鹅γ-干扰素基因真核表达载体的构建及在幼仓鼠肾细胞中的表达

Construction of goose gamma-interferon gene eukaryotic expression vector and its expression in BHK-21 cell lines

将克隆在pMD18-T载体中的鹅IFN-γ基因插入真核表达载体pcDNA3.1(+),得到真核表达质粒pcDNA3.1(+)-IFN-γ,经测序鉴定后,在室温条件下,质粒pcDNA3.1(+)-IFN-γ浓度20μg/mL,体积400μL,幼仓鼠肾细胞(BHK-21)细胞密度1×106/mL,电穿孔仪参数设为电压425 V、电容25μF、连续脉冲2次转染BHK-21细胞,待细胞贴壁后经过浓度为600μg/mL的G418连续筛选14 d后,出现抗性细胞克隆,将阳性BHK-21细胞上清液进行Western-blot鉴定,检测结果证明鹅IFN-γ在BHK-21细胞中得到表达。

The IFN y gene was inserted into the eukaryotic expression vector pcDNA3, 1（＋）,and the eukaryotic expression plasmid pcDNA3. 1 （＋） IFN -γ was constructed and identified by sequencing. Electroporation instrument was set to 425 V of voltage and 25 μF of capacitor,then pulsed 2 times consecutively under room temperature. 20μg/mL plasmid pcDNA3.1（ ＋）- IFNγ was transformed into 1 ×10^6 cells/mL BHK 21 in 400 μL of volume. After consecutive selection by 600 μg/mL G418 for 14 days, resistant cell clones were selected, and the cell supernatants were identified by Western-blot test. The results indicated that goose IFN-γ was expressed in BHK-21 cells.