[Construction and identification of the eukaryotic expression vector pIRES2-EGFP-IL-1ra-Fcepsilon].

Abstract

The cDNA of IgE constant domain of rat was cloned from the spleen of allergy asthma rat by RT-PCR. The IL-1ra segment was obtained from intermediate vector pBV220-IL-1ra. By overlap extension PCR, the fusion gene IL-1ra-Fcepsilon was cloned, then inserted into the eukaryotic expression plasmid pIRES2-EGFP to obtain a recombinant expression plasmid pIRES2-EGFP-IL-1ra-Fcepsilon. The recombinant expression plasmid was transfected into 293T cells using lipofectamin and instillated into the rat lung through trachea. The expression of IL-1ra-Fcepsilon was identified by Western blot, RT-PCR, and this protein could inhibit the activity of IL-1 in vitro. Green fluorescent protein could be detected in the transfected 293T cells and the rat lungs at different times. The research paved the way for the gene therapy of allergy asthma.