【摘要】 克隆小鼠IL-33基因构建其真核表达质粒,并转染COS-7细胞检测其表达。提取C57BL/6小鼠肺组织总RNA,经反转录聚合酶链式反应(RT-PCR)扩增小鼠IL-33基因,酶切后插入pc DNATM3.1/myc-His A构建其真核表达质粒pc DNA-3.1-IL-33,重组质粒转染COS-7细胞,RT-PCR和免疫印迹法(western blotting)检测目的基因表达。结果显示,pc DNA3.1-IL-33中插入的片段序列测定结果与小鼠IL-33c DNA序列一致,重组质粒转染COS-7细胞后检测到相应mRNA及蛋白表达。成功克隆了小鼠IL-33基因c DNA,并构建其真核表达质粒。更多还原

【Abstract】 Mouse IL-33 c DNA was cloned and constructed its eukaryotic expression plasmids and transfected into COS-7 cells to determine its expression. Total RNA of the lung of C57 BL /6 mouse was extracted and amplified mouse IL-33 c DNA by reverse transcription-polymerase chain reaction( RT-PCR). Then the resulting gene fragment was inserted into pc DNATM3. 1 / myc-His A vector after digested by restriction enzyme to construct eukaryotic expression plasmid pc DNA-3. 1-IL-33. The recombinant plasmid was transfected into COS-7 cells. The expression of target gene was detected by RT-PCR and Western blotting. The inserted DNA sequence in pc DNA3. 1-IL-33 was identical to mouse IL-33 c DNA,which was verified correctly by sequencing. The corresponding gene expression was detected in the recombinant plasmid-transfected COS-7 cells. The mouse IL-33 c DNA was successfully cloned and its eukaryotic expression plasmid was constructed.更多还原