SCN3A基因突变型表达载体的构建及在HEK 293细胞中的表达

Construction of SCN3A gene mutant expression vector and its expression in HEK 293 cells

目的体外构建含有SCN3A基因mRNA片段的质粒pCMV_6-XL_4-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pCMV_6-XL_4-SCN3A-N302S,并在基因水平上验证了能在HEK293细胞中表达。

Objective To construct the mutant N302S （c.905A〉G） of plasmid pCMV6-XLa-SCN3A containing the SCN3A gene mRNA fragment in vitro,and provide experimental basis for further functional studies. Methods The 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. Results Recombinant 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. Conclusion The SCN3A gene mutant eukaryotic expression vector pCMV6-XL4-SCN3A-N302S was successfully constructed and expressed in HEK293 cells at the gene level.