摘要： 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.coli TB1感受态细胞,在添加有50 mg/mLAMP、10μmol/L IPTG的LB平板上于37℃培养12～16 h后放置于25～30℃的培养箱中继续培养4～6 h,365 nmUV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365 nm UV照射下,可以直接从加有50 mg/mLAMP、10μmol/L IPTG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。

关键词: 绿色荧光蛋白, 原核表达载体, pMAL-c2X, 筛选标记

Abstract: The green fluorescent protein( GFP) exhibits bright green fluorescence when exposed to light in the blue to ultraviolet range and has no toxicity on host cells． In cell and molecular biology， the GFP is extensively used as a reporter or detection． In this paper， the GFP encoding sequence was proliferated by PCR under the direction of the primers designed according to the sequence obtained from NCBI website． Then the PCR product was inserted downstream the malE( maltose-binding protein) encoding gene between the EcoRⅠ and Hind Ⅲ sites after restriction endonuclease digestion，by which the gfp was fused with malE into one open reading frame． The recombinant prokaryotic expression vector of GFP was employed to transform the competent cells of E． coli TB1． The transformation mixture was cultured at 37 ℃ for 12 - 16 h on the LB plates with 50 mg /mL AMP and 10 μmol /L IPTG，and then cultured at 25 - 30 ℃ for 4 - 6 h on the same plate． The expression of GFP in TB1 was analysed by UV detection prior to SDS-PAGE． The GFP encoding gene infused dowmstream the malE gene can be expressed with green fluorescent excited by 365 nm UV under which the recombinant TB1 clone can be screened conveniently． Further analysis of SDS-PAGE showed that the GFP encoding gene was over expressed in E． coli host TB1． This result showed that the prokaryotic expression vector of GFP was successfully constructed and the malE fusion GFP could be expressed in high-level，which strongly supported the construction of the prokaryotic fusion expression vector with GFP as reporter and detection marker．

Key words: GFP, Prokaryotic expression, pMAL-c2X vector, Screening marker

中图分类号: 

• Q78