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GST-Smad4融合蛋白的表达与纯化

, PP. 134-138

Keywords: Smad4,原核表达,蛋白纯化

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Abstract:

旨在原核表达Smad4基因,纯化获得GST-Smad4融合蛋白。以人表皮HaCaT细胞的cDNA为模板,利用PCR扩增含有BamHI和SalI酶切位点的Smad4基因;然后将其克隆到pGEX-4T-1原核表达载体中,将正确的重组载体转入大肠杆菌BL21(DE3);用IPTG诱导表达,再利用MagneGSTparticles亲和纯化GST-Smad4融合蛋白;最后通过Westernblot鉴定此融合蛋白。结果显示,成功构建pGEX-4T-1-Smad4原核表达载体;30℃条件下,0.2mmol/L的IPTG能诱导出大量的可溶性GST-Smad4蛋白;经MagneGSTparticles纯化的GST-Smad4蛋白可被Smad4的抗体特异识别。纯化的GST-Smad4蛋白可用于后续的生物学研究。

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