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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。
The murine IL-33 gene was cloned into eukaryotic expression plasmid and transfected into COS-7 cells to detect its expression. The total RNA was extracted from the lung of C57BL / 6 mice and the mouse IL-33 gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and inserted into pcDNA3.1 / myc-His A The recombinant plasmid pcDNA-3.1-IL-33 was transfected into COS-7 cells. The expression of the target gene was detected by RT-PCR and western blotting. The results showed that the fragment of pcDNA3.1-IL-33 was sequenced and the result was consistent with that of mouse IL-33c. The expression of mRNA and protein was detected after transfection of COS-7 cells. The mouse IL-33 gene cDNA was successfully cloned and its eukaryotic expression plasmid was constructed.