应用RT-PCR构建人表皮生长因子基因真核表达质粒

Construction of the eukaryotic expression plasmid containing human epidermal growth factor gene with signal peptide

目的体外克隆及构建带信号肽的人表皮生长因子(hEGF)基因真核表达质粒。方法设计人表皮生长因子(hEGF)基因和信号肽基因引物,采用RT-PCR技术从胎肾组织总RNA中扩增出hEGF基因和信号肽基因序列,克隆入pGEM-T载体,酶切鉴定和序列分析后经T4连接酶连接并克隆入表达质粒pcDNA3.1(+),再次酶切鉴定和序列分析。结果RT-PCR扩增产物电泳获得约90 bp和180 bp条带,与hEGF及其信号肽cDNA大小符合;克隆入T载体后经序列分析与hEGF及信号肽基因一致,克隆入pcDNA3.1(+)质粒后双酶切获得约230 bp和5.4 kb条带,再次测序与hEGF cDNA一致。结论成功构建带信号肽的hEGF基因的真核表达质粒。

Objective To construct the eukaryotic expression plasmid containing human epidermal growth factor （hEGF） gene with signal peptide （SP）. Methods After two pairs of primers were designed and synthesized, the cDNA fragment of hEGF and SP genes were amplified from total RNAs. The amplified cDNA fragments were cloned into pGEM-T vector. The expression plasmids were verified by double endonuclease digestion and DNA sequence analysis. Results With RT-PCR using two pairs of primers, two bands （ about 90bp and 180bp） were obtained and confirmed as signal peptide and EGF cDNA fragment with electrophoresis analysis and DNA sequencing after cloned into pGEM-T vector. The SP and EGF eDNA fragments were inserted into plasmid peDNA3.1 （ ＋ ）. The bands of 240bp and 5.4kb were ohrained and identified as the full length of SP-EGF cDNA fragment by DNA sequence analysis. Conclusion The eukaryotic expression plasmids containing hEGF gene is successfully constructed.