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毛白杨PtoeIF5A4的原核表达、纯化及鉴定

, PP. 83-87

Keywords: 毛白杨,PtoeIF5A4,原核表达,蛋白纯化

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Abstract:

为获取大量高纯度的毛白杨PtoeIF5A4蛋白以便研究其特性和生物学功能,以pGEM-TEasy-PtoeIF5A4质粒为模板,PCR扩增毛白杨PtoeIF5A4基因的cDNA编码区,测序正确后,经酶切将其插入到原核表达载体pET28a上,然后将表达载体转入大肠杆菌表达菌株BL21(DE3),经不同浓度IPTG诱导培养后发现,0.1mmol/LIPTG,28℃诱导4h时目的蛋白最多,且该重组蛋白主要以可溶性的形式存在。采用亲和纯化从可溶性蛋白中纯化到大小为18kD的蛋白条带。Westernblotting分析发现亲和纯化所得蛋白可以与His单克隆抗体发生免疫交叉反应,表明纯化所得蛋白是PtoeIF5A4重组蛋白。

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