小鼠IL-33基因cDNA克隆与真核质粒的构建与表达

Cloning of mouse IL-33 gene cDNA and its eukaryotic plasmid construction and expression

目的克隆小鼠IL-33基因(mIL-33)cDNA构建其真核表达质粒,并转染EL-4细胞检测其表达。方法提取小鼠脑与肺组织总RNA,经逆转录-聚合酶链反应(RT-PCR)扩增小鼠IL-33基因cDNA,酶切后插入pcDNA-3.1构建其真核表达载体pcDNA-mIL-33,重组质粒转染EL-4细胞,RT-PCR与ELISA法检测目的基因表达。结果 pcDNA-mIL-33中插入的DNA序列测定结果与mIL-33 cDNA序列一致,重组质粒转染EL-4细胞后检测到相应基因表达。结论成功克隆小鼠IL-33基因cDNA,并构建其真核表达质粒。

Objective To clone and construct the eukaryotic expression plasmids containing mouse IL-33 cDNA and investigate their expression.Methods Total RNA was extracted from mouse lung and brain tissues to amplify mouse interleukin-33（mIL-33） cDNA by reverse transcription-polymerase chain reaction（RT-PCR）.The eukaryotic expression plasmids pcDNA-mIL-33 was constructed with the insertion of enzyme-digested pcDNA-3.1（＋） vector to construct eukaryotic expression vector pcDNA-mIL-33.The recombinant plasmids were transfected into EL-4 cells and the expression of target gene was detected by RT-PCR and ELISA.Results The inserted DNA sequence in pcDNA-mIL-33 was identical to the mIL-33 cDNA,which was verified correctly by sequencing.The corresponding gene expression was detected in the recombinant plasmid-transfected EL-4 cells.Conclusion The mouse IL-33 cDNA was successfully cloned and its eukaryotic expression plasmids were constructed.