pEGFP-N1-pAPN-SPC段基因真核表达载体的构建及其在Vero细胞中表达

Construction of Eukaryotic Expression Vector pEGFP-N1-pAPN-SPC and Its Expression in Vero Cells

为了构建能够表达猪pAPN-SPC段的真核细胞表达载体pEGFP-N1-pAPN-SPC,并在Vero细胞中进行表达,本试验从猪肠道黏膜组织基因组中扩增出pAPN-SPC段基因,插到带有EGFP基因的真核细胞表达载体pEGFP-N1中,经Bam HⅠ及EcoRⅠ酶切鉴定正确后转染Vero细胞,用荧光显微镜检测细胞中EGFP的表达,用RT-PCR和Western blot检测pAPN-SPC段基因的表达情况。结果成功构建了真核表达载体pEGFP-N1-pAPN-SPC,且pAPN-SPC段在Vero细胞中得到了有效表达,为研究pAPN-SPC段蛋白对病毒增殖效果的影响奠定了基础。

To construct the eukaryotic expression vector pEGFP-NI-pAPN-SPC, and to express pAPN-SPC in Vero cells,the pAPN-SPC gene was amplified by PCR using mucous membrane of small intestine ge- nomic DNA as a template, and confirmed by DNA sequencing. After enzyme digestion analysis and sequencing,the plasmid was transfected into the Vero cells using lipofectamine. The green fluorescence of EGFP was observed by fluorescent microscopy, and pAPN-SPC protein was found in the plasma and nuclear. A Vero cell strain for stable expression of fusion protein of pEGFP-NI-pAPN-SPC was successfully established, which laid a foundation for further study on function of pAPN-SPC.