人类胰岛素基因的克隆及其真核表达载体的构建  

Cloning of Human Insulin Gene and Construction of Eukaryotic Expression Vector pEGFP-N1-INS

作　　者：索剑飞[1] 雷雪芹[1] 徐廷生[1] 赵绍杰[2] 韦光辉[1] 

机构地区：[1]河南科技大学动物科技学院,河南洛阳471003 [2]河南科技大学第二附属医院,河南洛阳471003

出　　处：《河南科技大学学报（自然科学版）》2010年第2期77-80,共4页Journal of Henan University of Science And Technology:Natural Science

基　　金：河南省基础与前沿计划项目(072300430010)

摘　　要：从胎儿胰腺细胞中提取出总RNA作为模板,用特异性引物和RT-PCR法扩增INS基因的cDNA,PCR产物T-A克隆到T载体构建中间重组体pUC57-INS,其测序结果和Genbank公布的序列一致。HindIII和BamHI双酶切pUC57-INS后,将INS基因亚克隆到真核表达载体pEGFP-N1中,经PCR扩增、酶切分析和序列测定后证实了重组表达质粒pEGFP-N1-INS构建成功。这将为转人类胰岛素基因动物模型的建立及人类糖尿病的基因治疗奠定必要的基础。Cellular total RNA was extracted from human fetus pancreas as a template.The human INS cDNA was synthesized by RT-PCR with the specific primers.The human INS cDNA fragment directly cloned into T vector pUC-57 to generate middle recombinant.The resultant sequence was completely consistent with the Genbank published sequence.After PCR,restriction endonuclease Hind III and BamH I double digested it,the target fragment was subcloned into eukaryotic expression vector pEGFP-N1.The recombinant plasmid was verified with PCR,restriction analysis and sequencing.The eukaryotic expression vector pEGFP-N1-INS is constructed successfully.It is possible to study transgenic animal and gene therapy with INS.

关 键 词：人类胰岛素基因 克隆 真核表达载体 增强型绿色荧光蛋白 

分 类 号：Q782[生物学—分子生物学] Q819