【摘要】 【目的】构建以基因佐剂hGM-CSF基因为基础的真核表达质粒pIGM-CSF,并在宫颈癌细胞HeLa中表达与鉴定。【方法】采用聚合酶链反应从载体pORF-hGM-CSF中,扩增出hGM-CSF基因,克隆到双顺反子真核表达载体pIRES的多克隆位点B(MCSB)中,构建pIGM-CSF真核表达载体。然后采用脂质体转染法将其转染HeLa细胞,用RT-PCR在RNA水平和ELISA在蛋白水平检测其在真核细胞中的表达。【结果】所克隆hGM-CSF基因片段经测序完全正确;重组质粒转染Hela细胞后,检测有hGM-CSF表达。【结论】成功构建含基因佐剂真核表达载体pIGM-CSF,并在宫颈癌细胞中能有效表达,为继续克隆目的抗原从而构建双顺反子真核表达质粒奠定了基础。更多还原

【Abstract】 【Objective】 To construct eukaryotic expression plasmid pIGM-CSF based on gene adjuvant hGM-CSF,and express in Hela cells.【Methods】 Plasmid pORF-hGM-CSF was used as template to amplify hGM-CSF gene by PCR,and the product was inserted into multiple cloning site’s B of bicstronic eukaryotic expression vector pIRES to construct eukaryotic expression plasmid pIGM-CSF,then transfected into Hela cell by using liposome.Finally,the hGM-CSF gene expression of protein was detected by RT-PCR from RNA level and ELISA from protein level.【Results】 The length and sequence of the cloned hGM-CSF segment was correct.The hGM-CSF gene could be expressed in transfected Hela cells.【Conclusion】 The eukaryotic expression plasmid pIGM-CSF containing gene adjuvant was successfully constructed and expressed in Hela cells effectively,lays a foundation on constructing bicstronic eukaryotic co-expression plasmid.更多还原