真核表达载体pcDNA3.1-3a的构建及其在神经母细胞瘤SK-N-SH细胞中的表达  

Construction of pcDNA3.1-3a in pcDNA3.1 expression vector and its expression in neuroblastoma cell line SK-N-SH cells

作　　者：秦怀洲 张治国 王举磊 吴琳[2] 李立宏 高国栋 

机构地区：[1]第四军医大学第二附属医院神经外科,西安710038 [2]第四军医大学基础部生物化学与分子生物学教研室,西安710032

出　　处：《中国临床神经外科杂志》2011年第9期541-544,共4页Chinese Journal of Clinical Neurosurgery

摘　　要：目的在pcDNA3.1真核表达载体中构建表达融合myc/his标签的甲基化酶表达载体pcDNA3.1-3a,并在神经母细胞瘤细胞株SK-N-SH细胞中进行验证,以期获得可表达甲基化酶催化结构域的融合基因。方法以甲基化酶的pcDNA4.0-GBD-3a-myc/his质粒为模板,通过PCR的方法扩增获得融合有myc/his标签序列的目的区段--甲基化酶催化结构域3a,将其克隆入真核表达载体pcDNA3.1;以EcoRⅠ、EcoRⅤ双酶切和PCR方法对构建的表达载体进行鉴定,并通过测序证明载体构建正确,同时检测甲基化酶3a在SK-N-SH细胞中的表达。结果通过PCR方法获得了含有甲基化酶催化结构域的真核表达载体pcDNA3.1-3a,并通过免疫印记方法验证了在神经母细胞瘤细胞株SK-N-SH细胞样品中用抗his标签抗体可以特异性识别目的蛋白。结论正确构建了甲基化酶表达载体pcDNA3.1-3a,其可在神经母细胞瘤细胞株SK-N-SH细胞中表达。Objectives To clone and express the pcDNA3.1-3a gene fused with myc-6his tag in pcDNA3.1 eukaryotic expression system and to identify the protein of fused expression in order to derive the fused gene which may express the methyltransferase catalysis domain.Methods The methyltransferase catalysis domain 3a fused with myc-6his was derived from pcDNA4.0-GBD-3a-myc-6his plasmid with PCR amplification,and cloned into the eukaryotic expression vector pcDNA3.1.The vector was identified by PCR and double enzymes digestion.The expression product was analyzed with Western blot in Neuroblastoma cell line SK-N-SH cells.Results The pcDNA3.1-3a gene was amplified with PCR,and the interest protein could be specifically recognized by anti-myc-tag monoclonal antibody in the SK-N-SH cells.Conclusion The vector pcDNA3.1-3a may be cloned into the eukaryotic expression vector correctly and expressed successfully in Neuroblastoma cell line SK-N-SH cells.

关 键 词：DNA甲基化酶 真核表达载体 神经母细胞瘤 

分 类 号：R730.264[医药卫生—肿瘤] R730.231[医药卫生—临床医学]