人GM-CSF基因真核表达载体构建与表达  

Construction and Expression of Eukaryotic Expression Vector Containing Human GM-CSF Gene

作　　者：卢燕茹[1] 江振友[1] 

机构地区：[1]暨南大学医学院微生物学与免疫学教研室,广东广州510632

出　　处：《中山大学学报（医学科学版）》2007年第5期590-593,597,共5页Journal of Sun Yat-Sen University:Medical Sciences

基　　金：广东省中医药局科研基金(1060117);广东省医学科研课题(A2005340)

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。[Objective] To construct eukaryotic expression plasmid plGM-CSF based on gene adjuvant hGM- CSF, and express in Hela ceils. [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 plRES to construct eukaryotic expression plasmid plGM-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 plGM-CSF containing gene adjuvant was successfully constructed and expressed in Hela cells effectively, lays a foundation on constructing bicstronic eukaryotic co-expression plasmid.

关 键 词：真核表达载体 HGM-CSF 基因佐剂 双顺反子 

分 类 号：R392.9[医药卫生—免疫学]