犬MC4R突变体D90N真核表达载体的构建及表达  

Construction and Expression of Canis MC4R-D90N Mutation in Eukaryotic Cells

作　　者：魏嘉[1] 王光川[2] 武洁[3] 张轶博[4] 宋慧娟[1] 巴彩凤[2] 

机构地区：[1]辽宁医学院科学实验中心,辽宁锦州121001 [2]辽宁医学院实验动物中心,辽宁锦州121001 [3]辽宁医学院病理教研室,辽宁锦州121001 [4]辽宁医学院免疫与病原生物学教研室,辽宁锦州121001

出　　处：《安徽农业科学》2010年第12期6118-6121,6124,共5页Journal of Anhui Agricultural Sciences

基　　金：辽宁省科技基金项目(2007408001-6)

摘　　要：[目的]构建犬黑皮质素受体4(MC4R)突变体D90N真核表达载体,并在MDCK细胞中进行表达。[方法]以犬MC4R基因组DNA为模板,应用重叠PCR扩增MC4R突变体D90N目的基因,将扩增产物进行T-A克隆;酶切、测序鉴定成功后将目的基因亚克隆到真核表达载体pcDNA3.1-myc-His/A。重组体pcDNA3.1-myc-His/A-cMC4R-D90N经酶切和测序鉴定;采用FuGENEHD介导转染技术将重组体导入MDCK细胞;转染后继续培养72.0h,提取细胞内总RNA,用RT-PCR鉴定目的基因的表达;提取细胞总蛋白,用WesternBlot检测蛋白表达。[结果]构建带标签的pcDNA3.1-myc-His/A-cMC4R-D90N重组真核表达载体,测序结果显示含有D90N突变位点。RT-PCR和Western Blot检测到目的基因表达。[结论]成功构建犬真核表达载体pcDNA3.1-myc-His/A-cMC4R-D90N,重组体能在MD-CK细胞中表达。[Objective] The research aimed to construct the expressional vector with myc and His tags of canis MC4R-D90N gene and investigate its expression in MDCK cells.[Method] Using Canis genome DNA as template,the CDS fragment of MC4R-D90N was amplified by overlap PCR gene and then cloned into pMD18-T vector.After correct identification,the target gene was subcloned into pcDNA3.1-myc-His/A vector.The recombinant pcDNA3.1-myc-His/A-Canis MC4R-D90N was digested by double restriction enzymes and sequenced.Then it was transfected into the MDCK cells by FuGENE HD transfection reagent.After 72.0 hours culture,the expression of Canis MC4R-D90N gene was analyzed by RT-PCR and Western blot.[Result] The recombinant pcDNA3.1-myc-His/A-Canis MC4R-D90N mutation was achieved.MDCK cells that brought recombinant plasmid of D90N mutation were obtained by transfection reagent.The target sequence of Canis MC4R-D90N was amplified by RT-PCR and the expression of fusion protein was detected.[Conclusion] The construction and the expression of eukaryotic plasmid pcDNA3.1-myc-His/A-cMC4R-D90N were achieved successfully.And the recombination could be expressed in MDCK cells.

关 键 词：犬 黑皮质素受体4 真核表达载体 MDCK细胞 D90N 

分 类 号：Q786[生物学—分子生物学]