构建重组大鼠pEGFP-N1-IGF-1基因表达质粒

Construction of recombinant gene expression plasmid pEGFP-N1-IGF-1 in rats

目的:构建胰岛素样生长因子1基因的真核表达质粒(pEGFP-N1-IGF-1),为基因治疗脊髓损伤提供前提。方法:实验于2005-04/06在上海基康生物公司实验室完成。应用反转录-聚合酶链反应方法从大鼠肝脏组织总RNA中提取并扩增胰岛素样生长因子1基因的全长cDNA,并将该基因连接克隆到含有增强型绿色荧光蛋白报告基因的真核表达载体pEGFP-N1上,以构建重组质粒pEGFP-N1-IGF-1。结果:实验从大鼠肝脏组织中提取总RNA,以反转录-聚合酶链反应方法获取编码胰岛素样生长因子1基因的全序列cDNA。构建胰岛素样生长因子1cDNA真核表达质粒时将能发出绿色荧光的增强型绿色荧光蛋白报告基因融合在胰岛素样生长因子1基因,并经酶切后DNA电泳鉴定及DNA测序证实。结论:构建重组质粒pEGFP-N1-IGF-1成功,实验中将能发出绿色荧光的增强型绿色荧光蛋白报告基因融合在胰岛素样生长因子1基因的3'端,并以编码柔软肽段的核苷酸连接,即保留了胰岛素样生长因子1的神经营养活性,又便于基因治疗中可检测到蛋白表达。

AIM： To construct the eukaryotic expression plasmid of insulin-like growth factor-1 （pEGFP-NI-IGF-1）, so as to provide basis for gene treatment of spinal cord injury. METHODS： The experiment was conducted in Shanghai GeneCore Biotechnologies laboratory from April 2005 to June 2005. The cDNA of amplified IGF-1 gene extracted from the total RNA of rat livers by using RT-PCR method with total RNA. The IGF-1 gene was cloned into eukaryotic expression vector pEGFP-N1 of EGFP reported gene encoding green fluorescence protein in the form of fusion protein, so as to rebuild pEGFP-N 1 -IGF- 1. RESULTS： The total cDNA encoding of 1GF-1 gene was obtained by RT-PCR method with total RNA extracted from rat liver. IGF-1 gene was cloned into eukaryotic expression vector pEGFP-N1 of EGFP reported gene encoding green fluorescence protein in the form of fusion protein. IGF-1 gene in the recombinant plasmid pEGFP-NI-IGF-1 was exactly confirmed by DNA sequencing and DNA electrophoresis after enzyme restriction. CONCLUSION： The expression vector of recombinant plasmid pEGFP-N1- IGF-1 is successfully constructed. The 3＇ end of IGF-1 gene is fused with eukaryotic expression vector pEGFP-N1 of EGFP reported gene encoding green fluorescence protein, and connected by the nucleotide of encoding velvet peptide segment,ie, the neurotrophic activity of IGF-1 is kept, which can contribute to the detection of protein expression in gene therapy.