中文摘要：

背景：分化型胚胎软骨基因1可调控肿瘤生长、凋亡、衰老相关因子,与肿瘤的发生发展有着重要的联系。目的：构建针对人分化型胚胎软骨基因1的小干扰RNA表达载体。方法：从NCBI中查找人分化型胚胎软骨基因1基因全长mRNA序列,利用Katahdin提供的在线小干扰RNA模板序列设计软件,设计针对分化型胚胎软骨基因1的2条shRNA的DNA模板单链,合成靶向分化型胚胎软骨基因1基因转录可形成茎环结构的寡聚核苷酸,退火后与酶切后的pGreenPuroTM shRNA Cloning and Expression Lentivector质粒连接,在JM-109菌株中扩增,并进行质粒DNA琼脂糖凝胶电泳分析、紫外分光光度计检测、菌落PCR以及测序鉴定。结果与结论：将含有分化型胚胎软骨基因1目标序列25bp的双链DNA插入片段,连接到pGreenPuroTM shRNA Cloning and Expression Lentivector质粒形成重组质粒。质粒DNA琼脂糖凝胶电泳和紫外分光光度计分析结果确认所提质粒纯度较高,可用于后续实验。菌落PCR结果表明产物大小约为170bp,与预期相符。测序结果表明pGreenPuroTM shRNA Cloning and Expression Lentivector质粒已经插入人分化型胚胎软骨基因1的干扰合成片段,无碱基突变。成功构建了靶向分化型胚胎软骨基因1-小干扰RNA表达载体。

英文摘要：

BACKGROUND：Differentiated embryo-chondrocyte expressed gene 1（DEC1） is a basic helix-loop-helix transcription factor,which is closely associated with some malignant cancers.OBJECTIVE：To construct small interfering RNA（siRNA） expression plasmid target to DEC1.METHODS：The mRNA sequence of DEC1 gene was searched from NCBI.Utilize of Katahdin siRNA technology,DEC1-siRNA oligonucletides were inserted into pGreenPuroTM shRNA Cloning and Expression Lentivector,after annealing,then transformed into JM-109.The recombinant plasmid was identified by Agarose gel electrophoresis analysis,ultraviolet spectrophotometer analysis,PCR and DNA sequencing.RESULTS AND CONCLUSION：The recombinant plasmid pGreenPuroTM shRNA Cloning and Expression Lentivector-DEC1 was obtained by connecting 25 bp segment containing DEC1 sequence to pGreenPuroTM shRNA Cloning and Expression Lentivector.Agarose gel electrophorsis analysis and ultraviolet spectrophotometer analysis confirmed that plasmid DNA had higher purity.DNA sequencing showed Targeting siRNA oligonucleotides were correctly inserted into the eukaryotic expression vector pGreenPuroTM shRNA Cloning and Expression Lentivector without base mutation.The interference vector pGreenPuroTM shRNA cloning and expression lentivector-DEC1 was successfully constructed.