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pIRES2-EGFP-IL-1ra-Fcε真核表达载体的构建及鉴定 被引量:4

CONSTRUCTION AND IDENTIFICATION OF THE EUKARYOTIC EXPRESSION VECTOR pIRES2-EGFP-IL-1ra-Fcε
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摘要 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.
出处 《分子细胞生物学报》 CSCD 北大核心 2008年第4期309-316,共8页 Journal of Molecular Cell Biology
基金 国家科技支撑计划课题(2006BAI19B05-3)~~
关键词 IL-1ra-Fcε融合基因 转染 真核表达 鉴定 IL-1ra-Fcε fusion gene. Transfection. Eukaryotic expression. Identification
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