质粒经扩增纯化后瞬时转染到HEK293细胞中,24 h后用RT-PCR方法验证目的基因的表达。结果重组质粒经酶切鉴定及测序证实突变成功。突变质粒转入HEK293细胞系24 h后,RT-PCR可以检测到目的基因的表达。结论本实验成功地构建了SCN3A 基因突变型真核表达载体pCMV₆-XL₄-SCN3A-N302S,并在基因水平上验证了能在HEK293 细胞中表达。

Construction of SCN3A gene mutant expression vector andits expression in HEK 293 cells

Objective To construct the mutant N302S (c.905A>G) of plasmid pCMV₆-XL₄-SCN3A containing the SCN3A gene mRNA fragment in vitro,and provide experimental basis for further functional studies.MethodsThe primers with mutation sites were designed and the mutant plasmids were constructed by site-directed mutagenesis using wild-type plasmids as templates.The recombinant plasmid was transiently transfected into HEK293 cells,and the expression of the target gene was verified by RT-PCR after 24 hours.ResultsRecombinant plasmid digestion and sequencing confirmed that the mutation was successful.The expression of the target gene was detected by RT-PCR after transfection of recombinant SCN3A eukaryotic expression plasmid into HEK293 cell line.ConclusionThe SCN3A gene mutant eukaryotic expression vector pCMV₆-XL₄-SCN3A-N302S was successfully constructed and expressed in HEK293 cells at the gene level.