增强型绿色荧光蛋白原核表达载体的构建及表达

Construction and expression of eukaryotic expression vector pTWIN-EGFP

目的构建以增强型绿色荧光蛋白(EGFP)为标记的原核表达载体,并观察EGFP基因在大肠杆菌中的表达情况。方法据已知的EGFP基因序列,设计引物,并引入NdeⅠ和XhoⅠ酶切位点。采用PCR技术从含有EGFP的质粒pEGFP-C1中克隆EGFP编码序列;将其亚克隆入表达载体pTW IN1,得到以EGFP为标记的原核表达载体pTW IN-EGFP。转化大肠杆菌ER2566菌株,IPTG诱导EGFP基因表达。结果经过IPTG诱导后,细菌培养物在长波紫外线的照射下,发出明亮的绿色荧光。结论成功构建了原核表达载体pTW IN-EGFP,为应用EGFP作为报告基因和筛选标记奠定了实验基础。

Objective To construct the eukaryotic expression vector pTWIN-EGFP and detect the expression of enhanced green fluorescent protein（EGFP） in E. coil. Methods According to the open reading frame of known EGFP gene,a pair of prlmers,which contains two restriction enzyme sites Nde I and Xho I , were designed and synthesized. A specific fragment of EGFP gene was obtained by PCR amplification from pEGFP-C1 containing EGFP gene. Then the PCR products was subclaned into the expression vector pTWIN1, so the pTWIN-EGFP which contained EGFP,as a marker, was constructed. Then the pTWIN-EGFP was transformed into E. coil ER2566 to detect the EGFP. Results Induced by IPTG,the E. coil ER2566 showed brightly green fluorescent when excited by long-wave ultraviolet light. Conclusion The eukaryotic expression vector pTWIN-EGFP was constructed successfully,the experimental foundation for the usage of EGFP as a reporter and marker gene has been established.