重组人β-防御素基因在COS-7细胞的转染表达

Recombinant Human β-defensin-2 Gene Expression in Transfected COS-7 Cells

为探索建立持续稳定表达 h BD- 2的哺乳类动物型工程细胞的可能性 ,作者研究构建真核重组表达载体 p CMV.tag2 B/h BD- 2以进行 COS- 7细胞转染表达实验。将本室克隆获得的 h BD- 2全长 c DNA片段插入带有报告基因的真核表达质粒 p CMV.tag2 B构建出 h BD- 2的重组表达载体 p CMV.tag2 B/h BD- 2 ,并经 DNA测序证明h BD- 2 c DNA片段的插入方向和其全长 c DNA的碱基组成顺序均准确无误。通过脂质体转染法将 p CMV.tag2 B/h BD- 2导入 COS- 7细胞。RT- PCR法和免疫印迹法分别从 m RNA水平和蛋白质水平检测到被转染的 COS- 7细胞系能有效表达 h BD- 2。

This study was directed to establish a eukaryotic engineering cell line which can express human β defensin gene constitutively. The eukaryotic recombinant vector of human β defensin 2 gene pCMV.tag2B/hBD 2 was constructed with full hBD2 cDNA isolated from psoriatic skin and transfected into COS 7 cells by using FuGENE TM 6 Transfection Reagent. DNA sequencing confirmed the cDNA sequence and the orientation of the insert. The transfected cells were selected with the DEME medium containing high concentration antibiotic G418 (100μg/ml). To determine the expression of hBD2 gene at mRNA peptide levels, reverse transcription polymerase chain reaction (RT PCR) with T3/T7 primers and Western Blot were performed respectively. The results showed that COS 7 cells transfected by the recombinant pCMV.tag2B/hBD2 could express hBD2 gene effectively.