先天性长QT综合征KCNQ1基因真核表达载体的构建及表达

Construction of Long QT Syndrome KCNQ1 Gene Eukaryotic Expression Vector and Expression in HEK 293 Cell

目的 在原核表达载体pSP6 4 KCNQ1的基础上 ,构建KCNQ1的真核表达载体。方法 先将pSP6 4 KC NQ1通过限制性酶切得到KCNQ1cDNA ,将后者克隆入 pEGFP N1中 ,构建中间过渡载体 pEGFP KCNQ1;将pEGFP KCNQ1和 pIRES EGFP分别用NheⅠ和EcoRⅠ双酶切 ,把KCNQ1cDNA克隆到 pIRES EGFP ,即构建了pIRES EGFP KCNQ1。然后利用Effectene转染试剂介导将pIRES EGFP KCNQ1转染HEK2 93细胞。 结果 在原核表达载体 pSP6 4 KCNQ1的基础上 ,获得了KCNQ1的真核表达载体 pIRES EGFP KCNQ1,并使其在HEK 2 93细胞中成功表达。结论 该法可成功构建、表达KCNQ1的真核表达载体 ,为进一步的功能研究奠定了基础。

Objective To construct KCNQ1 gene eukaryotic expression vector on the basis of prokaryotic expression vector pSP64-KCNQ1. Methods KCNQ1 cDNA was obtained from pSP64-KCNQ1 by restriction enzyme digestion and inserted into the same restriction site of pEGFP-N1, thus pEGFP-KCNQ1 was constructed. KCNQ1 cDNA was obtained from pEGFP-KCNQ1 and inserted into the same restriction site of pIRES-EGFP in the same way, then pIRES-EGFP-KCNQ1 was constructed. With Effectene transfection reagent, pIRES-EGFP-KCNQ1 was transfected into HEK 293 cells. Results The eurkaryotic expression vector of KCNQ1 gene was constructed and expressed in HEK 293 cells correctly. Conclusion The eukaryotic expression vector expressing KCNQ1 gene could be successfully constructed by this method.