Flag标签MORC2-677丝氨酸位点突变载体构建及表达

构建Flag标签pcDNA3.1-MORC2的677丝氨酸位点突变重组质粒并完成在SGC-7901细胞中表达,更好地研究677丝氨酸位点所发生的功能.首先,构建含有KpnⅠ和XhoⅠ酶切位点Flag标签的pcDNA3.1空载体;再以含有KpnⅠ和XhoⅠ酶切位点的pcDNA3.1A-MORC2-WT野生型质粒为模板,在S677突变位点两侧设计重叠突变引物,进行三轮重叠延伸PCR完成定点突变,获得MORC2全长编码序列的S677位点的定点突变体载体(pcDNA3.1A-MORC2-S677A/S677E);再通过KpnⅠ和XhoⅠ酶切把pcDNA3.1A-MORC2-WT/S677A/S677E各载体定向克隆到已构建好的pcDNA3.1-Flag空载体中,酶切鉴定及测序正确后,转染到SGC-7901细胞中,利用Flag–标签抗体,Western blot检测其各突变载体的表达.成功构建了人Flag标签MORC2全长编码序列S677位点突变体真核表达载体并在SGC-7901细胞中获得带Flag标签融合蛋白表达产物,为进一步研究MORC2的功能奠定了基础.

Construction of the Eukaryotic Expression Plasmid of S677 Mutant MORC2 with Flag Tag and its Expression

The eukaryotic expression plasmids of the pcDNA3.1-MORC2(WT/S677A/S677E) with Flag tag were constructed and explored its expression in SGC-7901 cells,which will facilitate further functional study on MORC2 and its 677 mutant site.First,the pcDNA3.1-Flag vector was acquired with multiple cloning site containing KpnⅠ and XhoⅠ restriction endonuclease.Then,the coding sequence of MORC2 and its mu-tants were acquired from the plasmid pcDNA3.1A-MORC2-WT/S677A / S677E and subcloned into pcD-NA3.1-Flag expression vector by KpnⅠ and XhoⅠ to construct the eukaryotic expression plasmids pcD-NA3.1-Flag-MORC2(WT/S677A/S677E).After the restriction endonuclease digestion and DNA sequencing confirmation,the recombinant plasmids were transfected into SGC-7901 cells and the protein expression were identified by Western blot using flag-tagged antibody.The human MORC2 and its mutants recombinant plasmids with flag tag were obtained successfully and the fusion proteins were expressed successfully in SGC-7901 cell,which provide the basis for the further study on the biology functions of MORC2.