重组大鼠pEGFP-N1-IGF-1基因表达质粒的构建

目的构建胰岛素样生长因子1基因的真核表达质粒(pEGFP-N1IGF-1),为基因治疗脊髓损伤(SCI)提供前提。方法应用反转录聚合酶链反应(RTPCR)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(IGF1)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(EGFP)报告基因的真核表达载体pEGFPN1上,以构建重组质粒pEGFPN1IGF1。结果实验从大鼠肝脏组织中提取总RNA,以RTPCR方法获取编码IGF1基因的全序列cDNA。构建IGF1cDNA真核表达质粒时将能发出绿色荧光的EGFP报告基因融合在IGF1基因,并经酶切后DNA电泳鉴定及DNA测序证实结果。结论构建重组质粒pEGFPN1IGF1成功,实验中将能发出绿色荧光的EGFP报告基因融合在IGF1基因的3′端,并以编码柔软肽段的核苷酸连接,既保留了IGF1的神经营养活性,又便于基因治疗中可检测到蛋白表达。

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

Objective To construct the recombinant plasmid pEGFP-N1-IGF-1 for spinal cord injury (SCI) with gene therapy.Methods The cDNA encoding the mature rat insulin-like growth factor 1 was isolated by using 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.Results WTBZ]The cDNA encoding the mature rat insulin-like growth factor-1 (IGF-1) was successfully isolated by using 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-N1-IGF-1 was exactly confirmed with DNA sequencing and DNA electrophoresis after enzyme-incise.Conclusion The expression vector of recombinant plasmid pEGFP-N1-IGF-1 is successfully constructed,and could play an important role in gene therrapy of SCI.