小鼠IL-5真核表达质粒的构建及表达

Construction and Expression of Eukaryotic Expression Vector of Mouse IL-5

[目的]克隆小鼠白细胞介素-5(mIL-5)cDNA构建真核表达质粒,并转染293细胞检测其是否表达。[方法]以小鼠脾脏细胞总RNA,经逆转录-聚合酶链式反应(RT-PCR)扩增小鼠IL-5基因cDNA,酶切后插入pRc-CMV/Fc真核表达质粒中,构建重组真核表达质粒mIL-5/Fc-pRc-CMV,转染293细胞,RT-PCR与ELISA法检测目的基因表达。[结果]Fc-pRc-CMV中插入DNA序列与mIL-5 cDNA一致,重组质粒转染293细胞后,ELISA可检测出质粒能在293真核细胞中有效表达小鼠IL-5目的蛋白。[结论]该研究成功克隆了小鼠IL-5基因cDNA,并构建其真核表达质粒。

[Objective] The study aimed to clone and construct the eukaryotic expression plasmids containing mouse IL-5 cDNA and investigate whether they expressing in 293 cells.[Methods] Total RNA was extracted from mouse spleen to amplify mouse inerleukin-5（mIL-5） cDNA by reverse transcription-polymerase chain reaction（RT-PCR）.After sequence analysis,mIL-5 was cloned into expression vector Fc-pRc-CMV.The recombinant plasmids were transfected into 293 cells and the expression of target gene was detected by RT-PCR and ELISA.[Result] The inserted DNA sequence in Fc-pRc-CMV was identical to mIL-5 cDNA,which was verified correctly by sequencing.The corresponding gene expression was detected in recombinant plasmid transfected 293 cells.[Conclusion] The mouse IL-5 cDNA was successfully cloned and its eukaryotic expression plasmid was constructed.