重组犬IFN-γ在HEK293T细胞中的表达

Expression of recombinant canine IFN-γ gene in HEK 293T cells

为了建立一套犬IFN-γ(cIFN-γ)在HEK293T细胞中表达的方法,用伴刀豆球蛋白A(ConA)刺激犬脾细胞,通过RT-PCR方法从脾细胞中扩增犬IFN-γ基因,并将其克隆到pcDNA3.1A载体中,构建的真核表达载体pcDNA3.1A-cIFN-γ经磷酸钙介导转染HEK293T细胞进行表达。结果表明:克隆的犬IFN-γcDNA基因与GenBank上发表的序列一致,同源性100%。表达产物经Western-blot方法检测,证明克隆的犬IFN-γ基因能够在HEK293T细胞中进行表达,并且表达产物能够分泌到细胞外。

To establish the method of canine IFN-γ （cIFN-γ） expression in HEK293T cells, the canine spleen cells were stimulated by concanavalin A. The cDNA encoding cIFN-γ was amplified by RT- PCR from the stimulated cells. Then the cIFN-γ gene was inserted into eukaryotic ex- pression vector pcDNA3.1A. The recombinant pcDNA3.1A - cIFN-γ plasmids were transfected into HEK293T cells mediated by calcium phosphate. Results showed that the sequence of cIFN-γ cDNA amplified was identical to that published in GenBank, and the homology is 100 %. The expressed products were detected by Western - blot, which indicated that cIFN -γ gene could be ex pressed in HEK293T cells and secreted from the cells.