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禾谷镰饱菌β-微管蛋白基因的克隆及在原核细胞中的表达

DOI: 10.7685/j.issn.1000-2030.2011.01.009, PP. 51-56

Keywords: 禾谷镰孢菌,β-微管蛋白基因,原核表达,纯化,Western-blot

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Abstract:

分别扩增了禾谷镰孢菌(Fusariumgraminearum)对多菌灵(MBC)敏感菌株(MBCS)的β1-微管蛋白基因(β1-S)和β2-微管蛋白基因(β2-S),及多菌灵中抗菌株(MBCMR)和高抗菌株(MBCHR)的β2-微管蛋白基因(β2-MR和β2-HR),测序正确后连接到pET32a(+)原核表达载体上,转化至大肠杆菌Rosepta感受态中,经IPTG(1mmol.L-1)诱导,得到了N末端融合6×His纯化标签的表达产物。SDS-PAGE电泳分析表明在大肠杆菌中表达了相对分子质量约68×103的蛋白,和预测蛋白大小一致,表达的产物主要以包涵体的形式存在。表达的蛋白经HisTrapTMHPColumns纯化并做了Western-blot验证,结果表明,该蛋白能与抗His标签的单抗发生特异性反应。

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