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绿色荧光蛋白原核表达载体的构建

DOI: 10.3969/j.issn.1000-7091.2013.03.014, PP. 73-76

Keywords: 绿色荧光蛋白,原核表达载体,pMAL-c2X,筛选标记

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Abstract:

为开发以绿色荧光蛋白(GFP)为基因的原核表达载体,根据NCBI基因序列设计引物,通过PCR扩增获得GFP编码基因,经限制性核酸内切酶消化后将GFP定向克隆至原核表达载体pMAL-c2X中malE基因下游的EcoRⅠ与HindⅢ位点之间,与malE基因融合为一个表达框。重组产物转化E.coliTB1感受态细胞,在添加有50mg/mLAMP、10μmol/LIPTG的LB平板上于37℃培养12~16h后放置于25~30℃的培养箱中继续培养4~6h,365nmUV照射下挑取转化克隆,经扩大培养后用IPTG诱导GFP的表达并用SDS-PAGE检测表达效率。结果表明,融合在麦芽糖结合蛋白下游的GFP编码基因可以在宿主细胞中有效表达,在365nmUV照射下,可以直接从加有50mg/mLAMP、10μmol/LIPTG的LB平板上挑取发绿色荧光的重组克隆,SDS-PAGE分析结果显示其扩大培养物经IPTG诱导后可高效表达GFP。该结果为进一步构建以GFP为标记基因取代抗生素筛选标记的原核表达载体提供了切实的试验依据。

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