pIRES2-EGFP-IL-1ra-Fcε真核表达载体的构建及鉴定

CONSTRUCTION AND IDENTIFICATION OF THE EUKARYOTIC EXPRESSION VECTOR pIRES2-EGFP-IL-1ra-Fcε

采用RT-PCR方法从哮喘大鼠脾细胞中克隆IgE恒定区cDNA,同时从载体pBV220-IL-1ra中克隆IL-1ra基因,利用重叠延伸PCR技术构建IL-1ra-Fcε融合基因。将其克隆入真核表达载体pIRES2-EGFP,以脂质体法转染293T细胞,同时采用气管滴注方式滴注大鼠肺部。经Western blot、RT-PCR及荧光显微镜观察此融合基因在293T细胞及大鼠肺组织中实现了表达,为过敏性哮喘基因治疗奠定了基础。

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-Fcε was cloned, then inserted into the eukaryotic expression plasmid pIRES2-EGFP to obtain a recombinant expression plasmid pIRES2-EGFP-IL- 1ra-Fcε. The recombinant expression plasmid was transfected into 293T ceils using lipofectamin and instillated into the rat lung through trachea. The expression of IL-1ra-Fcε 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.