Flag标签MORC2-677丝氨酸位点突变载体构建及表达  

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

作　　者：王桂玲[1] 王孝会[1] 程正国[1] 宋艳艳[1] 李丰[1] 

机构地区：[1]中国医科大学基础医学院细胞生物学教研室教育部细胞生物学重点实验室,中国辽宁沈阳110001

出　　处：《生命科学研究》2012年第1期1-5,共5页Life Science Research

基　　金：国家自然科学基金资助项目(30871294);辽宁省自然科学基金资助项目(201102277)

摘　　要：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.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 Kpn I and Xho I restriction endonuclease. Then, the coding sequence of MORC2 and its mu- tants were acquired from the plasmid peDNA3.1A-MORC2-WT/S677A / $677E and subcloned into pcD- NA3.1-Flag expression vector by Kpn I and Xho I 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.

关 键 词：MORC2 突变体 真核表达载体 基因表达 胃癌细胞 

分 类 号：Q291[生物学—细胞生物学]